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[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "biochemistry and chemical biology" ]

[ [ "As animals rely on limited energy resources , they require regulatory mechanisms to decide how to best invest available energy and biomaterial .", "Some of the most extreme changes of energy expenditure are observed in so-called semelparous animals that reproduce only once and typically die afterwards .", "In both vertebrate and invertebrate representatives of semelparous animals , the switch to the reproductive state is accompanied by a massive breakdown of somatic tissue , in favor of forming and nourishing germ cells: migrating salmon break down about 50% of their white muscle mass while their gonad mass multiplies around six-fold during maturation ( Mommsen , 2004 ) .", "Likewise , female squids exhibit almost complete histolysis of mantle muscle ( Jackson and Mladenov , 1994 ) , and in mature female squids up to ~60% of the total body mass at spawning consists of eggs ( Rodrigues et al . , 2011 ) .", "Similarly , in the marine annelid worm Nereis virens , oocytes represent up to about 40% of the body mass of the spawning adult ( Hoeger et al . , 1999 ) .", "Insight into the molecules regulating these extreme shifts of energy expenditure has remained limited ( Cardon et al . , 1981; Mommsen , 2004; Hébert Chatelain et al . , 2008 ) .", "This can largely be attributed to the lack of an appropriate laboratory model system amenable to molecular research .", "In this study , we explore the marine annelid Platynereis dumerilii , a semelparous animal that can easily be reared in the laboratory ( Hauenschild and Fischer , 1969; Fischer and Dorresteijn , 2004 ) and has been established as a molecular model system over the last decade ( reviewed in Zantke et al . , 2014 ) .", "A series of studies show that Platynereis has retained various ancient-type characteristics ( Arendt et al . , 2004; Raible et al . , 2005; Denes et al . , 2007; Tessmar-Raible et al . , 2007; Keay and Thornton , 2009; Christodoulou et al . , 2010 ) , compatible with the notion that the hormones present in this species are also more broadly comparable with other clades .", "In Platynereis , the critical switch to the reproductive life stage has been linked to a hormonal activity released by the medial brain .", "This brain hormonal activity – also termed nereidin – was found to repress sexual maturation and to promote growth of the trunk and posterior regeneration ( Figure 1A ) : Decerebration of Platynereis leads to an accelerated sexual maturation and to the loss of regenerative abilities , whereas re-implantation of a juvenile head with neuroendocrine activity is able to reverse these effects ( Hauenschild , 1956a , 1956b , 1960 , 1966 ) .", "In the related species Nereis diversicolor , it could be shown that implantation of ganglia from immature animals into the coelomic cavity of maturing animals , reversed the effects of the decreasing nereidin concentration .", "In particular , germ cells were resorbed and the ability to regenerate lost segments was re-established ( Golding and Yuwono , 1994 ) .", "These findings highlight the exceptional importance of neuroendocrine signals in semelparous reproduction and suggest that the underlying molecular mechanisms are conserved across different annelids ( Hauenschild , 1956b , 1959; Golding , 1967a , 1967b; Durchon and Porchet , 1970; Hofmann , 1976; Cardon et al . , 1981; Golding , 1983; Golding and Yuwono , 1994 ) . 10 . 7554/eLife . 17126 . 003Figure 1 . Vitellogenin expression in eleocytes is a quantitative measure for the maturation stage of Platynereis , allowing for the establishment of a bioassay to purify the enigmatic brain hormone , nereidin .", "( A ) Scheme summarising the critical role of the brain hormone nereidin in energy expenditure , as derived from classical experiments: Before maturation ( left ) , high nereidin levels sustain somatic growth , but repress reproduction; drops in nereidin activity levels initiate the generation of germ cells and sexual maturation ( right ) .", "( B ) Eleocytes in the worm’s coelom have a central role in reproductive commitment , as they synthesise the yolk protein precursor Vitellogenin ( Vtg ) ; after release into the coelomic fluid , Vtg is endocytosed by the developing oocytes , leading to oocyte growth .", "( C ) Expression levels of vtg in eleocytes are significantly up-regulated during maturation , confirming vtg as a suitable molecular marker to quantify maturation state .", "qRT-PCR quantification of vtg levels in eleocytes sampled at different stages of sexual maturation ( as assessed by oocyte diameter ) .", "The increase in vtg transcripts from premature eleocytes ( oocyte diameter: 50 and 60 µm ) to those from mature eleocytes ( oocytes diameter: 170 µm ) is evident; expression levels were calculated against the arithmetic mean of the reference genes cdc5 and rps9 , and normalised to the expression of vtg in eleocytes from animals with an oocyte diameter of 50 µm .", "Statistical significance was tested an one-way ANOVA .", "***p<0 . 001 .", "n: number of biological replicates .", "( D ) Resulting bioassay for the purification of nereidin from head extracts: primary cell cultures were derived from coelomic cells of premature animals; vtg levels were quantified after overnight incubation with different fractions of head extract to determine those fractions containing significant inhibitory activity ( nereidin ) .", "Data for panel ( C ) provided in Figure 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 00310 . 7554/eLife . 17126 . 004Figure 1—source data 1 . Data for the graphs in Figure 1C , Figure 1—figure supplement 2A , Figure 1—figure supplement 2B ( vtg expression levels over the time course of maturation ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 00410 . 7554/eLife . 17126 . 005Figure 1—source data 2 . Alignment file for the phylogenetic tree of Vitellogenins ( Figure 1—figure supplement 1B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 00510 . 7554/eLife . 17126 . 006Figure 1—figure supplement 1 . Identification of a Platynereis Vitellogenin orthologue .", "( A ) Domain analysis for the protein encoded by the identified Platynereis cDNA ( top ) as well as chicken Vtg2 ( middle ) and mosquito Vit1 ( bottom ) reveals that Platynereis Vtg shares three conserved domains with other bilaterian Vitellogenins that are arranged in the same order: a Lipoprotein N-terminal Domain ( LPD-N , SMART accession SM000638 , blue ) , a DUF1943 domain ( DUF , SMART accession SM001169 , green ) , and a van Willebrand factor D domain ( VW , SMART accession SM000216 , red ) .", "( B ) Molecular phylogenetic analysis supports the orthology of Platynereis Vtg with representatives of other invertebrate and vertebrate Vitellogenins .", "Maximum likelihood tree constructed with IQtree 1 . 3 . 12 ( Minh et al . , 2013; Nguyen et al . , 2015 ) , using apolipophorins as an outgroup .", "By parameter optimisation , the LG+F+I+G4 substitution model was selected .", "Numbers at each node show confidence levels derived from 1000 replicates .", "Sequences accessions: Gallus Vtg2: NP_001026447 . 1; Xenopus VtgA2: P18709 . 1; Apis Vit1: Q868N5 . 1; Aedes Vit1: Q16927 . 2; Capitella Vtg: ELU00944 . 1; Haliotis Vtg: BAF98238 . 1; Platynereis Vtg: KU756287 ( this study ) ; Pecten Vtg: CAQ06469 . 2; Crassotrea Vtg: EKC30345 . 1; Xenopus ApoB: XP_002934538 . 2; Gallus ApoB: NP_001038098 . 1; Crassotrea ApoLp: EKC20363 . 1; Drosophila ApoLp: Q7KTG2; Aedes ApoLp: Q17BE3 .", "Alignment file for panel ( B ) provided as Figure 1—source data 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 00610 . 7554/eLife . 17126 . 007Figure 1—figure supplement 2 . Irrespective of the chosen reference gene , vitellogenin is regulated over the course of maturation . The figure shows the same data as Figure 1C , but with the results for the individual reference genes plotted separately .", "In the range relevant for the bioassay ( early premature stage ) , the observed relative expression of vitellogenin ( vtg ) in eleocytes is independent of the reference gene used for normalisation; differences in levels close to spawning are likely caused by the divergent role of rps9 and cdc5 in protein translation and the cell cycle , respectively .", "( A ) rps9 used as reference gene for normalisation .", "The vtg expression levels relative to the expression in eleocytes from animals with an oocyte diameter of 50 µm were 6 . 20 , 159 . 81 and 453 . 86 for eleocytes from animals with oocyte diameters of 60 , 100 and 170 µm , respectively .", "( B ) cdc5 used as reference gene for normalisation .", "The vtg expression levels relative to the expression in eleocytes from animals with an oocyte diameter of 50 µm were 6 . 36 , 212 . 01 and 225 . 80 for eleocytes from animals with oocyte diameters of 60 , 100 and 170 µm , respectively .", "Statistical test as in Figure 1C .", "****p<0 . 0001 .", "Data for panels ( A ) and ( B ) provided in Figure 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 007 Although nereidin activity was recognised as a critical cue more than 60 years ago , the molecular identity of nereidin still remains elusive .", "However , the results of classical experiments allow for several hypotheses concerning the function and molecular properties of this key brain hormone:", "( i ) When present , nereidin interferes with a critical step required for the animals to enter the cost-intensive process of maturation .", "( ii ) The effective hormone concentration decreases from young animals to mature animals , such that the drop in nereidin concentration allows maturation to proceed ( Hauenschild , 1956a , 1956b , 1966; Golding , 1983 ) .", "( iii ) As Nereidin-like activities have been identified in various annelids , this suggests that these substances could be chemically related .", "Histological analyses of the proposed biosynthetically active brain region ( Müller , 1973; Hofmann , 1976 ) were compatible with a fourth hypothesis , namely that nereidin is a neuropeptide .", "Based on this hypothesis , large-scale purifications have been performed from head extracts of N . diversicolor and Perinereis cultrifera ( Cardon et al . , 1981 ) .", "These finally resulted in the identification of a hexapeptide ( PPGPPG ) .", "However , this peptide did not display biological activity ( Durchon , 1984 ) .", "In this study , we take advantage of a newly developed , quantitative cell culture-based bioassay that measures transcript levels of the main Platynereis yolk precursor Vitellogenin ( Vtg ) .", "Using this assay , we demonstrate that nereidin activity is not sensitive to proteinase treatment , challenging the long-standing hypothesis that nereidin is a peptide .", "Instead , we correlate nereidin activity with methylfarnesoate ( MF ) , a substance belonging to the hormone class of sesquiterpenoids that has so far been assumed to be restricted to arthropods .", "We demonstrate that the concentration of MF drops in the course of maturation – as predicted from classical nereidin activity assays – and that eleocytes express the orthologue of a sesquiterpenoid receptor .", "This , together with our finding that MF exerts its action on vitellogenin production not only in cell culture , but also in vivo , establishes MF as a brain hormone orchestrating a critical metabolic switch in P . dumerilii .", "We finally show that insecticides targeting the sesquiterpenoid pathway interfere with Platynereis vitellogenesis , revealing a profound impact of insect growth regulators on a presumed non-target organism that serves as a key marine reference species ." ], [ "One of the major challenges faced by previous attempts to identify the brain hormone activity was the absence of a fast and sensitive bioassay to measure the inhibitory effect of nereidin on sexual maturation .", "Fractionated head extracts could be shown to interfere with the transition of cultured spermatogonia to spermatozoa ( Cardon , 1970; Durchon and Porchet , 1970; Cardon et al . , 1981 ) .", "However , the respective assay required more than a week of culturing and substantial input material ( thousands of sampled heads ) .", "Using this qualitative assay , and systematic fractionating of head extracts , the brain hormone activity could be associated with a small ( MR ≤ 2 . 000 ) , lipophilic molecule ( Cardon , 1970; Durchon and Porchet , 1970; Cardon et al . , 1981 ) .", "In order to establish a sensitive read-out for Platynereis maturation , we decided to focus on the yolk protein precursor Vtg .", "Yolk production underlies the significant increase in oocyte diameter observed in nereidids during maturation ( Fischer , 1984; Fischer and Hoeger , 1993 ) .", "Yolk protein is derived by endocytotic uptake of Vtg into the oocyte ( Fischer et al . , 1991 ) , where it accounts for more than 40% of protein ( Fischer and Schmitz , 1981 ) , reflecting its central role as reproductive investment .", "Vtg is synthesised in coelomic cells called eleocytes ( Figure 1B ) that are present in large numbers ( Baert and Slomianny , 1987; Hoeger , 1991 ) .", "Eleocytes and oocytes are freely floating in the coelomic cavity and can therefore easily be isolated and taken into primary ( co ) -culture , making them an excellent experimental model system to assess and monitor maturation in Platynereis and other nereidids , and to investigate the concomitant metabolic changes associated with this process .", "Based on the central role of eleocytes and Vtg for oocyte growth , we hypothesised that the expression level of vtg in eleocytes might be a suitable molecular read-out for maturation .", "As a first step , we identified a Platynereis vtg candidate in eleocyte transcriptome data ( Sven Schenk , Fritz Sedlazeck , Florian Raible , unpublished ) .", "Domain analyses and molecular phylogeny indicate that the protein encoded by this gene is an orthologue of Vtg identified in other animals ( insects , molluscs , amphibians and birds; Figure 1—figure supplement 1 ) .", "Next , we designed qRT-PCR primers to test if vtg transcript levels in P . dumerilii eleocytes increased over female maturation , as suggested by the increase of Vtg protein secretion in P . cultrifera ( Baert and Slomianny , 1992 ) .", "Indeed , we found a strong correlation between vtg-transcript abundance in eleocytes and the maturation state of the animal ( as assessed by oocyte diameter , Figure 1C and Figure 1—figure supplement 2 ) , indicating that vtg transcript levels could be used for a quantitative maturation assay .", "Given the central metabolic role of eleocytes in the reproductive switch , we further hypothesised that nereidin acts directly on eleocytes to regulate vtg production .", "We therefore designed a bioassay that takes coelomic cells into primary cell culture .", "After adding fractions of our purification scheme to small aliquots of this culture , and incubating these overnight , we extracted RNA from the cells and analysed them by qRT-PCR to determine the levels of vtg ( Figure 1D ) .", "After establishing this bioassay , we used it to chemically isolate the brain hormone-activity of Platynereis .", "Similar to the method of Cardon ( 1970 ) , a methanolic extract was prepared from 50 heads of premature animals , i . e . animals becoming sexually distinct , but before the irreversible onset of maturation .", "After solvent evaporation , the material was reconstituted in an aqueous phase and further fractionated via phenyl-solid phase extraction ( SPE , Figure 2A ) .", "Consistent with previous characterisations of nereidin , the 50% methanol eluate of this column showed clear activity ( Figure 2B ) when added to coelomocytes in vitro . 10 . 7554/eLife . 17126 . 008Figure 2 . Methylfarnesoate , not protein , constitutes the main nereidin/brain hormone activity .", "( A–C )", "Nereidin activity elutes in lipophilic fractions of head extracts but is not proteinaceous as previously hypothesised .", "( A ) Schematised fractionation of premature Platynereis head extracts , expected to recover the described nereidin activity in the methanolic eluate of SPE .", "( B ) Relative coelomocyte vtg levels after overnight incubation with flowthrough or eluate of", "( a ) , normalised against control samples .", "( C ) Insensitivity of nereidin activity against proteinase and heat treatment .", "Experiment as in", "( b ) , with pretreatment of eluate by proteinase K ( ‘ProtK’ ) and/or subsequent heat treatment ( 5’ , 95°C; ‘Heat’ ) .", "Results indicate that the repressive effect of nereidin on eleocyte vtg levels is neither sensitive to proteinase nor heat .", "( D–G )", "Identification of methylfarnesoate as a major component of nereidin .", "( D ) Further fractioning of the brain hormone activity ( eluate from", "b ) by C18-reverse phase column chromatography .", "Absorption of eluent at 280 nm ( red line ) and 215 nm ( black line ) during application of acetonitrile gradient ( dashed line , right ordinate ) .", "Peak 15 ( marked red ) , containing the nereidin activity , elutes at a retention time of 20 . 5 min . ( E ) Peak 15 , containing methylfarnesoate ( MF ) , significantly suppresses vtg levels in the coelomocyte bioassay when compared to the controls .", "( F ) Authentic MF recapitulates the observed effect at physiological concentration ranges; relative vtg levels in coelomocytes treated with 0 . 1 nM – 1000 nM MF in 0 . 01% DMSO , normalised to the control group of DMSO-treated cells .", "( G ) Specificity of the repressive effect caused by MF; comparison of coelomocyte vtg levels after incubation in 10 nM MF , palmitic acid ( PA ) or retinoic acid ( RA ) .", "Graphs in ( B , C , E , F , G ) show qRT-PCR quantification of vtg levels after overnight ( 20 hr ) treatment of primary coelomocyte cultures .", "Expressions values were calculated with respect to the reference gene rps9 , and normalised to the levels of controls ( untreated cells ) .", "Boxplots show the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "Whiskers denote the 10th and 90th percentile .", "Statistical significance was tested by a one-sided t-test of the treatment group against the control , or , in case of multiple comparisons , first with an ANOVA , followed by a one-sided t-test with adjustment of the p-values for multiple testing to assess differences between the different groups .", "*p<0 . 05 .", "**p<0 . 01 , ***p<0 . 001 .", "n: number of biological replicates .", "Raw data for panels B , C , E , F , G provided in Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 00810 . 7554/eLife . 17126 . 009Figure 2—source data 1 . Data for Figure 2B , C , E , F , G ( vtg expression levels normalised to the respective control treatment ) and Figure 2—figure supplement 2 ( stability of Ct values for rps9 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 00910 . 7554/eLife . 17126 . 010Figure 2—figure supplement 1 . Identification of MF as the principal component in peak 15 . ( A , B ) The panels shows the selected ion currents ( SIC ) in the range of m/z 251 . 0–251 . 5 ( top panels ) and the selected MS2-spectrum of the precursor ion at m/z 251 . 1 ( lower panels ) .", "( A ) Peak 15 .", "Note the single peak eluting at a retention time of 15 . 0 min in the upper panel , and the fragment pattern of the main peak .", "The fragment spectrum is characterised by the three major fragments at m/z 251 . 1 ( parent ion ) , 196 . 1 and 178 . 1 , with peak ratios of 0 . 13:1 . 00:0 . 41 , respectively .", "( B ) Authentic MF .", "Only one major fraction is eluting from the column with a retention time of 15 . 1 min ( upper panel ) .", "The derived MS2-spectrum again shows the characteristic ions at m/z 251 . 1 , 196 . 1 and 178 . 1 .", "with peak ratios of 0 . 11:1 . 00:0 . 44 , respectively .", "( C ) Structure of the observed ions at m/z 251 . 1 , 196 . 1 and 178 . 1 , and the neutral loss occurring during fragmentation . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01010 . 7554/eLife . 17126 . 011Figure 2—figure supplement 2 . Stability of the reference gene rps9 in coelomocyte culture . Ct values for rps9 were normalised to the mean of each experimental series .", "The box shows the first and third quartile and the median ( solid line ) .", "Whiskers denote the 1 . 5x fold range of the interquartile range .", "Blue dots denote the 12 outliers ( out of 292 data points ) .", "The minimum value is 0 . 876 , the maximum 1 . 134 .", "The mean is at 1 . 000 and the median at 0 . 999 , the standard deviation is 0 . 032 .", "The inter-quartile range ( 50% of data ) lies between 0 . 981 and 1 . 017 .", "Grubb`s test for outliers called the minimal and maximal values as statistically significant outliers .", "Data provided in Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 011 As outlined , it has long been speculated that nereidin is a peptide .", "This assumption was mainly based on the results of partial characterisation ( Durchon , 1984 ) , as well as histological observations , by which the brain regions producing nereidin were shown to contain highly active secretory cells ( Müller , 1973; Hofmann , 1976 ) .", "To assess whether the recovered nereidin activity was of proteinaceous nature , we treated the obtained eluate with proteinase K , and tested if this affected the ability of the eluate to repress vtg levels in cell culture .", "Our results demonstrate that nereidin is insensitive to proteinase K-treatment , thus strongly arguing against the notion that it is a neuropeptide hormone ( Figure 2C ) .", "To further characterise the recovered eluate , 250 premature worm heads were extracted and fractionated on a Phenyl-SPE-column .", "The recovered active fraction was then further fractionated by HPLC on a C18-column ( Figure 2D ) .", "A total of 16 fractions eluting from the column were collected and tested in the cell culture-based bioassay for their impact on vtg transcript levels .", "One peak ( peak 15 ) could be identified that robustly and significantly reduced vtg transcripts in two independent experiments and showed a reasonable chromatographic purity ( Figure 2D , E ) .", "Analysis of peak 15 by LC-MS/MS clearly demonstrated the presence of the sesquiterpenoid methylfarnesoate ( MF ) , when compared to an authentic standard run under the same conditions ( Figure 2—figure supplement 1 ) .", "To test if MF was indeed the active compound in peak 15 , we tested the effect of authentic MF on vtg transcript levels in the established cell culture assay .", "The results showed a clear down-regulation of vtg transcript levels ( Figure 2F ) .", "Strongest effects were observed in the range between 1 and 100 nM MF , in agreement with physiological concentrations expected for a hormone .", "To ascertain the specificity of MF in down regulation of vtg transcripts in vitro , coelomic cells were also challenged with structurally related compounds such as palmitic acid ( PA ) and all-trans-retinoic acid ( RA ) .", "As evident from Figure 2G , neither of these molecules showed a significant effect on vitellogenesis , thus establishing the specificity of MF on the observed reduction of vtg transcript levels .", "Based on the classical brain transplantation experiments , it was hypothesised that nereidin is present at high levels in young animals , whereas its concentration declines during maturation ( Hauenschild , 1956a , 1956b , 1966; Durchon and Porchet , 1970; Golding , 1983 ) .", "To test whether MF levels decrease over the course of maturation , we next set up a gas chromatography mass spectrometry ( GC-MS ) -based quantification assay .", "Our extraction method followed the method described by Westerlund and Hoffmann ( 2004 ) , where a methanolic extract is used in combination with a phase partition against an organic solvent to simultaneously denature degrading enzymes and to extract MF .", "However , as we used heads to extract MF rather than haemolymph , we used in a first step 80% methanol ( as in our initial extraction protocol , see above ) followed by liquid-liquid extraction into heptane , instead of directly diluting the sample with the methanolic-organic extraction solution ( Westerlund and Hoffmann , 2004 ) .", "The extraction efficiency of our method was determined to be >85% .", "With an estimated limit of detection of ~2 . 5 pg MF and a limit of quantification of ~15 pg MF , our assay is comparable to those used by other authors for the identification of MF and related compounds ( Westerlund and Hoffmann , 2004; Teal et al . , 2014 ) .", "This assay allowed us to unequivocally identify MF in Platynereis heads ( Figure 3—figure supplement 1 ) and to quantify the amount of MF by injecting 2 . 5 head equivalents into the GC-MS .", "Measuring MF levels present in heads of different developmental stages showed differences in MF content ( Figure 3A ) .", "Heads of premature animals were determined to possess ~5510 ± 660 pg MF/mg protein ( mean±standard error of the mean ) , whereas concentrations in the heads of mature animals were found to be significantly lower ( ~2870 ± 495 pg MF/mg protein ) .", "Calculated per head , the MF content is equivalent to ~34 . 6 ± 8 . 0 pg/head for premature heads and ~15 . 4 ± 2 . 5 pg/head for mature animals ( Figure 3—figure supplement 2 ) .", "Our results are therefore in agreement with the proposed decline of brain hormone concentrations during maturation . 10 . 7554/eLife . 17126 . 012Figure 3 . Levels of MF as well as the sesquiterpenoid receptor orthologue Met drop over the course of maturation .", "( A ) Significant decrease of MF levels between premature and mature heads; boxplots show amounts of MF normalised to the total protein content of the respective head sample .", "Heads of premature animals contain ~5510 ± 660 ( S . E . M ) pg MF per mg protein , whereas heads from mature animals contain ~2870 ± 495 pg MF per mg protein , thus roughly 50% of the amount found in heads of premature animals .", "Amounts are corrected for a MF recovery rate of 85% .", "The boxes show the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "Whiskers denote the 10th and 90th percentile .", "Significance was tested with a one-sided t-test , **p<0 . 01 .", "n: number of biological replicates .", "( B–D )", "Platynereis possesses an orthologue of arthropod sesquiterpenoid receptors that shows co-regulation with MF titres .", "( B ) Maximum Likelihood tree supporting the identity of Platynereis Met as orthologue of the validated arthropod sesquiterpenoid receptors Methoprene-tolerant ( Met ) and Germ cells expressed ( Gce ) .", "Phylogeny reconstructed with IQ-TREE 1 . 3 . 12 ( Minh et al . , 2013; Nguyen et al . , 2015 ) .", "By parameter optimisation , the LG+F+I+G4 substitution model was selected .", "Numbers refer to confidence levels ( in % ) of major nodes derived from 1000 replicates .", "Accessions: Drosophila Met: NP_511126 . 2; Drosophila Gce: NP_511160 . 2; Aedes Met: AAW82472 . 1; Nasonia Met: XP_001606775 . 2; Apis Met: XP_395005 . 4; Tribolium Met: NP_001092812 . 1; Daphnia pulex Met: BAM83853 . 1; Daphnia magna Met: BAM83855 . 1; Platynereis Met: KU756288 ( this study ) ; Lottia Met: XP_009046608 . 1 , Lymnaea Met: FX197139 . 1 .", "Drosophila Clock: NP_523964 . 2; Nasonia Clock: XP_008214438 . 1; Apis Clock: XP_394233 . 1; Aedes Clock: XP_001662706 . 1; Tribolium Clock: NP_001106937 . 1; Daphnia Clock: EFX7997 . 1; Platynereis Clock: AGX93013 . 1; Aplysia Clock: XP_005112430 . 1 .", "( C ) Eleocyte met levels drop by ~39% in the course of maturation .", "qRT-PCR analysis of met transcript levels in eleocytes sampled from premature and mature animals .", "Relative met levels were calculated with respect to the arithmetical mean of the reference genes sams and cim6pr , and normalised to the level of the premature sample .", "( D ) Eleocyte met levels are increased by the addition of MF .", "Similar analysis as in ( C ) , indicating that met levels in MF-treated cultured eleocytes ( right ) are ~30% higher than in untreated controls ( left ) .", "Relative met levels were calculated with respect to the reference gene rps9 , and normalised to the level of the control sample .", "Boxplots ( C , D ) show the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "Whiskers denote the 10th and 90th percentile .", "Statistical significance in ( B , C ) was determined by a two-sided t-test .", "*: p<0 . 05 .", "****p<0 . 0001 .", "n: number of biological replicates .", "Raw data for panels A , C , D provided in Figure 3—source data 1 .", "Alignment for panel B provided as Figure 3—source data 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01210 . 7554/eLife . 17126 . 013Figure 3—source data 1 . Data for the graphs in Figure 3A ( MF content in pg normalised to mg protein per head ) , Figure 3C ( met expression levels normalised to premature eleocytes ) and Figure 3D ( met expression levels normalised to the control treatment ) ; data for the graphs in Figure 3—figure supplement 2 and Figure 3—figure supplement 3 ( MF content in pg per head ) , Figure 3—figure supplement 4A and Figure 3—figure supplement 4B ( met expression levels quantified against individual reference genes ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01310 . 7554/eLife . 17126 . 014Figure 3—source data 2 . Alignment file for the phylogenetic tree of Met homologs ( Figure 3B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01410 . 7554/eLife . 17126 . 015Figure 3—figure supplement 1 . Quantification of MF in the heads of Platynereis by GC-MS . Representative Selected Ion Mode ( SIM ) chromatograms from premature Platynereis heads and authentic MF ( upper panel ) .", "The lower panel shows the selected ions at m/z 114 . 0 , 136 . 0 and 219 . 0 .", "( A ) SIM chromatogram of 2 . 5 premature Platynereis heads , the peak with a retention time of 9 . 09 min represents MF as evident from the selected ion mass spectrum ( lower panel ) .", "The ions at m/z 114 . 0 , 136 . 0 and 219 . 0 show peak ratios of 1 . 00:0 . 46:0 . 19 , respectively .", "( B ) SIM chromatogram of authentic MF eluting 9 . 09 min .", "Peaks eluting after 10 min are due to plasticisers .", "The base line jump at 10 min is due to a change in SIM ions from m/z 114 . 0 , 136 . 6 and 219 . 0 to ions at m/z 191 . 0 , 235 . 0 and 278 . 0 for the detection of methoprene ( not shown ) .", "The ions at m/z 114 . 0 , 136 . 0 and 219 . 0 have peak ratios of 1 . 00:0 . 34:0 . 17 , respectively ( lower panel ) .", "( C ) Structures of MF and the monitored SIM ions . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01510 . 7554/eLife . 17126 . 016Figure 3—figure supplement 2 . The levels of MF per head drop over the course of maturation . Significant decrease of MF levels between premature and mature heads; the boxplot shows the quantified amounts of MF normalised per head .", "Heads of premature animals contain on average ~ 34 . 6 ± 8 . 0 ( S . E . M ) pg MF per head , whereas heads from mature animals contain ~15 . 4 ± 2 . 5 pg MF per head , thus roughly 46% of the amount found in heads of premature animals .", "The amounts are corrected for a MF recovery rate of 85% .", "The box shows the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "The whiskers denote the 10th and 90th percentile .", "Significance was tested with a one-sided t-test , *p<0 . 05 .", "n: number of biological replicates .", "Raw data provided in Figure 3—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01610 . 7554/eLife . 17126 . 017Figure 3—figure supplement 3 . Methylfarnesoate is found in the heads of earthworms . The head of earthworms contain on average ~ 5 . 3 ± 1 . 9 ( S . E . M ) pg MF per head .", "The data range is between ~2 . 1 and ~9 . 8 pg MF per head .", "The amounts are corrected for a MF recovery rate of 85% .", "The box shows the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "The whiskers denote the data range .", "n: number of biological replicates .", "Raw data provided in Figure 3—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01710 . 7554/eLife . 17126 . 018Figure 3—figure supplement 4 . The choice of reference genes does not impact on the observed down-regulation of met . The figure shows the same data as Figure 3C , but with the results for the individual reference genes plotted separately .", "The observed relative expression of met in eleocytes is independent of the reference gene used for normalisation ( sams or cim6pr ) , ( A ) With sams used for normalisation , the expression levels of met in eleocytes from mature animals are ~30% lower than in the eleocytes from premature animals .", "( B ) With cim6pr used for normalisation , the expression levels of met in eleocytes from mature animals are ~45% lower than in the eleocytes from premature animals .", "Boxes show the first and third quartile , the median ( solid line ) , mean ( dashed line ) and the whiskers denote the 10th and 90th percentile .", "Statistical tests as in Figure 3C .", "***p<0 . 001 , ****p<0 . 0001 .", "Raw data provided in Figure 3—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 018 In summary , the results of our bioassay and purification scheme demonstrate that the brain hormone-activity present in the heads of P . dumerilii is not a peptide .", "Instead , we clearly identify MF as the major molecule in Platynereis heads that is able to suppress transcription of vtg in coelomocyte cultures , when added at physiological concentrations .", "Furthermore , MF concentration drops in the transition from the premature animal to a mature stage , in agreement with classical predictions on the regulation of nereidin .", "The occurrence of MF in the annelid Platynereis is counterintuitive , because sesquiterpenoid hormones have commonly been proposed to be restricted to arthropods , possibly as a secondary consequence of the reduction of a parallel biosynthetic pathway leading to the formation of sterols in this animal group ( Bellés et al . , 2005; Jindra et al . , 2015 ) .", "To determine whether the Platynereis MF system originated independently , or relies on common descent with arthropod sesquiterpenoid signalling systems , we investigated if Platynereis also possesses an orthologue of arthropod sesquiterpenoid receptors .", "Based on genetic screens in Drosophila , and subsequent biochemical and bioinformatics studies , homologues of the bHLH-PAS-domain-containing transcription factor Methoprene-tolerant ( Met ) have been identified as functional arthropod sesquiterpenoid receptors ( Miyakawa et al . , 2013; Jindra et al . , 2015 ) .", "In transcriptome data available from Platynereis eleocytes , we identified a transcript encoding a full-length bHLH-PAS-domain-containing transcription factor that showed strong similarity to insect Methoprene-tolerant receptors .", "Molecular phylogeny revealed this protein to be a clear orthologue of the insect Methoprene-tolerant and germ cell expressed ( Gce ) receptors , as well as the functionally confirmed Met receptors from the crustacean Daphnia .", "We therefore named the corresponding Platynereis gene methoprene-tolerant ( met ) .", "The phylogenetic analysis also revealed Met orthologues in the molluscs Lottia gigantea and Lymnaea stagnalis ( Figure 3B ) .", "Using qRT-PCR analysis , we could confirm the expression of Platynereis met in eleocytes .", "Furthermore , these analyses revealed that the expression level of this gene dropped significantly during maturation , reaching only ~ 61% of the level observed in cells from premature animals ( Figure 3C and Figure 3—figure supplement 4 ) .", "This parallels the observed decline in MF concentration in the head measured by GC-MS ( Figure 3A ) .", "Furthermore , treatment of cultured premature eleocytes with 10 nM MF induced the expression of Platynereis met , resulting in a 30% up-regulation ( Figure 3D ) .", "Together , these data confirm the expression of met in eleocytes by an independent method and are also consistent with a positive autoregulation of this putative receptor by MF .", "In vivo , eleocytes are freely floating in the coelomic cavity , compatible with the notion that they are accessible to hormonal factors like MF .", "However , coelomic fluid also represents a major metabolic compartment , where nutrients and metabolites are exchanged ( Fischer , 1979; Baert and Slomianny , 1987; Fischer and Hoeger , 1993; Hoeger and Kunz 1993; Hoeger et al . , 1999 ) .", "This raises the possibility that other factors in the coelomic fluid could modulate or even block the effect of MF that we observe in isolated cell culture and prompted us to investigate the effect of MF on eleocytes in their natural context .", "To reliably compare different animals , we used a classical decapitation paradigm .", "Decapitation leads to accelerated maturation , and , importantly , synchronisation of maturation across different individuals ( Hauenschild , 1966 , 1974 ) .", "We then quantified the maturation stage of MF-treated and DMSO-treated animals five days after decapitation , taking advantage of our established vtg qRT-PCR assay ( Figure 4A and Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 17126 . 019Figure 4 . MF also represses vtg expression in vivo , and sustains expression of a marker for caudal growth upon posterior amputation .", "( A ) Setup of experiment testing the ability of MF to interfere with vitellogenesis in vivo .", "Following decapitation , female individuals are known to start vitellogenesis , reflecting loss of nereidin; treatment with MF tests if the repressive effect of nereidin on vtg observed in primary cultures of coelomic cells also occurs in the normal context of these cells .", "( B ) qRT-PCR analysis of vtg expression in decapitated animals 5 days after decapitation .", "The animals were either treated with 0 . 1% DMSO ( control ) or in 100 nM MF in 0 . 1% DMSO for five days .", "Expression levels of vtg upon presence of MF are ~70% lower than in treated animals .", "vtg levels were related to the arithmetical mean of the reference genes rps9 and sams and normalised to the vtg-expression level of the control .", "( C ) MF sustains expression of hox3 , a marker diagnostic for caudal regeneration .", "qRT-PCR analysis of hox3 expression in Platynereis fragments 5 days after posterior amputation .", "Headless worm fragments treated with 100 nM MF in 0 . 1% DMSO ( right ) show twice as high levels of hox3 than headless control fragments ( left ) only treated with 0 . 1% DMSO .", "Hox3-expression is relative to that of the arithmetical mean of the reference genes rps9 and sams and normalised to the hox3-expression level of the untreated samples .", "( B , C )", "Boxplots show the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "Whiskers denote the 10th and 90th percentile .", "Statistical significance was tested by a one-sided ( B ) and a two-sided t-test ( C ) , respectively .", "*p<0 . 05 .", "****p<0 . 0001 .", "n: number of biological replicates .", "Raw data for panels B , C provided in Figure 4—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 01910 . 7554/eLife . 17126 . 020Figure 4—source data 1 . Data for the graphs in Figure 4B and Figure 4C ( vtg/hox3 expression levels normalised to the respective control treatment ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 02010 . 7554/eLife . 17126 . 021Figure 4—figure supplement 1 . The choice of reference genes does not impact on the observed down-regulation of vtg . The figure shows the same data as Figure 4B , but with the results for the individual reference genes plotted separately .", "The observed relative expression of vtg in whole animals treated with either with 0 . 1% DMSO ( control ) or 100 nM MF in 0 . 1% MF ( 100 nM ) is independent of the reference gene used for normalisation ( rps9 or sams ) .", "( A ) With rps9 used for normalisation , the expression levels of vtg are ~65% lower in treated animals than in the control group .", "( B ) With sams used for normalisation , the expression levels of vtg are ~75% lower in treated animals than in the control group .", "Boxes show the first and third quartile , the median ( solid line ) , mean ( dashed line ) and the whiskers denote the 10th and 90th percentile .", "Statistical tests as in Figure 4B .", "****p<0 . 0001 .", "Raw data provided in Figure 4—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 021 The results of these experiments showed that vtg levels were strongly down-regulated in MF-treated worms when compared to a non-treated control group ( Figure 4B ) .", "This establishes that MF does not only have an effect on isolated coelomocytes , but also exerts a similar effect in vivo , in spite of other physiological regulators that might be present in the coelomic fluid .", "Notably , our data are also reminiscent of classical decerebration and re-transplantation experiments that showed that implanted brains could reverse the accelerated maturation observed after decerebration ( Hauenschild , 1956a , 1956b , 1966; Golding , 1983 ) .", "This implicates MF as a major hormonal cue responsible for the anti-maturation effect of the juvenile brain .", "The sesquiterpenoid pathway is a prime target for a large variety of insecticides .", "After observing the effect of MF on annelid reproduction both in vivo and in vitro , we tested whether the juvenile hormone analogue methoprene is also capable of influencing vitellogenesis in Platynereis .", "We treated decapitated animals as before and assessed changes in vtg levels .", "In this experimental setup , we did not observe a significant decrease in vtg levels .", "In contrast , applying methoprene to eleocytes in our cell culture assay clearly led to reduced vtg levels at a concentration of 10 nM ( Figure 5 ) .", "To assess if this effect was specific to methoprene , we also tested the structurally unrelated , yet highly used insecticide pyriproxyfen for its ability to interfere with endogenous vtg transcript levels .", "Indeed , when applying pyriproxyfen at the same concentration ( 10 nM ) , we again observed a significant decrease in the abundance of vtg transcripts when compared to controls .", "This indicates a profound effect of presumed insect-specific growth regulators on a non-target species . 10 . 7554/eLife . 17126 . 022Figure 5 . Vitellogenin expression in worm coelomocytes is suppressed by the hormone agonists methoprene and pyriproxyfen that are considered to act exclusively on insects . Expression levels of vtg in coelomocytes are significantly down-regulated by treatment with 10 nM methoprene ( Meth ) and 10 nM pyriproxyfen ( Pyr ) in 0 . 01% DMSO .", "The graph shows qRT-PCR quantification of vtg levels after over night ( 20 hr ) treatment of primary coelomocyte cultures; expression levels are relative to those of the reference gene rps9 and are normalised to the vtg-expression level of the control ( DMSO-treated cells ) .", "The Boxplots show the first and third quartile , the median ( solid line ) , and the mean ( dashed line ) .", "Whiskers denote the 10th and 90th percentile .", "Statistical significance was tested first by an ANOVA , followed by a two-sided t-test with adjustment of the p-values for multiple testing to assess differences between the different groups .", "*p<0 . 05 .", "n: number of biological replicates .", "Raw data provided in Figure 5—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 02210 . 7554/eLife . 17126 . 023Figure 5—source data 1 . Data for the graph in Figure 5 ( vtg expression levels normalised to the control treatment ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 023 In addition to its central role in suppressing maturation , nereidin has also been classically defined by the ability of transplanted brain pieces to maintain caudal regenerative capacity ( Hauenschild , 1974; Hofmann , 1976 ) .", "Both functions have been used interchangeably in nereidin research .", "However , the lack of molecular candidates for nereidin has prevented the assessment of whether both functions rely on identical molecules , or if they can be separated .", "In a final set of experiments , we therefore tested if MF had any impact on regeneration of caudal body segments .", "To minimise inter-individual variations in regenerative capacities ( Golding , 1967b ) , we followed a similar approach as Hauenschild ( 1974 ) in which hormone-exposed and control fragments were derived from the same animal .", "In addition , as in our maturation assays , we aimed to develop a quantitative approach to assess the molecular effect of MF on caudal regeneration .", "For this , we established a qRT-PCR assay quantifying the expression of the homeobox gene hox3 .", "Hox3 expression has earlier been demonstrated to be a highly specific early on marker in caudal regeneration ( Pfeifer et al . , 2012 ) .", "More specifically , it has recently been linked to ectodermal cells of likely stem cell character ( ectoteleoblasts , Gazave et al . , 2013 ) involved in the regenerative process .", "Our results show that the expression of hox3 is significantly up-regulated ( ~2 fold ) after 5 days in test fragments treated with 100 nM MF when compared to DMSO-treated control fragments ( Figure 4C ) .", "This finding is consistent with the idea that besides its role in repressing maturation , MF may also contributes to sustaining putative stem cells involved in regeneration ." ], [ "Together , our results establish methylfarnesoate as a critical neurohormone suppressing maturation in a key lophotrochozoan reference species .", "Our data are fully consistent with properties classically attributed to nereidin: in light of the massive amounts of Vitellogenin that are produced by animals committed to reproduction , the ability of MF to suppress vtg levels is compatible with a central role of this compound in controlling reproduction-associated energy expenditure , as expected for nereidin .", "Likewise , our quantifications of MF levels over the course of reproductive development ( Figure 3A ) are consistent with the decline in nereidin activity present in transplanted heads of different stages .", "Although our data clearly argue against the long-held assumption that nereidin is a neuropeptide , they are still consistent with the chemical properties ( apparent mass , lipophilic nature ) that have been derived from previous purification attempts .", "Likewise , the data are consistent with the notion that related hormones are present in other annelids ( see below ) .", "Our findings not only reveal an overall role of this sesquiterpenoid in governing energy expenditure but also highlight critical mechanistic aspects:", "( i ) We show that eleocytes are direct target cells for MF , indicating a direct cross-talk between the brain and a highly relevant metabolic cell type .", "( ii ) Moreover , the observed effect of MF on vitellogenin levels is consistent with a direct function of MF in controlling this key factor for oocyte maturation .", "( iii ) The positive autoregulation of the sesquiterpenoid receptor orthologue Met that we observe is reminiscent of other hormone ligand-receptor systems , such as thyroxin or ecdysone ( Thummel , 1995; Bagamasbad and Denver , 2011 ) .", "In the biological context of semelparous reproduction , such autoregulation provides an attractive model how graded changes in MF levels can be amplified , contributing to sharpening the response of the target cells to above- vs . below-threshold levels .", "Future work on the fine-tuning of MF regulation , and its effect on target cells , will likely yield more detailed insight into the role of MF for eleocyte physiology .", "Such analyses will extend our understanding of molecular changes in the eleocyte , for instance to cover genes involved in nucleotide metabolism ( Hoeger et al . , 1999 ) .", "Moreover , they will resolve the question if residual levels of MF signalling are required for proper vitellogenesis , or if vitellogenesis only takes place in complete absence of MF .", "This addresses classical debates about the requirement of nereidin ( Hauenschild , 1966; Durchon and Porchet , 1970; Golding , 1983 ) but is also interesting for cross-comparisons between the annelid system and insects , where sesquiterpenoids do not only define juvenile stages – similar to Platynereis – but in later life play a – positive – role for vitellogenesis itself .", "A second phenomenon where the identification of MF provides important mechanistic insight , and opens up new experimental possibilities concerns the question of how regenerative abilities of Platynereis and other species are controlled .", "From a global perspective of organismal energy expenditure , it is plausible that semelparous reproduction ( investment in growth of germ cells ) and regeneration ( investment in re-growth of soma ) are anti-correlated , although it remains unclear exactly how this is achieved at the molecular level .", "Our finding that MF sustains expression of a marker ( hox3 ) characteristic for a population of putative stem cells in posterior segment addition ( Pfeifer et al . , 2012; Gazave et al . , 2013 ) is consistent with the idea that MF also plays a role in regenerative control , but in contrast to vtg regulation in eleocytes , it is less clear if this effect is direct or caused by other mediating factors .", "Due to the complexity of regeneration , reliable and quantitative short-term assays for regeneration will need to be established to pursue this question .", "The identification of MF in Platynereis heads challenges several hypotheses: most directly , it defeats the hypothesis that nereidin is a peptide , a notion that has been propagated in the literature for more than 30 years ( Cardon et al . , 1981; Durchon , 1984 ) .", "Furthermore , MF and related sesquiterpenoid hormones have so far only been found in arthropods ( Bellés et al . , 2005; Jindra et al . , 2015 ) , and their evolution has been hypothesised to be a secondary consequence of the loss of the sterol synthesis pathway in this clade ( Zandee , 1964; Bellés et al . , 2005 ) .", "As marine nereidid worms were previously shown to possess a functional sterol synthesis pathway ( Wootton and Wright , 1962 ) , and Platynereis even has a functional estrogen receptor ( Keay and Thornton , 2009 ) , our discovery makes it unlikely that loss of sterol synthesis preceded the evolution of sesquiterpenoid hormones .", "Rather , it suggests that , like sterols , bioactive sesquiterpenoids , could have been part of the ancient molecular repertoire available in the common ancestor of protostomes , if not even at earlier stages of animal evolution .", "Several lines of experimental evidence support this notion: Exogenous MF was found to affect larval development of the annelid Capitella sp .", "( Laufer and Biggers , 2001 ) , and annelid extracts displayed biological activity in assays for insect juvenile hormones ( Schneiderman and Gilbert , 1958 ) .", "The latter result would suggest that insect juvenile hormones might also be active in the annelid system , but this issue has not yet been addressed experimentally .", "Moreover , we have detected MF in head extracts of common earthworms ( Lumbricus sp . , Figure 3—figure supplement 3 ) .", "Beyond the annelid clade , our identification of Methoprene-tolerant orthologues in the limpet Lottia and the pond snail Lymnaea indicates that the sesquiterpenoid signalling pathway might even exist in molluscs .", "As annelids and molluscs are large groups within the lophotrochozoan superphylum ( Halanych et al . , 1995 ) , this implies a potential physiological role for sesquiterpenoids in a broad set of ( mostly aquatic ) species .", "What could such physiological roles be ?", "Semelparity , as it occurs in Platynereis , is considered an extreme form of 'capital breeding' , a reproductive strategy where females build up large energy stores in their bodies that are then invested into offspring at particular time points ( Drent and Daan , 1980 ) .", "Capital breeding strategies allow animals to time their reproduction more precisely and have larger clutches of offspring than could be afforded by daily nutrient uptake .", "It likely represents an ancient reproductive strategy that is very common in poikilothermic animals ( Bonnet et al . , 1998 ) .", "Therefore , it is plausible that the role for MF uncovered in the marine annelid Platynereis could reflect a more general role of sesquiterpenoids in regulating reproductive energy expenditure .", "The inhibitory function for maturation we uncover in Platynereis is reminiscent of the juvenoid functions of insect juvenile hormones: During pre-adult development , these provide a molecular cue for juvenile status , preventing insects from entering into adulthood , and thereby indirectly preventing reproductive energy expenditure as well .", "Likewise , MF treatment of larval stages of crustaceans can delay metamorphosis or prevent gonad formation ( Borst et al . , 1987; Tsukimura et al . , 2006 ) .", "During adulthood , however , insect and crustacean sesquiterpenoids have distinct functions .", "In contrast to our results in worms , juvenile hormones commonly act as positive regulators of vitellogenesis in the adult insect fat body , whereas their down-regulation is associated with diapause and longevity ( Gäde et al . , 1997; Wyatt , 1997; Hansen et al . , 2014 ) .", "Similarly , MF promotes adult maturation , vitellogenesis and mating behaviour in crustaceans ( Laufer et al . , 1987; Homola and Chang , 1997; Subramoniam , 2011 ) , with a separate function in sex determination ( Toyota et al . , 2015 ) .", "As Platynereis retains certain MF levels even in the mature stage ( see above ) , it will be a suitable model to address additional roles of MF in the life history of an annelid .", "Finally , our discovery of a sesquiterpenoid hormone in an annelid is not only relevant to understand hormone evolution but it likely also has significant ecological implications .", "Reproductive hormones are attractive targets for pesticides .", "Juvenile hormone analogues like methoprene and later pyriproxyfen have initially been designed to interfere with insect development and are broadly used to fight insect pests such as Aedes , a vector for dengue , chikungunya , Zika , and yellow fever viruses ( reviewed in Henrick , 2007 ) .", "The discovery the sesquiterpenoid MF in crustaceans by Laufer et al . ( 1987 ) already raised concerns if the effect of juvenile hormone analogues is restricted to insect development ( Walker et al . , 2005; Abe et al . , 2015 ) , calling for more specific tests with non-target species .", "Our results argue that aquatic lophotrochozoans should also be systematically included in specificity tests for compounds targeting sesquiterpenoid signalling pathway , and that such tests should not be restricted to mortality ( Henrick , 2007 ) , but include assays on reproductive fitness .", "For instance , in our studies , pyriproxyfen impacts on vtg levels at concentrations of 10 nM which lies in the range of pyriproxyfen found in surface waters ( Sullivan and Goh , 2008 ) .", "In turn , sesquiterpenoid analogues may also be suitable leads in the search for specific compounds targeting pest species outside the arthropod clade or factors to favourably affect the balance between reproductive and somatic growth in aqua- and mariculture .", "Taken together , our data argue for a critical role of sesquiterpenoid hormones outside arthropods and indicate that methylfarnesoate is the major constituent of the long-sought brain hormone nereidin , a key factor in regulating maturation in marine capital breeders .", "The discovery of this hormone in an annelid provides a new entry point to understand hormonal regulation of energy expenditure and sheds new light on the evolution of animal hormone systems ." ], [ "Platynereis dumerilii were kept in a continuous culture at the Marine Facility of the Max F . Perutz Laboratories in Vienna ( for details see Hauenschild and Fischer , 1969 ) .", "After anaesthetisation of the animals in a 1:1 ( v/v ) mixture of seawater and 7 . 5% ( w/v ) MgCl2 in distilled water , the animals were transferred into 500 µL Nereis balanced salt solution ( NBSS , see Fischer et al . , 1991 ) and the coelomic cavity was opened with fine scissors .", "The coelomic cells were gently squeezed out , passed through 136 , 70 and 30 µm gauze filters to select for eleocytes ( >90% pure as judged by microscopical inspection ) .", "Cells were then collected by centrifugation ( 5 min , 500 ×g , 0°C ) , and washed once with NBSS .", "For the isolation of coelomic cells ( eleocytes and oocytes ) , only the first gauze filtration was carried out .", "Coelomic cells were isolated as described above and cultured a total of volume of 50 µL of culture medium without hen egg ultra filtrate ( Schenk and Hoeger , 2010 ) overnight at 19°C on a rocking platform , in a BSA-coated 96-well microtitre plate .", "All animals were in the premature stage , with oocyte diameters between 50 and 65 µm , i . e . before the irreversible onset of maturation assumed to be beginning from oocyte diameters of 85 µm onwards ( Hauenschild , 1966 ) .", "Test substances were diluted in either 100 mM Na2PO4 , pH 7 . 5 , or in NBSS , and their final volume never exceeded 10% of the total , DMSO concentration was kept to a maximum of 0 . 01% .", "Controls received only the vehicle .", "After incubation , cells were harvested by centrifugation ( 5 min , 500 xg , 0°C ) and total RNA was isolated with a commercial kit ( RNeasy-Kit , Qiagen , Hilden , Germany ) according to the manufacturer’s protocol , stored at −80°C and used for qRT-PCR using primers against Platynereis vitellogenin ( sequences see Table 1 ) to assess changes in vitellogenin transcript abundance .", "As vitellogenin expression is strongly dependent on the maturation stage ( see results ) and as individual animals can show considerable variations in their physiology ( Fischer and Hoeger , 1993; Hoeger and Kunz 1993 ) the cells of at least two animals were pooled per treatment group .", "Each assay consisted of three biological replicates and was at least repeated once . 10 . 7554/eLife . 17126 . 024Table 1 . List of primers used for qRT-PCR , including predicted melting temperature . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 024NameSequence 5’→3’Tm / °CEfficiencyPdu vtg qPCR1 FACAGGCCATCACATTCACAA56 . 4101% Pdu vtg qPCR1 RTCTGCTCACGTCTCTTTCCA58 . 4Pdu met qPCR1 FGGATGATTATGATGTATACCTGCAAC62 . 9102% Pdu met qPCR1 RAGACCGAACTGGCGTTTG56 . 3Pdu hox3 qPCR1 FCTACCCCTGGATGAGGGAAT60 . 595% Pdu hox3 qPCR1 RACTTCCGGTTCCTGGTCC58 . 4Pdu rps9 FCGCCAGAGAGTTGCTGACT59 . 5102% Pdu rps9 RACTCCAATACGGACCAGACG60 . 5Pdu sams qPCR1 FCAGCAACGGTGAAATAACCA56 . 4101% Pdu sams qPCR1 RCATCACTCACTTGATCGCAAA57 . 5Pdu cim6pr qPCR1 FACTTCCCCTGCTGATGAGTG60 . 599% Pdu cim6pr qPCR1 RTTCGTAAGTCAGGTTTCCATTG58 . 4Pdu cdc5 FCCTATTGACATGGACGAAGATG60 . 1100% Pdu cdc5 RTTCCCTGTGTGTTCGCAAG57 . 5 Primers for qPCR were designed using the 'Universal Probe Library' web page provided by Hoffman-LaRoche ( http://lifescience . roche . com , see Table 1 ) .", "Available genome information ( http://4dx . embl . de/platy/ ) was used to choose primers across exon/intron junctions .", "Prior to use , all primers were tested for their dynamic range by serial dilutions ( 10x , 100x , 1000x ) of cDNA .", "These tests also included included a –RT-control ( no reverse transcription ) .", "All selected primers showed a linear amplification profile in the tested dilution range , and the primer efficiencies were calculated ( E = 101/slope× 100 , where \"E\" is the primer efficiency and \"slope\" is the calculated slope of the dilution series ) to be between 95% and 102% ( Table 1 ) , and amplification from –RT controls was not observed .", "In these primer tests , amplified products were subcloned and the sequence confirmed by Sanger sequencing as described below .", "In all assays , the same RNA amount was used for each group to be compared .", "Generally , this was 5 ng for the assessment of vtg , 150 ng for met , and 300 ng for hox3 .", "cDNA for qRT-PCR was synthesised using Qiagen’s QuantiTect-Kit and final dilution of the cDNA to 50 µL .", "Briefly , the desired amount of RNA was diluted to 12 µL with nuclease-free H20 , and 2 µL of the Kit`s gDNA-Whipeout buffer ( DNase I ) were added .", "The DNase digest was performed for 6 min at 42°C , following cooling to 0°C , cDNA was then synthesised in a total volume of 20 µL for 15 min at 42°C , the reaction was stopped by heat deactivation of the enzymes ( 5 min , 95°C ) ; finally , the reaction was centrifuged ( 1 min , 17 . 000 ×g ) , diluted to 50 µL with H2O and used for qRT-PCR .", "qRT-PCR was performed on a Applied Biosciences StepOne instrument using SybrGreen ( Thermo Fisher Scientific , Vienna , Austria ) as reporter dye .", "Five microlitres cDNA were measured in a volume of 20 µL , with two technical replicates per sample .", "After an initial denaturation for 10 min at 95°C , amplification and reading of the reporter dye were carried out at 60°C for 1 min over a total of 40 cycles with a 3 s denaturation step at 95°C at the beginning of each cycle , this was followed by recording a melting curve of the amplified product ( 60 . 0–95 . 0°C in 0 . 3°C increments ) to ascertain only a single amplified product .", "Suitable reference genes were chosen based on", "( i ) the stability of their expression values in the respective tissue and", "( ii ) similar Ct-values for these genes in relation to the target gene .", "Specifically , the target and the reference gene were aimed to be within a △Ct -range of ±5 .", "Generally , two reference genes were used for normalisation , and the target gene was normalised by the 2–△△Ct –method to the arithmetic mean of these two reference genes .", "For the coelomocyte cultures , as they were extensively used in the context of the brain hormone fractionation , the limited material available for each assay only allowed us to use a single reference gene , rps9 ( ribosomal protein small subunit 9 ) .", "However , we tested the stability of rps9 in coelomocytes against the reference gene cdc5 ( cell-cycle-dependent serine/threonine protein kinase ) when we first assessed vtg-transcript levels in eleocytes of different maturation stages ( Figure 1C ) .", "Separate quantification against either reference gene produced similar results ( Figure 1—figure supplement 2 ) .", "This is in line with the fact that both of these genes were previously shown to be suitable reference genes ( Dray et al . , 2010; Zantke et al . , 2013 ) .", "Additionally , we also assessed the stability of rps9 in cell culture by comparing the normalised Ct-values within each experimental series .", "These analyses demonstrated that there is a high stability of these values ( SD of 3% ) ( Figure 2—figure supplement 2 ) .", "For Platynereis S-adenosylmethionine synthetase ( sams; accession: KX907200 ) and cation-independent-mannose-6-phosphate-receptor ( cim6pr; accession: KX907201 ) , independent calculations with the respective second housekeeping gene support their suitability as additional reference genes ( Figure 3—figure supplement 4 , Figure 4—figure supplement 1 ) .", "Data were excluded from the analysis if the △Ct-values were ±1 . 0x of the mean of the respective treatment group , or if the melt curve analysis indicated ambiguous Tm for the PCR products .", "In any case , at least five biological replicates were used for analysis .", "Platynereis dumerilii were anaesthetised as described above and decapitated in front of the pharynx .", "The heads were suspended in 80% methanol containing 0 . 1% formic acid , and disrupted by three ultrasonic bursts for 30 s on ice and extracted twice for 15 min on ice .", "Insoluble material was pelleted for 15 min and 20 , 000 ×g at 0°C , after which the pellet was again extracted and centrifuged .", "To further eliminate insoluble macromolecules , the raw extract was left to precipitate for 30 min at −20°C , and was again centrifuged for 15 min and 20 , 000 ×g at 0°C .", "The resulting supernatant was mildly dried down in vacuo and the pellet suspended in 0 . 1% formic acid , by vigorous vortexing , followed by shaking at 1400 rpm at room temperature on a Thermomixer , and finally centrifuged ( 2 min , 17 , 000 ×g ) .", "The extract was then applied onto phenyl-extraction columns ( Alltech , 100 mg sorbent , ordered from Thermo Fisher Scientific , Vienna , Austria ) equilibrated in 0 . 1% formic acid .", "The flow-through was discarded , the column washed with 750 µL 0 . 1% formic acid , and eluted with 500 µL 50% methanol , 0 . 1% formic acid .", "The final eluate was dried down in vacuo and finally reconstituted at a concentration of 0 . 66 heads/µL in 100 mM Na2HPO4 , pH 7 . 4 , 10 min by shaking at 1400 rpm in a Thermomixer , and centrifuged ( 2 min 17 , 000 ×g ) .", "6 . 0 head equivalents were used per well in the cell culture assay .", "For proteinase treatment , an aliquot of the eluted active fraction was incubated with a final concentration of 1 mg/mL proteinase K ( EC 3 . 4 . 21 . 64; Merck Millipore , Vienna , Austria; 30 mU ) for 60 min at 56°C .", "The reaction was stopped by heating the sample to 95°C for 5 min and , after cooling to 0°C by making the sample 0 . 1 mM in phenylmethylsulphonyl fluoride , finally the samples were clarified by centrifugation .", "Controls without proteinase K were treated alike .", "Again , 6 . 0 head equivalents were used per cell culture assay .", "The brain hormone enriched fraction was dried down in vacuo , suspended in 0 . 1% trifluoroacetic acid and resolved on a Symmetry-ODS-column ( Waters ) equilibrated in 20% solvent B ( 0 . 1% TFA in acetonitrile ) and 80% solvent A ( 0 . 1% TFA in water ) , after an isocratic wash for 1 . 5 min , the column was developed by raising the concentration of B to 100% over 35 min , followed by a plateau of 3 . 5 min , with a flow rate of 0 . 5 mL/min .", "The eluent was monitored at 215 nm and 280 nm and a total of 16 fractions eluting were collected .", "The fractions were dried down in vacuo and stored at −20°C until use .", "To determine the biological activity of the collected peaks , each was reconstituted in 100 mM Na2HPO4 , pH 7 . 5 at a concentration of 0 . 66 heads per µL and used in the cell culture assay as described above .", "Peaks showing a significant reduction in vitellogenin transcript abundance in two independent experiments were further analysed by LC-MS/MS , and the obtained elution profiles and fragment patterns were compared with authentic standards .", "LC-MS/MS was performed on a LTQ Orbitrap ( Thermo Fisher Scientific ) equipped with a nanospray ion source connected to a Dionex Ultimate 3000 UHPLC ( Thermo Fisher Scientific ) , equipped with a Pepmap100 C18 guard column and a Pepmap100 C18 column ( 2 cm × 75 μm and 50 cm × 75 µm , respectively , 5 µm particle size , Thermo Fisher Scientific ) equilibrated in 1% B ( 80% acetonitrile , 0 . 2% formic acid ) and 99% A ( 0 . 2% formic acid in water ) .", "The samples were loaded onto the guard column with a flow rate of 10 μL/min and washed for 10 min using 100% A . The sample was the resolved on the analytical column with a flow of 300 nL/min .", "The column was developed for 10 min isocratic with 1% B; B was then raised to 20% in one min , before reaching 80% after 49 min .", "Following a 3-min plateau , the column was washed by raising B to 90% B in 2 min and washed for another 2 min .", "Mass spectra acquisition started after 10 min; precursor ions were scanned in the range of m/z 100–1000 , and the 12 most abundant ions were fragmented in the HCD cell with a normalised fragmentation energy of 40% ± 75% .", "Heads were collected as described above and stored at −80°C .", "They were thawed at room temperature , and subjected to 4 freeze-thaw cycle between liquid N2 and 55°C warm water .", "The heads were then transferred into 1 . 5 mL auto sampler vials containing a glass bead and covered with 400 µL 80% methanol , and the head capsule was broken with four cycles in an ultra sonic water bath at 55°C for ten minutes .", "The homogenate was then extracted with 1000 µL heptane for 5 min with vigorous shaking at 1400 rpm at room temperature in a Thermomixer followed by centrifugation at 2500 ×g for 10 min .", "The organic phase was recovered and the aqueous phase re-extracted with 500 µL as above .", "The pooled extract was mildly dried down in vacuo , dissolved in 300 µL heptane , transferred into a 300 µL auto sampler vial insert , dried down , and finally dissolved in 10 µL heptane .", "The remaining aqueous phase was dried down as well , and used for protein determination ( see below ) .", "Brain hormone amounts were quantified by GC-MS on an Agilent 6890N GC equipped with an Agilent 5973 mass spectrometer .", "The GC was operated in splitless mode with constant pressure of 68 . 6 kPa and an initial flow of 1 . 0 mL/min with He-gas as carrier .", "After injection ( inlet temperature: 250°C , purge flow: 50 mL/min , purge time: 2 . 0 min ) and an initial plateau phase of 1 min at 90°C , the temperature was raised from 90°C for 280°C at 20 °C/min followed by a plateau for 7 . 5 min .", "The eluting samples were ionised and fragmented by electron impact at an ionisation energy of 2376 eV and monitored in the range from m/z 50 . 0–400 . 0 with a sampling rate of 23/s in the total ion channel , and for identification and quantification in selected ion mode ( SIM ) at m/z 114 . 0 , 136 . 0 and 219 . 0 with 3 . 1 cycles/s and a dwell time of 100 ms per ion .", "The MS-source was operated at 230°C and the quadrupole at 150°C .", "One µL of the samples was injected , and the sample was resolved on a DB-5MS column ( J&W Scientific , Agilent; 30 m × 0 . 25 mm , 0 . 25 µm film thickness ) .", "The amount of methylfarnesoate was determined by comparison to authentic standards of methylfarnesoate ( Echelon Biosciences ) .", "For quantification , serial dilutions of a 2 . 5 ng/µL sock solution of MF in heptane were measured .", "Each data point was measured in triplicate , and a linear regression was fitted .", "Peak detection and quantification was carried out with the program , MSD ChemStation ( Agilent ) ; peaks had to fit into a specified retention time window ( ± 0 . 1 min of that of the standard ) and meet the qualifier ions .", "In rare cases , where automatic peak detection failed , peaks were manually integrated and the amount of MF calculated based on the regression generated .", "The extraction efficiency of our method was assessed by using the heads of 25 young animals as a matrix and spiking in a total of 12 . 5 ng MF ( out of a 12 . 5 ng/µL stock solution ) .", "The samples were then processed as described , and finally reconstituted in 25 µL heptane , out of which one µl was measured , resulting in an expected MF amount of 500 pg .", "The stock solution served as reference and was diluted 25-fold immediately before measurement .", "The extraction efficiency was thus estimated to be >85% .", "Under our conditions , the limit of detection was 10 fmole MF ( 2 . 5 pg MF/µL ) , and the limit of quantification ~ 60 fmole MF ( ~15 pg MF/µL ) .", "Earthworms Lumbricus spec .", "( Giant Canadian Night Crawlers , Baitmaster ) were purchased at a local fishing bait shop ( Angelsport Gangl , Wien ) .", "The animals were treated with antibiotics ( 125 mg/L ampicillin , 500 mg/L streptomycin sulphate ) for 5 min , before the antibiotics solution was allowed to soak into the soil the earthworms were covered in .", "After 24 hr in the antibiotics treated soil , the animals were anaesthetised in 15% ethanol at 4°C for 15 min .", "Two to six heads ( prostomium plus the next 4 segments ) were collected on ice , snap frozen in liquid N2 , and stored at −80°C until further use .", "For the extraction of MF , the samples were subjected to four freeze-thaw cycles between 55°C and liquid N2 .", "Thereafter , they were covered in 80% MeOH and processed as described for Platynereis heads , with adjustments accounting for the larger tissue size .", "The finally obtained dried extract was reconstituted in n-heptane to yield ~2 heads per µL .", "GC-MS measurements were carried out as described for Platynereis heads .", "For in vivo treatment of worms , premature female animals ( max . Oocyte diameter < 65 µm ) were selected and kept for 2 days in sterile filtered natural sea water ( NSW ) .", "They were then decapitated ( as described above ) to synchronise and to set in motion sexual maturation ( Hauenschild , 1966 ) .", "The animals were transferred in groups of 4–5 into glazed ceramic bowls ( treated with 6% PEG 6000 to prevent unspecific hydrophobic binding of the hormones to the glass coating ) containing 50 mL sterile filtered NSW supplemented with 0 . 125 mg/mL ampicillin and 0 . 500 mg/mL streptomycin sulphate .", "Worms were treated either with 100 nM methylfarnesoate , 100 nM methoprene ( Sigma , both added from a 100 µM stock in DMSO , resulting 0 . 1% DMSO final concentration ) , or 0 . 1% DMSO alone .", "After 24 hr , water was exchanged , and the antibiotics concentration reduced to 50% , after that , the water exchanged every 24 hr .", "After 5 days , the animals were snap-frozen in liquid N2 without any residual water and stored at −80°C until RNA extraction .", "Protein concentration was determined by the bicinchoninic acid assay with BSA as standard ( Smith et al . , 1985 ) .", "Fed , young premature ( 35–50 parapodial segments , pps ) worms were kept in sterile-filtered NSW supplemented with 0 . 125 mg/mL ampicillin and 0 . 500 mg/mL streptomycin for 24 hr , and further for an additional 24 hr in half concentrated ampicillin/streptomycin .", "Generally , the regeneration assay was carried out as described by Hauenschild ( 1974 ) .", "Briefly , worms were anesthetised as described and cut three times between segments .", "The first cut was after the 21st pps , the second after the 26th pps , and the third cut was after the 31st pps producing fragments of five segments .", "The test fragments from each individual animal used for assays consisted of pps 22–26 , and 27–31 .", "The fragments were then kept in 1 mL sterile-filtered NSW containing half concentrated ampicillin/streptomycin in GC-auto sampler vials containing either 100 nM methylfarnesoate ( added from a 100 µM stock solution in DMSO , fragment ‘+MF’ ) , or vehicle alone ( 0 . 1% DMSO , fragment ‘−MF’ ) ; during the course of the experiment , the water was exchanged every 24 hr , care was taken to not let the worm fragments dry out .", "To rule out any effect of the position within the animal on regenerative abilities , +MF and control were alternated between the anterior and posterior fragments .", "The anterior and posterior +MF- and control fragments of two individual worms were combined , to serve as one biological replicate for +MF , and –MF treatment groups , respectively .", "They were snap-frozen in liquid nitrogen , stored at −80°C and used for the quantification of hox3 via qRT-PCR .", "A transcript sequence encoding Platynereis Vitellogenin ( accession no . KU756287 ) was identified from an eleocyte-specific transcriptome dataset ( Schenk et al . , unpublished ) , and sequence-validated after PCR cloning using the primers 5’-ATGAAGACTCTCCTGATCTTCG-3’ and 5’-CTAGTAGTAGAATCTTGGTCCTTCAC-3’ ( for the list of used sequencing primers see Table 2 ) . 10 . 7554/eLife . 17126 . 025Table 2 . List of primers used for cloning and sequence validation . DOI: http://dx . doi . org/10 . 7554/eLife . 17126 . 025NameSequence 5’→3’Tm / °cPdu vtg 1F ATGAAGACTCTCCTGATCTTCG60 . 1Pdu vtg 1R CTAGTAGTAGAATCTTGGTCCTTCAC64 . 6Pdu vtg_seq 1F AGCCCTAGAAGCTGCCTCTG62 . 5Pdu vtg_seq 2F ATTGCTCAATCTGAACTCCCATGC63 . 6Pdu vtg_seq 3F GCTGTTCCACAGGAAATTGC58 . 4Pdu vtg_seq 4F GCTTTGGTCAGTGGACTTCC60 . 5Pdu vtg_seq 1R GGCAATCCTCTGATGTAAACATTCTC64 . 6Pdu vtg_seq 2R CAAGCGTTTCACGACCAAGAGG64 . 2Pdu vtg_seq 3R GAAGAGCTTCTTGCTGGAGC60 . 5Pdu vtg_seq 4R AAGACCAGCTGGCGCGTTATG63 . 2Pdu met FATGGAGCCGAATTCGGAGCAGAATTCGG71 . 8Pdu met RTCAACATGTCTCAGTTTCTTTTTGAGCG65 . 6Pdu hox3 FCCCCGGGGCTCTTGGTTTT61 . 6Pdu hox3 RGCCATCTCTATTCTCCTCGGCCG68 . 3 Similarly , we identified a transcript encoding Platynereis Methoprene-tolerant ( accession no . KU756288 ) that was confirmed by sequencing of a fragment sub-cloned with the primers 5’-ATGGAGCCGAATTCGGAGCAGAATTCGG-3’ and 5’-TCAACATGTCTCAGTTTCTTTTTGAGCG-3’ .", "For cloning , cDNA was prepared with the Transcriptor High Fidelity cDNA Synthesis Kit ( Roche ) according to the manufacturer’s instructions with oligo dT-priming from eleocyte or whole worm RNA ( isolated with the RNeasy Kit , Qiagen ) .", "PCR was performed using Phusion Taq ( Thermo ) , using the following cycle: 1x {98°C , 30”}; 35x {98°C , 7”; x°C , 20”; 72°C , 30”/kb}; 1x {72°C , 7’30”}; 10°C .", "The annealing temperature ‘x’ was determined by the web page ‘OligoCalc’ ( http://www . basic . northwestern . edu/biotools/oligocalc . html ) , using on the 'salt adjusted' algorithm .", "If necessary , annealing temperatures were optimized by gradient PCR and adjusted accordingly .", "The PCR products were run on agarose gels , stained with SybrSafe ( Invitrogen ) DNA-dye , the specific products were cut out and purified using the QIAquick gel extraction kit ( Qiagen ) .", "The purified PCR-products were sub-cloned into a pJET-vector ( CloneJet Kit , Thermo ) , and transformed into NEB-5-alpha ( New England Biolabs ) chemical competent E . coli .", "Bacteria plated on Luria-Bertani ( LB ) -medium agar plates supplemented with ampicillin ( amp , 50 µg/mL ) , after growth over night single colonies were selected and grown over night in LBAmp-medium .", "The next day , plasmids were purified ( Birnboim and Doly , 1979 ) and submitted for Sanger sequencing using the indicated primers .", "For domain analyses of Platynereis Vtg and lipoproteins representatives for other clades , we used the domain annotation implemented in SMART ( http://smart . embl . de; Schultz et al . , 1998; Letunic et al . , 2015 ) .", "For determining phylogenetic relationships , we aligned full-length protein sequences representative of distinct clades using MAFFT v7 . 221 ( Katoh and Standley , 2013 ) , and constructed Maximum Likelihood trees with IQ-TREE 1 . 3 . 12 ( Minh et al . , 2013; Nguyen et al . , 2015 ) .", "The consensus tree was visualized using iTOL ( http://itol . embl . de/ , Letunic and Bork , 2011 ) .", "For assessing the phylogenetic relationships of Platynereis Met and other bHLH-PAS domain proteins , BLASTP searches were performed against the nr section of the NCBI sequence repository .", "Sequences from representatives of selected clades were aligned using MAFFT v7 . 221 ( Katoh and Standley , 2013 ) , and maximum likelihood phylogenetic trees were constructed with IQ-TREE , using the built-in parameter optimisation ( Minh et al . , 2013; Nguyen et al . , 2015 ) .", "All statistical tests were performed with the program R ( R Core Team , 2015 ) .", "First , all data were tested for normal distribution using the Shapiro-Wilk normality test .", "Statistical significance was then tested with a t-test incorporating Welch`s correction to account for heteroscedasticity .", "If more than one comparison was to be assessed , statistical significance was first tested by a one-way analysis of variance test ( ANOVA ) , and if significance was given , pair wise comparisons were carried out by a ( Welch-corrected ) t-test and the obtained p-values were adjusted for multiple testing by the method of Benjamini and Hochberg implemented in R yielding adjusted p-values ( padj ) .", "A result was considered as statistically significant if p<0 . 05 in the case of t-tests , and p<0 . 10 in the case of ANOVA , the following t-test were then again considered to yield a statistically significant result if padj < 0 . 05 .", "Significance levels of p-values and adjusted p-values are indicated by *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 , if not stated otherwise ." ] ]

[ "Animals require molecular signals to determine when to divert resources from somatic functions to reproduction .", "This decision is vital in animals that reproduce in an all-or-nothing mode , such as bristle worms: females committed to reproduction spend roughly half their body mass for yolk and egg production; following mass spawning , the parents die .", "An enigmatic brain hormone activity suppresses reproduction .", "We now identify this hormone as the sesquiterpenoid methylfarnesoate .", "Methylfarnesoate suppresses transcript levels of the yolk precursor Vitellogenin both in cell culture and in vivo , directly inhibiting a central energy–costly step of reproductive maturation .", "We reveal that contrary to common assumptions , sesquiterpenoids are ancient animal hormones present in marine and terrestrial lophotrochozoans .", "In turn , insecticides targeting this pathway suppress vitellogenesis in cultured worm cells .", "These findings challenge current views of animal hormone evolution , and indicate that non-target species and marine ecosystems are susceptible to commonly used insect larvicides ." ]

[ "All organisms need energy to survive and grow .", "However , sources of energy are limited and so organisms need to decide how to spend the resources they have available .", "For instance , animals must choose whether they should continue to grow or if they should invest energy into reproduction instead .", "This decision becomes even more important for animals that reproduce in an “all-or-nothing” manner and invest all their available energy into reproduction and die soon after .", "Bristle worms live in coastal areas around world .", "In mass spawning events , thousands of individuals raise from the sea floor to the surface , to release sperm and eggs .", "While the fertilized eggs start to develop in the water , the parents invariably die .", "The female worms spend roughly half their body mass in producing eggs and supplying them with yolk as a source of energy .", "It has been known for decades that the brains of bristle worms produce a master hormone that promotes growth and suppresses reproduction .", "Yet the identity of this hormone that controls the life-or-death decision was not clear .", "Schenk et al . took advantage of new molecular tools to solve this puzzle .", "The experiments show that this hormone directly regulates how much yolk the female animals produce .", "This allowed Schenk et al . to design a new molecular assay that helped to identify the hormone itself .", "Unexpectedly , the hormone – called methylfarnesoate – belongs to a family of small molecules called sesquiterpenoids , which researchers previously thought were only found in insects and related groups .", "Hence , many insecticides have been developed to target sesquiterpenoid signaling and they are used in massive amounts to fight pests like the tiger mosquito ( which transmits the Zika virus ) .", "Schenk et al . also found that these insecticides also cause severe problems in bristle-worms .", "These findings challenge current views of how animal hormones have evolved and indicate that common insecticides may be harming bristle worms and other animals in marine environments .", "The next steps are to find out whether methylfarnesoate is found in other closely related animals , such as snails and mussels , and whether the insecticides are harmful to these animals too .", "Another future challenge will be to investigate how this hormone actually promotes animal growth ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology" ]

[ [ "The fitness landscape is a fundamental concept in evolutionary biology ( Kauffman and Levin , 1987; Poelwijk et al . , 2007; Romero and Arnold , 2009; Hartl , 2014; Kondrashov and Kondrashov , 2015; de Visser and Krug , 2014 ) .", "Large-scale datasets combined with quantitative analysis have successfully unraveled important features of empirical fitness landscapes ( Kouyos et al . , 2012; Barton et al . , 2015; Szendro et al . , 2013 ) .", "Nevertheless , there is a huge gap between the limited throughput of fitness measurements ( usually on the order of 102 variants ) and the vast size of sequence space .", "Recently , the bottleneck in experimental throughput has been improved substantially by coupling saturation mutagenesis with deep sequencing ( Fowler et al . , 2010; Hietpas et al . , 2011; Jacquier et al . , 2013; Wu et al . , 2014; Thyagarajan and Bloom , 2014; Qi et al . , 2014; Stiffler et al . , 2015 ) , which opens up unprecedented opportunities to understand the structure of high-dimensional fitness landscapes ( Jiménez et al . , 2013; Pitt and Ferré-D'Amaré , 2010; Payne and Wagner , 2014 ) .", "Previous empirical studies on combinatorially complete fitness landscapes have been limited to subgraphs of the sequence space consisting of only two amino acids at each site ( 2L genotypes ) ( Weinreich et al . , 2006; Lunzer et al . , 2005; O’Maille et al . , 2008; Lozovsky et al . , 2009; Franke et al . , 2011; Tan et al . , 2011 ) .", "Most studies of adaptive walks in these diallelic sequence spaces focused on “direct paths” where each mutational step reduces the Hamming distance from the starting point to the destination .", "However , it has also been shown that mutational reversions can occur during adaptive walks in diallelic sequence spaces such that adaptation proceeds via “indirect paths” ( DePristo et al . , 2007; Berestycki et al . , 2014; Martinsson , 2015; Li , 2015; Palmer et al . , 2015 ) .", "In sequence space with higher dimensionality ( 20L , for a protein sequence with L amino acid residues ) , the extra dimensions may further provide additional routes for adaptation ( Gavrilets , 1997; Cariani , 2002 ) .", "Although the existence of indirect paths has been implied in different contexts , it has not been studied systematically and its influence on protein adaptation remains unclear .", "Another underappreciated property of fitness landscapes is the influence of higher-order interactions .", "Empirical evidence suggests that pairwise epistasis is prevalent in fitness landscapes ( Kvitek and Sherlock , 2011; Kouyos et al . , 2012; O’Maille et al . , 2008; Lozovsky et al . , 2009 ) .", "Specifically , sign epistasis between two loci is known to constrain adaptation by limiting the number of selectively accessible paths ( Weinreich et al . , 2006 ) .", "Higher-order epistasis ( i . e . interactions among more than two loci ) has received much less attention and its role in adaptation is yet to be elucidated ( Weinreich et al . , 2013; Palmer et al . , 2015 ) ." ], [ "In this study , we investigated the fitness landscape of all variants ( 204 = 160 , 000 ) at four amino acid sites ( V39 , D40 , G41 and V54 ) in an epistatic region of protein G domain B1 ( GB1 , 56 amino acids in total ) ( Figure 1—figure supplement 1 ) , an immunoglobulin-binding protein expressed in Streptococcal bacteria ( Sjöbring et al . , 1991; Sauer-Eriksson et al . , 1995 ) .", "The four chosen sites contain 12 of the top 20 positively epistatic interactions among all pairwise interactions in protein GB1 , as we previously characterized ( Olson et al . , 2014 ) ( Figure 1—figure supplement 2 ) .", "Thus the sequence space is expected to cover highly beneficial variants , which presents an ideal scenario for studying adaptive evolution .", "Moreover , this empirical fitness landscape is expected to provide us insights on how high dimensionality and epistasis would influence evolutionary accessibility .", "Briefly , a mutant library containing all amino acid combinations at these four sites was generated by codon randomization .", "The “fitness” of protein GB1 variants , as determined by both stability ( i . e . the fraction of folded proteins ) and function ( i . e . binding affinity to IgG-Fc ) , was measured in a high-throughput manner by coupling mRNA display with Illumina sequencing ( see Materials and methods , Figure 1—figure supplement", "3 ) ( Roberts and Szostak , 1997; Olson et al . , 2012 ) .", "The relative frequency of mutant sequences before and after selection allowed us to compute the fitness of each variant relative to the wild type protein ( WT ) .", "While most mutants had a lower fitness compared to WT ( fitness < 1 ) , 2 . 4% of mutants were beneficial ( fitness > 1 ) .", "( Figure 1—figure supplement 4 ) .", "We note that this study does not aim to extrapolate protein fitness to organismal fitness .", "Although there are examples showing that protein fitness in vitro correlates with organismal fitness in vivo ( Natarajan et al . , 2013; Wu et al . , 2012 ) , this relation may not be linear and is likely to be system-specific due to the difference in selection pressures in vitro and in vivo ( Pál et al . , 2006; Hingorani and Gierasch , 2014 ) .", "To understand the impact of epistasis on protein adaptation , we first analyzed subgraphs of sequence space including only two amino acids at each site ( Figure 1A ) .", "Each subgraph represented a classical adaptive landscape connecting WT to a beneficial quadruple mutant , analogous to previously studied protein fitness landscapes ( Weinreich et al . , 2006; Szendro et al . , 2013 ) .", "Each variant is denoted by the single letter code of amino acids across sites 39 , 40 , 41 and 54 ( for example , WT sequence is VDGV ) .", "Each subgraph is combinatorially complete with 24 = 16 variants , including WT , the quadruple mutant , and all intermediate variants .", "We identified a total of 29 subgraphs in which the quadruple mutant was the only fitness peak .", "By focusing on these subgraphs , we essentially limited the analysis to direct paths of adaptation , where each step would reduce the Hamming distance from the starting point ( WT ) to the destination ( quadruple mutant ) .", "Out of 24 possible direct paths , the number of selectively accessible paths ( i . e . with monotonically increasing fitness ) varied from 12 to 1 among the 29 subgraphs ( Figure 1B ) .", "In the most extreme case , only one path was accessible from WT to the quadruple mutant WLFA ( Figure 1A ) .", "We also observed a substantial skew in the computed probability of realization among accessible direct paths ( Figure 1—figure supplement 5 ) , suggesting that most of the realizations in adaptation were captured by a small fraction of possible trajectories ( Weinreich et al . , 2006 ) .", "These results indicated the existence of sign epistasis and reciprocal sign epistasis , both of which may constrain the accessibility of direct paths ( Weinreich et al . , 2006; Tufts et al . , 2015 ) .", "Indeed , we found that these two types of epistasis were prevalent in our fitness landscape ( Figure 1C ) .", "Furthermore , we classified the types of all 24 pairwise epistasis in each subgraph and computed the level of ruggedness as fs⁢i⁢g⁢n+2⁢fr⁢e⁢c⁢i⁢p⁢r⁢o⁢c⁢a⁢l , where ft⁢y⁢p⁢e was the fraction of each type of pairwise epistasis .", "As expected , the number of selectively inaccessible direct paths , i . e . paths that involve fitness declines , was found to be positively correlated with the ruggedness induced by pairwise epistasis ( Figure 1—figure supplement 6 , Pearson correlation = 0 . 66 , p=1 . 0 × 10−4 ) ( Poelwijk et al . , 2007 ) . 10 . 7554/eLife . 16965 . 003Figure 1 . Direct paths of adaptation are constrained by pairwise epistasis .", "( A ) An example of subgraph that contains VDGV ( wild type , WT ) , the quadruple mutant WLFA and all intermediates between them .", "Each variant in the subgraph is represented by a node .", "Edges are drawn between nearest neighbors .", "The arrows in bold represent the only accessible direct path of adaptation from VDGV to WLFA .", "HD: Hamming distance .", "( B ) We identified a total of 29 subgraphs in which the quadruple mutant was the only fitness peak .", "The number of accessible direct paths from WT to the quadruple mutant is shown for each subgraph .", "The maximum number of direct paths is 24 .", "( C ) The fraction of three types of pairwise epistasis around WT ( 2091 out of 2166 ) , randomly sampled from the entire sequence space ( 105 in total ) , or in the neighborhood of the top 100 fitness variants and 100 lethal variants .", "We note that this analysis is different from previous studies on how epistasis changes along adaptive walks , where the quadruples are chosen such that the fitness values of genotype 00 , 01 and 11 are in increasing order ( Greene and Crona , 2014 ) .", "Sign epistasis and reciprocal sign epistasis , both of which can block adaptive paths , are prevalent in the fitness landscape .", "Classification scheme of epistasis is shown at the top .", "Each node represents a genotype , which is within a sequence space of two loci and two alleles .", "Green arrows represent the accessible paths from genotype “00” to a beneficial double mutant “11” ( colored in red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 00310 . 7554/eLife . 16965 . 004Figure 1—figure supplement 1 . The four-site sequence space of protein G .", "( A ) The locations of sites 39 , 40 , 41 , and 54 of protein GB1 are shown on the protein structure .", "PDB: 1PGA ( Gallagher et al . , 1994 ) .", "( B ) The WT sequence of the nucleotide template ( Olson et al . , 2014 ) .", "T7 promoter is highlighted in magenta .", "Randomized sites ( 39 , 40 , 41 , and 54 ) are highlighted in red .", "Poly-GS linkers are highlighted in green .", "FLAG-tag is highlighted in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 00410 . 7554/eLife . 16965 . 005Figure 1—figure supplement 2 . Positive epistasis is enriched in the four-site sequence space . The distribution of pairwise epistasis measured by Olson et al . ( Olson et al . , 2014 ) .", "The pairwise epistatic values among sites 39 , 40 , 41 , and 54 are ranked and represented by the red line .", "The pairwise epistatic values among other sites ( all but 39 , 40 , 41 , and 54 ) are ranked and represented by the black line . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 00510 . 7554/eLife . 16965 . 006Figure 1—figure supplement 3 . Workflow of mRNA display and data validation .", "( A ) The workflow of mRNA display is shown .", "This is adapted from ( Olson et al . , 2014 ) .", "( B ) The fitness values for all single substitution variants and double substitution variants in this study and in our previous study ( based on an independently constructed library ) ( Olson et al . , 2014 ) are compared .", "The high correlation ( Pearson correlation = 0 . 97 , p<2 . 2× 10−16 ) validates the fitness data obtained in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 00610 . 7554/eLife . 16965 . 007Figure 1—figure supplement 4 . Comparison of the wild type to the ensemble of possible genotypes .", "( A ) The distribution of fitness values of all non-lethal genotypes .", "The fitness rank of the wild type genotype is 2 . 4% in the distribution of all fitness measurements .", "19 . 7% of the measured genotypes are lethal ( fitness=0 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 00710 . 7554/eLife . 16965 . 008Figure 1—figure supplement 5 . Subgraph analysis . We calculated the relative probabilities to realize each accessible path ( Weinreich et al . , 2006 ) ( see Materials and methods ) .", "In all the subgraphs analyzed , we found that most of the realizations were captured by a few accessible paths , as demonstrated by the skew in the cumulative probability of realization among different paths .", "( A ) Cumulative probability of realization for mutational trajectories from WT ( VDGV ) to beneficial variants that have a Hamming distance ( HD ) of 4 from WT .", "This analysis only included those subgraphs with a reachable quadruple mutation variant ( HD = 4 from WT ) as the only fitness peak .", "Correlated Fixation Model is used .", "The diagonal line indicates the cumulative probability of a subgraph with equal probability of realization for all 24 possible trajectories .", "The bias of probability of realization in each subgraph was quantified using the Gini index ( see Materials and methods ) and is shown as a histogram in the inset .", "( B ) Same as panel A , except Equal Fixation Model is used instead .", "( C and D )", "Cumulative probability for mutational trajectories from a deleterious variants that have a Hamming distance ( HD ) of 4 from WT ( VDGV ) to WT .", "This analysis only included those subgraphs with WT being the only fitness peak and the quadruple variant has a fitness between 0 . 01 to 1 .", "The diagonal line indicates the cumulative probability of a subgraph with equal probability of realization for all 24 possible trajectories .", "The bias of probability of realization in each subgraph was quantified using the Gini index and is shown as a histogram in the inset .", "A total of 526 subgraphs were analyzed .", "( C ) Correlated Fixation Model is used .", "( D ) Equal Fixation Model is used .", "( E ) Number of accessible trajectory from a deleterious variants that have a Hamming distance ( HD ) of 4 from WT ( VDGV ) to WT in each subgraph is shown as a barplot .", "The maximum possible number of accessible trajectory is 24 .", "( F ) The distribution of number of accessible trajectory is shown as a box plot .", "“Adaptation from WT” indicates those subgraphs based on the adaptation from WT to a beneficial variant that has a Hamming distance of 4 from WT .", "“Adaptation to WT” indicates those subgraphs based on the adaptation from a deleterious variant that has a Hamming distance of 4 from WT to WT . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 00810 . 7554/eLife . 16965 . 009Figure 1—figure supplement 6 . Correlation between the number of selectively inaccessible direct paths and ruggedness . The number of inaccessible direct paths is positively correlated ( Pearson correlation = 0 . 66 , p=1 . 0 × 10−4 ) with the ruggedness induced by sign and reciprocal sign epistasis .", "The level of ruggedness is quantified as fs⁢i⁢g⁢n+2⁢fr⁢e⁢c⁢i⁢p⁢r⁢o⁢c⁢a⁢l , where ft⁢y⁢p⁢e denotes the fraction of each type of pairwise epistasis .", "The number inside a symbol indicates the number of subgraphs with identical properties . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 009 Our findings support the view that direct paths of protein adaptation are often constrained by pairwise epistasis on a rugged fitness landscape ( Weinreich et al . , 2005; Kondrashov and Kondrashov , 2015 ) .", "In particular , adaptation can be trapped when direct paths are blocked by reciprocal sign epistasis .", "However , crucially , this analysis was limited to mutational trajectories within a subgraph of the sequence space .", "In reality , the dimensionality of protein sequence space is higher .", "Intuitively , when an extra dimension is introduced , a local maximum may become a saddle point and allow for further adaptation – a phenomenon that is also known as “extra-dimensional bypass” ( Gavrilets , 1997; Cariani , 2002; Gutiérrez and Maere , 2014 ) .", "With our experimental data , we observed two distinct mechanisms of bypass , either using an extra amino acid at the same site or using an additional site , that allow proteins to continue adaptation when no direct paths were accessible due to reciprocal sign epistasis ( Figure 2 ) .", "The first mechanism of bypass , which we termed “conversion bypass” , works by converting to an extra amino acid at one of the interacting sites ( Palmer et al . , 2015 ) .", "Consider a simple scenario with only two interacting sites .", "If the sequence space is limited to 2 amino acids at each site , as in past analyses of adaptive trajectories , the number of neighbors is 2; however , if all 20 possible amino acids were considered , the total number of neighbors would be 38 .", "Some of these 36 extra neighbors may lead to potential routes that circumvent the reciprocal sign epistasis ( Figure 2A ) .", "In this case , a successful bypass would require a conversion step that substitutes one of the two interacting sites with an extra amino acid ( 00→20 ) , followed by the loss of this mutation ( 21→11 ) .", "This bypass is feasible only if the original reciprocal sign epistasis is changed to sign epistasis after the conversion .", "To test whether such bypasses were present in our system , we randomly sampled 105 pairwise interactions from the sequence space and analyzed the ~20 , 000 reciprocal sign epistasis among them ( see Materials and methods ) .", "More than 40% of the time there was at least one successful conversion bypass and in many cases multiple bypasses were available ( Figure 2B ) .", "The second mechanism of bypass , which we termed “detour bypass” , involves an additional site ( Figure 2C ) .", "In this case , adaptation can proceed by taking a detour step to gain a mutation at the third site ( 000→100 ) , followed by the later loss of this mutation ( 111→011 ) ( DePristo et al . , 2007; Palmer et al . , 2015 ) .", "Detour bypass was observed in our system ( Figure 2D ) , but was not as prevalent and had a lower probability of success than conversion bypass .", "Out of 38 possible detour bypasses for a chosen reciprocal sign epistasis , we found that there were on average 1 . 2 conversion bypasses and 0 . 27 detour bypasses available .", "We note , however , that the lower prevalence of detour bypass in our fitness landscape ( L=4 ) does not necessarily mean that it should be expected to be less frequent than conversion bypass in other systems .", "While the maximum number of possible conversion bypasses is always fixed ( 19×2-2=36 ) , the maximum number of possible detour bypasses ( 19 × ( L − 2 ) ) is proportional to the sequence length L of the entire protein ( whereas our study uses a subset L = 4 ) .", "The pervasiveness of extra-dimensional bypasses in our system contrasts with the prevailing view that adaptive evolution is often blocked by reciprocal sign epistasis , when only direct paths of adaptation are considered .", "The two distinct mechanisms of bypass both require the use of indirect paths , where the Hamming distance to the destination is either unchanged ( conversion ) or increased ( detour ) . 10 . 7554/eLife . 16965 . 010Figure 2 . Two distinct mechanisms of extra-dimensional bypass .", "( A ) Extra amino acids at one of the two interacting sites may open up potential paths that circumvent the reciprocal sign epistasis .", "The starting point is 00 and the destination is 11 ( in red ) .", "Green arrows indicate the accessible path .", "A successful bypass would require a “conversion” step that substitutes one of the two interacting sites with an extra amino acid ( 00→20 ) , followed by the loss of this mutation later ( 21→11 ) .", "The original reciprocal sign epistasis is changed to sign epistasis on the new genetic background after conversion .", "( B ) Among ~20 , 000 randomly sampled reciprocal sign epistasis , >40% of them can be circumvented by at least one conversion bypass ( i . e . success , inset ) .", "The number of available bypass for the success cases is shown as histogram .", "( C ) The second mechanism of bypass involves an additional site .", "In this case , adaptation involves a “detour” step to gain mutation at the third site ( 000→100 ) , followed by the loss of this mutation ( 111→011 ) .", "The original reciprocal sign epistasis is changed to either magnitude epistasis or sign epistasis on the new genetic background after detour ( Figure 2—figure supplement 1 ) .", "( D ) In comparison to conversion bypass , detour bypass has a lower probability of success ( <20% , inset ) and is less prevalent . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01010 . 7554/eLife . 16965 . 011Figure 2—figure supplement 1 . Three scenarios of extra-dimensional bypass via an extra site .", "( A ) Reciprocal sign epistasis may be bypassed via the involvement of a third site .", "( B–D )", "There are three possible scenarios .", "With a mutation at the third site , reciprocal sign epistasis between the first two sites can be changed to magnitude epistasis in ( B ) or sign epistasis in ( C–D ) .", "It can be proven that higher-order epistasis is required for the scenarios in ( B ) and ( C ) ( see Materials and methods ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 011 In order to circumvent the inaccessible direct paths via extra dimensions , reciprocal sign epistasis must be changed into other types of pairwise epistasis .", "For detour bypass , this means that the original reciprocal sign epistasis is changed to either magnitude epistasis or sign epistasis in the presence of a third mutation ( Figure 2—figure supplement 1A ) .", "There are three possible scenarios where detour bypass can occur ( Figure 2—figure supplement 1B–D ) .", "We proved that higher-order epistasis is necessary for the scenario that reciprocal sign epistasis is changed to magnitude epistasis , as well as for one of the two scenarios that reciprocal sign epistasis is changed to sign epistasis ( see Materials and methods ) .", "This suggests a critical role of higher-order epistasis in mediating detour bypass .", "To confirm the presence of higher-order epistasis , we decomposed the fitness landscape by Fourier analysis ( see Materials and methods , Figure 3—figure supplement", "1 ) ( Szendro et al . , 2013; Weinreich et al . , 2013; Neidhart et al . , 2013 ) .", "The Fourier coefficients can be interpreted as epistatic interactions of different orders ( Weinreich et al . , 2013; de Visser and Krug , 2014 ) , including the main effects of single mutations ( the first order ) , pairwise epistasis ( the second order ) , and higher-order epistasis ( the third and the fourth order ) .", "The fitness of variants can be reconstructed by expansion of Fourier coefficients up to a certain order ( Figure 3—figure supplement 2 ) .", "In our system with four sites , the fourth order Fourier expansion will always reproduce the measured fitness ( i . e . the fraction of variance in fitness explained equals 1 ) .", "When the second order Fourier expansion does not reproduce the measured fitness , it indicates the presence of higher-order epistasis .", "In this way , we identified the 0 . 1% of subgraphs with greatest fitness contribution from higher-order epistasis ( Figure 3A , red lines ) and visualized the corresponding quadruple mutants by the sequence logo plot ( Figure 3B ) .", "The skewed composition of amino acids in these subgraphs indicates that higher-order interactions are enriched among specific amino acid combinations of site 39 , 41 and 54 .", "This interaction among 3 sites is consistent with our knowledge of the protein structure , where the side chains of sites 39 , 41 , and 54 can physically interact with each other at the core ( Figure 1—figure supplement 1A ) and destabilize the protein due to steric effects ( Figure 3—figure supplement 3 ) .", "In the presence of higher-order epistasis , epistasis between any two sites would vary across different genetic backgrounds .", "We computed the magnitude of pairwise epistasis ( ε ) between each pair of amino acid substitutions ( see Materials and methods ) ( Khan et al . , 2011 ) , and observed numerous instances where the sign of pairwise epistasis depended on genetic background .", "For example , G41L and V54H were positively epistatic when site 39 was isoleucine [I] , but the interaction changed to negative epistasis when site 39 carried a tyrosine [Y] or a tryptophan [W] ( Figure 3C–D ) .", "Similar patterns were observed in other pairwise interactions among site 39 , 41 and 54 , such as G41F/V54A and V39W/V54H ( Figure 3—figure supplement 4 ) .", "The observed pattern of higher-order epistasis was consistent with the results of the Fourier analysis ( Figure 3B ) .", "For example , site 40 was mostly excluded from higher-order epistasis; tyrosine [Y] or tryptophan [W] at site 39 were involved in the most significant higher-order interactions , as they often changed the sign of pairwise epistasis .", "Higher-order epistasis can also switch the type of pairwise epistasis , such as shifting from reciprocal sign epistasis to magnitude or sign epistasis ( Figure 3—figure supplement 5 ) , which in turn is important for the existence of detour bypass . 10 . 7554/eLife . 16965 . 012Figure 3 . Evidence of higher-order epistasis .", "( A ) The fitness decomposition was performed on all subgraphs without missing variants .", "The fitness of variants can be reconstructed using Fourier coefficients truncated to a certain order .", "The fraction of variance in fitness explained by expansion of Fourier coefficients truncated to different orders ( from first to fourth ) is shown for each subgraph .", "The blue line corresponds to the median .", "The top 0 . 1% subgraphs with fitness contributions from higher-order epistasis ( the bottom 0 . 1% subgraphs ranked by fraction of variance explained at second order expansion ) are shown in red lines .", "( B ) A sequence logo was generated for the quadruple mutants corresponding to the top 0 . 1% subgraphs with higher-order epistasis .", "The skewed composition of amino acids indicates that higher-order interactions are enriched among specific amino acid combinations of site 39 , 41 and 54 .", "( C ) The magnitude of pairwise epistasis between G41L and V54H across different genetic backgrounds ( i . e . all combinations of amino acids at site 39 and", "40 ) is shown as a heat map .", "The amino acids of WT are boxed .", "Epistasis that cannot be determined due to missing variant is colored in grey .", "( D ) Altering the genetic background at site 39 changed the positive epistasis ( ε>0 ) between G41L and V54H to negative epistasis ( ε<0 ) .", "The fitness of each variant is indicated in the parentheses . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01210 . 7554/eLife . 16965 . 013Figure 3—figure supplement 1 . The fraction of variance explained by Fourier coefficients at each order . For the 109 , 235 complete subgraphs that we analyzed , the fraction of the variance in fitness explained by each order is shown .", "Although the first order ( main effects ) and the second order Fourier coefficients ( pairwise epistasis ) can explain most of the variance in fitness , higher-order epistasis is present in certain combination of mutations . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01310 . 7554/eLife . 16965 . 014Figure 3—figure supplement 2 . Fourier analysis decomposes the fitness landscape into epistatic interactions of different orders . Here we show two examples where the fitness contribution from higher-order epistasis is small ( bottom 0 . 1% , from VDGV to CSPA ) in ( A ) and large ( top 0 . 1% , from VDGV to WLLH ) in ( B ) .", "The fitness of variants can be reconstructed using Fourier coefficients truncated to a certain order .", "The Fourier coefficients can be interpreted as epistatic interactions of different orders , including the main effects of single mutations ( the first order ) , pairwise epistasis ( the second order ) , and higher-order epistasis ( the third and the fourth order ) .", "( C ) In our system with four sites , the reconstructed fitness by expansion to the fourth order Fourier coefficients will always reproduce the measured fitness ( i . e . Pearson correlation equals 1 ) .", "If expansion to the second order Fourier coefficients did not reproduce the measured fitness ( i . e . Pearson correlation less than 1 ) , it would indicate the presence of higher-order epistasis . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01410 . 7554/eLife . 16965 . 015Figure 3—figure supplement 3 . Relationship between fitness , size of the protein core , and predicted ΔΔG .", "( A and B )", "The relationship between fitness and the total volume of residue 39 , 41 , and 54 is shown as a scatter plot for ( A ) all variants , and ( B ) variants with HD of 4 from WT .", "The blue line indicates the volume of WT ( VDGV ) .", "The red line indicates the fraction of beneficial variants within a sliding window of ± 20 Å3 .", "We postulated that the observed higher-order epistasis was , at least partially , due to the steric effect among site 39 , 41 , and 54 .", "This was evidenced by the enrichment of beneficial variants when the total volume of these three interacting residues was between ~200 Å3 and ~300 Å3 .", "As the total volume further increased , proportion of beneficial variants dropped .", "( C and D )", "The relationship between the predicted Δ⁢ΔG and the total size of residue 39 , 41 , and 54 for all variants is shown as a scatter plot for ( C ) all variants ( Pearson correlation = 0 . 41 ) , and ( D ) variants with HD of 4 from WT ( Pearson correlation = 0 . 34 ) .", "The purple line represents the linear regression .", "The predicted Δ⁢ΔG increased as the total volume of core residues increased , indicating that the protein would be destabilized ( i . e . decrease in fitness ) when the core was overpacked .", "Therefore , the higher-order epistasis observed in this study could be partially attributed to steric effect .", "Nonetheless , we acknowledged that entropic effect and conformational effect in IgG-FC binding may also contribute to higher-order epistasis . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01510 . 7554/eLife . 16965 . 016Figure 3—figure supplement 4 . Alteration of pairwise epistatic effect under different genetic backgrounds .", "( A ) Pairwise epistatic effect of each substitution pair under each genetic background ( total possible genetic backgrounds for each substitution pair = 20 × 20 = 400 ) was quantified .", "For each substitution pair , the range of epistasis across different genetic backgrounds is shown in the top panel ( brown ) .", "For each substitution pair , the standard deviation of epistasis across different genetic backgrounds is shown in the middle panel ( green ) .", "For each substitution pair , the maximum epistatic value across different genetic backgrounds ( magenta ) , the minimum epistatic value across different genetic backgrounds ( cyan ) , and the epistatic value under WT background ( grey ) are shown .", "Substitution pair is ranked by the range of epistasis ( maximum epistatic value minus minimum epistatic value ) .", "( B ) Epistasis between G41F and V54A across different genetic backgrounds ( different combination of amino acids in sites 39 and", "40 ) is shown .", "The epistasis value is color coded .", "Amino acids of WT are boxed .", "Epistasis that cannot be determined due to missing variant is colored in grey .", "( C ) Epistasis between V39W and V54H across different genetic backgrounds ( different combination of amino acids in sites 40 and", "41 ) is shown .", "The epistasis value is color coded .", "Amino acids of WT are boxed .", "Epistasis that cannot be determined due to missing variant is colored in grey . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01610 . 7554/eLife . 16965 . 017Figure 3—figure supplement 5 . Higher-order epistasis can change the type of pairwise epistasis . The type of pairwise interaction could be changed in the presence of higher-order epistasis .", "( A ) Reciprocal sign epistasis between G41L-V54G is changed to magnitude epistasis given the mutation at site 39 ( K39W ) .", "( B ) Reciprocal sign epistasis between G41L-V54G is changed to sign epistasis given the mutation at site 39 ( Q39Y ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01710 . 7554/eLife . 16965 . 018Figure 3—figure supplement 6 . Higher-order epistasis increases the ruggedness of fitness landscapes . To further study the effect of higher-order epistasis on landscape ruggedness , we performed additional analyses on the subgraphs identified in Figure 1B .", "We applied Fourier decomposition and set all higher-order coefficients to 0 .", "( A ) Among the subgraphs in which the quadruple mutant was still the only fitness peak , we found that the ruggedness score did not show a systematic trend after removal of higher-order epistasis .", "( B ) In the meantime , on average the number of accessible direct paths seemed to decrease .", "Each data point is color-coded so that individual subgraphs can be matched between panel A and B . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 018 Our analysis on circumventing reciprocal sign epistasis revealed how indirect paths could open up new avenues of adaptation .", "To study the impact of indirect paths at a global scale , we performed simulated adaptation in the entire sequence space of 160 , 000 variants .", "The fitness landscape was completed by imputing fitness values of the 10 , 639 missing variants ( i . e . 6 . 6% of the sequence space ) that had fewer than 10 sequencing read counts in the input library .", "Our model of protein fitness incorporated main effects of single mutations , pairwise interactions , and three-way interactions among site 39 , 41 and 54 ( see Materials and methods , Figure 4—figure supplement 1 ) .", "We used predictor selection based on biological knowledge , followed by regularized regression , which has been demonstrated to ameliorate possible bias in the inferred fitness landscape ( Otwinowski and Plotkin , 2014 ) .", "In the complete sequence space , we identified a total of 30 fitness peaks ( i . e . local maxima ) ; among them 15 peaks had fitness larger than WT and their combined basins of attraction covered 99% of the sequence space ( Figure 4A ) . 10 . 7554/eLife . 16965 . 019Figure 4 . Indirect paths promote evolutionary accessibility .", "( A ) 15 peaks had fitness larger than WT and their combined basins of attraction accounted for 99% of the entire sequence space .", "The size of each basin of attraction is identified by the Greedy Model ( see Materials and methods ) .", "The area of each node is in proportion to the size of the basin of attraction of the corresponding fitness peak .", "An edge is drawn between fitness peaks that are separated by a Hamming distance of 2 .", "( B ) A possible adaptive path starting from WT ( VDGV ) to the fitness peak LYGV .", "( C ) The frequency of different types of mutational step are shown .", "Three models , including the Greedy Model ( green ) , Correlated Fixation Model ( blue ) and Equal Fixation Model ( red ) , are used to simulate 1000 adaptive paths starting from each variant in the sequence space .", "All the adaptive paths end at a fitness peak .", "( D ) The distribution of the length of the adaptive path initiated at different starting points .", "For Correlated Fixation Model and Equal Fixation Model , the length was computed by averaging over 1000 simulated paths for each starting point .", "The scale on the left is for Greedy Model .", "The scale on the right is for Correlated Fixation Model and Equal Fixation Model .", "( E ) Indirect paths increased the number of genotypes accessible to each fitness peak .", "The 15 peaks are ordered by increasing fitness ( from left to right ) .", "( F ) A large fraction of beneficial variants in the sequence space ( fitness > 1 ) were accessible from WT only via indirect paths .", "Beneficial variants were classified by their Hamming distance ( HD ) from WT . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 01910 . 7554/eLife . 16965 . 020Figure 4—figure supplement 1 . Lasso regression . Coefficients of the statistical model were fit by lasso regression on the measured fitness values of 119 , 884 non-lethal variants ( see Materials and methods ) .", "( A ) 10-fold CV ( cross-validation ) MSE ( mean squared errors ) of lasso regression with varying penalty parameter λ .", "The black line indicates the 10-fold CV MSE of ordinary least squares regression ( i . e . penalty parameter is zero ) .", "The red lines indicate the standard deviation .", "λ=10-4 is chosen for imputing the fitness values of missing variants .", "( B ) The number of nonzero coefficients in the model with varying penalty parameter λ .", "The line indicates the number of nonzero coefficients given by ordinary least squares regression .", "( C ) Comparison between the predicted fitness values and the measured fitness values ( Pearson correlation=0 . 93 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 02010 . 7554/eLife . 16965 . 021Figure 4—figure supplement 2 . Indirect paths in adaptation .", "( A ) A mutational trajectory initiated from PIWI under Greedy Model , which ended at the fitness peak , FWLG .", "( B ) One of the shortest mutational trajectories from WT ( VDGV ) to a beneficial mutation ( VHGL ) .", "( C ) Histogram of the number of fitness accessible from a given genotype .", "The fraction of genotypes accessible to 15 fitness peaks increased substantially when indirect paths are allowed in adaptation . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 02110 . 7554/eLife . 16965 . 022Figure 4—figure supplement 3 . Evolutionary accessibility under constraints imposed by the standard genetic code .", "( A ) To study evolutionary accessibility under constraints imposed by the standard genetic code , we restricted amino acid substitutions to those that were differed by a single nucleotide .", "This constraint is shown as a symmetric matrix , where red represents substitution that is allowed and white represents substitution that is not allowed .", "( B ) Beneficial variants ( fitness > 1 ) were classified by their Hamming distance ( HD ) from WT at the amino acid level .", "Accessibility to these beneficial variants from WT was determined under the constraints imposed by the standard genetic code . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 02210 . 7554/eLife . 16965 . 023Figure 4—figure supplement 4 . Delay of commitment in mutational trajectories involving extra-dimensional bypass . An entropy of evolutionary outcome was calculated for each of the 160 , 000 variants .", "Given a variant v with n accessible fitness peaks , the entropy of evolutionary outcome was then computed as follow:E⁢n⁢t⁢r⁢o⁢p⁢yv=∑i=1n-Pi×ln⁡ ( Pi ) where Pi represented the frequency of reaching the fitness peak i among 1000 simulated mutational trajectories from variant v following Correlated Fixation Model . The entropy of evolutionary fates at each step along an adaptive path is shown .", "Adaptive paths with the same number of steps are grouped together .", "We observed that many mutational trajectories that involved extra-dimensional bypass did not fully commit to a fitness peak ( entropy = 0 ) until the last two steps .", "Each grey line represents a mutational trajectory in each category .", "Only 100 randomly sampled trajectories are shown due to the difficulty in visualizing a large number of lines on the graph .", "The median entropy at each step in each category is represented by the red line . DOI: http://dx . doi . org/10 . 7554/eLife . 16965 . 023 We then simulated adaptation on the fitness landscape using three different models of adaptive walks ( see Materials and methods ) , namely the Greedy Model ( de Visser and Krug , 2014 ) , Correlated Fixation Model ( Gillespie , 1984 ) , and Equal Fixation Model ( Weinreich et al . , 2006 ) .", "In the Greedy Model , adaptation proceeds by sequential fixation of mutations that render the largest fitness gain at each step .", "The other two models assign a nonzero fixation probability to all beneficial mutations , either weighted by ( Correlated Fixation Model ) or independent of ( Equal Fixation Model ) the relative fitness gain .", "The Greedy Model represents adaptive evolution of a large population with pervasive clonal interference ( de Visser and Krug , 2014 ) .", "The Correlated Fixation Model represents adaptive evolution of a population under the scheme of strong-selection/weak-mutation ( SSWM ) ( Gillespie , 1984 ) , which assumes that the time to fixation is much shorter than the time between mutations and the fixation probability of a given mutation is proportional to the improvement in fitness .", "The Equal Fixation Model represents a simplified scenario of adaptation where all beneficial mutations fix with equal probability ( Weinreich et al . , 2006 ) .", "Among all the possible adaptive paths to fitness peaks , many of them involved indirect paths , i . e . they employed mechanisms of extra-dimensional bypass ( Figure 4B , Figure 4—figure supplement 2 ) .", "We classified each step on the adaptive paths into three categories based on the change of Hamming distance to the destination ( a fitness peak , in this case ) : “towards ( -1 ) ” , “conversion ( 0 ) ” , and “detour ( +1 ) ” ( Figure 4C ) .", "Conversion was found to be pervasive during adaptation in our fitness landscape ( 17% of mutational steps for Greedy Model , 41% for Correlated Fixation Model , 59% for Equal Fixation Model ) .", "The use of detour was less frequent ( 0 . 1% of mutational steps for Greedy Model , 1 . 3% for Correlated Fixation Model , 3 . 7% for Equal Fixation Model ) , in accordance with the previous observation that detour bypass was less available than conversion bypass in our fitness landscape with L=4 .", "A conversion step would increase the length of an adaptive path by 1 , while a detour step would increase the length by 2 .", "As a result , an indirect path can be substantially longer than a direct path consisting of only “towards” steps .", "We found that many of the adaptive paths required more than 4 steps , which was the maximal length of a direct path between any variants in this landscape ( Figure 4D ) .", "Interestingly , because indirect adaptive paths involved more variants of intermediate fitness , the use of conversion and detour steps depended on the strength of selection .", "Consistent with previous studies ( Orr , 2002 , 2003 ) , when mutations conferring larger fitness gains were more likely to fix ( e . g . Greedy Model and Correlated Fixation Model ) , adaptation favored direct moves toward the destination , thus leading to a shorter adaptive paths ( Figure 4C–D ) .", "This suggests that the strength of selection interacts with the topological structure of fitness landscapes to determine the length and directness of evolutionary trajectories .", "Given that extra-dimensional bypasses can help proteins avoid evolutionary traps , we expect that their existence would facilitate adaptation in rugged fitness landscapes .", "Indeed , we found that indirect paths increased the number of genotypes with access to each fitness peak ( Figure 4E ) .", "In addition , the fraction of genotypes with accessible paths to all 15 fitness peaks increased from from 34% to 93% when indirect adaptive paths were allowed ( Figure 4—figure supplement 2C ) .", "We also found that a substantial fraction of beneficial variants ( fitness > 1 ) in the sequence space were accessible from WT only if indirect paths were used ( Figure 4F ) .", "We repeated the analysis in Figure 4F with the consideration of the constraints imposed by the standard genetic code ( Figure 4—figure supplement 3A ) .", "The constraints from the genetic code decreased the number of accessible variants due to the reduction in connectivity .", "However , this reduction in connectivity did not alter our core finding that indirect paths substantially increase evolutionary accessibility ( Figure 4—figure supplement 3B ) .", "Taken together , these results suggest that indirect paths promote evolutionary accessibility in rugged fitness landscapes .", "This enhanced accessibility would allow proteins to explore more sequence space and lead to delayed commitment to evolutionary fates ( i . e . fitness peaks ) ( Palmer et al . , 2015 ) .", "Consistent with this expectation , our simulations showed that many mutational trajectories involving extra-dimensional bypass did not fully commit to a fitness peak until the last two steps ( Figure 4—figure supplement 4 ) ." ], [ "In our analysis , we have limited adaptation to the regime where fitness is monotonically increasing via sequential fixation of one-step beneficial mutants .", "When this assumption is relaxed , adaptation can sometimes proceed by crossing fitness valleys , such as via genetic drift or recombination ( de Visser and Krug , 2014; Weissman et al . , 2009; Ostman et al . , 2012; Poelwijk et al . , 2007; Weissman et al . , 2010 ) .", "Another simplification in most of our analyses is to treat all sequences in a “protein space” ( Smith , 1970 ) , where two sequences are considered as neighbors if they differ by a single amino-acid substitution .", "In practice , amino acid substitutions occurring via a single nucleotide mutation are limited by the genetic code , so the total number of one-step neighbors would be reduced from 19L to approximately 6L ( Figure 4—figure supplement 3 ) .", "We also expect fitness landscapes of different systems to have different topological structure .", "Even in our system ( with >93% coverage of the genotype space ) , the global structure of the fitness landscape is influenced by the imputed fitness values of missing variants , which can vary when different fitness models or fitting methods are used .", "Our analysis also ignored measurement errors , but the measurement errors are expected to be very small due to the high reproducibility in the data ( Figure 1—figure supplement 3B ) .", "Both imputation of missing variants and measurement errors can lead to slight mis-specification of the topological structure of the fitness landscape .", "Finally , we note that the four amino acids chosen in our study are in physical proximity and have strong epistatic interactions .", "While the availability of conversion bypass only depends on the dimensionality at each site , the degree of higher-order epistasis and the availability of detour bypasses can be quite different in other fitness landscapes .", "Although the details of a particular fitness landscape can influence the quantitative role of different bypass mechanisms , this does not undermine the generality of our conceptual findings on extra-dimensional bypass , higher-order epistasis , and their roles in protein evolution .", "Higher-order epistasis has been reported in a few biological systems ( Wang et al . , 2013; Pettersson et al . , 2011; Palmer et al . , 2015 ) , and is likely to be common in nature ( Weinreich et al . , 2013 ) .", "In this study , we observed the presence of higher-order epistasis and systematically quantified its contribution to protein fitness .", "Our results suggest that higher-order epistasis can either increase or decrease the ruggedness induced by pairwise epistasis , which in turn determines the accessibility of direct paths in a rugged fitness landscape ( Figure 3—figure supplement 6 ) .", "We also revealed the important role of higher-order epistasis in mediating detour bypass , which could promote evolutionary accessibility via indirect paths .", "Our work demonstrates that even in the most rugged regions of a protein fitness landscape , most of the sequence space can remain highly accessible owing to the indirect paths opened up by high dimensionality .", "The enhanced accessibility mediated by indirect paths may provide a partial explanation for some observations in viral evolution .", "For example , throughout the course of infection HIV always seems to find a way to revert to the global consensus sequence , a putatively “optimal” HIV-1 sequence after immune invasion ( Zanini et al . , 2015 ) .", "As we pointed out , the possible number of detour bypasses scales up with sequence length , so it will be interesting to study how extra-dimensional bypass influences adaptation in sequence space of even higher dimensionality .", "For example , it is plausible that the sequence of a large protein may never be trapped in adaptation ( Gavrilets , 1997 ) , so that adaptive accessibility becomes a quantitative rather than qualitative problem .", "Given the continuing development of sequencing technology , we anticipate that the scale of experimentally determined fitness landscapes will further increase , yet the full protein sequence space is too huge to be mapped exhaustively .", "Does this mean that we will never be able to understand the full complexity of fitness landscapes ?", "Or perhaps big data from high-throughput measurements will guide us to find general rules ?", "By coupling state-of-the-art experimental techniques with novel quantitative analysis of fitness landscapes , this work takes the optimistic view that we can push the boundary further and discover new mechanisms underlying evolution ( Fisher et al . , 2013; Desai , 2013; Szendro et al . , 2013] ) ." ], [ "Two oligonucleotides ( Integrated DNA Technologies , Coralville , IA ) , 5’-AGT CTA GTA TCC AAC GGC NNS NNS NNK GAA TGG ACC TAC GAC GAC GCT ACC AAA ACC TT-3’ and 5’-TTG TAA TCG GAT CCT CCG GAT TCG GTM NNC GTG AAG GTT TTG GTA GCG TCG TCG T-3’ were annealed by heating to 95°C for 5 min and cooling to room temperature over 1 hr .", "The annealed nucleotide was extended in a reaction containing 0 . 5 µM of each oligonucleotide , 50 mM NaCl , 10 mM Tris-HCl pH 7 . 9 , 10 mM MgCl2 , 1 mM DTT , 250 µM each dNTP , and 50 units Klenow exo- ( New England Biolabs , Ipswich , MA ) for 30 mins at 37°C .", "The product ( cassette I ) was purified by PureLink PCR Purification Kit ( Life Technologies , Carlsbad , CA ) according to manufacturer’s instructions .", "A constant region was generated by PCR amplification using KOD DNA polymerase ( EMD Millipore , Billerica , MA ) with 1 . 5 mM MgSO4 , 0 . 2 mM of each dNTP ( dATP , dCTP , dGTP , and dTTP ) , 0 . 05 ng protein GB1 wild type ( WT ) template , and 0 . 5 µM each of 5’-TTC TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA TAT CCA CCA TG-3’ and 5’-AGT CTA GTA TCC TCG ACG CCG TTG TCG TTA GCG TAC TGC-3’ .", "The sequence of the WT template consisted of a T7 promoter , 5’ UTR , the coding sequence of Protein GB1 , 3’ poly-GS linkers , and a FLAG-tag ( Figure 1—figure supplement 1B ) ( [Olson et al . , 2014 ) .", "The thermocycler was set as follows: 2 min at 95°C , then 18 three-step cycles of 20 s at 95°C , 15 s at 58°C , and 20 s at 68°C , and 1 min final extension at 68°C .", "The product ( constant region ) was purified by PureLink PCR Purification Kit ( Life Technologies ) according to manufacturer’s instructions .", "Both the purified constant region and cassette I were digested with BciVI ( New England Biolabs ) and purified by PureLink PCR Purification Kit ( Life Technologies ) according to manufacturer’s instructions .", "Ligation between the constant region and cassette I ( molar ratio of 1:1 ) was performed using T4 DNA ligase ( New England Biolabs ) .", "Agarose gel electrophoresis was performed to separate the ligated product from the reactants .", "The ligated product was purified from the agarose gel using Zymoclean Gel DNA Recovery Kit ( Zymo Research , Irvine , CA ) according to manufacturer’s instructions .", "PCR amplification was then performed using KOD DNA polymerase ( EMD Millipore ) with 1 . 5 mM MgSO4 , 0 . 2 mM of each dNTP ( dATP , dCTP , dGTP , and dTTP ) , 4 ng of the ligated product , and 0 . 5 µM each of 5’-TTC TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA TAT CCA CCA TG-3’ and 5’-GGA GCC GCT ACC CTT ATC GTC GTC ATC CTT GTA ATC GGA TCC TCC GGA TTC-3’ .", "The thermocycler was set as follows: 2 min at 95°C , then 10 three-step cycles of 20 s at 95°C , 15 s at 56°C , and 20 s at 68°C , and 1 min final extension at 68°C .", "The product , which is referred as “DNA library” , was purified by PureLink PCR Purification Kit ( Life Technologies ) according to manufacturer’s instructions .", "Affinity selection by mRNA display ( Roberts and Szostak , 1997; Olson et al . , 2012 ) was performed as described ( Figure 1—figure supplement 3A ) ( Olson et al . , 2014 ) .", "Briefly , The DNA library was transcribed by T7 RNA polymerase ( Life Technologies ) according to manufacturer’s instructions .", "Ligation was performed using 1 nmol of mRNA , 1 . 1 nmol of 5’-TTT TTT TTT TTT GGA GCC GCT ACC-3’ , and 1 . 2 nmol of 5’-/5Phos/-d ( A ) 21- ( C9 ) 3-d ( ACC ) -Puromycin by T4 DNA ligase ( New England Biolabs ) in a 100 µL reaction .", "The ligated product was purified by urea PAGE and translated in a 100 µL reaction volume using Retic Lysate IVT Kit ( Life Technologies ) according to manufacturer’s instructions followed by incubation with 500 mM final concentration of KCl and 60 mM final concentration of MgCl2 for at least 30 min at room temperature to increase the efficiency for fusion formation ( Liu et al . , 2000 ) .", "The mRNA-protein fusion was then purified using ANTI-FLAG M2 Affinity Gel ( Sigma-Aldrich , St . Louis , MO ) .", "Elution was performed using 3X FLAG peptide ( Sigma-Aldrich ) .", "The purified mRNA-protein fusion was reverse transcribed using SuperScript III Reverse Transcriptase ( Life Technologies ) .", "This reverse transcribed product , which was referred as “input library” , was incubated with Pierce streptavidin agarose ( SA ) beads ( Life Technologies ) that were conjugated with biotinylated human IgG-FC ( Rockland Immunochemicals , Limerick , PA ) .", "After washing , the immobilized mRNA-protein fusion was eluted by heating to 95°C .", "The eluted sample was referred as “selected library” .", "PCR amplification was performed using KOD DNA polymerase ( EMD Millipore ) with 1 . 5 mM MgSO4 , 0 . 2 mM of each dNTP ( dATP , dCTP , dGTP , and dTTP ) , the selected library , and 0 . 5 µM each of 5’-CTA CAC GAC GCT CTT CCG ATC TNN NAG CAG TAC GCT AAC GAC AAC G-3’ and 5’-TGC TGA ACC GCT CTT CCG ATC TNN NTA ATC GGA TCC TCC GGA TTC G-3’ .", "The underlined “NNN” indicated the position of the multiplex identifier , GTG for input library and TGT for post-selection library .", "The thermocycler was set as follows: 2 min at 95°C , then 10 to 12 three-step cycles of 20 s at 95°C , 15 s at 56°C , and 20 s at 68°C , and 1 min final extension at 68°C .", "The product was then PCR amplified again using KOD DNA polymerase ( EMD Millipore ) with 1 . 5 mM MgSO4 , 0 . 2 mM of each dNTP ( dATP , dCTP , dGTP , and dTTP ) , the eluted product from mRNA display , and 0 . 5 µM each of 5’-AAT GAT ACG GCG ACC ACC GAG ATC TA CAC TCT TTC CCT ACA CGA CGC TCT TCC G-3’ and 5’-CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG-3’ .", "The thermocycler was set as follows: 2 min at 95°C , then 10 to 12 three-step cycles of 20 s at 95°C , 15 s at 56°C , and 20 s at 68°C , and 1 min final extension at 68°C .", "The PCR product was then subjected to 2 × 100 bp paired-end sequencing using Illumina HiSeq 2500 platform .", "We aimed to obtain at least 20 million paired-end reads for each input library and post-selection library such that the average coverage for each variant would be more than 100 paired-end reads .", "There were 89 , 075 , 246 paired-end reads obtained for the input library and 45 , 587 , 128 paired-end reads obtained for the post-selection library .", "Raw sequencing data have been submitted to the NIH Short Read Archive under accession number: BioProject PRJNA278685 .", "We were able to compute the fitness for 93 . 4% of all variants from the sequencing data .", "The fitness measurements in this study were highly consistent with our previous study on the fitness of single and double mutants in protein GB1 ( Pearson correlation = 0 . 97 , Figure 1—figure supplement 3B ) ( Olson et al . , 2014 ) .", "The first three nucleotides of both forward read and reverse read were used for demultiplexing .", "If the first three nucleotides of the forward read were different from that of the reverse read , the given paired-end read would be discarded .", "For both forward read and reverse read , the nucleotides that were corresponding to the codons of protein GB1 sites 39 , 40 , 41 , and 54 were extracted .", "If coding sequence of sites 39 , 40 , 41 , and 54 in the forward read and that in the reverse read did not reverse-complement each other , the paired-end read would be discarded .", "Subsequently , the occurrence of individual variants at the amino acid level for site 39 , 40 , 41 , and 54 in both input library and selected library were counted , with each paired-end read represented 1 count .", "Custom python scripts and bash scripts were used for sequencing data processing .", "All scripts have been deposited to https://github . com/wchnicholas/ProteinGFourMutants .", "The fitness ( w ) for a given variant i was computed as: ( 1 ) wi=c⁢o⁢u⁢n⁢ti , s⁢e⁢l⁢e⁢c⁢t⁢e⁢d/c⁢o⁢u⁢n⁢ti , i⁢n⁢p⁢u⁢tc⁢o⁢u⁢n⁢tW⁢T , s⁢e⁢l⁢e⁢c⁢t⁢e⁢d/c⁢o⁢u⁢n⁢tW⁢T , i⁢n⁢p⁢u⁢t where counti , selected represented the count of variant i in the selected library , counti , input represented the count of variant i in the input library , countWT , selected represented the count of WT ( VDGV ) in the selected library , and countWT , input represented the count of WT ( VDGV ) in the input library .", "Therefore , the fitness of each variant , wi , could be viewed as the fitness relative to WT ( VDGV ) , such that = 1 .", "Variants with countinput<10 were filtered to reduce noise .", "The fraction of all possible variants that passed this filter was 93 . 4% ( 149 , 361 out of 160 , 000 all possible variants ) .", "The fitness of each single substitution variant was referenced to our previous study ( Olson et al . , 2014 ) , because the sequencing coverage of single substitution variants in our previous study was much higher than in this study ( ~100 fold higher ) .", "Hence , our confidence in computing fitness for a single substitution variant should also be much higher in our previous study than this study .", "Subsequently , the fitness of each single substitution in this study was calculated by multiplying a factor of 1 . 159 by the fitness of that single substitution computed from our previous study ( Olson et al . , 2014 ) .", "This is based on the linear regression analysis between the single substitution fitness as measured in our previous study and in this study , which had a slope of 1 . 159 and a y-intercept of ~0 .", "The fitness of each profiled variant is shown in Supplementary file 1 .", "The three types of pairwise epistasis ( magnitude , sign and reciprocal sign ) were classified by ranking the fitness of the four variants involved ( Greene and Crona , 2014 ) .", "To quantify the magnitude of epistasis ( ε ) between substitutions a and b on a given background variant BG , the relative epistasis model ( Khan et al . , 2011 ) was employed as follows: ( 2 ) εa⁢b , B⁢G=ln⁡ ( wa⁢bwB⁢G ) -ln⁡ ( wawB⁢G ) -ln⁡ ( wbwB⁢G ) where wab represents the fitness of the double substitution , ln ( wa ) and ln ( wb ) represents the fitness of each of the single substitution respectively , and wBG represents the fitness of the background variant .", "As described previously ( Olson et al . , 2014 ) , there exists a limitation in determining the exact fitness for very low-fitness variants in this system .", "To account for this limitation , several rules were adapted from our previous study to minimize potential artifacts in determining ε ( Olson et al . , 2014 ) .", "We previously determined that the detection limit of fitness ( w ) in this system is ~0 . 01 ( Olson et al . , 2014 ) .", "Rule", "1 ) if max ( wa⁢bwB⁢G , wawB⁢G , wbwB⁢G ) < 0 . 01 , εa⁢b , B⁢G , a⁢d⁢j⁢u⁢s⁢t⁢e⁢d = 0 Rule", "2 ) if min ( wa , wb , wawB⁢G , wbwB⁢G ) < 0 . 01 , εa⁢b , B⁢G , a⁢d⁢j⁢u⁢s⁢t⁢e⁢d = max ( 0 , εa⁢b , B⁢G ) Rule", "3 ) if min ( wab , wa⁢bwB⁢G ) < 0 . 01 , εa⁢b , B⁢G , a⁢d⁢j⁢u⁢s⁢t⁢e⁢d = min ( 0 , εa⁢b , B⁢G ) Rule 1 prevents epistasis being artificially estimated from low-fitness variants .", "Rule 2 prevents overestimation of epistasis due to low fitness of one of the two single substitutions .", "Rule 3 prevents underestimation of epistasis due to low fitness of the double substitution .", "Of note , εa⁢b , B⁢G , a⁢d⁢j⁢u⁢s⁢t⁢e⁢d was set to 0 if both Rule 2 and Rule 3 were satisfied .", "To compute the epistasis between two substitutions , a and b , on a given background variant BG , εa⁢b , B⁢G , a⁢d⁢j⁢u⁢s⁢t⁢e⁢d would be used if any one of the above three rules was satisfied .", "Otherwise , εa⁢b , B⁢G would be used .", "Fitness decomposition was performed on all subgraphs without missing variants ( 109 , 235 subgraphs in total ) .", "We decomposed the fitness landscape into epistatic interactions of different orders by Fourier analysis ( Stadler , 1996; Szendro et al . , 2013; Weinreich et al . , 2013; Neidhart et al . , 2013 ) .", "The Fourier coefficients given by the transform can be interpreted as epistasis of different orders ( Weinreich et al . , 2013; de Visser and Krug , 2014 ) .", "For a binary sequence z→ with dimension L ( zi equals 1 if mutation is present at position i , or 0 otherwise ) , the Fourier decomposition theorem states that the fitness function f⁢ ( z→ ) can be expressed as ( Weinberger , 1991 ) : ( 3 ) f⁢ ( z→ ) =∑k→f^k→⁢ ( -1 ) z→⋅k→ The formula for the Fourier coefficients f^k→ is then: ( 4 ) f^k→=12L⁢∑z→f⁢ ( z→ ) ⁢ ( -1 ) z→⋅k→ For example , we can expand the fitness landscape up to the second order , i . e . with linear and quadratic terms ( 5 ) f ( σ→ ) =f0^+∑if^ei→σi+∑i<jf^ei→+ej→σiσj+⋯ where σi≡ ( -1 ) zi∈{+1 , -1} , and ei→ is a unit vector along the it⁢h direction .", "In our analysis of subgraphs , there are a total of 24=16 terms in the Fourier decomposition , with ( 4i ) terms for the it⁢h order ( i=0 , 1 , 2 , 3 , 4 ) .", "We can expand the fitness landscape up to a given order by ignoring all higher-order terms in Equation 3 .", "In this paper , we refer to higher-order epistasis as non-zero contribution to fitness from the third order terms and beyond .", "The fitness values for 10 , 639 variants ( 6 . 6% of the entire sequence space ) were not directly measured ( read count in the input pool = 0 ) or were filtered out because of low read counts in the input pool ( see section “Calculation of fitness” ) .", "To impute the fitness of these missing variants , we performed regularized regression on fitness values of observed variants using the following model ( Hinkley et al . , 2011; Otwinowski and Plotkin , 2014 ) : ( 6 ) l⁢o⁢g⁢ ( f ) =α0+∑i=1NMβi⁢Mi+∑j=1NPγj⁢Pj+∑k=1NTδk⁢Tk Here , f is the protein fitness .", "α0 is the intercept that represents the log fitness of WT; βi represents the main effect of a single mutation , i; Mi is a dummy variable that equals 1 if the single mutation i is present in the sequence , or 0 if the single mutation is absent; and NM=19× ( 41 ) =76 is the total number of single mutations .", "Similarly , γj represents the effect of interaction between a pair of mutations; Pj is the dummy variable that equals either 1 or 0 depending on the presence of that those two mutations; and NP=192× ( 42 ) =2166 is the total number of possible pairwise interactions .", "In addition to the main effects of single mutations and pairwise interactions , the three-way interactions among sites 39 , 41 and 54 are included in the model , based on our knowledge of higher-order epistasis ( Figure 3 ) .", "δk represents the effect of three-way interactions among sites 39 , 41 and 54; Tk is the dummy variable that equals either 1 or 0 depending on the presence of that three-way interaction; and NT=193=6859 is the total number of three-way interactions .", "Thus , the total number of coefficients in this model is 9102 , including main effects of each site ( i . e . additive effects ) , interactions between pairs of sites ( i . e . pairwise epistasis ) , and a subset of three-way interactions ( i . e . higher-order epistasis ) .", "Out of the 149 , 361 variants with experimentally measured fitness values , 119 , 884 variants were non-lethal ( f>0 ) and were used to fit the model coefficients using lasso regression ( Matlab R2014b ) .", "Lasso regression adds a penalty term λ⁢∑|θ| ( θ stands for any coefficient in the model ) when minimizing the least squares , thus it favors sparse solutions of coefficients ( Figure 4—figure supplement 1B ) .", "We calculated the 10-fold cross-validation MSE ( mean squared errors ) of the lasso regression for a wide range of penalty parameter λ ( Figure 4—figure supplement 1A ) .", "λ=10-4 is chosen .", "For measured variants , the model-predicted fitness values were highly correlated with the actual fitness values ( Pearson correlation=0 . 93 , Figure 4—figure supplement 1C ) .", "We then used the fitted model to impute the fitness of the 10 , 639 missing variants and complete the entire fitness landscape .", "Imputed fitness values for missing variants are listed in Supplementary file 2 .", "Python package “networkx” was employed to construct a directed graph that represented the entire fitness landscape for sites 39 , 40 , 41 , and 54 .", "A total of 204 = 160 , 000 nodes were present in the directed graph , where each node represented a 4-site variant .", "For all pairs of variants separated by a Hamming distance of 1 , a directed edge was generated from the variant with a lower fitness to the variant with a higher fitness .", "Therefore , all successors of a given node had a higher fitness than the given node .", "A fitness peak was defined as a node that had 0 out-degree .", "Three models , namely the Greedy Model ( de Visser and Krug , 2014 ) , the Correlated Fixation Model ( Gillespie , 1984 ) , and the Equal Fixation Model ( Weinreich et al . , 2006 ) , were employed in this study to simulate the mutational steps in adaptive trajectories .", "Under all three models , the probability of fixation of a deleterious or neutral mutation is 0 .", "Considering a mutational trajectory initiated at a node , ni with a fitness value of wi , where ni has M successors , ( n1 , n2 , … nM ) with fitness values of ( w1 , w2 , … wM ) .", "Then the probability that the next mutational step is from ni to nk , where k ∈ ( 1 , 2 , … M ) , is denoted Pi→k and called the probability of fixation , and can be computed for each model as follows .", "For the Greedy Model ( deterministic model ) , ( 7 ) if⁢wk=m⁢a⁢x⁢ ( w1 , w2 , …⁢wM ) ⁢ , ⁢Pi→k=1 ( 8 ) otherwise , ⁢Pi→k=0 For the Correlated Fixation Model ( non-deterministic model ) , ( 9 ) Pi→k=wk−wi∑n=1M ( wn−wi ) For the Equal Fixation Model ( non-deterministic model ) , ( 10 ) Pi→k=1M To compute the shortest path from a given variant to all reachable variants , the function “single_source_shortest_path” in “networkx” was used .", "If the shortest path between a low-fitness variant and a high-fitness variant does not exist , it means that the high-fitness variant is inaccessible .", "If the length of the shortest path is larger than the Hamming distance between two variants , it means that adaptation requires indirect paths .", "Under constraints imposed by the standard genetic code , the connectivity of the directed graph that represented the fitness landscape was restricted according to the matrix shown in Figure 4—figure supplement 3A .", "The genetic distance between two variants was calculated according to the matrix shown in Figure 4—figure supplement 3A .", "If the length of the shortest path is larger than the genetic distance between two variants , it means that adaptation requires indirect paths .", "In the subgraph analysis shown in Figure 1—figure supplement 4 , the fitness landscape was restricted to 2 amino acids at each of the 4 sites ( the WT and adapted alleles ) .", "There was a total of 24 variants , hence nodes , in a given subgraph .", "Only those subgraphs where the fitness of all variants was measured directly were used ( i . e . any subgraph with missing variants was excluded from this analysis ) .", "Mutational trajectories were generated in the same manner as in the analysis of the entire fitness landscape ( see subsection “Simulating adaptation using three models for fixation” ) .", "In a subgraph with only one fitness peak , the probability of a mutational trajectory from node i to node j via intermediate a , b , and c was as follows: ( 11 ) Pi→a→b→c→j=Pi→a×Pa→b×Pb→c×Pc→j To compute the Gini index for a given set of mutational trajectories from node i to node j , the probabilities of all possible mutational trajectories were sorted from large to small .", "Inaccessible trajectories were also included in this sorted list with a probability of 0 .", "This sorted list with t trajectories was denoted as ( Pi→j , 1 , Pi→j , 2 , … Pi→j , t ) , where Pi→j , 1 was the largest and Pi→j , t was the smallest .", "This sorted list was converted into a list of cumulative probabilities , which is denoted as ( Ai→j , 1 , Ai→j , 2 , … Ai→j , t ) , where Ai→j , t = ∑n=1tPi→j , t .", "The Gini index for the given subgraph was then computed as follows: ( 12 ) Gini index =2×∑n=1t−1 ( Ai→j , n ) +Ai→j , t−tt−1 Sequence logo was generated by WebLogo ( http://weblogo . berkeley . edu/logo . cgi ) ( Crooks et al . , 2004 ) .", "The visualization of basins of attraction ( Figure 4A ) was generated using Graphviz with “fdp” as the option for layout .", "The Δ⁢ΔG prediction was performed by the ddg_monomer application in Rosetta software ( Das and Baker , 2008 ) using the parameters from row 16 of Table I in Kellogg et al . ( Kellogg et al . , 2011 ) .", "Here we prove that higher-order epistasis is required for two possible scenarios of extra-dimensional bypass via an additional site ( Figure 2—figure supplement 1 ) .", "For a fitness landscape defined on a Boolean hypercube , we can expand the fitness as Taylor series ( Weinberger , 1991 ) .", "( 13 ) f000=α0f001=α0+α1f010=α0+α2f100=α0+α3f011=α0+α1+α2+α12f101=α0+α1+α3+α13f110=α0+α2+α3+α23f111=α0+α1+α2+α3+α12+α13+α23+α123 To prove that higher-order epistasis is present is equivalent to prove that α123≠0 .", "The fitness difference between neighbors is visualized by the directed edges that go from low-fitness variant to high-fitness variant , thus each edge represents an inequality .", "No cyclic paths are allowed in this directed graph .", "The reciprocal sign epistasis ( Figure 2—figure supplement 1A ) gives , ( 14 ) 000←001:α1 < 0 ( 15 ) 000←010:α2 < 0 ( 16 ) 001→011:α2+α12 > 0 ( 17 ) 010→011:α1+α12 > 0 The detour step ( 000→100 ) and the loss step ( 111→011 ) are required for extra-dimensional bypass , ( 18 ) 000→100:α3 > 0 ( 19 ) 011←111:α3+α13+α23+α123 < 0 For the remaining 6 edges , there are 3 possible configurations ( Figure 2—figure supplement 1B–D ) .", "For the scenario illustrated in ( B ) , we have ( 20 ) 100→101:α1+α13 > 0 ( 21 ) 100→110:α2+α23 > 0 Combining inequality ( 14 ) and ( 20 ) gives ( 22 ) α13 > 0 Combining inequality ( 15 ) and ( 21 ) gives ( 23 ) α23 > 0 Combining the above two inequalities with ( 18 ) and ( 19 ) , we arrive at ( 24 ) α123 < 0 For the scenario in ( C ) , the proof of higher-order epistasis is similar .", "We have ( the yellow edge ) ( 25 ) 001→101:α3+α13 > 0 Combining the above inequality with ( 15 ) , ( 19 ) and ( 21 ) , we arrive at ( 26 ) α123 < 0 For the scenario in ( D ) , when α3+α13 < 0 , all the inequalities can be satisfied with α123=0 .", "So higher-order epistasis is not necessary in this case ." ] ]

[ "The structure of fitness landscapes is critical for understanding adaptive protein evolution .", "Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence , involving mostly single and double mutants , or a combinatorially complete subgraph involving only two amino acids at each site .", "In reality , the dimensionality of protein sequence space is higher ( 20L ) and there may be higher-order interactions among more than two sites .", "Here we experimentally characterized the fitness landscape of four sites in protein GB1 , containing 204 = 160 , 000 variants .", "We found that while reciprocal sign epistasis blocked many direct paths of adaptation , such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations .", "These indirect paths alleviate the constraint on adaptive protein evolution , suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve ." ]

[ "Proteins can evolve over time by changing their component parts , which are called amino acids .", "These changes usually happen one at a time and natural selection tends to preserve those changes that make the protein more efficient at its specific tasks , while discarding those that impair the protein’s activity .", "However the effect of each change depends on the protein as a whole , and so two changes that separately make the protein worse can make it much better if they occur together .", "This phenomenon is called epistasis and in some cases it can trap proteins in a sub-optimal form and prevent them from improving further .", "Proteins are made from twenty different kinds of amino acid , and there are millions of different combinations of amino acids that could , in theory , make a protein of a given length .", "Studying protein evolution involves making variants of the same protein , each with just a few changes , and comparing how efficient , or “fit” , they are .", "Previous studies only measured the fitness of a few variants and showed that epistasis could block protein evolution by requiring the protein to lose some fitness before it could improve further .", "However , new techniques have now made it easier to study protein evolution by testing many more protein variants .", "Wu , Dai et al . focused on four amino acids in part of a protein called GB1 and tested the efficiency of every possible combination of these four amino acids , a total of 160 , 000 ( 204 ) variants .", "Contrary to expectations , the results suggested that the protein could evolve quickly to maximise fitness despite there being epistasis between the four amino acids .", "Overcoming epistasis typically involved making a change to one amino acid that paved the way for further changes while avoiding the need to lose fitness .", "The original change could then be reversed once the epistasis was overcome .", "The complexity of this solution means it can only be seen by studying a large number of protein variants that represent many alternative sequences of protein changes .", "Wu , Dai et al . conclude that proteins are able to achieve a higher level of fitness through evolution by exploring a large number of changes .", "There are many possible changes for each protein and it is this variety that , despite epistasis , allows proteins to become naturally optimised for the tasks that they perform .", "While the full complexity of protein evolution cannot be explored at the moment , as technology advances it will become possible to study more protein variants .", "Such advances would therefore hopefully allow researchers to discover even more about the natural mechanisms of protein evolution ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "developmental biology" ]

[ [ "The radiation of the vascular plants was underpinned by the innovation of a branching growth habit in the shoots of their last shared common ancestor around 430 million years ago ( Langdale and Harrison , 2008; Edwards et al . , 2014 ) .", "The earliest vascular plants branched by bifurcation , involving the even partitioning of the growing tip into two new shoot tips ( Sussex and Kerk , 2001; Harrison et al . , 2005 , 2007; Langdale and Harrison , 2008; Harrison and Langdale , 2010; Ligrone et al . , 2012; Edwards et al . , 2014; Tomescu et al . , 2014 ) .", "Subsequent diversification within the seed plants was underpinned by the evolution of lateral ( axillary ) branching ( Sussex and Kerk , 2001; Langdale and Harrison , 2008 ) , in which buds initiate in leaf axils but may then become dormant until receiving environmental or internal cues to promote their activation and growth .", "Lateral branching thus permits finely tuned regulation of plant architecture and space filling in response to the environment ( Bergamini and Peintinger , 2002; Domagalska and Leyser , 2011 ) .", "Whilst branching by bifurcation is prevalent in non-seed vascular plant sporophytes and many bryophyte gametophytes , a capacity for lateral branching evolved by convergence in mosses and primed their diversification ( Farge-England , 1996 ) .", "The mechanisms regulating lateral branching are well studied in flowering plant sporophytes , in which the initiation of axillary meristems is regulated by a drop in auxin levels and a rise in cytokinin levels as leaves initiate from the apical meristem ( Wang et al . , 2014a , 2014b ) .", "Classical decapitation experiments showed that the main shoot apex exerts an inhibitory effect over subsequent branch outgrowth , a phenomenon known as apical dominance .", "Replacing decapitated apices with lanolin impregnated with phytohormones showed that auxin can mediate this inhibition , and that its action can be antagonized by cytokinin application to buds ( Thimann and Skoog , 1933; Wickson and Thimann , 1958; Cline , 1991 ) .", "Strigolactone has recently been identified as a third hormonal regulator of branching , exerting an inhibitory or stimulatory effect on branch outgrowth depending on the auxin transport status of the plant ( Gomez-Roldan et al . , 2008; Umehara et al . , 2008; Shinohara et al . , 2013 ) .", "The mechanism by which the co-ordinated action of auxin , cytokinin and strigolactone regulates branching is not yet fully clear , but regulated auxin transport plays a key role ( Crawford et al . , 2010; Domagalska and Leyser , 2011 ) .", "Auxin is synthesized in young expanding leaves and is transported basipetally by several transporters including PIN-FORMED1 ( PIN1 ) polar auxin efflux carriers to generate the polar auxin transport stream in the stem ( Gälweiler et al . , 1998; Ljung et al . , 2001 ) .", "The suppressive action of auxin on lateral branch outgrowth is mediated indirectly ( Morris , 1997; Booker et al . , 2003 ) , leading to the hypothesis that a second messenger ( for example cytokinin ) could act as an intermediary between auxin and bud activation ( Morris , 1997; Booker et al . , 2003 ) .", "However , a dominant bud need not be apical , and more recent work suggests that buds compete to export auxin into the main auxin transport stream and that such competition is enhanced by a suppressive action of strigolactones on PIN1-mediated auxin transport ( Crawford et al . , 2010; Prusinkiewicz et al . , 2010; Shinohara et al . , 2013 ) .", "Thus , PIN-mediated auxin transport is a key integration point in the regulation of branching patterns .", "Despite the pivotal contribution of branching pattern innovations to the evolution of plant architecture , the mechanisms underlying the evolution of branching are poorly understood ( Sussex and Kerk , 2001 ) .", "Auxin transport assays and decapitation experiments used in conjunction with pharmacological treatments in the lycophyte , Selaginella , suggest that auxin ( acting via polar transport ) and cytokinin are conserved regulators of branching in vascular plant sporophytes ( Williams , 1937; Wochok and Sussex , 1973 , 1975; Sanders and Langdale , 2013 ) .", "Although bryophyte sporophytes do not normally branch , there is a detectable basipetal auxin transport stream in mosses ( Fujita et al . , 2008 ) .", "In Physcomitrella , disruption of auxin transport by application of polar transport inhibitors or perturbations in PIN function can induce a branching form ( Fujita et al . , 2008; Bennett et al . , 2014b ) that closely resembles branching forms in the early fossil record , and such branching forms have also been reported as rare natural liverwort variants ( Bower , 1935 ) .", "These data suggest that bulk basipetal polar auxin transport is a conserved regulator of land plant sporophyte development , and point to a potential contribution of PIN-mediated polar auxin transport to the evolution of sporophytic branching ( Fujita et al . , 2008; Bennett et al . , 2014b ) .", "The extent of conservation between sporophytic and gametophytic branching mechanisms is unknown .", "Classical decapitation experiments revealed apical dominance in mosses and showed that as in flowering plants , a suppressive role of the apex on lateral branching acts via auxin and can be antagonized by cytokinin ( von Maltzahn , 1959 ) .", "Whilst Physcomitrella PIN proteins are plasma-membrane targeted polar auxin transporters with many roles in gametophore development ( Bennett et al . , 2014a , 2014b; Viaene et al . , 2014 ) , bulk basipetal auxin transport is not detected with radiolabelled auxin transport assays in moss gametophores ( Fujita et al . , 2008; Fujita and Hasebe , 2009 ) , suggesting that auxin transport patterns are not shared between sporophytes and gametophytes .", "The contribution of cytokinins to gametophore branching has not been extended beyond the pharmacological approaches mentioned above ( von Maltzahn , 1959 ) , and strigolactone biosynthesis ppccd8 mutants have increased branching in protonemal tissues but no reported gametophore defects ( Proust et al . , 2011 ) .", "These data have led to the hypothesis that distinct developmental mechanisms have been recruited to regulate gametophyte and sporophyte shoot architecture in evolution ( Fujita et al . , 2008; Fujita and Hasebe , 2009 ) .", "Here , we investigated the hormonal regulation of lateral gametophore branching in the moss Physcomitrella patens .", "We present a simple model in which apical auxin sources interact via non-polar auxin transport with hormonally regulated global and local sensitivities to auxin elsewhere in the moss gametophore , accurately reproducing observed branching patterns .", "Our work suggests that three conserved hormonal cues have been recruited independently in evolution to produce a convergent branching morphology , but that their co-ordinated action is integrated by different mechanisms to those in flowering plants ." ], [ "To determine the manner of branch initiation in Physcomitrella , we first undertook a morphological and histological analysis .", "We found rhizoids initiating in all leaf axils beyond a certain distance from the main gametophore apex ( Figure 1A–D ) but branches were present in only a subset of axils ( Figure 1E–P ) .", "In contrast to reports from other species that have identified stereotypical and taxon-specific branch initiation patterns ( Berthier et al . , 1965; Berthier , 1970 ) , we found no regular spacing in the pattern of initiation of Physcomitrella branches .", "We were unable to detect any evidence of dormancy , suggesting that branch initiation and outgrowth are not distinct developmental processes ( Figure 1A–D ) .", "At the earliest stage of branch initiation that we were able to detect , branches were manifest as a single apical cell surrounded by leaf initials and adjacent to rhizoids ( Figure 1E–H ) , appearing to differentiate from the epidermis ( Figure 1G–H ) .", "At later stages of development , more rhizoids developed at the base of each branch and newly formed leaves were distinguishable ( Figure 1I–L ) .", "Well developed lateral branches were morphologically similar to the main gametophore axis ( Figure 1M ) , and transverse sections cut at the base of lateral branches showed that each branch persisted as a superficial projection; there was no continuity between the conducting tissue of the lateral branches and the conducting tissue of the main stem ( Figure 1N–P ) .", "We reasoned that the largest branches would have initiated first and evaluated the sequence of branch initiation by dissecting branches from 6 week old gametophores , and ranking their size relative to their position ( Figure 1Q ) .", "These morphological data suggest that in Physcomitrella branches initiate de novo from the epidermis of the gametophore axis and that initiation is usually , but not invariably , acropetal . 10 . 7554/eLife . 06808 . 003Figure 1 . Branches initiate from the epidermis or outermost cortical cell layer in Physcomitrella .", "( A–D )", "Although all leaf axils contained axillary rhizoids , branches were absent in most .", "( A ) Light micrograph , ( B ) scanning electron microscope ( SEM ) micrograph , ( C ) transverse histological section and ( D ) corresponding line drawing showing a rhizoid ( blue ) in the axil of a leaf ( yellow ) .", "Gametophore conducting tissues are shown in red .", "The label C in ( A ) indicates the approximate plane of section in ( C ) .", "( E–H )", "At the earliest detectable stage of branching , each branch comprised a single apical cell surrounded by leaf initials and was adjacent to one or several developing rhizoids .", "( E ) Light micrograph , ( F ) SEM micrograph , ( G ) transverse histological section and ( H ) corresponding line drawing showing a branch apical cell ( pink ) surrounded by leaf initials ( green ) and adjacent to an initiating rhizoid ( blue ) .", "The label G in ( E ) indicates the approximate plane of section in ( G ) .", "( I–L )", "At a later stage , growing buds had well-developed leaves .", "( I ) Light micrograph , ( J ) SEM micrograph , ( K ) transverse histological section and ( L ) corresponding line drawing showing a well-developed bud ( green ) and its apical cell ( pink ) in the axil of the leaf ( yellow ) .", "The label K in ( I ) indicates the approximate plane of section in ( K ) .", "( M–P )", "Well-developed branches persisted as superficial projections , there was no continuity between the conducting tissue system of the branch and the gametophore axis .", "( M ) Light micrograph of a lateral branch on a gametophore whose leaves have been removed .", "( N ) Transverse histological section at the junction point of the lateral branch with the main gametophore axis ( indicated by N in [M] ) , the red arrowhead shows cortical tissue and the absence of continuity between conducting tissue systems .", "( O ) Transverse histological section and ( P ) corresponding line drawing above the junction point ( indicated by O in [M] ) where the conducting tissues ( red ) of the lateral branch ( green ) and the main axis ( white ) can be seen .", "( Q ) The sequence of branch initiation in thirty 6 week old gametophores .", "For all images , arrows show lateral buds , dotted lines indicate the level of corresponding histological sections , asterisks mark rhizoids , dashed lines mark the boundary between the stem and the detached leaf .", "Scale bars = 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 003 To determine how branches are distributed in Physcomitrella , the leaves were removed from 60 wild-type ( WT ) gametophores grown on sterile BCD + AT medium for 5 or 7 weeks ( Figure 2A ) .", "The position of lateral branches was recorded ( Figure 2B ) , showing an uneven distribution ( Figure 2C ) .", "The formation of a minimum of 18 leaves was required prior to branch initiation , and an apical portion devoid of branches was maintained at a similar length throughout development .", "We termed the barren apical portion of the gametophore the apical inhibition zone ( AIZ ) , and the portion of the gametophore in which branches initiated was termed the branching zone ( BZ ) .", "Reasoning that each of these aspects of the branch distribution might be under regulatory control , we sought to determine how the branch initiation pattern deviated from a random distribution .", "We simulated a dataset of 60 gametophores in which branching occurs stochastically with a probability of 5% to obtain a similar number of branches to WT ( Figure 2D , ‘Materials and methods’ ) .", "We defined a gametophore as a 1D series of metamers where each metamer consists of a section of the main gametophore axis and a leaf , and growth proceeds at a fixed rate adding new metamers as the simulation progresses .", "Although it was immediately apparent that the simulated dataset lacked apical inhibition , potential differences in branch spacing in the branching zone between WT and simulated datasets were not obvious .", "We therefore calculated and compared the mean minimum distance between branches in the branching zone of WT ( 4 . 48 ± 1 . 48 ) and simulated stochastically branching shoots ( 3 . 62 ± 0 . 41 ) .", "In the WT dataset , branches were more evenly dispersed than expected at random ( p-value < 0 . 05; Figure 2D ) , supporting the notion that the distribution of branches within the BZ is regulated .", "Even if an apical inhibition zone is introduced into a random model of branching , for example by assuming a branching competency that requires a minimum metamer age , it cannot produce a realistic branching pattern ( Figure 2E , Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 06808 . 004Figure 2 . Branching patterns are non-random .", "( A ) A wild-type gametophore before ( left ) and after ( right ) removing the leaves .", "Asterisks indicate lateral branches .", "Scale bar = 1 mm . ( B ) Each gametophore is represented as 1-D series of metamers ( light green squares ) and lateral branch position is indicated in dark green .", "( C–E )", "Branching patterns of 60 gametophores ordered by increasing size showing the apical inhibition zone ( AIZ ) and branching zone ( BZ ) .", "Data from wild-type ( C ) , stochastic simulation ( D ) and stochastic simulation with imposed apical inhibition ( E ) had similar branch numbers but different branch distributions .", "( F–I )", "Assumptions and replacement rules of the computational model .", "( F ) Each gametophore starts off as an apex ( red ) , which produces auxin and grows by producing metamers ( grey ) containing auxin at concentration c .", "( G ) illustrates replacement rules used in simulations .", "1: an apex ( red ) regularly produces metamers ( grey ) .", "2: if auxin concentration c in a metamer falls below the auxin sensitivity threshold T , then the metamer becomes a branch and an auxin source ( red ) .", "3: if auxin concentration c is higher than T , then branch formation is inhibited .", "( H ) The auxin concentration c within a metamer reflects acropetal transport defined by the constant KA and basipetal transport defined by the constant KB .", "( I ) Stages of growth in a single simulated gametophore showing that , as new metamers are added , the ratio of c/T drops towards the base , allowing a branch to initiate ( compare iii to", "iv ) .", "The new apex becomes an auxin source and can export auxin both up and down the gametophore", "( v ) .", "This results in the formation of an auxin minimum further up the gametophore axis and a second branch initiates ( vi to vii ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 00410 . 7554/eLife . 06808 . 005Figure 2—figure supplement 1 . Comparison of the WT branching pattern plot with different model outputs .", "( A–C )", "Model C ( directionally unbiased transport , with basal inhibitor ) best approximates the distribution of branches observed in WT .", "Model A , in which an apical inhibition zone is specified to match WT but there is a random branch distribution in the branching zone , does not capture WT branch distribution .", "Model B , in which there is no basal inhibitor and transport is directionally unbiased , shows a shift in branch distribution due to the constitutive basal activation .", "Lines indicate the best fitting polynomial of the seventh degree .", "For model B , a polynomial of the fifth degree was selected to fit the data points .", "Table: comparison of WT branching patterns to different model outputs .", "Model C is the best fit to WT branch number , apical inhibition zone size and minimum distance of a branch to its closest neighbour in comparison to models A and B . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 00510 . 7554/eLife . 06808 . 006Figure 2—figure supplement 2 . Stages of growth in a single simulated gametophore showing that as new metamers are added , the ratio of c/T drops towards the base , allowing a branch to initiate ( compare v to", "vi ) .", "The new apex becomes an auxin source and can export auxin both up and down the gametophore", "( vi ) .", "This results in the formation of an auxin minimum further up the gametophore axis and a second branch initiates ( vii , A ) .", "The zone of basal inhibition is indicated by green metamer outlines ( B ) .", "Minima of c/T can initiate out of acropetal series ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 006 Previous studies in mosses have shown that the main gametophore apex exerts an inhibitory effect over branching that acts via auxin ( von Maltzahn , 1959; Nyman and Cutter , 1981 ) .", "To help us understand branch patterning , we generated a regulatory model of moss branching which assumes the main apex and newly forming branch apices are sources of auxin ( assumption 1; Figure 2F ) .", "The terminal apex is represented by a red square and produces new metamers represented by grey squares ( Figure 2G; rule 1 ) .", "The bottom metamer is always defined as the first of the 1D metamer file representing the gametophore in the model .", "The concentration of auxin in a metamer is expressed with the parameter c , and auxin levels in the terminal apex and lateral apices are set to fixed values of Hapex and H respectively; thus auxin is produced to a constant concentration .", "Biologically Hapex and H represent a combination of inputs to regulate auxin levels including auxin synthesis , transport and decay , and any potential feedback between these processes .", "We assume that auxin moves from sites of production into neighboring metamers , changing the auxin concentration in those metamers ( assumption 2; Figure 2H ) .", "Such changes per metamer are expressed in the model with the following equation: ( 1 ) dcidt=KB ( ci+1−ci ) +KA ( ci−1−ci ) −vci , where c is the concentration of auxin , t is the time interval between simulation steps , K represents an auxin transport constant , i represents metamer indexing and v represents auxin decay .", "Since metamer indexing increases from basal to apical metamers , parameter KB in equation ( Langdale and Harrison , 2008 ) always represents basipetal movement of auxin and parameter KA represents acropetal movement of auxin .", "If KA = KB , auxin movement between metamers is not biased acropetally or basipetally , and equation ( Langdale and Harrison , 2008 ) conforms to Fick's second law of diffusion .", "If KA ≠ KB , auxin movement is directionally biased .", "A lateral branch can only initiate if the concentration of auxin in a metamer falls below a threshold T ( assumption 3 ) , or c/T < 1 ( Figure 2G; rules 2 and 3 ) .", "Points of branch initiation and insertion are abstracted as red squares in the metamer file produced by the main apex ( Figure 2I ) .", "Although branch outgrowth is not depicted , it is also simulated as illustrated in Figure 2—figure supplement 2 .", "The ratio c/T is represented by blue hexagons ( Figure 2F , G , I ) .", "Parameters Hapex , H and T are determined for each gametophore in a series before simulations start and are stochastically attributed values within a specified range .", "Parameter values may therefore differ between , but not within gametophores .", "The model incorporates a constant notional level of auxin decay represented by parameter v ( see ‘Materials and methods’ ) .", "Therefore metamers that are further away from the apex are older , and have less auxin derived from the terminal apex than newer metamers , and a smaller value of c/T ( Figure 2I , Figure 2—figure supplement 2 ) .", "This accounts for branch initiation towards the gametophore base .", "The new lateral branch apices produce auxin , which moves to metamers adjacent to the branch insertion point ( Figure 2Iv ) .", "As the gametophore grows , a local auxin minimum appears between active apices ( Figure 2Ivi ) and another branch initiates ( Figure 2Ivii ) .", "To determine whether the model can capture WT branching patterns , a series of 60 gametophores with 20–40 leaves was simulated .", "In line with published data showing bulk basipetal auxin transport to be undetectable in moss gametophores , parameter exploration showed that the WT branch distribution was best approximated by ratios of KA/KB = 1 ( Table 1 ) .", "A small bias in the direction of transport had a major impact on the distribution of branches Figure 3 ) .", "Where KA/KB > 1 , branches initiated both acropetally and basipetally and apical inhibition was lost ( Figure 3A , B , Videos 1 , 2 ) .", "Where KA/KB < 1 , the branch density in the basal portion of each gametophore and the size of the apical inhibition zone were both significantly increased ( Figure 3C , D , Videos 3 , 4 ) .", "When KA/KB was set to reflect levels of basipetal transport detected in Arabidopsis ( KA/KB = 1/100 ) , basal branches activated consecutively in the branching zone ( Figure 3D ) .", "Thus bidirectional transport was required in order to generate an apical inhibition zone comparable in metamer number to those in real plants , and to generate a branching zone with a branch distribution similar to the distribution in real plants ( compare Figures 2C to Figure 3E , F , and see Figure 2—figure supplement 1C for quantitative comparison . See also Video 5 ) . 10 . 7554/eLife . 06808 . 007Table 1 . Parameter values for the models shown in Figures 3 , 6 , 7DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 007Model wild-typeModel shi2-1Model SHI ox-5Model CKX2oeModel IPT1oe ( Figure 3F ) ( Figure 6A ) ( Figure 6B ) ( Figure 7A ) ( Figure 7B ) Hapexµ = 80 , σ = 20µ = 48 , σ = 12µ = 240 , σ = 60Hµ = 20 , σ = 4 . 5µ = 12 , σ = 2 . 7µ = 60 , σ = 13 . 5Tµ = 3 . 0 , σ = 0 . 8µ = 0 . 45 , σ = 0 . 2µ = 5 . 4 , σ = 0 . 8ν0 . 01KA0 . 05KB0 . 05Parameter µ represents the mean and σ the variance of the normally distributed stochastic variables H and T . Parameter values left blank are identical to the wild-type model . 10 . 7554/eLife . 06808 . 009Figure 3 . Simulated branching patterns are sensitive to changes in the direction of auxin transport and predict a basal branching inhibitor .", "( A–B )", "Output of simulations with mainly acropetal auxin transport , set to KA/KB = 3 ( A ) or KA/KB = 100 ( B ) .", "( C–D )", "Output of simulations with predominantly basipetal transport , set to KA/KB = 1/3 ( C ) or KA/KB = 1/100 ( D ) .", "( E , F )", "Output of simulations with bi-directional auxin transport set to KA/KB = 1 .", "Without local basal reduction of the branching threshold ( T , black arrow ) , the most basal metamer always produced a branch ( E ) .", "A reduction of the branching threshold in the basal metamers was required to generate a realistic WT branching pattern ( F ) .", "For all insets , arrow sizes indicate the relative amount of basipetal and acropetal auxin transport .", "Red indicates gametophore and branch apices , and blue indicates the auxin concentration ( c ) relative to T . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 00910 . 7554/eLife . 06808 . 010Video 1 . ( corresponds to Figure 3A ) Simulation of branching activation pattern with mainly acropetal auxin transport ( set to KA/KB = 3 ) and no basal branching inhibitor . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 01010 . 7554/eLife . 06808 . 011Video 2 . ( corresponds to Figure 3B ) Simulation of branching activation pattern with mainly acropetal auxin transport ( set to KA/KB = 100 ) and no basal branching inhibitor . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 01110 . 7554/eLife . 06808 . 012Video 3 . ( corresponds to Figure 3C ) Simulation of branching activation pattern with mainly basipetal auxin transport ( set to KA/KB = 1/3 ) and no basal branching inhibitor . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 01210 . 7554/eLife . 06808 . 013Video 4 . ( corresponds to Figure 3D ) Simulation of branching activation pattern with mainly basipetal auxin transport ( set to KA/KB = 1/100 ) and no basal branching inhibitor . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 01310 . 7554/eLife . 06808 . 014Video 5 . ( corresponds to Figure 3E ) Simulation of branching activation pattern with equal basipetal and acropetal auxin transport ( set to KA/KB = 1 ) and no basal branching inhibitor . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 014 A point of convergence between the model and branch distribution data from real plants was that in both , branch initiation in adjacent metamers was occasionally observed ( compare Figure 3F to Figures 4B , C , D , 5A , 8A ) .", "We note that the frequency of such initiation is lower in data from real plants ( 8/210 gametophores using combined data from Figures 2C , 4B , 5A , 8A ) than in the model ( 7/60 gametophores with data shown in Figure 3F ) , and it is possible that there are mechanisms to prevent adjacent initiations in plants .", "A further point of comparison relates to the sequence of branch activation ( Figure 1Q ) , which we originally assumed to be acropetal . Although branches normally initiate in an acropetal series , the model predicted that minima of c/T should also form out of series ( see Figure 2—figure supplement 2 ) , and we also found that branches can initiate out of series in real plants ( Figure 1Q ) . 10 . 7554/eLife . 06808 . 015Figure 4 . The main Physcomitrella gametophore apex is an auxin source that suppresses branching .", "( A ) Gametophores were isolated from wild-type colonies ( top ) , and the six top metamers were excised at the dotted line ( middle ) and replaced with lanolin or lanolin plus 1 mM auxin ( bottom ) .", "Scale bar = 1 mm . ( B ) Branching pattern of 30 intact gametophores ordered by increasing size .", "AIZ , apical inhibition zone .", "BZ , branching zone .", "( C ) Branching pattern of 30 gametophores 5 days after excision .", "Lateral branches activated in the portion of gametophore corresponding to the apical inhibition zone before excision , coloured in red .", "( D ) Branching pattern of 30 gametophores 5 days after excision and replacement of the apex by a source of auxin .", "( E ) The number of normal and defective branches formed in the apical inhibition zone of mock and IAA-treated gametophores .", "( F ) Mean minimum metamer number between lateral branches was not affected by gametophore apex excision .", "( G ) shows mean auxin levels quantified from five biological replicates; levels were are highest at the tip of the gametophore and decreased toward the base .", "( H ) GH3::GUS expression was high in the apical inhibition zone ( left ) and was strongly reduced after decapitation ( right ) .", "Scale bar = 1 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 01510 . 7554/eLife . 06808 . 016Figure 5 . Globally applied exogenous auxins inhibit branch initiation .", "( A–D )", "Branching patterns in wild-type gametophores grown for 5 weeks , immersed for 24 hr in mock ( A ) , 1 μM IAA ( B ) , 100 nM NAA ( C ) or 1 μM 2 , 4-D ( D ) and grown for another 2 weeks .", "( E–H ) mock ( E ) , IAA ( F ) , NAA ( G ) and 2 , 4-D ( H ) treated gametophores before ( left ) and after ( right ) removing the leaves , with asterisks indicating lateral branches .", "Scale bar = 1 mm . ( I–K ) Bubble plots showed that branch number decreased in response to IAA ( I ) , NAA ( J ) and 2 , 4-D ( K ) compared with mock-treated gametophores .", "Gametophore length is depicted as the number of metamers and the bubble area is proportional to the number of gametophores with a similar branch number at a particular length .", "Ordinary least squares regression was used to test whether the relationship between branch number and gametophore leaf number depended on treatment ( see ‘Material and methods’ ) , and for ( I ) the best fitting model was B = ( −4 . 45 + 2 . 52X ) + ( 0 . 2 − 0 . 12X ) L meaning that IAA treatment significantly differed from mock treatment ( p < 0 . 01 ) .", "For ( J ) the best fitting model was B = ( −4 . 45 + 2 . 87X ) + ( 0 . 2 − 0 . 13X ) L; NAA treatment significantly differed from mock treatment ( p < 0 . 001 ) .", "For ( K ) the best fitting model was B = ( −4 . 45 + 2 . 8X ) + ( 0 . 2 − 0 . 13X ) L; 2 , 4-D treatment significantly differed from mock treatment ( p < 0 . 001 ) .", "( L ) The apical inhibition zone size increased in response to IAA , NAA and 2 , 4-D ( mean ± SD; bilateral t-test different from mock control , *p < 0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 016 A point of divergence between branch patterns predicted by the model and data from real plants was that given bidirectional auxin transport , model simulations constitutively activated branches in the basal metamer ( Figures 2I , 3E ) .", "A phenomenon that was not observed in real plants ( compare Figure 2C to Figure 3E ) .", "This led us to hypothesize that branching could be locally suppressed at the gametophore base by an unknown inhibitory signal .", "We adjusted the model by locally reducing the branching threshold in the most basal metamers of the gametophore ( assumption 4; Figure 2—figure supplement 2 ) .", "As a decrease in the value of T drives up the ratio c/T , basal metamers less frequently reached conditions where c < T than in simulations without the basal inhibitor .", "The frequency of branch initiation in the bottom five metamers of subsequent simulations was similar to the frequency of branch initiation in those metamers in real plants ( Figure 3F , Figure 2—figure supplement 1C and Video 6 ) . 10 . 7554/eLife . 06808 . 018Video 6 . ( corresponds to Figure 3F ) Simulation of branching activation pattern with equal basipetal and acropetal auxin transport ( set to KA/KB = 1 ) and with basal branching inhibitor . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 018 Therefore , the model attained branch distribution patterns that were comparable to data from real plants ( Figure 2—figure supplement 1 ) , and generated testable hypotheses relating to the regulation of branch initiation .", "To test the assumptions of our model we undertook a series of experiments in Physcomitrella .", "We first sought to identify potential contributions of the main gametophore apex and auxin to the branching pattern by performing surgical experiments in which thirty gametophores were teased out of several colonies and decapitated by removal of the top six metamers ( the top third of the apical inhibition zone ) .", "The excised apices were replaced with lanolin or lanolin impregnated with indole-3-acetic acid ( IAA ) ( Figure 4A ) .", "Thirty further gametophores were isolated and left intact as a control , and all sets were left to grow for a week .", "Whilst control gametophores continued to grow as normal ( Figure 4B ) , the gametophores which had their apices replaced by lanolin discontinued growth of the main axis and initiated branches in the portion of the stem originally comprising the apical inhibition zone beneath the cut site ( Figure 4C , E ) .", "In contrast , the gametophores that had their apices replaced by IAA impregnated lanolin had impaired branch initiation and development ( Figure 4D , E ) .", "Whilst a total of around 70 branches initiated from lanolin treated control gametophores , around 50 branches initiated from IAA treated gametophores , and of these less than 10 showed normal development .", "The remainder were shorter than normal and were unable to initiate leaves ( Figure 4E ) , a defect that we have previously observed in the main apex in gametophores treated with high concentrations of auxin ( Bennett et al . , 2014b ) .", "No difference in the spacing of branches in control or experimental treatments was detected ( Figure 4F ) .", "These results suggest the hypothesis that the shoot apex suppresses branch initiation by acting as an auxin source , which contradicts the low auxin activity levels in gametophore tips reported by GH3::GUS signal accumulation .", "However , the GH3::GUS reporter reflects downstream transcriptional outputs of auxin signalling , and may not accurately reflect the auxin distribution ( Brunoud et al . , 2012 ) .", "We therefore quantified IAA levels along the gametophore axis by sampling the top six metamers , the apical inhibition zone minus the tip and the branching zone with the branches removed ( Figure 4A ) .", "Despite some variability between replicates , we found that mean IAA levels were highest in the tip .", "We also undertook decapitation experiments in the GH3::GUS reporter line and found that the signal intensity dropped substantially in decapitated plants , consistent with the notion that the apex can affect auxin levels elsewhere in the gametophore .", "In combination , data shown in Figure 4 support assumption one of our model by", "( i ) showing that the main apex can regulate the branching pattern ,", "( ii ) providing evidence that the main apex is an auxin source , and", "( iii ) showing that apically applied auxin can suppress branching .", "To determine whether the suppression of branching by auxin is position dependent or whether auxin can act as a global suppressor of branching , 5 week old gametophores were immersed in an auxin solution for 24 hr and grown for a further 2 weeks prior to analysis of branching patterns ( Figure 5 ) .", "A natural auxin , IAA , and two synthetic auxins with different metabolic and transport properties , 1-Naphthaleneacetic acid ( NAA ) and 2 , 4-Dichlorophenoxyacetic acid ( 2 , 4-D ) , were tested .", "For all the treatments , the size of the apical inhibition zone significantly increased , the branch number was strongly reduced and branch initiation in the branching zone significantly decreased , suggesting that auxin can act as a global suppressor of branching .", "Although our model assumes that both the main and lateral apices can act as auxin sources ( Hapex and H respectively ) , we were unable to isolate the effect of lateral apices in surgical experiments due to technical constraints .", "We therefore hypothesized that the effects of altering global levels of auxin synthesis could capture aspects of variance in both the primary and lateral gametophore tips ( Figure 6 , Figure 6—figure supplement 1 ) .", "To test this hypothesis , we grew Physcomitrella mutants or transgenics in which auxin synthesis is diminished or increased ( Eklund et al . , 2010 ) , and quantified their branching patterns with respect to model predictions and WT controls .", "A global decrease in auxin levels was simulated by reducing the values of Hapex and H , and the predicted outcome was a reduction of the apical inhibition zone size , accompanied by an increase in branch density within the branching zone ( Figure 6A ) .", "The branching phenotype of Ppshi2-1 , an auxin-deficient mutant in which auxin synthesis at the gametophore apex is reduced ( Eklund et al . , 2010 ) , matched this predicted outcome ( Figure 6C , E , G , I , J , Figure 6—figure supplement 1 , Video 7 ) .", "Conversely , a global increase in auxin levels was simulated by increasing the values of Hapex and H , generating the predictions that the apical inhibition zone size should increase and the branch density should decrease ( Figure 6B ) .", "The branching pattern of PpSHI ox-5 , a transgenic line with elevated auxin levels ( Eklund et al . , 2010 ) , again matched the model output ( Figure 6D , F , H , I , J , Figure 6—figure supplement 1 , Video 8 ) .", "Thus , by generating a decreased inhibition zone with more branches in the branching zone or an increased inhibition zone with fewer branches in the branching zone respectively , the effects of depleting or elevating SHI-mediated auxin biosynthesis captures predicted effects of changing the target auxin concentrations Hapex and H in the model . 10 . 7554/eLife . 06808 . 019Figure 6 . Auxin biosynthesis mutants and transgenics match predicted effects of changing parameter values of Hapex and H .", "( A–B )", "Model simulations of branching patterns with Hapex and H values reduced from 80 ± 20 to 48 ± 12 and from 20 ± 4 . 5 to 12 ± 2 . 7 respectively ( A ) or Hapex and H values increased from 80 ± 20 to 240 ± 60 and from 20 ± 4 . 5 to 60 ± 13 . 5 ( B ) .", "( C–D )", "Branching patterns in shi2-1 mutants with reduced auxin biosynthesis levels ( C ) and SHI ox-5 transgenics with elevated auxin biosynthesis levels ( D ) .", "( E–F ) shi2-1 ( E ) and SHI ox-5 ( F ) gametophores before ( left ) and after ( right ) removing the leaves , with asterisks indicating lateral branches .", "Scale bar = 1 mm . ( G–H ) Bubble plots showed that branch number increased in shi2-1 ( G ) and diminished in SHI ox-5 ( H ) compared with WT gametophores .", "Gametophore length is depicted as the number of metamers and the bubble area is proportional to the number of gametophores with a similar branch number at a particular length .", "For ( G ) the best fitting model was B = ( −3 . 27 + 1 . 16X ) + 0 . 18L; shi2-1 significantly differed from WT ( p < 0 . 001 ) .", "For ( H ) the best fitting model was B = ( −3 . 29 + 2 . 05X ) + ( 0 . 18 − 0 . 11X ) L , SHI ox-5 significantly differed from WT ( p < 0 . 001 ) .", "( I ) Apical inhibition zone size was reduced in shi2-1 and increased in SHI ox-5 ( mean ± SD; bilateral t-test different from WT , *p < 0 . 05 ) .", "( J ) Minimum distance between lateral branches was reduced in shi2-1 ( mean ± SD; bilateral t-test different from WT , *p < 0 . 05 ) .", "n . d . , not determined because branch number was insufficient . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 01910 . 7554/eLife . 06808 . 020Figure 6—figure supplement 1 . Comparison of mutant branching pattern plots with model outputs . Every model closely approximates the branch distribution as a function of metamer position , the total branch number , the apical inhibition zone size and the minimum distance of a branch to its closest neighbour for every corresponding mutant .", "n . d . , not determined because branch number was insufficient .", "Table: comparison of mutant branching patterns with model outputs . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02010 . 7554/eLife . 06808 . 021Video 7 . ( corresponds to Figure 6A ) Simulation of branching activation pattern in the shi2-1 mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02110 . 7554/eLife . 06808 . 022Video 8 . ( corresponds to Figure 6B ) Simulation of branching activation pattern in the SHI ox-5 transgenic line . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 022 A second assumption of our regulatory model is that the sensitivity to auxin ( parameter T ) modulates the branching pattern .", "In bud treatment experiments in flowering plants and moss , cytokinin antagonizes the suppressive effects of auxin in promoting branching ( Thimann and Skoog , 1933; Wickson and Thimann , 1958; von Maltzahn , 1959 ) , and we postulated that cytokinin could modulate auxin sensitivity with levels approximating to values of T ( Figure 7 , Figure 6—figure supplement 1 ) .", "Therefore , decreasing the values of T in our model should have the converse effect to an increase in parameter values of H , and this effect should be observed in moss shoots by global reduction of cytokinin levels .", "To test this hypothesis , we grew Physcomitrella transgenics in which cytokinin degradation is increased , thereby decreasing cytokinin levels ( von Schwartzenberg et al . , 2007 ) , and quantified their branching patterns ( Figure 7 ) .", "The predicted outcome of the model was an increase of the apical inhibition zone size and a reduction in branch number ( Figure 7A ) .", "The branching pattern of a transgenic line that constitutively expresses the Arabidopsis cytokinin degradation gene , CYTOKININ OXIDASE-2 ( CKX2oe ) ( von Schwartzenberg et al . , 2007 ) , was quantified and found to be similar to the model output in having fewer branches and a larger apical inhibition zone ( Figure 7C , E , G , I , J , Figure 6—figure supplement 1 , Video 9 ) .", "Conversely , increasing the values of T in the model should have similar effects to a global increase in cytokinin levels .", "To test this hypothesis , we generated Physcomitrella ISOPENTENYL TRANSFERASE-1 ( von Schwartzenberg et al . , 2007 ) transgenics ( IPT1oe ) in which cytokinin levels are upregulated ( Figure 7—figure supplement 1 ) and quantified their branching patterns .", "The predicted outcome of the model was a reduction of the apical inhibition zone size and an increase in the branch number ( Figure 7B , Video 10 ) , and the branching pattern in the IPT1oe line was similar ( Figure 7D , F , H , I , J , Figure 6—figure supplement 1 ) .", "Moreover , cytokinin application to gametophores was sufficient to promote meristematic cell formation and proliferation along the gametophore axis ( Figure 7K–N ) .", "Thus cytokinin is necessary for and promotes branching and lateral meristem activity . 10 . 7554/eLife . 06808 . 023Figure 7 . Cytokinin biosynthesis mutants and transgenics match predicted effects of changing parameter values of T and exogenous cytokinin treatment is sufficient for lateral meristem formation .", "( A ) Model simulation of branching patterns with T values reduced from 3 ± 0 . 8 to 0 . 45 ± 0 . 2 .", "( B ) Model simulation of branching patterns with T values increased from 3 ± 0 . 8 to 5 . 4 ± 0 . 8 .", "( C–D )", "Branching patterns in CKX2oe transgenics with reduced cytokinin levels ( C ) and IPToe transgenics with increased cytokinin levels ( D ) .", "( E–F )", "CKX2oe ( E ) and IPToe ( F ) gametophores before ( left ) and after ( right ) removing the leaves , with asterisks indicating lateral branches .", "Scale bar = 1 mm . ( G–H ) Bubble plots showed that branch number diminished in CKX2oe ( G ) and increased in IPToe ( H ) compared with WT gametophores .", "Gametophore length is depicted as the number of metamers and the bubble area is proportional to the number of gametophores with a similar branch number at a particular length .", "( G ) The best fitting model was B = ( −3 . 28 + 3 . 19X ) + ( 0 . 18 − 0 . 17X ) L , CKX2oe significantly differed from WT ( p < 0 . 001 ) .", "( H ) The best fitting model was B = ( −3 . 69 + 1 . 28X ) + 0 . 2L , IPToe significantly differed from WT ( p < 0 . 001 ) .", "( I ) Apical inhibition zone size was reduced in IPT1oe ( mean ± SD; bilateral t-test different from WT , *p < 0 . 05 ) .", "( J ) Minimum distance between lateral branches was reduced in IPT1oe ( mean ± SD; bilateral t-test different from WT , *p < 0 . 05 ) .", "n . d . , not determined because branch number was insufficient .", "( K–N )", "Exogenous cytokinin treatment promoted branch initiation .", "WT gametophores 1 week after immersion in mock ( K ) or 1 μM BAP ( L ) solution for 24 hr .", "Cytokinin promoted development of callus-like structures ( M ) .", "Scale bar = 1 mm . ( N ) Confocal microscope image showing that cytokinin-induced structures were mainly constituted of leaves ( white asterisks ) resulting from the proliferation of ectopic meristematic cells .", "Scale bar = 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02310 . 7554/eLife . 06808 . 024Figure 7—figure supplement 1 . Molecular characterization and cytokinin profiling of the PpIPT1 overexpressing line .", "( A ) Schematic of the genetic construct pTHUBI-PpIPT1 ( modified from pTHUBI-Gateway [Perroud et al . , 2011] ) .", "Used to generate PpIPT1 overexpressing lines .", "( B ) RT-PCR analysis detected strong expression of PpIPT1 and HYG genes in wild-type plants genetically transformed with pTHUBI-PpIPT1 ( IPToe ) , but not in untransformed wild-type plants ( WT ) .", "PpUBI gene was used as internal control .", "( C ) Cytokinin ( CK ) profiling showed a global increase in CK levels in IPToe transgenics in comparison with WT .", "CK levels represent the mean ( ± s . d . ) of four biological replicates and are expressed in pmol per gram of fresh weight ( pmol/g F . W . ) .", "iP , N-isopentenyladenine; iPR , N-isopentenyladenosine; iPR5′MP , N-isopentenyladenosine-5′-monophosphate; tZ , trans-zeatin; tZR , trans-zeatin riboside; tZR5′MP , trans-zeatin riboside-5′-monophosphate; tZOG , trans-zeatin O-glucoside; tZROG , trans-zeatin riboside O-glucoside; cZ , cis-zeatin; cZR , cis-zeatin riboside; cZR5′MP , cis-zeatin riboside-5′-monophosphate; cZOG , cis-zeatin O-glucoside; cZROG , cis-zeatin riboside O-glucoside . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02410 . 7554/eLife . 06808 . 025Video 9 . ( corresponds to Figure 7A ) Simulation of branching activation pattern in the CKX2oe transgenic line . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02510 . 7554/eLife . 06808 . 026Video 10 . ( corresponds to Figure 7B ) Simulation of the branching activiation pattern in the IPT1oe transgenic line . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 026 A third assumption of our regulatory model is that auxin moves between metamers , and the output of the model was found to be very sensitive to changes in the direction of auxin transport .", "Realistic branching patterns were only generated when there was relatively even basipetal and acropetal transport within simulated gametophore axes ( Figure 3 ) , a result that is consistent with the absence of detectable bulk basipetal transport in Physcomitrella gametophores ( Fujita et al . , 2008 ) .", "The molecular functions of canonical PIN proteins are conserved between flowering plants and mosses ( Bennett et al . , 2014b; Viaene et al . , 2014 ) , and Physcomitrella PINs have been demonstrated to localise both proximally and distally within leaf cells ( Viaene et al . , 2014 ) .", "PINs therefore provide one potential mechanism to account for bi-directional auxin transport in the regulation of branching .", "We previously engineered Physcomitrella pin mutants in a GH3::GUS background ( Bennett et al . , 2014b ) , and found that whilst branching patterns in the GH3::GUS line were similar to WT controls ( Figure 8A ) , pin mutants had mildly disrupted branching patterns ( Figure 8A–H ) .", "The apical inhibition zone was shorter in pina pinb mutants than in GH3::GUS controls ( Figure 8I ) .", "Branch number was slightly increased in pinb and pina pinb double mutants ( Figure 8J–L ) and the mean minimum distance between branches was reduced in pina , pinb and pina pinb double mutants ( Figure 8M ) .", "To rule out the possibility that residual PIN-mediated auxin transport could be responsible for the mild effects of pin mutants on branching patterns , mutants were grown on medium supplemented with 5 μM NPA ( Figure 8N–W ) .", "In both GH3::GUS and pina pinb lines , branching patterns were found to be similar in mock and NPA-treated plants .", "Thus PIN-mediated auxin transport is not a major contributor to branching patterns . 10 . 7554/eLife . 06808 . 017Figure 8 . PIN-mediated auxin transport is a minor contributor to branching patterns .", "( A–D )", "Branching patterns in GH3::GUS , pina , pinb and pina pinb mutants .", "( E–H )", "GH3::GUS ( E ) , pina ( F ) , pinb ( G ) and pina pinb ( H ) gametophores before ( left ) and after ( right ) removing leaves , with asterisks indicating lateral branches .", "Scale bar = 1 mm . ( I ) Apical inhibition zone size is significantly reduced in the pina pinb double mutant but not in pina or pinb single mutants ( mean ± SD; bilateral t-test different from GH3::GUS , *p < 0 . 05 ) .", "( J–L )", "Bubble plots showed that branch number increases in pinb ( K ) and pina pinb ( L ) but not pina ( J ) compared with GH3::GUS gametophores .", "Gametophore length is depicted as the number of metamers and the bubble area is proportional to the number of gametophores with a similar branch number at a particular length .", "The best fitting model for ( J ) was B = −2 . 8 + 0 . 15L , pina was not significantly different from GH3::GUS .", "The best fitting model for ( K ) was B = ( −2 . 47 − 1 . 40X ) + ( 0 . 14 + 0 . 07X ) L , pinb was significantly different from GH3::GUS ( p < 0 . 01 ) .", "The best fitting model for ( L ) was B = ( −2 . 96 + 0 . 52X ) + 0 . 15L , pina pinb significantly differed from GH3::GUS ( p < 0 . 01 ) .", "( M ) The minimum distance between lateral branches is reduced in pina , pinb and pina pinb mutants with respect to GH3::GUS controls ( mean ± SD; bilateral t-test different from GH3::GUS , *p < 0 . 05 ) , thus branch density in the branching zone is higher in all the mutants .", "( N–O )", "Branching patterns in GH3::GUS mutants treated without ( N ) or with ( O ) 5 μM NPA .", "( P–R ) 5 μM NPA treatment did not affect the branch number in GH3::GUS transgenics ( P ) , the apical inhibition zone size ( Q ) and the minimum distance between lateral branches ( R ) .", "For ( P ) , the best fitting model was B = −3 . 64 + 0 . 2L; NPA treatment was not significantly different from the mock treatment .", "( S–T )", "Branching patterns in pina pinb mutants treated without ( S ) or with ( T ) 5 μM NPA .", "( U–W ) 5 μM NPA treatment did not affect the branch number in pina pinb mutants ( U ) , the apical inhibition zone size ( V ) and the minimum distance between lateral branches ( W ) .", "For ( U ) , the best fitting model was B = −1 . 99 + 0 . 14L; NPA treatment was not significantly different from the mock treatment .", "( X–Y )", "Branching patterns in WT treated without ( X ) or with ( Y ) 5 μM BUM .", "( Z–AB ) 5 μM BUM treatment did not affect the branch number in WT ( Z ) , the apical inhibition zone size ( AA ) and the minimum distance between lateral branches ( AB ) ( mean ± SD ) .", "For ( Z ) , the best fitting model was B = −1 . 24 + 0 . 07L; BUM treatment was not significantly different from the mock treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 017 A second class of auxin efflux transporters controlling development which interact with PIN proteins to regulate auxin distributions are the ATP-binding cassette protein subfamily B ( ABCB/PGP ) transporters ( Cho and Cho , 2013 ) .", "ABCB/PGP proteins have non-polar plasma membrane localizations and could therefore putatively generate the bi-directional auxin transport required by the model to regulate branching .", "10 genes have been identified in the Physcomitrella genome ( Carraro et al . , 2012 ) .", "To test the hypothesis that ABCB/PGPs regulate Physcomitrella branching , we analysed branching patterns in WT gametophores grown on an ABCB/PGP inhibitor ( Kim et al . , 2010 ) , 2-[4- ( diethylamino ) -2-hydroxybenzoyl]benzoic acid ( BUM ) at a 5 μM concentration ( Figure 8X–AB ) .", "We found no difference between mock and BUM-treated plants suggesting that ABCB/PGP proteins do not regulate branching in Physcomitrella .", "Although the contributions of plasma membrane-targeted PIN and ABCB/PGP auxin transporters to plant development are well accepted ( Petrásek and Friml , 2009 ) , recent work in Arabidopsis has suggested that symplastic auxin transfer via plasmodesmata may also contribute to development by maintaining auxin concentration gradients between cells ( Han et al . , 2014 ) .", "Symplastic permeability is under active control via callose deposition such that increased deposition blocks plasmodesmatal connections , and decreased deposition opens them up to allow greater permeability and higher rates of diffusion ( Han et al . , 2014 ) .", "As the apolar auxin transport required to generate realistic branching patterns in our model is equivalent in principle to diffusion , we hypothesized that a callose-dependent mechanism could regulate branching .", "To test this hypothesis , we grew Physcomitrella on a callose biosynthesis inhibitor , 2-deoxy-glucose ( DDG ) , and found that its application did not cause general defects at the concentrations used .", "We therefore compared the branching patterns to model predictions arising from a simulated increase in the rate of auxin movement ( Figure 9 , Table 2 ) .", "Simulations predicted that stepwise increases in the rate of movement should progressively reduce branching ( Figure 9B , C , Videos 11 , 12 , 13 ) , and in DDG treated plants branching was similarly reduced ( Figure 9D–M ) .", "These data suggest callose-gated plasmodesmal connectivity as a plausible mechanism to regulate branching patterns in Physcomitrella . 10 . 7554/eLife . 06808 . 027Figure 9 . A callose synthesis inhibitor regulates branching .", "( A–C )", "Model simulation of branching patterns with K values increased from 0 . 025 ( A ) to 0 . 045 ( B ) and 0 . 07 ( C ) .", "( D–F )", "Branching patterns in wild-type gametophores treated with mock ( D ) , 10 μM 2-deoxy-glucose ( DDG ) ( E ) or 25 μM DDG ( F ) .", "( G–I ) mock ( G ) , 10 μM DDG ( H ) or 25 μM DDG ( I ) treated gametophores before ( left ) and after ( right ) removing the leaves , with asterisks indicating lateral branches .", "Scale bar = 1 mm . ( J–K ) Bubble plots show that branch number diminishes in 10 μM DDG ( J ) and is strongly reduced in 25 μM DDG ( K ) treated gametophores compared with mock treatment .", "Gametophore length is depicted as the number of metamers and the bubble area is proportional to the number of gametophores with a similar branch number at a particular length .", "The best fitting model for ( J ) was B = ( −3 . 82 + 1 . 41X ) + ( 0 . 21 − 0 . 08X ) L , 10 μM DDG was significantly different from mock treatment ( p < 0 . 001 ) .", "The best fitting model for ( K ) was B = ( −3 . 82 + 2 . 47X ) + ( 0 . 21 − 0 . 14X ) L , 25 μM DDG was significantly different from mock treatment ( p < 0 . 001 ) .", "( L ) Apical inhibition size is significantly increased in response to DDG treatment ( mean ± SD; bilateral t-test different from WT mock , *p < 0 . 05 ) .", "( M ) The minimum distance between lateral branches is not different in 10 μM DDG treated gametophores with respect to mock controls ( mean ± SD ) .", "n . d . , not determined because branch number was insufficient . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02710 . 7554/eLife . 06808 . 028Figure 9—figure supplement 1 . Comparison of branching pattern plots from pharmacologically treated plants with model outputs . Every model closely approximates the branch distribution as a function of metamer position , the total branch number , the apical inhibition zone size and the minimum distance of a branch to its closest neighbour for every corresponding mutant .", "n . d . , not determined because branch number was insufficient .", "Table: comparison of branching patterns from pharmacologically treated plants with model outputs' .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02810 . 7554/eLife . 06808 . 008Table 2 . Refitted parameter values for the models shown in Figure 9DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 008Model wild-typeModel 10 µM DDGModel 25 µM DDG ( Figure 9A ) ( Figure 9B ) ( Figure 9C ) Hapexμ = 30 , σ = 10Hμ = 5 , σ = 1 . 5Tμ = 0 . 7 , σ = 0 . 2ν0 . 002KA0 . 0250 . 0450 . 07KB0 . 0250 . 0450 . 0710 . 7554/eLife . 06808 . 029Video 11 . ( corresponds to Figure 9A ) Simulation of the WT branching activation pattern with refitted parameters . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 02910 . 7554/eLife . 06808 . 030Video 12 . ( corresponds to Figure 9B ) Simulation of branching activation pattern with K values increased by 100% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 03010 . 7554/eLife . 06808 . 031Video 13 . ( corresponds to Figure 9C ) Simulation of branching activation pattern with K values increased by 200% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 031 A final testable assumption of our model is that branching is locally suppressed at the base of the gametophore , expressed as a local reduction in T in our model ( Figure 3F ) .", "We noted from published literature ( Proust et al . , 2011 ) that the strigolactone biosynthesis gene ppccd8 is expressed at the base of gametophores .", "ppccd8 mutants are strigolactone deficient and although no branching defects were previously detected in mutant gametophores ( Proust et al . , 2011 ) , we reasoned that strigolactones could be the repressive signal .", "We therefore quantified the branching patterns in ppccd8 mutant gametophores relative to WT ( Figure 10 ) .", "We found strongly increased branch activation at the base of gametophores ( number of branching gametophores: WT , 4/60; ppccd8 mutant , 29/60 ) ( Figure 10A , B ) .", "To confirm that the ppccd8 mutant phenotype was caused by a deficiency in strigolactone production , we quantified the branching pattern in ppccd8 mutants grown on medium supplemented with 1 μM GR24 , a synthetic strigolactone analogue , and found that GR24 was able to suppress basal branch formation ( Figure 10C ) .", "To evaluate whether strigolactone can act as a global branch suppressor , we analysed the branching pattern in Physcomitrella mutants that over express the pea strigolactone synthesis gene , RMS1 ( Proust et al . , 2011 ) .", "We found that branching was very strongly suppressed in this transgenic line ( Figure 10D , Figure 10—figure supplement 1 ) .", "We used gametophores with fewer than 20 leaves for this experiment because comparison of model predictions ( Figure 3E , F ) to WT data ( Figure 2C ) showed that we should most clearly detect a difference at this stage of development , and ppccd8 mutants make few gametophores .", "Our inferences were supported in a similar but scaled down experiment using fully grown gametophores ( Figure 10—figure supplement 1 ) .", "Thus basal expression of a strigolactone biosynthetic gene can account for the predicted requirement of our model for a local basal inhibitor of branching . 10 . 7554/eLife . 06808 . 032Figure 10 . Expression levels of a strigolactone biosynthesis gene modulates branching .", "( A–D )", "Branching patterns of 60 gametophores with fewer than 20 leaves in ( A ) WT , ( B ) strigolactone-deficient ppccd8 mutants , ( C ) ppccd8 mutants treated with 1 μM GR24 and ( D ) RMS1oe transgenics with increased strigolactone levels .", "( E–H )", "WT ( E ) , ppccd8 ( F ) , GR24-treated ppccd8 ( G ) and RMS1oe ( H ) gametophores before ( left ) and after ( right ) removing the leaves , with asterisks indicating lateral branches .", "Scale bar = 1 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 03210 . 7554/eLife . 06808 . 033Figure 10—figure supplement 1 . Branching patterns of gametophores with more than 20 leaves and branch proportion in the five most basal metamers .", "( A–C )", "Branching patterns of 40 gametophores with more than 20 leaves in ( A ) WT , ( B ) strigolactone-deficient ppccd8 mutants and ( C ) RMS1oe transgenic lines with increased strigolactone levels .", "( D–E )", "Bubble plots show that branch number increases at the base of ccd8 mutants ( D ) and is strongly reduced in RMS1oe transgenics ( E ) compared with WT gametophores .", "Gametophore length is depicted as the number of metamers and the bubble area is proportional to the number of gametophores with a similar branch number at a particular length .", "The best fitting model for ( D ) was B = ( −2 . 84 + 0 . 73X ) + 0 . 16L , ccd8 was significantly different from WT ( p < 0 . 01 ) .", "The best fitting model for ( E ) was B = ( −4 . 25 + 2 . 71X ) + ( 0 . 21 − 0 . 15X ) L , RMS1oe was significantly different from WT ( p < 0 . 001 ) .", "( F ) Branch proportion in the five most basal metamers is similar between the ‘no basal branching inhibitor’ model output ( Figure 3E ) and ppccd8 mutants ( B , D ) .", "Addition of a basal branching inhibitor in the model simulations ( Figure 3F ) captures basal branch proportion of WT gametophores ( A , Figure 2C ) .", "Increase of strigolactone biosynthesis suppresses basal branching in RMS1oe gametophores ( C , E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06808 . 033" ], [ "Hormonal signalling by auxin , cytokinin and strigolactone evolved before plants' transition to land ( Cooke et al . , 2002; De Smet et al . , 2011; Delaux et al . , 2013; Gruhn and Heyl , 2013 ) , and was recruited to regulate axillary bud initiation ( auxin and cytokinin; [Wang et al . , 2014a , 2014b] ) and lateral branch outgrowth in flowering plant sporophytes ( Domagalska and Leyser , 2011 ) .", "We have shown that these three ancient hormonal cues were recruited independently to regulate lateral branching in gametophores of the moss Physcomitrella .", "In combination our model and experimental data lead us to a notion of branching whereby the ratio c/T determines the fate of epidermal cells in the gametophore axis .", "If the ratio drops below a threshold level , an epidermal cell can be respecified as a lateral apical cell thereby triggering branch initiation .", "Such a drop usually occurs in leaf axils independently of the leaf initiation process at the meristem ( Harrison et al . , 2009 ) as a result of competing hormonal cues dispersed across the gametophore .", "However , cells elsewhere in the metamer are also competent to form branch initials , and do so if the c/T ratio is perturbed , for instance by changing auxin ( Figures 5 , 6 ) , strigolactone ( Figure 9 ) or cytokinin levels ( Figure 7 ) .", "Although this process has some similarities to the branch initiation process in Arabidopsis in which a drop in auxin levels in the axils of initiating leaves coupled with a rise in cytokinin levels primes the initiation of the axillary meristem ( Wang et al . , 2014a , 2014b ) , there are many differences in the regulation of lateral branching between flowering plant sporophytes and Physcomitrella gametophytes .", "A first key difference is the mechanism underlying apical dominance , which requires PIN-mediated bulk basipetal polar auxin transport in Arabidopsis ( Gälweiler et al . , 1998; Ljung et al . , 2001 ) .", "Our model for branching in Physcomitrella assumes that the main gametophore apex acts as the source of a cue that can move into the gametophore axis to suppress branching .", "Decapitation experiments show that the main gametophore apex inhibits branching and that this inhibitory effect can be maintained if the apex is substituted by a source of auxin ( Figure 4 ) .", "We detect higher levels of auxin in the apex than elsewhere in the gametophore , and removal of the apex diminishes levels of expression of an auxin responsive reporter in the gametophore axis , thereby implicating auxin as an apical cue and indicating a requirement for auxin transport away from the apex in the regulation of branching patterns ( Figure 4 ) .", "Although these results are qualitatively similar to the outcome of such experiments in Arabidopsis ( Thimann and Skoog , 1933; Wickson and Thimann , 1958; Cline , 1991 ) , no bulk basipetal auxin transport has been detected in Physcomitrella ( Fujita et al . , 2008 ) .", "Our model predicts a requirement for bi-directional , or diffusion-like transport to generate realistic patterns ( Figure 3 ) , and suggests that not only the direction ( Figure 3 ) , but also the rate of transport has a significant impact on branch patterning ( Figure 9 ) .", "Our experiments with pin mutants and pharmacological inhibitors show that membrane-targeted auxin transporters belonging to PIN and ABCB/PGP families are not significant contributors to branch patterning ( Figure 8 ) .", "Patterning is sensitive to the application of the callose synthesis inhibitor DDG , and incremental increases in the concentration of applied DDG have similar effects on branch patterning as incremental increases in the rates of transport implemented in our model ( Figure 9 ) .", "These data suggest that auxin may move with diffusive properties via callose-gated plasmodesmatal connections between cells ( Han et al . , 2014 ) .", "Further distinctions relate to variation in conceptualized notions of global and local sensitivities to auxin that can be produced in moss by varying the activity of cytokinin biosynthesis and degradation , and strigolactone biosynthesis pathways respectively .", "Although the role of cytokinin in gametophore induction is well characterized in Physcomitrella ( Ashton et al . , 1979 ) , and cytokinin has previously been shown to antagonize auxin in branch formation ( von Maltzahn , 1959 ) , little is known about where and how cytokinin acts later in gametophore development .", "Our results are consistent with a homogenous effect of cytokinin within the gametophore axis ( Figure 8 ) , and further analysis of cytokinin distributions , for instance with a modified TCS reporter ( Müller and Sheen , 2008 ) , may be informative .", "Upregulation of cytokinin levels by overexpressing IPT1 , and exogenous cytokinin applications show that cytokinin is sufficient to promote lateral meristem formation and therefore indicate that cytokinin directly promotes branching ( Figure 7 ) .", "The mechanism by which strigolactone acts with auxin in the regulation of branching is likely to differ between Arabidopsis and Physcomitrella .", "Whereas strigolactone is thought to suppress branch outgrowth in Arabidopsis by dampening PIN mediated polar auxin transport , the mode of action of strigolactone in Physcomitrella is unclear .", "Notably , key strigolactone signalling components required for the inhibition of sporophyte branching in higher plants are not present in mosses ( Delaux et al . , 2013; de Saint Germain et al . , 2013 ) .", "For instance , components such as D14 ( involved in strigolactone perception ) and D53 are absent in Physcomitrella ( Challis et al . , 2013; Delaux et al . , 2013; Zhou et al . , 2013 ) .", "Similarly the Physcomitrella homologue of the F-Box protein MAX2 may not be involved in strigolactone signalling , despite its central role in many strigolactone responses in flowering plants ( de Saint Germain et al . , 2013 ) .", "The modulation of branching by PpCCD8 and RMS1 ( Figure 10 , Figure 10—figure supplement 1 ) was unexpected as previously published work identified roles for PpCCD8 in protonemal but not gametophore branching patterns ( Proust et al . , 2011 ) .", "The basal expression domain of PpCCD8 ( Proust et al . , 2011 ) is consistent with a local site of hormone action , but the mechanism underlying the ppccd8 and RMS1oe mutant phenotypes remains to be seen .", "Although there is a substantial body of evidence that strigolactone-like signals are active across the plant kingdom , the ancestral function of strigolactone and/or these strigolactone-like signals is thought to be in rhizoid elongation ( Delaux et al . , 2013; de Saint Germain et al . , 2013 ) .", "The absence of distinct branch initiation and outgrowth processes in Physcomitrella implicates strigolactone in branch initiation , and the relative unimportance of PIN-mediated auxin transport in the regulation of Physcomitrella branching patterns suggests that strigolactone is unlikely to act via PIN-mediated auxin transport .", "Diverse branching forms are evident in both the gametophytic ( Taylor et al . , 2005 ) and sporophytic plant fossil record ( Gerrienne et al . , 2006; Edwards et al . , 2014 ) .", "Although plant phylogenies show that gametophytic branching forms have several independent evolutionary origins , the innovation of sporophytic branching maps once onto the plant tree of life ( Langdale and Harrison , 2008 ) and occurred in pre-vascular plants such as Partitatheca ( Edwards et al . , 2014 ) .", "Exogenous hormone treatments in Selaginella have shown that the antagonistic relationship between auxin and cytokinin in the regulation of branching is conserved within the vascular plants ( Sanders and Langdale , 2013 ) .", "Furthermore , auxin transport assays and pharmacological treatments used in combination with decapitation in Selaginella ( Williams , 1937; Wochok and Sussex , 1973 , 1975 ) have shown that auxin transport-mediated apical dominance is a homology of vascular plant sporophytes .", "Disruption of PIN-mediated polar auxin transport in moss sporophytes can induce a branching form that is intermediate between living bryophytes and vascular plants ( Fujita et al . , 2008; Bennett et al . , 2014b ) , and resembles the earliest branching forms in the sporophytic fossil record ( Edwards et al . , 2014 ) , suggesting that auxin transport-regulated branching may be a homology of the sporophyte generation .", "Although the regulation of lateral branching by three key regulatory hormone pathways in moss gametophytes indicates deep homology ( Scotland , 2010 ) , the mode and basis of auxin transport are a key divergence point in branch patterning mechanisms between land plant sporophytes and gametophytes ." ], [ "All computational models were created using the VVe modeling environment ( Abley et al . , 2013 ) .", "The moss gametophore is represented in the model as a graph of vertices and connecting edges .", "Each vertex represents either a metamer or an apex .", "The graph representing the moss is dynamic .", "Periodically , new vertices are added to the graph representing growth of the gametophore ( Figure 2G ) .", "The number of simulation steps between the addition of new vertices in the model constitutes 1 plastochron , which we assume to be approximately 1 day for wild-type Physcomitrella patens grown on agar medium .", "The differential equations calculating the rate of change of auxin concentration per vertex ( Equation 1 ) of the graph are solved numerically using the forward Euler integration scheme .", "The time step Δt is set to 0 . 005 , there are thus 200 simulation steps per plastochron .", "For all simulation results presented , a number of gametophores have been simulated where growth is delayed by a random amount of simulation steps between two neighboring axes , and the simulation stops after a set number of total simulation steps has been reached .", "All simulated gametophores initiate as one apical vertex visualized in red , and a vertex representing the first metamer , visualized in grey .", "Separate lateral axes are simulated , and their initiation point is represented as a red cell .", "Higher order branching is not represented and was rarely observed in experiments .", "For the most important parameters such as auxin concentrations , auxin movement or auxin decay rates there were no direct estimates available to this project .", "Nevertheless , we assume that auxin movement in the gametophore of Physcomitrella is slower than in Arabidopsis shoot tissue ( ∼20 mm/hr ) and faster than in NPA treated Arabidopsis shoot tissue ( ∼0 . 1 mm/hr ) choosing an arbitrary value inside these bounds for our simulations .", "Our parameter value settings , given that we have 200 simulation steps per plastochron , correspond to an auxin movement rate of approximately 1 mm/hr in the gametophore .", "However , we found for several auxin movement rate values inside these bounds for which simulation results could be reproduced by readjusting other parameter values .", "For example , the simulations represented in Figure 9 use a smaller time step Δt of 0 . 001 compared to all other simulations .", "Ordinary least squares regression was used to test whether the relationship between branch number and gametophore leaf number depended on genotype .", "A linear model B = ( a0 + a1 X ) + ( a2 + a3 X ) L , where a0 , a1 , a2 and a3 are coefficients , L is the number of leaves , B the number of branches , and X is an indicator variable depending on genotype ( 0 corresponded to a mutant , 1 corresponded to WT ) was fitted .", "Backwards stepwise elimination was used to find the minimal model with support from the experimental data .", "A mutant was considered as different from control genotype if either the interaction term ( a3 ) or the term corresponding to genotype ( a1 ) remained in the minimal model ." ] ]

[ "Shoot branching is a primary contributor to plant architecture , evolving independently in flowering plant sporophytes and moss gametophytes .", "Mechanistic understanding of branching is largely limited to flowering plants such as Arabidopsis , which have a recent evolutionary origin .", "We show that in gametophytic shoots of Physcomitrella , lateral branches arise by re-specification of epidermal cells into branch initials .", "A simple model co-ordinating the activity of leafy shoot tips can account for branching patterns , and three known and ancient hormonal regulators of sporophytic branching interact to generate the branching pattern- auxin , cytokinin and strigolactone .", "The mode of auxin transport required in branch patterning is a key divergence point from known sporophytic pathways .", "Although PIN-mediated basipetal auxin transport regulates branching patterns in flowering plants , this is not so in Physcomitrella , where bi-directional transport is required to generate realistic branching patterns .", "Experiments with callose synthesis inhibitors suggest plasmodesmal connectivity as a potential mechanism for transport ." ]

[ "Most land plants have shoots that form branches and plants can regulate when and where they grow these branches to best exploit their environment .", "Plants with flowers and the more ancient mosses both have branching shoots , but these two groups of plants evolved to grow in this way independently of each other .", "Most studies on shoot branching have focused on flowering plants and so it is less clear how branching works in mosses .", "Three plant hormones—called auxin , cytokinin and strigolactone—control shoot branching in flowering plants .", "Auxin moves down the main shoot of the plant to prevent new branches from forming .", "This movement is controlled by the PIN proteins and several other families of proteins .", "On the other hand , cytokinin promotes the growth of new branches; and strigolactone can either promote or inhibit shoot branching depending on how the auxin is travelling around the plant .", "Coudert , Palubicki et al . studied shoot branching in a species of moss called Physcomitrella patens .", "The experiments show that cells on the outer surface of the main shoot are essentially reprogrammed to become so-called ‘branch initials’ , which will then develop into new branches .", "Next , Coudert , Palubicki et al . made a computational model that was able to simulate the pattern of shoot branching in the moss .", "Further experiments supported the predictions made by the model .", "Coudert , Palubicki et al . found that , as in flowering plants , auxin from the tip of the main shoot suppresses branching in the moss , and cytokinin promotes branching .", "The experiments also showed that strigolactone inhibits shoot branching , but its role is restricted to the base of the shoots .", "The model predicts that , unlike in flowering plants , auxin must flow in both directions in moss shoots to produce the observed patterns of shoot branching .", "Also , the experiments suggest that the PIN proteins and another group of proteins that control the movement of auxin do not regulate shoot branching in moss .", "Instead , it appears that auxin may move through microscopic channels that link one moss cell to the next .", "Coudert , Palubicki et al . 's findings suggest that both flowering plants and mosses have evolved to use the same three hormones to control shoot branching , but that these hormones interact in different ways .", "One key next step will be to find out how auxin is transported during shoot branching in moss by manipulating the opening of the channels between the cells .", "A further challenge will be to find out the precise details of how the hormones control the activity of the branch initial cells ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "computational and systems biology", "microbiology and infectious disease" ]

[ [ "Both the oral cavity and large intestine accommodate unique microbiomes that are relevant to human health and disease ( Lynch and Pedersen , 2016; Wade , 2013 ) .", "Mouth and gut are linked by a constant flow of ingested food and saliva along the gastrointestinal tract ( GIT ) , yet they host distinct microbial communities ( Ding and Schloss , 2014; Segata et al . , 2012 ) in distinct microenvironments ( Savage , 1977 ) , and have been reported to harbor locally adapted strains ( Lloyd-Price et al . , 2017 ) .", "The segregation of oral and intestinal communities is thought to be maintained by various mechanisms , such as gastric acidity ( Howden and Hunt , 1987; Martinsen et al . , 2005 ) and antimicrobial bile acids in the duodenum ( Ridlon et al . , 2014 ) .", "Failure of this oral-gut barrier has been proposed to lead to intestinal infection ( Martinsen et al . , 2005 ) , and the prolonged usage of proton pump inhibitors can result in an enrichment of particular oral microbes in the gut ( Imhann et al . , 2016 ) .", "Increased presence of specific oral taxa in the intestine has in turn been linked to several diseases , including rheumatoid arthritis ( Zhang et al . , 2015 ) , colorectal cancer ( Flynn et al . , 2016; Zeller et al . , 2014 ) and inflammatory bowel disease ( IBD , ( Gevers et al . , 2014 ) ) .", "While it remains unclear whether disease-associated strains are indeed acquired endogenously ( from the oral cavity ) or from the environment , it was recently shown that Klebsiella strains originating from salivary samples of two IBD patients triggered intestinal inflammation in gnotobiotic mice ( Atarashi et al . , 2017 ) .", "This suggests that the presence of oral commensals in the gut is a rare , aberrant event as a consequence of ectopic colonization ( i . e . , ‘in the wrong place’ ) , and hence a hallmark of disease .", "Outside a disease context , however , possible links between the oral and gut microbiome remain poorly characterized .", "Several genera were shown to be prevalent at both sites ( Segata et al . , 2012 ) , with community types in one being weakly predictive of the other ( Ding and Schloss , 2014 ) , and with similar gene content in particular species ( Franzosa et al . , 2014 ) , but with distinct , locally adapted strains ( Lloyd-Price et al . , 2017 ) .", "We hypothesized that this picture is incomplete , and that microbial transmission along the GIT is more common than previously appreciated: that despite oral-gut barrier effects , some microbes freely and frequently traverse the GIT and colonize different niches , forming continuous populations that shape the human microbiome ." ], [ "To test this hypothesis , we assembled and analyzed a dataset of 753 public and 182 newly sequenced saliva and stool metagenomes from 470 healthy and diseased individuals ( diagnosed with rheumatoid arthritis , colorectal cancer or type-1 diabetes ) from Fiji ( Brito et al . , 2016 ) , China ( Zhang et al . , 2015 ) , Luxembourg ( Heintz-Buschart et al . , 2016 ) , France ( Zeller et al . , 2014 ) , and Germany ( Voigt et al . , 2015 ) ( see Materials and methods , Figure 1 , and Supplementary file 1 ) .", "For these samples we profiled 310 prevalent species , accounting for 99% of classifiable microbial abundance in both saliva and stool ( see Materials and methods and Supplementary file 2 ) .", "We reasoned that if transmission between the oral and gut microenvironments is frequent , we would expect salivary and fecal microbial populations to be more similar within an individual than between individuals .", "Conversely , under a strong barrier model with restricted transmission , intra- and inter-individual similarities would be equivalent .", "We found that at species level , community composition was consistent with distinct populations occupying the oral and intestinal microenvironments .", "By prevalence across subjects , the 310 profiled species fell into three categories ( Figure 2A ) : 44% were predominantly fecal ( observed in ≥10% of fecal , but <10% of saliva samples ) , including core members of the gut microbiome , such as Clostridium sp .", ", Ruminococcus sp .", "and Bacteroides sp .", "; 16% of species were predominantly oral .", "Although the remaining 125 ( 40% ) species were prevalent in ≥10% of saliva and stool samples , their relative abundances differed greatly between the two habitats .", "The overall oral and fecal microbiome compositions appeared independent of each other ( between-subject Bray-Curtis dissimilarities per site , ρPearson=-0 . 03 ) , and the compositional overlap between mouth and gut of the same subject was not found to be significantly different when compared to a between-subject background ( Wilcoxon test , Bray-Curtis dissimilarities , p=0 . 46 ) .", "However , to accurately establish and quantify microbial transmission , it is necessary to track populations at the resolution of strains rather than species , as demonstrated previously in fecal microbiota transplantation ( Li et al . , 2016 ) or seeding of the infant microbiome ( Asnicar et al . , 2017 ) ; Korpela et al . , 2018 ) .", "We therefore profiled microbial single nucleotide variants ( SNVs ) across metagenomes , as a proxy for strain populations ( Li et al . , 2016 ) .", "We formulated a transmission score for each species per subject , based on the likelihood that the observed intra-individual SNV overlap was generated by an inter-individual background model ( see Materials and methods ) .", "Of the 125 species prevalent in both mouth and gut , 77% showed evidence of oral-fecal transmission .", "Out of these , 74 species ( 59% ) showed significantly higher intra-individual SNV similarity across all subjects compared to cohort-wide background SNV frequencies ( Benjamini-Hochberg-corrected Wilcoxon tests on transmission scores , p<0 . 05 , see Materials and methods; Figure 2B , Figure 2—figure supplement 1 , Supplementary file 2 ) .", "This suggests that they form coherent strain populations along the GIT in most subjects , subject to frequent oral-fecal microbial transmission .", "Strains of Streptococcus , Veillonella , Actinomyces and Haemophilus , among other core oral taxa , fell into this category .", "An additional 22 species ( 18% ) showed evidence of at least occasional transmission , with individually significant oral-fecal SNV overlap in some , but not across all subjects , as did 18 species that were generally prevalent in either the mouth or the gut ( but not both ) .", "All 21 members of the Prevotella genus , an important clade of the gut microbiome , were among these occasionally transmitted species .", "The remaining 29 ( 23% ) species , which were prevalent in both sites , did not show signs of transmission under the strict thresholds we applied .", "The fecal abundance of all species with paired observations exceeded lower-bound physiologically predicted levels ( i . e . , the detection of salivary bacteria in stool purely as the result of ingestion ) by several orders of magnitude , even with conservative estimates ( Figure 1C , Figure 1—figure supplement 1 ) .", "An average person swallows an estimated 1 . 5 * 1012 oral bacteria per day ( Humphrey and Williamson , 2001; Sender et al . , 2016 ) .", "Passage through the stomach reduces the viable bacterial load by 5–6 orders of magnitude ( Giannella et al . , 1972; Sender et al . , 2016 ) , a reduction that is expected to be mirrored at the DNA level , given that free DNA , released from dead bacterial cells , is degraded within seconds to minutes in saliva , the stomach and the intestine ( see for example Mercer et al . , 1999 and Liu et al . , 2015 ) .", "Relative to the ~3 . 8*1013 bacterial cells in the large intestine , ‘passive’ transmission without subsequent colonization in the gut would therefore account for a reduction in relative abundance by ~4*10−7 from saliva to feces ( Figure 1C ) .", "Thus , the observed overlap of microbial SNVs could not be explained by passive translocation , but was indeed caused by active colonization in the gut .", "Moreover , transmission scores across species and subjects were independent of technical covariates , such as the horizontal or vertical coverage of genome mappings ( Figure 2—figure supplement 2 ) .", "Average transmission scores across subjects did not correlate with prevalence in stool across all taxa ( ρSpearman =0 . 05 ) , whereas an association was evident when considering only transmitters ( ρ=0 . 67 ) .", "In saliva , prevalence was globally indicative of transmission scores ( ρ=0 . 6 ) , reinforcing the notion that core oral taxa tended to be transmitted .", "Given the limited microbial read depth of salivary metagenomes ( due to high fractions of human DNA ) , this result also indicates that our estimates of oral-fecal transmissibility were quite conservative , with potentially high rates of false negatives .", "It was recently shown that during early life , infants are colonized by maternal strains from both the oral cavity and gut ( Ferretti et al . , 2018 ) , and that strains from the latter can persist in the infant gut at least into childhood ( Korpela et al . , 2018 ) .", "Therefore , to determine whether the observed intra-individual overlap of selected strain populations was due to continuous oral-gut transmission or rare colonization events with subsequent independent expansion in each site , we focused on a subset of 46 individuals for whom longitudinal data was available ( with sampling intervals ranging from 1 week to >1 year; mean 79 days ) .", "We found that both oral and fecal strain populations were usually stable , even over extended periods of time ( Figure 2—figure supplement 3 ) , in line with earlier observations for each individual body site ( Lloyd-Price et al . , 2017; Schloissnig et al . , 2013 ) .", "Oral and fecal longitudinal SNV patterns were coupled for transmitted species ( see Materials and methods ) : oral SNVs observed at an initial time point were significantly enriched among fecal SNVs that were newly gained over time , but generally not vice versa ( Figure 2—figure supplement 4 ) .", "Moreover , oral-fecal transmission rates ( i . e . , the fraction of fecal strain turnover attributable to oral strains; see Materials and methods ) significantly exceeded background expectation for frequently transmitted taxa ( Figure 2C ) .", "These findings orthogonally support the oral-gut transmission hypothesis as they strongly suggest that transmission is in the direction of mouth to gut , and not vice versa; and they imply that oral-intestinal transmission is indeed a frequent and continuous process in which oral strain populations constantly re-colonize the gut .", "Oral-fecal transmissibility , as a trait , generally aligned with phylogenetic clade boundaries ( phylogenetic signal , λPagel=0 . 76 ) , although transmitting groups were found across bacterial phyla ( Figure 2DE , Figure 2—figure supplement 1 , Supplementary file 2 ) .", "Transmission scores were negatively correlated with genome size ( ρSpearman=-0 . 6 ) , indicating that transmitted species generally had smaller genomes than non-transmitted ones .", "Moreover , oxygen tolerant species ( aerobes and facultative anaerobes ) showed 7-fold higher scores than anaerobes on average ( ANOVA , p=10−16 ) .", "In contrast , no association was observed for sporulation and motility .", "To account for possible bias in the species reference and the phylogenetic signal of oral-fecal transmissibility , we confirmed that these signals were robust to phylogenetic regression ( Supplementary file 2 ) .", "Viewed across individuals , we found that seeding of the gut microbiome from the oral cavity was extensive , with high levels of variation ( Figure 3A ) .", "On average , potentially transmissible species ( i . e . , frequent and occasional transmitters ) accounted for 75% of classifiable microbes in saliva , ranging up to 99% in some subjects .", "However , not all of these were detectable in the matched fecal samples , and oral-fecal strain overlap was generally incomplete .", "We therefore quantified the fraction of realized transmission based on paired observations of species and intra-individual SNV overlap ( see Materials and methods ) .", "With these criteria , on average 35% of classifiable salivary microbes were transmitted strains that could be traced from mouth to gut within subjects .", "Similarly , on average 45% ( range 2–95% ) of classifiable fecal microbes were potential transmitters .", "These included common fecal species ( e . g . , Prevotella copri ) that were detectable in a subset of salivary samples and showed only occasional transmission .", "Nevertheless , on average only 2% of classifiable fecal microbes could be confidently ascribed to transmitted strains , ranging to >30% in some subjects .", "Between-subject variation in the relative abundance of transmitted oral and fecal microbes was found to be independent of subject sex , age and body mass index , although moderate differences were observed between study cohorts ( ANOVA , p=0 . 002; Figure 3B; Supplementary file 3 ) .", "Levels of transmitted microbial abundance in mouth and gut were found to correlate with each other ( ρSpearman=0 . 48 ) and with fecal species richness , but salivary transmitted abundance negatively correlated with oral species richness .", "This is in line with the observation that core oral species are transmissible , with higher richness implying the increased presence of non-transmitted taxa .", "Conversely , transmission would add species to a mostly non-transmissible core community in the gut .", "Although there was no overall association to community composition , levels of transmission correlated with oral or fecal abundances of individual genera ( Supplementary file 3 ) .", "To test whether specific oral and gut microbiome features were predictive of transmission , we categorized individuals based on total transmitted abundance in saliva and stool as ‘high’ or ‘low’ transmission individuals ( Materials and methods ) .", "We found that models based on salivary species abundances were mildly predictive of both oral ( AUC = 0 . 738 ) and fecal ( AUC = 0 . 642 ) transmission levels ( Supplementary file 4 , Figure 3—figure supplement 1 ) .", "Gut species models , in contrast , were very strong predictors of transmission in both mouth ( AUC = 0 . 951 ) and gut ( AUC = 0 . 971 ) .", "This signal was largely driven by the enrichment of transmitting species in stool ( Supplementary file 4 ) , but surprisingly robust to an elimination of all detected transmitters from the model ( AUC = 0 . 835 for the stool transmission group ) , again implying that the true extent of oral-intestinal transmission may indeed exceed our conservative estimates .", "Fusobacterium nucleatum subsp .", "animalis and nucleatum stood out among non-trivial gut markers enriched in high-transmission individuals , in line with existing hypotheses that Fusobacterium nucleatum subspecies may enable synergistic colonization of oral bacteria in the gut , in association with certain diseases ( see for example Flynn et al . , 2016 ) .", "In general , the fecal enrichment of specific oral microbes has repeatedly been associated with various diseases ( Zeller et al . , 2014; Zhang et al . , 2015 ) .", "However , due to insufficient taxonomic resolution , oral provenance has so far remained impossible to distinguish from an influx of closely related but distinct strains from the environment .", "We therefore defined a list of disease states with putative links to oral-fecal transmission and annotated known associations in the literature to all species in our dataset ( Figure 4A; Supplementary file 2 ) .", "Transmission scores were significantly increased for known opportunistic pathogens ( ANOVA , p=0 . 016 ) , causative agents of dental caries ( p=10−9 ) , and plaque-dwelling bacteria ( p=0 . 002 ) .", "Likewise , species associated with periodontitis showed increased evidence for transmission ( p=0 . 002 ) , though this signal was mostly due to mildly periodontic species , while core drivers , such as Tannerella forsythia , Treponema denticola and Porphyromonas gingivalis ( Socransky et al . , 1998 ) , showed little or no indication of oral-fecal transmission .", "Endocarditis-associated species showed significantly increased transmission scores upon phylogenetic regression ( p=0 . 007 ) , mostly driven by Haemophilus , Aggregatibacter and viridans Streptococci .", "This overall elevated transmissibility of taxa known to colonize ectopically in various habitats across the body ( i . e . , opportunistic pathogens ) , in particular via the bloodstream and associated with inflammation ( i . e . , endocarditis- or periodontitis-associated species ( Hajishengallis , 2015 ) ) , may provide first cues to possible mechanisms of oral-fecal transmission .", "Our dataset included metagenomes from case-control studies for rheumatoid arthritis ( RA , ( Zhang et al . , 2015 ) ) , colorectal cancer ( CRC , ( Zeller et al . , 2014 ) ) and type-1 diabetes ( T1D , ( Heintz-Buschart et al . , 2016 ) ) , totaling 299 individuals , including 172 with salivary and fecal samples .", "Treatment-naïve CRC patients , sampled before colonoscopy , showed increased transmission scores across all taxa ( average per-taxon Cohen’s d = 0 . 27; ANOVA p=10−23; Figure 4B ) , as well as for transmitted taxa only ( d = 0 . 23; p=10−10 ) .", "The effect was even more pronounced for species previously described ( Zeller et al . , 2014 ) to be enriched in the feces of CRC patients ( d = 0 . 33; p=10−4; Figure 4—figure supplement 1 ) , including Fusobacterium nucleatum spp .", ", Parvimonas micra and Peptostreptococcus stomatis .", "These findings are in line with a recent report that the oral and fecal microbiome are linked in the context of CRC ( Felmer et al . , 2018 ) , and support the hypothesis ( Flynn et al . , 2016 ) that CRC-associated species are sourced intra-individually from the oral cavity .", "Treatment-naïve RA patients displayed mildly elevated transmissibility across all taxa ( d = 0 . 03 , p=0 . 01 ) and transmissible taxa only ( d = 0 . 07 , p=0 . 08 ) .", "Interestingly , species that were orally depleted in RA patients showed markedly increased transmission scores ( d = 0 . 61; p=10−21 ) .", "In contrast , a trend towards decreased transmission in T1D patients was not statistically significant .", "Our results demonstrate that influx of oral strains from phylogenetically diverse microbial taxa into the gut microbiome is extensive in healthy individuals , with a high degree of variation between subjects .", "We showed that the vast majority of species prevalent in both the oral cavity and gut form connected strain populations along the gastrointestinal tract .", "Furthermore , by leveraging longitudinal data , we established that transmission from the mouth to the gut is a constant process .", "Approximately one in three classifiable salivary microbial cells colonize in the gut , accounting for at least 2% of the classifiable microbial abundance in feces .", "This puts oral-fecal transmission well in the range of other factors that determine human gut microbiome composition ( Schmidt et al . , 2018 ) .", "Moreover , we note that by using saliva and feces as metagenomic readouts , we may underestimate colonization by oral microbes of the mucosa , given that fecal microbiome composition is not fully representative of the gastrointestinal tract ( see for example Zmora et al . , 2018 ) .", "Therefore , and considering that our estimates of both the number of transmissible species and of the fraction of transmissible microbial abundance are conservative lower bounds due to strict thresholding and current detection limits of metagenomic sequencing , we posit that true levels of transmission are likely even higher , and that virtually all known oral species can translocate to the intestine at least under some circumstances .", "Finally , we found increased transmission linked to some diseases , and showed for colorectal cancer and rheumatoid arthritis that disease-associated strains of several species enriched in the intestine are indeed sourced endogenously , that is from the patient’s oral cavity , and not from the environment .", "These results may extend to other diseases beyond those tested here , calling for revised models of microbiome-disease associations that consider the gastrointestinal microbiome as a whole rather than a sum of parts , with important implications for disease prevention , diagnosis , and ( microbiome-modulating or -modulated ) therapy .", "While our findings are observational and do not reveal oral-intestinal transmission routes or mechanistic insights , they challenge current ecological and physiological models of the gastrointestinal tract that assume the oral cavity and large intestine to harbour mostly independent and segregated microbial communities .", "Instead , most strain populations appear to be continuous along the gastrointestinal tract , originating from the oral cavity , an underappreciated reservoir for the gut microbiome in health and disease ." ], [ "Publicly available raw sequence data was downloaded from the European Nucleotide Archive ( ENA ) for the FJ-CTR ( FijiCOMP , project accession PRJNA217052 ) ( Brito et al . , 2016 ) and CN-RA ( PRJEB6997 ) ( Zhang et al . , 2015 ) cohorts .", "Sample metadata was parsed from ENA and the respective study publications .", "For the LU-T1D ( PRJNA289586 ) ( Heintz-Buschart et al . , 2016 ) cohort , newly generated salivary and fecal metagenomes were added under the existing project accession .", "For the FR-CRC ( ERP005534 ) ( Zeller et al . , 2014 ) and DE-CTR ( ERP009422 ) ( Voigt et al . , 2015 ) cohorts , newly generated metagenomes were uploaded under project accession PRJEB28422 ( samples ERS2692266-ERS2692323 ) .", "German healthy controls ( DE-CTR ) .", "Salivary samples were collected at home before dental hygiene and breakfast in the early morning .", "Donors collected 2–3 ml of saliva and immediately mixed with 15 ml of RNAlater ( Sigma-Aldrich ) .", "Samples were transported to the laboratory on ice or dry ice and stored at −80C until further processing .", "French colorectal cancer cohort ( FR-CRC ) .", "Subject recruitment and cohort characteristics were described previously ( Zeller et al . , 2014 ) .", "Saliva samples were collected in 1 . 5 ml saline and stored at −80C until further processing .", "Luxembourg type-1 diabetes cohort ( LU-T1D ) .", "Donors collected 2–3 ml of saliva at home before dental hygiene and breakfast in the early morning .", "Samples were immediately frozen on dry ice , transported to the laboratory and stored at −80C until further processing .", "DE-CTR and FR-CRC .", "After thawing on ice , 1–2 ml of each sample were centrifuged directly ( FR-CRC ) or after dilution in RNALater ( DE-CTR ) .", "Cell pellets were washed 3x in sterile Dulbecco’s PBS ( PAA Laboratories ) and DNA was extracted using the using the GNOME DNA Isolation Kit ( MP Biomedicals ) .", "Briefly , cell pellets were lysed using a multi-step process of chemical cell lysis/denaturation , bead-beating and enzymatic digestion as described previously ( Zeller et al . , 2014 ) .", "DE-CTR samples were processed in duplicates , with one replicate being enriched for microbial DNA using the NEBNext Microbiome DNA Enrichment Kit ( NEB , Ipswich , USA ) following the manufacturer’s instructions .", "LU-T1D .", "After thawing on ice , two 500 µl aliquots of each sample were centrifuged .", "Cell pellets were frozen in liquid nitrogen and lysed by cryo-milling and chemical lysis in RLT buffer ( QIAGEN ) .", "Cell debris was passed through QiaShredder columns ( QIAGEN ) , before DNA was isolated using the QIAGEN AllPrep kit according to the manufacturer’s instructions , as described previously ( Heintz-Buschart et al . , 2016 ) .", "Libraries for salivary samples of the French and German cohorts were prepared using the NEBNext Ultra DNA Library Prep kit ( New England Biolabs , Ipswich ) using a dual barcoding system , and sequenced at 125 bp paired-end on an Illumina HiSeq 2000 .", "For the additional LU-T1D samples , libraries were likewise prepared using a dual barcoding system , and sequenced at 150 bp paired-end on Illumina HiSeq 4000 and Illumina NextSeq 500 machines .", "Raw reads were quality trimmed and filtered against the human genome issue 19 to exclude host sequences using MOCAT2 , as described previously ( Kultima et al . , 2016 ) .", "For taxonomic profiling , reads were mapped against a database of 10 universal marker genes for 1753 species-level genome clusters ( specI clusters , ( Mende et al . , 2013 ) ) , using NGless ( Coelho et al . , 2018 ) .", "A maximum likelihood-approximate phylogenetic tree ( with the JTT model , ( Jones et al . , 1992 ) ) for representative genomes of the same 1753 clusters was inferred based on protein sequences of 40 near-universal marker genes ( Mende et al . , 2013 ) using the ETE3 toolkit ( Huerta-Cepas et al . , 2016 ) , with default parameters for ClustalOmega ( Sievers et al . , 2011 ) and FastTree2 ( Price et al . , 2010 ) .", "Metagenomic reads were mapped at 97% sequence identity ( across at least 45nt ) against full cluster-representative genomes , using the Burrows-Wheeler Aligner ( Li and Durbin , 2009 ) , as implemented in NGless .", "Reads mapping to multiple genomes at ≥97% identity were discarded from the analysis .", "Average vertical coverage ( sequencing depth ) and horizontal coverage ( breadth ) per microbial genome in each sample were quantified using the qaCompute utility in metaSNV ( Costea et al . , 2017 ) .", "Two cohorts ( CN-RA ( Zhang et al . , 2015 ) and DE-CTR ( Voigt et al . , 2015 ) ) contained technical replicates for several salivary samples; these were pooled after the read mapping step .", "The dataset was filtered to include taxa satisfying the following criteria in ≥10% of samples ( see Figure 2—figure supplement 5 for details ) : horizontal coverage ( breadth ) of ≥0 . 05; average vertical coverage ( depth ) ≥0 . 25; specI cluster relative abundance of ≥10−6 .", "These criteria excluded taxa representing 0 . 8 ± 1 . 2% of gut and 1 . 2 ± 1 . 9% of oral total mapped abundance .", "For the remaining 310 taxa , general phenotypes ( Gram stain , sporulation , motility , oxygen requirement , among others ) were annotated using the PATRIC database ( accessed Dec 2015 ) ( Wattam et al . , 2017 ) , and missing values were amended manually .", "Host and disease association phenotypes ( including opportunistic pathogenicity and periodontitis association ) were annotated manually , based on published literature and the MicrobeWiki website ( https://microbewiki . kenyon . edu/index . php/MicrobeWiki , accessed June 2017 ) .", "Per taxon summary statistics and annotated metadata are available from Supplementary file 2 .", "Microbial Single Nucleotide Variants ( SNVs ) were called using metaSNV ( Costea et al . , 2017 ) .", "Each potential SNV required support by at least two non-reference sequencing reads ( relative to the specI cluster representative genomes ( Mende et al . , 2013 ) ) at a base call quality of Phred ≥ 20 .", "The resulting sets of raw SNVs per taxon were filtered differentially for the various downstream analyses , as detailed below .", "To distinguish intra-individual microbial transmission from random drift , we calculated a transmission score ( ST ) per subject and microbial taxon .", "In short , ST quantifies how much the similarity between oral and gut SNV profiles within an individual deviates from an inter-individual background .", "To calculate ST , we first filtered the set of informative SNVs ( all SNVs at a given genome position ) by applying the following criteria:", "( i ) observation ( read coverage ≥1 ) at focal position in ≥10 oral and ≥10 gut samples;", "( ii ) SNV observation in ≥1 oral and ≥1 gut sample .", "Next , we calculated the global background incidence of each allele across oral ( foral ) and gut ( fgut ) samples .", "From these , we calculated the background probabilities for each of the four possible cases in paired oral and gut observations: any given allele i could either be present in both samples ( p1 , 1 ) , absent in both samples ( p0 , 0 ) , or present in one but absent in the other sample ( p1 , 0 and p0 , 1 ) :p1 , 1", "( i ) =foral", "( i ) ∗fgut", "( i ) p0 , 1", "( i ) = ( 1−foral", "( i ) ) ∗ ( 1−fgut", "( i ) ) p1 , 0", "( i ) =foral", "( i ) ∗ ( 1−fgut", "( i ) ) p0 , 1", "( i ) = ( 1−foral", "( i ) ) ∗fgut", "( i ) For every permuted oral-gut pair of samples , we then calculated the raw summed log-likelihood of the observed SNV profile overlap ( Lobs ) across all alleles with shared coverage:Lobs= ( ∑i1 , 1log ( p1 , 1", "( i ) ) +∑j0 , 0log ( p0 , 0", "( j ) ) ) - ( ∑k1 , 0log ( p1 , 0", "( k ) ) +∑l0 , 1log ( p0 , 1", "( l ) ) ) In other words , Lobs quantifies how likely the observed average allele profile agreement between two samples is , given the respective background allele incidence frequencies .", "Similarly , we computed the log-likelihood of the least likely agreement case ( Lmin ) per allele:Lmin=∑imin ( log ( p1 , 1", "( i ) ) , log ( p0 , 0", "( i ) ) ) From these values , we calculated a raw probability score ( Praw ) for the observed allele agreement between a given pair of oral and gut samples:praw=Lobs/Lmin Praw scales the likelihood of the observed agreement by the likelihood of the theoretically most extreme cases of agreement across all observed alleles .", "In particular , the shared observation of very rare alleles ( very low foral and fgut ) has a strong impact on Praw , whereas the shared observation of very common variants is downweighted .", "We computed Praw for all pairwise permutations of oral and gut samples in the dataset with observations ( reads ) at ≥20 matching positions .", "We defined the transmission score ST ( t , s ) for taxon t in subject s as a standard Z score of the intra-individual ( within subject ) observation against an inter-individual ( between subjects ) background:ST= ( Praw ( s ) −μraw ) /σraw We tested for potential effects of the choice of background observations by calculating ST against", "( i ) a global background of all pairwise inter-individual oral-gut comparisons , across all cohorts;", "( ii ) a cohort-specific background per subject;", "( iii ) a global background , but taking only subject-specific comparisons into account ( the focal subject’s oral sample vs all gut samples , and vice versa ) ;", "( iv ) a within-cohort subject-specific background .", "Oral-gut comparisons for the same individual across different timepoints , within families ( information available for LU and CN cohorts ) and within village ( for the Fijian cohort ) were excluded from the background sets .", "Although smaller background sets ( iii and", "iv ) provided generally noisier scores , overall trends between these backgrounds were very consistent; in particular , cohort-specific vs global backgrounds did not impact trends in our findings ( data not shown ) .", "All results discussed in the main text therefore refer to scores against a cohort-specific background", "( ii ) .", "To quantify oral-gut transmission per individual , we defined a set of potentially transmissible species to include both frequently and occasionally transmitting species .", "Frequent transmitters encompassed a set of 74 species for which intra-individual transmission scores ST across subjects were significantly higher than inter-individual background ( Benjamini-Hochberg-adjusted one-sided Wilcoxon p<0 . 05 ) .", "Occasional transmitters did not satisfy this global criterion , but showed significant evidence for oral-fecal strain overlap in at least one individual ( Benjamini-Hochberg-adjusted Z test p<0 . 05 ) .", "To quantify the transmitted microbial abundance per individual , we adjusted the observed relative oral and fecal abundance of each given species by oral-fecal SNV overlap .", "In other words , the potentially transmissible abundance in the oral cavity was defined as the total abundance of potentially transmitting species , and the realized transmitted abundance was defined to include only species for which overlapping strain populations could be confidently traced within individuals .", "This included frequent transmitters that were observable ( above detection limits ) in matched oral-fecal sample pairs , and occasional transmitters satisfying the additional criterion that significant transmission scores were required in the focal individual for ( i . e . , an occasional transmitter such as Prevotella denticola would only be considered in individuals in which it showed significant transmission scores ) .", "For these species , relative oral and fecal abundances were adjusted for total strain population overlap , estimated as the Jaccard overlap of SNVs observed in the oral cavity and gut of the focal individual .", "Longitudinal data ( 2–3 timepoints , see Supplementary file 1 ) was available for 46 individuals from three cohorts ( Heintz-Buschart et al . , 2016; Voigt et al . , 2015; Zhang et al . , 2015 ) .", "To quantify site-specific temporal stability of strain populations , we contrasted within-subject SNV profile similarity over time to between-subject similarities .", "Moreover , we tested the longitudinal coupling of strain populations between a putative source site ( e . g . , oral cavity ) and sink site ( e . g . , gut ) .", "For this , we required shared observations ( read coverage ≥1 ) for at least 100 SNV positions across three samples ( see Figure 1 ) :", "( i ) source site at the initial time point ( t0 ) ;", "( ii ) sink site at t0;", "( iii ) sink site at a later time point t1 .", "We defined source SNVs as present in sample", "( i ) , and newly gained sink SNVs as present in sample", "( iii ) but not", "( ii ) , and performed Fisher’s exact tests ( followed by Benjamini-Hochberg correction ) to test for associations between these SNV sets .", "In other words , we tested for the association of strain populations present in the source site at t0 with strains newly gained in the sink site over time , by proxy of SNV profiles .", "We considered two sites to be longitudinally coupled in the source - > sink direction if the tested odds ratio was >1 at a ( corrected ) p≤0 . 05 .", "Significant odds ratios < 1 indicated unconnected sites in the tested directionality .", "Tests were performed independently for oral-to-gut ( oral as source , gut as sink ) and gut-to-oral coupling , per each taxon .", "Longitudinal data was also leveraged to estimate oral-fecal transmission rates , here defined as the fraction of fecal strain turnover attributable to the corresponding salivary sample .", "For each subject and taxon , the absolute fecal strain turnover was quantified as described above , as the difference in SNV profiles between fecal samples at t0 and t1 ( samples ii and iii in the previous section ) .", "Though sampling intervals ranged from 1 week to >1 year , they were relatively consistent within cohorts ( see Supplementary file 1 ) .", "Transmission rates were then quantified as the fraction of fecal alleles gained between t0 and t1 that were also observed in the paired oral sample at t0 .", "Arguably , this provides a conservative lower estimate: oral-fecal transmission could account for both newly gained fecal alleles and for the enhanced stability of existing alleles in the fecal strain population due to a constantly exerted dispersal pressure .", "However , since the latter effect cannot reliably be quantified from sparse longitudinal metagenomic data , the transmission rates reported in the main text only encompass the former ( newly gained alleles ) .", "To test whether transmission rates per taxon were statistically significant across subjects , we compared observed rates to two distinct randomized backgrounds: by shuffling fecal samples at t1 within cohorts , subject-specific longitudinal background sets on fecal strain turnover were generated; shuffling oral samples at t0 provided subject-specific coupled backgrounds .", "For each taxon and subject , we Z-transformed observed transmission rates against either of these subject-specific backgrounds; the resulting standard scores ( in unit standard deviations ) are reported in Figure 2C .", "Per-sample community richness was calculated from the average of 100 rarefactions to normalised marker gene-based abundances of 1000 .", "Between-sample community compositional similarities were computed as Bray-Curtis and TINA indices , as described previously ( Schmidt et al . , 2017 ) .", "Distance-based Redundancy Analyses to associate community composition to levels of oral-fecal transmission were performed using the R package vegan ( Oksanen et al . , 2015 ) .", "The association of transmission scores with taxa phenotypes ( oxygen requirement , sporulation , etc . ) and taxa disease annotations ( opportunistic pathogenicity , etc . ) were tested using ANOVA of a combined linear model ( ‘naïve’ ANOVA in Supplementary file 2 ) .", "To correct for potentially confounding phylogenetic signals of the tested variables , an ANOVA of a phylogenetically regressed model of the same formulation was performed using the R package caper ( Orme et al . , 2018 ) .", "Associations of total transmitted classifiable abundance in saliva and stool per subject with subject variables ( sex , BMI , age ) were tested using ANOVAs on linear models blocked by cohort .", "The association of transmission scores per subject with disease status was tested using ANOVAs per disease cohort , on linear models accounting for taxon baselines , as well as effects of subject sex , BMI and age .", "To test for links between microbiome composition and the amount of transmitted abundance in saliva and stool , we trained machine learning models to classify samples into ‘high’ and ‘low’ transmission groups .", "These groups were defined as the top and bottom quartiles of the fraction of transmitted abundance , independently for stool and saliva samples .", "For model training , relative abundances were log-transformed and standardized as z-scores .", "In a 10 times-repeated 10-fold cross-validation setting , L1-regularized ( LASSO ) logistic regression models ( Tibshirani , 1996 ) were trained on the training set and then evaluated on the test set within each fold .", "In a second step , all species defined as frequent transmitters ( see Quantification of Intra-Individual Microbial Transmission above ) were eliminated as features before preprocessing and training .", "All steps ( data preprocessing , model building , and model evaluation ) were performed using the SIAMCAT R package ( https://bioconductor . org/packages/SIAMCAT , version 1 . 1 . 0; see also Zeller et al . , 2014 ) .", "All statistical analyses were performed in R . Analysis code is available online ( see below ) .", "All generated raw sequence data has been uploaded to the European Nucleotide Archive under the project accessions PRJEB28422 ( French CRC , ( Zeller et al . , 2014 ) and German German healthy controls , ( Voigt et al . , 2015 ) ) and PRJNA289586 ( Luxembourg T1D , ( Heintz-Buschart et al . , 2016 ) ) .", "Sample metadata is available from Supplementary file 1 .", "Processed data ( taxonomic profiles , taxa annotations , etc . ) and full analysis code are available via a gitlab repository ( https://git . embl . de/tschmidt/oral-fecal-transmission-public; copy archived at https://github . com/elifesciences-publications/oral-fecal-transmission-public- ) ." ] ]

[ "The gastrointestinal tract is abundantly colonized by microbes , yet the translocation of oral species to the intestine is considered a rare aberrant event , and a hallmark of disease .", "By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries , we found that transmission to , and subsequent colonization of , the large intestine by oral microbes is common and extensive among healthy individuals .", "We found evidence for a vast majority of oral species to be transferable , with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and , more generally , for species described as opportunistic pathogens .", "This establishes the oral cavity as an endogenous reservoir for gut microbial strains , and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease ." ]

[ "Trillions of bacteria and other microbes live in the human body .", "The mouth and the gut in particular , are microbial hot spots at either end of the digestive tract .", "Every day , humans swallow around 1 . 5 liters of saliva , along with millions of oral microbes .", "Scientists believe that more than 99% of these microbes die as they pass through the acidic environment of the stomach and later the small intestine , which act as a barrier between the bacteria of the mouth and gut .", "Failure of this barrier can lead to overgrowth of oral microbes in the gut .", "This may contribute to diseases like bowel cancer , rheumatoid arthritis and inflammatory bowel diseases .", "But even in healthy people , low levels of microbes usually found in the mouth are often found in stool .", "It is unclear if these microbes cross the barrier or if they are similar microbes that originate in the gut .", "Now , Schmidt , Hayward et al . show that in healthy people at least one in three oral microbial cells pass through the digestive tract to settle the gut in healthy people .", "This challenges the notion of a mouth-gut barrier .", "In the experiments , the genetic material of all the microbes in the saliva and stool of several hundred people from three continents was analyzed .", "This allowed Schmidt , Hayward et al . to determine whether strains found in the gut originate from the mouth , or are closely related but specialized gut types of the same species .", "The results also showed that patients with bowel cancer and rheumatoid arthritis had more mouth-to-gut microbial transmission than their healthy counterparts .", "The experiments suggest that the mouth is a microbial reservoir that constantly replenishes the gut flora .", "Some of the gut-traveling oral bacteria trigger inflammation when they grow in other parts of the body like the lining of the heart .", "This , along with the discovery that patients with certain diseases have more oral bacteria in the gut , may suggest that the transmission of these microbes contributes to disease .", "The experiments also indicate that finding ways to influence oral bacteria might affect the ones in the gut .", "More studies are needed to understand how mouth microbes survive the trip to the gut and are able to thrive in this competitive environment , and what role they play in health and disease ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "stem cells and regenerative medicine" ]

[ [ "The postnatal development of the mammary gland comprises two distinct morphogenetic events: the growth and branching of epithelial ducts during puberty and the lobulo–alveolar development during pregnancy .", "The mammary epithelium is embedded in a fatty connective tissue and organized as a bilayer , with a basal layer of myoepithelial cells and a luminal epithelial layer .", "During lactation , the luminal cells produce milk , whereas myoepithelial cells serve for milk expulsion .", "The luminal cell layer is characterized by the expression of keratins 8/18/19 and comprises a subset of hormone-sensing cells that express estrogen , progesterone , and prolactin receptors ( ER , PR , and PrlR , respectively ) ( Brisken , 2013 ) .", "Basal myoepithelial cells express keratins 5 and 14 , P-cadherin , the transcription factor p63 , and smooth muscle-specific contractile proteins ( Moumen et al . , 2011 ) .", "This compartment specifically displays Slug/Snail2 ( Guo et al . , 2012; Nassour et al . , 2012 ) , a key transcription factor coordinating the epithelial–mesenchymal ( EMT ) transition program ( Thiery et al . , 2009; Nieto and Cano , 2012; Gonzalez and Medici , 2014 ) .", "The adult mammary basal compartment harbors multipotent stem cells able to fully regenerate the gland upon transplantation ( Shackleton et al . , 2006; Stingl et al . , 2006 ) .", "The luminal cell layer contains clonogenic cells , the luminal progenitors ( Asselin-Labat et al . , 2007; Sleeman et al . , 2007 ) .", "This population is heterogeneous , consisting of hormone receptor-positive and hormone receptor-negative clonogenic cells that are considered to be functionally distinct ductal and alveolar progenitors ( Beleut et al . , 2010; Regan et al . , 2012; Shehata et al . , 2012; Fu et al . , 2014 ) .", "Considerable interest has recently focused on luminal progenitors .", "According to lineage-tracing studies , these cells are able to drive the expansion of the luminal cell population in mammary ducts and alveoli during postnatal development ( Van Keymeulen et al . , 2011; Fu et al . , 2014; Rios et al . , 2014 ) .", "Moreover , luminal progenitors are thought to be at the origin of the triple-negative , basal-like , Brca1-associated breast cancers , indicating that they display phenotypic plasticity and an ability to upregulate basal markers ( Lim et al . , 2009; Molyneux et al . , 2010; Proia et al . , 2011 ) .", "Of note , ectopic expression of Snail2 endows luminal progenitors with basal cell features ( Guo et al . , 2012 ) , supporting an important role of EMT in the regulation of luminal progenitor plasticity .", "The Met tyrosine kinase receptor and its major ligand , HGF , are known to control numerous epithelial cell functions and trigger EMT ( Trusolino et al . , 2010; Gherardi et al . , 2012 ) .", "Met signaling has been implicated in mammary development and tumorigenesis .", "Although not investigated in detail yet , Met-deficient mammary glands of adult MMTV-Cre:Metf/f mice showed defects in branching morphogenesis ( Garner et al . , 2011 ) .", "Aberrant Met activation occurs in breast cancers , in particular in the triple-negative , basal-like subtype ( Gastaldi et al . , 2010; Ponzo and Park , 2010 ) .", "Expression of activated Met or over-expression of HGF in mouse mammary gland induce tumors , including those of the basal-like subtype ( Graveel et al . , 2009; Ponzo et al . , 2009; Holland et al . , 2013; Knight et al . , 2013 ) .", "Several in vitro studies performed with mammary organoids or immortalized cell lines have implicated HGF/Met signaling in the regulation of mammary epithelial cell growth , morphogenesis , and differentiation ( Soriano et al . , 1995; Yang et al . , 1995; Haslam et al . , 2008; Lee et al . , 2011 ) .", "One recent work indicated that luminal progenitors express Met and that , when forced to express HGF , display an enhanced in vivo regenerative potential , a property attributed to basal-type stem cells ( Gastaldi et al . , 2013 ) .", "However , in situ , mammary luminal cells do not express HGF , suggesting that Met signaling in mammary epithelial cells is activated in a paracrine manner .", "HGF-mediated paracrine and autocrine Met stimulation can trigger distinct biological responses in epithelial cells ( Mai et al . , 2014 ) .", "How Met-expressing luminal progenitors respond to the physiological paracrine action of HGF has not yet been investigated at the cellular and molecular levels .", "Met activation requires adhesion molecules as co-receptors .", "Two co-receptors for Met have been identified in hepatocytes: CD44v6 , the CD44 isoform containing the exon v6 and ICAM-1 ( CD54 ) , a member of the immunoglobulin superfamily of cell adhesion molecules ( Orian-Rousseau et al . , 2002; Olaku et al . , 2011 ) .", "In this study , we investigated the expression of ICAM-1 , CD44v6 , HGF , and Met in the mouse mammary epithelium .", "We found that ICAM-1 is a useful marker for obtaining highly enriched preparations of clonogenic luminal progenitors that express Met , and that HGF is produced by both stromal and myoepithelial cells .", "Using ICAM-1 as a surface marker , we further characterized Met-expressing luminal progenitors and analyzed their cellular and molecular responses to HGF .", "Our data suggest that HGF can act as a paracrine regulator of luminal progenitor function and fate during mammary development and tumorigenesis ." ], [ "We examined ICAM-1 expression at representative stages of mammary gland development , including puberty , maturity , early , and late gestation .", "Freshly isolated mammary cells , double-stained for CD24 and ICAM-1 , were analyzed by flow cytometry ( Figure 1A , Figure 1—figure supplement 1A ) .", "From puberty to late gestation , ICAM-1 was differentially expressed in the mammary epithelium .", "Staining for this marker identified three epithelial populations , negative ( ICAM1-neg ) or displaying low ( ICAM1-low ) or high ( ICAM1-hi ) levels of expression ( Figure 1A , Figure 1—figure supplement 1B ) .", "Gene expression analysis by qPCR showed that , at all stages of development , K5-expressing basal myoepithelial cells were located in the ICAM1-hi population , while K8-positive luminal cells were distributed in ICAM1-neg and ICAM1-low-cell subsets ( Figure 1B ) .", "Thus , ICAM-1 can be used to identify the basal cell compartment , and separate two populations of luminal cells , referred to hereafter as Lu-pos and Lu-neg cells . 10 . 7554/eLife . 06104 . 003Figure 1 . ICAM-1 expression discriminates basal and luminal cell compartments and defines a luminal population highly enriched in clonogenic progenitors .", "( A ) Flow cytometry dot plots showing CD24 and ICAM-1 expression in cells isolated from mouse mammary glands taken at representative stages of development: V-6w , 6-week-old virgin; V-12w , 12-week-old virgin; P-8d , 8-day pregnant mice; P-16d , 16-day pregnant mice .", "Within the CD24-positive epithelial cell population , ICAM-1 discriminated three distinct fractions , negative ( Neg ) , low-expressing ( Low ) , and high-expressing ( Hi ) cells .", "( B ) Levels of Icam1 and lineage-specific gene expression in ICAM1-neg , ICAM1-low , and ICAM1-hi epithelial cells as determined by q-PCR analysis .", "Cells were isolated from mammary glands at different stages of development , as shown in panel A . The values were normalized to Gapdh expression and represent mean values from at least two distinct cell preparations .", "Data obtained with adult virgin mice ( V-12w ) are from four independent groups of cell samples and presented as mean ±S . E . M . ( C ) Colony formation by ICAM1-neg ( Lu-neg ) and ICAM1-low ( Lu-pos ) mammary luminal cells .", "Left panel: hematoxylin and eosin ( H&E ) staining of clonal colonies after 8 days in culture .", "Right panel: percentages of clonogenic cells .", "Cells were isolated from mature virgin mice ( V ) and early pregnant females ( P-8d ) .", "The results are from two ( P-8d ) or three ( V ) independent cell preparations ( each of which with three separate wells ) , and presented as mean values ±S . E . M . ( D ) q-PCR analysis of relative gene expression levels in Lu-neg and Lu-pos cells isolated from mammary glands of mature virgin females .", "Mean ratios ( ±S . E . M ) of values normalized to Gapdh expression are shown .", "Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in left and right panels , respectively .", "Results are from three independent cell preparations . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 00310 . 7554/eLife . 06104 . 004Figure 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 00410 . 7554/eLife . 06104 . 005Figure 1—figure supplement 1 . Gating procedure for flow cytometry analysis .", "( A ) Sequential steps of gating procedure for flow cytometry analysis and sort of mammary epithelial cells stained with anti-CD31 , anti-CD45 , anti-CD24 and anti-ICAM-1 antibodies .", "From left to right: exclusion of debris by gating cells on forward ( FSC-A ) and side scatter ( SSC-A ) parameters , exclusion of doublets by gating cells on SSC-A and SSC-W parameters , exclusion of CD31/CD45-expressing cells , luminal and basal cell separation using CD24 and ICAM-1 expression .", "( B ) Purity control of the sorted ICAM1-neg , ICAM1-low , and ICAM1-hi CD24-positive epithelial cell populations .", "Cell purity was ≥97% .", "( C ) Percentages of ICAM1-neg , ICAM1-low , and ICAM1-hi mammary epithelial cells at puberty , maturity , early- , and late pregnancy .", "Data are expressed as the mean ( ±S . E . M ) of three flow cytometry analyses . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 00510 . 7554/eLife . 06104 . 006Figure 1—figure supplement 2 . Isolation of mammary luminal progenitors from adult virgin C57Bl/6J and Blg-Cre; R26 females using ICAM-1 . ( A ) Isolation of clonogenic luminal progenitors from adult virgin C57Bl/6J mice using ICAM-1 .", "Left panel: flow cytometry analysis of ICAM-1 and CD24 expression in freshly isolated mammary epithelial cells .", "Middle panel: H&E staining of clonal colonies obtained from Lu-neg and Lu-pos luminal cells after 8 days in culture .", "Right panel: percentages of clonogenic cells .", "The results are from triplicates obtained with one cell preparation and presented as mean values ±S . E . M . ( B ) Flow cytometry analysis of ICAM-1 and CD24 expression in mammary epithelial cells freshly isolated from adult virgin Blg-Cre; R26 females .", "( C ) Sections through Blg-Cre; R26 mouse mammary gland Xgal-stained in whole mount .", "Blue and white arrows indicate LacZ-positive luminal cells and LacZ-negative basal cells , respectively .", "Bar , 15 μm .", "( D ) Icam-1 and Cre expression in Lu-neg , Lu-pos , and basal cells , as determined by q-PCR .", "The values normalized to Gapdh expression are from one representative experiment performed with 3 pooled adult virgin Blg-Cre; R26 mice .", "( E ) Clonogenic potential Lu-neg and Lu-pos luminal cells isolated from adult virgin Blg-Cre; R26 mice using ICAM-1 .", "Left panel: Xgal staining of colonies counterstained with fast red .", "Right panel: percentages of clonogenic cells .", "The results are from triplicates obtained with one cell preparation and presented as mean values ±S . E . M . ( F ) q-PCR analysis of gene expression levels in Lu-neg and Lu-pos cells isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice .", "Ratios of values normalized to Gapdh expression are shown .", "Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in left and right panels , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 006 Throughout development , the basal myoepithelial cell population constitutively expressed ICAM-1 at high level ( Figure 1A ) .", "By contrast , the luminal cells displayed temporal changes in ICAM-1 expression ( Figure 1A , Figure 1—figure supplement 1C ) .", "In virgin mice , most of the luminal cells were negative for ICAM-1 at puberty , whereas 25–30% of the luminal cells expressed ICAM-1 in the mature gland of 11 to 14-week-old cycling females .", "Notably , the Lu-pos cell subset was amplified early in gestation ( P-8d ) , during the expansion of the alveolar buds .", "Later , at P-16d , when the alveoli were well developed , almost the entire luminal compartment was positive for ICAM-1 .", "Thus , in the luminal cell population , ICAM-1 expression is developmentally regulated and correlates temporally with changes in hormone signaling .", "We investigated the functional properties of the luminal cell populations separated on the basis of ICAM-1 expression , by plating purified Lu-pos and Lu-neg cells at low density and assessing their ability to form colonies in two-dimensional cultures .", "In adult virgin mice , almost all the colony-forming activity was associated with the Lu-pos cell subset , demonstrating that ICAM-1 expression delineates a luminal population highly enriched in clonogenic progenitors ( Figure 1C ) .", "Notably , ICAM-1 efficiently marked mammary luminal progenitors from adult virgin Balb/c ( Figure 1C ) , C57Bl/6J ( Figure 1—figure supplement 2A ) and Blg-Cre; R26 mice ( Figure 1—figure supplement 2B–E ) .", "As expected from previous findings ( Selbert et al . , 1998; Molyneux et al . , 2010 ) , LacZ activity , as assessed by X-gal staining , was undetectable in basal myoepithelial cells and present in a subset of luminal cells in the mammary gland of adult virgin Blg-Cre; R26 females ( Figure 1—figure supplement 2C ) .", "An analysis of Cre expression by qPCR indicated that Blg ( β-Lactoglobulin ) drove recombination primarily in the luminal progenitor population identified by ICAM-1 ( Figure 1—figure supplement 2D ) .", "Accordingly , 98% of the colonies formed by ICAM1-expressing Blg-Cre; R26 luminal cells were LacZ-positive ( Figure 1—figure supplement 2E ) .", "We next compared the molecular characteristics of the luminal cell populations defined by ICAM-1 by analyzing the expression of a panel of genes by qPCR ( Figure 1D , Figure 1—figure supplement 2F ) .", "The non-clonogenic Lu-neg population had high level of transcripts for the hormone receptors , ERα , PR , and PrlR , and for genes encoding secreted hormonal mediators implicated in the control of mammary development such as amphiregulin ( encoded by Areg ) , RankL ( encoded by Tnfsf11 ) and Wnt4 .", "These genes , along with Ly6a ( encoding Sca-1 ) , are characteristic of mature mammary luminal cells ( Mulac-Jericevic et al . , 2003; Kendrick et al . , 2008; Cai et al . , 2014 ) .", "The clonogenic Lu-pos population expressed Mki67 , a marker of cell proliferation , more strongly than the Lu-neg fraction .", "It exhibited high levels of expression for genes encoding essential regulators of mammary development including Elf5 , the Notch effector , Hey1 , RANK ( encoded by Tnfrsf11a ) , and the local mediator R-spondin1 ( encoded by Rspo1 ) ( Fata et al . , 2000; Bouras et al . , 2008; Oakes et al . , 2008; Cai et al . , 2014 ) .", "Lu-pos cells also over-expressed Kit and Itga2 , two markers previously used to obtain mammary cell populations enriched in luminal progenitors ( Regan et al . , 2012; Shehata et al . , 2012 ) .", "The Lu-neg and Lu-pos cell populations isolated from the mammary glands of females in early pregnancy had functional properties and gene expression patterns similar to those purified from adult virgin glands ( Figure 1D , Figure 2—figure supplement 1A ) .", "ICAM-1 thus appeared to be a robust surface marker for the enrichment in mammary luminal progenitors from adult virgin and early pregnant females .", "We then used q-PCR to examine the expression of Met and its major cytoplasmic effector , Gab1 , in the mammary cell populations separated by ICAM-1 .", "In adult virgin mice , Met transcript levels were five and ten times higher in the Lu-pos population than in the Lu-neg and myoepithelial cells , respectively; Gab1 , like Met , was strongly expressed only in the Lu-pos fraction ( Figure 2A ) .", "Similarly , the clonogenic Lu-pos population isolated from the mammary glands of early pregnant females was greatly enriched in Met-expressing cells ( Figure 3—figure supplement 1B ) .", "Thus , in adult virgin females and during early pregnancy , the target cells for Met signaling mostly belonged to the luminal progenitor-enriched population defined by ICAM-1 expression . 10 . 7554/eLife . 06104 . 007Figure 2 . ICAM-1 identifies Met-expressing clonogenic luminal progenitors .", "( A ) Icam-1 , Met , Gab1 , and Hgf expression in Lu-neg , Lu-pos , and basal/myoepithelial cells isolated from mammary glands of mature virgin mice by flow cytometry .", "The q-PCR values were normalized to Gapdh expression and represent mean values ±S . E . M from three independent preparations .", "( B ) Levels of Hgf expression in stromal and basal/myoepithelial cells .", "Left panel: flow cytometry dot plot showing mammary basal ( Ba ) and stromal ( S ) cell compartments isolated from a 14-week-old virgin mice .", "Right panel: q-PCR analysis of Hgf expression in basal and stromal cells .", "The values were normalized to Gapdh expression and represent mean values ±S . E . M from three independent preparations .", "( C ) Characteristics of HGF-treated and untreated spheres derived from purified Lu-pos cells cultured in the absence or presence of HGF for 10 days .", "Left panels: Representative phase contrast images of HGF-treated and untreated spheres ( bar , 400 μm ) and H&E staining of sections through HGF-treated and untreated spheres ( bar , 150 μm ) .", "Right panel: Sphere size distribution ( in arbitrary units ) in HGF-treated and untreated cultures .", "At least 250 spheres were analyzed per conditions .", "( D ) Average percentages ( ±S . E . M ) of clonogenic cells in non-stimulated and HGF-stimulated cultures of Lu-pos cells .", "Data from two independent cell preparations ( each of which with three separate wells analyzed at day 10 ) are shown .", "*p < 0 . 0004 .", "( E ) Caspase-3 and caspase-7 activity in non-stimulated and HGF-stimulated Lu-pos cells after 1 and 2 days in culture as measured by a luminescent assay .", "Data are the mean ( ±S . E . M ) of three measurements from separate wells .", "*p < 0 . 003 at day 2 , not significant ( n . s ) at day 1 .", "A . U . , arbitrary unit .", "( F ) BrdU incorporation in Lu-pos cells grown in the absence or presence of HGF for 1 , 2 , and 4 days .", "Left panel: percentage of BrdU-positive cells .", "Mean values from two distinct cell preparations are shown .", "Right panel: representative images of cells cytocentrifuged and immunostained with anti-BrdU antibody after 2 days in culture .", "Nuclei were counterstained with DAPI .", "Arrowheads indicate proliferating BrdU-positive cells .", "Bar , 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 00710 . 7554/eLife . 06104 . 008Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 00810 . 7554/eLife . 06104 . 009Figure 2—figure supplement 1 . Molecular and phenotypic characteristics of luminal progenitors isolated from virgin and early pregnant females .", "( A ) q-PCR analysis of gene expression levels in mammary Lu-neg and Lu-pos cells isolated from early pregnant females ( P-8d ) .", "Mean ratios of values normalized to Gapdh expression are shown .", "Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in upper and lower panels , respectively .", "Data are from two independent cell preparations .", "( B ) Icam-1 , Met , and Hgf expression in Lu-neg , Lu-pos , and basal/myoepithelial cells , as determined by q-PCR .", "Cells were isolated from mammary glands of 8-day-pregnant mice .", "The q-PCR values were normalized to Gapdh expression and represent mean from two independent preparations .", "( C ) Immunodetection of CD44v6 and K8 in cytocentrifuged Lu-neg and Lu-pos cells freshly isolated from mammary glands of mature virgin mice .", "In upper panels , nuclei were stained with DAPI .", "Bar , 15 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 009 As expected ( Niranjan et al . , 1995; Yang et al . , 1995 ) , HGF , the major Met ligand , was strongly expressed in the CD24-negative Pdgfr-expressing stromal cell population ( Figure 2B ) .", "In addition , HGF was expressed at high level in the mammary basal cell population of mature virgin and early pregnant mice ( Figure 2A , B , Figure 2—figure supplement 1B ) , suggesting paracrine interactions between basal myoepithelial cells and luminal progenitors .", "As both ICAM-1 and CD44v6 may serve as co-receptors for Met ( Olaku et al . , 2011 ) , we analyzed the expression of CD44v6 in freshly isolated Lu-pos and Lu-neg cells .", "Most cells of both populations were stained with the anti-CD44v6 antibody ( Figure 2—figure supplement 1C ) .", "Thus , CD44v6 is broadly expressed in the luminal compartment and cannot , therefore , be used for the enrichment in Met-expressing progenitors .", "We used ICAM-1 to isolate Met-expressing luminal progenitors and investigated their response to the paracrine action of HGF .", "Purified Lu-pos cells were cultured in suspension in the presence of 2% Matrigel as previously described ( Spike et al . , 2012; Chiche et al . , 2013 ) .", "We found that HGF-treated Lu-pos cells formed enlarged spheres after 10 days in culture ( Figure 2C ) .", "These spheres had multiple lumens , with an overall lumen space larger than that of the untreated spheres ( Figure 2C ) .", "Quantitative analysis revealed that HGF-treated cultures of purified Lu-pos cells contained twice as many spheres as non-stimulated cultures ( Figure 2D ) , suggesting a role for HGF in favoring cell survival and/or proliferation .", "Measurements of caspase activity showed that HGF-treated cells had lower levels of activated caspase-3 and caspase-7 than untreated cells after 2 days in culture ( Figure 2E ) .", "BrdU-incorporation assays performed after 1 , 2 , and 4 days in culture indicated that HGF treatment increased cell proliferation , particularly on day 2 ( Figure 2F ) .", "Thus , in 3D cultures , HGF stimulated the clonogenic activity of ICAM1-expressing luminal progenitors , by promoting cell survival and proliferation early in culture .", "We then investigated the long-term effects of HGF on purified luminal progenitors .", "Using q-PCR , we analyzed proliferation and lineage-specific gene expression in Lu-pos cells cultured in the presence or absence of HGF for 9–10 days ( Figure 3A ) .", "Mki67 was down-regulated in HGF-stimulated cultures whereas Cdkn1a , which encodes p21 , a negative regulator of the cell cycle ( Abbas and Dutta , 2009 ) , was upregulated by a factor of 2 . 5 .", "The basal-specific keratin , Krt5 , undetectable in untreated spheres , was clearly expressed in HGF-stimulated spheres , whereas no significant decrease was observed in expression of the luminal-specific keratin , Krt18 .", "Thus , in the long-term , HGF/Met signaling biased luminal progenitors toward a basal cell fate while attenuating cell cycle progression . 10 . 7554/eLife . 06104 . 010Figure 3 . HGF promotes acquisition of basal-specific keratins in luminal progenitors identified by ICAM-1 . ( A ) Analysis of Mki67 , Cdkn1 , Krt18 and Krt5 expression in spheres derived from untreated and HGF-treated Lu-pos cells , as determined by q-PCR .", "Lu-pos cells were isolated from mammary glands of mature virgin mice .", "The values , normalized to Gapdh expression , represent mean values ±S . E . M . from at least three independent sphere preparations harvested after 10 days in culture .", "*p < 0 . 05 , p < 0 . 001 , p < 0 . 007 for Mki67 , Cdkn1 , and Krt5 , respectively .", "n . s , not significant ( Krt18 ) .", "( B ) Double immunofluorescence labeling of non-stimulated and HGF-stimulated Lu-pos cells grown for 10 days .", "Left panel: K8 and K5 staining of spheres sections .", "Bar , 60 μm .", "Middle panel: K8 and K5 staining of cytocentrifuged cells derived from HGF-stimulated cultures .", "Arrowheads indicate a group of double-positive K5/K8 cells .", "Bar , 45 μm .", "Right panel: Average percentages ( ±S . E . M ) of K5-expressing cells in 10- to 12-day-old spheres derived from untreated and HGF-treated Lu-pos cells .", "1000 cells at least were counted per sample .", "Percentages of K5-positive cells in untreated and HGF-treated cultures were 0 . 8% ± 0 . 4 and 8 . 4% ± 2 . 2 , respectively .", "Data are from four independent cell preparations .", "*p < 0 . 002 .", "( C ) BrdU incorporation in 10-day-old HGF-treated spheres .", "Sections were stained with anti-BrdU antibody combined either to anti-K8 ( left panels ) or anti-K5 ( right panels ) antibodies .", "Pictures at low magnification and enlarged views of defined areas are shown .", "Bar , 45 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 01010 . 7554/eLife . 06104 . 011Figure 3—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 01110 . 7554/eLife . 06104 . 012Figure 3—figure supplement 1 . α-SMA expression in untreated and HGF-treated spheres . Absence of α-SMA expression in untreated and HGF-treated spheres derived from Lu-pos cells grown for 10 days .", "Left panel: Acta2 expression determined by q-PCR .", "The values normalized to Gapdh expression represent mean ±S . E . M . from three independent sphere preparations .", "Right panel: double immunofluorescence labeling of a HGF-treated sphere section with anti-K5 and anti-α-SMA antibodies .", "Nuclei were stained with DAPI .", "Bar , 45 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 012 Consistent with the q-PCR data , immunofluorescence labeling showed that the percentages of K5-positive cells in spheres stimulated with HGF for 9–10 days were six times higher that that of untreated cultures ( Figure 3B ) .", "Numerous K5-expressing cells stained positive for K8 ( Figure 3B ) , and none contained the myoepithelial-specific protein α-SMA ( Figure 3—figure supplement 1 ) .", "Interestingly , BrdU incorporation assays showed that , unlike K5-negative luminal cells , K5-expressing cells did not cycle following HGF stimulation ( Figure 3C ) .", "Thus , paracrine Met activation controls the survival and proliferation of ICAM1-expressing luminal progenitors and favors the acquisition of basal-specific keratin .", "To prove that K5-expressing cells in HGF-treated spheres originated from luminal progenitors , we isolated Lu-pos cells from Blg-Cre; R26 mouse mammary glands and stimulated them with HGF for 9–10 days .", "As in Balb/c mice , Met was strongly expressed in the Blg-Cre; R26 luminal progenitor population purified with ICAM-1 ( Figure 4—figure supplement 1A ) .", "Notably , in HGF-stimulated cultures , 85% of the K5-positive cells expressed LacZ and were therefore of luminal origin ( Figure 4A , Figure 4—figure supplement 1B ) .", "Control HGF-treated basal cells did not stain positive for X-gal . 10 . 7554/eLife . 06104 . 013Figure 4 . HGF triggers activation of EMT program in ICAM1-expressing luminal progenitors .", "( A ) K5 and β-galactosidase expression in cells isolated from HGF-treated spheres .", "Lu-pos ( upper panels ) and basal cells ( lower panels ) were purified from mammary glands of mature virgin Blg-Cre; R26 mice and stimulated with HGF for 13 days .", "Left panels: X-gal staining with fast red counterstaining .", "Bar , 15 μm .", "Middle and right panels: correlated images of K5 immunostaining and X-gal staining .", "Arrows indicate LacZ-positive cells expressing K5 .", "Quantitative data are shown in Figure 4—figure supplement 1B .", "Bar , 10 μm .", "( B ) Comparative expression levels of basal-specific , epithelial–mesenchymal transition ( EMT ) -associated and luminal-specific genes in spheres derived from untreated and HGF-treated Lu-pos cells as determined by q-PCR .", "Lu-pos cells isolated from mammary glands of mature virgin Balb/c mice were cultured in the absence or presence of HGF for 10 days .", "Results are expressed as Log2 ratios of values normalized to Gapdh and represent mean values ±S . E . M . from at least three independent sphere preparations .", "The comparator values were those obtained with untreated spheres .", "( C-D )", "Immunodetection of p63 ( C ) and Snail2 ( D ) in sections through untreated and HGF-stimulated spheres .", "Nuclei were counterstained with DAPI .", "Bar , 25 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 01310 . 7554/eLife . 06104 . 014Figure 4—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 01410 . 7554/eLife . 06104 . 015Figure 4—figure supplement 1 . Met expression in luminal progenitors isolated from Blg-Cre; R26 mice .", "( A ) Icam-1 , Met , and Hgf expression in Lu-neg , Lu-pos , and basal/myoepithelial cells , as determined by q-PCR .", "Cells were isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice .", "The q-PCR values were normalized to Gapdh expression .", "( B ) Percentages of K5-positive cells ( upper panel ) and K5-positive cells expressing LacZ ( lower panel ) in HGF-treated spheres derived from Lu-pos and basal cells isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice .", "Data are from one cell preparation .", "At least 500 cells were counted per sample . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 015 We investigated the molecular events induced by the paracrine action of HGF in luminal progenitors , by analyzing the expression of basal-specific and luminal-specific regulatory genes in untreated and HGF-treated spheres derived from purified Lu-pos cells ( Figure 4B ) .", "Upon stimulation with HGF , the expression of essential regulators of the luminal cell fate such as Elf5 , Hey1 , and Gata3 was repressed , whereas that of several basal-specific genes increased markedly .", "These genes included Cdh3 ( encoding P-cadherin ) , Trp63 , and the EMT inducer Snai2 .", "Other genes associated with EMT program—Snai1 , Sox9 , and Cdh2 ( encoding N-cadherin ) —were also upregulated .", "In agreement with gene expression data , immunodetection studies revealed that HGF-treated spheres contained numerous p63- and Snail2-positive cells , whereas untreated cells were negative for these basal-specific markers ( Figure 4C , D ) .", "Snai2 and Snai1 can directly repress the transcription of Cdh1 ( encoding E-cadherin ) and claudins ( reviewed in Thiery et al . , 2009; Gonzalez and Medici , 2014 ) .", "Noticeably , Cdh1 expression was not significantly reduced upon HGF treatment , whereas claudin-1 and claudin-3 transcript levels decreased strongly ( Figure 4B ) .", "Consistent with the q-PCR data , immunofluorescence staining revealed E-cadherin in the vast majority of cell–cell contacts in untreated and HGF-treated spheres ( Figure 5A ) .", "Notably , numerous Snail2-positive cells present in the HGF-stimulated spheres displayed E-cadherin at their surface ( Figure 5B ) .", "Claudin-1 distribution was more heterogeneous than that of E-cadherin , but it was clearly altered following HGF treatment .", "Untreated spheres contained a majority of cells with claudin-1 at their junctions , whereas HGF-treated spheres displayed large cell areas lacking claudin-1 expression ( Figure 5C ) . 10 . 7554/eLife . 06104 . 016Figure 5 . HGF treatment perturbs cell–cell adhesion in ICAM1-expressing luminal progenitors .", "( A ) Immunofluorescence labeling of sphere sections with anti-E-cadherin antibody .", "Low- and high-magnification views of untreated ( left panels ) and HGF-treated ( right panels ) spheres .", "Nuclei were stained with DAPI .", "Bars , 60 μm and 30 μm .", "( B ) Double immunodetection of Snail2 and E-cadherin in a HGF-treated sphere .", "Arrowheads indicate Snail2-positive cells expressing E-cadherin at their surface .", "Nuclei were stained with DAPI .", "Bar , 20 μm .", "( C ) Double immunofluorescence labeling of sphere sections with anti-claudin-1 and anti-E-cadherin antibodies .", "Nuclei were stained with DAPI .", "Bar , 75 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 016 Thus , the persistent stimulation of Met signaling by exogenous HGF in luminal progenitors repressed luminal-regulatory gene expression , upregulated basal-specific markers , and triggered an EMT program , including Snai2 and Snai1 expression and decrease in claudin levels .", "To examine whether the HGF/Met signaling effects on luminal progenitors persist after HGF withdrawal , primary HGF-treated spheres obtained from purified Lu-pos cells were dissociated and then cultured either with or without HGF for 10 additional days .", "Both clonogenic activity and level of expression of the basal-specific keratin Krt5 were much lower in secondary mammospheres deprived of HGF ( Figure 6A , B ) .", "Consistently , immunofluorescence stainings revealed that , like untreated control cultures , secondary mammospheres deprived of HGF contained less than 1% K5-positive cells ( Figure 6C , D ) .", "HGF-treated secondary mammospheres contained twice more K5-positive cells than HGF-treated primary spheres .", "Many K5-positive cells co-expressed K8 , as in primary spheres , ( Figure 6C ) .", "Experiments performed with mammary luminal progenitors purified from BlgCre; R26 mice confirmed that , in HGF-treated secondary mammospheres , K5-expressing cells were derived from LacZ-positive luminal cells ( Figure 6E ) .", "Gene expression analysis showed that Trp63 , Snai2 , and Cdkn1a were upregulated whereas Hey1 , Elf5 , and Mki67 were down-regulated only in HGF-treated secondary mammospheres ( Figure 6F ) . 10 . 7554/eLife . 06104 . 017Figure 6 . Persistent stimulation with HGF is required to sustain effects on luminal progenitors .", "( A ) Microphotographs of primary ( MS1 ) and secondary ( MS2 ) mammospheres derived from purified Lu-pos cells .", "Primary spheres obtained after 11 days of culture in the presence of HGF were dissociated and 5000 cells were replated and grown either in the presence or absence of HGF for an additional period of 10 days .", "( B ) Krt18 and Krt5 expression levels in MS1 and MS2 cultures derived from Lu-pos cells as determined by q-PCR .", "HGF-treated MS2 cultures ( + ) grown either in the presence or absence of HGF are labeled +/+ and +/− , respectively .", "Untreated primary ( − ) and secondary ( −/− ) spheres of Lu-pos cells served as controls .", "The values were normalized to Gapdh expression .", "Data are presented as mean values ±S . E . M . of three independent experiments .", "( C ) K5-expressing cells in MS1 and MS2 cultures derived from Lu-pos cells .", "Immunodetection of K5 and K8 in cytocentrifuged cells isolated from MS1 and MS2 .", "Bar , 30 μm .", "( D ) Percentages of K5-expressing cells in MS1 and MS2 cultures according to their treatment with HGF .", "2000 cells were counted per analyzed cytospots .", "Data from two independent experiments ( three separate cytospots ) are presented as mean values ±S . E . M . ( E ) Correlated images of K5 immunostaining and X-gal staining in cells isolated from HGF-treated secondary spheres .", "Lu-pos cells were purified from mammary glands of mature virgin Blg-Cre; R26 mice and continuously stimulated with HGF .", "Arrows indicate two groups of LacZ-positive cells expressing K5 .", "Bar , 15 μm .", "( F ) Modulation of Trp63 , Snai2 , Hey1 , Elf5 , Cdkn1a , and Mki67 expression levels in MS2 cultures derived from Lu-pos cells as determined by q-PCR .", "The q-PCR values were normalized to Gapdh expression .", "Results are shown as mean values ±S . E . M . of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 01710 . 7554/eLife . 06104 . 018Figure 6—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 018 Thus , the continuous activation of HGF/Met signaling is required to sustain the response of luminal progenitors in vitro and leads to the accumulation of cells with basal characteristics .", "To compare the stem cell activity of untreated and HGF-treated luminal progenitors , we transplanted either single-cell suspensions or intact spheres into cleared mammary fat pads of recipient mice ( Figure 7 ) .", "Untreated cells isolated from primary spheres failed to repopulate the fat pad or gave rise to very limited outgrowths .", "In contrast , HGF-treated cultures formed ductal-type outgrowths in seven of the eight transplanted fat pads ( Figure 7A , Supplementary file 1 ) .", "Serial passages of luminal progenitor-derived mammospheres stimulated by HGF led to an important enrichment in K5-positive cells ( Figure 6C ) ; we therefore compared the regenerative potential of secondary spheres from untreated and HGF-stimulated cultures .", "Strikingly , these transplantation assays showed that only HGF-treated spheres were able to form outgrowths ( Figure 7B ) .", "6-week-old outgrowths were composed of well-organized ducts and growing buds comprising K8-positive luminal cells and basally located α-SMA-expressing myoepithelial cells ( Figure 7C , D ) . 10 . 7554/eLife . 06104 . 019Figure 7 . HGF-treated luminal progenitors display regenerative potential .", "( A ) Regenerative properties of cells isolated from untreated and HGF-treated cultures of luminal progenitors .", "Primary spheres were dissociated and single cell suspensions were transplanted at 2000 , 1000 , and 500 cells/fat pad .", "Left panels: Representative images of carmine-stained outgrowths obtained 12 weeks after transplantation of 2000 cells and take rates .", "Bar , 0 . 2 mm .", "Right panels: Diagrams showing take rate and fat pad filling .", "( B ) Representative images of carmine-stained outgrowths obtained 5 weeks after transplantation of intact secondary spheres harvested from untreated and HGF-treated cultures of luminal progenitors .", "Take rates are indicated .", "Bar , 2 mm . ( C ) Enlarged view of the outgrowth shown in B . Bar , 0 . 8 mm . ( D ) Double immunofluorescence stainings of sections through the outgrowth shown in C . Immunodetection of K5/K8 in a duct ( left panel ) and α-SMA/TK in a duct and a growing bud ( middle and right panels , respectively ) .", "Bar , 45 μm .", "The following supplementary file is available for Figure 7: Supplementary file 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 019 These data demonstrate that paracrine Met activation induced multipotent stem cell properties in luminal progenitors .", "Differential Sca-1 expression in the luminal compartment has been reported to delineate populations enriched in hormone receptor-positive and hormone receptor-negative cells , both containing clonogenic progenitors ( Sleeman et al . , 2007; Regan et al . , 2012 ) .", "We characterized Met-expressing luminal progenitors further , by analyzing ICAM-1 and Sca-1 distribution in the luminal compartment at different developmental stages by flow cytometry ( Figure 8A , Figure 8—figure supplement 1A ) . 10 . 7554/eLife . 06104 . 020Figure 8 . Met-expressing clonogenic cells are distributed in hormone-receptor-positive and negative luminal cell populations .", "( A ) Representative flow cytometry dot-plots showing Sca-1 and ICAM-1 expression in the mammary luminal cell population isolated from mice at different stages of development: V-6w , 6-week-old virgin; V-13w , 13-week-old virgin; P-8d , 8-day pregnant; P-16d , 16-day pregnant mice .", "Combined with Sca-1 , ICAM-1 discriminated four cell populations referred to as Lu1 , Lu2 , Lu3 , and Lu4 .", "( B ) Colony formation by Lu1 , Lu2 , Lu3 , and Lu4 cell subsets isolated from mammary glands of mature virgin mice .", "Upper panel: H&E staining of colonies after 8 days in culture .", "Lower panel: percentages of clonogenic cells .", "The results from two independent cell samples ( each of which with three separate wells ) are presented as mean values ±S . E . M . ( C ) Heat map of qPCR gene expression analysis performed on Lu1 , Lu2 , Lu3 , and Lu4 cells freshly isolated from mammary glands of mature virgin mice .", "The qPCR values were normalized to Gapdh expression .", "Mean values from three independent cell preparations were used to establish the map and determine relationships between the luminal subsets by unsupervised hierarchical clustering .", "( D ) q-PCR analysis of Icam1 , Ly6a , and Met expression in Lu1 , Lu2 , Lu3 , and Lu4 cell populations freshly isolated from mammary glands of mature virgin mice .", "The values were normalized to Gapdh expression and represent mean values ±S . E . M from three independent preparations .", "( E ) Representative phase contrast images of spheres derived from Lu2 and Lu4 cell populations grown in the absence or presence of HGF for 10 days .", "Bar , 150 μm .", "( F ) Comparative expression levels of basal-specific , EMT-associated and luminal-specific genes in spheres derived from Lu2 and Lu4 cells , as determined by q-PCR .", "Cells were grown as described above in ( E ) .", "Results are expressed as Log2 ratios of values normalized to Gapdh .", "The comparator values were those obtained with cell preparations grown in the absence of HGF . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 02010 . 7554/eLife . 06104 . 021Figure 8—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 02110 . 7554/eLife . 06104 . 022Figure 8—figure supplement 1 . Characterization of luminal cell populations isolated from virgin and pregnant mice using ICAM-1 and Sca-1 expression .", "( A ) Flow cytometry dot plots showing the gating procedure of the four luminal cell subsets ( Lu1 , Lu2 , Lu3 , and Lu4 ) identified by Sca-1 and ICAM-1 .", "Cells were isolated from mammary glands of mature virgin mice .", "( B ) qPCR analysis of Krt18 and Krt14 expression in Lu1 , Lu2 , Lu3 , and Lu4 cells isolated from mammary glands of mature virgin mice as shown in ( A ) .", "Mean values ±S . E . M . from three independent cell preparations are shown .", "( C ) Percentages of clonogenic cells in Lu1 , Lu2 , Lu3 , and Lu4 populations isolated from mammary glands of mice taken at different stages of development: V-6w , 6-week-old virgin; P-8d , 8-day pregnant; P-16d , 16-day pregnant mice .", "Corresponding flow cytometry dot plots are shown in Figure 8A .", "Two separate cell preparations ( distributed in three wells ) were analyzed at each stages of development .", "The results are presented as mean values ±S . E . M . ( * ) No cell population .", "( D ) Heat map of qPCR gene expression analysis performed on Lu1 , Lu2 , Lu3 , and Lu4 cells freshly isolated from mammary glands of 8-day pregnant mice ( P8-d ) .", "The qPCR values were normalized to Gapdh expression .", "Mean values from two independent cell preparations were used to establish the map and determine relationships between the luminal subsets by unsupervised hierarchical clustering .", "( E ) q-PCR analysis of Icam-1 and Met expression in Lu1 , Lu2 , Lu3 , and Lu4 cell populations freshly isolated from mammary glands of 8-day pregnant mice ( P8-d ) .", "The values were normalized to Gapdh expression and represent means from two independent preparations . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 022 In adult virgin mammary gland , ICAM-1 separated both Sca-1-positive and Sca-1-negative cell populations , revealing four cell subsets , Lu1 ( ICAM1-neg/Sca-1-pos ) , Lu2 ( ICAM1-pos/Sca-1-pos ) , Lu3 ( ICAM1-neg/Sca-1-neg ) , and Lu4 ( ICAM1-pos/Sca-1-neg ) accounting for 39 . 8 ± 4 . 6% , 9 . 1 ± 1 . 4% , 15 . 0 ± 2 . 5% , and 25 . 4 ± 6 . 1% of the luminal compartment , respectively ( Figure 8A ) .", "We assessed the colony-forming potential of these cell subsets .", "Lu1 was considered as non-clonogenic ( <0 . 5% colony-forming cells ) , Lu3 cells , negative for ICAM-1 , were poorly clonogenic whereas both Lu2 and Lu4 cells , positive for ICAM-1 , were highly clonogenic ( Figure 8B ) .", "All four luminal cell subsets expressed K8 and displayed similar high levels of Krt18 and very low levels of the basal-specific gene Krt14 ( Figure 8A , Figure 8—figure supplement 1B ) .", "We characterized the four cell subsets further by carrying out qPCR-based gene expression analyses on a panel of luminal-specific genes .", "Unsupervised hierarchical clustering divided the populations into two main branches ( Figure 8C ) .", "Lu1 , Lu2 , and Lu3 clustered together , sharing strong expression of hormone receptors , ERa , PR , and PrlR .", "The second branch contained only Lu4 , which displayed very low levels of hormone receptor transcripts .", "Only Lu4 , the major clonogenic subset devoid of HR-positive cells , expressed the milk protein β-casein ( Figure 8C ) .", "It also strongly expressed Elf5 and Tnfrsf11a , two crucial regulators of alveologenesis ( Fata et al . , 2000; Oakes et al . , 2008 ) .", "Interestingly , flow cytometry analysis on mammary epithelial cells isolated at different stages of development showed that Lu4 was absent at puberty; this subset appeared in the glands of sexually mature cycling females and displayed massive expansion early in pregnancy ( Figure 8A ) .", "By contrast , Lu2 and Lu3 were detected at all stages of development .", "As in mature virgin glands , Lu2 and Lu4 were highly clonogenic during early pregnancy , whereas Lu3 was not ( Figure 8—figure supplement 1C ) .", "The gene expression patterns of Lu1 , Lu2 , Lu3 , and Lu4 cells isolated from mature virgin and early pregnant mice were similar ( Figure 8—figure supplement 1D , E ) .", "The q-PCR analysis showed that Met was strongly expressed in the clonogenic populations , Lu2 and Lu4 ( Figure 8D ) .", "These populations responded similarly to HGF treatment in 3D cultures .", "On stimulation , both produced larger spheres , displaying an upregulation of basal-specific genes ( Snai2 , Trp63 , and Cdh3 ) and a down-regulation of luminal-specific regulatory genes ( Gata3 , Elf5 , and Hey1 ) ( Figure 8E , F ) .", "These data show that luminal progenitors defined by ICAM-1 expression include hormone receptor-positive and hormone receptor-negative cells , all of which are potential targets of HGF/Met signaling .", "To complement the flow cytometry data , we analyzed ICAM-1 localization on histological sections of mammary gland .", "Immunohistochemical studies confirmed that in the pubertal gland , basal myoepithelial rather than luminal cells expressed ICAM-1 while at late pregnancy , the whole epithelium was positive ( Figure 9A ) .", "At the onset of lactation , ICAM-1 was down-regulated both in ducts and alveoli ( Figure 9A ) . 10 . 7554/eLife . 06104 . 023Figure 9 . ICAM-1 localization in the developing and adult mammary gland .", "( A ) Immunohistochemical detection of ICAM-1 in sections through mammary glands from mice at puberty ( V-6w; left panel ) , late pregnancy ( P-16d; middle panel ) , and onset of lactation ( L-2d; right panel ) .", "Left panel: bar , 60 μm .", "Middle and right panels: bar , 100 μm .", "( B ) Double immunofluorescence labeling of ICAM-1 and K8 in a mammary duct of a 15-week-old virgin female ( V-15w ) .", "The right panel shows an enlarged view of the area defined on the left .", "The arrows indicate a cluster of ICAM1-positive luminal cells .", "Bars , 40 μm .", "( C ) Double immunofluorescence staining of ICAM-1 and K8 in a large mammary duct at early gestation ( P-8d ) .", "The right panel shows enlarged view of the area defined on the left .", "The arrows point to myoepithelial cells expressing ICAM-1 at their apical and lateral surfaces .", "The arrowheads indicate luminal layer negative for ICAM-1 .", "Bars , 40 μm .", "( D and E )", "Sections through an alveolus and a small duct from a mammary gland of 8-day-pregnant mouse ( P-8d ) .", "( D ) Double immunofluorescence labeling of ICAM-1 and α-SMA ( SMA ) ; ( E ) ICAM-1 and ER .", "DAPI served to stain the nuclei .", "The arrows point to clusters of ICAM1-positive cells .", "The arrowheads indicate clusters of ER-positive cells negative for ICAM-1 .", "Bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 02310 . 7554/eLife . 06104 . 024Figure 9—figure supplement 1 . Response of luminal progenitors isolated from Icam1-deficient mammary epithelium to HGF stimulation .", "( A ) Representative images of carmine-stained mammary glands from adult wild-type ( WT ) and Icam1-KO mice .", "V-12w , 12-week-old virgin and P-16d , 16-day pregnant females .", "Bar , 1 . 5 mm . ( B ) Flow cytometry analysis of CD24 , Itga6 , and ICAM-1 expression in mammary epithelial cells freshly isolated from adult virgin WT and Icam1-KO females .", "Upper panels: Dot plots of CD24 and Itga6 distribution showing luminal and basal cell compartments .", "The percentages of luminal cells were equal to 74 ± 9% and 67 ± 9% ( data from four independent sorting experiments ) in WT and KO epithelium , respectively .", "Lower panels: Corresponding histograms of ICAM-1 expression in the whole mammary cell population .", "( C ) Flow cytometry analysis of CD24 , Itga6 and Sca-1 expression in mammary epithelial cells isolated from adult virgin Icam1-KO females .", "Left panel: Dot plots of CD24 and Itga6 distribution .", "Right panel: Dot plots of CD24 and Sca-1 expression in the luminal cell population .", "( D ) Representative microphotographs of primary mammospheres derived from purified Icam1-KO Sca1-neg luminal cells and grown in the presence or absence of HGF for 12 days .", "( E ) Characteristics of primary mammospheres derived from purified Icam1-KO Sca1-neg luminal cells .", "Left panel: average percentages ( ±S . E . M ) of clonogenic cells in non-stimulated and HGF-stimulated cultures .", "Data from one cell preparation with three separate wells are shown .", "Right panel: sphere size distribution ( in arbitrary units ) in HGF-treated and untreated cultures .", "At least 250 spheres were analyzed per conditions .", "( F ) Krt18 and Krt5 expression levels in spheres derived from untreated and HGF-treated Icam1-KO Sca1-neg luminal cells .", "The values were normalized to Gapdh expression .", "( G ) Comparative expression levels of basal-specific , EMT-associated and luminal-specific genes in spheres derived from untreated and HGF-treated Icam1-KO Sca1-neg luminal cells .", "The q-PCR data are expressed as log2 ratios of values normalized to Gapdh .", "The comparator values were those obtained with untreated spheres . DOI: http://dx . doi . org/10 . 7554/eLife . 06104 . 024 ICAM-1 localization in the adult mammary epithelium was further investigated using double immunofluorescence labeling with anti-ICAM-1 and anti-K8 antibodies .", "In agreement with the flow cytometry data , mammary basal cells in adult virgin mice displayed high levels of ICAM-1 , whereas the luminal compartment comprised ICAM1-negative and ICAM1-positive cells ( Figure 9B ) .", "ICAM1-positive luminal cells , enriched in clonogenic cells , often formed cell clusters within the ducts .", "At early pregnancy , myoepithelial cells lining large ducts strongly expressed ICAM-1 , whereas luminal cells were devoid of ICAM-1 ( Figure 9C ) .", "In contrast , in the developing alveoli and small ducts , the luminal layer , presumably enriched in alveolar progenitors , contained clusters of cells positive for ICAM-1 ( Figure 9D ) .", "Consistently with qPCR data , ER-positive cells were mostly ICAM1-negative ( Figure 9E ) .", "Notably , in adult virgin and early pregnant mice , ICAM-1 expression was restricted to cell–cell contacts , basal-to-basal , basal-to-luminal , and luminal-to-luminal ( Figure 9B–E ) .", "To analyze a possible contribution of ICAM-1 in mammary development and HGF/Met signaling , we investigated the mammary phenotype of Icam1-deficient ( Icam1-KO ) mice .", "The Icam1-KO females are viable , fertile , and able to feed their pups ( Xu et al . , 1994 ) .", "Whole-mount analysis of mammary glands from wild-type and Icam1-KO adult virgin and pregnant mice indicated that lack of ICAM-1 did not induce visible defects in mammary morphogenesis ( Figure 9—figure supplement 1A ) .", "Flow cytometry analysis showed that the percentages of basal and luminal cells were not significantly affected in the Icam1-KO epithelium of adult virgin mice ( Figure 9—figure supplement 1B ) .", "For HGF stimulation assays , we isolated Icam1-KO mammary epithelial cells and used the Sca-1-negative luminal cell population enriched in clonogenic progenitors ( Figure 9—figure supplement 1C ) .", "We found that Icam1-KO Sca-1-negative luminal cells responded to HGF by increased clonogenicity and production of large spheres ( Figure 9—figure supplement 1D , E ) .", "In addition , as previously observed in the presence of ICAM-1 , HGF treatment led to the induction of the basal-specific genes Krt5 , Trp63 , and Snai2 , with a concomitant decrease in the regulators of the luminal lineage , Elf5 , Gata3 , and Hey1 ( Figure 9—figure supplement 1F , G ) .", "Thus , ICAM-1 is not mandatory for mammary development and HGF/Met signaling in luminal progenitors ." ], [ "Our study provides the first evidence showing that ICAM-1 is differentially distributed , both spatially and temporally , in the mouse mammary epithelium .", "In mature virgin mice and early in pregnancy , the luminal cell population comprises ICAM1-negative and ICAM1-positive cell fractions , highly enriched in hormone-sensing cells and clonogenic progenitors , respectively .", "The luminal progenitors identified by ICAM-1 staining strongly express Elf5 , Hey1 , Tnfrsf11a , and Rspo1 genes encoding critical regulators of mammary development ( Fata et al . , 2000; Bouras et al . , 2008; Lee et al . , 2011; Cai et al . , 2014 ) .", "Several recent studies have indicated that the luminal progenitor population includes cells with different phenotypes ( Oliver et al . , 2012; Regan et al . , 2012; Shehata et al . , 2012; Lafkas et al . , 2013; Sale et al . , 2013; Rodilla et al . , 2015 ) .", "The differential expression of Sca-1 and CD61 was first used to obtain partial enrichment in luminal progenitor cells ( Asselin-Labat et al . , 2007; Sleeman et al . , 2007; Rios et al . , 2014 ) .", "Subsequently , the combined use of Sca-1 and c-kit or CD49b revealed that the luminal progenitors in the mature virgin gland belong to two distinct populations , one consisting essentially of HR-positive cells and the other mostly of HR-negative cells ( Regan et al . , 2012; Shehata et al . , 2012 ) .", "We found that in virgin glands and during early pregnancy , combined ICAM-1 and Sca-1 expression discriminated these two major luminal clonogenic cell subsets .", "ICAM-1 clearly appeared more efficient than Sca-1 for separating non-clonogenic and clonogenic luminal populations .", "Importantly , ICAM-1 marked the luminal progenitor population in different mouse strains , including C57Bl6/J , and in mixed backgrounds , such as that of BlgCre; R26 mice , whereas neither CD61 nor c-kit can be used for luminal progenitor enrichment from C57Bl6/J mouse mammary glands ( Visvader and Stingl , 2014 ) .", "Furthermore , differential ICAM-1 expression within the Sca-1-negative luminal cell population separated a poorly clonogenic ICAM1-negative subset enriched in HR-positive cells ( Lu3 ) from a major clonogenic cell fraction positive for ICAM-1 and lacking HR expression ( Lu4 ) .", "Such separation was not possible with CD49b , the most recently described marker for characterizing luminal cell subsets ( Shehata et al . , 2012; De Silva et al . , 2015 ) .", "The clonogenic luminal cell population is thought to comprise distinct ductal- and alveolar-restricted progenitors , as yet not fully characterized ( Regan et al . , 2012; Shehata et al . , 2012 ) .", "Cells committed to the alveolar fate are expected to expand in adult females particularly at the onset of pregnancy , to express Tnfrsf11a and Elf5 strongly , and most probably , to lack ER and PR ( Fata et al . , 2000; Lee et al . , 2011; Fu et al . , 2014; Rodilla et al . , 2015 ) .", "Interestingly , the Sca-1-negative ICAM1-positive clonogenic cell population ( Lu4 ) absent from prepubertal mammary epithelium fulfills these criteria .", "In addition , these cells have high levels of transcript for the milk protein β-casein .", "By contrast , the Sca-1- and ICAM1-positive clonogenic subset containing HR-expressing cells ( Lu2 ) present throughout mammary development may preferentially contain ductal-restricted progenitors .", "Thus , ICAM-1 is a new robust marker for separating basal and luminal cells and analyzing the heterogeneity of the luminal cell compartment in the adult mammary gland using flow cytometry .", "In addition , ICAM-1 can be used to localize and further characterize luminal progenitors in situ .", "The target cells for Met signaling in the mammary epithelium remain poorly characterized .", "A recent work reported the expression of Met predominantly in the Sca-1-negative luminal progenitor-enriched population ( Gastaldi et al . , 2013 ) .", "In agreement with these data , we found high-Met transcript levels in the Sca-1-negative ICAM1-positive clonogenic cell population ( Lu4 ) .", "Additionally , we observed strong Met expression in the Sca-1-positive ICAM1-positive luminal progenitors ( Lu2 ) .", "Thus , the two major clonogenic populations of luminal progenitors , enriched in HR-negative and HR-positive cells , are potential targets of HGF/Met signaling .", "Met was also expressed in the Sca-1-negative ICAM1-negative cell subset ( Lu3 ) , which was weakly clonogenic and enriched in HR-positive cells .", "Our data thus highlight the phenotypic diversity of Met-expressing cells in the luminal population .", "Consistently , a knockin mouse model with a mutationally activated Met has been reported to develop various types of mammary tumors , including ER-positive and ER-negative tumors ( Graveel et al . , 2009 ) .", "We found that ICAM1-expressing luminal progenitors responded to HGF stimulation by basal marker upregulation .", "Importantly , using the Blg-Cre; R26 reporter mouse , we demonstrated that these basal marker-expressing cells were of luminal origin .", "Triple-negative basal-like breast cancers are thought to originate from luminal progenitors aberrantly expressing basal-specific markers ( Lim et al . , 2009; Molyneux et al . , 2010; Proia et al . , 2011 ) .", "The highest levels of Met have been associated with the basal subtype of breast cancers ( Garcia et al . , 2007; Graveel et al . , 2009; Ponzo et al . , 2009; Gastaldi et al . , 2010; Knight et al . , 2013 ) .", "Moreover , amplifications of the Met locus and Met overexpression occur in Brca1/p53-deficient mouse tumors , a model of basal-like breast cancers ( Smolen et al . , 2006; Gastaldi et al . , 2013 ) .", "Thus , consistent with these reports , our results strongly indicate that Met signaling may contribute to the phenotypic characteristics of certain basal-like breast carcinomas .", "We found that in the mouse mammary epithelium , Met-expressing luminal progenitors displayed both ICAM-1 and CD44v6 at their surface .", "In mouse hepatocytes , CD44v6 acts as main co-receptor for Met , however , ICAM-1 can trigger Met activation in the absence of CD44 ( Orian-Rousseau et al . , 2002; Olaku et al . , 2011 ) .", "Our data showed that loss of ICAM-1 did not severely affect mammary development , HGF/Met signaling , and basal cell differentiation process in luminal progenitors .", "This suggests that CD44v6 rather than ICAM-1 serve as main co-receptor for Met in the mouse mammary epithelium and that identical compensation mechanisms may be involved in Met activation in liver and mammary gland .", "Distribution of Met and its co-receptors in the human mammary gland remains poorly defined; however , strong expression of Met , ICAM-1 , and CD44v6 has been associated with triple-negative breast cancers and correlated with poor prognosis ( Charpin et al . , 2009; Gastaldi et al . , 2010; Ponzo and Park , 2010; Schroder et al . , 2011 ) .", "Moreover , ICAM-1 has recently been identified as a molecular target for triple-negative breast cancers ( Guo et al . , 2014 ) .", "Stromal cells are usually considered to be the only source of HGF in the mammary gland ( Niranjan et al . , 1995; Yang et al . , 1995; Gastaldi et al . , 2013 ) .", "However , we show here that mammary myoepithelial cells also produce HGF .", "It is worth mentioning that in the postnatal mammary epithelium , ductal luminal cells are in direct contact with myoepithelial rather than stromal cells .", "In alveoli , the myoepithelial cell layer is discontinuous , so luminal cells may also come into contact with the basement membrane ( Moumen et al . , 2011 ) .", "HGF produced by both myoepithelial and stromal cells may therefore contribute to the control of the luminal progenitor activity , favoring either their normal expansion during development or aberrant amplification in cancers .", "Paracrine basal-to-luminal signaling mediated by the Nrg1-Erbb4 ligand–receptor pair has recently been implicated in the regulation of luminal cell maturation during lobulo–alveolar development ( Forster et al . , 2014 ) .", "We found that HGF had multiple effects on Met-expressing luminal progenitors .", "Early in culture , HGF promoted cell survival and proliferation .", "Consistent with its role in tubulogenesis ( Rosario and Birchmeier , 2003 ) , HGF then favored lumen formation .", "Finally , HGF treatment affected the fate of luminal progenitors , leading to the accumulation of a cell population expressing basal markers , including cells co-expressing basal and luminal keratins .", "Such dual luminal/basal cells are seldom in the adult mammary epithelium but are abundant in fetal mammary rudiments ( Spike et al . , 2012 ) .", "The accumulation of cells co-expressing basal and luminal keratins has been observed in the mammary epithelium of Elf5-null mice , which have high-Snail2 levels and display EMT-like phenotypic changes ( Chakrabarti et al . , 2012a , 2012b ) .", "Several regulators of the balance between mammary luminal and basal phenotypes have recently been identified .", "Notch signaling specifies a luminal cell fate , whereas ΔNp63 is required for the maintenance of basal cell characteristics ( Bouras et al . , 2008; Yalcin-Ozuysal et al . , 2010 ) .", "Forced expression of Snail2 endows mammary luminal progenitor cells with molecular characteristics and functional properties of basal-type stem cells , whereas Elf5 , through the direct repression of Snai2 , promotes luminal cell differentiation ( Guo et al . , 2012; Chakrabarti et al . , 2012a , 2012b ) .", "Consistent with these data , we found that HGF induced the expression of Trp63 whilst repressing that of Hey1 , a major Notch effector , in luminal progenitors .", "Concomitantly , we observed important hallmarks of EMT , including an increase in Snai1 and Snai2 levels , Elf5 repression and claudin down-regulation .", "The transcription of P-cadherin and N-cadherin was induced upon HGF treatment , but E-cadherin expression was not repressed , suggesting a partial , reversible EMT process ( Nieto and Cano , 2012; Gonzalez and Medici , 2014 ) .", "Consistently , alterations of the luminal progenitor phenotype did not persist in vitro after HGF withdrawal .", "Altogether , these data suggest that microenvironmental signals , such as HGF , may modulate the phenotype of luminal progenitors , by affecting the expression of antagonistic regulators of the basal and luminal fates .", "In addition to their role in triggering EMT , transcription factors of the Snail family have been reported to attenuate cell cycle progression by increasing p21 expression ( Vega et al . , 2004 ) .", "At late time points , Cdkn1a , Snai1 , and Snai2 were upregulated in HGF-treated cultures of luminal progenitors , whereas Mki67 was down-regulated .", "These changes were accompanied by the accumulation of K5-expressing quiescent cells .", "How p21 and its major upstream regulator p53 are involved in the response of luminal progenitors to HGF remains to be unraveled .", "A recent analysis of transgenic mouse models showed that Met can synergize with p53 loss to promote the formation of triple-negative mammary tumors with a claudin-low , EMT-type molecular signature ( Knight et al . , 2013 ) .", "Furthermore , the loss of p53 from the mammary epithelium leads to the expansion of clonogenic mammary luminal progenitors ( Chiche et al . , 2013 ) .", "In brief , our data provide new insights into the cellular and molecular mechanisms underlying the phenotypic plasticity of luminal progenitors .", "They also suggest that mammary basal and stromal cells may both be involved in controlling luminal progenitor function via the paracrine activation of Met during mammary development and tumorigenesis ." ], [ "BALB/cByJ JAX , C57BL/6 , and BALB/c-Nude JAX females were purchased from Charles Rivers ( L'arbresle , France ) .", "Transgenic mice expressing the Cre recombinase under the control of the β-Lactoglobulin promoter ( Blg-Cre ) were from the Jackson Laboratories ( Sacramento , CA , USA ) and purchased from Charles Rivers .", "Rosa26-LacZ reporter strain was provided by Soriano ( 1999 ) .", "Icam1-deficient mice ( B6 . 129S4-Icam1tm1Jcgr/J ) were from the Jackson Laboratories .", "The mature virgin females used in the experiments were 11–25 weeks old .", "The care and use of animals used here was strictly applying European and National Regulation for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes in force ( facility licence #C75-05-18 ) .", "It complies also with internationally established principles of replacement , reduction , and refinement in accordance with Guide for the Care and Use of Laboratory animals ( NRC , 2011 ) .", "All experimental procedures were ethically approved ( Ethical approval 02265 . 01/CEEA-IC 118 ) .", "Single cells were prepared from 2 to 8 inguinal mammary glands .", "Glands taken from virgin or pregnant females were minced , first with scissors and then with scalpels .", "Minced tissues were transferred to a digestion solution containing 3 mg/ml collagenase ( Roche Diagnostics , Meylan , France ) , 100 units/ml hyaluronidase ( Sigma–Aldrich , Saint Louis , MO , USA ) in CO2-independent medium ( Gibco , Life Technologies , St Aubin , France ) completed with 5% fetal bovine serum ( FBS , Lonza , Basel , Switzerland ) and 2 mM L-glutamine ( Sigma–Aldrich ) , and incubated for 90 min at 37°C with shaking ( 150 rpm ) .", "Digested samples were centrifuged at 450×g for 5 min and the supernatant eliminated .", "Pellets were washed once with CO2-independent medium , treated with a prewarmed 0 . 25% trypsin/0 . 1% versen ( Life Technologies; Biochrom , Berlin , Germany ) solution in Phosphate Buffered Saline ( PBS ) for 1 min and rinsed with CO2-independent medium containing 5% FBS .", "Pellets were then resuspended in a solution of 5 mg/ml dispase II ( Roche ) in CO2-independent medium containing 5% FBS and DNAseI ( Sigma–Aldrich ) was added to a final concentration of 0 . 1 mg/ml .", "After 5 min incubation at 37°C , cells were rinsed once in CO2-independent medium containing 5% FBS and the pellets were treated with a cold ammonium chloride solution ( Stem Cell Technologies , Grenoble , France ) .", "Cell suspensions were centrifuged , resuspended in CO2-independent medium , and filtered through a nylon mesh cell strainer with 40-μm pores ( Fisher Scientific , Illkirch , France ) before immunolabeling .", "Freshly isolated cells were incubated at 4°C for 20 min with the following conjugated antibodies: anti-CD24-FITC ( clone M1/69 ) and anti-CD49f-PE ( clone GoH3 ) from BD Biosciences ( BD France , Le Pont-de-Claix , France ) , anti-CD54-PE ( clone YN1/1 . 7 . 4 ) , anti-CD45-APC ( clone 30-F11 ) , and anti-CD31-APC ( clone MEC13 . 3 ) from Biolegend ( San Diego , CA , USA ) , anti-Ly6A/E-PE-Cy5 ( clone D7 ) from eBiosciences ( San Diego , CA , USA ) .", "Labeled cells were sorted on a FACSVantage flow cytometer ( BD Biosciences , San Jose , CA , USA ) , and data analyzed using FlowJo software .", "Sorted cell populations were routinely re-analyzed and found to be 96–98% pure .", "As estimated by trypan blue exclusion , cell viability after sorting was between 83% and 92% .", "For two-dimensional clonogenic assays , sorted luminal cells were plated on irradiated 3T3 cell feeders on 24-well plates at a density of 250–500 cells per well and cultured in DMEM/F12 medium supplemented with 10% FBS , 5 μg/ml insulin ( Sigma–Aldrich ) , 10 ng/ml EGF ( Invitrogen , Life Technologies ) , and 100 ng/ml cholera toxin ( ICN Biochemicals , Irvine , CA , USA ) for 7 days as described elsewhere ( Sleeman et al . , 2007 ) .", "For mammosphere cultures , freshly isolated luminal cells were seeded on ultra-low adherence 24-well plates ( Corning , NY , USA ) at the density of 2000–5000 cells/well , in mammosphere media: DMEM/F12 medium supplemented with 2% B27 , 20 ng/ml EGF , 20 ng/ml bFGF ( Gibco , Life Technologies ) , 4 μg/ml heparin ( Sigma–Aldrich ) , 10 μg/ml insulin , and 2% growth-factor-reduced Matrigel ( BD Biosciences ) as described elsewhere ( Spike et al . , 2012; Chiche et al . , 2013 ) .", "When specified , mammospheres were treated with 25–50 ng/ml recombinant mouse HGF ( R&D Systems Europe , Lille , France ) every 2 days .", "ImageJ software was used to count the colonies and the mammospheres and evaluate their size in pixels .", "Single cell suspensions were obtained from mammospheres by treatment with 0 . 05% trypsin ( Gibco , Life Technologies ) .", "Caspase-3/7 activity was assessed in cells harvested 1 and 2 days after plating them in mammosphere medium , using the Caspase-Glo 3/7 Assay System ( Promega , Madison , WI , USA ) according to the manufacturer's protocol .", "Freshly isolated cells were cytocentrifuged onto slides and fixed in cold methanol for 10 min .", "Cells were incubated with primary antibodies at 37°C for 1 hr , with secondary antibodies at room temperature for 1 hr and mounted in Prolong Gold antifade reagent with DAPI ( Invitrogen , Life Technologies ) .", "Mammospheres were resuspended in 50 μl Matrigel and incubated at 37°C for 2 hr prior to embedding either in OCT ( Tissue-Tek , Sakura Finetek Europe , Leiden , Netherlands ) for cryosections or in paraffin .", "Cryosections were post-fixed with 4% paraformaldehyde for 10 min and treated with 0 . 5% triton X-100 for 5 min before immunostaining .", "Spheres were fixed in Methacarn ( 1:3:6 mixture of acetic acid:chloroform:methanol ) before embedding in paraffin .", "Sections were dewaxed , processed for acidic antigen retrieval , and immunolabeled , as described elsewhere ( Chiche et al . , 2013 ) .", "For cell proliferation assays , mammospheres were incubated with BrdU ( 5 μg/ml; Sigma–Aldrich ) for 1 hr before being fixed and processed .", "The following primary antibodies were used: anti-K5 and anti-K8 ( Covance , Princeton , NJ , USA ) , anti-BrdU , anti-CD44v6 ( AbD Serotec , Oxford , UK ) , anti-E-cadherin ( ECCD2; Life Technologies ) , anti-p63 and anti-claudin-1 ( Abcam , Cambridge , UK ) , anti-snail2/slug ( Cell Signaling Technology , Danvers , MA , USA ) , anti-ICAM-1 ( Proteintech , Chicago , IL , USA ) , anti-ER ( Dako France , Les Ulis , France ) , and Cy3-conjugated anti-α-SMA ( Sigma–Aldrich ) .", "Alexafluor-conjugated secondary antibodies ( Molecular Probes , Life Technologies ) were used .", "For the immunohistochemical detection of ICAM-1 , we used the EnVision System from Dako .", "Image acquisition was performed using a Leica DM 6000B microscope ( Wetzlar , Germany ) and MetaMorph software .", "Confocal images were acquired with a Nikon Confocal A1r microscope using a 60× CFI Plan oil objective ( Apo VC/NA 1 . 4/WD 0 . 13 ) .", "Mammospheres established from Blg-Cre; R26 mammary cells were dissociated and isolated cells were pre-fixed with 2 . 5% paraformaldehyde for 5 min at 4°C and X-gal stained in suspension at 37°C overnight .", "Cells were cytocentrifuged , post-fixed with 4% paraformaldehyde for 10 min at room temperature , and processed either for Fast Red staining or immunostaining .", "For whole-mount X-gal staining , mammary glands from Blg-Cre; R26 females were fixed in 2 . 5% paraformaldehyde for 1 hr at 4°C and stained overnight at 30°C .", "Glands were embedded in paraffin and sections counterstained with Fast Red .", "Isolated cells derived from primary spheres or intact secondary spheres were transplanted into the inguinal fat pads of 3-week-old BALB/c-Nude JAX females cleared of endogenous epithelium as described elsewhere ( Deome et al . , 1959; Moumen et al . , 2012; Chiche et al . , 2013 ) .", "Untreated and HGF-treated cells or spheres were resuspended in 10 μl of 25% growth factor-reduced Matrigel before being grafted in the contralateral fat pads of host mice .", "For secondary sphere transplantations , 5000 ICAM1-positive luminal progenitors per well were plated , grown with or without HGF for 14 days , then , dissociated and replated at 5000 cells per well as for primary spheres .", "After 15 days in culture , the content of each culture well was grafted in a separate mammary fat pad .", "To analyze the outgrowths , dissected mammary fat pads were spread onto glass slides , fixed in Methacarn , and stained with carmine alum ( Stem Cell Technologies ) , as described elsewhere ( Moumen et al . , 2012; Chiche et al . , 2013 ) .", "RNA was reverse-transcribed with MMLV H ( − ) Point reverse transcriptase ( Promega , Madison , WI , USA ) , and quantitative PCR ( qPCR ) was performed by monitoring , in real time , the increase in fluorescence of the SYBR Green dye on an LightCycler 480 Real-Time PCR System ( Roche Applied Science , Basel , Switzerland ) .", "The primers used for qPCR analysis were purchased from SABiosciences/Qiagen ( Hilden , Germany ) or designed using Oligo 6 . 8 software and synthesized by Eurogentec ( Seraing , Belgium ) ( Supplementary file 2 ) .", "p values were determined using Student's test with two-tailed distribution and unequal variance ." ] ]

[ "HGF/Met signaling has recently been associated with basal-type breast cancers , which are thought to originate from progenitor cells residing in the luminal compartment of the mammary epithelium .", "We found that ICAM-1 efficiently marks mammary luminal progenitors comprising hormone receptor-positive and receptor-negative cells , presumably ductal and alveolar progenitors .", "Both cell populations strongly express Met , while HGF is produced by stromal and basal myoepithelial cells .", "We show that persistent HGF treatment stimulates the clonogenic activity of ICAM1-positive luminal progenitors , controlling their survival and proliferation , and leads to the expression of basal cell characteristics , including stem cell potential .", "This is accompanied by the induction of Snai1 and Snai2 , two major transcription factors triggering epithelial–mesenchymal transition , the repression of the luminal-regulatory genes Elf5 and Hey1 , and claudin down-regulation .", "Our data strongly indicate that paracrine Met signaling can control the function of luminal progenitors and modulate their fate during mammary development and tumorigenesis ." ]

[ "Throughout the life of a female mammal , the mammary glands undergo different phases of development to prepare for , and adapt to , feeding offspring .", "Luminal cells line the inside of branch-like structures throughout the mammary gland and are responsible for producing milk .", "When the mammary gland grows , new luminal cells develop from a kind of cell called luminal progenitor cells .", "However , these progenitor cells are also thought to be the source of certain types of breast cancer .", "Recently , it has been suggested that luminal progenitor cells display a receptor protein called Met on their surface .", "When Met and ‘co-receptor’ proteins bind to a molecule called HGF , this triggers a cascade of signals that can cause certain cells to change their properties .", "This is known as the epithelial–mesenchymal transition .", "Although this transition is important for new tissues to develop , it can also result in cancerous tumors forming if it is not correctly controlled .", "Luminal cells do not produce HGF themselves , which suggests that Met signaling in these cells is triggered by the HGF released from neighboring cells .", "However , neither the mechanisms behind this signaling nor the effects of signaling on the luminal progenitor cells are well understood .", "Di-Cicco et al . set out to identify where Met , its co-receptors and HGF are located in the mouse mammary gland during different phases of development .", "This revealed that one of the co-receptors—called ICAM-1—can be used as a marker to identify certain types of luminal progenitor cell .", "Di-Cicco et al . found that these progenitor cells display Met on their surface , and other types of mammary cell—called stromal cells and myoepithelial cells—produce HGF .", "When exposed to HGF , luminal progenitor cells grown in culture in the laboratory proliferated and went through the epithelial–mesenchymal transition .", "These findings suggest that myoepithelial and stromal cells regulate luminal progenitor cells by producing HGF to activate Met signaling in these cells .", "Such interactions could be of great importance during mammary development and tumorigenesis .", "The next big challenge will be to determine the circumstances under which luminal progenitor cells stimulated by HGF can give rise to breast cancers .", "This work will allow us to better define the cell population that should be targeted by anti-cancer drugs ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Sleep is a global vigilance state with well-known behavioral , electroencephalographic and neuromodulatory attributes .", "However , cerebral correlates of non-rapid-eye-movement sleep ( NREMS ) and REMS , notably several major EEG sleep rhythms , occur variably at different times in different brain regions ( Massimini et al . , 2004; Nir et al . , 2011; Siclari and Tononi , 2017 ) .", "This suggests that , on top of a global regulation , forebrain pacemakers with regionally specific oscillatory properties shape sleep across the cortex ( Krueger et al . , 2013 ) .", "Such local aspects probably underlie the sleeping brain’s decreased capacity to integrate external information and enable plasticity in specific neural circuits ( Siclari and Tononi , 2017; Crunelli et al . , 2018 ) .", "For example , sleep-dependent memory consolidation is linked to spatially confined regulation of NREMS rhythms in the brain areas involved in recent learning ( Rasch and Born , 2013 ) .", "Furthermore , sleep disorders may arise from a pathologically altered spatial heterogeneity that negatively impacts sleep as a global state ( Krueger et al . , 2013; Siclari and Tononi , 2017 ) .", "Prototype cellular pacemakers for NREMS rhythms , notably for the slow-oscillation ( SO ) ( <1 Hz ) , delta waves ( 1–4 Hz ) and sleep spindles ( 10–15 Hz ) have been known for decades ( for review , see ( Steriade et al . , 1993; Astori et al . , 2013; Sanchez-Vives et al . , 2017 ) ) .", "However , the spatiotemporal variability of cortical rhythms challenges the idea that these are homogeneous sources across the cortical surface ( Piantoni et al . , 2016; Siclari and Tononi , 2017 ) .", "For example , ‘fast’ and ‘slow’ human sleep spindles distribute variably and correlate differentially with memory consolidation , which has prompted a search for at least two , if not several , separately active spindle generators ( Schabus et al . , 2007; Frauscher et al . , 2015 ) .", "Interestingly , anatomical and functional boundaries of cortical areas go in parallel with variations of sleep rhythms ( Fernandez et al . , 2017; Piantoni et al . , 2017 ) .", "Moreover , sleep rhythms can show local singularities within a single cortical area according to developmental stage , or as a result of experience and learning during the day ( Huber et al . , 2004; Kurth et al . , 2010; Johnson et al . , 2012; Laventure et al . , 2016 ) .", "There is also evidence for the localized appearance of sleep-related patterns in individual cortical columns ( Pigarev et al . , 1997; Rector et al . , 2005 ) .", "Therefore , modality-specific thalamocortical loops could account for diversity in local NREMS .", "Moreover , there should be powerful local tuning mechanisms to locally switch between different NREMS rhythms .", "The current view is dominated by cortical focal influences as essential to shape local rhythms ( Contreras et al . , 1996; Piantoni et al . , 2017; Siclari and Tononi , 2017 ) .", "In contrast , thalamic oscillators are seen as broad and relatively homogeneous sources of oscillatory power that can spread focally or globally to cortex ( Bonjean et al . , 2012 ) , but that have little bearing on their ultimate cortical correlates .", "This view is becoming revised as novel observations on heterogeneous thalamic pacemaker mechanisms are reported .", "Of interest is the thalamic reticular nucleus ( TRN ) , typically referred to as an inhibitory shell of highly oscillatory , burst-prone cells surrounding the dorsal thalamus ( Pinault , 2004; Fogerson and Huguenard , 2016 ) .", "Burst discharge generates large inhibitory synaptic potentials ( Herd et al . , 2013; Rovó et al . , 2014 ) that entrain thalamocortical neurons into rhythmicity .", "Bursting is based on the CaV3 . 3 channel that is crucial for sleep spindle generation ( Astori et al . , 2011; Pellegrini et al . , 2016 ) , but many studies indicate that not all TRN cells burst equally ( Contreras et al . , 1992; Brunton and Charpak , 1997; Lee et al . , 2007; Kimura et al . , 2012; Clemente-Perez et al . , 2017; Higashikubo and Moore , 2018 ) .", "Then , the TRN is parcellated into at least five modality-specific sectors , sensory , motor or limbic ones , according to their innervation by a particular dorsal thalamic nucleus and the reciprocally connected cortical area ( Crabtree , 1999; Pinault , 2004 ) .", "Moreover , optogenetic activation of TRN promotes cortical spindles or delta waves ( Halassa et al . , 2011; Lewis et al . , 2015 ) , suggesting that the exact patterns of TRN cell activity may determine the contribution of these two rhythms at the cortical surface .", "Most recently , various discharge propensity in TRN cells was linked to the differential expression of parvalbumin ( PV ) or somatostatin ( Clemente-Perez et al . , 2017 ) .", "The number of PV-expressing cells is smaller in TRN of schizophrenic patients and mouse models ( Steullet et al . , 2018 ) in which reduced sleep spindle density is a common observation ( Manoach et al . , 2016 ) .", "Together , there is accruing evidence for marked molecular and functional TRN cell heterogeneity .", "However , whether TRN heterogeneity is relevant for brain correlates of NREMS has so far not been tested .", "This study shows that the heterogeneous cellular properties of optogenetically identified TRN sectors are a major source of local NREMS rhythms in sensory cortices .", "We identify the ionic mechanisms underlying this heterogeneity and study its impact on major NREMS correlates in sensory and non-sensory cortices using genetic and chemogenetic approaches .", "We thereby unravel novel organizing principles of NREMS topography in mouse and show that heterogeneity in TRN sectors accounts for a previously unrecognized enrichment of fast and large sleep spindles in somatosensory cortices that are coupled to the SO .", "We also find that TRN heterogeneity enables rapid switching between different forms of NREMS rhythms , suggesting that this could underlie the regulation of local NREMS by use and experience ." ], [ "To identify TRN cells belonging to a sensory sector , we used stereotaxic injections of AAV-ChR2_EYFP into somatosensory ( S1 , barrel field ) or auditory cortex ( AC ) of 3- to 4-week-old mice .", "Non-sensory TRN sectors related to associative areas , such as the medial prefrontal cortex ( PFC ) , were instead targeted through injections into the mediodorsal ( MD ) thalamic nucleus that is the input structure forming reciprocal loops with several areas of the PFC ( Mátyás et al . , 2014; Delevich et al . , 2015; Collins et al . , 2018 ) and that forms reciprocal circuits with the TRN ( Mitchell , 2015 ) .", "Enhanced yellow fluorescent protein ( EYFP ) fluorescence was present at injection sites and in restricted portions of TRN 3–4 weeks after injection , as verified on coronal sections stained immunohistochemically for PV to delineate the dorso-ventral extent of the TRN ( Figure 1 ) .", "The observed sectorial portions coincided with the ones established previously ( Pinault and Deschênes , 1998; Pinault , 2004 ) .", "Thus , injections into S1 revealed fibers navigating through the postero-dorsal portion of the TRN that terminated in elongated fluorescent spots in the thalamic ventral posterior medial nucleus corresponding to thalamic barreloids ( Bourassa et al . , 1995 ) .", "AC injections resulted in fluorescent labeling of postero-central regions of the TRN that are anterior to the medial geniculate nucleus .", "Finally , MD injections labeled antero-ventral portions of the TRN , overlapping with TRN areas innervating motor and intralaminar nuclei ( Pinault and Deschênes , 1998; Pinault , 2004 ) .", "In acute coronal slices prepared from injected animals , TRN cells recorded in the green fluorescent areas through whole-cell patch clamp recordings reliably ( > 85 % of the cells across sectors ) responded to optogenetic stimulation ( 470 nm light , pulse duration ≤1 ms , 0 . 16 mW/mm2 ) with rapid excitatory postsynaptic currents ranging between -42 to -938 pA ( Figure 2A ) .", "Paired stimuli ( interstimulus interval: 100 ms ) yielded paired-pulse facilitation for cortical afferents ( S1-innervated TRN cells: 203 ± 12% , n = 12 , Wilcoxon signed rank-test , p=4 . 9x10−4 for 2nd vs 1st stimulus; for AC-innervated TRN cells: 175 ± 21% , n = 6 , p=0 . 03 ) , as described previously ( Astori and Lüthi , 2013 ) , whereas paired responses to MD stimulation showed comparable size ( 105 ± 4 % , n = 13 , p=0 . 27 ) .", "Cells showed values for resting membrane potential and capacitance consistent with previous data ( Figure 2B; Astori et al . , 2011 ) .", "Rebound action potential discharge was elicited in response to square somatic current injections ( negative current injections of -50 to -300 pA for 500 ms to hyperpolarize the somatic membrane potential < -100 mV , yielding cell input resistance values of 344 ± 18 MΩ for all three sectors ) .", "Rebound oscillatory bursting hallmarks the capacity of TRN cells to engage in rhythm generation ( Astori et al . , 2011; Wimmer et al . , 2012; Clemente-Perez et al . , 2017 ) .", "S1- and AC-innervated TRN cells showed the rhythmic , repetitive burst discharge described previously ( Figure 2C; Cueni et al . , 2008; Astori et al . , 2011 ) , evident as several groups of high-frequency action potentials each riding on a triangular-shaped membrane depolarization and followed by a pronounced afterhyperpolarization .", "Repetitive burst discharge strongly depended on the membrane voltage , showing an inverted U-shaped voltage dependence that peaked at -65 to -60 mV for S1-innervated cells ( Figure 2D1 , E ) .", "Only 1/12 S1-innervated TRN cells was a non-repetitive bursting cell ( Figure 2F ) .", "Similar burst propensity was found for TRN cells innervated from AC ( Figure 2E ) , but 4/13 cells were non-repetitive bursters ( Figure 2F ) .", "In TRN cells responding to MD inputs , repetitive bursting was weak ( Figure 2C , D1 ) and 9/14 cells discharged maximally one burst ( Figure 2F ) .", "These results show that repetitive burst propensity is stronger in sensory compared to non-sensory TRN sectors .", "Within sensory sectors , the somatosensory sector displayed the highest density of strongly bursting cells , whereas the auditory sector had a smaller proportion of cells with rhythmic bursting .", "To test whether the heterogeneity of burst discharge across TRN sectors depended on CaV3 . 3 channels , the optogenetic strategy described above was applied to animals with a genetic deletion of the Cacna1i ( CaV3 . 3 , α1I ) gene ( Astori et al . , 2011 ) .", "This channel is primarily responsible for burst discharge in TRN cells , while co-expressed CaV3 . 2 channels play a minor role ( Pellegrini et al . , 2016 ) .", "Accordingly , TRN cells of these animals are unable to burst repetitively , whereas tonic action potential discharge is preserved .", "Cells patched in acute slices from Cacna1i-/- ( CaV3 . 3 KO ) animals showed passive properties comparable to those in C57BL/6J ( WT ) cells of the corresponding sectors ( Figure 2B ) , although S1-innervated cells had a smaller capacitance indicative of reduced cell size , perhaps resulting from morphological alterations in these constitutive knock-outs .", "Light-evoked synaptic responses showed a similar range of amplitudes ( −10 to −1076 pA ) and a short-term plasticity that was comparable to the one found for WT cells ( S1-innervated TRN cells: 188 ± 32% , n = 7 , p=0 . 016; for AC-innervated TRN cells: 203 ± 13% , n = 8 , p=0 . 008; for MD-innervated TRN cells: 87 ± 8 , n = 10 , p=0 . 31; two-way ANOVA with factors ‘genotype’ and ‘sector’ , p=0 . 28 for interaction ) .", "However , the vigorous bursting in somatosensory and auditory TRN cells was suppressed , thus abolishing the dependence of repetitive burst discharge propensity on TRN sector type ( Kruskal-Wallis with factor ‘sector’ , p=3×10−4 for WT , p=0 . 14 for CaV3 . 3 KO ) ( Figure 2D2 , E ) .", "Together , these data indicate that the CaV3 . 3 channel endows superior bursting capacity to sensory over non-sensory TRN cells .", "It has been shown that TRN bursting capacities are sensitive to cortical lesions ( Paz et al . , 2010 ) .", "To exclude the possibility that the viral injections compromised TRN discharge , we also recorded from TRN cells in slices prepared from uninjected animals and identified putative sensory and non-sensory sectors in horizontal slices along the anteroposterior axis .", "These experiments confirmed the different burst propensity in sensory vs non-sensory sectors ( Figure 2—figure supplement 1 ) .", "We next monitored local NREMS in cortical areas connected to the TRN sectors studied in vitro .", "Under stereotaxic guidance , animals were implanted for in vivo multi-site recordings of local field potentials ( LFPs ) in the same cortical areas that were previously targeted for the anterograde viral tracing of TRN sectors , along with electroencephalography/electromyography ( EEG/EMG ) ( Figure 3A , B ) .", "For S1 and AC , electrodes were positioned in deep layers ( layers 5 and 6 ) , whereas infra-/prelimbic cortical areas ( collectively referred to as PFC ) were implanted in middle layers ( layers 3 and 5 , see Materials and methods for exact stereotaxic coordinates ) , according to the majority of thalamocortical input received in the respective areas .", "Additionally , the secondary somatosensory cortex ( S2 ) was implanted ( layers 2/3 and 4 ) to monitor NREMS in an associative sensory cortical area with strong reciprocal connections to S1 ( Zingg et al . , 2014 ) .", "We chose high-impedance electrodes ( ~10–12 MΩ ) for LFP recordings to maximize detection of local signals .", "Simultaneous EEG/EMG recordings on the contralateral hemisphere were used for vigilance state scoring ( Figure 3A , B ) .", "Animals were recorded in head-restrained mode , which yields a sleep profile comparable to that in freely moving conditions , as previously shown ( Fernandez et al . , 2017; Lecci et al . , 2017 ) .", "Each mouse was recorded for 2–3 hr/day and spontaneously switched between periods of wakefulness , NREMS and REMS with power spectra typical for each vigilance state ( Figure 3—figure supplement 1A ) .", "NREMS was accompanied by distinct LFP waveforms across cortical areas of WT animals ( Figure 3C ) .", "In S1 and S2 , prominent activity in the SO ( 0 . 5–1 . 5 Hz , frequency band chosen based on visual inspection of the power spectrum ) and the sleep spindle ( sigma , 10–15 Hz ) frequency ranges was visible ( Figure 3—figure supplement 1B ) .", "Activity in the delta ( 1 . 5–4 Hz ) frequency range was evident as large positive deflections ( Figure 3—figure supplement 1B ) .", "Similar , yet weaker rhythmic activity was observed in AC and PFC .", "The PFC showed signals dominated by slow events , as reported ( Fernandez et al . , 2017 ) , which included a component around 4 Hz resembling a respiratory-related rhythm in frontal brain areas ( Figure 3C , D; Zhong et al . , 2017 ) .", "Power spectral analyses over total NREMS times of 2200–6100 s per animal ( average 4487 ± 603 s , concatenated from NREMS bouts across recording days ) showed that NREMS in all recorded areas had broadly elevated power in the low-frequency range covering both the SO and the delta range ( 0 . 5–4 Hz ) , whereas a ‘shoulder’ in the sigma band was present only in somatosensory areas ( n = 9 for S1 and n = 8 for S2 ) but not in AC ( n = 6 ) and PFC ( n = 6 ) ( Figure 3D ) .", "NREMS in animals lacking CaV3 . 3 channels showed several marked changes that were apparent in both the raw traces and in characteristic alterations of the power spectra ( NREMS recording times of 1800–7000 s per animal , average 3459 ± 421 s ) .", "First , there was a striking lack of visually recognizable sleep spindle activity in S1 and S2 , and a sigma power shoulder was not present in the power spectrum ( Figure 3C , D , bottom panel showing enlarged portions of the power spectrum and Figure 3—figure supplement 1B ) .", "Second , activity in the delta range was augmented , whereas the SO was less prominent in LFP traces from S1 , S2 and AC .", "These visual observations manifested as a rightward shift of the low-frequency activity in the power spectrum , with a clear power peak present in the delta band that dominated over power values < 1 . 5 Hz .", "The power spectra for WT and CaV3 . 3 KO animals intersected in the slow frequencies around 1 . 6–1 . 8 Hz for S1 , S2 and AC , suggesting a consistent spectral border between the SO and the delta bands .", "Separate quantification of total power in the SO , the delta and the sigma frequency band confirmed these observations ( Figure 3E ) .", "After Bonferroni correction , the sigma power reduction in S1 appeared as a trend .", "However , this is an underestimation because the difference between the two curves extends up to ~18 Hz .", "In PFC , in contrast , no alterations in the power spectrum were observed , with in particular no significant change in the sigma power and in the SO peak .", "The delta band was not analyzed in this area due to the superposition of the respiratory rhythm on top of the delta waves .", "Together , the lack of CaV3 . 3 channels altered the spectral mix of NREMS in sensory circuits , whereby power in the delta frequency band became overrepresented compared to the SO .", "This shift was greatest in S1 and S2 , where in addition sigma power activity was suppressed .", "In contrast , the PFC did not show these alterations in two of the three major frequency bands , suggesting a minor dependence on CaV3 . 3 channels .", "The results from CaV3 . 3 KO animals suggest that strong CaV3 . 3-dependent burst discharge is required for spindle-enriched NREMS .", "Therefore , acute reduction of TRN excitability to suppress bursting should also deplete spindles and lead to a NREMS enriched in delta waves .", "To test this , we hyperpolarized TRN cells with a chemogenetic approach , whereby we expressed the inhibitory DREADD receptor hM4Di in VGAT-Ires-Cre animals ( Vong et al . , 2011 ) through bilateral injection of AAV-hM4D ( Gi ) _mCherry or AAV-hM4D ( Gi ) _IRES_mCitrine in the region of the somatosensory TRN sector ( Figure 4—figure supplement 1A ) .", "In acute slices prepared 3 weeks after injection , bath application of the DREADD-ligand Clozapine N-oxide ( CNO , 10 μM ) induced a marked membrane hyperpolarization ( ΔV = −13 . 9 ± 1 . 5 mV , n = 10 , paired t-test , p=6×10−6 ) of fluorescent cells held in whole-cell current-clamp at resting membrane potentials ranging from −50 to −70 mV ( Figure 4A , B ) .", "Cellular input resistance was reduced and rebound burst discharge suppressed in the continuous presence of CNO .", "Bursting could be recovered upon direct current ( d . c . ) injection to restore the original membrane potential ( d . c . injection tested in n = 3 cells , Figure 4A ) .", "Non-fluorescent cells in the vicinity of the injected area did not respond to CNO ( Figure 4B ) .", "This result is consistent with CNO-induced activation of K+ conductances and shows that TRN neurons become silenced without impairing rebound bursting and action potential firing .", "Treatment with CNO thus overall reduces TRN excitability , and in particular specifically reproduces the decreased burst propensity of TRN cells found in the CaV3 . 3-KO animals that is relevant for the altered NREMS spectral properties .", "Similarly injected animals were also implanted in vivo for S1 LFP and EEG/EMG freely moving recordings and treated with CNO ( i . p . 1 mg/kg ) or NaCl at 2 hr into the light phase ( ZT2 ) .", "The latencies to fall asleep were comparable after drug or NaCl injections ( 31 . 4 ± 3 . 7 min for CNO , 24 . 8 ± 2 . 1 min for NaCl; n = 5 , paired t-test , p=0 . 16 ) .", "NREMS analysis was done for the time period of 20–65 min after drug injection , which is the period where drug effects peak ( Figure 4—figure supplement 1B ) .", "Total time spent in NREMS was not different between CNO and NaCl injections in the analysis period ( 24 . 1 ± 1 . 7 min for CNO , 27 . 7 ± 1 . 9 min for NaCl injections , Wilcoxon signed rank-test , p=0 . 22 ) and mean NREMS bout durations were comparable ( 93 . 3 ± 12 . 3 s for CNO , 91 . 2 ± 32 . 5 s for NaCl , Wilcoxon signed rank-test , p=0 . 31 ) .", "Following CNO injections , S1 LFP signals during NREMS showed reduced spindle activity and instead became enriched in activity in the delta frequency range ( Figure 4C , D , repeated-measures ANOVA with factors ‘frequency’ and ‘treatment’ , p=7 . 7×10−5 ) .", "Compared to NaCl injections , total power in the delta frequency range was increased , whereas the SO and spindle activity were suppressed ( Figure 4D , E ) .", "Control animals injected with AAV8 carrying a DREADD-unrelated optogenetic construct ( see Materials and methods ) did not respond to CNO ( Figure 4—figure supplement 1C–F ) .", "The CNO-induced acute membrane hyperpolarization thus reproduced the major power spectral changes observed in NREMS of the CaV3 . 3 KO mouse: the suppression of the SO and sleep spindle power , and the enhancement of delta power .", "As CNO-induced hyperpolarization suppressed burst discharge ( see also Figure 2D1 ) , the joint results from the genetic and the chemogenetic manipulations identify decreased TRN bursting as the primary factor relevant for the enrichment of delta power at the expense of sigma and SO power in NREMS .", "Given the importance of CaV3 . 3-dependent TRN burst discharge for NREMS in sensory cortices , the question remains open of how this discharge pattern controls the properties of local , discrete spindle events .", "The exact sources of regional spindle properties have recently received considerable attention ( Schabus et al . , 2007; Frauscher et al . , 2015; Piantoni et al . , 2017 ) .", "Therefore , we developed an algorithm to isolate discrete spindle events in NREMS of WT and CaV3 . 3 KO mice .", "We followed a previously established thresholding approach in rat that successfully characterized spindles in both rodent and human ( Mölle et al . , 2009 ) complemented with additional criteria , as detailed in the Materials and methods ( Figure 5—figure supplement 1 ) .", "For the band-pass filtering , we were guided by the observation that in both S1 and S2 of the CaV3 . 3 KO animals , power was attenuated beyond the widely used sigma band of 10–15 Hz .", "Therefore , we chose 9–16 Hz to allow for the possible inclusion of comparatively slow and fast spindles .", "We isolated 727–2289 events per WT mouse and area that showed the typical spindle-shaped , waxing-waning waveform ( Figure 5A ) .", "Spindles in S1 and S2 showed large amplitudes that were comparable to that of the SO , whereas those in AC and PFC were less prominent ( Figure 5A ) .", "In the CaV3 . 3 KO animals , the large spindle events were reduced in S1 and S2 , but remained comparable in AC .", "There was also a reduction of event amplitude in PFC ( Figure 5B1 ) .", "Cumulative probability density curves showed a marked leftward shift of the amplitude distribution in S1 and S2 , but not in AC and PFC ( Figure 5B2 ) .", "We also analyzed the intra-spindle frequencies , one of the major markers of spindle heterogeneity ( Figure 5C ) .", "The frequency of detected events was distributed according to a Gaussian between 9 and 16 Hz , with a maximum around 10–12 Hz , yielding means of 11 . 6 ± 0 . 09 Hz for S1; 11 . 7 ± 0 . 05 Hz for S2; 11 . 2 ± 0 . 05 Hz for AC; 11 . 5 ± 0 . 03 Hz for PFC ( Figure 5C1 ) .", "All distributions showed a tail indicating a small proportion of events with a frequency >14 Hz ( Figure 5C2 ) .", "In the CaV3 . 3 KO animals , frequencies were specifically attenuated in S1 and S2 , but remained comparable in AC and PFC .", "The greater activity of CaV3 . 3 channels in the sensory sectors of TRN thus correlated strongly with higher amplitudes and frequencies of individual spindles in S1 and S2 .", "Sleep spindles are temporally grouped by the active state of the SO , which reflects the strong role of corticothalamic volleys in recruiting thalamic circuits ( Contreras et al . , 1996 ) .", "This grouping is key for the promotion of sleep-dependent memory consolidation ( Mölle et al . , 2009 ) and it may be disrupted in some cases of schizophrenia ( Manoach et al . , 2016 ) .", "We determined the onset times of detected spindles with respect to the phase of the SO ( Figure 6A ) and confirmed such time-locking throughout all areas , with ~60% and 40% of detected spindles initiating during the cortical active and silent states ( also named UP and DOWN states ) , respectively ( Figure 6B , C ) .", "Remarkably , the same analysis in the CaV3 . 3 KO animals showed that the time-locking of sleep spindles to the SO was altered for S1 and S2 ( Figure 6B , C ) , but not for AC and PFC .", "In both S1 and S2 , the presence of spindles on the active state was diminished in favor of spindles in the silent state .", "In S1 from CaV3 . 3 KO animals , the occurrence of spindles was comparable between active and silent states , whereas events occurred preferentially in the silent state in S2 .", "This difference in the coupling was not due to a less faithful detection of the smaller spindles in the CaV3 . 3 KO animals , because the analysis produced similar results when it was limited to WT spindles with amplitudes corresponding to those of the CaV3 . 3 KO animals .", "Therefore , the presence of CaV3 . 3 ensures the timely occurrence of sleep spindles with respect to the cortical active state ." ], [ "We identify a novel mechanism underlying local cortical correlates of NREMS .", "It arises through heterogeneity in thalamic circuits and correlates with variable oscillatory bursting propensity across TRN sectors .", "Strong CaV3 . 3-dependent bursting in the somatosensory TRN sector led to local NREMS with fast and large spindles coupled to the SO , whereas this was not the case for cortical areas corresponding to TRN sectors with weakly bursting cells .", "Intriguingly , high burst propensity was also coupled to a dual regulation of local NREMS , such that the SO and spindles could be suppressed in favor of delta waves .", "Sectorial attributes of TRN circuits thus emerge as a powerful source for local NREMS correlates .", "They additionally facilitate a tuning of NREMS between major spectral correlates , offering a candidate mechanism for local sleep modulation , possibly in response to use , experience and learning .", "A sectorial heterogeneity of TRN will also advance insight into a proposed common vulnerability of TRN circuitry for both sleep as well as wakefulness-driven selective attention in neuropsychiatric disorders ( Krol et al . , 2018 ) .", "The TRN has been traditionally regarded as a homogeneous sleep rhythm pacemaker ( Fuentealba and Steriade , 2005; Fogerson and Huguenard , 2016 ) that underlies global thalamic inhibition ( Halassa and Acsády , 2016 ) .", "Although its molecular and functional heterogeneity is increasingly recognized , consequences for NREMS are so far poorly explored and the role of sector-specific TRN cell characteristics is unknown .", "Thus , it is not clear whether heterogeneous cellular discharge properties ( Contreras et al . , 1992; Brunton and Charpak , 1997; Lee et al . , 2007; Kimura et al . , 2012; Higashikubo and Moore , 2018 ) or variable expression of PV and somatostatin are important for local sleep ( Clemente-Perez et al . , 2017 ) .", "Furthermore , sensory but not limbic TRN cells preferentially engage in sleep-related activity ( Halassa et al . , 2014 ) , but it is not known whether they are cellularly distinct .", "Therefore , this study is the first to bring together TRN heterogeneities at both the in vitro and the in vivo levels using optogenetically assisted mapping of TRN sectors .", "We show that cellular heterogeneities align with identified TRN sectors , and TRN sectors with area-specific NREMS , thus establishing the TRN sector-specific cellular properties as a source for local and tunable NREMS .", "Based on a previous study demonstrating that PV-positive cells are stronger bursters than cells enriched in somatostatin ( Clemente-Perez et al . , 2017 ) , we propose that PV-positive TRN cells are primary determinants of the unique sleep spindle properties in somatosensory cortex .", "The focus on NREMS in specific cortical areas using LFP recordings , combined with genetic and chemogenetic manipulation and cellular analysis , reveals novel and surprising topographical aspects of mouse NREMS ( Terrier and Gottesmann , 1978; Kim et al . , 2015; Fernandez et al . , 2017 ) .", "One remarkable observation is that somatosensory cortices showed a clear sigma power ‘shoulder’ and strong , fast , CaV3 . 3-dependent spindle events .", "The widespread idea from EEG recordings that sleep spindle generation in mouse NREMS is weak ( Astori et al . , 2011 ) should thus be revised as we now show that full-fledged spindle activity is generated in local regions of the mouse brain .", "The highly focal synaptic organization of the barrel system , where TRN-thalamic , thalamocortical and corticothalamic projections between barreloid and barrel topographically match on a cell-to-cell basis ( Desîlets-Roy et al . , 2002; Wimmer et al . , 2010 ) , together with a high density of PV-positive , bursty cells in the TRN somatosensory sector ( Clemente-Perez et al . , 2017 ) , are likely important anatomical substrates enabling strong local spindles .", "The tight alignment of thalamic unit and S1 LFP activity recorded simultaneously during spindles in urethane anaesthesia ( Rovó et al . , 2014 ) supports this interpretation .", "We also observed prominent sigma power and high-amplitude spindles in S2 that exceeded levels in S1 .", "High reciprocal cortical connectivity between S1 and S2 ( Feldmeyer et al . , 2013 ) , and a differential recruitment of first- and higher order thalamic nuclei in these two areas , could be some of the reasons behind this difference .", "In contrast to S1 and S2 , no discernable sigma power shoulder was present in AC , and detected spindles showed no dependence on the CaV3 . 3 channel .", "We found a tendency for a decreased density of highly burst-prone cells in mouse , which possibly reduces the strength of spindle generation in auditory sectors .", "It also remains to be seen whether there exist functional and anatomical differences in the overall connectivity of each sensory TRN sector ( Crabtree , 1999 ) .", "Similarly , the spectral composition of NREMS in pre- and infralimbic portions of the PFC showed no prominent sigma power and no overall dependence on CaV3 . 3 channels , consistent with a minor expression of CaV3 . 3 channels in the MD-innervated sector and with the presence of a majority of somatostatin-positive , weakly bursting cells in these areas of the TRN ( Clemente-Perez et al . , 2017 ) .", "However , discrete spindles could be detected in mouse and they are well-described for rat PFC ( Siapas and Wilson , 1998; Peyrache et al . , 2011; Maingret et al . , 2016 ) .", "Discrete spindle events were accompanied by a phase entrainment of TRN discharge in sleeping rats , although mean firing rates seemed low ( Gardner et al . , 2013 ) .", "There could thus be a spindle-generating circuitry independent of the powerful CaV3 . 3-dependent mechanisms in sensory TC loops , in which anterior and midline thalamic nuclei and hippocampus are involved .", "Such ideas remain to be tested also regarding the notion of distinct frontal spindles in both human ( Schabus et al . , 2007 ) and mouse ( Kim et al . , 2015 ) .", "Power in the SO band was consistently co-modulated with sleep spindle activity in both the CaV3 . 3 KO mouse and during chemogenetic TRN inhibition .", "This could be evidence in favor of a role of TRN in the generation of the SO ( Crunelli et al . , 2015 ) .", "Our chemogenetic results generally support the idea that the TRN sustains low-frequency activity in cortico-thalamocortical loops , an effect which could possibly also result secondarily from its phasic recruitment by cortex to generate sleep spindles .", "Also , the increase of delta power could have de-emphasized the relative presence of SOs .", "Additionally , possible roles of CaV3 . 3 channels in cortically generated rhythms could arise from a subgroup of cortical interneurons ( Liu et al . , 2011 ) .", "The genetic removal of the CaV3 . 3 channel enhanced delta power in S1 and S2 at the expense of sleep spindles .", "Chemogenetic hyperpolarization reproduced this observation closely .", "Although the chemogenetic approach suppresses excitability in a manner that is not limited to bursting , the close correspondence with the observations in the CaV3 . 3-KO animals , in which only bursting but not tonic firing is impaired ( Astori et al . , 2011 ) , strongly suggests that suppressed bursting , which is the common denominator of both experimental approaches , is the principal mechanism involved in the effects at the level of NREMS .", "While the reduction in sleep spindle activity under these conditions is expected ( Astori et al . , 2011 ) , an enhancement and/or unmasking of delta wave-generating activity in thalamocortical circuits has now become apparent thanks to the locality of our recordings .", "It is reminiscent of previous observations of an opposite regulation of slow wave/delta and spindle power during NREMS ( Dijk et al . , 1993; Steriade et al . , 1993; Franken et al . , 1998 ) , which has been explained based on the different membrane potential polarizations of TC cells involved in spindle or delta rhythm generation ( Nuñez et al . , 1992 ) .", "Here , we identify TRN cell membrane potential polarization as a determinant for such regulation in somatosensory thalamocortical circuits during NREMS .", "Remarkably , the capability of TRN in generating either spindles or delta waves was also evident based on whether brief or prolonged optogenetic stimulation was applied ( Halassa et al . , 2011; Lewis et al . , 2015 ) .", "This underscores the power of TRN-dependent inhibition in controlling thalamocortical synchrony according to discharge patterns .", "We add to this the capability of a local , switchable tuning of NREMS spectral properties in somatosensory cortex .", "In agreement with pioneering work on delta waves , we propose that TRN hyperpolarization liberates TC cells from phasic hyperpolarization to engage in a clock-like rhythm at delta frequencies ( Steriade et al . , 1993 ) .", "Results consistent with this interpretation were also obtained in animals doubly deficient in CaV3 channels ( Pellegrini et al . , 2016 ) .", "Recently , it was also shown that optogenetic inhibition of anterior TRN cells may suppress rather than strengthen low-frequency EEG activity ( Herrera et al . , 2016 ) , yet this effect occurred with a 10-s-long delay after acute inhibition of TRN cell discharge .", "The powerful control of delta power by TRN-dependent mechanism could be relevant for the role of delta waves in the homeostatic regulation of NREMS .", "The increase in low-frequency power of NREMS over the 0 . 75–4 Hz power band , referred to as slow-wave activity , is the most widely used marker to quantify homeostatic sleep pressure ( Borbély and Tobler , 2011 ) .", "We find here that slow-wave activity contains a thalamically controlled component that can be rapidly and bidirectionally modulated through TRN membrane polarization .", "Such mechanisms could contribute to sleep-deprivation induced boosting of the high- but not the low-frequency component of slow-wave activity ( Achermann and Borbély , 1997; Huber et al . , 2000 ) .", "Bidirectional regulation of slow-wave activity in local brain areas according to use dependence has also been described ( Kattler et al . , 1994; Pigarev et al . , 1997; Vyazovskiy et al . , 2000; Miyamoto and Hensch , 2003; Huber et al . , 2006 ) .", "More complex localized alterations in NREMS are observed following exposure to learning tasks that involve enhanced power in both the low-frequency range ( SO and delta waves in the slow wave activity frequency range of 0 . 5–4 Hz ) and in the fast sleep spindle range ( Huber et al . , 2004 ) .", "Learning tasks involving motor cortex increase the density of individual spindle events in a manner specifically restricted to motor cortex ( Johnson et al . , 2012 ) or augmented both slow wave and sleep spindle power in supplementary motor cortex ( Tamaki et al . , 2013 ) .", "Therefore , cortical modules are capable of generating qualitatively different forms of local NREMS spontaneously and according to recent use and experience .", "Membrane potential polarization within TRN sectors could represent a powerful addition to previously proposed mechanisms that primarily imply changes in cortical synaptic strength ( Tononi and Cirelli , 2014 ) .", "Both ascending brainstem and basal forebrain ( McCormick and Bal , 1997; Beierlein , 2014 ) as well as descending cortical inputs ( McCormick and von Krosigk , 1992; Zhang et al . , 2012 ) regulate TRN membrane potential and burst propensity .", "Whether differential neuromodulation of TRN sectors contributes to use- and experience-dependent sleep regulation remains an intriguing topic for further study .", "Compromised sleep spindle generation is a promising read-out for neuropsychiatric disorders involving aberrant sensory percepts and attentional deficits , such as schizophrenia ( Manoach et al . , 2016 ) .", "In large-scale genome-wide association studies , the gene encoding CaV3 . 3 channels ranks in the top list of candidate risk genes in schizophrenia , together with several genes that are highly enriched in TRN and implied in repetitive burst discharge ( Krol et al . , 2018 ) .", "Based on our data , we propose that wake-related deficits in some of these patients may show a specificity for certain sensory modalities that co-vary with local deficits in sleep spindles and their coupling to the SO .", "This could help to further refine the classification and diagnosis of these complex disorders ." ], [ "Mice from the C57BL/6J line ( also referred to as wild-type , WT ) , the CaV3 . 3 KO line and the VGAT-Ires-Cre line ( Jackson Labs , generated by Dr . B . Lowell , Beth Israel Deaconess Medical Center , Harvard ) ( Vong et al . , 2011 ) were bred on a C57BL/6J background and housed in a temperature- and humidity-controlled animal house with a 12 hr/12 hr light-dark cycle ( lights on at 9 am ) .", "Food and water were available ad libitum .", "For viral injections , 3- to 4-week-old mice of either sex were transferred to a P2 safety level housing room with identical conditions 1 day prior to injection .", "Then , for in vitro experimentation , animals were transferred 3 to 4 weeks later to a housing room with identical conditions , 3–5 days prior to sacrifice .", "For in vivo experimentation , animals were brought to the recording room at least one week prior to experimentation .", "All experimental procedures complied with the Swiss National Institutional Guidelines on Animal Experimentation and were approved by the Swiss Cantonal Veterinary Office Committee for Animal Experimentation .", "Mice 3- to 4-week-old were anaesthetized using Ketamine-Xylazine ( 83 and 3 . 5 mg/kg , respectively ) .", "Mice were placed on a heating blanket to maintain the body temperature at 37°C .", "An initial dose of analgesic was administrated at the beginning of the surgery ( Carprofen i . p . 5 mg/kg ) .", "The animal was head-fixed on a stereotactic apparatus equipped with a head adaptor for young animals ( Stoelting 51925 , Wood Dale , IL ) .", "A small incision was made on the skin and the bone exposed at the desired injection site .", "Viruses were injected with a thin glass pipette ( 5-000-1001-X , Drummond Scientific , Broomall , PA ) pulled on a vertical puller ( Narishige , Tokyo , Japan ) .", "WT and CaV3 . 3 KO mice were injected bilaterally with a virus encoding ChR2-EYFP ( 500 nl of AAV1-hSyn-ChR2 ( H134R ) _eYFP-WPRE-hGH , 1012 GC , ~100–200 nl/min ) for one of the following sites ( in stereotaxic coordinates , relative to bregma: anteroposterior , lateral , depth from surface ) : S1 ( -1 . 7 , ±3 . 1 , -0 . 8 ) , AC ( -2 . 5 , ±4 , -1 . 1 ) , MD ( -1 . 7 , ±0 . 4 , -3 . 2 ) .", "VGAT-Ires-Cre mice were injected bilaterally with a virus encoding DREADD-mCherry ( 500 nl of AAV8-hSyn-DIO-hM4D ( Gi ) _mCherry , 6 . 4x1012 GC ) , or DREADD-IRES-mCitrine ( 500 nl of ssAAV8/2-hSyn1-dlox-HA_hM4D ( Gi ) _IRES_mCitrine-dlox-WPRE-hGHp ( A ) , 3 . 1x1012 GC ) or a control AAV8 encoding a DREADD-unrelated construct ( 500 nl of AAV8-hSyn-FLEX-Jaws_KGC_GFP_ER2 , 3 . 2x1012 GC ) in the sensory sector of the TRN ( -1 . 7 , ±2 . 25 , -2 . 9 ) .", "Adult WT , CaV3 . 3 KO and VGAT-Ires-Cre mice ( 3–4 weeks post viral injection ) , 7- to 9-week-old , were briefly anaesthetized with isoflurane and their brains quickly extracted .", "Acute 300-µm-thick coronal brain slices were prepared in ice-cold oxygenated sucrose solution ( which contained in mM: NaCl 66 , KCl 2 . 5 , NaH2PO4 1 . 25 , NaHCO3 26 , D-saccharose 105 , D-glucose 27 , L ( + ) -ascorbic acid 1 . 7 , CaCl2 0 . 5 and MgCl2 7 ) , using a sliding vibratome ( Histocom , Zug , Switzerland ) .", "Slices were kept for 30 min in a recovery solution at 35°C ( in mM: NaCl 131 , KCl 2 . 5 , NaH2PO4 1 . 25 , NaHCO3 26 , D-glucose 20 , L ( + ) -ascorbic acid 1 . 7 , CaCl2 2 , MgCl2 1 . 2 , myo-inositol 3 , pyruvate 2 ) before being transferred to room temperature for at least 30 more min before starting the recording .", "Recording glass pipettes were pulled from borosilicate glass ( TW150F-4 ) ( World Precision Instruments , Sarasota , FL ) with a DMZ horizontal puller ( Zeitz Instruments , Martinsried , Germany ) to a final resistance of 2–4 MΩ .", "Pipettes were filled with a K+-based intracellular solution that contained in mM: KGluconate 140 , Hepes 10 , KCl 10 , EGTA 0 . 1 , phosphocreatine 10 , Mg-ATP 4 , Na-GTP 0 . 4 , pH 7 . 3 , 290–305 mOsm , supplemented with ~2 mg/ml of neurobiotin ( Vector Labs , Servion , Switzerland ) .", "Slices were placed in the recording chamber of an upright microscope ( Olympus BX50WI , Volketswil , Switzerland ) and continuously superfused at room temperature with oxygenated ACSF containing in mM: NaCl 131 , KCl 2 . 5 , NaH2PO4 1 . 25 , NaHCO3 26 , D-glucose 20 , L ( + ) -ascorbic acid 1 . 7 , CaCl2 2 and MgCl2 1 . 2 , picrotoxin 0 . 1 , glycine 0 . 01 .", "Cells were visualized with differential interference contrast optics and 10X and 40X immersion objectives .", "Infrared images were acquired with an iXon Camera X2481 ( Andor , Belfast , Northern Ireland ) .", "Signals were amplified using a Multiclamp 700B amplifier , digitized via a Digidata1322A and sampled at 10 kHz with Clampex10 . 2 ( Molecular Devices , San José , CA ) .", "Immediately after gaining whole-cell access , cell capacitance Cm was measured in voltage-clamp at −60 mV through applying 500 ms-long , 20 mV hyperpolarizing steps ( 5 steps/cell ) .", "Whole-field blue LED ( Cairn Res , Faversham , UK ) stimulation ( 455 nm , duration: 0 . 1 to 1 ms , maximal light intensity 0 . 16 mW/mm2 ) in voltage-clamp ( −60 mV ) was used to assess the connectivity of TRN neurons through fibers arising from the previously injected area ( S1 , AC or MD ) .", "Once identified , squared somatic current injections ( −50 to −300 pA for 500 ms , 4 injections/cell and membrane potential ) hyperpolarized neurons below −100 mV from membrane potentials between −90 and −50 mV ( corrected for a liquid junction potential of 10 mV ) and induced repetitive burst discharge in TRN neurons .", "For comparison between neurons from different TRN sectors , the response to this current injection was also used to assess cellular input resistance Ri .", "For CNO application , a stable baseline of at least 2 min was recorded before bath application of water-soluble CNO ( 10 µM , Tocris , Bristol , UK ) for at least 2 min until its hyperpolarizing effect reached a plateau .", "Washout of CNO did not reverse the hyperpolarizing effect for up to 10 min of washout .", "Two VPM and two TRN neurons outside the visually identified fluorescent site of injection were used as control for CNO’s effect on membrane potential .", "Cell parameters were calculated on Clampfit v . 10 . 2 .", "and IgorPro Wavemetrics ( Lake Oswego , OR ) .", "Input and access resistances were evaluated all along the recordings .", "Neurons presenting a variation of the access resistance >20% or a holding current at −60 mV < −150 pA were excluded from analysis .", "Light-evoked excitatory postsynaptic current ( EPSC ) amplitudes were measured in traces presenting monophasic synaptic events that occurred at fixed latency after LED onset and that were not contaminated by asynchronous release .", "In the subset of neurons that underwent paired-pulse stimulation , traces containing spontaneous activity between the two LED stimulations were discarded .", "Paired-pulse ratios are expressed as percentage of the second over the first EPSC amplitude ( 6 paired-stimuli/cell ) .", "The bursting of TRN neurons was assessed by counting the number of Ca2+ spikes following a 500 ms hyperpolarization below −100 mV .", "Ca2+ spikes were counted as bursts if they generated triangular-shaped membrane depolarizations followed by a clear afterhyperpolarization , regardless of the presence of high-frequency action potentials on top of the Ca2+ spikes .", "Surgery was performed as recently described ( Lecci et al . , 2017 ) .", "Briefly , animals were subjected to gas anaesthesia ( isoflurane supplemented with a mixture O2 and N2O ) and small craniotomies ( 0 . 3–0 . 5 mm ) were performed at the location for implantation of high-impedance fine tungsten LFP microelectrodes ( 10–12 MΩ , 75 µm shaft diameter , FHC , Bowdoin , ME ) at the following sites ( in stereotaxic coordinates , relative to bregma: anteroposterior , lateral , depth from surface ) : sensory regions S1 ( −1 . 7 , 3 . 0 , -1 . 0 ) , S2 ( −0 . 7 , 4 . 2 , -1 . 1 ) , and AC ( −2 . 5 , 4 . 0 , -1 . 1 ) , limbic areas of PFC ( +1 . 8 , 0 . 3 , -1 . 85 ) .", "Implantations were guided through calculating the corresponding interaural coordinates .", "As a neutral reference for LFP electrodes , a silver wire ( Harvard Apparatus , Holliston , MA ) was inserted in the bone over lateral portions of the cerebellum .", "On the contralateral skull site , two conventional gold-coated low-impedance electrodes were implanted over the dura mater through frontal and parietal bones for differential surface EEG recordings .", "Two gold pellets inserted into the muscles of the neck served as EMG electrodes .", "Multi-site recordings were carried out in head-restrained conditions , for which a light-weight metal head-post ( Bourgeois Mécanique SAS , Lyon , France ) was glued and cemented onto the midline skull in order to perform painless head-fixed recording sessions ( Fernandez et al . , 2017; Lecci et al . , 2017 ) .", "Carprofen ( 5 mg/kg , i . p . ) and paracetamol ( 2 mg/mL , drinking water ) were provided during the pre- and post-operative periods .", "Mice were gently and gradually habituated to a custom-made head-fixation system ( Bourgeois Mécanique SAS , Lyon , France ) by increasing the amount of time spent in head fixation daily from 10 to 30 min to 2–3 hr/day .", "Mice sat within a cardboard roll such that only the head protruded .", "Occasionally , a heating pad was placed underneath the cardboard .", "After each head-restrained period , mice were rewarded with ad libitum drops of sweetened water .", "Mice typically started sleeping spontaneously after 7–14 d of habituation , generating periods of both NREMS and REMS .", "LFP and EEG/EMG signals were amplified 1000x through a 16-channel Multiple Acquisition Processor System ( Plexon Inc . , Dallas , TX ) , high- and low-pass filtered at 0 . 8 and 300 Hz , respectively , and digitized at 1 kHz .", "For multi-site recordings , 5 WT and 6 CaV3 . 3 KO animals had all four recording sites histologically confirmed , 1 WT ( S1 , S2 , PFC ) and 3 CaV3 . 3 KO ( 2x S1 , S2 , AC; 1x S1 , S2 , PFC ) animals had three confirmed recording sites , 3 WT ( 1x S1 , AC; 2x S1 , S2 ) and 4 CaV3 . 3 KO ( S1 , S2 ) animals had two confirmed sites .", "Four animals were excluded because recording sites could not be identified or because brain appearance was not satisfying ( ventricle dilatation or damaged brain slice ) .", "After 1 week of recovery from viral injection , bilateral S1 LFP electrodes , and EEG/EMG electrodes were implanted as described above , followed by a week of recovery .", "After 4 days of habituation to the tethering cable , baseline activity was recorded in freely moving conditions during 3 to 4 days .", "3 weeks after viral injections , intra-peritoneal injection of CNO ( water-soluble diluted in NaCl 0 . 9% , dosis 1 mg/kg , Ref . 6329 , Tocris , Bristol , UK ) or NaCl 0 . 9% was performed at Zeitgeber time ZT2 in a random cross-over design , with the experimenter blind to the injection .", "Recordings under each condition took place on 4–5 successive days , with 2–3 CNO and 1–2 NaCl injections per animal .", "Signals were acquired at 1 kHz with an Intan digital RHD2132 amplifier board and a RHD2000 USB Interface board , with a high-pass filter set at 0 . 8 Hz ( Intan Technologies , Los Angeles , CA ) .", "Data were acquired in Matlab using the RHD2000 Matlab toolbox and customized display software in Matlab .", "To assess the time course of CNO action , 3 DREADD-mCherry and 3 AAV8-control animals were recorded starting at ZT0 .", "Based on these dynamics , we chose a window of 45 min ( starting 20 min after the injection ) for comparison of CNO and NaCl effects ( Figure 4—figure supplement 1B , D ) .", "The average power spectrum per animal was calculated as mean between recording sites and repetition-days for the two conditions ( CNO or NaCl ) .", "Spectral analysis of the signals was performed as described in the data analysis section .", "For chemogenetic recordings , a total of 3 DREADD-mCherry and 2 control AAV8 mice had bilateral S1 recording sites histologically confirmed , 2 DREADD-mCherry and 1 control AAV8 had one confirmed S1 recording sites .", "After completion of patch-clamp recordings in vitro , slices were post-fixed in paraformaldehyde ( 4% ) for >24 hr .", "An immunostaining on free-floating sections was used to outline PV-positive ( PV+ ) neurons in the TRN and to recover neurobiotin-filled neurons .", "To ensure proper staining of PV+ neurons , a 5-day incubation at 4°C of the primary antibody ( mouse anti-PV , 1/4000 , Swant Inc . , Marly , Switzerland ) in 1% Triton was required .", "The secondary antibody ( goat anti-mouse CY5 , 1/500 , Jackson ImmunoResearch , Ely , Nevada ) and Streptavidin ( coupled with Alexa Fluor 594 , 1/8000 , Jackson ImmunoResearch ) were incubated at 4°C for 24 hr .", "Sections were observed with an Axiovision Imager Z1 ( Carl Zeiss ) microscope equipped with an AxioCam MRc5 camera .", "Objectives were EC-Plan Neofluar 2 . 5x/0 . 075 ∞/0 . 17 , 5x/0 . 16 ∞/0 . 17 .", "The AxioVision Rel .", "4 . 7 and Adobe Photoshop CS5 software were used to merge micrographs from the different channels .", "The nomenclature of the location of TRN sectors in Figure 1 follows the descriptions established by Pinault and Deschênes ( 1998 ) .", "After completion of in vivo recordings ( multi-site or chemogenetics recordings ) , recording sites were marked through electro-coagulation ( 50 μA , 8–10 s ) during deep pentobarbital anaesthesia ( 80 mg/kg ) before transcardiac perfusion ( 4% paraformaldehyde in 0 . 1 M phosphate buffer ) .", "After >24 hr post-fixation , 100 µm coronal brain sections were cut and imaged to confirm electrocoagulation sites of LFP implantation or fluorescent expression of viral injection site .", "Data were analyzed using IgorPro ( Wavemetrics , v7 , Lake Oswego , OR ) , MatLab ( MathWorks ) and Excel ( Microsoft ) .", "In vitro .", "Statistical analysis was done using R programming language ( 2 . 15 . 0 , R Core Team ) [The R Development Core Team , The R Foundation for Statistical Computing ( www . r-project . org/foundation ) , 2007] .", "The normality of the data sets was assessed using Shapiro-Wilk normality test .", "Comparisons between paired conditions ( amplitude of 1st versus 2nd EPSCs during paired-stimulation and effect of CNO on membrane potential ) were done using Wilcoxon signed rank-test and paired Student’s t-test , respectively .", "Comparisons between unpaired conditions in non-normally distributed datasets ( passive cellular properties , sector effect on repetitive bursting in WT , sector effect on repetitive bursting in CaV3 . 3 KO , genotype effect on repetitive bursting ) were done using Mann-Whitney or Kruskal-Wallis H tests .", "Comparisons between unpaired conditions in normally distributed datasets ( sector and genotype effect onto PPR and CNO effect onto WT vs CaV3 . 3 KO ) were done using unpaired Student’s t-tests or two-way ANOVAs .", "Bonferroni correction was applied whenever more than two comparisons were done for the same data set .", "The proportion of repetitive bursting neurons in the different TRN sectors was compared using Chi-square test for independence followed by a pairwise proportion test with Holm’s adjustment method .", "Exact significant p-values are indicated .", "In vivo .", "Statistical analysis was done using IgorPro , R programming language and Matlab .", "The normality of the data sets was assessed using Shapiro-Wilk normality test .", "Comparisons between genotypes per site of recording were done using Student’s t-test ( parametric data set ) or Mann-Whitney ( non-parametric unpaired data set ) .", "Comparisons between paired conditions ( CNO versus NaCl , or SO Active state versus Silent state ) were done using paired Student’s t-test ( parametric data set ) or Wilcoxon signed-rank test ( non-parametric paired data ) .", "Bonferroni correction was applied when more than two comparisons were done for the same data set ." ] ]

[ "Sleep affects brain activity globally , but many cortical sleep waves are spatially confined .", "Local rhythms serve cortical area-specific sleep needs and functions; however , mechanisms controlling locality are unclear .", "We identify the thalamic reticular nucleus ( TRN ) as a source for local , sensory-cortex-specific non-rapid-eye-movement sleep ( NREMS ) in mouse .", "Neurons in optogenetically identified sensory TRN sectors showed stronger repetitive burst discharge compared to non-sensory TRN cells due to higher activity of the low-threshold Ca2+ channel CaV3 . 3 .", "Major NREMS rhythms in sensory but not non-sensory cortical areas were regulated in a CaV3 . 3-dependent manner .", "In particular , NREMS in somatosensory cortex was enriched in fast spindles , but switched to delta wave-dominated sleep when CaV3 . 3 channels were genetically eliminated or somatosensory TRN cells chemogenetically hyperpolarized .", "Our data indicate a previously unrecognized heterogeneity in a powerful forebrain oscillator that contributes to sensory-cortex-specific and dually regulated NREMS , enabling local sleep regulation according to use- and experience-dependence ." ]

[ "Falling asleep affects our behavior immediately and profoundly .", "During sleep , large electrical waves appear across the brain in areas responsible for consciousness , sensation and movement .", "In the cortex – the outer layer of the brain – sleep waves arise from networks that connect to the thalamus , a deeper structure within the brain .", "However , not all areas of the brain sleep equally .", "We know this intuitively because sensory stimuli , such as an alarm clock or a baby’s cry , can still wake us up .", "By contrast , we typically do not move much or take major decisions while we sleep .", "Therefore , the brain areas involved in sensation should not be expected to sleep in the same way as areas involved in movement or reasoning .", "Neighboring brain areas generally show very different sleep waves .", "The brain regions that we use during the day can also affect how sleep varies from one area to the next .", "It is not well understood what determines these ‘local’ sleep properties .", "By studying the brains of mice , Fernandez et al . now show that the networks between the cortex and thalamus are much more varied than previously thought , in particular regarding a thalamic nucleus that is relevant for sleep wave generation .", "These previously unrecognized differences deep within the brain are part of the origin of local sleep in the outer layer of the brain .", "Sleep wave activity differed depending on whether the networks were involved in sensory or non-sensory roles .", "The networks allow sensory areas to switch efficiently between different forms of local sleep .", "This might underlie how the brain’s sensory activity during the day can influence local sleep at night .", "There is growing evidence that major sleep disorders are due to disturbances to local sleep .", "Techniques to modify or restore specific sleep waves locally in the brain could help to develop new sleep therapies .", "For example , having a detailed map of electrical waves within the sleep-disordered brain could help researchers to apply transcranial stimulation techniques in ways that might help to treat these debilitating disorders ." ]

[ "Introduction", "Results", "Discussion", "Methods" ]

[ "epidemiology and global health", "medicine" ]

[ [ "When a girl reaches menarche she may be regarded by some societies as ‘ready’ to start sex and to marry ( Sommer , 2009 ) .", "This may be linked to initiation ceremonies , recognising the transition to adulthood ( Munthali and Zulu , 2007 ) .", "Puberty may change her own desires , and change the behaviour of those around her .", "In diverse settings , earlier age at menarche has been associated with earlier sexual debut and earlier marriage ( Buga et al . , 1996; Mensch et al . , 2001; Downing and Bellis , 2009; Glynn et al . , 2010; Boden et al . , 2011 ) .", "Earlier menarche is also associated with school drop-out in some settings ( Biddlecom et al . , 2008; Glynn et al . , 2010; Boden et al . , 2011 ) .", "An association between early menarche and sexually transmitted infections might therefore be expected , but there is limited direct evidence to support this .", "In Croatia earlier menarche was associated with Chlamydial infection among sexually active adolescents attending outpatients ( Hirsl-Hecej et al . , 2006 ) ; in Tanzania earlier menarche was associated with human papilloma virus ( ter Meulen et al . , 1992 ) ; and in a prospective study in New Zealand earlier menarche was associated with attendance at an STI clinic before age 18 ( Boden et al . , 2011 ) .", "A small study in Uganda found no link between age at menarche and HIV incidence .", "( Quigley et al . , 2000 )", "Since first coitus before menarche has been identified as a risk factor for sexually transmitted infections ( Duncan et al . , 1994 ) , earlier menarche could also be protective in certain situations .", "In many populations the prevalence of Herpes simplex type-2 ( HSV-2 ) , an important co-factor for HIV acquisition ( Glynn et al . , 2009 ) , rises quickly after onset of sexual activity , so it provides a useful marker of unprotected sex , especially in women in whom the prevalence can reach high levels ( Obasi et al . , 1999 ) .", "In a study in rural northern Malawi we have previously shown that earlier menarche leads to earlier sexual debut , earlier marriage , and earlier school drop-out ( Glynn et al . , 2010 ) .", "Here we investigate the associations with HSV-2 and HIV infection .", "In this area , unlike southern Malawi ( Morris , 2000; Munthali and Zulu , 2007 ) , there are no initiation rituals associated with menarche .", "Girls experiencing their first period are traditionally sent to stay with an aunt or other female relative for instruction , and it is likely to become known in the community ." ], [ "Of 4772 women aged 15–30 who were eligible , 650 were not found and 146 refused to take part , leaving 3976 , of whom 3965 were interviewed ( 83% of all those eligible ) .", "HIV results were available for 3651 , and HSV-2 results for 3419 .", "Those with missing HIV and HSV2 data were , on average , older , more educated , lived in better houses and experienced slightly later menarche .", "Approximately one quarter of the women had menarche at each of <14 , 14 , 15 or ≥16 years of age .", "The associations between age at menarche , age at sexual debut and age at marriage are summarised in Figure 1 .", "The age of sexual debut and the age at marriage increased with age at menarche .", "For example , among those with early menarche ( age <14 ) , 60% were married and only 16% were not yet sexually active at age 17 , compared to 11% married and 71% not yet sexually active among those with late menarche ( age ≥16 ) . 10 . 7554/eLife . 01604 . 003Figure 1 . Multistate lifetables showing proportion of young women who are still virgins , have started sex but are unmarried , and are married , by age and age at menarche . Karonga District , Malawi . DOI: http://dx . doi . org/10 . 7554/eLife . 01604 . 003 Overall 25 . 5% of those tested were HSV-2 positive and 5 . 6% were HIV positive .", "The risk of both infections increased rapidly with age ( Figure 2 ) .", "After adjusting for current age , HSV-2 infection was less common in those with later menarche than in those with earlier menarche ( p-trend = 0 . 001 ) but there was no evidence for a linear trend between age at menarche and HIV infection ( p-trend = 0 . 8 ) ( Figure 3; Table 1 ) . 10 . 7554/eLife . 01604 . 004Figure 2 . Prevalence of HSV-2 and HIV infection in women by age , Karonga District , Malawi . DOI: http://dx . doi . org/10 . 7554/eLife . 01604 . 00410 . 7554/eLife . 01604 . 005Figure 3 . Association between age at menarche and ( A ) HSV-2 and ( B ) HIV , adjusted for current age . Karonga District , Malawi . DOI: http://dx . doi . org/10 . 7554/eLife . 01604 . 00510 . 7554/eLife . 01604 . 006Table 1 . Risk factors for HSV2 and HIV infection in 15–30 year old women in a population survey , Karonga District , Malawi 2007–8DOI: http://dx . doi . org/10 . 7554/eLife . 01604 . 006HSV2HIVn/N%OR ( 95% CI ) Age adjustedn/N%OR ( 95% CI ) Age adjustedOR ( 95% CI ) OR ( 95% CI ) All870/341925 . 5203/36515 . 6Age 15–9126/124010 . 2118/13141 . 41 20–24276/107225 . 83 . 1 ( 2 . 4–3 . 9 ) 64/11285 . 74 . 3 ( 2 . 6–7 . 4 ) 25–30468/110742 . 36 . 5 ( 5 . 2–8 . 1 ) 121/120910 . 08 . 0 ( 4 . 8–13 . 2 ) Age at menarche <14245/81530 . 11151/8615 . 911 14195/76925 . 40 . 79 ( 0 . 63–0 . 99 ) 0 . 89 ( 0 . 71–1 . 1 ) 43/8195 . 30 . 88 ( 0 . 60–1 . 3 ) 1 . 0 ( 0 . 66–1 . 5 ) 15225/96023 . 40 . 71 ( 0 . 58–0 . 88 ) 0 . 71 ( 0 . 57–0 . 89 ) 41/10304 . 00 . 66 ( 0 . 43–1 . 0 ) 0 . 68 ( 0 . 44–1 . 0 ) ≥16157/62225 . 20 . 79 ( 0 . 62–0 . 99 ) 0 . 69 ( 0 . 54–0 . 89 ) 51/6657 . 71 . 3 ( 0 . 88–2 . 0 ) 1 . 2 ( 0 . 81–1 . 8 ) Age at first sex Never51/6398 . 00 . 21 ( 0 . 15–0 . 29 ) 0 . 48 ( 0 . 33–0 . 69 ) 10/6841 . 50 . 23 ( 0 . 12–0 . 44 ) 0 . 69 ( 0 . 32–1 . 3 ) <16296/101429 . 21166/10766 . 111 16–17235/79729 . 51 . 0 ( 0 . 83–1 . 2 ) 0 . 99 ( 0 . 80–1 . 2 ) 53/8476 . 31 . 0 ( 0 . 70–1 . 4 ) 0 . 99 ( 0 . 68–1 . 5 ) 18+273/93029 . 41 . 0 ( 0 . 83–1 . 2 ) 0 . 76 ( 0 . 62–0 . 93 ) 71/10027 . 11 . 2 ( 0 . 82–1 . 7 ) 0 . 90 ( 0 . 63–1 . 3 ) Marital status Never76/8269 . 21119/8852 . 211 Current681/233029 . 24 . 1 ( 3 . 2–5 . 2 ) 1 . 7 ( 1 . 3–2 . 3 ) 133/24815 . 42 . 6 ( 1 . 6–4 . 2 ) 0 . 78 ( 0 . 44–1 . 4 ) Previous113/26343 . 07 . 4 ( 5 . 3–10 . 4 ) 3 . 4 ( 2 . 4–5 . 0 ) 51/28517 . 99 . 9 ( 5 . 8–17 . 2 ) 3 . 2 ( 1 . 7–5 . 9 ) Lifetime no .", "of partners 051/6398 . 00 . 33 ( 0 . 24–0 . 46 ) 0 . 76 ( 0 . 53–1 . 1 ) 10/6841 . 50 . 49 ( 0 . 25–0 . 98 ) 1 . 4 ( 0 . 66–3 . 2 ) 1304/147520 . 61146/15742 . 911 2320/90035 . 62 . 1 ( 1 . 8–2 . 6 ) 2 . 0 ( 1 . 7–2 . 4 ) 82/9515 . 63 . 1 ( 2 . 2–4 . 5 ) 2 . 9 ( 2 . 0–4 . 2 ) 3139/28948 . 13 . 6 ( 2 . 7–4 . 6 ) 3 . 2 ( 2 . 4–4 . 2 ) 41/31313 . 15 . 0 ( 3 . 2–7 . 8 ) 4 . 4 ( 2 . 8–6 . 8 ) ≥453/10749 . 53 . 8 ( 2 . 5–5 . 6 ) 3 . 5 ( 2 . 3–5 . 3 ) 24/12020 . 08 . 3 ( 4 . 9–14 . 2 ) 7 . 4 ( 4 . 3–12 . 8 ) Distance from road <1 km410/158925 . 811100/15956 . 311 >1 km460/183025 . 10 . 97 ( 0 . 83–1 . 1 ) 0 . 94 ( 0 . 80–1 . 1 ) 62/18353 . 40 . 52 ( 0 . 38–0 . 72 ) 0 . 50 ( 0 . 36–0 . 70 ) Schooling None/Primary 1–5101/34729 . 1116/3784 . 211 Primary 6–7215/88924 . 20 . 78 ( 0 . 59–1 . 0 ) 0 . 88 ( 0 . 66–1 . 2 ) 38/9454 . 00 . 95 ( 0 . 52–1 . 7 ) 1 . 1 ( 0 . 59–2 . 0 ) Primary 8271/101126 . 80 . 89 ( 0 . 68–1 . 2 ) 0 . 89 ( 0 . 67–1 . 2 ) 58/10695 . 41 . 3 ( 0 . 74–2 . 3 ) 1 . 3 ( 0 . 75–2 . 4 ) Secondary 1–3218/86025 . 40 . 83 ( 0 . 63–1 . 1 ) 0 . 88 ( 0 . 65–1 . 2 ) 70/9167 . 61 . 9 ( 1 . 1–3 . 3 ) 2 . 0 ( 1 . 2–3 . 6 ) Secondary 4/Tertiary36/19618 . 40 . 55 ( 0 . 36–0 . 84 ) 0 . 42 ( 0 . 27–0 . 66 ) 18/2158 . 42 . 1 ( 1 . 0–4 . 1 ) 1 . 8 ( 0 . 87–3 . 6 ) Mother schooling <=Primary716/286125 . 011151/30215 . 011 Secondary80/35222 . 70 . 88 ( 0 . 68–1 . 1 ) 1 . 0 ( 0 . 76–1 . 3 ) 30/3827 . 91 . 6 ( 1 . 1–2 . 4 ) 1 . 8 ( 1 . 2–2 . 8 ) Father schooling <=Primary517/204625 . 31194/21584 . 411 Secondary260/109523 . 70 . 92 ( 0 . 78–1 . 1 ) 0 . 96 ( 0 . 80–1 . 1 ) 81/11667 . 01 . 6 ( 1 . 2–2 . 2 ) 1 . 7 ( 1 . 3–2 . 3 ) Housing quality 1 ( Best ) 148/66222 . 41171/7329 . 711 2149/56526 . 41 . 2 ( 0 . 96–1 . 6 ) 1 . 2 ( 0 . 89–1 . 5 ) 32/5985 . 40 . 53 ( 0 . 34–0 . 81 ) 0 . 48 ( 0 . 31–0 . 75 ) 3317/123825 . 61 . 2 ( 0 . 96–1 . 5 ) 1 . 0 ( 0 . 96–1 . 3 ) 60/12964 . 60 . 45 ( 0 . 32–0 . 65 ) 0 . 38 ( 0 . 26–0 . 55 ) 4 ( Worst ) 227/84326 . 91 . 3 ( 1 . 0–1 . 6 ) 1 . 2 ( 0 . 96–1 . 6 ) 38/9034 . 20 . 41 ( 0 . 27–0 . 61 ) 0 . 37 ( 0 . 25–0 . 56 ) Occupation Farmer652/222929 . 311143/23696 . 011 In education63/7208 . 80 . 23 ( 0 . 18–0 . 31 ) 0 . 55 ( 0 . 40–0 . 76 ) 10/7671 . 30 . 21 ( 0 . 11–0 . 39 ) 0 . 58 ( 0 . 28–1 . 2 ) Not working23/7729 . 91 . 0 ( 0 . 63–1 . 7 ) 1 . 1 ( 0 . 66–1 . 9 ) 8/889 . 11 . 6 ( 0 . 74–3 . 3 ) 1 . 6 ( 0 . 77–3 . 5 ) Other102/27237 . 51 . 5 ( 1 . 1–1 . 9 ) 1 . 3 ( 1 . 0–1 . 7 ) 38/29313 . 02 . 3 ( 1 . 6–3 . 4 ) 2 . 1 ( 1 . 4–3 . 1 ) Times of insufficient food in household in last year No674/257526 . 211162/27485 . 911 Yes166/72822 . 80 . 83 ( 0 . 69–1 . 0 ) 0 . 83 ( 0 . 67–1 . 0 ) 39/7755 . 00 . 85 ( 0 . 59–1 . 2 ) 0 . 85 ( 0 . 59–1 . 2 ) Times when can’t afford soap in last year No519/202125 . 711136/21666 . 311 Yes321/128025 . 10 . 97 ( 0 . 82–1 . 1 ) 0 . 97 ( 0 . 82–1 . 1 ) 65/13554 . 80 . 75 ( 0 . 56–1 . 0 ) 0 . 75 ( 0 . 55–1 . 0 ) OR = odds ratio Some women who reported that they were not yet sexually active tested positive for HSV-2 and/or HIV ( as previously noted ) ( Glynn et al . , 2011 ) .", "For both HSV-2 and HIV , the proportion positive increased with the lifetime number of partners , and was highest in those who had previously been married ( divorced or widowed ) and least common in those who had never married ( Table 1 ) .", "HIV infection was more common near the main road; in those with more schooling , and whose parents had had more schooling; in those living in better-built houses; and in those working in the cash-economy .", "HSV-2 infection was equally common in the more distant areas as in the areas close to the road and was not associated with parental schooling or type of housing .", "It was least common among those with secondary or further education , and among those still in education .", "It was slightly less common among those who reported food insecurity .", "These trends persisted after adjusting for current age ( Table 1 ) .", "Table 2 shows the effect of additionally adjusting the association of age at menarche with HSV-2 or HIV for socio-economic variables and sexual behaviour variables .", "It is restricted to those with no missing data for the factors included to allow possible mediation by these factors to be fully explored .", "As shown in Table 2 , adjustment for socio-economic and behavioural factors made no difference to the associations with HIV and only slightly weakened the trend with HSV-2 .", "The factors included are those that were independently associated with HSV-2 or HIV infection in a full model , or that confounded the association between age at menarche and HSV2 or HIV .", "Additional adjustment for the other factors shown in Table 1 made no further difference to the associations with HSV-2 or HIV . 10 . 7554/eLife . 01604 . 007Table 2 . Association between age at menarche and HSV2 and HIV , adjusted for other variables , among 15–30 year-old women in Karonga District MalawiDOI: http://dx . doi . org/10 . 7554/eLife . 01604 . 007HSV2 ( N = 3034 ) *HIV ( N = 3231 ) *Adjusted for ageAlso adjusted for socio-economic variablesAlso adjusted for behavioural variablesAdjusted for ageAlso adjusted for socio-economic variablesAlso adjusted for behavioural variablesOR ( 95% CI ) OR ( 95% CI ) †OR ( 95% CI ) ‡OR ( 95% CI ) OR ( 95% CI ) †OR ( 95% CI ) ‡Age at menarche<14111111140 . 91 ( 0 . 72–1 . 2 ) 0 . 92 ( 0 . 73–1 . 2 ) 0 . 92 ( 0 . 72–1 . 2 ) 0 . 97 ( 0 . 63–1 . 5 ) 0 . 94 ( 0 . 61–1 . 4 ) 0 . 96 ( 0 . 62–1 . 5 ) 150 . 73 ( 0 . 58–0 . 91 ) 0 . 76 ( 0 . 60–0 . 95 ) 0 . 77 ( 0 . 61–0 . 98 ) 0 . 68 ( 0 . 44–1 . 0 ) 0 . 64 ( 0 . 41–0 . 98 ) 0 . 65 ( 0 . 42–1 . 0 ) ≥160 . 70 ( 0 . 54–0 . 89 ) 0 . 75 ( 0 . 58–0 . 97 ) 0 . 77 ( 0 . 59–1 . 0 ) 1 . 2 ( 0 . 79–1 . 8 ) 1 . 1 ( 0 . 69–1 . 6 ) 1 . 1 ( 0 . 70–1 . 7 ) *To allow the effects of these potential mediators to be explored fully , the analysis is restricted to individuals with no missing data for the factors included .", "†age , schooling , occupation , lack of food .", "‡age , schooling , occupation , lack of food , marital status , lifetime number of partners ." ], [ "Age at menarche in this population has a profound influence on adolescent trajectories through sexual debut and marriage .", "We have previously shown that early menarche is associated with school drop-out ( Glynn et al . , 2010 ) .", "Here we show an association with HSV-2 infection , which is a marker of unprotected sex .", "The serological tests for HSV-2 have imperfect sensitivity and specificity ( van Dyck et al . , 2004 ) , and since the resultant bias should be non-differential the true association with menarche could be stronger .", "In our study this association was not explained by measured socio-economic or behavioural variables .", "This suggests that it was influenced by unmeasured factors , such as the risk groups of the partners , or by factors that we have measured imprecisely .", "Limitations in reported sexual behaviour are well known , with a tendency for women to under-report onset of sexual activity and number of partners ( Buve et al . , 2001; Glynn et al . , 2011; Soler-Hampejsek et al . , 2013 ) .", "The failure to explain the association may also be explained by the limitations of a cross-sectional study .", "The outcome was HSV-2 prevalence , not incidence , and the time of the infection was unknown .", "The age at menarche depended on recall .", "This would not be influenced by HSV-2 status , but could be influenced by ( recalled ) age at marriage or sexual debut .", "If the association between early menarche and HSV-2 infection is due to riskier behaviour or higher risk partners , an association with HIV infection might also be expected .", "Especially as HSV-2 infection and HIV infection are strongly associated , in this population ( not shown ) and elsewhere .", "( Glynn et al . , 2009 )", "However , as shown in Table 1 , HIV is much less uniformly distributed in the population and much less common , so the risk of a woman acquiring HIV depends less directly on her own or her partner’s behaviour , and more on the local prevalence ( and the probability that a given risk behaviour will lead to exposure to a partner with HIV infection ) , and on chance .", "The important role of population level rather than individual level risk factors for HIV has been shown in other settings ( Gabrysch et al . , 2008 ) .", "Early age at menarche appears to be a serious disadvantage to a girl’s life chances in this and other populations ( Biddlecom et al . , 2008; Downing and Bellis , 2009; Glynn et al . , 2010 ) .", "As nutrition improves , age at puberty decreases ( Bellis et al . , 2006 ) .", "Unless society’s expectations of a girl’s role once she reaches puberty changes , increases in early sex , marriage and sexually transmitted infections can be expected .", "We have already discussed the need for community and individual level interventions to encourage and enable adolescent girls with early menarche to stay in school throughout puberty and beyond .", "To this should be added specific concerns over risky sexual behaviour in this group ." ], [ "Demographic surveillance was established in a population of approximately 33 , 000 individuals living in one area of Karonga District , northern Malawi , in 2002 .", "The surveillance uses local key informants who register births and deaths continuously , and report them periodically to project staff .", "There was a re-census 2 years after registration was completed , and then annually thereafter .", "Full details are provided elsewhere .", "( Jahn et al . , 2007; Crampin et al . , 2012 )", "In 2007 , after extensive piloting , a sexual behaviour survey was added to follow the annual re-census , together with a serosurvey for HIV and HSV-2 .", "Each sexual behaviour interview was conducted in the local language , in private .", "Written informed consent was requested separately for the interview and the serosurvey ( Glynn et al . , 2011 ) .", "Ethics permission for the study was received from the Malawi Health Sciences Research Committee and the ethics committee of the London School of Hygiene & Tropical Medicine .", "Data from the first sexual behaviour survey round were used for this analysis .", "A question on age at menarche for women was added midway through the first round .", "For those with missing data on age at menarche from the first round , the response from the subsequent round was used .", "Other questions included the participant’s age at first sex , age at marriage , and number of partners , as well as schooling level and other socio-economic characteristics .", "HSV-2 tests were conducted for women aged 15–30 , using the type-2 specific enzyme immunoassay ( Kalon Biological Ltd , Surrey , UK ) which has the highest sensitivity and specificity of commercially available assays when used on African samples ( van Dyck et al . , 2004 ) .", "HIV testing used rapid tests with the results immediately available to the participants ( Floyd et al . , 2013 ) .", "We followed a parallel testing algorithm with Determine and Unigold , and SD-bioline as a tiebreaker if results were discordant , which we have previously shown to have very high sensitivity and specificity in this setting ( Molesworth et al . , 2010 ) .", "The association between age at menarche and ages at sexual debut and marriage were described using multistate life tables .", "The associations between age at menarche and other risk factors with HSV-2 and HIV were investigated using logistic regression .", "The extent to which the association between age at menarche and HSV-2 or HIV could be explained by measured behavioural and socio-economic factors was explored in multi-variable analyses , including these factors in the models , as appropriate .", "Analyses were done using STATA 12 . 1 ( StataCorp , College Station , TX ) .", "The data come from the Karonga Prevention study which has been conducting research in this population for more than 30 years .", "We have recently been awarded funding from the Wellcome Trust for a 4-year project to make our extensive data ( on ∼300 , 000 individuals and >1 million participant contacts ) more accessible , and to ensure that , as we continue to add to this unique resource , it remains usable , flexible and available to local and international researchers .", "This involves making major changes to the data structure , the user interface and the way new data are collected , whilst maintaining the integrity and relationships in the existing database .", "Meanwhile , a procedure for data sharing is in place , which requires the consent of the programme director in consultation with senior science staff .", "No reasonable data sharing requests are turned down and the programme is committed to ensuring as much access as possible to the data , while maintaining full confidentiality .", "Enquiries to Dr Mia Crampin ( mia . crampin@lshtm . ac . uk ) ." ] ]

[ "Remarkably little is known about associations between age at menarche and sexually transmitted infections , although girls with earlier menarche tend to have earlier sexual debut and school drop-out , so an association might be expected .", "In a population-based survey of >3000 women aged 15–30 in northern Malawi we show that those with earlier menarche had earlier sexual debut , earlier marriage and were more often Herpes simplex type-2 ( HSV-2 ) positive .", "Compared to those with menarche aged <14 , the age-adjusted odds ratios for HSV-2 were 0 . 89 ( 95%CI 0 . 71–1 . 1 ) , 0 . 71 ( 0 . 57–0 . 89 ) and 0 . 69 ( 0 . 54–0 . 89 ) for menarche aged 14 , 15 and 16+ respectively .", "This association persisted after adjusting for socio-economic factors , including schooling , and for sexual behaviour .", "No such association was seen with HIV infection , which is much less common and less uniformly distributed than HSV-2 in this population .", "The extra vulnerability of girls with earlier menarche needs to be recognised ." ]

[ "For many girls in sub-Saharan Africa their first menstrual cycle can mean an abrupt end to childhood because a girl’s first period is often taken as a signal that she is ‘ready’ for sex and marriage .", "Most girls in this region have their first menstrual cycle between the ages of 13 and 18 , with the median being around 15 , and this is likely to get earlier as nutrition improves .", "The age at which a girl in sub-Saharan Africa has her first period can have dramatic effects on her future prospects .", "Previous research in rural Malawi showed that more than half of those young women who had their first period before their 14th birthday never finish primary school and have sex before they are 16 years old .", "By comparison , 70% of women whose first menstrual cycle occurs at age 16 or older finish primary school and delay sex until after age 18 .", "To assess whether early menstruation might also be associated with the risk of contracting a sexually transmitted infection , Glynn et al . surveyed more than 3000 women between the ages of 15 and 30 in northern Malawi .", "Women who had their first menstrual cycle at an older age were less likely than those with an earlier onset of menstruation to be infected with Herpes simplex type-2 .", "This difference persisted after adjustment for age at first sex and marriage , and for differences in socio-economic position , education and number of sexual partners .", "This may suggest that women with earlier onset of menstruation tended to have higher risk partnerships .", "There was no relationship between HIV and age at first menstrual cycle , probably because HIV is much less common than Herpes in the areas where the women lived .", "Community and individual level interventions are needed to encourage and enable adolescent girls with early onset of menstruation to stay in school throughout puberty and beyond , and to help them reduce sexual risk taking from before their first sexual experiences ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "The N-acetylglucosaminyltransferase MGAT1 ( GlcNAc-TI or GnT-1 ) catalyzes the transfer of GlcNAc from UDP-GlcNAc to Man5GlcNAc2Asn of glycoproteins in the medial Golgi to initiate the synthesis of complex and hybrid N-glycans ( Robertson et al . , 1978; Tabas et al . , 1978; Kornfeld and Kornfeld , 1985 ) .", "In experiments to identify the activity of murine cDNA 41334120Rik , two transcripts were characterized in the mouse ( Huang and Stanley , 2010 ) .", "The longer transcript encodes a membrane-bound protein that inhibits MGAT1 in transfected cells , and is termed GlcNAcT-I Inhibitory Protein , Long form , GnT1IP-L ( Huang and Stanley , 2010 ) .", "GnT1IP-L is a Type II membrane glycoprotein with sequence homology to glycosyltransferase genes in family 54 in the CaZY database ( Cantarel et al . , 2009 ) .", "A rat testis membrane-bound form has been termed GL54D but its activity has not been determined ( Au et al . , 2015 ) .", "The mouse homologue of GL54D is the shorter transcript , previously termed GnT1IP-S ( Huang and Stanley , 2010 ) , and recently designated MGAT4D by the Human Genome Nomenclature Committee .", "When the N-terminus of GnT1IP-S is extended by a Myc or HA tag , it becomes membrane-bound and inhibits MGAT1 in cultured cells , similar to GnT1IP-L .", "In male germ cells mouse GnT1IP-S is probably membrane-bound like its rat homologue GL54D ( Au et al . , 2015 ) .", "The sequence of GnT1IP-L ( Genbank accession HM067443 ) is identical to GnT1IP-S with an additional 44 N-terminal amino acids .", "Our previous study ( Huang and Stanley , 2010 ) showed that transfection of a cDNA encoding GnT1IP-L inhibits endogenous or co-transfected MGAT1 activity in Chinese hamster ovary ( CHO ) cells .", "However , cell lysates of GnT1IP-L transfectants with low MGAT1 activity exhibit normal levels of B4GALT1 and MGAT3 activities ( the latter in LEC10 CHO cells [Campbell and Stanley , 1984] ) .", "Co-immunoprecipitation experiments showed that GnT1IP-L interacts physically with MGAT1 , but does not interact with trans Golgi B4GALT1 , nor the trans Golgi network sialyltransferase , ST8SIA2 .", "Deletion mutagenesis experiments showed that removal of 39 amino acids from the C-terminus of membrane-bound Myc-GnT1IP-S , or removal of the stem domain from Myc-GnT1IP-L , abrogates MGAT1 inhibitory activity ( Huang and Stanley , 2010 ) .", "In this paper , we investigate whether GnT1IP-L inhibits MGAT1 via its luminal or cytoplasmic and transmembrane ( TM ) domain , and whether GnT1IP-L retained in the endoplasmic reticulum ( ER ) can inhibit MGAT1 .", "The specificity of GnT1IP-L for MGAT1 compared to related medial Golgi GlcNAc-transferases , and the interactions of GnT1IP-L in the ER and Golgi , were investigated by dynamic fluorescent resonance energy transfer ( FRET ) and bimolecular fluorescence complementation ( BiFC ) experiments that previously identified homomeric and heteromeric interactions between Golgi glycosyltransferases ( Rivinoja et al . , 2009; Hassinen et al . , 2010 , 2011; Hassinen and Kellokumpu , 2014 ) .", "The combined results show that GnT1IP-L inhibitory activity lies in its luminal domain , that it forms homomers in the ER , and in the Golgi it forms heteromers specifically with MGAT1 .", "Interestingly , data extracted from published RNA-seq and microarray experiments reveal differential and complementary expression of mouse Mgat1 and GnT1IP/Mgat4d genes in male Sertoli and germ cells , and show that transcripts of human GnT1IP/MGAT4D are markedly reduced in testis biopsies of men with impaired spermatogenesis ." ], [ "To investigate whether the TM or luminal domain of GnT1IP-L is important for inhibition of MGAT1 in CHO cells , different mutant and chimeric expression plasmids were constructed ( Figure 1 and Table 1 ) .", "Constructs were transfected into CHO cells and stable populations selected for hygromycin resistance were examined for resistance to the toxicity of Phaseolus vulgaris leukoagglutinin ( L-PHA ) , and/or binding of the lectin Galanthus nivalis agglutinin ( GNA ) .", "Resistance to L-PHA , accompanied by increased expression of cell surface oligomannose N-glycans detected by GNA , are hallmarks of inhibition of MGAT1 activity in CHO cells ( Chen and Stanley , 2003; Huang and Stanley , 2010 ) .", "The subcellular localization of each construct was investigated by transient transfection of HeLa cells and analysis of immunofluorescence using antibodies to Myc or HA , Golgi α-mannosidase II ( MAN2A1 ) , or GM130 , or ER protein disulfide isomerase ( PDI ) .", "In initial experiments , five Phe residues in the GnT1IP-L TM domain were all replaced with either Leu ( similar hydrophobicity index to Phe ) or Ala ( hydrophobicity reduced ∼50% compared to Phe or Leu ) .", "Transfectants expressing GnT1IP-L ( F/L ) or GnT1IP-L ( F/A ) ( Table", "1 ) at similar levels based on western analysis , had an increased ability to bind GNA , and exhibited resistance to the toxicity of L-PHA ( Figure 2B and data not shown ) .", "Thus , replacement of five Phe residues with Ala in the TM domain of GnT1IP-L did not markedly reduce its MGAT1 inhibitory activity . 10 . 7554/eLife . 08916 . 003Figure 1 . Expression constructs . Mouse GnT1IP-L ( 417 aa ) contains an N-terminal cytoplasmic domain of 48 aa , a transmembrane ( TM ) domain of 21 aa ( shaded ) , and a luminal domain of 348 amino acids .", "The location of the Myc tag ( red ) is shown for each construct .", "Chimeric constructs contained the cytoplasmic and TM domain of MGAT1 ( green ) linked to the luminal domain of GnT1IP-L ( blue ) , or the cytoplasmic and TM domain of GnT1IP-L linked to the luminal domain of MGAT1 , or N-terminal aa 1–47 of human Invariant chain p33 ( Iv; beige ) linked to aa 45 to 417 of GnT1IP-L .", "Predicted TM domains are shown in darker colors .", "Numbers on top of each chimera are aa from the N-terminal domain and underneath are aa from the luminal domain . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 00310 . 7554/eLife . 08916 . 004Table 1 . Primers for expression constructsDOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 004GnT1IP-L-Myc For: 1301: ( HindIII , Kozak ) CAGATCAAGCTTCCACCATGTGCCTGGGAGAAAGTGTTGGGGACC Rev: 1346: ( Myc , BamH1 ) GACTAGGGATCCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTAATAATTATCCTTGAGGTGCMyc-GnT1IP-L For: 1312: ( HindIII , Kozak , Myc ) CAGATCAAGCTTCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGTGCCTGGGAGAAAGTGTTGGGGACC Rev: 1313: ( BamH1 ) GCCTGTGGATCCCTAGTAATAATTATCCTTGAGGTGCTGHA-GnT1IP-L For: 1068: ( HindIII , Kozak , HA-GnT1IP-L ) GGAACTAAGCTTCCACCATGTACCCTTATGACGTCCCCGATTACGCCAGCCTGTGCCTGGGAGAAAGTG TTGGGG Rev: 1313GnT1IP-L ( F/L ) For: 1431: CTCTTAGCCTTAGTTGCCGTCCTGCTCTTAGGTTTATCGTGTTTATGC Rev: 1432: GCATAAACACGATAAACCTAAGAGCAGGACGGCAACTAAGGCTAAGAGGnT1IP-L ( F/A ) For: 1467: CTCGCCGCCGCCGTTGCCGTCCTGCTCGCTGGTGCCTCGTGTGCCTGC Rev: 1468: GCAGGCACACGAGGCACCAGCGAGCAGGACGGCAACGGCGGCGGCGAGGnT1IP-L-Myc-KDEL For: 1301 Rev: 1471: ( BamH1 Myc-KDEL ) GGATCCCTACAACTCATCTTTCAGATCCTCTTCTGAGATGAGMGAT1/GnT1IP-L-Myc For: 1282 ( HindIII , Kozak ) GGACCGAAGCTTCCACCATGCTGAAGAAGCAGTCTGCAGGGCInternal: MGAT1/GnT1IP-L Rev: 1434: TTGATTATTGGTTTGGTTCATCCTCCAGAAGAAGAGGAGCAGCAG For: 1433: CTGCTGCTCCTCTTCTTCTGGAGGATGAACCAAACCAATAATCAA Rev: 1346: ( BamH1 , Myc ) GACTAGGGATCCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTAATAATTATCCTTGAGGTGCMGAT1/GnT1IP-L-Myc-KDEL For: 1282 Rev: 1471: ( BamH1 , Myc-KDEL ) GGAGTCGGATCCCTACAACTCATCTTTCAGATCCTCTTCTGAGATGAGIv/GnT1IP-L-Myc For: 1435: ( HindIII , Kozak ) GGACCGAAGCTTCCACCATGCACAGGAGGAGAAGCAGGInternal Iv/GnT1IP-L Rev: 1436: CAAGTTAACGTTCTTGGCCTTCATTCCGCGGCTGCACTTGCTCTC For: 1437: GAGAGCAAGTGCAGCCGCGGAATGAAGGCCAAGAACGTTAACTTG Rev: 1346: ( BamH1 , Myc ) Iv/GnT1IP-L-Myc-KDEL For: 1435 Rev: 1471GnT1IP-L/MGAT1-Myc For: 161: ( Kozak ) GCCACCATGTGCCTGGGAGAAAGTGTTGGGGACCTGInternal ( MGAT1/GnT1IP-L ) Rev: 162: GTCTGAGGGCAGCCTGCCAGGTGCTGGGCGGGAGATGCAGAAACACGAGAAACCAAAGAG For: 163: CTCTTTGGTTTCTCGTGTTTCTGCATCTCCCGCCCAGCACCTGGCAGGCTGCCCTCAGAC Rev: 164: ( MGAT1-Myc ) CTACAGATCTTCTTCAGAAATAAGTTTTTGTTCATTCCAGCTAGGATCATAGCCAGTCCATGT10 . 7554/eLife . 08916 . 005Figure 2 . The luminal domain of GnT1IP-L inhibits MGAT1 . ( A ) HeLa cells transiently expressing the chimera MGAT1/GnT1IP-L-Myc or MGAT1/GnT1IP-L-Myc-KDEL were analysed for expression of Myc , MAN2A1 , GM130 and protein disulfide isomerase ( PDI ) .", "Each result is representative of 40–50 cells examined .", "( B ) Resistance to L-PHA of the same chimeric proteins along with Myc-GnT1IP-L ( F/L ) and Myc-GnT1IP-L ( F/A ) in Chinese hamster ovary ( CHO ) transfectant populations selected for hygromycin resistance , compared to CHO and Lec1 cells .", "Independent transfectant populations gave the same results in 2–3 replicate assays .", "( C ) Western analysis of lysates corresponding to CHO populations numbered in panel B . The blot was probed with anti-Myc antibodies .", "* non-specific band loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 005 To investigate the GnT1IP-L luminal domain , the TM and cytoplasmic domains of GnT1IP-L were replaced with the cytoplasmic and TM domains of MGAT1 to create the construct MGAT1/GnT1IP-L-Myc ( Figure 1 and Table 1 ) .", "The chimeric protein was localized to the Golgi compartment ( Figure 2A ) , was well expressed , and conferred resistance to L-PHA in stable CHO transfectant populations ( Figure 2B , C ) .", "The L-PHA resistance assay in Figure 2B shows transfectants or control cells that were stained by methylene blue after ∼3 days of growth from 2000 cells plated in the presence of increasing concentrations of L-PHA .", "Plates were stained when wells incubated in medium alone ( no L-PHA ) had become confluent .", "The variability seen in the proportion of transfectants highly resistant to L-PHA in populations expressing GnT1IP-L mutant or chimeric proteins is due to variable expression levels of cDNAs and is also observed with wild-type GnT1IP-L ( see Figure 5B; Huang and Stanley , 2010 ) .", "The important parameter is the proportion of cells in a transfectant population that consistently resist the toxicity of L-PHA .", "Homogenous mutant Lec1 CHO cells that completely lack MGAT1 , or cells selected for high expression of GnT1IP-L ( Huang and Stanley , 2010 ) , are uniformly resistant to L-PHA ( Figure 2B ) .", "When a C-terminal KDEL retention sequence ( Cancino et al . , 2013 ) was added to the MGAT1/GnT1IP-L-Myc chimera , resistance to L-PHA was reduced ( Figure 2B ) , consistent with reduced localization to the Golgi ( Figure 2A ) .", "This result suggests that the luminal domain of GnT1IP-L is responsible for its ability to inhibit MGAT1 .", "An important control was to examine the reverse chimera—the cytoplasmic and TM domains of GnT1IP-L linked to the luminal domain of MGAT1 , termed GnT1IP-L/MGAT1-Myc ( Figure 1 and Table 1 ) .", "This chimera did not cause stable transfectants to become resistant to L-PHA ( Figure 3A ) , and did not induce hypersensitivity to Con A ( Figure 3B ) , in two independent clones with equivalent expression ( Figure 3C ) .", "In addition , the activity of MGAT1 in the GnT1IP-L/MGAT1-Myc transfectant lysates was 6 . 1 or 15 . 5 nmol/mg protein/hr , respectively , compared to 7 . 7 nmol/mg/hr in a CHO cell lysate and 0 . 5 nmol/mg protein/hr in a Lec1 lysate .", "The activity of B4GALT1 in the same lysates was equivalent ( 16–21 nmol/mg protein/hr ) .", "A separate experiment with the same extracts gave qualitatively similar results .", "The fact that one GnT1IP-L/MGAT1-Myc transfectant did not have increased MGAT1 activity may reflect the efficiency of active enzyme formation when the chimeric protein was overexpressed .", "Nevertheless , it is clear that GnT1IP-L/MGAT1-Myc does not significantly inhibit MGAT1 activity whereas MGAT1/GnT1IP-L is inhibitory .", "Thus , the GnT1IP-L luminal domain is active when localized by the MGAT1 cytoplasmic and TM domain , and the luminal domain of GnT1IP-L is necessary to inhibit MGAT1 activity . 10 . 7554/eLife . 08916 . 007Figure 3 . The TM and cytoplasmic domain of GnT1IP-L does not inhibit MGAT1 . ( A ) Lectin-resistance test of cloned CHO cells stably expressing GnT1IP-L/MGAT1-Myc compared to CHO cells and Lec1 CHO cells that lack MGAT1 ( n = 2 ) .", "( B ) The same cloned GnT1IP-L transfectant lines were compared to CHO and Lec1 cells for resistance to Con A ( n = 2 ) .", "( C ) Western analysis of CHO cell lysates from the cloned transfectants in ( A ) and ( B ) .", "* non-specific band shows equal loading . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 007 MGAT1 is a resident of the medial Golgi , along with other GlcNAc-transferases of the N-glycan pathway MGAT2 , MGAT3 , MGAT4 and MGAT5 .", "Previous experiments have shown that GnT1IP-L does not inhibit MGAT3 activity , although it interacted with MGAT3 in immunoprecipitation assays in CHO cell lysate .", "MGAT1 and MGAT2 form a complex ( Nilsson et al . , 1993 , 1994; Opat et al . , 2000 ) , and their interactions have been directly observed in a dynamic FRET assay ( Hassinen et al . , 2010 , 2011; Hassinen and Kellokumpu , 2014 ) .", "To determine if GnT1IP-L inhibits medial Golgi glycosyltransferases other than MGAT1 , CHO stable transfectants expressing HA-GnT1IP-L were assayed for MGAT2 and MGAT5 activities .", "A specific acceptor for MGAT4 was not available for assay .", "Relative expression was determined as a ratio to B4GALT1 activity , which is not affected by GnT1IP-L ( Huang and Stanley , 2010 ) .", "It can be seen in Table 2 ( Source code", "1 ) that whereas GnT1IP-L inhibited MGAT1 activity by ∼75% , MGAT2 and MGAT5 activities were not markedly inhibited in CHO cells .", "We also found that GnT1IP-L inhibits MGAT1 activity in COS-7 cells , and induces increased expression of GNA binding reflecting increased expression of oligomannose N-glycans at the surface of COS-7 cells , as expected when MGAT1 is inhibited ( data not shown ) . 10 . 7554/eLife . 08916 . 008Table 2 . Glycosyltransferase activities in CHO cells expressing HA-GnT1IP-LDOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 008CellsB4GALT1 nmol/mg/hrMGAT1 nmol/mg/hrMGAT2 nmol/mg/hrMGAT5 nmol/mg/hrCHORatio to B4GALT115 . 6 ( 11–23 . 8 ) –4 . 2 ( 3 . 2–5 . 5 ) 0 . 270 . 41 ( 0 . 4–0 . 42 ) 0 . 0260 . 34 ( 0 . 35–0 . 48 ) 0 . 022CHO/HA-GnT1IP-LRatio to B4GALT111 . 5 ( 9 . 7–13 . 2 ) –1 . 1 ( 0 . 9–1 . 3 ) 0 . 0960 . 38 ( 0 . 16–0 . 61 ) 0 . 0330 . 33 ( 0 . 24–0 . 41 ) 0 . 028Ratio activityGnT1IP-L:CHO0 . 740 . 260 . 930 . 97Glycosyltransferase assays were performed as described in ‘Materials and methods’ on cell extracts from CHO cells and CHO cells stably expressing HA-GnT1IP-L .", "Each assay was performed in duplicate , and activity ( with range ) is given as the average of duplicates for 2–3 independent assays .", "To further investigate the specificity of GnT1IP-L for MGAT1 , a dynamic FRET assay was employed .", "Previous assays of glycosyltransferases tagged at the C-terminus by monomeric cerulean ( mCer ) or monomeric venus ( mVen ) determined FRET interactions by flow cytometry and showed that numerous glycosyltransferases of the N- and O-glycan pathways form homomers in the ER and heteromers in the Golgi ( Rivinoja et al . , 2009; Hassinen et al . , 2010 , 2011; Rivinoja et al . , 2012; Hassinen and Kellokumpu , 2014 ) .", "The latter heteromeric interactions occur between glycosyltransferases that act sequentially in the same glycan pathway and in the same compartment of the Golgi .", "Interactions between GnT1IP-L and medial Golgi GlcNAc-transferases MGAT1 to MGAT5 were investigated by the same methods via transient transfection into COS-7 or Lec1 CHO cells stably expressing GnT1IP-L , or transient co-transfection of GnT1IP-L cDNA with an individual MGAT cDNA .", "Mouse GnT1IP-L-mVen was investigated for interactions with mouse or human MGAT1 , MGAT2 , MGAT3 , MGAT4B and MGAT5 .", "First , it was shown by fluorescence microscopy that each of the human GlcNAc-transferases tagged with mChe localized correctly to the Golgi when transfected into COS-7 or Lec1 CHO cells stably expressing GnT1IP-L-mVen ( Figure 4A and data not shown ) .", "Measurements of FRET efficiencies revealed that GnT1IP-L interacted with MGAT1 but not with MGAT2 , MGAT3 , MGAT4B or MGAT5 in COS-7 or Lec1 CHO cells which lack endogenous MGAT1 ( Chen and Stanley , 2003 ) ( Figure 4B and Figure 4—source data 1 ) .", "The same results were obtained with the mouse GlcNAc-transferases ( Figure 4C and Figure 4—source data 1 ) .", "The lower FRET efficiencies of mouse enzymes may reflect species differences as lower expression of all mouse MGAT constructs was observed .", "If the data are expressed as a percentage of the MGAT1/GnT1IP-L interaction , no differences are evident between mouse and human interactions .", "These data also show that GnT1IP-L forms homomers with itself as well as heteromers with MGAT1 .", "The specificity of the FRET signal between GnT1IP-L and MGAT1 was further demonstrated by inhibition of complex formation by overexpression of either GnT1IP-L-HA or MGAT1-HA ( Figure 4D and Figure 4—source data 1 ) .", "As expected , overexpression of HA-tagged MGAT2 , MGAT3 , MGAT4B or MGAT5 did not inhibit the FRET signal induced by co-transfection of GnT1IP-L and MGAT1 ( Figure 4D ) . 10 . 7554/eLife . 08916 . 009Figure 4 . GnT1IP-L interacts specifically with MGAT1 in the Golgi .", "( A ) Fluorescence microscopy of COS-7 cells stably expressing GnT1IP-L-mVen and transiently expressing medial Golgi GlcNAc-transferases MGAT1 to MGAT5 conjugated to mChe at their C-terminus compared to the Golgi marker GM130 .", "( B ) GnT1IP-L interaction with human GlcNAc-transferases .", "COS-7 or Lec1 CHO cells stably expressing GnT1IP-L-mVen were transfected with human cDNAs encoding MGAT1 to MGAT5 C-terminally tagged with mChe , and fluorescent resonance energy transfer ( FRET ) efficiencies were determined .", "( C ) GnT1IP-L interaction with mouse GlcNAc-transferases .", "COS-7 cells transiently expressing mouse GnT1IP-L-mVen and GnT1IP-L-mChe or mouse MGAT1-mChe , MGAT2-mChe , MGAT3-mChe , MGAT4B-mChe or MGAT5-mChe and FRET efficiencies determined .", "( D ) COS-7 cells stably expressing GnT1IP-L-mVen were co-transfected with mouse MGAT1-mChe together with competitive cDNA encoding mouse MGAT1 to MGAT5 C-terminally tagged with HA .", "( E ) Transiently co-expressed MGAT1-mVen , MGAT2-mChe and GnT1IP-L-HA are localized in the Golgi .", "( F ) COS-7 cells were transiently expressed with MGAT1-mVen , MGAT2-mChe and GnT1IP-L-HA , and FRET efficiencies were determined .", "Bars represent the mean ± STDEV ( n = 10 cells ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 00910 . 7554/eLife . 08916 . 010Figure 4—source data 1 . GnT1IP-L interactions with human and mouse MGATs in the Golgi of COS-7 and CHO Lec1 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 010 MGAT1 and MGAT2 have previously been shown by FRET analyses to form heteromers in the Golgi ( Hassinen et al . , 2010 , 2011; Hassinen and Kellokumpu , 2014 ) .", "To determine if GnT1IP-L inhibits formation of MGAT1/MGAT2 heteromers , tagged human or mouse MGAT1 and MGAT2 were transiently expressed with competitive GnT1IP-L-HA in COS-7 cells , and FRET efficiencies measured .", "All proteins localized to the Golgi ( Figure 4E ) , and the presence of GnT1IP-L-HA did not interfere with the formation or stability of MGAT1/MGAT2 heteromers ( Figure 4F and Figure 4F—source data 1 ) .", "When GnT1IP-L is overexpressed , Golgi-localized MGAT1 is markedly relocated to the ER ( Huang and Stanley , 2010 ) .", "To determine if GnT1IP-L interacts with MGAT1 in the ER prior to exit for the Golgi , chimeric proteins were constructed using the N-terminal ER retention signal from human invariant chain p33 ( termed Iv ) ( Nilsson et al . , 1991 ) , with and without a C-terminal KDEL retention sequence .", "Transfection of Iv/GnT1IP-L-Myc into HeLa cells gave predominant expression in the ER , whereas co-transfected MGAT1-HA was largely localized to the Golgi compartment ( Figure 5A ) .", "Expression in wild type CHO cells was robust but did not lead to resistance to L-PHA when either Iv/GnT1IP-L-Myc or Iv/GnT1IP-L-Myc-KDEL were overexpressed in CHO cells ( Figure 5B , C ) .", "Therefore , GnT1IP-L that is largely localized to the ER does not inhibit MGAT1 activity . 10 . 7554/eLife . 08916 . 006Figure 5 . ER-localized GnT1IP-L does not inhibit MGAT1 . ( A ) HeLa cells transiently expressing GnT1IP-L-Myc and MGAT1-HA , or the chimera Iv/GnT1IP-L-Myc with and without MGAT1-HA were analysed for expression of Myc , HA and PDI .", "Each result is representative of 40–50 cells examined .", "( B ) Lectin-resistance test comparing the various GnT1IP-L stable transfectant populations with CHO cells for resistance to L-PHA .", "( C ) Western analyses of CHO and stable transfectant populations expressing GnT1IP-L chimeric proteins probed with anti-Myc antibody .", "Lanes are numbered according to the corresponding cell populations in panel B . Lanes ( 1 ) and ( 2 ) were cropped from the same blot .", "Lane 5 is CHO cells expressing MGAT1-HA .", "* non-specific band loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 006 MGAT1 , MGAT2 and other glycosyltransferases undergo homomeric interactions in the ER and heteromeric interactions in the Golgi ( Hassinen et al . , 2011; Hassinen and Kellokumpu , 2014 ) .", "This paradigm was also investigated for GnT1IP-L and MGAT1 using BiFC analysis with N-terminal Venus ( VN ) or C-terminal Venus ( VC ) fragments attached to the C-terminus of MGAT1 or GnT1IP-L .", "Expression and Golgi localization of VN and VC fusion proteins were confirmed by staining with rabbit anti-GFP Ab ( detected with anti-rabbit Ab conjugated to Alexa Fluor 594 ) , and goat anti-GFP Ab ( detected with anti-goat Ab conjugated to Alexa Fluor 405 ) .", "Confocal microscopy was performed with a filter set that detected BiFC signal ( green ) , VN signal ( red ) and VC signal ( blue ) .", "To examine complex formation in the ER , COS-7 cells were treated with the microtubule disruptor nocodazole for 8 hr to prevent exit from the ER , as previously described ( Hassinen and Kellokumpu , 2014 ) .", "The effect of nocodazole treatment on interactions between GnT1IP-L-VN and GnT1IP-L-VC is seen in Figure 6A .", "Compared to the control in which both proteins were localized in the Golgi , nocodazole treatment ( added 8 hr post-tranfection for 16 hr ) , caused both to accumulate in the ER .", "A BiFC signal was observed indicating GnT1IP-L homomer formation ( Figure 6A , Nocodazole ) .", "In the absence of nocodazole ( Control ) , homomers were transported to the Golgi .", "By contrast , when GnT1IP-L–VN was co-transfected with MGAT1-VC , a BiFC signal was not detected in nocodazole-treated cells ( Figure 6B ) , indicating that heteromer formation did not take place in the ER ( Figure 6B ) .", "However , in the absence of nocodazole ( Figure 6B , Control ) , GnT1IP-L-VN and MGAT1-VC were transported to the Golgi with the concomitant emergence of the BiFC signal due to heteromer formation . 10 . 7554/eLife . 08916 . 011Figure 6 . GnT1IP-L and MGAT1 form homomers in the endoplasmic reticulum ( ER ) and heteromers in the Golgi .", "( A ) COS-7 cells were co-transfected with mouse GnT1IP-L-VN and GnT1IP-L-VC and , after 8 hr in culture , one set of plates was treated with 1 µg/ml nocodazole overnight .", "Cells were examined by fluorescence microscopy ( 50 cells/view ) for expression and bimolecular fluorescence complementation ( BiFC ) signal .", "The VN tag was detected with rabbit anti-GFP and anti-rabbit Ab conjugated to Alexa Fluor 594 , and the VC tag was detected with goat anti-GFP and anti-goat Ab conjugated to Alexa Fluor 405 .", "Confocal imaging detected the BiFC signal ( green ) , VN signal ( red ) and VC signal ( blue ) .", "( B ) COS-7 cells were co-transfected with mouse GnT1IP-L-VN and human MGAT1-VC , treated with nocodazole as in ( A ) , and examined by fluorescence microscopy for expression and BiFC signal .", "All cells expressing GnT1IP-L-VN with GnT1IP-L-VC gave a BiFC signal in the presence and absence of nocodazole .", "In contrast , no signal was detected in nocodazole-treated cells expressing GnT1IP-L-VN with MGAT1-VC . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 011 To further investigate GnT1IP-L/MGAT1 heteromer assembly , we utilized the dynamic FRET assay .", "COS-7 cells co-transfected with GnT1IP-L-mVen and MGAT1-mChe were treated with nocodazole at 16 hr post-transfection .", "Golgi-localized heteromers of GnT1IP-L and MGAT1 ( Figure 7A , Control ) were relocated to the ER ( Figure 7A , Nocodazole ) with a concomitant reduction of the FRET signal ( Figure 7B and Figure 7—source data 1 ) .", "Removal of nocodazole allowed reformation of heteromeric complexes in the Golgi ( Figure 7A , B , Recovery ) .", "The histogram shows that , compared to the GnT1IP-L/MGAT1 heteromeric FRET signal , nocodazole treatment reduced heteromer formation , and removal of nocodazole partially rescued heteromer formation , whereas GnT1IP-L homomers were not affected by nocodazole treatment ( Figure 7B ) . 10 . 7554/eLife . 08916 . 012Figure 7 . Golgi-localized GnT1IP-L and MGAT1 heteromers are disrupted in the ER following nocodazole treatment and reform after recovery .", "( A ) COS-7 cells stably expressing GnT1IP-L-mVen were transfected with human MGAT1-mChe and , after 16 hr , treated with 1 µg/ml nocodazole .", "After 4 hr of treatment , nocodazole was removed from half the samples and recovery allowed to occur for 4 hr .", "Samples were examined by fluorescence microscopy .", "( B ) FRET efficiencies of COS-7 cells stably expressing GnT1IP-L-mVen and transfected with either MGAT1-mChe or GnT1IP-L-mChe ( as in A ) were determined by FRET microscopy .", "FRET efficiencies ( mean ± STDEV; n = 10 cells ) are given as % of control .", "Control samples ( 100% ) gave a FRET efficiency of 38 ± 3% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 01210 . 7554/eLife . 08916 . 013Figure 7—source data 1 . Disruption of Golgi-localized GnT1IP-L and MGAT1 heteromers in the ER following nocodazole treatment and their recovery in the Golgi after drug removal . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 013 Previous experiments showed that increasing the pH of the Golgi by ∼0 . 4 units following incubation for 4–16 hr in chloroquine inhibits heteromer formation and favors homomer formation between glycosyltransferases ( Hassinen and Kellokumpu , 2014 ) .", "To determine if Golgi acidity is also important for the formation of GnT1IP-L heteromers with MGAT1 , COS-7 cells stably expressing GnT1IP-L-mVen were co-transfected either with GnT1IP-L-mChe or MGAT1-mChe , and treated with 40 μM chloroquine for 4 hr ( added 16 hr post-transfection ) , or for 16 hr ( added at 8 hr post-transfection ) .", "Compared to untreated controls , either treatment with chloroquine did not significantly reduce GnT1IP-L homomers , but caused an ∼60% reduction in GnT1IP-L/MGAT1 heteromers ( Figure 8A and Figure 8—source data 1 ) .", "To evaluate whether this reduction in heteromers is accompanied by an increase in the amount of GnT1IP-L homomers , MGAT1-HA and GnT1IP-L-mChe were co-transfected into cells stably expressing GnT1IP-L-mVen , and treated with chloroquine .", "The proportion of homomers increased almost twofold , presumably due to the disruption of heteromer formation ( 16 hr treatment ) , or their disassembly ( 4 hr treatment ) at the higher Golgi pH ( Figure 8B and Figure 8—source data 1 ) .", "Chloroquine treatment did not impair the Golgi localization of any of the test proteins ( Figure 8C ) .", "Therefore GnT1IP-L , like MGAT1 as shown previously ( Hassinen and Kellokumpu , 2014 ) , forms homomers in the ER and heteromers with MGAT1 in the acidic Golgi lumen , where it inhibits MGAT1 activity . 10 . 7554/eLife . 08916 . 014Figure 8 . Chloroquine inhibits the formation of GnT1IP-L and MGAT1 heteromers and favors homomers .", "( A ) COS-7 cells stably expressing GnT1IP-L-mVen were transfected with mouse GnT1IP-L-mChe or human MGAT1-mChe and treated after 8 or 16 hr with 40 μM chloroquine for 16 hr or 4 hr , respectively .", "Untreated control cells ( 100% ) gave FRET efficiencies of 32% for the GnT1IP-L homomer and 38% for the heteromer with MGAT1 .", "( B ) The same experiment as in ( A ) was performed except that , in addition , MGAT1-HA was added as a competitor .", "Bars in ( A ) and ( B ) show mean ± STDEV ( n = 10 cells ) .", "( C ) Fluorescence microscopy of the transfected cells in the presence or absence of 40 µM chloroquine . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 01410 . 7554/eLife . 08916 . 015Figure 8—source data 1 . Disruption of GnT1IP-L and MGAT1 heteromers and enhanced formation of homomers following chloroquine treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 015 We previously identified a potential function for GnT1IP-L in testis based on the observation that cells expressing GnT1IP-L , Myc-GnT1IP-S or lacking MGAT1 bind more tightly to a Sertoli cell line ( Huang and Stanley , 2010 ) .", "Lectin histology experiments in mouse and rat have shown that spermatocytes bind low levels of L-PHA ( Jones et al . , 1992; Batista et al . , 2012 ) , reflecting low expression of complex N-glycans due potentially to inhibition of MGAT1 by GnT1IP-L ( Jones et al . , 1992; Batista et al . , 2012 ) .", "The GnT1IP/Mgat4d gene is very highly expressed in mouse testes compared to all other tissues ( see Mgat4d BioGPS microarray data [Wu et al . , 2009 , 2013] ) .", "In mouse germ cells , expression of GnT1IP/Mgat4d based on microarray and RT-PCR data is very low in spermatogonia , highest in spermatocytes and intermediate in spermatids ( Chalmel et al . , 2007; Huang and Stanley , 2010 ) .", "This expression pattern in mouse germ cells is complementary to Mgat1 that is high in spermatogonia , and greatly reduced in spermatocytes ( Chalmel et al . , 2007 ) .", "Very similar results are evident from an analysis of mouse RNA-Seq data that we interrogated for GnT1IP/Mgat4d and Mgat1 transcripts ( Gene Expression Omnibus Dataset GSE43717; [Soumillon et al . , 2013a , 2013b] ) .", "Mapping the relative expression values of GnT1IP/Mgat4D , ( ENSMUSG00000035057 ) and Mgat1 ( ENSMUSG00000020346 ) onto the expression values of all 36 , 823 transcripts for different mouse germ cell subtypes clearly indicates that GnT1IP/Mgat4D ( Figure 9 , blue ) is exclusively expressed in post-meiotic germ cells ( Figure 9—source data 1 ) .", "In contrast , Mgat1 ( Figure 9 , red ) is expressed at lower levels in all germ cell types , as well as somatic Sertoli cells .", "These results , as well as the observation that antibodies to rat GnT1IP ( GL54D ) detect signals in spermatocytes and spermatids but not spermatogonia ( Au et al . , 2015 ) , suggest post-meiotic transcriptional activation of the GnT1IP/Mgat4d gene .", "Interestingly , examination of the Soumillon et al . RNA-Seq data for the 130 nucleotides upstream of the Mgat4d start site which encode the sequence specific to GnT1IP-L , revealed very low numbers of reads that were not significant ( data not shown ) .", "This may reflect the regulated expression of GnT1IP-L during spermatogenesis ( Iguchi et al . , 2006; Huang and Stanley , 2010 ) . 10 . 7554/eLife . 08916 . 016Figure 9 . RNA-Seq data for GnT1IP/Mgat4d and Mgat1 in mouse germ cells . Histogram overlay plot for GnT1IP/Mgat4D ( blue ) and Mgat1 ( red ) gene expression in isolated mouse germ cell subtypes as described in Soumillon et al . ( 2013a ) .", "( A ) Sertoli cells , ( B ) Spermatogonia , ( C ) Spermatocytes , ( D ) Spermatids , ( E ) Spermatozoa .", "The grey histogram reflects the log2-transformed Fragments Per Kilobase of transcript per Million mapped reads ( FPKM ) values of all 36 , 823 transcripts identified by RNA sequencing and deposited in the GEO database as GSE43717 .", "Red and blue overlayed vertical lines depict the expression values for Mgat1 ( ENSMUSG00000020346 ) and GnT1IP/Mgat4D ( ENSMUSG00000035057 ) , respectively .", "Note the absence of GnT1IP/Mgat4D transcripts in Sertoli cells , spermatogonia and spermatozoa . DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 01610 . 7554/eLife . 08916 . 017Figure 9—source data 1 . R code and comma-delimited data files for generating Figure 9 . The R code files import the data files and exactly reproduce Figure", "9 . The data file contains the log2 FPKM data for the different testicular cell types from Soumillon et al . ( Cell Reports 3 , 2179–2190 , 2013 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 017 GnT1IP/MGAT4D is also very highly expressed in human testis compared to 26 other tissues examined by RNA-Seq ( ArrayExpress E-MTAB-1733 MGAT4D ENSG00000205301 [Fagerberg et al . , 2013 , 2014] ) .", "In another study , MGAT4D transcripts were shown to be highly enriched in human testis ( 29 fragments per kilobase of transcript per million mapped reads ( FPKM ) compared to a maximum FPKM of 0 . 1 for MGAT4D transcripts in 26 human tissues [Djureinovic et al . , 2014] ) .", "To determine GnT1IP/MGAT4D expression in human germ cell subtypes , we investigated microarray data ( ArrayExpress E-TABM-234 ) ( Cappallo-Obermann et al . , 2008 ) of human testis biopsies from men with different testicular phenotypes of impaired spermatogenesis ( Spiess et al . , 2007 ) with respect to the expression of GnT1IP/MGAT4D and MGAT1 .", "The data show that GnT1IP/MGAT4D transcripts are very poorly expressed in all testicular phenotypes in which there are no pre-meiotic germ cells in the germinal epithelium ( Tubular atrophy , TA; Sertoli cell only syndrome , SCO ) , or only pre-meiotic germ cells ( only spermatogonia present ( SPG ) ) ( Figure 10A; Figure 10—source data 1 ) .", "It is also evident that the phenotype of meiotic arrest ( MA ) , which in the majority of cases occurs at the level of pachytene spermatocytes in the human , exhibits no significant GnT1IP/MGAT4D expression .", "However , testicular phenotypes presenting with reduced ( hypospermatogenesis , HYS ) or normal and unimpaired levels of round and elongated spermatids ( full spermatogenesis , FS ) , display a massive increase in GnT1IP/MGAT4D expression .", "These findings point to a clear post-meiotic expression of GnT1IP during human spermatogenesis that occurs earliest at the level of secondary spermatocytes ( mitotic phase of meiosis ) or spermatids . 10 . 7554/eLife . 08916 . 018Figure 10 . GnT1IP/MGAT4D and MGAT1 transcripts in testis biopsies from men with impaired spermatogenesis . Transcript levels of GnT1IP/MGAT4D and MGAT1 were determined from the microarray data of Spiess et al . ( 2007 ) .", "( A ) Boxplot of GnT1IP/MGAT4D log2 fluorescence in human testicular biopsies presenting with different types of spermatogenic impairment ( tubular atrophy ( TA ) , Sertoli cell only ( SCO ) , presence of spermatogonial cells ( SPG ) , meiotic arrest ( MA ) at the level of primary spermatocytes , hypospermatogenesis with decreased numbers of round/elongated spermatids ( HYS ) , and full spermatogenesis with normal numbers of round/elongated spermatids ( FS ) ) .", "Sample size ( n ) for each group is given below the abscissa .", "( B ) Same as in ( A ) , but for MGAT1 .", "Note the decreasing transcript abundance , which is a common observation for somatic transcripts in the presence of increasing germ cell content ( Cappallo-Obermann et al . , 2013 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 01810 . 7554/eLife . 08916 . 019Figure 10—source data 1 . R code and comma-delimited data files for generating Figure 10 . The R code files import the data files and exactly reproduce Figure", "10 . The data file contains the log2-transformed expression values for MGAT4D and MGAT1 for sample replicates of different spermatogenic arrest states , as described in Spiess et al . ( Hum Reprod 22 , 2936–2946 , 2007 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08916 . 019 By contrast , MGAT1 transcripts concomitantly decrease with increasing germ cell differentiation ( Figure 10B ) , with highest expression in testicular phenotypes without germ cells ( TA , SCO ) .", "This indicates an expression largely restricted to testicular somatic cell types .", "A small increase at the level of spermatogonia suggests that , in humans , MGAT1 is expressed in spermatogonia whereas GnT1IP/MGAT4D is not , tallying with the data obtained from mouse microarray studies ( Chalmel et al . , 2007 ) .", "The overall decline of MGAT1 expression throughout spermatogenesis reflects a typical somatic transcript dilution effect ( compare Figure 1 in Cappallo-Obermann et al . , 2013 ) , due to increasing numbers of germ cell-specific transcripts ." ], [ "In this paper we show that the MGAT1 inhibitory activity of GnT1IP-L requires its luminal domain .", "Thus , mutations in the TM domain from Phe to Leu or Ala , or swapping the cytoplasmic and TM domain with that of MGAT1 , do not significantly reduce GnT1IP-L inhibitor activity .", "The requirement for the luminal domain is consistent with our previous findings that removal of the C-terminal 39 aa of membrane-bound GnT1IP-S , or the stem domain of GnT1IP-L , abrogate inhibitor activity ( Huang and Stanley , 2010 ) .", "The specificity of GnT1IP-L for MGAT1 vs other GlcNAc-transferases of the medial Golgi was investigated here using BiFC and a dynamic FRET assay .", "These experiments showed no significant FRET activity between GnT1IP-L and MGAT2 , MGAT3 , MGAT4B or MGAT5 .", "The only substantial FRET signal was obtained between GnT1IP-L and MGAT1 , and this signal could only be inhibited by overexpression of either GnT1IP-L or MGAT1 .", "As this result implies , and as shown previously for MGAT1 , GnT1IP-L forms homomers with itself , as well as heteromers with MGAT1 .", "These interactions were further defined using BiFC and FRET experiments following treatment with nocodazole or chloroquine .", "The combined data show that GnT1IP-L preferentially forms homomers in cells treated with nocodazole when it is confined to the ER , and heteromers with MGAT1 following nocodazole removal and a recovery period when it moves to the Golgi .", "GnT1IP-L homomers are also formed preferentially when the Golgi pH is elevated by treatment with chloroquine .", "Therefore , GnT1IP-L behaves like the glycosyltransferases previously studied ( Hassinen and Kellokumpu , 2014 ) , interacting with itself in the ER and primarily with MGAT1 in the Golgi .", "It is interesting that GnT1IP-L showed no FRET interaction with MGAT2 which is predicted to be in a ‘kin recognition’ complex with MGAT1 ( Nilsson et al . , 1993 , 1994 ) , and which has been shown to form heteromers with MGAT1 using the dynamic FRET assay used here ( Hassinen et al . , 2010 , 2011; Hassinen and Kellokumpu , 2014 ) .", "GnT1IP-L also did not inhibit or disrupt the formation of MGAT1/MGAT2 heteromers in a competition assay .", "Therefore , it may be concluded that when GnT1IP-L is in a complex with MGAT1 , MGAT2 is not excluded from that complex , and that GnT1IP-L binds to a different site on MGAT1 than MGAT2 .", "In addition , our data show that overexpression of MGAT2 did not disrupt GnT1IP-L/MGAT1 heteromers .", "The same lack of competition was observed for overexpression of MGAT3 , MGAT4B and MGAT5 .", "A recent study of rat testis identified GL54D , the rat homologue of mouse GnT1IP-S , as the most abundant species amongst membrane proteins in Golgi preparations ( Au et al . , 2015 ) .", "Immunohistochemistry showed that rat GL54D is confined to spermatocytes and spermatids , consistent with the expression pattern of GnT1IP transcripts in purified mouse germ cells ( Figure 9; [Soumillon et al . , 2013a] ) .", "Thus , while the GL54D homologue GnT1IP-S is secreted from CHO cells ( Huang and Stanley , 2010 ) , it is likely to be membrane-bound in mouse germ cells , similar to GL54D in rat ( Au et al . , 2015 ) .", "While it is possible that GnT1IP may also have a glycosyltransferase activity , as suggested by its recent designation as MGAT4D , we have observed no evidence of such an activity in transfected cells .", "In characterizing the CHO N-glycans generated following expression of membrane-bound GnT1IP , no complex N-glycans that might reflect GlcNAc transfer were identified by mass spectrometry , but rather a great increase in the abundance of the Man5GlcNAc2 substrate of MGAT1 was observed ( Huang and Stanley , 2010 ) .", "In addition , we have shown that GnT1IP-L induces increased binding of GNA reflecting enhanced expression of oligomannose N-glycans , increased resistance to lectins that bind complex N-glycans , and/or inhibition of MGAT1 activity , in a variety of cell lines including CHO , COS-7 , HeLa cells ( Huang and Stanley , 2010; this work ) and PC3 cells ( unpublished observations ) .", "Of course , a glycan , protein or lipid substrate may be present in only very low quantities or not expressed in CHO cells .", "Thus it cannot be ruled out that GnT1IP-L has an activity other than its ability to specifically inhibit MGAT1 .", "Importantly however , another example of a gene that has homology to , and the protein domain structure of a glycosyltransferase , but an activity that is distinct , is C1GALT1C1 , originally called COSMC ( Ju and Cummings , 2002; Wang et al . , 2010 ) .", "This protein is a specific chaperone dedicated to the Gal-transferase C1GALT1 , and essential for its activity ( Wang et al . , 2010 ) .", "Thus , although GnT1IP-L ( or MGAT4D ) has homology to family 54 glycosyltransferases , the only activity yet identified for membrane-bound GnT1IP is as a specific inhibitor of MGAT1 .", "A functional role for GnT1IP-L has been proposed in testis based on the following: the gene encoding GnT1IP-L is most highly expressed in testis compared to other mouse tissues ( Wu et al . , 2009 , 2013; Djureinovic et al . , 2014; Fagerberg et al . , 2014 ) , the expression of the transcript encoding GnT1IP-L is developmentally regulated ( Huang and Stanley , 2010 ) ; GnT1IP is well expressed in spermatocytes and spermatids but not in spermatogonia ( Chalmel et al . , 2007; Huang and Stanley , 2010 ) ; and cells expressing GnT1IP-L and oligomannose N-glycans bind more tightly to TM4 Sertoli cells than cells expressing MGAT1 and complex N-glycans ( Huang and Stanley , 2010 ) .", "Most interestingly , the gene encoding MGAT1 is expressed in a complementary manner to GnT1IP-L in male germ cells ( Figure 9 and [Chalmel et al . , 2007] ) .", "We are currently investigating the hypothesis that membrane-bound GnT1IP functions to down-regulate MGAT1 activity in spermatocytes and potentially spermatids , thereby enhancing their ability to bind to Sertoli cells .", "It is therefore of interest that men with impaired spermatogenesis exhibit greatly reduced expression of GnT1IP in microarray studies of testis biopsies ( Figure 10 ) .", "The degree of reduction appears to reflect the proportion of the remaining population of germ cells .", "Interestingly , MGAT1 transcripts were not reduced but were slightly increased reflecting robust expression of MGAT1 in Sertoli cells ( Chalmel et al . , 2007 ) .", "Germ cell expression of MGAT1 is however , essential for spermatogenesis in mice since conditional deletion of the Mgat1 gene in spermatogonia blocks spermatogenesis and results in infertile males ( Batista et al . , 2012 ) .", "Ongoing studies with conditional knockout mice , and mice overexpressing GnT1IP-L or MGAT1 in specific germ cell populations , should reveal roles for GnT1IP-L and MGAT1 in spermatogenesis ." ], [ "The plasmids Myc-GnT1IP-L , HA-GnT1IP-L , GnT1IP-L-Myc were prepared from mouse GnT1IP-L cDNA ( accession number HM067443 ) using the primers given in Table 1 that include HindIII or BamH1 restriction sites for insertion into pCDNA3 . 1 containing a hygromycin resistance cassette .", "The TM mutations F/L and F/A were made by site-directed mutagenesis using the primers shown in Table 1 to generate Myc-GnT1IP-L ( F/L or F/A ) or HA-GnT1IP-L ( F/L or F/A ) .", "Chimeric proteins were generated using a set of primers that included internal primers covering the boundaries of the sequences to be linked as shown in Table 1 .", "The GnT1IP-L/MGAT1-Myc chimera was made similarly except that PCR fragments were subcloned into pStrata and the full length PCR product was cloned into pCDNA3 . 1 containing a zeomycin resistance cassette .", "All constructs were verified by DNA sequencing .", "Full-length cDNA clones encoding mouse or human GlcNAc-transferases MGAT1 to MGAT5 were obtained from Imagenes GmbH ( Berlin , Germany ) , or Open Biosystems Inc . ( Huntsville , AL ) ( moue Mgat2 and Mgat4b ) or cloned by us ( mouse Mgat1; [Kumar et al . , 1992] ) .", "Constructs for BiFC were pCDNA3-based and possessed C-terminal mYFP fragments VN or VC as described earlier ( Hassinen et al . , 2010 , 2011 ) .", "FRET plasmids with C-terminal monomeric Venus ( mVen ) or monomeric mCherry ( mChe ) , as well as cDNAs C-terminally tagged with HA or Myc , were prepared as described ( Hassinen et al . , 2011; Hassinen and Kellokumpu , 2014 ) .", "All constructs were sequence-verified with the ABI3500xL Genetic Analyzer before use .", "Mouse anti-HA mAb ( HA . 11 ) and mouse anti-Myc mAb ( 9E10 ) were from Covance ( Princeton , NJ ) , rabbit anti-HA polyclonal antibody ( pAb ) ( Y-11 ) was from Santa Cruz Biotechnology Inc ( Dallas , TX ) , mouse anti-beta actin mAb ( AC-15 ) was from Abcam ( Cambridge , MA ) , rabbit anti-human GM130 pAb was from EMD Millipore ( Billerica , MA ) , mouse anti-rat Golgi GM130 mAb ( 35/GM130 ) was from BD Biosciences ( San Jose , CA ) , goat horse radish peroxidase ( HRP ) -conjugated anti-mouse secondary antibody was from Thermo Fisher Scientific Inc . ( Rockford , IL ) , rabbit anti-bovine PDI pAb and mouse anti-rat PDI mAb ( 1D3 ) were from Stressgen Biotechnologies Corp ( San Diego , CA ) , and rabbit anti-human MAN2A1 pAb was a gift of Kelly Moremen ( University of Georgia , GA ) .", "Secondary antibodies conjugated to Alexa-488 ( green; goat anti-rabbit or anti-mouse ) , or Alexa-568 ( red; goat anti-mouse IgG ( H + L ) ) were from Invitrogen Life Technologies ( Grand Island , NY ) .", "P . vulgaris leukoagglutinin ( L-PHA ) , concanavalin A ( Con A ) , GNA and GNA-FITC were from Vector Laboratories ( Burlingame , CA ) .", "CHO cells were grown in suspension or on plates in alpha-modified Eagle's medium with 10% FBS ( Gemini BioProducts , Sacramento , CA ) in 5% CO2 at 37°C .", "HeLa and COS-7 cells were grown on plates in Dulbecco's modified Eagle's medium with 10% FBS in 5% CO2 at 37°C ( HyClone , Thermo Scientific , Waltham , MA ) .", "Expression plasmids were transfected using FuGENE 6 ( Promega Corp , Fitchburg , WI ) according to the manufacturer's instructions .", "To obtain stable transfectant populations , antibiotic selection was initiated 24–48 hr post-transfection by adding ∼106 transfectants to selection media containing 1 mg/ml active G418 ( Gemini Bio-Products ) for 5 to 7 days or 1 . 4 mg/ml hygromycin ( EMD Millipore ) for 1 day before switching to 0 . 7 mg/ml hygromycin for 4–6 days .", "Resistant colonies were pooled and characterized or sorted by fluorescence-activated cell sorting ( FACS ) for expression of GFP or binding of GNA-FITC prior to use .", "For FRET and BiFC experiments , cells cultured for 1 day were transfected using 0 . 5 µg of each plasmid cDNA and FuGENE 6 according to the supplier's protocol ( Promega Corp ) .", "After 24 hr , cells were processed either for fluorescence microscopy , BiFC or FRET measurements ( see below ) .", "Where noted , chloroquine ( CQ ) from Sigma Aldrich ( St . Louis , MO ) was added to the culture medium at 40 µM , or nocodazole ( 1 µg/ml , Sigma Aldrich ) was added at different times as described in ‘Results’ .", "For FRET and BiFC experiments , COS-7 and CHO cells were prepared for immunofluorescence microscopy as described previously ( Hassinen et al . , 2010 ) .", "Briefly , after fixation with 4% paraformaldehyde for 15 min at room temperature , cells were permeabilized with 0 . 1% saponin in PBS and stained with anti-GM130 ( BD Biosciences ) , mono- or polyclonal anti-HA ( Sigma Aldrich ) , anti-FLAG ( Sigma Aldrich ) , anti-Myc ( Abcam ) , anti-PDI ( 5B5 , M877 , Dakopatts a/s , Denmark ) , rabbit anti-N-terminal GFP ( Affinity Bioreagents , Golden , CO and goat anti-C-terminal GFP ( Santa Cruz Biotechnology , Inc ) antibodies .", "After washing , cells were treated with relevant Alexa fluor 405- , 488- and 594-conjugated anti-mouse , anti-rabbit and anti-goat secondary antibodies ( Invitrogen , Carlsbad , CA .", "After staining , cells were mounted and imaged using the Zeiss LSM 700 confocal microscope , Zen2009 software ( Carl Zeiss AG , Oberkochen , Germany ) , 63× or 100× Plan-Apo oil immersion objectives and appropriate filter sets for each dye .", "For immunofluorescence experiments , HeLa cells ( 3 × 105 ) were added to coverslips coated with poly-L-lysine in a 6-well dish and incubated at 37°C in 5% CO2 .", "After 16 hr cells were washed with PBS , fixed in 3% paraformaldehyde , and incubated in 0 . 2% Triton X-100 , 1% FBS and 0 . 5% ( wt/vol ) bovine serum albumin ( BSA , fraction V ) in PBS containing 1 mM CaCl2 and 1 mM MgCl2 as described ( Huang and Stanley , 2010 ) .", "Following first and secondary antibody incubations in the same buffer , nuclei were stained with blue DAPI ( 1 μg/ml , Sigma–Aldrich ) .", "Coverslips were mounted using Fluoromount ( SouthernBiotech , Birmingham , AL ) and fluorescent images acquired on an inverted microscope ( Zeiss Axiovert 200M ) coupled to a 12-bit cooled charge-coupled device camera ( Zeiss AxioCam MRm Rev . 3 ) controlled by Axiovision software ( Zeiss AxioVs40 , Version: 4 . 7 . 2 . 0 ) , using a 100× 1 . 3 NA oil immersion objective ( Zeiss EC Plan-NeoFluar ) , and saved as tif files ( 1388 × 1040 , 8 bit ) .", "FRET microscopy measurements were performed using the Zeiss LSM700 confocal microscope , mVen and mChe variants as the donor/acceptor FRET pair and the acceptor bleaching protocol with appropriate filter sets for mVen and mChe ( Hassinen and Kellokumpu , 2014 ) .", "Samples were fixed before analysis as described for immunofluorescence ( see above ) .", "The samples were subjected to iterative bleaching ( 30 cycles , 20 iterations , 555 nm , 70% laser intensity ) during which the intensity values of the mVen were recorded .", "Background values were subtracted from the measured intensity values .", "FRET % was calculated from the acceptor-corrected intensities ( a macro package from Zeiss ) using the formulaFRET %= ( Dpost−Bpost ) − ( Dpre−Bpre ) ( Dpost−Bpost ) ×100 Where the D = donor intensity and B = background intensity .", "For BiFC experiments , expression and localization of VN and VC fusion proteins were determined by confocal microscopy following staining with polyclonal rabbit anti-GFP ( 1:1000 dilution; Affinity BioReagents , Golden , CO ) and goat anti-GFP ( 1:500; Santa Cruz Biotechnology , Inc . , Santa Cruz , CA ) antibodies followed by anti-rabbit secondary Ab conjugated with Alexa Fluor 594 and anti goat secondary Ab conjugated with Alexa Fluor 405 .", "BiFC microscopy was performed using a Zeiss LSM 700 confocal microscope equipped with a 63× oil immersion objective and appropriate filter set for the BiFC signal ( green ) , and the VN ( red ) or VC ( blue ) fusion proteins .", "Resistance to the lectins L-PHA and Con A was determined as described ( Stanley and Sundaram , 2014 ) .", "Briefly , 2000 CHO cells were added to each well of a 96-well plate in 100 µl culture medium , followed by 100 µl lectin at increasing concentrations and incubation at 37°C in a 5% CO2 incubator .", "When control wells were confluent ( ∼4 days ) , medium was removed and cells remaining attached to the plate were stained with Methylene Blue in 50% methanol .", "For flow cytometry , 5 × 105 cells were washed with 1 ml FACS binding buffer ( Hank's buffered salt solution containing 1 mM CaCl2 , 1 mM MgCl2 , 0 . 05% or 0 . 1% sodium azide , and 2% BSA Fraction V [Sigma] ) at 4°C and incubated with the mannose binding lectin from G . nivalis ( GNA ) conjugated to FITC at 12 µg/ml in FACS buffer on ice .", "After 30 min cells were washed with 1 ml FACS buffer , resuspended in 0 . 5 ml FACS buffer , without BSA , and 7-Amino-actinomycin D ( BD Biosciences ) was added prior to analysis in a FACSscan ( BD Biosciences ) flow cytometer .", "Flowjo software ( Tree Star Inc . , Ashland , OR ) was used to obtain profiles after 7-AAD-positive cells were gated out .", "For cell sorting , FACS binding buffer without sodium azide was used .", "Cells were resuspended in 0 . 5 ml FACS buffer containing penicillin ( 100 units ) and streptomycin ( 100 µg/ml , Invitrogen ) and amphotericin B ( 2 . 50 µg/ml , Invitrogen ) and subjected to flow cytometry ( DakoCytomation MoFlo and Dako MoFlo XDP , Beckman Coulter , Jersey City , NJ ) to sort GFP- or GNA- binding cells and remove 7-AAD-positive cells .", "Exponentially growing cells were washed three times and lysed ( 107 cells/75 μl ) in 1 . 5% Triton X-100 in distilled water containing protease inhibitor cocktail ( Roche , Nutley , NJ ) .", "MGAT1 and B4GALT1 were assayed as described previously ( Huang and Stanley , 2010 ) using Man5GlcNAc2Asn and UDP-3H-GlcNAc for MGAT1 , and GlcNAc with UDP-3H-Gal for B4GALT1 .", "To determine MGAT2 and MGAT5 activities , synthetic glycan acceptors specific for MGAT2 ( GlcNAcβ1 , 2Manα1 , 3 ( Manα1 , 6 ) Manβ1 , 4GlcNAcβ1 , octyl ) or ( MGAT5 Manα1 , 6Manβ1 , 4GlcNAcβ1 , octyl ) respectively , were kindly provided by Dr Ole Hindsgaul .", "Assays were performed in a final volume of 50 µl containing ∼50 µg cell lysate protein incubated in duplicate at 37°C for 2 hr with 20–40 µg substrate in the 62 . 5 mM 2- ( N morpholino ) ethanesulfonate ( MES ) ( pH 6 . 25–6 . 5 ) , 25 mM MnCl2 , and 0 . 75 mM UDP-[3H]-GlcNAc ( 10 , 000–20 , 000 cpm/nmol; Perkin Elmer , Inc . , Waltham , MA ) .", "MGAT1 reactions were stopped by adding 0 . 5 ml of Con A buffer ( 0 . 1 M sodium acetate , 1 . 0 M NaCl , 10 mM MgCl2 , 10 mM CaCl2 , 10 mM MnCl2 , and 0 . 02% sodium azide ) .", "After centrifugation in a microfuge , the supernatant was added to a 1 ml column of Con A-Sepharose ( GE Healthcare , Piscataway , NJ ) .", "For MGAT1 , the column was washed with Con A buffer and the product eluted with 200 mM α-methylmannoside in Con A buffer .", "For MGAT2 and MGAT5 assays , reaction products were separated on a SepPak column to which the octyl moiety of the acceptor bound .", "Specific activities ( nmol transferred per mg protein per hour ) were determined from 3H-GlcNAc incorporated into products in the presence vs the absence of acceptor , or by comparison with boiled extract .", "ß4GalT activity was assayed using GlcNAc as acceptor as described ( Lee et al . , 2001 ) .", "Transfectants were washed with PBS and lysed in distilled water containing 75 µl of 1 . 5% Triton X-100 ( Sigma–Aldrich ) with protease inhibitor cocktail ( Roche ) per 107 cells .", "Protein was determined by Dc protein assay ( Bio-Rad , Hercules , CA ) and ∼50–100 μg protein electrophoresed in a 10% Tris-HCl polyacrylamide gel at 10–30 mA for 2 hr .", "Transfer to polyvinylidene difluoride ( PerkinElmer , Inc . ) membrane was performed overnight at 50 mA in buffer containing 10% methanol .", "Antibodies were diluted in Tris buffered saline ( 10 mM Tris HCl , pH 7 . 4 , 150 mM NaCl ) containing 0 . 05% Tween 20 ( Sigma Aldrich ) and 3% nonfat dry milk supplemented with 3% BSA ( Fraction V ) or 3% nonfat dry milk , respectively .", "Antibody dilutions were: anti-Myc mAb ( 9E10 ) 1:500 , anti-HA mAb ( HA . 11 ) 1:1000 , anti-beta actin mAb 1:5000 , and HRP-conjugated goat anti-mouse secondary antibody , 1:5000–10 , 000 .", "After washing in Tris buffered saline containing 0 . 05% Tween 20 , membrane was incubated with Super Signal West Pico chemiluminescence reagent ( Thermo Scientific ) and exposed to film ( Denville Scientific , Inc . , South Palinfield , NJ ) .", "Mouse RNA sequencing data ( Soumillon et al . , 2013a ) containing the FPKM values for all five germ cell subtypes were downloaded from the GEO database at http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ?", "acc=GSE43717 .", "The frequency of log2-transformed FPKM values of all 36 , 823 transcripts were displayed as histograms and the log2-transformed FPKM values for Mgat1 ( ENSMUSG00000020346 ) and GnT1IP/Mgat4d ( ENSMUSG00000035057 ) mapped as vertical bars in order to visualize their gene expression levels .", "All analyses and visualizations were conducted using the statistical programming environment R ( www . r-project . org; Figure 9—source data 1 ) .", "Human testis microarray data containing the log2-transformed fluorescence values from Spiess et al . ( 2007 ) were downloaded from the ArrayExpress database at http://www . ebi . ac . uk/arrayexpress/experiments/E-TABM-234/ .", "Expression values for GnT1IP ( MGAT4D; LOC152586 , Probeset 1569995_at ) and MGAT1 ( Probeset 201126_s_at ) were extracted for all replicates of spermatogenic subtypes , log2-transformed , and displayed as boxplots .", "In detail , these were TA , SCO , presence of SPG , MA at the level of primary spermatocytes , hypospermatogenesis with decreased numbers of round/elongated spermatids ( HYS ) , and full spermatogenesis with normal numbers of round/elongated spermatids ( FS ) ." ] ]

[ "Mouse GnT1IP-L , and membrane-bound GnT1IP-S ( MGAT4D ) expressed in cultured cells inhibit MGAT1 , the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans .", "However , it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1 , nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi .", "We show here that the luminal domain of GnT1IP-L contains its inhibitory activity .", "Retention of GnT1IP-L in the endoplasmic reticulum ( ER ) via the N-terminal region of human invariant chain p33 , with or without C-terminal KDEL , markedly reduced inhibitory activity .", "Dynamic fluorescent resonance energy transfer ( FRET ) and bimolecular fluorescence complementation ( BiFC ) assays revealed homomeric interactions for GnT1IP-L in the ER , and heteromeric interactions with MGAT1 in the Golgi .", "GnT1IP-L did not generate a FRET signal with MGAT2 , MGAT3 , MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases .", "GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse , and are reduced in men with impaired spermatogenesis ." ]

[ "Proteins are made up of chains of amino acids that fold into three-dimensional shapes and many are assembled in a cell compartment known as the endoplasmic reticulum .", "From here , these new proteins move to another compartment called the Golgi , where they may be further modified before they are transported to their final destination in the cell .", "One way that proteins may be modified is known as glycosylation , in which sugar molecules are attached to specific amino acids .", "Some sugar molecules can act as labels that ensure the new proteins are transported to the correct destination in the cell .", "For proteins that are delivered to the surface of the cell , the sugar molecules can also play important roles in communication with other cells .", "A simple sugar molecule , or a complex arrangement of many sugar molecules , may be attached to an amino acid by glycosylation .", "An enzyme called MGAT1 controls the synthesis of sugars called complex N-glycans in the Golgi .", "In 2010 , researchers reported that a glycoprotein called GnT1IP-L binds to MGAT1 and inhibits its activity , thereby blocking the production of complex N-glycans .", "GnT1IP-L was found in the endoplasmic reticulum and Golgi , but it was not clear how it inhibits MGAT1 .", "Huang et al . —including some of the researchers from the 2010 study—have now investigated the activity of GnT1IP-L in cells grown in the laboratory using several biochemical techniques .", "The experiments show that GnT1IP-L only binds to MGAT1 when both proteins are in the Golgi .", "There are three sections ( or ‘domains’ ) in GnT1IP-L , but Huang et al . found that only the domain that is on the inside of the Golgi is involved in this interaction .", "Previous work indicated that GnT1IP-L may be involved in the formation of sperm in mice .", "Huang et al . have now analyzed previously published data on samples of testis tissue from human patients and found that the gene that encodes GnT1IP-L is present in very low amounts in patients whose sperm do not develop properly .", "Huang et al . 's findings suggest that GnT1IP-L may inhibit MGAT1 to control the glycosylation of proteins in the Golgi of developing sperm .", "The next step is to test this hypothesis by generating mutant mice that lack GnT1IP-L , or to make GnT1P-L in other cells in which it is not normally made , to find out if this affects the production of sperm ." ]

[ "Introduction", "Materials and methods", "Results", "Discussion" ]

[ "ecology" ]

[ [ "Individuals require information to reduce uncertainty about their environment .", "Information can be acquired in two ways ( Dall et al . , 2005 ) : by interacting with the environment directly ( personal information ) or by attending to the behaviour of others ( social information ) .", "Individuals benefit from both personal and social information in myriad contexts , including foraging , predator avoidance , and mate choice ( Giraldeau et al . , 2002 ) .", "Their use is moderated by their expense and reliability: personal information is usually reliable but costly and time consuming to collect , social information is less costly but more likely to become outdated and unreliable ( Giraldeau et al . , 2002; Laland , 2004 ) .", "The low costs of social information also allow it to disseminate more rapidly across groups , such that it can play an important role in the formation of traditions and cultures ( Whiten , 2000; Castro and Toro , 2004 ) .", "Despite the fitness benefits of information use , we have very little understanding of how individuals vary in their ability to capture these benefits .", "Indeed , theory developed to explain the costs and benefits of information use usually assumes homogeneity within a population ( e . g . Pradhan et al . , 2012 ) .", "To understand individual variation in information use , either personal or social , we suggest it is helpful to decompose the process into a sequence of three steps: the acquisition of information , its application , and the exploitation of its benefits ( Table 1 ) .", "Up until now , many studies have implicitly assumed that these three steps are synonymous , but recent evidence indicates that information use is substantially more complex .", "In particular , Carter et al . ( 2014 ) found that the time spent acquiring social information about a task did not correlate with subsequent performance ( information application ) in wild baboons ( Papio ursinus ) , while Atton et al . ( 2012 ) found differences in individual performance between task discovery ( information acquisition ) and task solving ( information exploitation ) in three-spine sticklebacks ( Gasterosteus aculeatus ) .", "The recognition of three sequential steps allows us to begin unpacking the complexity of information use , and to explore variation in the performance of different individuals at different points along the sequence .", "Distinct sensory and motor capabilities are likely to be involved at each stage , leading to different phenotypic constraints .", "As a result , individuals who are effective at one step may be less so at another , with significant implications for who captures the most benefits . 10 . 7554/eLife . 13125 . 003Table 1 . The information use sequence: definitions and examples . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 003StageDefinitionExample ( s ) of stageAcquisitionAn individual gains knowledge1 .", "Gaining knowledge of the location of a food patch .", "2 .", "Gaining knowledge of the location or form of a novel task . ApplicationAn individual uses the information that it has acquired in a relevant ( but not necessarily successful ) way1 .", "Entering a food patch .", "Because information can become outdated , ‘application’ can occur even after the patch has been fully depleted , leading to no reward .", "2 .", "Using stimulus or local enhancement to manipulate a novel task , but not necessarily successfully . ExploitationAn individual successfully uses information that it has acquired and applied to gain a benefit1 .", "Gaining food from a patch .", "2 .", "Solving a novel task .", "The range of phenotypic constraints operating at each step might include cognitive , social , behavioural , ecological and demographic characteristics .", "The importance of these constraints is likely to differ not only between individuals but also between populations and species .", "Here , we focus on social , behavioural and demographic constraints on the social information use sequence .", "To begin with , we consider the social phenotype , i . e . , phenotypic traits that emerge from social interactions with others and are likely to be under selection , in this case individual dominance rank ( Moore , 1993 ) and position in the social network ( Aplin et al . , 2015 ) .", "Dominant animals can aggressively monopolise resources such as mates ( Cowlishaw and Dunbar , 1991 ) and food ( Koenig , 2002 ) , limiting the opportunities for others to apply and exploit information that they have acquired either personally or socially about these resources .", "This can further lead to voluntary inhibition in the use of information by subordinate animals , e . g . , low-ranked rhesus monkeys ( Macaca mulatta ) only performed a socially-learnt task when high-ranked monkeys were not present ( Drea and Wallen , 1999 ) .", "The social network will likely manifest constraints on different stages of the information use sequence depending on the type of association indexed by the network , i . e . , associations according to spatiotemporal proximity or direct interactions .", "For instance , positions in proximity networks may affect an individual’s opportunities for information acquisition , assuming individuals are more likely to acquire information from others with whom they are more frequently in visual contact ( Coussi-Korbel and Fragaszy , 1995; Voelkl and Noë , 2008 , 2010 ) , e . g . , stickleback proximity networks predict the flow of information about the location of a novel task ( Atton et al . , 2012 ) .", "Similarly , positions in interaction networks may limit the application and exploitation of information about resources if social bonds are required to gain access to those resources ( Henzi and Barrett , 2002; Clarke et al . , 2010 ) , e . g . , vervet monkeys ( Chlorocebus aethiops ) allocate their social effort to access food provided by others ( Fruteau et al . , 2009 ) .", "The behavioural phenotype , specifically personality , may also be important in mediating individuals’ acquisition , application and exploitation of social information .", "We have previously shown that personality can affect both the first and second steps of the social information use sequence: calmer baboons were more likely to acquire social information but bolder individuals were more likely to apply it ( Carter et al . , 2014 ) .", "In geese ( Branta leucopsis ) , personality affected the final step in the sequence: shyer geese were more likely to exploit social information to forage where other geese were successfully foraging ( Kurvers et al . , 2010 ) .", "Similarly , fast exploring great tits ( Parus major ) were more likely to apply social information and change their foraging behaviour to mirror a demonstrator’s ( Marchetti and Drent , 2000 ) .", "Finally , individual demographic characteristics , particularly age and sex , may affect each step of the social information use sequence .", "Juveniles may be more reliant on social information because adults have already acquired the necessary information to survive to adulthood ( Galef and Laland , 2005 ) .", "This prediction is supported in baboons , where juveniles spend more time than adults acquiring social information about a novel food ( Carter et al . , 2014 ) .", "Similarly , in Japanese macaques ( Macaca fuscata ) , novel socially-transmitted behaviours were more likely to be adopted by juveniles ( Huffman et al . , 1996 ) .", "Sex differences in the social information use sequence are also likely due to , e . g . , sex-specific costs of competition at resources ( e . g . Aragón , 2009 ) .", "A further challenge involved in elucidating the social information use sequence is the identification of the relevant network through which information diffuses during the acquisition phase .", "Researchers have usually assumed information transfers primarily between individuals who are in close spatial proximity ( for examples , see Kendal et al . , 2010; Aplin et al . , 2012; Claidiére et al . , 2013 ) .", "However , individuals may preferentially acquire information from others besides those to whom they are associated as neighbours .", "For instance , individuals may be more attentive to those with whom they have strong affiliative bonds ( Coussi-Korbel and Fragaszy , 1995 ) or to lower ranking animals from whom they can scrounge resources ( King et al . , 2009 ) .", "The need to consider alternative networks in the identification of information diffusion paths is well illustrated by Boogert et al . ( 2014 ) , who showed that the spread of solutions to a novel foraging task in captive starlings ( Sturnus vulgaris ) was better predicted by a network based on perching associations than foraging associations .", "In this study , we explore phenotypic limitations on social information use .", "We examined information transmission among wild chacma baboons ( Papio ursinus ) by experimentally introducing ephemeral patches of a highly preferred food while the troops foraged naturally .", "We first compared which of five networks best predicted the diffusion of information through the troops about the location of a highly preferred food .", "Next , we investigated how individuals’ social , behavioural and demographic phenotypes affected their abilities to successfully acquire , apply and exploit this social information ." ], [ "We studied two habituated troops ( J , L ) of wild chacma baboons at Tsaobis Nature Park , Namibia ( 15° 45’E , 22° 23’S ) from May to July 2014 .", "Two habitat types make up the Tsaobis terrain: open desert and riparian woodland .", "The open desert is characterised by small herbs and shrubs , such as Monechma cleomoides , Sesamum capense , and Commiphora virgata , in a mosaic of alluvial plains and steep-sided hills surrounding the ephemeral Swakop River .", "The riparian woodland along the Swakop is characterised by large trees and bushes , such as Faidherbia albida , Prosopis glandulosa , and Salvadora persica ( see Cowlishaw and Davies , 1997 for more details ) .", "The baboons’ diet largely consists of berries , flowers , seedpods , and immature leaves ( Cowlishaw , 1997 ) .", "The baboons’ main predator , the leopard ( Panthera pardus ) , is rare at Tsaobis and the risk of predation is low .", "The baboon troops were followed daily from dawn until dusk .", "We collected data on all baboons over 2 years of age , who were individually recognisable by marks ( ear notches ) ( NJ = 46 , NJ adult female = 18 , NJ adult male = 8 , NJ juvenile female = 6 , NJ juvenile male = 14; NL = 48 , NL adult female = 19 , NL adult male = 10 , NL juvenile female = 2 , NJ juvenile male = 17 ) .", "Individuals younger than 2 years did not have marks , were not individually recognisable and did not form part of the study .", "Dominance ranks were assessed through aggressive interactions , recorded ad libitum , using Matman 1 . 1 . 4 ( Noldus Information Technology 2003 ) .", "These data included all displacements , supplants , threats , chases and attacks that occurred for which we could identify both the actor and recipient .", "If more than one dominance behaviour occurred in one event , such as a threat followed by a chase , only one interaction was recorded .", "The dominance hierarchies were strongly linear ( Landau’s corrected linearity index: h’J troop = 0 . 162 , h’L troop = 0 . 183 , NJ = 618 , NL = 856 , p<0 . 001 in both cases ) .", "Dominance rank was expressed relatively ( which controls for group size ) , using the formula 1-[ ( 1-r ) / ( 1-n ) ] where r is the individual’s absolute rank and n is the group size , and ranges from 0 ( lowest rank ) to 1 ( highest rank ) .", "Personality was indexed by boldness , estimated by presenting individuals with a novel food ( 2 cm2 pieces of potato or sweet potato dyed blue ) while foraging naturally alone and quantifying the time that the individuals spent investigating—handling and smelling—the novel food ( for further details , see Carter et al . , 2012b ) .", "Individuals who investigated the novel food for longer were considered bolder .", "We tested individuals’ boldness only once during the study period , but have previously found this test to be repeatable over three years ( Carter et al . , 2012b ) and correlated with subjective ratings of boldness ( Carter et al . , 2012a ) .", "Age ( in years ) was estimated from a combination of known birth dates and dental patterns of tooth eruption and wear ( see below ) .", "Unmarked immigrant males’ ages were estimated at 9 years old when they appeared in the study troops , as this is the age most males were observed to transfer from our study groups .", "The Tsaobis baboons are a wild population that has been under study every austral winter since 2000 .", "The field site is on private land , and the baboons have minimal contact with people other than the research team .", "The troops forage entirely naturally , except during specific research events that involve troop capture or feeding experiments .", "These occur very rarely ( five occasions over the past 10 years ) , are short in duration ( 2–4 weeks ) , and entail the provisioning of the entire troop with corn kernels at a single site at dawn ( e . g . , King et al . 2008; Carter et al . , 2013 ) .", "Since 2009 , individuals foraging alone have also been given the opportunity to sample a small , novel , food item ( e . g . , a slice of apple ) at a random place and time , on average once per year , as a personality test ( Carter et al . , 2013 ) .", "During troop captures , all troop members are captured at dawn in individual cages baited with corn .", "They are sequentially anaesthetised using tiletamine–zolazepam and the entire troop is processed within a day , to be released together the following morning when fully awake .", "While the baboons are anaesthetised , age is estimated through dentition .", "Tooth eruption schedules are used to assign age up to molar eruption ( Kahumbu and Eley , 1991 ) , while age beyond this point is estimated from molar wear .", "Validation of this approach using individuals captured on multiple occasions ( N = 19 over periods of 1–5 years ) to compare estimated versus known age differences between captures indicates these estimates are robust ( the mean difference between the observed and estimated time periods does not differ from zero: one-sample t-test , p>0 . 05; G . Cowlishaw , unpublished data ) .", "Individual associations were quantified using three proximity measures and two interaction measures .", "We experimentally assessed the diffusion of information about the location of newly discovered food resources by introducing patches of a highly preferred food , maize kernels , to the baboons .", "Two considerations were key to the design of the patch presentations: first , that the presentations were representative of naturalistic diffusions of information , such as about the location of a nest of eggs , and second , that the baboons did not learn to associate the observers with food .", "As such , one observer ( AJC ) created food patches by moving ahead of a foraging troop and scattering 52 . 9 ± 5 . 3 g of maize kernels over a 0 . 5 m2 core area ( with a little surrounding scatter enlarging this area to no more than 1 m2 ) , in the direction of travel of the troop .", "To avoid the baboons observing the patch being created , the observer either", "( i ) quickly scattered the kernels as she was walking or", "( ii ) pretended to get something from her field backpack while scattering the kernels behind her bag .", "In all cases , the baboons did not see the kernels being placed .", "Furthermore , because the observer was present in the troops for many hours preceding and following these trials , the baboons did not associate the camera nor the waiting behaviour of the observer with the presence of the patches .", "Because the foraging paths of the baboons are unpredictable , and the baboons typically have to be within 2 m of the patch to see the corn kernels ( median , range 0–8 m , N = 38 trials with recorded detection distances ) , there was variation in the spatial position of the individual who discovered the patch .", "In 28 of the 50 experiments ( 56% ) , it was an individual at the leading edge of the group that found the patch .", "In 11 experiments ( 22% ) , it was an individual at the side periphery and in a further 11 ( 22% ) an individual in the middle-back of the troop .", "In total , 37 different baboons ( J = 19 , L = 18 ) discovered the patches ( median = 1 time , min = 1 , max = 5 ) .", "Not every patch that was put out was found by a baboon , either because passing individuals failed to detect it or the troop changed their direction of travel ( N < 10 ) .", "In such cases , the patches were picked up by the observer after the baboons had left the area , and excluded from the analysis .", "In total , we performed 50 successful information diffusion experiments ( 25 per troop ) .", "One or two observers ( which always included AJC ) initially stood 15 m away from the patch and recorded each experiment using a video camera trained on the patch and surrounding area to dictate the identity and behaviour of any individuals coming within 25 m of the patch .", "The observer moved as required during the experiment to identify baboons .", "We recorded the identities of all individuals who", "( i ) gazed at an individual in the patch ,", "( ii ) entered the patch and", "( iii ) ate food from the patch ( see Videos 1 and 2 ) .", "These data were used to quantify social information", "( i ) acquisition ,", "( ii ) application and", "( iii ) exploitation , respectively .", "Note that the prolonged gazes observed during information acquisition were clearly distinct from the brief glances used in the routine monitoring of conspecifics , with most animals approaching and halting at the patch to watch its occupants ( e . g . , Videos 1–3 ) .", "The experiment was conducted 25 times per troop .", "We subsequently counted the number of times each individual was recorded performing", "( i ) –", "( iii ) above , excluding those cases when they discovered the patch .", "On two occasions ( in 50 experiments ) an individual who acquired social information could not be identified before they left the vicinity of the patch , but otherwise we were able to identify all individuals who acquired , applied and exploited social information . 10 . 7554/eLife . 13125 . 007Video 1 . Information diffusion experiments . The video shows rapid diffusion of information about the location of the food patch after its initial discovery .", "In the first experiment , several individual baboons successively enter the patch and are supplanted by more dominant individuals .", "In the second experiment , the patch is discovered by a low ranking female and then monopolised by high ranking juvenile male .", "The diffusion path is comparatively short .", "Please note that the videos were used to facilitate data extraction by dictating identities during rapid diffusions; we did not aim to capture all activity in the field of view of the camera . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 00710 . 7554/eLife . 13125 . 008Video 2 . Information acquisition , application and exploitation . The video shows an adult male monopolising the food patch , surrounded by juveniles who are obviously aware of the location of the patch , but cannot enter because of their lower rank .", "After the patch is depleted , the adult male exits the patch and many of the individuals subsequently apply the information they have acquired , even though it is outdated .", "One juvenile female ( just off the bottom of the screen ) , has the lowest rank in the troop and could not apply the social information she had acquired .", "In this case , there was no social information exploitation , because the patch discoverer ( the adult male ) depleted the patch . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 00810 . 7554/eLife . 13125 . 009Video 3 . Tolerated queuing , co-feeding and vocal protest . The first video ( tolerated queuing ) shows an adult male monopolising the food patch , with adult females and juvenile males and females queuing to check the patch after the male leaves .", "They enter the patch in the same order in which they were queuing .", "Two lower ranking adult females leave the area after queuing without entering the patch , demonstrating these females’ unwillingness to apply the social information they had acquired after patch depletion .", "The second video ( tolerated co-feed followed by protest ) shows the initial patch discovery by an adult female who has come within 1 m of the patch ( directly after she has been startled by the higher-ranking juvenile male foraging behind her ) .", "The pair subsequently co-feed in the patch , before the juvenile male vocally protests with pant-grunting . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 009 To identify the phenotypic constraints on the social information use sequence , we investigated whether individuals’", "( i ) acquisition ,", "( ii ) application and", "( iii ) exploitation of social information were affected by their phenotypes .", "Phenotypes were quantified according to social traits ( dominance rank , network centrality ) , behavioural traits ( personality ) , and demographic traits ( age , sex ) .", "Two different measures of network centrality were used , namely the individual strength scores for the 10 m proximity and directed grooming networks , generating six phenotypic predictors in total .", "Individual betweenness scores were not used because they were strongly correlated with their corresponding strengths in almost all cases ( Table 2; Figure 2—figure supplement 1 ) .", "We chose to use the strengths and betweennesses from the 10 m proximity and directed grooming networks , because these were the best proximity and interaction network predictors of information diffusion respectively ( see below ) .", "The 10 m proximity and grooming strengths were only marginally correlated with each other ( r = -0 . 29 ) and below the level of collinearity concern ( Dormann et al . , 2013 ) .", "All other combinations of phenotypic variables were similarly below the level of collinearity concern ( Table 3 ) . 10 . 7554/eLife . 13125 . 010Table 2 . Results of Spearman rank correlations testing whether there is a correlation between strengths and betweennesses in social networks created with different proximity and interaction rules .", "Presented is the rule , test statistic ( S ) , rho ( ρ ) , and p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 010RuleSρp5 m chain217907 . 6-0 . 57<0 . 00110 m220939 . 0-0 . 59<0 . 001Nearest neighbour directed81586 . 80 . 41<0 . 001Nearest neighbour9218 . 60 . 430 . 003Groom directed98412 . 90 . 300 . 005Groom15346 . 00 . 050 . 72Dominance directed67342 . 10 . 51<0 . 001Dominance7950 . 40 . 51<0 . 001 We ran three generalised linear mixed models ( GLMMs ) in the lme4 package ( Bates and Sarkar , 2007 ) with a Poisson link with the count of social information", "( i ) acquisition ,", "( ii ) application and", "( iii ) exploitation as the responses and troop as a random effect .", "For each response , we started with a full model comprising all six phenotypic predictors , and used backwards elimination of non-significant terms until we obtained the minimal model .", "Dropped terms were added to the minimal models to check significance .", "All data used in these analyses are available online ( Carter et al . , 2015 ) ." ], [ "We found widespread evidence for the social transmission of information about the location of food patches ( Table 4 ) .", "All proximity networks and grooming networks had strong support for predicting the diffusion of information between group members in comparison to the asocial transmission model ( ΔAICC for the social transmission model with the lowest AICC versus the asocial model = 192 . 9 ) .", "In all cases , the multiplicative models had a better fit than the additive models .", "The dominance networks , however , had little support in either case .", "The social network that best predicted the transmission of information was the 10 m network ( AICC = 3968 . 9 ) , followed by the 5 m chain network ( AICC = 3993 . 4 , ΔAICC = 24 . 5 ) .", "These were followed by the undirected nearest neighbour network ( ΔAICC = 82 . 1 ) , which performed better than the directed neighbour network ( ΔAICC = 114 . 4 ) .", "All proximity networks were better at predicting diffusion than the grooming networks ( Table 4 ) .", "For both grooming and dominance , there was minimal difference in the performance of the models between the directed and undirected networks ( ΔAICC grooming = 3 . 6 , dominance = 0 . 2 ) .", "The social transmission parameter of the best multiplicative model ( 10 m proximity , s = 0 . 999 ) suggests that , following patch discovery , all subsequent discoveries were via social information .", "This was confirmed when comparing all possible combinations of individual-level variables in the 10 m model as all four of the best candidate models ( ΔAICC = 0 ) were multiplicative and the best additive model was comparatively poor ( ΔAICC = 9 . 85 ) ( Supplementary file 1 ) .", "Model-averaged estimates of the individual-level variables calculated from the multiplicative OADA models indicated that rank ( β = 0 . 26 ) , sex ( β = 0 . 15 ) and age ( β = -0 . 01 ) affected information diffusion , while boldness did not ( β = 0 . 00 ) , such that more dominant , younger male baboons were more likely to learn asocially about patch locations ( see Table 5 for a list of the parameter estimates of the competing models ) . 10 . 7554/eLife . 13125 . 012Table 3 . Correlation matrix of the phenotypes used in the analyses .", "Presented are the Spearman’s rank correlation ( S ) estimates . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 012PhenotypeSexaAgeRankBoldnessProximitybGroomcSex1Age-0 . 491Rank0 . 430 . 161Boldness0 . 16-0 . 55-0 . 241Proximity0 . 29-0 . 60-0 . 020 . 511Groom-0 . 580 . 630 . 15-0 . 46-0 . 291acoded as an integer: females = 0 , male = 1 . b , cRefer to strength in the identified network . 10 . 7554/eLife . 13125 . 013Table 4 . Comparisons of the additive and multiplicative OADA models with social transmission versus the asocial learning model . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 013ModelAdd/MultiPredictor networkdfLogLikAICCSocial transmissionAdd10 m51984 . 73979 . 4Social transmissionMulti51979 . 43968 . 9Social transmissionAdd5 m51992 . 93995 . 9Social transmissionMulti51991 . 73993 . 4Social transmissionAddNN directed52037 . 44085 . 0Social transmissionMulti52036 . 64083 . 3Social transmissionAddNN52024 . 44059 . 0Social transmissionMulti52020 . 54051 . 0Social transmissionAddGroom directed52045 . 74101 . 4Social transmissionMulti52043 . 04096 . 1Social transmissionAddGroom52045 . 24100 . 6Social transmissionMulti52044 . 84099 . 7Social transmissionAddDom directed52076 . 84163 . 8Social transmissionMulti52076 . 84163 . 8Social transmissionAddDom52076 . 64163 . 3Social transmissionMulti52076 . 74163 . 6Asocial learning--42076 . 84161 . 8The predictor networks were the 10 m rule ( 10 m ) , 5 m chain rule ( 5 m ) , both of which were undirected , directed and undirected nearest neighbour rule ( NN ) , directed and undirected grooming interactions ( Groom ) and directed and undirected dominance interactions ( Dom ) .", "Presented are the models , degrees of freedom ( df ) , -log-likelihoods ( LogLik ) , corrected Akaike information criteria ( AICC ) .", "Add/Multi refers to whether the model was additive ( Add ) or multiplicative ( Multi ) . 10 . 7554/eLife . 13125 . 014Table 5 . Parameter estimates of individual-level variables of the competing OADA models for asocial effects on social transmission in the 10 m networks . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 014ModelCoefficientEstimateS . E . 1Social transmission0 . 999Sex0 . 1320 . 107Rank0 . 4980 . 174Age-0 . 0240 . 0122Social transmission0 . 999Boldness0 . 0010 . 001Age-0 . 0200 . 0113Social transmission0 . 999Sex0 . 3280 . 0864Social transmission0 . 999Sex0 . 2500 . 093Rank0 . 3530 . 158Presented are the bounded social transmission estimates ( for completeness ) , the fixed effects in the models and their standard errors ( S . E . ) .", "We found that individuals’ phenotypes limited the acquisition , application and exploitation of social information about the location of food patches ( Table 6 , Figure 3 ) .", "Proximity strength was the only predictor of information acquisition: more central baboons acquired social information more frequently .", "Proximity strength also showed a similar pattern with information application , but not exploitation .", "Information application and exploitation were further limited by sex and grooming strength , with males and more central individuals in the grooming network more likely both to apply and exploit acquired information .", "In combination with sex , dominance rank played a further limiting role on individuals’ exploitation of information , such that females of low rank were most limited in their exploitation of patches .", "Finally , individuals’ behavioural phenotypes , i . e . , boldness , also influenced information exploitation .", "Overall , individuals that were better connected in the 10 m network were more likely to acquire social information , but it was higher ranking males who were more likely to exploit this information . 10 . 7554/eLife . 13125 . 015Table 6 . Parameter estimates of the minimal models investigating the effect of proximity and grooming strength on social information", "( i ) acquisition ,", "( ii ) application and", "( iii ) exploitation . DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 015ResponsePredictorEffect sizeS . E .", "tPSocial information acquisitionIntercept0 . 230 . 211 . 110 . 27Proximity strength0 . 660 . 078 . 87<0 . 001Social information applicationIntercept-1 . 300 . 38-3 . 40<0 . 001Proximity strength0 . 650 . 106 . 45<0 . 001Grooming strength0 . 01<0 . 0014 . 74<0 . 001Sexa0 . 840 . 155 . 48<0 . 001Social information exploitationIntercept-2 . 430 . 39-6 . 17<0 . 001Grooming strength0 . 02<0 . 0014 . 67<0 . 001Sexa0 . 730 . 332 . 210 . 03Boldness0 . 01<0 . 0013 . 74<0 . 001Rank1 . 390 . 512 . 710 . 01Presented are the predictor variables , their effect sizes , standard errors ( S . E . ) , t values and p-values . aReference category: female10 . 7554/eLife . 13125 . 016Figure 3 . The relationships between social network centrality and successive steps of the social information process . The relationships between social information ( c ,", "d ) acquired ,", "( e ) applied and", "( f ) exploited by wild baboons and their degree strengths in the social networks .", "Presented are the proximity networks from which degree strengths were calculated for", "( a ) J and", "( b ) L troops , where nodes represent individuals , node size represents the rank of the individual , and node luminance represents the number of times the individual acquired information ( darker nodes acquired social information on more occasions; this colouration is conserved throughout the figure ) .", "Lines connecting nodes represent the strengths of the connections between dyads where thicker lines are stronger connections ( see legend ) .", "Presented below the networks is the relationship between ( c ,", "d ) social information acquired in", "( c ) J and", "( d ) L troops ,", "( e ) social information applied and", "( f ) social information exploited ( both troops plotted together ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13125 . 016 As a result of these constraints on successive steps in the social information use sequence , individuals only acquired social information about the patches on average ( median ) 6 times , applied social information 2 . 5 times , and exploited social information once ( across a total of 25 trials per group ) .", "However , because of phenotypic variation , there was a substantial range around these medians .", "Thus , while the average individual acquired and exploited social information on <25% and <5% of occasions , respectively , others were able to acquire and exploit information on >50% and >35% of occasions , or not at all , depending on their phenotype ." ], [ "Our study suggests that the route of information flow about ephemeral food patches is most closely matched by the 10 m proximity network , although the 5 m chain and nearest neighbour networks ( both directed and undirected ) also provided a good approximation .", "These results are consistent with several previous studies indicating the importance of spatiotemporal associations for information flow ( e . g . , great tits: Aplin et al . , 2012; three-spine sticklebacks: Atton et al . , 2012 ) .", "While the most appropriate proximity network to capture information flow will vary depending on the type of information , the social forager , and the environment , our study emphasises the need to recognise that not all networks will be equally applicable .", "These findings build on the suggestions of previous authors ( Lehmann and Ross , 2011; Madden et al . , 2011; Castles et al . , 2014 ) , and provide the first quantitative demonstration that multiple networks should be considered when studying information transmission .", "In the present analysis , the grooming networks also provided a good match to the pattern of information flow , suggesting that individuals may be more likely to monitor those with whom they share strong social bonds , irrespective of the symmetry of those bonds ( the model results for the undirected and directed networks were very similar ) .", "However , the absence of an effect of grooming strength on information acquisition at the individual level ( Table 6 ) suggests that such effects are relatively weak in comparison to those of spatial proximity .", "In contrast , the dominance networks were entirely unrelated to information flow .", "Since information flow required visual observation , this suggests that the monitoring of conspecifics is independent of dominance rank .", "A recent study of social attention in wild vervet monkeys reported a similar pattern ( Renevey et al . , 2013 ) .", "This may reflect the fact that dominants and subordinates monitor each other equally , albeit for different reasons: dominant animals seek to scrounge the food discoveries of others , while subordinates seek to avoid aggression .", "In our analysis of phenotypic constraints on social information use , we identified three steps in the information use sequence: acquisition , application , and exploitation .", "In the first step , we found that information acquisition was independent of almost all phenotypic traits tested , i . e . , age , sex , rank , personality , and social bonds ( grooming strength ) .", "The only important trait was individual centrality in the 10 m proximity network .", "This suggests that visual information about the patch was inexpensive to collect , and limited only by an individual’s spatial associations with other group members .", "Where the acquisition of social information is more costly , we might expect other phenotypic traits to become important .", "For instance , among juvenile chimpanzees ( Pan troglodytes ) , sex differences in attentiveness are believed to explain why females spend more time observing , and are faster to learn , the challenging skill of ‘termite fishing’ ( Lonsdorf , 2005 ) .", "In the second and third steps of the information use sequence , the application and exploitation of information were closely linked .", "Both steps involved patch entry , but the latter also involved the successful capture of foraging benefits from the patch .", "The similarities and dissimilarities in phenotypic predictors between the two models are highly informative , not only with respect to understanding the different constraints that can operate along the information use sequence , but also in elucidating how information use mediates the acquisition of monopolisable resources .", "The latter is possible because our experimental design makes information exploitation synonymous with resource acquisition .", "Clearly , in many other cases , information exploitation will be unrelated to monopolisable resources but rather involve other types of knowledge , such as foraging skills , predation risk , and mate compatibility .", "In these instances , the phenotypic constraints on information application and exploitation may be quite different to those observed here .", "We begin our assessment of phenotypic constraints on information application and exploitation with dominance rank .", "Dominant animals were far more likely to successfully exploit social information , because they were able to monopolise food patches .", "Indeed , as information about the patch spread , increasingly dominant animals would become informed and enter the patch , supplanting lower ranked occupants and preventing others from entering subsequently until the patch was exhausted .", "This pattern is consistent with how dominant animals scrounge from others in this population ( King et al . , 2009; Marshall et al . , 2012 ) and across social foragers generally ( Barta and Giraldeau , 1998 ) .", "Surprisingly , however , dominance did not predict information application .", "The reason for this is that many subordinates also entered the patch , but only after the dominant animal had left ( see Video 2 ) .", "Some of these animals were late arrivals , but a large number would be waiting ( ‘queuing’ ) nearby for the dominant animal to leave .", "Since the patch was largely depleted when the dominant animal left , the subsequent patch entries by subordinates imply the application of outdated information , at a surprisingly large scale given over half of those entering the patch failed to exploit it .", "One possible explanation for such apparently maladaptive behaviour is that , while baboons are able to collect social information about patch location , they are unable to collect ‘public’ information about patch quality .", "A similar pattern has been observed in three-spined sticklebacks ( Coolen et al . , 2003 ) .", "However , other experimental work in this population indicates that baboons are able to collect such information ( Lee , 2015 ) .", "A more likely explanation is that lower ranking individuals are aware that the patch is largely empty , but there is minimal cost in entering it and sifting through the sand in case any food items remain .", "Indeed , in a small number of cases , late arrivals did find a small number of kernels that had been overlooked .", "Proximity strength remained a strong predictor for information application , as might be expected: individuals had to find the patch before they could enter it .", "However , because only a very small fraction of those who acquired and applied the information were able to benefit from it , the effect of proximity strength was lost in information exploitation .", "Grooming strength , however , was important for both information application and exploitation , possibly reflecting the role that social bonds play in tolerance at feeding sites , both in this baboon population ( Sick et al . , 2014 ) and in primates more generally ( Ventura et al . , 2006; Tiddi et al . , 2011 ) .", "Nevertheless , tolerance of direct co-feeding at the patch ( patch sharing ) was rare in this experiment: of the 293 patch entries observed in the 44 experiments for which these data could be extracted , only 14 ( 4 . 8% ) resulted in co-feeding ( where two or more individuals fed simultaneously from the patch ) .", "Moreover , in 4 of these cases ( including one group of 3 co-feeders ) , there were vocal protests of intolerance from one or both parties ( see Video 3 for an example of a vocal protest during co-feeding ) .", "Thus toleration of co-feeding could be said to occur on only 9 occasions ( 3 . 1% ) .", "Instead , the tolerance observed in this experiment was primarily of close proximity of individuals to the patch , which allowed individuals to queue for and quickly enter the patch on a dominant’s exit ( see Video 3 ) .", "There are thus two strategies for information application to occur , both of which may rely on social bonds: tolerated co-feeding and tolerated ( close-proximity ) queuing .", "A change in the dimensions of the patch to allow more foragers concurrently to occupy it , and thus a greater possibility of tolerated co-feeding , may show a greater effect of grooming strength on both social information exploitation and application .", "Sex also played a role: females were less likely to apply and exploit social information .", "As we control for rank in the analyses , this finding may reflect female reproductive constraints .", "Previous work on nine-spine sticklebacks ( Pungitius pungitius ) has indicated that female gravidity can influence the use of social information ( Webster and Laland , 2010 ) .", "In our case , many of the observed females were either pregnant or lactating , and females in these states experience higher foraging demands ( Silk , 1987; Barrett et al . , 2006 ) , potentially making them less willing either to forego valuable foraging time to queue for patch entry ( information application ) or to spend excessive time searching for food once in the patch ( information exploitation ) .", "Shy animals also showed lower rates of information exploitation , presumably because they were more nervous and therefore spent more time in the social monitoring of conspecifics ( Edwards et al . , 2013 ) .", "The observation that bolder animals were more likely to successfully exploit information is consistent with the finding that bolder animals are also more likely to demonstrate social learning in this population ( Carter et al . , 2014 ) .", "In comparison , Harcourt et al . ( 2010 ) found no effect of boldness on social information use in three-spined sticklebacks .", "However , that study only went as far as the information application step of the information use sequence .", "We only found an effect of personality in the final information exploitation step .", "Phenotypic variation was also observed in asocial learning .", "Younger , more dominant males were more efficient at finding food patches .", "The most likely explanation for this pattern is that dominant juveniles were more likely to be at the leading edge of a foraging group , and therefore the first to encounter the patches .", "Similarly , juvenile ring-tailed coatis ( Nasua nasua ) occupy positions at the leading edge of their foraging groups ( Hirsch , 2011 ) .", "The multiplicative effect further suggests that the probability of younger dominant males occupying this spatial position increased the better connected they were in their social network .", "Notably , we found no effect of age on social information use .", "While this is not surprising at the information acquisition stage , where there were no phenotypic constraints other than network position , it is more surprising for information application and exploitation , especially since juveniles appear to show greater propensity for social learning than adults , both in baboons ( Carter et al . , 2014 ) and more generally ( e . g . , meerkats , Suricata suricatta: Thornton and Malapert , 2009 ) .", "However , this propensity usually refers to social learning tasks involving novelty or complexity , reflecting the tendency for juveniles to be more curious and exploratory about their environment ( Reader and Laland , 2001; Kendal et al . , 2005; Benson-Amram and Holekamp , 2012 ) .", "In our case , the task simply involved entering and exploiting a patch of familiar food , and for such a task it might be expected that individuals of all ages would show equal abilities .", "Our study raises a further point about the type of social information that is transmitted .", "We provided individuals with the opportunity to acquire social information about the location of a highly preferred food source in a novel location .", "These novel patches were rapidly depleted and the social information quickly became outdated .", "Such short-lived , ephemeral information may be easy and cheap to acquire and , as we have found , more likely be transmitted through proximity rather than interaction networks .", "Whilst our results support previous findings that proximity social networks are important for the transmission of ephemeral and/or easy-to-acquire information among individuals in the wild ( Aplin et al . , 2012 ) , evidence from starlings suggests that more complex information diffuses through alternative networks .", "Boogert et al . ( 2014 ) showed that information about novel foraging skills diffused through the perching rather than foraging social networks .", "Whilst it is challenging to determine the difficulty with which species acquire different types of information ( Griffin et al . , 2015 ) , research is needed to elucidate how the type of social information influences how quickly and through which networks it is transmitted .", "In the baboon system , we might similarly expect that social information that takes longer to acquire and/or process will transmit through different networks to those that transmit easy-to-acquire information .", "Our finding that phenotype limits information use builds on previous work indicating that individual state , such as uncertainty or the possession of outdated information , can influence social learning strategies ( reviewed in Rendell et al . , 2011 ) .", "Our study extends this work , revealing fundamental individual differences in the ability to use social information .", "Decomposing social information use into three constituent steps has further illuminated how these individual differences limit information use .", "The implications of such sequential phenotypic constraints are manifold .", "We conclude by considering two points .", "First , only a small number of individuals who acquire or apply social information may successfully exploit it .", "Similarly , Racine et al . ( 2012 ) report that ring-billed gulls ( Larus delawarensis ) may acquire social information about the location of food resources but be unable to apply it because of parenting constraints .", "Second , where phenotypic traits are expressed in relation to conspecifics ( e . g . , dominance rank , network position ) , social information use will vary markedly between social environments .", "For instance , in a captive setting where competition is minimised ( e . g . by testing individuals when isolated and not at risk of aggression: Call et al . , 2005 ) ( c . f . Drea and Wallen , 1999 ) , subordinate animals may show a propensity for copying others ( e . g . Kendal et al . , 2015 ) , but in the wild , where subordinates are more vulnerable to aggression and where food resources are relatively more valuable , subordinates may rarely copy others .", "Together , these two points highlight a critical disjunction between an ability to acquire information and to capture its benefits .", "This disconnection is likely to have a fundamental impact on selection for social information use , such that even in social information-rich environments , only a small number of individuals of a particular phenotype may be selected to use it ." ] ]

[ "Social information allows the rapid dissemination of novel information among individuals .", "However , an individual’s ability to use information is likely to be dependent on phenotypic constraints operating at three successive steps: acquisition , application , and exploitation .", "We tested this novel framework by quantifying the sequential process of social information use with experimental food patches in wild baboons ( Papio ursinus ) .", "We identified phenotypic constraints at each step of the information use sequence: peripheral individuals in the proximity network were less likely to acquire and apply social information , while subordinate females were less likely to exploit it successfully .", "Social bonds and personality also played a limiting role along the sequence .", "As a result of these constraints , the average individual only acquired and exploited social information on <25% and <5% of occasions .", "Our study highlights the sequential nature of information use and the fundamental importance of phenotypic constraints on this sequence ." ]

[ "Animals need information to make decisions , and a quick way to get this information is to watch what others are doing .", "Animals , like humans , have different social networks that they could acquire this kind of ‘social information’ from , yet we know little about which networks they actually use .", "In addition , once an animal has obtained social information , some aspect of their lives , such as their sex or social rank , could prevent them from using it .", "Once again , however , we know very little about the impact of these personal constraints .", "Carter et al . found that information about the location of a highly preferred food flowed through a social network of wild baboons that was based on who was regularly in close proximity to whom .", "However , while individuals with more neighbours were better at obtaining social information about food location , they were not better at using it .", "Rather , individuals were more likely to successfully exploit such information if they were dominant , bold , male , and had good social bonds with others .", "Carter et al . ’s results show that the use of social information is a process with several stages – from information acquisition , to its application , and finally its exploitation .", "Furthermore , the characteristics of an individual can limit their success at each of these stages .", "The next step is to figure out whether different types of social information – whether short- or long-lived , easy to acquire or more complex – flow through the same networks and have the same personal constraints on who can use them ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "Herpes simplex viruses ( HSV ) are major global health problems .", "HSV serotype 1 ( HSV-1 ) is associated primarily with oral mucocutaneous disease , sporadic encephalitis , and is a leading cause of corneal blindness worldwide , whereas HSV serotype 2 ( HSV-2 ) is the leading cause of genital ulcerative disease and a major cofactor in fueling the HIV epidemic ( Gray et al . , 2001; Wald and Link , 2002; Freeman et al . , 2006 ) .", "The World Health Organization estimated that over 500 million people are infected with HSV-2 worldwide with approximately 20 million new cases annually ( Looker et al . , 2008 ) .", "The extremely high prevalence of HSV-2 in sub-Saharan Africa ( ∼70% ) ( Looker et al . , 2008 ) may contribute more to the spread of HIV-1 than number of sex partners or other sexually transmitted infections ( Freeman et al . , 2006; Chen et al . , 2007 ) .", "Moreover , as HSV establishes latency in neurons with frequent subclinical or clinical reactivations , there is a lifelong impact of infection .", "Notably , HSV-1 has emerged as a predominant cause of genital disease in the developed world ( Roberts et al . , 2003; Xu et al . , 2006; Horowitz et al . , 2011; Bernstein et al . , 2013 ) .", "These epidemiological findings highlight the urgency to develop a safe and effective vaccine that protects against both serotypes .", "Subunit vaccines comprised of the HSV-2 viral envelope glycoprotein D ( gD-2 ) alone or combined with other envelope glycoproteins and with different adjuvants have predominated the HSV vaccine field for nearly 20 years ( Mertz et al . , 1990; Corey et al . , 1999; Stanberry et al . , 2002; Bernstein et al . , 2005; Belshe et al . , 2012; Leroux-Roels et al . , 2013 ) .", "The rationale for gD-2 subunit vaccines included their safety and ability to elicit neutralizing antibody ( Ab ) responses in vitro .", "However , the clinical trial outcomes have been disappointing .", "For example , in the most recent efficacy trial , an HSV-2 gD adjuvanted subunit vaccine provided no protection against HSV-2 genital disease , but surprisingly provided 35% cross-protection against HSV-1 infection and 58% cross-protection against HSV-1 disease ( Belshe et al . , 2012 ) .", "Neutralizing Abs ( 1:422 against HSV-1 and 1:97 against HSV-2 ) were detected in the serum , but were not measured in mucosal sites ( Awasthi et al . , 2014; Belshe et al . , 2014 ) .", "The gD-2 subunit vaccine also elicited CD4+ , but not CD8+ T cell responses; however , the cellular responses did not correlate with HSV-1 protection ( Belshe et al . , 2014 ) .", "These findings suggest the need for more complex immunogens to elicit protective immune responses against both serotypes .", "The efficacy of the live attenuated varicella zoster virus ( VZV ) ( a related herpes virus ) vaccine suggests that a similar approach for HSV may prove effective .", "However , preclinical studies with attenuated HSV vaccine candidates have yielded variable but incomplete protection against primary infection and latency ( Boursnell et al . , 1997; Spector et al . , 1998; Hoshino et al . , 2008; Shin and Iwasaki , 2012 ) .", "For example , Shin and Iwasaki found that a ‘prime-pull’ strategy , in which mice were first primed subcutaneously with an attenuated HSV deleted in thymidine kinase and then treated intravaginally with chemokines to recruit protective CD8+ T cells into the genital tract , was required for protection ( Shin and Iwasaki , 2012 ) .", "We adopted a different attenuated vaccine strategy based on an HSV-2 virus genetically deleted for the gene that encodes gD-2 ( US6 ) and complemented the virus to allow a first round of replication by growing it on Vero cells encoding HSV-1 US6 ( gD-1 , VD60 cells ) ( Cheshenko et al . , 2013 ) .", "The HSV-1 gD complemented HSV-2 mutant virus ( designated HSV-2 ΔgD−/+gD−1 ) replicates in VD60 cells to high titers , but a single passage through non-complementing cells yields non-infectious progeny , reflecting the requirement for gD in entry and cell-to-cell spread .", "We evaluated this virus for safety , immunogenicity , and vaccine efficacy against intravaginal challenge and in a skin scarification model in different strains of mice .", "Subcutaneous prime and boost of mice with HSV-2 ΔgD−/+gD−1 protected against lethal challenge in both models and prevented the establishment of latency .", "Adoptive transfer experiments showed that protection was mediated by antibodies ( Abs ) and required both the Fcγ receptor and neonatal Fc receptor ( FcRn ) ." ], [ "We reasoned that deletion of gD-2 , an envelope glycoprotein required for viral entry and cell-to-cell spread , would restrict infection to a single round of viral replication and thus provide a safe , highly attenuated candidate vaccine .", "Therefore , we constructed a variant of HSV-2 ( G ) deleted for US6 ( gD-2 ) ( Cheshenko et al . , 2013 ) .", "The virus was subsequently subjected to three rounds of plaque purification and grew to high titers ( 108 pfu/ml ) when phenotypically complemented by growing the virus on VD60 cells , which express HSV-1 gD under the control of the gD-1 promoter .", "No plaques were observed when three independent plaque-purified isolates were grown to 108 pfu on VD60 cells and then plated on non-complementing Vero cells .", "Moreover , no recombinants were detected by PCR , most likely reflecting differences in the regions flanking gD in VD60 cells and in HSV-2 ( Figure 1A ) .", "Western blots show similar levels of expression of other HSV viral proteins when the deletion virus is grown on VD60 or Vero cells ( Figure 1B ) . 10 . 7554/eLife . 06054 . 003Figure 1 . Characterization of the ΔgD−/− virus .", "( A ) Alignment of the upstream and downstream regions of gD located within the HSV-1 BamHI J fragment encoded in VD60 cells and within the genome of ΔgD−/− using LALIGN ( ExPASy ) ( Myers and Miller , 1988 ) .", "Global alignments assess end-to-end sequences and local pairwise alignments search for regions with high identity .", "( B ) Western blots of dextran gradient-purified virus isolated 24 hr after infection of VD60 ( ΔgD−/+gD−1 ) and Vero ( ΔgD−/− ) cells .", "Protein expression was assessed for viral glycoproteins B ( gB , UL27 ) , gC ( UL44 ) , gD ( Us6 ) , gE ( Us8 ) , gH ( Us22 ) , gL ( UL1 ) , VP5 ( UL19 ) and VP16 ( UL48 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 003 To assess whether HSV-2 ΔgD−/+gD−1 is safe in vivo , severe combined immunodeficiency ( SCID ) mice were inoculated subcutaneously or intravaginally with the complemented HSV-2 ΔgD−/+gD−1 strain or parental HSV-2 ( G ) virus ( Figure 2A ) .", "SCID mice inoculated intravaginally with 107 pfu of HSV-2 ΔgD−/+gD−1 manifested no signs of disease , whereas animals inoculated with 104 pfu of HSV-2 ( G ) quickly succumbed to the infection and manifested severe HSV-2-induced epithelial and neurological disease ( Figure 2B , C ) .", "Subcutaneous inoculation with wild-type virus also induced disease ( 60% mortality with 105 pfu ) , while no evidence of disease was observed following exposure to high doses of HSV-2 ΔgD−/+gD−1 in SCID mice .", "Moreover , no virus was detected in the neural tissue of SCID mice inoculated intravaginally with HSV-2 ΔgD−/+gD−1 by qPCR ( Figure 2D ) . 10 . 7554/eLife . 06054 . 004Figure 2 . HSV-2 ΔgD−/+gD−1 is attenuated in severe combined immunodeficiency ( SCID ) mice .", "( A ) Survival of SCID mice inoculated with up to 107 pfu of HSV-2 ΔgD−/+gD−1 or up to 105 pfu of the parental HSV-2 ( G ) strain either intravaginally ( ivag ) or subcutaneously ( sc ) .", "Statistical significance was measured by log-rank Mantel–Cox test; **p < 0 . 01 for ΔgD and WT after ivag inoculation .", "( B ) Epithelial and ( C ) Neurological disease scores for SCID mice inoculated with the different viruses at indicated doses .", "( D ) HSV-2 DNA ( qPCR , UL30 gene ) in genital tract and neural tissue samples at day 5 post-virus inoculation .", "The Ct cut off was determined with HSV-uninfected naïve samples .", "Statistical significance was measured by two-way ANOVA with Sidak's multiple comparisons test for ( B , C and D ) ; ***p < 0 . 001 .", "HSV-2 ΔgD−/+gD−1 and its parental strain are abbreviated as ΔgD and WT , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 004 To determine whether vaccination with HSV-2 ΔgD−/+gD−1 induces protective immunity to wild-type HSV-2 intravaginal challenge , mice were primed and boosted subcutaneously ( sc ) with 5 × 106 pfu ( 100 µl total volume ) or with an uninfected VD60 cell lysate as a control .", "3 weeks after the boost , the mice were challenged intravaginally with a clinical isolate of HSV-2 ( strain 4674 ) ( Segarra et al . , 2011 ) equivalent to an LD90 ( 5 × 104 pfu/mouse ) and 10 times the LD90 ( 5 × 105 pfu/mouse ) .", "Vaccinated mice were completely protected from lethal disease , whereas all of the control mice succumbed by day 8 post–challenge ( Figure 3A ) .", "The majority of the vaccinated mice ( 14/20 ) showed no evidence of epithelial disease ( Figure 3B ) and none showed any signs of neurological disease ( Figure 3C ) .", "The mice with epithelial signs recovered fully and HSV was detected by plaque assay in vaginal washes from vaccinated mice only on day 2 post–challenge but was cleared by day 4 post–challenge ( Figure 3D ) .", "These findings suggest that the epithelial signs observed may reflect a local immune response and not ongoing viral replication .", "Moreover , no infectious virus was recovered from vaginal or neural tissue in HSV-2 ΔgD−/+gD-1-vaccinated mice at day 5 post-infection , but virus was readily detected from all of these sites in control-vaccinated mice ( Figure 3E ) .", "Furthermore , we were unable to reactivate any virus from dorsal root ganglia ( DRG ) cultured ex vivo for 21 days ( Figure 3F , G ) .", "In contrast , virus reactivated from DRG extracted from all control-vaccinated animals .", "In addition , HSV DNA ( US6 gene ) was not detected by qPCR in vaccinated mice ( limit of detection 3 HSV-2 genome copies ) , but was detected in all controls ( Figure 3H ) .", "Similar results were obtained using primers for a different viral gene , UL30 ( data not shown ) .", "These findings suggest that the vaccine prevents establishment of latency . 10 . 7554/eLife . 06054 . 005Figure 3 . Vaccination with HSV-2 ΔgD−/+gD−1 protects mice against intravaginal lethal challenge . C57BL/6 mice were subcutaneously primed and boosted 3 weeks apart either with HSV-2 ΔgD−/+gD−1 or VD60 cell lysate ( Control ) .", "21 days after boost , mice were challenged with an LD90 of wild-type HSV-2 ( 4674 ) or 10 × LD90 .", "( A ) Survival , ( B ) Epithelial and ( C ) Neurological disease scores were followed daily after challenge .", "( D ) Viral titers in vaginal washes at days 2 and 4 after challenge ( n = 10 mice pooled two per group , lines indicate means ) .", "( E ) Viral titers in vaginal and neural tissue ( including the dorsal root ganglia , DRG ) at day 5 after challenge ( n = 5 , lines indicate means ) .", "( F ) Ex vivo reactivation of neural tissue obtained from challenged mice ( n = 5 per group ) .", "( G ) HSV-2 pfu in the media of ex vivo reactivated neural tissue obtained from challenged mice ( n = 5 mice per group , lines indicate means ) .", "( H ) HSV-2 DNA ( qPCR , US6 gene ) of genital tissue and neural tissue at day 5 after challenge ( n = 5 , lines indicate means ) .", "( I ) Survival of BALB/c mice that were primed , boosted and challenged as the C57BL/6 mice described above .", "( J ) HSV-2 DNA ( qPCR , US6 gene ) in Control- and ΔgD-vaccinated BALB/c neural tissue at day 7 ( n = 5 , lines indicate means ) and ΔgD-vaccinated BALB/c neural tissue at day 21 ( n = 9 , lines indicate means ) after challenge .", "HSV-2 ΔgD−/+gD−1-vaccinated group vs Control-vaccinated group were compared by log-rank Mantel–Cox test ( A , I ) , two-way ANOVA with Sidak's multiple comparisons test ( D , E , G , H ) or unpaired t-test ( J ) ; ***p < 0 . 001 .", "HSV-2 ΔgD−/+gD−1 and Control are abbreviated as ΔgD and Ctrl , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 00510 . 7554/eLife . 06054 . 006Figure 3—figure supplement 1 . ΔgD−/+gD−1-vaccinated cycling mice are protected against intravaginal HSV-2 challenge . C57BL/6 mice were treated ( w/MPA ) or not ( w/o MPA ) with medroxyprogesterone ( MPA ) 5 days previous to subcutaneous prime and boost 3 weeks apart either with HSV-2 ΔgD−/+gD−1 ( ΔgD ) or VD60 cell lysate ( Control ) .", "16 days after boost , all mice were treated with MPA and 5 days later challenged intravaginally with an LD90 of wild-type HSV-2 ( 4674 ) ( n = 5 mice per group ) .", "Statistical significance was measured by log-rank Mantel–Cox test; ***p < 0 . 001 , treatments vs Control . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 006 Mice were pretreated with medroxyprogesterone ( MPA ) to hormonally synchronize them prior to immunization in the efficacy studies .", "However , MPA can modulate innate and adaptive immune responses ( Kaushic et al . , 2003; Vicetti Miguel et al . , 2012 ) .", "Therefore we conducted additional experiments with cycling and MPA treated mice .", "The vaccine protected 100% of mice independent of hormonal treatment ( Figure 3—figure supplement 1 ) .", "In addition , mouse strains can differ in susceptibility and immune responses to pathogens .", "Therefore we also tested the efficacy of the vaccine against HSV-2 intravaginal infection in BALB/c mice .", "Consistent with the findings in C57BL/6 mice , 100% of HSV-2 ΔgD−/+gD−1-vaccinated mice survived intravaginal challenge ( Figure 3I ) and no infectious virus or viral DNA was recovered from neural tissues at day 7 and 21 post-infection ( Figure 3J ) .", "The majority of clinical HSV lesions are observed on the external genital skin rather than cervicovaginal sites .", "Therefore , we tested the vaccine for efficacy in a modified skin scarification model against both serotypes .", "The flank skin of C57BL/6 mice was depilated and scarified prior to direct inoculation of the skin with 5 × 104 pfu ( HSV-2 ( 4674 ) ) or 1 × 107 pfu ( HSV-1 ( 17 ) ) in 5 µl volume .", "The control-vaccinated ( VD60 lysate ) mice developed zosteriform lesions , neurologic disease and succumbed to infection following challenge with HSV-2 , whereas HSV-1 produced more modest signs ( Figure 4A , B and Figure 4—figure supplement 1 ) .", "More importantly , the HSV-2 ΔgD−/+gD−1-vaccinated mice showed minimal signs of epithelial disease ( maximal disease score 1 ) , no neurological signs and 100% survival after challenge with either serotype ( Figure 4C and Figure 4—figure supplement 1 ) .", "No HSV was detected by titering or qPCR in day 14 skin biopsies or neural tissue from HSV-2 ΔgD−/+gD−1-vaccinated mice; whereas virus was detected in 100% of control mice challenged with HSV-2 ( Figure 4D , E ) . 10 . 7554/eLife . 06054 . 007Figure 4 . Vaccination with HSV-2 ΔgD−/+gD−1 protects mice infected with HSV-2 and HSV-1 in a skin scarification model . Mice were subcutaneously primed and boosted 3 weeks apart either with HSV-2 ΔgD−/+gD−1 , Control VD60 cell lysate or PBS .", "3 weeks later , mice were depilated and challenged in the flank skin with PBS ( mock ) , ( A ) 5 × 104 pfu HSV-2 ( 4674 ) or ( B ) , 1 × 107 pfu HSV-1 ( 17 ) .", "Representative images are shown .", "( C ) Skin disease scores for HSV-2 ( 4674 ) -challenged mice at days 3–11 .", "( D ) Viral titers from biopsies of skin or neural tissue obtained on day 6–7 ( Control mice ) and day 14 ( HSV-2 ΔgD−/+gD−1-vaccinated mice ) ( n = 5 mice per group , lines indicate means ) .", "( E ) HSV-2 DNA ( qPCR , US6 gene ) in skin biopsies and neural tissue of Control mice ( day 6–7 ) and HSV-2 ΔgD−/+gD−1-vaccinated mice ( day 14 ) challenged with virus ( 5 mice per group , lines indicate means ) .", "Statistical significance was measured by two-way ANOVA with Sidak's multiple comparisons test ( D and E ) ; ***p < 0 . 001 , ΔgD−/+gD−1-vaccinated group vs control group .", "HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 00710 . 7554/eLife . 06054 . 008Figure 4—figure supplement 1 . Vaccination with HSV-2 ΔgD−/+gD−1 protects mice infected with HSV-1 in a skin scarification model . Mice were subcutaneously primed and boosted 3 weeks apart either with HSV-2 ΔgD−/+gD−1 or Control VD60 cell lysate .", "3 weeks later , mice were depilated and challenged in the flank skin with1 × 107 pfu HSV-1 ( 17 ) .", "Skin disease scores shown for challenged mice at days 3–11 .", "Viral titers from biopsies of skin obtained on day 7–8 ( Control mice ) and day 14 ( HSV-2 ΔgD−/+gD−1-vaccinated mice ) ( n = 5 mice per group , lines indicate means ) .", "Statistical significance was measured by unpaired t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 008 To determine if protection could be transferred to naïve mice , 250 μl of serum ( containing 750 μg of immunoglobulin administered intraperitoneally ) or 3 × 106 T cells ( administered intravenously ) isolated from the blood or spleen and lymph nodes of immunized mice , respectively , were administered to C57BL/6 mice .", "48 hr later , the mice were challenged with 105 pfu of HSV-2 ( 4674 ) intravaginally .", "Mice treated with a single dose of immune serum displayed modest epithelial signs ( mean score 2 ) , but no neurologic disease with 100% survival ( Figure 5A and Figure 5—figure supplement 1A , B ) .", "In contrast , mice that received T cells from immune mice or serum or T cells from the controls succumbed to disease .", "Depletion of immunoglobulins from the immune serum by passage on a Protein L column abolished the protective capacity .", "HSV-specific Abs were detected in vaginal washes in mice that received the immune , but not control serum ( Figure 5B ) . 10 . 7554/eLife . 06054 . 009Figure 5 . Serum from HSV-2 ΔgD−/+gD−1-vaccinated mice protects naïve mice against HSV-2 intravaginal and skin challenge . Mice were subcutaneously primed and boosted 3-weeks apart either with HSV-2 ΔgD−/+gD−1 or VD60 cell lysate ( Control ) .", "21 days later , blood and spleen were collected for serum and T cell purification and transferred intraperitoneally and intravenously , respectively , into naïve wild-type C57BL/6 mice .", "24 hr and 48 hr after serum and T cell transfer , respectively , mice were challenged intravaginally with LD90 of HSV-2 ( 4674 ) and followed for survival ( n = 5 mice per group ) .", "Serum immunoglobulins were depleted using a Protein L column ( A ) .", "( B ) Transferred anti-HSV-2-antibodies were assessed by ELISA in vaginal washes of recipient mice ( washes pooled from five mice in three independent experiments ) .", "( C ) Pooled serum from Control- or HSV-2 ΔgD−/+gD−1-vaccinated mice was transferred into naïve wild-type C57BL/6 mice .", "24 hr after serum transfer , mice were depilated in the flank skin and challenged with HSV-2 ( 4674 ) and followed for survival ( n = 5 mice per group ) .", "( D ) Viral titers in skin biopsies and neural tissue of mice receiving Control-serum ( day 7 ) and ΔgD−/+gD−1-serum ( day 14 ) ( n = 5 mice per group ) .", "( E ) HSV-2 DNA ( qPCR , US6 gene ) in skin biopsies and neural tissue of mice receiving Control-serum ( day 7 ) and ΔgD−/+gD−1-serum ( day 14 ) ( n = 5 mice per group ) .", "Statistical significance was measured by log-rank Mantel–Cox test ( A and C ) , t-test ( B ) and two-way ANOVA with Sidak’s multiple comparisons test ( D ) ; **p < 0 . 01 , ***p < 0 . 001 , treatment vs control .", "HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 00910 . 7554/eLife . 06054 . 010Figure 5—figure supplement 1 . Serum from HSV-2 ΔgD−/+gD−1-vaccinated mice protects naïve mice against epithelial and neurological disease after HSV-2 intravaginal and skin challenge . Serum from Control- ( VD60 cell lysate ) , ΔgD−/+gD−1-vaccinated mice , or ΔgD−/+gD−1 serum depleted of immunoglobulins using a Protein L column was transferred intraperitoneally into naïve wild-type C57BL/6 mice .", "24 hr later , mice were challenged intravaginally with LD90 of HSV-2 ( 4674 ) and followed for ( A ) epithelial and ( B ) neurological disease ( n = 5 mice per group ) .", "( C ) Serum from Control- or HSV-2 ΔgD−/+gD−1-vaccinated mice was transferred intraperitoneally into naïve wild-type C57BL/6 mice .", "24 hr later , mice were depilated and challenged in the flank skin with HSV-2 ( 4674 ) and followed for epithelial disease . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 010 Intraperitoneal administration of serum also protected mice from skin disease .", "The immune sera treated mice survived challenge ( Figure 5C ) and developed moderate signs of epithelial disease ( mean score of 2 . 2 ) , which peaked on day 6 ( Figure 5—figure supplement 1C ) .", "Moreover , no neurological signs were observed and no virus was detected in neural tissue by titering or qPCR ( Figure 5D , E ) .", "In contrast , mice that received control serum developed severe epithelial and neurological disease and succumbed to the infection with 100% lethality .", "HSV-specific Ab responses were consistently observed with serum dilutions as high as 1:800 , 000 7 days post-boost ( Figure 6A ) and were detected in vaginal washes within 4 days of challenge , indicating transport of HSV Abs into the vaginal lumen ( Figure 6B ) .", "The serum HSV-specific Abs exhibited low levels of neutralizing activity ( 1:5 dilution ) ( Figure 6C ) , but did display ADCC activity , which was reduced in the presence of anti-FcγR Ab ( Figure 6D ) .", "The HSV-specific Abs in serum and vaginal washes were comprised predominantly of IgG2a and IgG2b ( Figure 6E and Figure 6—figure supplement 1A ) and recognized multiple viral proteins on Western blots with HSV-2-infected cell lysates as the immunogen ( Figure 6F ) .", "Serum from control-immunized and subsequently HSV-2 infected mice ( obtained at day 8 post–challenge ) recognized a more restricted set of proteins .", "Western blots with purified HSV-2 ΔgD−/+gD−1 or HSV-2 ΔgD−/− indicate that the predominant antigen recognized by control-immunized , HSV-2-challenged , but not ΔgD−/+gD−1-vaccinated mice , is gD ( Figure 6—figure supplement 1B ) .", "Blots with recombinant glycoprotein B-1 identify the ∼100 kDa band recognized by the ΔgD−/+gD−1-vaccinated immune serum as gB ( Figure 6—figure supplement 1C ) . 10 . 7554/eLife . 06054 . 011Figure 6 . Vaccination with HSV-2 ΔgD−/+gD−1 induces protective mucosal antibodies targeting multiple HSV proteins with ADCC activity .", "( A ) Anti-HSV-2 antibodies detected by ELISA in serum samples at day 7 post-boost in mice subcutaneously primed and boosted 3-weeks apart with ΔgD−/+gD−1 or VD60 lysate ( Control ) ( 4 independent pools of serum from 5–10 mice each , results shown as means ± SD ) .", "( B ) Anti-HSV-2 antibodies detected by ELISA in vaginal washes at day 7 post-boost and day 4 post–challenge with HSV-2 ( 4674 ) ( n = 5 mice per group , lines indicate means ) .", "( C ) In vitro neutralizing activity of serum antibodies ( 1:5 dilution ) obtained from HSV-2 ΔgD−/+gD−1- or Control-vaccinated mice against HSV-2 ( left ) and HSV-1 ( right ) ( n = 5 mice per group , lines indicate means ) .", "( D ) Antibody-dependent cell-mediated cytotoxicity ( ADCC ) using mouse splenocytes , HSV-2-infected Vero cells and serum obtained either from Control- ( VD60 cell lysate ) or HSV-2 ΔgD−/+gD−1-vaccinated mice conducted in the absence or presence of anti-CD16/CD32 Ab to FcγRIII and FcγRII .", "% ADCC is defined as the percentage of dead ( Live/Dead+ ) target cells within HSV-2 GFPHigh positive cells .", "A representative dot blot is shown in the upper panel and lower panel shows results for five mice per group ( lines indicate means ) .", "( E ) Isotype of anti-HSV-2 serum antibodies obtained from five mice each that were either HSV-2 ΔgD−/+gD−1-vaccinated and HSV-2 ( 4674 ) -challenged or Control-vaccinated and HSV-2 ( 4674 ) -challenged ( results shown as means ± SD ) .", "( F ) Western blots of cellular lysates infected with HSV-2 ( 4674 ) and probed with dilutions of sera obtained from VD60 lysate-vaccinated and then subsequently infected mice ( Control-Challenged ) or dilutions of sera from HSV-2 ΔgD−/+gD−1-vaccinated mice 7 days post boost ( ΔgD-Vaccinated ) ; blots are representative of five independent experiments .", "HSV-2 ΔgD−/+gD−1-vaccinated groups were compared to control-vaccinated mice by two-way ANOVA with Sidak's multiple comparisons test ( A , B , D and E ) and unpaired t-test ( C ) ; *p < 0 . 05; **p < 0 . 01 , ***p < 0 . 001 .", "HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 01110 . 7554/eLife . 06054 . 012Figure 6—figure supplement 1 . Characterization of vaginal wash and serum antibodies .", "( A ) Isotypes of anti-HSV-2 antibodies in vaginal washes obtained at day 4 and day 8 post intravaginal challenge in mice that were immunized with HSV-2 ΔgD−/+gD−1 ( ΔgD ) or VD60 cell lysate ( Control ) .", "Antibody responses are shown as means ± SD ( n = 5 per group ) and were compared by two-way ANOVA; *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 .", "( B ) Western blots with sucrose gradient-purified ΔgD−/+gD−1 and ΔgD−/− viruses ( equivalent particle numbers based on a Western blot for VP5 ) probed with an anti-gD mAb ( mAb ( + ) ) , sera from HSV-2 ΔgD−/+gD−1-vaccinated mice day 7 post-boost ( ΔgD ) or sera from VD60 lysate-vaccinated mice day 7 post-intravaginal challenge with HSV-2 ( 4674 ) ( Ctrl + Challenge ) .", "( C ) Western blots with purified recombinant glycoprotein B-1 ( 2 μg per lane ) probed with an anti-gB mAb ( mAb ( + ) ) , sera from VD60 lysate -vaccinated mice ( Ctrl ) , HSV-2 ΔgD−/+gD−1-vaccinated mice day 7 post boost ( ΔgD ) or VD60 lysate-vaccinated mice day 7 post-intravaginal challenge with HSV-2 ( 4674 ) ( Ctrl + Challenge ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 012 Importantly , antibody-mediated protection was lost when serum was transferred to FcγR and FcRn knock-out mice ( Figure 7A , B and Figure 7—figure supplement 1A , B ) .", "HSV-specific Abs were detected in vaginal washes of the FcγR−/− mice , but failed to provide protection , whereas Abs were not detected in vaginal washes from FcRn−/− mice consistent with the role of FcRn in transport of Abs from serum to the mucosa ( Li et al . , 2011 ) ( Figure 7C , D ) . 10 . 7554/eLife . 06054 . 013Figure 7 . Antibody-mediated protection requires FcγR and FcRn expression . Survival of ( A ) FcγR−/− and ( B ) FcRn−/− mice that were either transferred serum obtained from Control- ( VD60 cell lysate ) or HSV-2 ΔgD−/+gD−1-vaccinated wild-type mice and then challenged intravaginally with HSV-2 ( 4674 ) ( n = 5 mice per group ) .", "Detection of HSV-specific Abs by ELISA in pooled vaginal washes ( n = 3 pools ) of ( C ) FcγR−/− and ( D ) FcRn−/− mice receiving serum ( intraperitoneally ) from control- ( VD60 cell lysate ) or HSV-2 ΔgD−/+gD−1-vaccinated wild-type mice .", "Survival curves were compared using log-rank Mantel–Cox test ( A and B ) and antibody titers using unpaired t-test ( C and D ) ; ***p < 0 . 001 .", "HSV-2 ΔgD−/+gD−1 is abbreviated as ΔgD . DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 01310 . 7554/eLife . 06054 . 014Figure 7—figure supplement 1 . Antibody-mediated protection requires FcγR and FcRn expression . Epithelial and neurological disease scores in ( A , B ) FcγR−/− and ( C , D ) FcRn−/− mice receiving serum from control- or HSV-2 ΔgD−/+gD−1-vaccinated wild-type mice and then challenged intravaginally with HSV-2 ( 4674 ) ( n = 5 mice per group ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06054 . 014" ], [ "The HSV vaccine field has focused most of its efforts on the induction of neutralizing Abs to gD as the major determinant of protection .", "The current study challenges this paradigm .", "We found that a live attenuated virus deleted in HSV-2 gD protects mice from vaginal , skin , and neuronal disease .", "Specifically , we observed no evidence of neurological disease in any model and could not detect virus in DRG of vaccinated mice by ex vivo plaque assays or by qPCR with a lower limit of detection of three viral genomes .", "The vaccine was effective in C57BL/6 and BALB/c mice and elicited high titers of HSV Abs ( 1:800 , 000 ) that were capable of passively protecting mice from both intravaginal and skin challenges .", "Notably , the Abs had low levels of neutralizing activity but did elicit ADCC and were able to passively transfer protection with a single dose of immune serum .", "The HSV-specific Abs could not be detected in vaginal washes of vaccinated mice , but were rapidly recruited into the vaginal lumen following intravaginal challenge and were associated with rapid clearance of the virus .", "Notably , the vaginal wash Abs were predominantly IgG , not IgA ( data not shown ) , which is consistent with prior studies that have shown that while the predominant immunoglobulin by weight in the vaginal mucus of immune mice is secretory IgA , HSV-specific Abs are almost entirely IgG ( Parr and Parr , 1998; Parr et al . , 1998 ) .", "The HSV-specific Abs were likely critical for protection as evidenced by the studies with FcRn knockout mice .", "Passive protection was also lost in FcγR knockout mice highlighting the role of Fc-mediated effector mechanisms .", "These findings differ from those obtained with other subunit or attenuated vaccine candidates .", "The first attenuated vaccine to be evaluated clinically was a virus deleted in glycoprotein H ( gH , UL22 ) .", "Unlike the data presented here with the HSV-2 ΔgD virus , the gH deletion viral vaccine did not prevent the establishment of latency in mice ( Speck et al . , 1996 ) , was not shown to passively transfer protection and failed to reduce recurrences when evaluated as a therapeutic vaccine in a clinical trial ( de Bruyn et al . , 2006 ) .", "More recently , a virus deleted in HSV-2 UL5 and UL29 , which is defective in HSV DNA replication , has been advanced to a Phase 1 study .", "This deletion virus also differs from the HSV-2 ΔgD−/+gD−1 vaccine candidate described here in that it does not induce as high an Ab response , has not been shown to passively protect , and did not prevent the establishment of latency as low levels of viral DNA were detected in the DRG following both immunization and viral challenge ( Hoshino et al . , 2005 , 2008 , 2009; Dudek et al . , 2008 ) .", "Notably , the UL5/UL29 deletion virus expresses gD at levels similar to that of wild-type virus , but lower levels of gB ( Da Costa et al . , 2000 ) .", "Thymidine kinase ( tk ) deletion viruses , which have been used to study HSV immunogenicity because they are attenuated in mice ( but not humans ) , express the full repertoire of viral proteins including gD .", "Protection following immunization with tk-deletion viruses requires anti-HSV-2 T cells in the genital tract ( Milligan et al . , 2004; Shin and Iwasaki , 2012 ) and serum from mice immunized with this tk-deletion virus has not been shown to passively transfer protection ( Morrison et al . , 2001; Milligan et al . , 2004 ) .", "Possibly , deletion of gD , which is immunodominant as evidenced by the immune response in control , HSV-2 challenged mice ( Figure 6F ) and by clinical data showing that the majority of HSV-infected individuals have neutralizing Abs to gD ( Cairns et al . , 2014 ) , unmasks viral antigens important in generating a protective humoral response .", "Additionally , gD may modulate immune responses through its interactions with herpesvirus entry mediator ( HVEM ) on immune cells ( La et al . , 2002; Aubert et al . , 2009; Stiles et al . , 2010; Kopp et al . , 2012; Grauwet et al . , 2014; Sharma et al . , 2014 ) .", "Interactions between gD and HVEM may skew the immune response towards neutralizing Abs whereas in the absence of the gD-HVEM interaction or yet undiscovered immunosuppressive functions of gD , the host may mount a polyantigenic IgG2 dominant response capable of eliciting FcR-mediated protection .", "This hypothesis will be tested in future studies by: ( 1 ) comparing , for example , the immune response to virus in mice deficient for HVEM , ( 2 ) examining the immune response to the HSV-2 ΔgD−/+gD−1 vaccine when combined with the gD subunit vaccine , or ( 3 ) isolating mutant forms of gD that have lost immunosuppressive functions .", "The inability of gD to elicit protective immunity against HSV-2 is highlighted by the results of the clinical trials with an adjuvanted gD subunit vaccine ( Mertz et al . , 1990; Corey et al . , 1999; Kohl et al . , 2000; Bernstein et al . , 2005; Belshe et al . , 2012 ) .", "An additional mechanism that may contribute to the protection observed with the HSV-2 ΔgD−/+gD−1 vaccine and to the observation that it induces Abs to multiple viral proteins is that by deleting the gene for gD , which is required for cell-to-cell spread , the cells initially infected by the complemented virus may function as a ‘factory’ generating viral proteins and ‘defective’ particles that are immunogenic .", "Consistent with this is the observation that heat-killed HSV-2 ΔgD−/+gD−1 failed to protect mice from vaginal challenge ( data not shown ) .", "In addition to its unique ability to generate high levels of Abs capable of translocating to mucosal sites and to prevent virus from reaching neuronal tissue following intravaginal or skin challenge , the HSV-2 ΔgD−/+gD−1 virus is also an attractive vaccine because of its safety .", "The virus induced no disease in SCID mice inoculated with 1000 times the lethal dose of wild-type virus and to date we have observed no recombinants either by plaque assay or PCR .", "This likely reflects that the virus is propagated on an HSV-1 and not an HSV-2 complementing cell line .", "The flanking regions of gD in HSV-1 and HSV-2 share approximately 54% and 73% identity within the 5′ and 3′ region of the US6 gene , respectively ( HSV-1 ( KOS ) vs HSV-2 ( HG52 ) , NCBI genome , Figure 1A ) .", "This level of homology makes recombination highly unlikely .", "It remains to be determined whether pre-existing immunity to HSV-1 will impact the immunogenicity of the HSV-2 ΔgD−/+gD−1 vaccine as was shown with the gD subunit vaccine ( Stanberry et al . , 2002 ) This may be less of a problem with HSV-2 ΔgD−/+gD−1 as it elicits a polyantigenic response whereas infection primarily elicits Abs to gD as evidenced by the immunoblots .", "If future studies suggest this is a problem , an alternative strategy is to complement the HSV-2 ΔgD−/+gD−1 virus with gD from a nonhuman herpes virus or a chimeric gD ( Zago et al . , 2004 ) .", "Ideally the development of new mouse models of HSV latency and reactivation will allow us to explore the efficacy of HSV-2 ΔgD−/+gD−1 as a therapeutic vaccine .", "The demonstration that this vaccine strain confers protective Abs against skin and intravaginal challenge that function through the Fc receptor highlights the importance of ADCC and antibody-mediated phagocytosis .", "Consistent with this concept are the results of the HIV RV144 vaccine trial , which found that non-neutralizing antibodies that mediate ADCC and antibody-mediated phagocytosis contributed to protection against HIV ( Bonsignori et al . , 2012 ) .", "Similar findings have been observed in nonhuman primate studies of SHIV ( Florese et al . , 2009; Bialuk et al . , 2011 ) .", "Moreover , recent studies suggest that Ab–FcγR interactions are required to mediate protection against influenza ( Dilillo et al . , 2014 ) .", "Together these findings suggest that the HSV-2 ΔgD−/+gD−1 vaccine provides a model to elicit such protective responses and indicate that it may be an ideal vaccine vector for other pathogens that enter through these sites ." ], [ "Experiments were carried out with approval from the Albert Einstein College of Medicine Institutional Animal Care and Use Committee , Protocol #20130913 .", "Female C57BL/6 ( wild-type ) , BALB/c ( wild-type ) , severe combined immunodeficiency ( SCID , BALB/c background ) , and FcRn−/− ( B6 . 129X1-Fcgrttm1Dcr/DcrJ ) mice were purchased from the National Cancer Institute ( NCI , Frederick , MD ) or Jackson Laboratory ( JAX , Bar Harbor , ME ) at 4–6 weeks of age .", "Female FcγR−/− ( B6 . 129P2-Fcer1gtm1Rav N12 ) mice were purchased from Taconic ( Albany , NY ) at 4–6 weeks of age .", "CaSki ( human cervical epithelial cell line; CRL-1550; American Type Culture Collection [ATTC] , Manassas , VA , USA ) , Vero ( green monkey kidney cell line; CCL-81; ATCC ) cells , human keratinocytes ( HaCAT , provided by David Johnson , Oregon Health & Sciences University , Portland , OR ) , and VD60 cells ( Vero cells encoding gD-1 under the endogenous gene promoter [Ligas and Johnson , 1988] ) were passaged in DMEM supplemented with 10% fetal bovine serum ( FBS , Gemini Bio-Products , West Sacramento , CA ) .", "Splenocytes were obtained by generating a single cell suspension from harvested spleens from animals and subsequently , red blood cells were lysed with ACK lysis buffer ( Gibco , Grand Island , NY ) .", "Splenocytes were then resuspended in complete RPMI media until use .", "Construction of the HSV-2 ΔgD−/+gD−1 virus in the HSV-2 ( G ) ( Ejercito et al . , 1968 ) genetic background has been previously described by our group ( Cheshenko et al . , 2013 ) .", "Briefly , 1 , 000 bp upstream and downstream regions of the US6 gene were PCR-amplified using viral genomic DNA ( HSV-2 ( G ) ) with primers LL-V91I-US6 TTTTTT TTCCAT AAATTG GAAAGG GAACAG CGACCA AATGTC AC and LR-V91I-US6 TTTTTT TTCCAT TTCTTG GTGATA CGCGAT GCACAC GAAAAA CG for the upstream region of US6 and primers RL-V91I-US6 TTTTTT TTCCAT AGATTG GTTCCC CGCTCC CGTGTA CC and RR-V91I-US6 TTTTTT TTCCAT CTTTTG GCGGGG GCGCCT GTATCG G for the downstream region of this gene .", "Stocks of HSV-2 ΔgD−/+gD−1 virus were propagated on complementing VD60 cells and titered on VD60 and Vero cells .", "HSV-2 ( 4674 ) ( Nixon et al . , 2013 ) was propagated on HaCAT cells .", "HSV-1 ( 17 ) ( Brown et al . , 1973 ) , HSV-2 ( G ) ( Ejercito et al . , 1968 ) , HSV-1 ( F ) ( Ejercito et al . , 1968 ) and HSV-2 ( 333 ) ZAG ( Nixon et al . , 2013 ) , a recombinant virus expressing the green fluorescent protein ( GFP ) were propagated on Vero cells .", "Concentrated viral stocks were stored at −80°C and diluted in PBS to the desired concentration when needed .", "5 days prior to immunization , mice were treated subcutaneously with 2 . 5 mg of medoxyprogesterone acetate ( MPA; Sicor Pharmaceuticals , Irvine , CA ) .", "All prime immunizations were given subcutaneously ( sc , medial to the hind limb and pelvis ) with 5 × 106 pfu of HSV-2 ΔgD−/+gD−1 or an equal amount of VD60 cell lysate ( Control ) .", "3 weeks after initial immunizations , a boosting dose of 5 × 106 pfu of ΔgD−/+gD−1 or VD60 cell lysate was administered .", "For adoptive transfer of T cells , spleens were harvested from immunized mice 3 weeks after the boosting dose .", "Splenocytes obtained by single cell suspensions from harvested spleens were then treated with ACK Lysing buffer ( Gibco ) to remove erythrocytes and resuspended in MACs buffer ( 2% heat-inactivated FBS , 2 mM EDTA in PBS ) .", "T cells were magnetically purified via negative selection ( Pan T Cell Isolation Kit II , Miltenyi Biotec , Germany ) using LS columns .", "Before inoculation into recipient mice , purity and viability of cells were verified by flow cytometry and trypan blue staining .", "A total of 3 × 106 T cells were transferred into recipient mice intravenously through the tail .", "Alternatively , serum was collected from blood and a volume of 250 µl containing 750 µg of total IgG ( detected via anti-mouse IgG ELISA ) was transferred into recipient mice intraperitoneally 24 hr before intravaginal challenge of HSV-2 .", "In select experiments , serum Ig was depleted by passing the serum through columns containing Protein L ( GE healthcare Life Sciences , Piscataway Township , NJ ) .", "Mice were treated with MPA and challenged 5 days later with HSV-2 ( 4674 ) 3 weeks after the vaccine boosting dose with 5 × 104 plaque forming units ( pfu , LD90 ) or 5 × 105 pfu ( 10× LD90 ) of HSV-2 ( 4674 ) intravaginally and monitored and scored for disease for 14 days as previously described ( Nixon et al . , 2013 ) .", "For safety studies , unimmunized SCID mice were treated with MPA and inoculated 5 days later intravaginally or subcutaneously with HSV-2 ( 4674 ) or ΔgD−/+gD−1 either with 30 µl or 100 µl total volume , respectively .", "150 µl of sterile PBS containing protease inhibitor ( F Hoffmann-La Roche AG , Switzerland ) was used to perform intravaginal washes for antibody and viral load quantification .", "Epithelial disease for intravaginal infections was scored as follows: ( 1 ) mild erythema , ( 2 ) hair loss , erythema , edema , ( 3 ) severe edema , hair loss , lesion formation , ( 4 ) severe ulcerations , multiple lesions and ( 5 ) death .", "Neurological disease for intravaginal infection was scored as follows: ( 1 ) urinary retention , ( 2 ) urinary retention and constipation , hind-limp paresis , ( 3 ) hind-limb paralysis ( one leg ) , ( 4 ) complete hind limb paralysis ( both legs ) , and ( 5 ) death .", "Mice were euthanized at a score of 3 or 4 and assigned a score of 5 on subsequent days for statistical analyses .", "For HSV skin infections , a protocol modified from Goel et al . ( 2002 ) was used .", "Briefly , mice were depilated on the right flank with Nair and allowed to rest for 24 hr .", "Depilated mice were anesthetized with isoflurane ( Isothesia , Henry-Schein ) , then abraded on the exposed skin with a disposable emory board for 20–25 strokes and subsequently challenged with 5 × 104 pfu of HSV-2 ( 4674 ) or 1 × 107 pfu of HSV-1 ( 17 ) in 5 µl deposited on the abraded skin .", "Mice were then anesthetized for an additional 5 min to allow the virus inoculum to dry .", "Mice were monitored for 14 days and scored for signs of disease .", "Epithelial disease in the skin scarification model was scored as follows: ( 1 ) primary lesion or erythema , ( 2 ) distant site zosteriform lesions , mild edema/erythema , ( 3 ) severe ulceration and edema , increased epidermal spread , ( 4 ) hind-limb paralysis and ( 5 ) death .", "Mice that were euthanized at a score of 4 were given a value of 5 the next day .", "For pfu detection in tissue samples of mice inoculated and/or challenged with HSV , genital tract , skin , or dorsal root ganglia ( including sciatic nerve from hind limb to spinal cord ) were weighed and homogenized in serum-free Dubecco's Modified Eagles Medium ( DMEM , Gibco ) using RNase-free pestles .", "Homogenized tissues were then sonicated for 30 s at maximum strength and centrifuged at 10 , 000×g for 5 min .", "Supernatants were then overlaid on confluent Vero cell monolayers ( 2 × 105 cells/well in a 48-well plate ) for 1 hr .", "Wells were washed with PBS , then 199 medium ( Gibco ) containing 1% heat-inactivated FBS and overlaid with 0 . 5% methylcellulose and incubated at 37°C for 48 hr .", "Cells were fixed with 2% paraformaldehyde , stained with a crystal violet solution and the number of plaque forming units ( pfu ) were quantified .", "Vaginal washes from mice obtained after virus inoculation were plated neat or diluted in PBS on confluent Vero cell monolayers ( 2 × 105 cells/well in a 48-well plate ) and viral pfu were quantified as described above .", "Data are presented as log10 pfu per gram of tissue .", "For ex-vivo HSV reactivation , neural tissue obtained at day 5 post–challenge was excised , cut into 3–4 pieces and cultured with confluent Vero cell monolayers in serum-free DMEM in 60 × 15 mm tissue culture dishes ( Corning ) .", "Dishes were observed daily up to 21 days post co-culture for cytopathic effect with media exchanged every 2 days .", "Supernatants were harvested every other day to measure viral pfu by standard plaque assay .", "DNA was extracted from weighed tissue samples using DNeasy Blood and Tissue ( Qiagen ) following the manufacturer's recommendations .", "Extracted DNA was then normalized to 10 ng of DNA per reaction and viral DNA quantified using real-time quantitative PCR ( RT-qPCR , qPCR ) using ABsolute qPCR ROX Mix ( Thermo Scientific ) .", "Primers for HSV-2 gD ( US6 ) and polymerase ( UL30 ) were purchased from Integrated DNA Technologies ( US6 cat: 117920498 and UL30 cat: 1179200494 ) and used to detect viral genome DNA .", "To determine the number of HSV-2 genome copies , HSV-2 viral DNA was used as a standard curve ( Cheshenko and Herold , 2002 ) .", "Samples that read three or less copy numbers were considered negative .", "Data are presented as log10 HSV-2 copy numbers per gram of tissue .", "Serum was obtained from individual mice at 1 week post-prime , at boost , 3 weeks post-boost , and 8 days after intravaginal challenge with HSV-2 .", "Vaginal washes were collected in 150 µl PBS containing protease inhibitor at different time points after sc boost with ΔgD−/+gD−1 or intravaginal challenge with HSV-2 .", "For HSV-2 specific antibody detection , Vero cells were mock-infected or infected with HSV-2 ( 4674 ) and 24 hr later sonicated for 30 s and then coated on 96 well MaxiSorp ELISA plates ( Nunc , NY ) at a concentration of 45 µg cell lysate/well at 4°C overnight .", "Cell lysates were then further permeabilized with PBS/0 . 1% Triton X-100 buffer and fixed with 1% formaldehyde .", "Serum and vaginal washes obtained from individual mice were serially diluted and overlaid in duplicates onto cell lysate-coated wells and incubated at RT for 2 hr .", "Wells were washed with PBS/0 . 05% Tween 20 buffer five times and incubated with purified biotin anti-mouse Ig κ or biotin anti-mouse IgG1 , IgG2a , IgG2b , or IgG3 at 1 µg/ml ( Becton Dickenson , San Diego ) for an additional 2 hr at RT .", "Next , cell lysates were washed as above and incubated with HRP-conjugated streptavidin ( Becton Dickenson , San Diego ) for 30 min at RT and then developed with TMB substrate ( BD OptEIA ) for 5 min .", "The reaction was stopped with 2 N H2SO4 .", "Wells were read on a SpectraMax ( M5 series ) ELISA plate reader at an absorbance of 450 nm .", "The resulting absorbance was determined by subtracting values obtained for uninfected cell lysates to values obtained with infected cell lysates at a serum dilution of 1:2500 or 1:100 ( total Ig or isotype specific , respectively ) and at a 1:5 dilution for vaginal washes .", "Serial dilutions of control and immune serum from 1 week post-boost were inactivated ( 56°C for 30 min ) and then incubated at a 1:1 ratio with 50 pfu of HSV-2 ( 4674 ) or HSV-1 ( F ) in a 96-well plate at 37°C for 1 hr .", "Virus-antibody complexes were then overlaid onto confluent Vero cells in a 48-well plate for 1 hr at 37°C .", "Wells were then washed with PBS and subsequently with 500 µl 199 medium ( Gibco ) containing 1% heat-inactivated FBS with 0 . 5% methyl cellulose and incubated for 37°C for 48 hr .", "Plaques were quantified and percent neutralization was determined as the reduction of HSV-2 plaques compared to control ( 50 pfu HSV-2 in media ) .", "Vero cells ( used as target cells ) were infected with HSV-2:GFP ( 333 ) ZAG at an MOI of 1 pfu/cell .", "18 hr post-infection cells were harvested via CellStripper ( Corning ) and labeled with CellTrace Violet ( Invitrogen , Grand Island , NY ) at 1 . 25 µM .", "Labeled infected cells were then incubated with heat-inactivated serum from control or vaccinated individual mice at a 1:2 dilution for 15 min .", "Splenocytes from naïve mice were treated with ACK Lysing Buffer ( Gibco ) to remove erythrocytes and used as effector cells .", "Where indicated , effector cells were either treated or not with FCR-4G8 ( anti-FcγRIII/II , anti-mouse CD16/32 , Invitrogen , Grand Island , NY ) at 2 µg/106 cells .", "Co-cultures were performed at an effector cell to target cell ratio of 25:1 for 4 hr .", "Effector/target and target cells were then stained with Live/Dead Red fixable dye ( Invitrogen ) for 15 min and subsequently washed and resuspended in FACs buffer ( 2% heat-inactivated FBS , 2 mM EDTA in PBS ) .", "The percentage of dead HSV-2-infected target cells was determined on a 5-laser LSRII flow cytometer ( Becton Dickenson , San Diego , CA ) at the Einstein Flow Cytometry Core Facility .", "Final analysis was carried out using FlowJo software version 10 ( Tree Star , Inc . , Ashland , OR ) .", "Stocks of HSV-2 ΔgD−/+gD−1 and ΔgD−/− were propagated on VD60 or Vero cells and purified using dextran or sucrose gradients as previously described ( Cheshenko et al . , 2013 ) and then analyzed for viral protein expression by performing Western blots .", "Equivalent numbers of viral particles ( based on Western blot probing with antibody to the viral capsid protein , VP5 [Cheshenko et al . , 2007] ) were loaded on gels , proteins separated by SDS-PAGE , transferred to nitrocellulose and immunoblotted with anti-HSV-2; gC ( H1196 ) , anti-gE ( 101 ) , anti-VP16 ( VA16 ) , anti-VP5 ( 3B6 ) , anti-gB ( 10B7 ) , anti-gD ( 0191 ) from Santa Cruz Biotechnology ( Santa Cruz , CA ) and anti-gH-gL ( CH31 , gift from R Eisenberg and G Cohen , University of Pennsylvania ) as previously reported ( Cheshenko et al . , 2014 ) .", "Serum immune-target profiling was done by performing Western blots with 10 µg of HSV-2 ( 4674 ) infected Vero cell lysates per lane or equivalent particle numbers ( based on Western blots for VP5 ) of HSV-2 ΔgD−/+gD−1 and ΔgD−/− purified virus , and probing with serial dilutions of immune or control serum obtained 1 week post-boost or 8 days post-intravaginal challenge , respectively .", "Alternatively , Western blots were performed using 2 µg of recombinant glycoprotein B-1 produced in baculovirus and purified on a CL-68 sepharose column ( 17-0467-01 , GE Healthcare , Sweden ) as previously reported ( Cheshenko et al . , 2004 ) .", "The anti-gB-1/gB-2 monoclonal antibody 10B7 and the anti-gD-1 monoclonal antibody 0191 ( Santa Cruz Biotechnology ) were used as positive controls .", "Blots were incubated with immune and control serum for 16 hr at 4°C .", "After repetitive washes , bound antibodies were detected using goat anti-mouse-HRP secondary antibody ( Bio Rad , Hercules , CA ) .", "Results were compared by two-way analysis of variance ( two-way ANOVA ) with multiple comparisons or unpaired t tests using GraphPad Prism version 6 ( San Diego , CA ) .", "Mantel–Cox survival curves were compared by log rank tests .", "p values <0 . 05 ( * ) , <0 . 01 ( ** ) , <0 . 001 ( *** ) were considered significant ." ] ]

[ "Subunit vaccines comprised of glycoprotein D ( gD-2 ) failed to prevent HSV-2 highlighting need for novel strategies .", "To test the hypothesis that deletion of gD-2 unmasks protective antigens , we evaluated the efficacy and safety of an HSV-2 virus deleted in gD-2 and complemented allowing a single round of replication on cells expressing HSV-1 gD ( ΔgD−/+gD−1 ) .", "Subcutaneous immunization of C57BL/6 or BALB/c mice with ΔgD−/+gD1 provided 100% protection against lethal intravaginal or skin challenges and prevented latency .", "ΔgD−/+gD1 elicited no disease in SCID mice , whereas 1000-fold lower doses of wild-type virus were lethal .", "HSV-specific antibodies were detected in serum ( titer 1:800 , 000 ) following immunization and in vaginal washes after intravaginal challenge .", "The antibodies elicited cell-mediated cytotoxicity , but little neutralizing activity .", "Passive transfer of immune serum completely protected wild-type , but not Fcγ-receptor or neonatal Fc-receptor knock-out mice .", "These studies demonstrate that non-neutralizing Fc-mediated humoral responses confer protection and support advancement of this attenuated vaccine ." ]

[ "Herpes simplex virus 2 ( or HSV-2 ) infects millions of people worldwide and is the leading cause of genital diseases .", "The virus initially infects skin cells , but then spreads to nerve cells where it persists for life .", "Often , the virus remains in a dormant state for long periods of time and does not cause any symptoms .", "However , HSV-2 can periodically re-activate , leading to repeated infections; this can be life-threatening in patients who suffer from a weak immune system .", "There is no cure for Herpes simplex virus infection , and there are currently no vaccines that would prevent the virus from infecting humans .", "HSV-2 contains a protein on its surface known as ‘glycoprotein D’ which it needs to enter host cells .", "The interaction between glycoprotein D and the host is also essential for cell-to-cell spread of the virus .", "Vaccines that contain glycoprotein D trigger the production of antibodies that bind to this viral protein .", "These vaccines have been tested in several large clinical trials , but the results have so far been disappointing .", "As such , new vaccines that provide effective protection against HSV-2 are urgently needed .", "Live attenuated vaccines are commonly used to prevent diseases such as measles mumps and chicken pox or shingles .", "These vaccines contain a harmless or weakened version of the disease-causing virus .", "Petro , González et al . have now developed a new potential vaccine that contains live attenuated HSV-2 , which completely lacks glycoprotein D and thus cannot spread from cell-to-cell .", "When this weakened virus was administered to mice that have a poor immune system , the mice remained healthy .", "On the other hand , when Petro , González et al . treated similar mice with the wild-type HSV-2 virus instead , many mice died within a few days .", "Petro , González et al . then went on to show that mice that had been treated with the weakened virus as a vaccine were completely protected from a later infection with wild-type HSV-2 and did not develop any symptoms of the disease .", "Furthermore , no virus was detected in the nerve cells of these mice—which is where the virus would normally persist in its dormant state .", "Finally , Petro , González et al . showed that blood serum from immunized mice could be used to completely protect other mice from exposure to wild-type virus .", "These results demonstrate that a live attenuated HSV-2 virus that lacks glycoprotein D ( the main component of other failed vaccines ) elicits a different type of immune response and is a safe and effective vaccine in mouse models of virus infection .", "With further work , these findings may eventually lead to a preventative treatment to combat HSV-2 infections in humans ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "The diversity and specificity of cellular responses rely on the precise integration of biochemical and physical cues from the microenvironment .", "Cells generate a coordinated response through interactions among signaling pathways – from ligands and receptors to intracellular effectors .", "Receptors are a particularly versatile locus of control since they undergo regulated microdomain clustering , internalization and homo/hetero-meric multimerization .", "Because these mechanisms affect ligand binding , enzymatic activity , and effector recruitment , receptors play a crucial role in defining signal intensity , duration , location , and quality ( Bethani et al . , 2010; Di Guglielmo et al . , 2003; Groves and Kuriyan , 2010; Salaita et al . , 2010 ) .", "However , many questions remain about the mechanisms by which receptors participate in the concerted cellular response to a multitude of concurrent cues .", "The TGFβ signaling pathway exemplifies the importance of regulated receptor multimerization .", "TGFβ signals through a heterotetrameric complex of transmembrane receptor kinases .", "Once the TGFβ ligand is activated from its latent form , it binds directly to a dimer of type II receptors ( TβRII ) ( Annes , 2003; Munger et al . , 1999; Wipff et al . , 2007; Munger and Sheppard , 2011 ) .", "The ligand-bound TβRII complex recruits and phosphorylates two type I receptors ( TβRI ) – either Alk5 or Alk1 ( Wrana et al . , 2008 ) .", "TβRI , in turn , phosphorylates and activates canonical Smad proteins and multiple non-canonical effectors , such as RhoA , TAK1 and Akt ( Massague , 1998; Feng and Derynck , 2005 ) .", "Specifically , recruitment of Alk5 to the TβRII complex stimulates phosphorylation of Smad2/3 , whereas Alk1 recruitment drives activation of Smad1/5/8 ( Lin et al . , 2008 ) .", "The inappropriate shift of TβRII multimerization partner from Alk5 to Alk1 underlies disease processes ranging from vascular disorders to osteoarthritis ( Blaney Davidson et al . , 2009; Goumans , 2002 ) .", "Not only do TGFβ receptors associate with one another , but also with a number of other receptor families , notably integrins ( Scaffidi et al . , 2004; Garamszegi et al . , 2010 ) .", "Garamszegi et al . revealed a physical interaction between integrin α2β1 and TGFβ receptors involved in collagen-induced Smad phosphorylation ( Garamszegi et al . , 2010 ) .", "TGFβ receptor interactions alter ligand specificity and effector selection , offering a regulatory mechanism to calibrate TGFβ signaling based on the cellular microenvironment .", "Integrins , another class of multimeric receptors , are central to cellular mechanotransduction .", "Upon integrin binding to the extracellular matrix , the formation of focal adhesions stimulates actomyosin contractility to generate cellular tension ( DuFort et al . , 2011; Giancotti , 1999; Ingber , 1997 ) .", "Through this Rho/ROCK-dependent mechanism , cells establish tensional homeostasis with the physical features of the extracellular environment ( DuFort et al . , 2011 ) .", "Cellular tension can amplify , alter , or suppress cellular responses to growth factor signaling ( Allen et al . , 2012; McBeath et al . , 2004; Wang et al . , 2012 ) .", "The functional state of many intracellular effectors , including β-catenin , YAP/TAZ , and MAPK , is modulated by cellular tension ( Samuel et al . , 2011; Dupont et al . , 2011; Wang et al . , 1998 ) .", "In the case of TGFβ signaling , we and others have identified several mechanosensitive responses ( Allen et al . , 2012; Wang et al . , 2012; Leight et al . , 2012 ) .", "The activation of latent TGFβ ligand , as well as the phosphorylation , nuclear translocation and transactivation of Smads is regulated by cellular tension in a Rho/ROCK-dependent manner ( Allen et al . , 2012; Wipff and Hinz , 2008 ) .", "However , the mechanisms by which changes in cellular tension modulate effector activity remain unclear .", "The effect of cellular tension on the multimerization of receptors other than integrins is largely unexplored .", "In spite of the established tension-sensitive regulation of downstream signaling effectors , the effect of physical cues on growth factor receptor interactions is unknown .", "This gap in understanding is partly due to the fact that until recently , studies of cell surface receptor colocalization and physical interactions have mostly utilized biochemical , biophysical , or fluorescence imaging approaches .", "While invaluable , these approaches are limited by their inability to discriminate spatially discrete molecular interactions that occur in specific cellular domains .", "Novel super-resolution imaging approaches provide the capability to visualize receptor responses to biochemical and physical cues at the single molecule level with spatial and temporal specificity ( Coelho et al . , 2013; Manley et al . , 2008; Rossier et al . , 2012; Calebiro et al . , 2013; Xia et al . , 2013 ) .", "To elucidate mechanisms by which physical cues regulate growth factor signaling , we utilize high-resolution imaging , single particle tracking , mass spectrometry and biochemical assays to test the hypothesis that cellular tension regulates TGFβ receptor multimerization .", "We find that cellular tension controls the spatial organization , multimerization and activity of a discrete population of TGFβ receptors at integrin-rich focal adhesions , suggesting a novel mechanism by which physical cues calibrate the activity of the TGFβ signaling pathway ." ], [ "To investigate the spatiotemporal control of TGFβ receptors , we evaluated the localization of endogenous and fluorescently tagged TβRII and TβRI in ATDC5 chondroprogenitor cells and NIH3T3 fibroblasts .", "Immunofluorescence of TβRII in both wildtype and transfected ATDC5 cells yielded similar results , revealing specific punctate staining that did not provide structural information ( Figure 1A , Figure 1—figure supplement 1 ) .", "Proceeding with fluorescently tagged TβRII allowed for visualization of fine structural features in static and dynamic conditions .", "Spinning disc confocal microscopy of TβRII-mEmerald allowed visualization of its spatial organization , revealing shadowed regions where TβRII expression is completely absent ( indicated by arrows , Figure 1B–D ) .", "Total internal reflection fluorescence ( TIRF ) microscopy improves visualization of transmembrane proteins by examining a thin section of the sample at the adherent cell surface .", "Switching from widefield microscopy ( Figure 1E ) to TIRF on the same cell vividly revealed segregated domains of TβRII ( Figure 1F ) and TβRI ( Figure 1G , H ) .", "The sequestration of TβRII from TβRI was present with either the canonical ( Alk5 ) or non-canonical ( Alk1 ) type I TGFβ receptors ( Figure 1G , H ) .", "Indeed , when co-expressed in the same cell , TβRII is enriched at the boundary of discrete TβRI domains , demonstrating a novel spatial segregation of these signaling partners ( Figure 1I–L ) . 10 . 7554/eLife . 09300 . 003Figure 1 . Spatial segregation of TβRII from TβRI . Spinning disc confocal imaging of endogenous TβRII ( A , Figure 1—figure supplement 1 ) demonstrates punctate staining .", "Imaging of mEmerald-labeled TβRII ( B ) reveals TβRII-absent domains in ATDC5 ( B , C ) and NIH3T3 ( D ) cells expressing mEmerald-TβRII .", "Switching from widefield ( E ) to TIRF mode imaging ( F ) on the same cell unveils a specific spatial organization of TβRII , which is discrete from that of TβRI ( Alk5 and Alk1 ) ( G , H ) .", "ATDC5 cells co-expressing mEmerald-TβRII and mCherry-TβRI ( Alk1 ) reveal that TβRII surrounds specific domains of TβRI ( I-L ) .", "Quantitative profile plot of expression intensity demonstrates separate and distinct localization patterns of TβRI and TβRII ( L ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 00310 . 7554/eLife . 09300 . 004Figure 1—figure supplement 1 . Endogenous staining of TβRII insufficient for spatial organization visualization . Wildtype ATDC5 cells ( A-C ) and ATDC5 cells expressing TβRII-mCherry were stained with two different anti-TβRII ( A , D and B , E ) antibodies and an IgG control ( C , F ) .", "Staining of TβRII between wildtype and transfected cells differs only in intensity and not structurally , indicating that the observed spatial organization is not due to expression constructs alone .", "Although TβRII antibodies showed specificity relative to IgG staining , this bright punctate staining is insufficient for visualizing fine structural organization . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 004 Since dynamic recruitment of TβRI to TGFβ-bound TβRII complexes stimulates downstream effectors , we sought to determine if spatial segregation of TGFβ receptors affects receptor mobility .", "Single-particle tracking photoactivated localization microscopy ( sptPALM ) resolves the dynamics of individual molecules in live single cells .", "Using sptPALM , we captured thousands of trajectories of individual TβRI ( Alk5 ) and TβRII proteins labeled with photoswitchable mEos2 ( Figure 2A , B ) ( McKinney et al . , 2009 ) .", "The large number of long duration molecular trajectories ( Figure 2C ) allowed us to visualize single molecule track behavior and describe molecular environments within individual cells .", "For both TβRI and TβRII , individual receptors showed a range of mobility , resulting in groups of immobile , confined , or freely diffusive receptors ( representative tracks , Figure 2D ) .", "Mobility of each group of TβRI did not differ significantly from TβRII ( Figure 2E ) , but the diffusion coefficient of TβRI was slightly higher ( Figure 2F ) , perhaps because of its lower molecular weight ( TβRI/Alk5 56 kDa vs . TβRII 65 kDa ) .", "Relative to whole cell TGFβ receptor dynamics , TβRI and TβRII are significantly less mobile in cellular domains enriched with clusters of spatially organized receptors ( Figure 2F ) .", "Thus , this spatially organized population of TGFβ receptors is slower and more confined , possibly due to interactions with other proteins . 10 . 7554/eLife . 09300 . 005Figure 2 . Limited TβRI ( Alk5 ) and TβRII mobility in areas of receptor spatial organization . All mEos2-tagged TβRI and TβRII sptPALM single molecule trajectories with durations of at least 5 frames ( 500 ms ) are plotted for representative ATDC5 cells , in which each color represents a different track ( A , B ) .", "Cellular domains outside the imaging plane appear black .", "The histogram represents the distribution within a single cell of trajectory durations for individual TβRI and TβRII molecules ( C ) .", "Representative individual TβRI sptPALM single molecule trajectories exhibiting immobile ( red ) , confined ( green ) , and freely diffusive ( blue ) movement are plotted in ( D ) , with calculated mean squared displacement ( MSD ) plots for each population of TβRI and TβRII shown in E ( mean ± SEM ) .", "Comparison of diffusion coefficients for TβRI and TβRII ( F , mean ± SEM ) in whole cells relative to areas of segregated TβRI/TβRII identify a less mobile population of TGFβ receptors in these regions of interest ( ROI ) .", "See Source code 1 and Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 00510 . 7554/eLife . 09300 . 006Figure 2—source data 1 . sptPALM single molecule trajectories DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 006 The distinct localization of TGFβ receptors could result from physical interactions with any number of known TGFβ receptor-associated proteins .", "Among these , integrins bind to TβRI and TβRII and functionally interact with the TGFβ pathway at multiple levels ( Wrana et al . , 2008; Scaffidi et al . , 2004 ) .", "The primary integrins in chondrocytes are integrins α2 and αV , which bind collagen and vitronectin/fibronectin ( Loeser , 2000 ) .", "Both integrins interact with the TGFβ pathway ( Scaffidi et al . , 2004; Garamszegi et al . , 2010 ) .", "TIRF imaging of mCherry-labelled integrin α2 revealed the presence of focal adhesions at these TβR-rich sites .", "Specifically , TβRII is absent from sites of adhesions and forms a peripheral ring surrounding integrin α2 , resulting in distinct patterns of spatial localization ( Figure 3A ) .", "Interestingly , this spatial organization is absent in cells grown on poly-l-lysine-coated substrates that facilitate integrin-independent cell adhesion ( Figure 3—figure supplement 1 ) .", "Therefore , TβRII organization at sites of adhesion is dependent upon integrin activity .", "Profile plots of intensity and a custom analysis ( Figure 3Ai , Bi , Ci ) were utilized to quantify colocalization between TβRs and integrin α2 across multiple cells .", "The slope of the regression line can be used as a metric , in which higher values indicate increased colocalization of two proteins .", "TβRI ( Alk5 and Alk1 ) is precisely colocalized with integrin α2 within focal adhesions , such that adhesions appear yellow ( Figure 3B , C ) and regression line slopes ( Figure 3Bi , Ci ) are higher relative to TβRII ( Figure 3A , Ai ) .", "This analysis reveals that integrin α2 colocalizes significantly more with Alk5 and Alk1 than with TβRII ( Figure 3D ) .", "The specific localization of TβRII near focal adhesions is apparent in cells of both mesenchymal ( ATDC5 , Figure 3A–C , E; Saos-2 , Figure 3F ) and epithelial ( MCF10A , Figure 3G ) origin and is observed whether integrin α2 or integrin αV is tagged with a fluorescent protein ( Figure 3 ) .", "Furthermore , this observation still holds if the fluorescent labels for TβRII and integrin α2 are switched , as well as if TβRII is expressed and imaged alone ( Figure 3E , H ) .", "The overall spatial organization of TGFβ receptors at sites of adhesion is not affected upon stimulation with exogenous TGFβ ( Figure 3I ) , suggesting that this spatiotemporal organization is regulated through mechanisms independent from TGFβ ligand addition .", "Given the critical role of integrins in mechanotransduction and the known sensitivity of TGFβ signaling to cellular tension ( Allen et al . , 2012 ) , the unique pattern of TGFβ receptor and integrin localization could prime TGFβ receptors for regulation by elements of the mechanotransduction pathway . 10 . 7554/eLife . 09300 . 007Figure 3 . Focal adhesions sequester TβRI from TβRII . TIRF mode imaging and a custom colocalization analysis were used to evaluate localization of TβRII ( A ) , Alk5 ( B ) , or Alk1 ( C ) with integrin α2 in ATDC5 cells .", "TβRII surrounds integrin α2 ( A ) , whereas both subtypes of TβRI , Alk5 ( B ) and Alk1 ( C ) , are included within integrin-rich focal adhesions , as reflected by profile plots and the slope values of the regression lines ( Ai , Bi , Ci ) .", "Quantification of colocalization reveals that Alk5 and Alk1 are significantly more colocalized with integrin α2 relative to TβRII ( **p < 0 . 001 , mean ± SD , D , Figure 3—source data 1 ) .", "This organization is also present in ATDC5 cells when the fluorescent labels for TβRII and integrin α2 have been switched ( E ) , in osteosarcoma Saos-2 cells ( F ) , or in epithelial MCF10A cells ( G ) , when labeling focal adhesions with integrin αV ( G ) , and when TβRII is expressed and imaged alone ( H ) .", "TβRII spatial organization is unaffected by addition of TGFβ , indicated by red outlines in the same cellular region following 15 min of TGFβ treatment ( I ) .", "See Source code 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 00710 . 7554/eLife . 09300 . 008Figure 3—source data 1 . Colocalization Index DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 00810 . 7554/eLife . 09300 . 009Figure 3—figure supplement 1 . Focal adhesion formation and TβRII spatial organization are dependent on integrin activity . TβRII spatial organization ( A , B , E , F ) and focal adhesion formation ( C , D , G , H ) are absent on poly-l-lysine ( B , D , F , H ) coated glass substrates relative to collagen II ( A , C , E , G ) .", "Spinning disc confocal microscopy at 40x ( A-D ) and TIRF microscopy at 100x ( E–H ) of ATDC5 cells expressing TβRII-mEmerald and integrin α2-mCherry demonstrate a loss of TβRII depleted regions ( A , B ) , TβRII peripheral ring formations ( E , F ) , and integrin α2 developed adhesions ( C , D , G , H ) on poly-l-lysine relative to collagen II-coated substrates . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 009 To investigate the effect of focal adhesions on TβRI ( Alk5 ) and TβRII dynamics , we used sptPALM to visualize TGFβ receptor trajectories near or within these vinculin-rich domains ( Figure 4A , B ) .", "SptPALM shows , both qualitatively and quantitatively , that TβRI is preferentially enriched and TβRII is preferentially excluded at sites of adhesion ( Figure 4A–C ) .", "Analysis of individual TβRII trajectories shows that TβRII ‘bounces’ around the edges of individual focal adhesions ( Figure 4E ) but is rarely incorporated within the focal adhesion , as is common for TβRI ( Figure 4D , i–ii ) .", "To determine if focal adhesions shifted the fractions of freely diffusive , confined , or immobile receptors , TβRI and TβRII trajectories near sites of adhesion were mapped based on receptor mobility .", "Trajectory maps reveal that TβRII mobility is confined near focal adhesions , which sequester and immobilize TβRI ( Figure 4F , G ) .", "Indeed , a higher fraction of immobilized TβRI is present inside adhesions relative to outside ( Figure 4H ) .", "Accordingly , the diffusion coefficient for TβRI decreases for tracks inside adhesions compared to those outside , demonstrating that this spatial organization specifically limits TβRI mobility ( Figure 4I ) .", "The differential localization and dynamics of TβRI and TβRII in adhesion-rich domains , relative to one another and to the whole cell TGFβ receptor population , indicates that this spatial control has functional implications for TGFβ signaling and for mechanotransduction . 10 . 7554/eLife . 09300 . 010Figure 4 . Dynamic interaction of TβRs with integrins facilitate spatial organization . Representative trajectories for TβRI ( Alk5 ) overlaid with the tagged focal adhesion marker vinculin are consistent with TIRF results showing a colocalization and interaction between integrin-based adhesions and TβRI ( A ) but not TβRII ( B ) .", "Quantification of these regions shows that TβRI is preferentially enriched inside adhesions relative to outside , and that TβRII is preferentially excluded at these same sites ( *p < 0 . 01 , mean ± SD , C ) .", "Representative single molecule trajectories show sequestration of TβRI in focal adhesions ( D , i-ii ) and free diffusion outside adhesions ( D , iii-iv ) , whereas TβRII bounces around the edges of focal adhesions in a freely diffusive ( E , i-ii ) or confined ( E , iii-iv ) manner .", "Analyzing TβR trajectories at focal adhesions based on diffusion ( Red: Immobile , Green: Confined , Blue: Freely Diffusive ) shows a higher density of tracks inside adhesions for TβRI ( F ) compared to TβRII ( G ) , and demonstrates a higher fraction of immobile TβRI tracks inside relative to outside adhesions ( H ) .", "The diffusion coefficient of TβRI trajectories decreases inside adhesions ( mean ± SD , I ) .", "See Source code 1 and Figure 4—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 01010 . 7554/eLife . 09300 . 011Figure 4—source data 1 . Enrichment Ratio and Diffusion Coefficient DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 011 To determine whether these changes in receptor mobility at sites of adhesion are due to direct or indirect physical interactions with other proteins , we performed mass spectrometry and co-immunoprecipitation experiments .", "Mass spectrometric analysis of proteins that precipitate with Flag-tagged TβRI ( Alk5 ) and TβRII revealed hundreds of proteins , several of which were specifically enriched compared with precipitates of untransfected ( mock ) cells .", "The analysis identified proteins already known to interact with TβRs , such as PRMT5 and PRMT1 ( Xu et al . , 2013 ) .", "TβRs also precipitated several adhesion-related proteins , including integrin αV and endogenous cofilin , as shown in the annotated spectra ( Figure 5A , B ) .", "The peptide counts ( graph insets ) indicate that integrin αV associates with both TβRI and TβRII , and that cofilin preferentially associates with TβRII ( Figure 5A , B ) .", "Cofilin is an actin-binding protein that severs ADP-actin filaments at the leading edge of migratory cells ( Pollard and Borisy , 2003 ) .", "Previous reports implicate cofilin as a target of TGFβ-activated RhoA , which promotes actin reorganization through ROCK , LIMK and cofilin ( Vardouli et al . , 2005; Lamouille et al . , 2014 ) .", "However , this is the first report , to our knowledge , of a complex between TGFβ receptors and cofilin .", "To confirm these mass spectrometry findings , we performed co-immunoprecipitation on cells expressing Flag-tagged TβRI/II and tagged integrin αV or cofilin ( Figure 5C , D ) .", "Consistent with the mass spectrometry peptide counts , integrin αV forms a complex with both TβRI and TβRII , whereas cofilin primarily interacts with TβRII .", "Although the novel finding of a complex formation , either through direct or indirect interactions , between TβRII and cofilin remains to be further explored , it suggests a potential mechanism underlying the discrete spatial organization of TβRII at focal adhesions . 10 . 7554/eLife . 09300 . 012Figure 5 . TβRs form complexes with integrin αV and cofilin . High-energy collision dissociation–tandem mass spectra obtained from precursor ions with mass 549 . 7775+2 ( A ) and 669 . 3185+2 ( B ) found in tryptic digests of immunoaffinity pulldowns of TβRI/II , corresponding to peptides spanning residues Y153-K165 of human integrin αV ( A ) and Y82-K92 of human cofilin ( B ) .", "b- and y- type ion series are labeled in the figure .", "Insets show the sequences of the peptides as well as representative peptide counts for integrin αV ( A ) and cofilin ( B ) for mock ( M ) , TβRI ( RI , Alk5 ) , and TβRII ( RII ) pulldowns .", "Co-immunoprecipitation of Flag-tagged TβRI and TβRII demonstrate the presence of integrin αV and cofilin in these complexes ( C , D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 012 Integrins transmit changes in the physical microenvironment across the plasma membrane to modulate cellular tension and signaling .", "The presence of a focal adhesion-associated TGFβ-receptor population suggests a novel mechanism by which cellular tension may regulate TGFβ signaling .", "To test the hypothesis that TGFβ receptor organization at focal adhesions is sensitive to cellular tension , we treated ATDC5 cells with the ROCK inhibitor Y27632 or the myosin II inhibitor blebbistatin .", "Within 15 min of adding Y27632 ( Figure 6A , B ) or blebbistatin ( Figure 6C , D ) , the peripheral ring of TβRII completely collapses .", "The segregation of TβRII from TβRI and integrin α2 at sites of adhesion is dynamically released , such that TβRII ( Video 1 ) converges and colocalizes with integrin α2 ( Video 2 , Video 3 ) .", "Quantitative analysis demonstrates that TβRII is significantly more colocalized with integrin α2 after addition of Y27632 and blebbistatin ( Figure 6E ) . 10 . 7554/eLife . 09300 . 013Figure 6 . Tension-sensitive regulation of TβR spatial organization . Within 15 min of disrupting cellular tension by adding the ROCK inhibitor Y27632 ( A , B ) or the myosin II inhibitor blebbistatin ( C , D ) , the peripheral ring of TβRII-mEmerald around focal adhesions ( A , C ) completely collapses ( B , D ) .", "Colocalization quantification ( Ai , Bi , Ci ) demonstrates that TβRII is significantly more colocalized with integrin α2 post-treatment ( Y27632 , blebbistatin ) relative to pre-treatment ( **p < 0 . 001 , mean ± SD , E , Figure 6—source data 1 ) .", "Disruption of tension with Y27632 enhances integrin αV association with TβRI but reduces its association with TβRII ( F ) .", "See Source code 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 01310 . 7554/eLife . 09300 . 014Figure 6—source data 1 . Colocalization Index ( vehicle and treatment ) DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 01410 . 7554/eLife . 09300 . 015Video 1 . Disruption of cellular tension leads to dynamic disassembly of TβRII spatial organization at sites of adhesion ( Figure 6 ) .", "TβRII-mEmerald spatial organization collapses within 15 min of adding ROCK inhibitor Y27632 in ATDC5 cells ( 45 min , 7 fps ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 01510 . 7554/eLife . 09300 . 016Video 2 . Disruption of cellular tension leads to dynamic disassembly of TβRII spatial organization at sites of adhesion ( Figure 6 ) .", "Integrin α2-mCherry adhesions disassemble within 15 min of adding ROCK inhibitor Y27632 in ATDC5 cells ( 45 min , 7 fps ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 01610 . 7554/eLife . 09300 . 017Video 3 . Disruption of cellular tension leads to dynamic disassembly of TβRII spatial organization at sites of adhesion ( Figure 6 ) .", "Composite of TβRII and integrin α2 ( Video 3 ) demonstrate a tension-sensitive collapse of this discrete spatial organization at sites of adhesion and a reorganization at the cell periphery . DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 017 To assess the effect of cellular tension on physical associations among TβRI , TβRII and integrins , we performed co-immunoprecipitation experiments .", "We find that cellular tension not only regulates the spatial organization of integrins and TGFβ receptors , but also affects their physical associations with each other; though these interactions may be direct or indirect .", "Specifically , while disruption of tension with the ROCK inhibitor enhanced integrin αV association with TβRI , it almost completely blocked the association between integrin αV and TβRII ( Figure 6F ) .", "Since a reduction in cellular tension drives colocalization of TβRI and TβRII , we sought to determine if this change in spatial organization had functional consequences for TGFβ signaling .", "We first evaluated the effect of reduced cellular tension on TβRI/TβRII heteromerization using co-immunoprecipitation .", "Release of this discrete spatial segregation of TGFβ receptors at focal adhesions allows the receptor subunits to interact such that ROCK-inhibition stimulates formation of heteromeric TβRI/TβRII complexes ( Figure 7A ) .", "To examine the effect of manipulating cellular tension under physiological conditions , we cultured cells on polydimethylsiloxane ( PDMS ) substrates of varying stiffness .", "A reduction in cellular tension through culture on compliant substrates significantly drives TβRI/TβRII complex formation ( Figure 7B ) .", "Therefore , a reduction in cellular tension , due to pharmacologic ROCK inhibition or changes to the stiffness of the microenvironment , drives formation of a multimeric TβRI/TβRII complex that is required for the activation of downstream TGFβ effectors . 10 . 7554/eLife . 09300 . 018Figure 7 . Disruption of tension-sensitive TβR segregation increases TβRI/TβRII multimerization and phosphorylation of Smad3 . ROCK inhibition releases the discrete spatial organization of TβRs at focal adhesions and drives the formation of heteromeric TβRI/TβRII complexes within 15 min of Y27632 exposure ( A ) , as shown by Flag co-immunoprecipitation ( IP ) and immunoblotting ( IB ) .", "Likewise , manipulation of cellular tension through culturing cells on collagen II-coated glass or 0 . 5 kPa PDMS substrates increases co-immunoprecipitation of TβRI with Flag-tagged TβRII ( p < 0 . 05 , B ) .", "In cells grown on collagen II-coated compliant ( 0 . 5 kPa , p < 0 . 05 ) or stiff ( 16 kPa ) PDMS substrates , endogenous Smad3 phosphorylation is increased ( C ) .", "The effect of TGFβ on Smad3 phosphorylation is substrate-dependent , such that maximal TGFβ-inducibility is observed on 0 . 5 kPa substrates ( p < 0 . 05 ) , consistent with a tension-sensitive calibration of TβR localization and activity ( C ) .", "See Figure 7 – source data .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 01810 . 7554/eLife . 09300 . 019Figure 7—source data 1 . Western Quantitative Analysis DOI: http://dx . doi . org/10 . 7554/eLife . 09300 . 019 To determine the effect of tension-sensitive TβR localization and heteromerization on downstream TGFβ effectors , we evaluated the phosphorylation of Smad3 .", "Culturing cells on compliant substrates leads to significantly increased endogenous Smad3 phosphorylation ( Figure 7C ) .", "Interestingly , the effect of TGFβ on Smad3 phosphorylation is substrate-dependent , such that TGFβ induces Smad3 phosphorylation on 0 . 5 kPa substrates but not on 16 kPa substrates ( Figure 7C ) .", "This is consistent with the established non-linear response of TGFβ signaling and other cellular behaviors to cellular tension ( Allen et al . , 2012; Rape et al . , 2015 ) .", "Thus the spatial organization of TβRI and TβRII by integrins at focal adhesions affords tension-sensitive control of TβRI and TβRII multimerization and activation of Smad3 , providing a mechanosensitive mechanism by which cells calibrate their response to TGFβ ." ], [ "Here we show that cellular tension regulates TGFβ receptor spatial organization and interactions at focal adhesions , providing a novel mechanism for the cellular integration of signaling by physical and biochemical cues .", "We observe a novel spatiotemporal regulation of the TGFβ pathway such that TβRII is segregated from TβRI and integrins at sites of adhesions .", "Single particle tracking reveals the dynamics of individual TGFβ receptor molecules , and identifies populations of TGFβ receptors with distinct behaviors and mobility near and far from sites of focal adhesions .", "The confined population of TGFβ receptors at focal adhesions has lower mobility than the freely diffusive receptor population far from sites of adhesion .", "TGFβ receptors associate with several adhesion-related proteins , including the actin-binding protein cofilin , which preferentially associates with TβRII relative to TβRI .", "This novel spatial organization of TβRI and TβRII at sites of adhesion provides mechanosensitive control of TGFβ receptor multimerization and function independently of TGFβ ligand stimulation .", "Overall , this reveals the potential of two differentially regulated populations of TGFβ receptors – one that is TGFβ-sensitive and one that is tension-sensitive – a finding that may contribute to the context-dependent signaling outcomes of this pathway .", "This tension-dependent mechanism for the regulation of TGFβ receptors has a number of interesting functional implications .", "At the level of the TGFβ ligand , integrins activate TGFβ from its latent form through cellular tension generated by actomyosin contraction ( Wipff et al . , 2007; Munger and Sheppard , 2011; Wells and Discher , 2008; Giacomini et al . , 2012 ) .", "The observed recruitment of TGFβ receptors to focal adhesions would enrich their access to this reservoir of integrin-activated TGFβ .", "At the receptor level , focal adhesions may sequester TβRI from TβRII to limit their activity in the presence of ligand .", "The extent to which this sequestration is cofilin-dependent requires further investigation .", "This sequestration of TβRI may contribute to its slow internalization , relative to TβRII , following TGFβ stimulation ( Vizan et al . , 2013; Ma et al . , 2007 ) .", "Alternatively , focal adhesions may create structured TβRI and TβRII boundaries that prime a robust response when cells encounter the correct combination of physical and biochemical cues .", "We demonstrate tension-sensitive regulation of endogenous downstream Smad3 phosphorylation by cellular tension and TGFβ .", "In addition , chondrocytes grown in TGFβ on 0 . 5 MPa substrates induce differentiation markers far beyond levels induced by either cue alone ( Allen et al . , 2012 ) .", "We and others have reported that the effect of substrate stiffness or cellular tension/Rho/ROCK activity on downstream TGFβ signaling is synergistic and nonlinear ( Allen et al . , 2012; Wang et al . , 2012; Leight et al . , 2012 ) .", "Therefore , it is possible that lower cell tension in one cell type may have a differential effect on Smad phosphorylation , nuclear localization , and transactivation than in another cell type .", "It would be interesting to examine this effect utilizing a substrate system that provides independent and continuous gradients of ligand density and substrate stiffness ( Rape et al . , 2015 ) .", "The mechanisms responsible for such synergy have been unclear , but this newly described regulation of TGFβ receptor multimerization and downstream signaling may couple the mechanosensitive activity of the TGFβ pathway to physical cues .", "Fully understanding the functional implications of this spatially-distinct TGFβ receptor population will require the development of new imaging tools , such as those that can dynamically visualize TGFβ effector activity locally at focal adhesions .", "The current study of TGFβ receptors opens the possibility that tension-sensitive receptor multimerization may underlie mechanosensitive signaling by other pathways .", "Cellular tension impacts the activation , translocation , and function of intracellular effectors including small GTP-ases , kinases and transcriptional regulators such as Smads and YAP/TAZ ( Allen et al . , 2012; Wang et al . , 2012; Dupont et al . , 2011; Leight et al . , 2012 ) .", "However , known mechanisms are insufficient to explain the ability of physical cues to modulate cell-type specific responses to BMP , EGF , and other growth factors ( Wang et al . , 2012; Paszek et al . , 2005 ) .", "Several receptor families share features with TGFβ receptors that may contribute to their mechanosensitivity , such as their association with integrin-rich focal adhesions and their potential for the formation of stable receptor clusters by geometric constraints ( Bethani et al . , 2010; Salaita et al . , 2010; Hartman and Groves , 2011 ) .", "Previous studies have established important physical and functional links between focal adhesion components and growth factor receptors .", "EGF receptor binds actin and colocalizes with integrin α2β1 ( Alam et al . , 2007 ) , while the receptor CD44 interacts with several components of the focal adhesion complex , such that hyaluronan-bound CD44 activates c-Src and Rac1 ( Turley et al . , 2002 ) .", "Aside from TGFβ , shown herein , the extent to which these physical associations contribute to mechanosensitive control of receptor multimerization or downstream signaling remains to be determined .", "Nonetheless , others have postulated receptor multimerization as a mechanism for mechanocoupling of TGFβ , ephrin , and T cell receptor signaling ( Hartman and Groves , 2011; Hynes , 2009 ) .", "In each case , the solid state presentation of the ligand is thought to play a critical role in structuring multimeric receptor clusters .", "In T cell receptor and ephrin signaling , the solid state is provided by ligands on the neighboring cell , which create geometric constraints that mechanically trap receptors to induce clustering ( Bethani et al . , 2010; Salaita et al . , 2010; Hartman and Groves , 2011 ) .", "For growth factors like TGFβ , BMP , and EGF , the ECM serves as the solid state ( Hynes , 2009 ) .", "ECM proteins such as collagen II bind both TGFβ and integrin α2β1 ( Zhu , 1999 ) , imposing geometric constraints that may structure receptor clusters .", "Therefore , growth factor receptor multimerization at focal adhesions , controlled by receptor interactions with integrins and with solid state growth factors , provide focal adhesions with the capability to integrate signaling between physical and biochemical cues .", "Understanding the mechanosensitive regulation of TGFβ signaling has significant biological implications .", "We find that focal adhesions segregate TβRI from TβRII in both epithelial and mesenchymal cell lineages , opening the possibility that this is a general cellular mechanism for the control of TGFβ signaling .", "It will be interesting to determine if TGFβ receptor multimerization at focal adhesions responds to physical cues that aberrantly promote TGFβ-induced epithelial-mesenchymal transition ( EMT ) in cancer or the loss of chondrocyte homeostasis in osteoarthritis .", "On stiff substrates , TGFβ preferentially activates PI3K to induce EMT instead of apoptosis ( Leight et al . , 2012 ) .", "In osteoarthritis , the material properties of the cartilage ECM deteriorate as chondrocytes inappropriately shift the balance from canonical ( Alk5/Smad2/3 ) to non-canonical ( Alk1/Smad1/5/8 ) TβRI signaling ( Blaney Davidson et al . , 2009 ) .", "In each case , the extent to which changing the physical environment alters TGFβ effector selection through differential TGFβ receptor multimerization remains to be determined .", "Applied physical cues , such as compression or shear flow , also regulate TGFβ signaling in cartilage , vasculature , and other tissues ( Li et al . , 2010; Sakai et al . , 1998; Streuli , 1993 ) .", "Whether similar mechanisms operate in response to exogenous physical cues remains to be elucidated .", "In conclusion , we utilized novel high-resolution imaging and single particle tracking microscopy coupled with biochemical assays to explore the spatial organization of TGFβ signaling at the receptor level .", "At focal adhesions , TβRII is uniquely segregated from its TβRI counterpart .", "Cellular tension modulates the spatial organization , multimerization , and downstream signaling of TGFβ receptors at sites of adhesion , suggesting the existence of a functionally distinct subpopulation of TGFβ receptors .", "Overall , this finding provides a new mechanism by which cellular tension and physical cues exert control of growth factor signaling at the cellular membrane ." ], [ "The plasmids pRK5 TGFβ type I receptor Flag and pRK5 TGFβ type II receptor Flag were gifts from Rik Derynck ( Addgene plasmids 14 , 831 ( Feng and Derynck , 1996 ) , 31719 ) .", "The plasmid pRK5 TGFβ type I receptor Myc was also a gift from Rik Derynck .", "All fluorescent protein expression vectors are available in the Michael Davidson Fluorescent Protein Collection on Addgene .", "All fluorescent protein expression vectors were constructed using C1 or N1 ( Clontech-style ) cloning vectors and initially characterized using the advanced EGFP variant mEmerald to verify proper localization of the fusions .", "To construct the N-terminal ( with respect to the fluorescent protein ) human integrin alpha2 ( NM_002203 . 3 ) fusions , the following primers were used to amplify the integrin alpha 2 , and create the 18-amino acid linker ( GSAGGSGVPRARDPPVAT ) : XhoI forward: CTC CGT CTC GAG ACC GCC ATG GGG CCA GAA CGG ACA GGG GCC KpnI reverse: CGG AAC GGT ACC CCG CTT CCG CCT GCG CTG CCG CTA CTG AGC TCT GTG GTC TCA TCA ATC TCA TCT GGA TTT TTG GTC Following digestion and gel purification , the PCR product was ligated into a similarly digested mEmerald-N1 cloning vector to produce mEmerald-Integrin alpha2-N-18 .", "The resulting fusion , along with mCherry-N1 cloning vector , was sequentially digested with AgeI and NotI to yield mCherry-Integrin alpha2-N-18 .", "To generate the N-terminal human integrin alpha V ( NM_002210 . 4 ) fusions , the following primers were used to amplify the protein and create the 25-amino acid linker ( PGSRAQASNSAVDGTAGPGSPPVAT ) : AgeI forward: CCC GGG ATC CAC CGG TCG CCA CCA TGG CTT TTC CGC CGC GGC GAC GGC TGC GCC TCG GTC HindIII reverse: AAT TGA AGC TTG AGC TCG AGA TCC CGG AAG TTT CTG AGT TTC CTT CAC CAT TTT CAT GAG GTT GAA GCT GCT CCC TTT CTT GTT CTT CTT GAG The PCR product was digested , gel purified , and ligated into a similarly treated mEmerald-N1 or mCherry-N1 cloning vector to yield the mEmerald-Integrin alphaV-25 or mCherry-Integrin-alphaV-25 fusions .", "To construct the N-terminal tagged human vinculin ( NM_003373 . 3 ) plasmids , the following primers were used to PCR-amplify and create a 21-amino acid linker ( SGGSGILQSTVPRARDPPVAT ) : NheI forward: GTC AGA TCC GCT AGC ACC GCC ACC ATG CCA GTG TTT CAT ACG CGC ACG ATC GAG AGC EcoR1 reverse: CGA CTG CAG AAT TCC GCT GCC ACC GGA CTG GTA CCA GGG AGT CTT TCT AAC CCA GCG CAG The PCR product was digested and ligated into a similarly cut mEmerald-N1 or mCherry-N1 cloning vector to yield mEmerald-Vinculin-N-21 or mCherry-Vinculin-N-21 expression vectors .", "To construct the C-terminal human TβR2 ( NM_001024847 . 2 ) fusion plasmids , the following primers were used to amplify the TβR2 and generate an 18-amino acid linker ( SGLRSRESGSGGSSGSGS ) : XhoI forward: GAC GAG CTC GAG AGA GTG GCT CTG GTG GGT CGA GTG GAA GTG GCA GCG GTC GGG GGC TGC TCA GGG GCC TG BamHI reverse: CGT CTA GGA TCC CTA TTT GGT AGT GTT TAG GGA GCC GTC TTC AGG AAT CTT CTC C Following digestion and gel purification , the PCR product was ligated into a similarly digested mEmerald-C1 cloning vector , to produce mEmerald-TβRII-C-18 .", "The fusion , along with mCherry-C1 and mEos2-C1 cloning vectors , was sequentially digested with AgeI and BamHI and ligated to yield mCherry-TβRII-C-18 and mEos2-TβRII-C-18 .", "To generate the N-terminal human TβRII plasmids and create an 18-amino acid linker ( SSGGASAASGSADPPVAT ) , the following primers were used: NheI forward: CGA TCC GCT AGC GCC ACC ATG GGT CGG GGG CTG CTC AGG GGC BamHI reverse: CCT GTA CGG ATC CGC GCT ACC ACT GGC TGC GCT TGC TCC ACC GCT GCT TTT GGT AGT GTT TAG GGA GCC GTC TTC AGG AAT CTT CTC C The PCR fragment was digested , gel purified , and ligated with a similarly treated mEmerald-N1 cloning vector to produce mEmerald-TβRII-N-18 .", "The resulting fusion , along with mCherry-N1 and mEos2-N1 , was double digested with BamHI and NotI to yield mCherry-TβRII-N-18 and mEos2-TβRII-N-18 respectively .", "To construct the N-terminal human Alk1 ( NM_000020 . 2 ) expression vectors , the following primers were used to amplify the plasmid , and create a 13-amino acid linker ( GSAGGSGDPPVAT ) : EcoRI forward: GCG TTG AAT TCA CCG CCA TGA CCT TGG GCT CCC CCA GGA AAG GCC BamHI reverse: CGG AAC GGA TCC CCG CTT CCG CCT GCG CTG CCT TGA ATC ACT TTA GGC TTC TCT GGA CTG TTG CTA ATT TTT TGT AGT GTC TTC TTG ATC Following amplification , the PCR fragment was digested , purified , and ligated to a similarly treated mEmerald-N1 cloning vector to yield mEmerald-Alk1-N-13 .", "Upon sequence verification , the resulting fusion , along with mCherry-N1 , was digested with BamH1 and NotI and ligated to yield mCherry-Alk1-N-13 .", "To generate the C-terminal human Alk5 ( NM_004612 . 2 ) expression vectors , the following primers were used to amplify the plasmid , and create an 18-amino acid linker ( SGLRSGSSAGSASGGSGS ) : BglII forward: GAC TCG AGA TCT GGC TCC AGC GCA GGC AGC GCA TCC GGC GGA AGC GGA AGC GAG GCG GCG GTC GCT GCT CCG CGT C HindIII reverse: CGG TCA AAG CTT TTA CAT TTT GAT GCC TTC CTG TTG ACT GAG TTG CGA TAA TGT TTT CTT AAT CCG C Following amplification , the PCR fragment was digested , purified , and ligated to a similarly treated mEmerald-C1 cloning vector to yield mEmerald-Alk5-C-18 .", "The resulting fusion , along with mCherry-C1 and mEos2-C1 , was double digested with BglII and NheI to yield mCherry-Alk5-C-18 and mEos2-Alk5-C-18 .", "To construct the N-terminally labeled Alk5 fusions , the following primers were used to amplify the Alk5 and generate a 13-amino acid linker ( GSGGAGGGGPVAT ) : BglII forward: GTC TGT AGA TCT GCC ACC ATG GAG GCG GCG GTC GCT GCT CCG AgeI reverse: CGG TCA ACC GGT CCT CCG CCG CCC GCA CCC CCG GAA CCC ATT TTG ATG CCT TCC TGT TGA CTG AGT TGC GAT AAT GTT TTC TTA ATC CGC Following amplification , the resulting fragment was digested , purified , and ligated to a similarly treated mEmerald-N1 cloning vector , resulting in mEmerald-Alk5-N-13 .", "Following sequence verification , the plasmid , along with mCherry-N1 and mEos2-N1 cloning vectors , was sequentially digested with AgeI and NotI and ligated to produce mCherry-Alk5-N-13 and mEos2-Alk5-N-13 fusions .", "All DNA for transfection was prepared using the Plasmid Maxi kit ( QIAGEN , Valencia , CA ) and characterized by transfection in HeLa cells ( CCL2 line; ATCC , Manassas , VA ) using Effectene ( QIAGEN ) followed by observation under widefield fluorescence illumination to ensure proper localization .", "The sequences for all vectors were confirmed using Big Dye technology ( The Florida State University Bioanalytical and Molecular Cloning DNA Sequencing Laboratory Tallahassee , FL ) .", "Studies were performed using ATDC5 murine chondroprogenitor cells ( RCB0565 , RIKEN , Wako , Japan ) , NIH3T3 fibroblasts , MCF10A human mammary epithelial cells , and Human Embryonic Kidney ( HEK ) 293 cells .", "Cells were treated as indicated with TGFβ1 ( 5 ng/ml , HumanZyme , Chicago , IL ) , Y27632 ( 10 μM , Sigma-Aldrich , St . Louis , MO ) , and blebbistatin ( 10 μM , Cayman Chemical , Ann Arbor , MI ) .", "For imaging experiments , glass-bottom imaging wells ( Cellvis , Mountain View , CA ) were coated with collagen II ( 1 mg/ml in acetic acid diluted 1:100 in PBS ) , fibronectin ( 1 mg/ml diluted 1:100 in PBS ) , or poly-l-lysine ( 0 . 1 mg/ml ) .", "ATDC5 , NIH3T3 , and MCF10A cells were transfected using Nucleofection ( Lonza , Basel , Switzerland ) or Effectene ( Qiagen , Valencia , CA ) , and then plated on to the imaging wells .", "For biochemical assays , 293 cells were plated in 100 mm cell-culture dishes and transfected using Effectene ( Qiagen ) at 80% confluency .", "For co-immunprecipitation experiments , 293 cells were transfected with integrin αV-mCherry , cofilin-mEmerald , TβRI-Flag , TβRII-Flag , and/or TβRI-Myc .", "24 hr after transfection , cells were treated with TGFβ or Y27632 for 15 min .", "Cells were lysed ( 50 mM Tris pH 7 . 5 , 150 mM NaCl , 2 mM EDTA , 0 . 5% Igepal , 0 . 25% sodium deoxycholate , supplemented with protease and phosphatase inhibitor tablets ) and immunoprecipitated with anti-Flag M2 beads ( Sigma-Aldrich ) overnight prior to Western analysis ( Alliston , 2001 ) .", "For immunoblotting experiments of downstream endogenous proteins , 293 cells were cultured on plastic or fibronectin-coated gel substrates ( Advanced BioMatrix , Carlsbad , CA ) , treated with TGFβ as indicated for 30 min and then lysed .", "Blots were probed with anti-Flag ( F3165 , Sigma-Aldrich ) , anti-CD51 , integrin αV ( 611013 , BD Biosciences , San Jose , CA ) , anti-cofilin ( ACFL02 , Cytoskeleton , Denver , CO ) , anti-pSmad3 ( gift from E . Leof , Mayo Clinic , Rochester , MN ) , anti-β-actin ( ab8226 , Abcam , Cambridge , MA ) and anti-TβRI ( sc-398 , Santa Cruz Biotechnology , Santa Cruz , CA ) , and anti-mouse anti-rabbit secondary antibodies that were conjugated to 680 or 800CW IRDye fluorophores for detection using a LI-COR infrared imaging system ( LI-COR Biosciences , Lincoln , NE ) .", "Blots shown are representative of multiple technical replicates of at least three independent experiments for each condition ( N≥3 ) .", "Where indicated , quantitative analysis was performed using ImageJ ( National Institutes of Health , Bethesda , MD ) .", "Band intensity for proteins of interest was normalized to band intensity of controls ( Flag for co-IP and β-actin for whole cell extract ) .", "Fold change in band intensity was calculated relative to plastic control samples .", "ANOVA followed by Bonferroni correction and student’s t test were used to evaluate statistical significance .", "For immunofluorescence studies , ATDC5 cells were cultured on collagen II-coated glass substrates in 8 well Lab-Tek chamber slides ( Nunc/Thermo Fisher Scientific , Waltham , MA ) .", "Cells were fixed ( 4% paraformaldehyde ) and permeabilized ( 0 . 5% Triton X-100 in PBS ) .", "Primary antibodies included anti-TβRII ( sc-1700 , sc-400 , Santa Cruz ) and anti-TβRI ( sc-398 , sc-9048 , Santa Cruz ) .", "Cells were imaged as described below .", "Cells expressing TβRII-Flag or TβRI-Flag and integrin αV-mCherry in 10 cm cell culture dishes were lysed as above and affinity-purified with M2-Flag magnetic beads ( Sigma-Aldrich ) , followed by on-bead trypsin digestion ( Kean et al . , 2012 ) and mass spectrometry approaches to study associated proteins ( N=3 ) .", "Peptides recovered were analyzed by reversed-phase liquid chromatography-electrospray tandem mass spectrometry ( LC-MS/MS ) as described ( Duong et al . , 2015 ) .", "Briefly , peptides were separated by nano-flow chromatography in a C18 column , and the eluate was coupled to a hybrid linear ion trap-Orbitrap mass spectrometer ( LTQ-OrbitrapVelos , Thermo Fisher Scientific , Waltham , MA ) equipped with a nanoelectrospray ion source .", "Following LC-MS/MS analysis , peak lists generated from spectra were searched against the human subset of the SwissProt database using in-house ProteinProspector ( Clauser et al . , 1999 ) .", "For analysis , peptide counts of each protein were normalized by the total protein content in the sample and the molecular weight of the respective protein .", "This provided an abundance index for each protein that served as a comparison between pulldowns .", "The ratio between abundance indices for TβR pulldowns to untransfected control ( mock ) pulldowns was used to screen candidate proteins .", "ATDC5 , NIH3T3 , and MCF10A cells were transiently transfected with fluorescently labeled expression plasmids and plated on collagen II , fibronectin or poly-l-lysine-coated glass-bottom imaging wells .", "Cells were imaged 24 hr after transfection , and treated with Y27632 , blebbistatin , or TGFβ as indicated .", "Confocal images were obtained on a motorized Yokogawa CSU-X1 spinning disk confocal unit on an inverted microscope system ( Ti-E Perfect Focus System , Nikon , Tokyo , Japan ) , with either a 100X/NA 1 . 49 oil-immersion objective ( CFI Apo TIRF , Nikon ) or a 40X/NA 1 . 15 water-immersion objective ( CFI Apo LWD , Nikon ) , on a front illuminated CMOS camera ( Zyla sCMOS , Andor , Belfast , United Kingdom ) .", "For TIRF and sptPALM , imaging was performed on a motorized objective-type TIRF inverted microscope system ( Ti-E Perfect Focus System , Nikon ) with activation and excitation lasers of 405 nm , 488 nm , and 561 nm , and an electron-multiplying charged-coupled device camera ( QuantEM 512 , Photometrics , Tuscon , AZ ) , a 100X/NA 1 . 49 oil-immersion objective ( CFI Apo TIRF , Nikon ) , a stage top incubator ( Okolab , Burlingame , CA ) , and controlled by NIS-Elements software ( Nikon ) .", "Cells expressing mEos2-tagged constructs were simultaneously activated with a 405 nm laser and excited with a 561 nm laser .", "Laser intensities were adjusted to maintain a constant sparse population of activated molecules that were spaced well enough for accurate localization and tracking .", "Prior to each sptPALM imaging sequence and photoconversion of mEos2 , the mEmerald signal from mEmerald fusions of vinculin was imaged to localize focal adhesions .", "NIS-Elements software ( Nikon ) was used for the acquisition of images at 10 fps .", "Individual receptors were localized and tracked using a previously described algorithm ( Sbalzarini and Koumoutsakos , 2005 ) written in MosaicSuite for ImageJ and available at ( www . mosaic . mpi-cbg . de ) .", "All images were processed using ImageJ with a 0 . 6 gaussian blur filter to remove noise .", "Images shown are representative of multiple cells ( N≥5 ) for at least three independent experiments for each condition .", "TIRF mode imaging was used to obtain intensity profiles of two distinct molecules over adhesion-rich regions of interest ( for example , regions shown in Figure 3A–C ) .", "The similarity of the two profiles was quantified to provide a measure of colocalization , specifically by comparing pixel intensities ( 8-bit grayscale ) at each point across the two profiles .", "For each pixel , an ordered pair containing the intensities at that particular coordinate from both images was plotted .", "Values closer to the line y=x refer to coordinates that have very similar intensities in both profiles .", "Values further from y=x are coordinates that have a mismatch in intensities .", "By reflecting all points in the top half of this graph across y=x , a distribution of points is created between y=x and the x-axis , but the distance of individual points from y=x is preserved .", "The magnitude of the slope of the regression line through these points can be used as a quantitative metric of colocalization .", "The greater this slope , the higher the degree of colocalization .", "Plots are representative of multiple cells ( N≥3 ) and multiple regions of interest ( N=5 ) .", "ANOVA followed by Bonferroni correction was used to evaluate statistical significance .", "Each sptPALM imaging sequence generates tens of thousands of molecule trajectories per cell ( N=6 cells for each TβR ) .", "From these , only trajectories lasting between 0 . 5 seconds and 2 seconds ( 5 to 20 frames at 10 fps ) were selected for analysis .", "Tracks that were not confined to either inside or outside focal adhesions were not considered in the quantitative analysis .", "For each individual track , a series of parameters were calculated to quantify receptor dynamics .", "These parameters include mean squared displacement ( MSD ) , diffusion coefficient ( D ) , and radius of confinement ( rconf ) .", "MSD was computed as per Equation 1 ( Rossier et al . , 2012 ) : ( 1 ) MSD ( τ=n·∆t ) =∑i=1N−n ( xi+n−xi ) 2+ ( yi+n−yi ) 2N−n .", "Where xi and yi are the coordinates of the molecule at time i*∆t and N is the number of frames for which the trajectory persisted .", "The radius of confinement ( rconf ) of a track is defined to be the magnitude of the radius of the smallest circle that encloses all points in that track .", "D is defined as one-fourth of the slope of the regression line fitted to the first four values of the MSD as per Equation 2 .", "( 2 ) MSD ( τ ) =4Dτ .", "Using these variables , trajectories were pooled into three fractions: immobile , confined , and freely diffusive .", "Immobile molecules were defined as being restricted to a radius of confinement equal to one pixel ( rconf < 0 . 166 μm ) .", "Confined molecules were defined as non-immobile tracks with a diffusion coefficient D of less than 0 . 2 µm2/s , and the remaining tracks were considered freely diffusive .", "Custom routines written for Python were used for track quantification , analysis , and visualization ( source code ) .", "To account for variability in these large data sets consisting of tens of thousands of tracks , we report mean and standard error of the mean ( SEM ) .", "ANOVA followed by Bonferroni correction and student’s t test were used to evaluate statistical significance .", "To quantify the diffusive behavior of TβRI and TβRII around focal adhesions , we calculated an enrichment ratio to compare track densities in several ( N≥5 ) focal adhesion-rich regions ( where vinculin covered more than 25% of total area ) across at least three cells .", "The enrichment ratio was defined as the ratio of the track density ( tracks/µm2 ) inside adhesions to the track density outside adhesions within a given area .", "Student’s t test was used to evaluate statistical significance .", "For colocalization quantification ( Figure 3 and Figure 6 ) and enrichment ratio ( Figure 4C ) , we report mean and standard deviation ( SD ) .", "There are three circumstances in which it was more statistically appropriate to report the standard error of the mean ( SEM ) .", "Specifically , to account for variability in large sptPALM data sets ( consisting of tens of thousands of tracks ) , we report mean and SEM ( Figure 2E , F and Figure 4I ) .", "Significance was calculated with ANOVA followed by Bonferroni correction and student's t test , with significance defined as p<0 . 01 ." ] ]

[ "Cell surface receptors are central to the cell's ability to generate coordinated responses to the multitude of biochemical and physical cues in the microenvironment .", "However , the mechanisms by which receptors enable this concerted cellular response remain unclear .", "To investigate the effect of cellular tension on cell surface receptors , we combined novel high-resolution imaging and single particle tracking with established biochemical assays to examine TGFβ signaling .", "We find that TGFβ receptors are discretely organized to segregated spatial domains at the cell surface .", "Integrin-rich focal adhesions organize TβRII around TβRI , limiting the integration of TβRII while sequestering TβRI at these sites .", "Disruption of cellular tension leads to a collapse of this spatial organization and drives formation of heteromeric TβRI/TβRII complexes and Smad activation .", "This work details a novel mechanism by which cellular tension regulates TGFβ receptor organization , multimerization , and function , providing new insight into the mechanisms that integrate biochemical and physical cues ." ]

[ "Cells constantly encounter diverse physical and biological signals in their surroundings .", "Information contained in these signals is transmitted from the cell surface to the interior to trigger coordinated changes in the cell’s behavior .", "Physical signals include the forces generated by cells pulling on one another or on their surroundings .", "These pulling forces calibrate the cell’s response to biological signals through mechanisms that remain unclear .", "The cell surface contains many different proteins that are specialized to sense these signals and guide the cell’s response .", "In animals , these membrane proteins include the receptors that detect a small signaling protein known as TGFβ .", "TGFβ first binds to one of these receptors ( called TβRII ) .", "Next another receptor ( called TβRI ) is recruited to the complex .", "Once this complex is formed , the TGFβ receptors activate a complicated signaling pathway that controls how cells grow and divide .", "Previous work has shown that the TGFβ pathway can also sense and respond to mechanical forces .", "But it remains poorly understood how pulling forces ( or tension ) impact TGFβ receptors at the cell surface .", "Rys , DuFort et al . have now used cutting-edge microscopy and biochemical techniques to analyze individual TβRI and TβRII receptors and observe how they respond to mechanical forces in real-time .", "This revealed that TβRI and TβRII exist in discrete regions on the cell surface .", "Rys , DuFort et al . observed that TβRI is enriched at assemblies of molecules called focal adhesions .", "Focal adhesions are the sites on cell surfaces that allow cells to adhere to one another and to the molecular scaffolding in their surroundings .", "Unlike TβRI , TβRII was often excluded from these sites and more commonly appeared to ‘bounce’ around the edges of individual focal adhesions .", "Therefore , focal adhesions limit the interactions between TβRI and TβRII , by sequestering one away from the other .", "Rys , DuFort et al . next treated cells with a chemical that disrupts tension , and saw that the physical separation between TβRI and TβRII collapsed , which permitted these two receptors to interact and form a working signaling complex .", "Further work is needed to understand how physical control of TGFβ receptor interactions helps cells coordinate their tasks in response to the myriad biological and physical signals in their surroundings ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease", "genetics and genomics" ]

[ [ "Invasive fungal infections are an increasing problem worldwide , contributing to 1 . 6 million deaths annually ( Almeida et al . , 2019; Bongomin et al . , 2017; Brown et al . , 2012 ) .", "These problematic infections are difficult to treat for many reasons .", "Delayed diagnoses , the paucity and toxicity of antifungal drugs , and the already immunocompromised state of many patients result in mortality rates of up to 90% ( Brown et al . , 2012; Pianalto and Alspaugh , 2016; Scorzoni et al . , 2017 ) .", "To date , there are only four classes of antifungals , which primarily target the fungal cell envelope ( cell wall and plasma membrane ) ( Coelho and Casadevall , 2016; Odds et al . , 2003; Pianalto and Alspaugh , 2016; Scorzoni et al . , 2017 ) .", "The population of immunocompromised individuals is growing due to medical advancements , such as immunosuppression for transplants and chemotherapy .", "Emerging fungal pathogens are simultaneously increasing in both clinical burden and the number of causal species due to human activity such as agricultural drug use ( Berger et al . , 2017 ) and global warming ( Almeida et al . , 2019; Garcia-Solache and Casadevall , 2010 ) .", "Thus , the need for more and better antifungal therapeutics is evident .", "Cryptococcosis is among the most common invasive mycoses , causing 220 , 000 life-threatening infections and 180 , 000 deaths annually worldwide ( Rajasingham et al . , 2017 ) .", "Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis , though nearly 95% of cases are caused by C . neoformans ( Brown et al . , 2012; Maziarz and Perfect , 2016 ) .", "As C . neoformans is globally distributed throughout the environment , most individuals are exposed by two years of age ( Goldman et al . , 2001 ) .", "However , systemic disease primarily occurs in the immunocompromised , particularly those with decreased T helper-1 cell reactions ( Maziarz and Perfect , 2016 ) .", "Accordingly , HIV/AIDS patients account for 80% of cryptococcal cases ( Maziarz and Perfect , 2016; Rajasingham et al . , 2017 ) .", "The primary treatment for cryptococcosis involves three different classes of antifungals .", "Standard care is a combination of amphotericin B ( polyene class ) and 5-fluorocytosine ( 5-FC; pyrimidine analog ) for two weeks , followed by high dose azole treatment ( e . g . fluconazole ( FLZ ) ) for at least 8 weeks , and finally a low dose oral FLZ for at least 6 months ( Cox and Perfect , 2018; Mourad and Perfect , 2018 ) .", "Despite this , mortality rates remain as high as 80% for cryptococcal meningitis ( Rajasingham et al . , 2017 ) .", "This is mainly due to the difficulty of obtaining ideal treatment standards .", "5-FC is unavailable in 78% of countries , mostly due to licensing issues ( Kneale et al . , 2016; Mourad and Perfect , 2018 ) .", "Without the inclusion of 5-FC in the treatment regiment , mortality increases by up to 25% ( Kneale et al . , 2016 ) .", "Amphotericin B is administered intravenously , requiring hospitalization .", "Treatment with amphotericin B is therefore particularly challenging in areas such as sub-Saharan Africa , which has the highest burden of cryptococcal disease ( Rajasingham et al . , 2017 ) .", "Due to these therapeutic hurdles , many patients are treated with FLZ alone , which decreases survival rates from 75% to 30% in high burden areas ( Kneale et al . , 2016 ) .", "Additional treatment options are thus needed to prevent these unnecessary deaths .", "One theoretical approach to improve treatment is synergistic combination therapy .", "Synergistic interactions occur when the combined effect of two drugs is greater than the sum of each drug’s individual activity ( Cokol et al . , 2011; Kalan and Wright , 2011 ) .", "This is a powerful treatment option which has been utilized for a variety of infections ( Kalan and Wright , 2011; Robbins et al . , 2015; Spitzer et al . , 2011; Zheng et al . , 2018 ) .", "For instance , the common combination of sulfamethoxazole and trimethoprim for bacterial infections is a synergistic interaction that works to inhibit sequential steps in bacterial folic acid biosynthetic pathway ( Kalan and Wright , 2011 ) .", "In addition , Amphotericin B and 5-FC act synergistically , and mortality rates increase dramatically when one is unavailable ( Beggs , 1986; Kneale et al . , 2016; Schwarz et al . , 2006 ) .", "Synergistic interactions can also cause fungistatic drugs to switch to fungicidal , providing a more effective treatment option ( Cowen et al . , 2009 ) .", "Additionally , molecules can interact antagonistically to decrease therapeutic efficacy ( Caesar and Cech , 2019; Roberts and Gibbs , 2018 ) .", "Bacterial growth increases when antibiotics that inhibit DNA synthesis are used in combination with protein synthesis inhibitors ( Bollenbach et al . , 2009 ) .", "Such antagonistic interactions further complicate already challenging infections ( Khandeparkar and Rataboli , 2017; Vadlapatla et al . , 2014 ) , since immunocompromised patients are frequently treated with multiple drugs .", "56% of AIDS patients experience polypharmacy , or greater than five medications ( Siefried et al . , 2018 ) .", "Polypharmacy doubles the risk of antiviral therapy nonadherence to 49% of HIV+ patients ( Lohman et al . , 2018 ) and increases mortality by 68% in HIV+ and 99% in HIV- patients ( Cantudo-Cuenca et al . , 2014 ) .", "Better understanding of the molecular mechanisms underlying both synergistic and antagonistic drug interactions will allow us to improve identification and selection for or against these interactions .", "The overlap2 method ( O2M ) uses at least one known synergistic drug pair and a large-scale chemical-genetics dataset to predict synergistic and antagonistic drug interactions rapidly and on large scales ( Brown et al . , 2014; Wambaugh and Brown , 2018; Wambaugh et al . , 2017 ) .", "Each molecule of the synergistic pair induces a growth phenotype in a precise set of mutants ( enhanced or reduced growth ) .", "Since these mutants exhibit the same phenotype in the presence of both molecules in a synergistic pair , we hypothesize that any other molecule eliciting the same phenotype in those mutants will also synergize with each molecule in the original pair .", "This method can be used against multiple microbes and applied to any published chemical-genetics dataset ( Brown et al . , 2014; Wambaugh et al . , 2017 ) .", "In this study , we utilized previously identified synergy prediction mutants ( Brown et al . , 2014 ) to screen a library of small molecules enriched for Federal Drug Administration ( FDA- ) -approved molecules .", "We non-discriminately identified 59 molecules that interact with FLZ , either synergistically or antagonistically .", "When validating these new combinations , we found that even though the analysis used a C . neoformans dataset ( Brown et al . , 2014 ) , our synergistic and antagonistic combinations acted against pathogenic fungi from multiple phyla .", "These include C . deuterogattii , Candida species , and multiple clinical and environmental strains of C . neoformans , as well as clinical isolates of the increasingly problematic and multi-drug resistant species Candida auris ( Chowdhary et al . , 2017 ) .", "Furthermore , we elucidated molecular mechanisms underlying the interaction with FLZ for a few of our most clinically relevant combinations .", "We also demonstrate these effects in an in vivo model of cryptococcosis .", "A particularly promising synergistic combination , dicyclomine hydrochloride and FLZ , almost doubled time-to-endpoint in a murine infection model .", "In sum , our high-throughput method , O2M , identifies FLZ interacting molecules with potential clinical impacts ." ], [ "We previously demonstrated that O2M identifies genes whose knockout mutants , termed synergy prediction mutants , exhibit phenotypes that are indicative of synergistic interactions between small molecules ( Brown et al . , 2014; Wambaugh et al . , 2017 ) .", "O2M identifies synergy prediction mutants by using a chemical-genetics dataset , in which a library of knockout mutants is grown in the presence of >100 small molecules .", "We calculated quantitative growth scores ( slower or faster growth ) for each mutant/molecule combination .", "This large number of phenotypes produces a ‘chemical genetic signature’ for each molecule in the dataset .", "We then compared the ‘signatures’ for fluconazole and known fluconazole synergizers to identify knockout mutants that exhibit significant phenotypes ( i . e . statistically significant slow or fast growth ) in all chemical genetic signatures ( Brown et al . , 2014 ) .", "The rationale was that similarities between chemical-genetic signatures of known synergistic pairs contain information that is indicative of the synergistic interaction with fluconazole .", "We term these ‘synergy prediction mutants’ because their knockout phenotype predicts additional fluconazole-synergizing molecules .", "We then identified genes whose mutant exhibited significant growth scores ( |Z| > 2 . 5 ) when compared to wild-type growth for fluconazole and each of its known synergizers .", "We compared the significant gene set for fluconazole and each synergizer and identified common genes .", "We called these ‘synergy prediction mutants’ ( Figure 1A ) .", "Since fluconazole and its known synergizers induce a growth phenotype from these mutants , we hypothesized that any molecule eliciting the same growth phenotype would also act synergistically with fluconazole .", "This was completed and tested in our previous publication ( Brown et al . , 2014 ) using FLZ and its known synergistic interacting partners fenpropimorph and sertraline ( Jansen et al . , 2009; Zhai et al . , 2012 ) .", "Our work identified three synergy prediction mutants ( cnag_00573Δ , cnag_03664Δ , and cnag_03917Δ ) ( Brown et al . , 2014 ) .", "Since each chemical-genetic signature contains phenotypes from ~1400 gene knockouts , there does not have to be considerable overlap between chemical-genetic signatures to identify synergy prediction mutants .", "Here we use synergy prediction mutants to rapidly screen for small molecules that synergize with fluconazole and can be quickly moved into clinical use .", "CNAG 00573 encodes a NADH dehydrogenase ( Janbon et al . , 2014 ) , CNAG 03664 encodes NIC1 , a high-affinity nickel-transporter ( Singh et al . , 2013 ) , and CNAG 03917 encodes a nuclear pore complex protein homologous to Nup75 ( Liu et al . , 2008; Stajich et al . , 2012 ) .", "Using these gene mutants , we performed a high-throughput screen for synergistic interactions .", "Our assay is simple: differential growth between wild-type and synergy prediction mutants is indicative of a synergistic interaction with FLZ or any other starting drug .", "It does not require multi-drug assays , as the ‘synergy prediction mutant’ substitutes for one of the small molecules in the interaction , phenocopying the FLZ-small molecule interaction to produce synthetic lethality .", "We screened the Microsource Spectrum Collection , a small-molecule library of 2000 compounds enriched for FDA-approved molecules .", "We grew C . neoformans wild-type and synergy prediction mutants ( cnag_00573Δ and cnag_03917Δ ) in the presence of each small molecule ( 1 µM ) , identifying those that caused a significant difference in growth between the wild-type and both synergy prediction mutants after 48 hr of growth ( Figure 1B ) .", "The mutant cnag_03664Δ was not used due to its inherent slow growth .", "The small molecule concentration of 1 µM gave the lowest false discovery rate when testing molecules known to synergize or not synergize with fluconazole .", "Using these synergy prediction mutants , we identified 313 putative FLZ synergistic molecules ( Supplementary file 1 ) .", "We validated potential synergistic interactions in checkerboard assays , for which serial dilutions of each drug are crossed in a 96-well plate ( Figure 1B ) .", "Synergistic interactions are defined as a ≥ 4 fold decrease in the minimum inhibitory concentration ( MIC ) of each small molecule in the pair , resulting in a fractional inhibitory concentration index ( FICI ) of ≤0 . 5 ( Johnson et al . , 2004; Odds , 2003 ) .", "We tested the 129 molecules with single agent efficacy against C . neoformans growth in the preferred checkerboard assay .", "We found that 40 molecules were synergistic with FLZ , meaning 31% of these molecules were correctly predicted by O2M ( Figure 2A ) .", "However , checkerboard assays require that both small molecules in the pair are able to inhibit growth of C . neoformans individually , which was not the case with all our putative synergistic molecules .", "In those cases , we performed Bliss Independence , which identifies whether molecules enhance the action of FLZ ( Tang et al . , 2015 ) .", "In a 96-well plate , we created a gradient of FLZ combined with 10 µM and 100 nM concentrations of the 55 small molecules that could not inhibit C . neoformans alone .", "We found 6 of these molecules enhanced the action of FLZ at both 10 µM and 100 nM concentrations and were deemed synergistic ( Figure 2—figure supplement 1 ) .", "The FLZ-synergistic molecules belonged to a wide range of bioactive categories including antidepressants , adrenergic agonists , as well as antiinfectives ( Figure 2B and Table 1 ) .", "Additionally , our screen identified antagonistic interactions .", "These interactions are defined by a minimum 4-fold increase in MICs causing increased fungal growth and a FICI ≥4 ( Cetin et al . , 2013 ) .", "We identified 19 antagonistic interactions with FLZ ( Figure 2C ) .", "Of note , many antagonists were documented antiinfectives , including some antifungals ( Figure 2D and Table 1 ) .", "The remaining 70 molecules did not interact with FLZ and represent false positives of our screen ( Figure 2—figure supplement 2A ) .", "Overall , the O2M screen predicted that 16% of the library’s small molecules would interact with FLZ .", "Of the predicted interactions , 46% were validated by checkerboard assay to truly interact with FLZ ( synergistic or antagonistic ) ( Figure 2E ) .", "All small molecules that interact with FLZ and inhibit fungal cell growth ( i . e . were tested in checkerboard assays ) are listed with their MICs in Table", "1 . We determined a MIC for 90% growth inhibition ( MIC90 ) for most molecules .", "For a subset , we were only able to determine a MIC for 50% growth inhibition ( MIC50 ) .", "In some cases , molecule solubility in aqueous solutions are too low to reach concentrations necessary to inhibit fungal growth .", "Alternatively , the inability to determine a MIC90 could result from trailing growth , which has been seen in fungal species ( Arthington-Skaggs et al . , 2002 ) .", "FLZ non-interacting molecules are listed in Supplementary file", "2 . The remaining 129 molecules predicted to interact were not tested due to unavailability , or known toxicities that would have made them impractical treatments .", "During the screening process , wild-type C . neoformans is grown in each of the small molecules alone , allowing us to identify general anti-C .", "neoformans molecules ( Figure 1B ) .", "These molecules had MICs ranging from 16 nM to 760 µM , and were mostly listed as antifungals ( Supplementary file 3 ) .", "However , the phenotype of a general anti-C .", "neoformans molecule can overshadow any synergistic phenotypes in the synergy prediction mutant .", "Therefore , we also tested these molecules for synergistic interactions in the standard checkerboard assay .", "Two of the general anti-C .", "neoformans molecules , sulconazole nitrate and tacrolimus , were synergistic with FLZ ( Figure 2—figure supplement 2B ) .", "Once we identified new FLZ-based synergistic pairs , we sought to predict additional FLZ-synergizing molecules in order to increase our chances of identifying a promising therapeutic combination .", "We hypothesized that structural similarity with our newly-identified FLZ-synergizing molecules would predict additional FLZ synergizers .", "We chose four new FLZ synergizers that contain large ring structures , which are common among our synergizers: dicyclomine HCl , desipramine HCl , sertraline HCl , and diphenhydramine HCl .", "We used both ChemSpider ( Pence and Williams , 2010 ) and ChemMine MSC Similarity Tool to identify structurally similar molecules ( Backman et al . , 2011 ) .", "We chose three molecules ( proadifen , drofenine , and naftidrofuryl ) with ≥50% structural similarity to dicyclomine HCl , and all were synergistic with FLZ in checkerboard assays ( Figure 3A–D , M ) .", "We tested three molecules ( impramine , mianserine , and lofepramine ) with >40% structural similarity to desipramine , two of which synergized with FLZ ( Figure 3E–H , M ) .", "Lofepramine , which was not synergistic , is the prodrug to desipramine .", "Sibutramine , shares >40% structural similarity with sertraline , and is synergistic with FLZ as well ( Figure 3I , J , M ) .", "Lastly , we tested citalopram , which shares >45% structural similarity to diphenhydramine , but was not synergistic with FLZ ( Figure 3K–M ) .", "Overall , 75% of the structurally similar molecules acted synergistically with FLZ , demonstrating that structure can serve as a powerful basis to predict additional synergistic combinations prior to elucidating mechanism of action .", "As our goal is to identify potential antifungal therapies with broad efficacy , we tested our new synergistic pairs against additional strains of C . neoformans and other medically important fungi .", "Since we identified numerous fluconazole interaction molecules ( Figure 2A ) , we focused on several promising synergistic molecules based on either low MICs or interesting bioactivity .", "We tested these interactions against the C . neoformans lab strain KN99 and 10 environmental or clinical C . neoformans isolates ( Chen et al . , 2015 ) .", "We also tested our combinations against Cryptococcus deuterogattii , Candida albicans , Candida glabrata , and two strains of Candida auris ( Supplementary file 4 ) .", "Our FLZ-synergizing small molecules displayed similar MICs across the different C . neoformans strains and fungal species ( Supplementary file 5 ) .", "Of the FLZ-interacting pairs , sertraline hydrochloride ( HCl ) , clomipramine HCl , benzalkonium chloride , berbamine HCl and dicyclomine HCl synergistically inhibited the growth of most of the strains/species ( Figure 4A , B , C , D , E ) .", "We also chose two antagonistic interactions to test in these additional strains and species based on bioactivities relevant to cryptococcosis patient populations .", "We chose nafcillin sodium , a common antibiotic , and Ivermectin , an antiparasitic drug ( Table 1 and Figure 2C ) .", "Both bacterial and parasitic infections are common among immunocompromised patients ( Kaplan et al . , 2009 ) .", "Nafcillin sodium was antagonistic with FLZ in most of the strains/species ( Figure 4M ) .", "The other synergistic or antagonistic molecule pairs had more variable results among the different strains/species ( Figure 4 ) , highlighting the importance of testing putative antimicrobials against multiple species and strains when considering therapeutic relevance .", "Upon identifying FLZ interacting combinations that are consistent among other fungal species and strains , we sought to investigate a potentially harmful combination for treating fungal infections .", "Among the antagonists , we were particularly interested in the antagonistic interaction between nafcillin sodium ( nafcillin ) and FLZ that emerged in multiple fungal strains and species ( Figure 4M ) .", "Nafcillin is a common penicillinase-resistant penicillin antibiotic ( Letourneau , 2019; Nathwani and Wood , 1993 ) .", "Furthermore , the cryptococcosis patient population , which consists of mainly HIV/AIDS patients , is at high risk for multiple infections , increasing the likelihood that they could require overlapping treatments for bacterial and fungal infections ( Kaplan et al . , 2009 ) .", "We first tested the nafcillin + FLZ combination on additional clinical isolates of either C . neoformans or Candida auris that are considered FLZ resistant ( Supplementary files 4 and 6 ) .", "The FLZ MIC for resistant C . neoformans strains ranged from 1 to 32 µg/mL and was 256 µg/mL for C . auris upon initial resistance characterization .", "In all 7 C . neoformans strains , nafcillin antagonized FLZ activity .", "Nafcillin acted antagonistically with FLZ against one of the three C . auris strains ( Figure 4—figure supplement 1 and Figure 4—source data 5 ) .", "We next asked whether other beta-lactam antibiotics also antagonize FLZ activity ( Figure 5A–L ) .", "Antagonism with FLZ was not a universal attribute among penicillin antibiotics but was evident for oxacillin and methicillin ( Figure 5M ) .", "We also tested a first-generation cephalosporin , cefazolin , that is often prescribed in place of nafcillin ( Letourneau , 2019; Miller et al . , 2018 ) and a second-generation cephalosporin , cefonicid .", "We found that both of these molecules also antagonize FLZ ( Figure 5M ) .", "Of the beta-lactams tested , those that are often used to treat Staphylococcus aureus were antagonistic with FLZ ( Fowler and Holland , 2018; Letourneau , 2019 ) .", "We therefore decided to test additional antibiotics used to treat S . aureus infections .", "These included vancomycin and linezolid ( Fowler and Holland , 2018; Lowy , 2019 ) , neither of which interacted with FLZ ( Figure 5M ) .", "To investigate the mechanism of antagonism , we looked to other known drug interactions involving nafcillin .", "In particular , nafcillin antagonizes warfarin and other drugs in vivo by inducing cytochrome P450 enzymes through an unknown mechanism , which increases warfarin metabolism ( King et al . , 2018; Wungwattana and Savic , 2017 ) .", "FLZ inhibits a fungal cytochrome P450 enzyme , 14α-demethylase , which halts ergosterol biosynthesis and fungal growth ( Figure 5N; Odds et al . , 2003 ) .", "We hypothesized that nafcillin may also induce cytochrome P450 enzymes , such as 14α-demethylase , in C . neoformans , counteracting FLZ’s mechanism of action .", "To test this hypothesis , we examined whether nafcillin affects ergosterol biosynthesis .", "We extracted sterols from C . neoformans cells grown in the presence of nafcillin , FLZ , nafcillin + FLZ , or vehicle .", "We found an increase in ergosterol with a high-dose nafcillin treatment ( Figure 5O ) .", "Ergosterol levels in nafcillin + FLZ are not statistically different from the control treatment ( Figure 5O ) .", "Next , we tested whether nafcillin is synergistic with the antifungal amphotericin B . Since amphotericin B kills target cells by binding and extracting ergosterol from the plasma membrane ( Anderson et al . , 2014 ) , we hypothesized that if nafcillin increases ergosterol levels in C . neoformans , nafcillin would act synergistically with amphotericin B by increasing amphotericin B binding sites ( i . e . ergosterol ) .", "Indeed , amphotericin B was synergistic with nafcillin in checkerboard assays giving an average FICI score of 0 . 5 ( Figure 5—source data 2 ) , further suggesting that nafcillin increases ergosterol levels in C . neoformans .", "Lastly , ketoconazole is an imidazole antifungal that , like fluconazole , inhibits ergosterol synthesis ( Marichal et al . , 1985; Odds et al . , 2003 ) and is also a potent inhibitor of antiretroviral drug metabolism through its inhibition of cytochrome P450 enzymes ( Dooley et al . , 2008; Kaeser et al . , 2009; Polk et al . , 1999 ) .", "Ketoconazole is used in certain areas worldwide in order to increase the half-life of antivirals in blood ( Dooley et al . , 2008 ) .", "We found that nafcillin also antagonizes ketoconazole activity , producing an average FICI score of 5 against C . neoformans ( Figure 4—source data 2 ) .", "Overall , nafcillin and other beta-lactam antibiotics could decrease treatment efficacy when combined with antifungals FLZ and ketoconazole .", "Finally , we investigated a promising fluconazole-synergizer that was effective against multiple fungal species and strains: dicyclomine HCl ( dicyclomine ) , an anticholinergic agent ( Table 1 ) .", "To further evaluate the potential of the combination , we first tested this molecule in the FLZ-resistant C . neoformans and Candida auris strains ( Supplementary files 4 and 6 ) .", "Dicyclomine was synergistic with FLZ against each of these resistant strains and species ( Figure 4—figure supplement 2 ) .", "We also tested whether dicyclomine synergizes with ketoconazole .", "As previously mentioned , ketoconazole has effects in combination with antiretroviral therapies and maybe used in certain areas of HIV endemicity .", "We found the dicyclomine + ketoconazole combination was also synergistic in a checkerboard assay , giving an average FICI score of 0 . 313 ( Figure 4—source data 4 ) .", "We next investigated the molecular mechanism underlying the dicyclomine + FLZ interaction .", "Dicyclomine is an FDA-approved drug , also known as Bentyl , used to treat urinary incontinence ( Malone and Okano , 1999; Page and Dirnberger , 1981 ) .", "It targets a G-protein coupled receptor ( GPCR ) encoded by the CHRM1 gene ( UniProt Consortium . , 2018; Kilbinger and Stein , 1988 ) .", "C . neoformans does not have a CHRM1 ortholog , but there are a large number of GPCRs in fungi that could be potential targets ( Xue et al . , 2008 ) .", "When we screened a deletion library of C . neoformans for mutants resistant to dicyclomine , we found that 44% of the annotated dicyclomine-resistant mutants were involved in transport and trafficking , suggesting that those processes may be related to dicyclomine’s mechanism ( Figure 6A and Supplementary file 7 ) .", "Thus , we hypothesized that dicyclomine alters Golgi transport .", "In Saccharomyces cerevisiae , simultaneously inhibiting Golgi trafficking and blocking ergosterol synthesis leads to mislocalization of essential plasma membrane transporters ( Estrada et al . , 2015 ) .", "We hypothesized that the combination of dicyclomine and FLZ will phenocopy this effect in C . neoformans ( Figure 6B ) .", "Using propidium iodide internalization as a measure of cell permeability , we observed that dicyclomine , similar to FLZ , permeabilizes fungal cells at high doses ( Figure 6C , D , F , G ) .", "Furthermore , we saw a greater than additive increase in permeability when fungal cells were treated with low concentrations of dicyclomine and FLZ in combination ( Figure 6E and H ) .", "Dicyclomine-induced permeability appeared to be independent of significant changes to cell wall chitin ( Figure 6—figure supplement 2A-F ) .", "We next tested if dicyclomine + FLZ disrupted nutrient transporter function by measuring uptake of amino acids .", "If amino acid permeases are not localized to the plasma membrane , fungal cells are resistant to toxic amino acid analogs ( Roberg et al . , 1997 ) .", "Using the same low doses of FLZ and DIC that alone do not permeabilize cells , C . neoformans is susceptible to the effect of either 5-fluoroanthranilic acid ( 5-FAA ) or 5-methyl-tryptophan ( 5-MT ) .", "When the dose is combined , cells now show resistance ( Figure 6I–L and Figure 6—figure supplement 1G–J ) .", "This demonstrates that certain amino acid transporters’ function is decreased and they thus may be mislocalized , conferring resistance to toxic forms of tryptophan .", "Finally , we tested whether dicyclomine is able to kill C . neoformans cells , either alone or when combined with FLZ .", "FLZ is fungistatic , inhibiting the growth the C . neoformans cells but not killing them ( Hazen , 1998; Klepser et al . , 1998 ) .", "Since synergistic interactions can cause fungistatic drugs to switch to fungicidal ( Cowen et al . , 2009 ) , we tested whether our new synergistic combination of dicyclomine + FLZ killed C . neoformans cells .", "Using the stain BCECF-AM to identify dead cells ( McMullan et al . , 2015 ) , we evaluated various doses of dicyclomine or FLZ individually or in combination .", "As a control , we tested the fungicidal drug amphotericin B ( Gray et al . , 2012 ) .", "Dicyclomine only caused cell death after 3 hr at a very high dose ( Figure 6—figure supplement 2A , E ) .", "We saw no increase in death of C . neoformans cells after treatments of FLZ after 3 hr ( Figure 6—figure supplement 2B , F ) .", "However , when treating cells with 8 µg/mL amphotericin B nearly 100% of cells died within 3 hr ( Figure 6—figure supplement 2D , H ) .", "After a 3 hr treatment , we did not observe an increase in cell death with the combination ( Figure 6—figure supplement 2C , G ) .", "However , since dicyclomine + FLZ increases cell permeability and decreases nutrient uptake , we hypothesized that it may cause a slower death of cells than a fungicidal drug such as amphotericin B . Thus , we also sought to determine fungal cell death after 3 , 9 , and 24 hr of treatment .", "After 9 hr of treatment , we found that C . neoformans cells treated with all synergistic combinations and doses of FLZ 1/3 MIC and higher exhibited increased cell death ( Figure 6—figure supplement 2J ) .", "After 24 hr of treatment , all synergistic combinations exhibited nearly 100% cell death .", "Additionally , high doses of FLZ also had killed almost all cells ( Figure 6—figure supplement 2K ) .", "When evaluating the combination with the lowest FLZ + dicyclomine doses , we did not observe cell death at 3 hr .", "However , after 9- and 24 hr treatment , the combination had significantly more cell death than untreated or single agent-treated cells ( Figure 6—figure supplement 2M; Figure 6M ) .", "Thus , our synergistic combination is fungicidal .", "Since dicyclomine + FLZ combination exhibit a potent synergistic interaction in vitro , we tested its efficacy in a mouse model of cryptococcal meningitis .", "We intranasally inoculated outbred CD-1 mice ( Charles River ) with C . neoformans and allowed the infection to progress for 8 days .", "Colony forming unit data indicated that at this point 100% of the mice exhibited fungal dissemination to the liver , and 40% exhibited dissemination to the brain ( Figure 7—figure supplement 1 ) .", "A disseminated infection is consistent with human patients at treatment onset , as patients often don’t seek treatment until C . neoformans has disseminated to the brain ( Zhu et al . , 2010 ) .", "Between 8- and 40 days post-inoculation ( d . p . i ) , we intraperitoneally administered dicyclomine , FLZ , dicyclomine + FLZ , or PBS ( vehicle ) daily .", "We used doses of both FLZ and dicyclomine which were within the range of doses given to humans ( Lexicomp , 2019a; Lexicomp , 2019b ) .", "We sacrificed mice when they reached 80% of their initial mass ( survival endpoint ) .", "Dicyclomine alone did not affect mouse survival compared to PBS-treatment .", "However , dicyclomine in combination with FLZ significantly improved survival over FLZ alone in a dose-dependent manner ( Figure 7 ) , indicating that dicyclomine is not effective at treating cryptococcosis on its own , but could well be of therapeutic benefit when combined with FLZ ." ], [ "Synergistic combination therapies are increasingly important clinical options , especially for drug resistant microbes ( Cowen and Lindquist , 2005; Kalan and Wright , 2011; Uppuluri et al . , 2008; Zheng et al . , 2018 ) .", "Traditionally , synergistic drug pairs were discovered serendipitously , but new methods are improving our ability to uncover important interactions ( Brown et al . , 2014; Cokol et al . , 2011; Cokol et al . , 2018; Jansen et al . , 2009; Robbins et al . , 2015; Spitzer et al . , 2011; Wambaugh et al . , 2017; Wildenhain et al . , 2015 ) .", "In this study , we identified a wide variety of molecules that interact synergistically with the antifungal FLZ to inhibit fungal growth .", "We do so without the use of noisy multi-drug assays , allowing for rapid and scalable screening .", "We also identify and investigate antagonistic interactions , which are clinically important ( Khandeparkar and Rataboli , 2017; Vadlapatla et al . , 2014 ) but have not been investigated in a systematic manner .", "This work and our application of O2M to antibiotic trimethoprim ( Wambaugh et al . , 2017 ) suggests that O2M’s synergy prediction genes tend to identify synergizers with mechanism of action functionally related to those of the starting synergizer .", "Here , one of the known synergizers used to identify synergy prediction genes , sertraline , inhibits phospholipase activity in Saccharomyces cerevisiae , which inhibits vesicle formation Rainey et al . , 2010 ) .", "This could limit the range of synergizers identified by O2M .", "However , our studies on the trimethoprim demonstrates that identifying synergizers that phenocopy the downstream effect of known synergistic pairs , but target different factors , will bypass resistance to the starting synergizers due to the new targets ( Wambaugh et al . , 2017 ) .", "Of the 59 FLZ interacting molecules we identified , 10 have been previously described in various fungi .", "Of those 10 , three were reported as synergists but antagonized FLZ activity in our assays ( Ahmad et al . , 2010; Butts et al . , 2014; Cardoso et al . , 2016; Eldesouky et al . , 2018; Kang et al . , 2010; Li et al . , 2018; Marchetti et al . , 2000; Quan et al . , 2006; Robbins et al . , 2015; Spitzer et al . , 2011; Zhai et al . , 2012 ) .", "Prior work found that a single small molecule can both synergize with and antagonize the activity of a second small molecule depending on the concentration ( Meletiadis et al . , 2007 ) .", "This phenomenon could explain the difference in some of our results compared to previous published interactions .", "An important step in drug discovery is the ability to improve upon the efficacy of known drugs through synthetic modification and/or identification of structurally related molecules .", "We found that structural similarity predicts synergistic interactions ( Figure 3 ) , just as drugs of similar structure have similar function .", "These data demonstrate that many additional synergizers and antagonizers can be identified from a single example .", "Furthermore , we identified broad spectrum interactions .", "All the combinations tested showed efficacy against multiple clinical and environmental isolates of C . neoformans , as well as C . deuterogattii , a related species which can cause disease in apparently immunocompetent individuals ( Applen Clancey et al . , 2019 ) .", "We also tested our combinations against common Candida species that often develop multi-drug resistance ( Colombo et al . , 2017 ) , including the emerging MDR pathogen Candida auris ( Figure 4 ) .", "Our data demonstrate that FLZ synergizers and antagonizers exhibit broad activities against multiple species and isolates .", "We investigated the antagonistic interaction between FLZ and nafcillin , a beta-lactam antibiotic commonly used against Staphylococcus aureus and other difficult-to-treat bacterial infections ( Letourneau , 2019 ) .", "Patients with these infections include some of the same patients at risk for cryptococcosis ( HIV and cancer patients ) ( Kaplan et al . , 2009; Utay et al . , 2016 ) and other fungal infections , including C . auris ( Rudramurthy et al . , 2017 ) .", "Nafcillin can reach mean plasma concentrations in humans of 30 µg/mL after a single 500 mg dose ( FDA , 2020 ) .", "This is above the concentration needed to achieve antagonism in vitro and could prove problematic in patients .", "When we examined nafcillin-related molecules , we found that methicillin and oxacillin also antagonize FLZ .", "Furthermore , two cephalosporins often used in place of nafcillin , cefonicid and cefazolin , also unexpectedly antagonize fluconazole activity .", "Nafcillin has previously been shown to adversely affect patients on the drug warfarin due to nafcillin’s induction of cytochrome P450 ( King et al . , 2018 ) , which decreases warfarin concentration .", "This has been seen in a similar combination of FLZ and the antibiotic rifampicin ( Panomvana Na Ayudhya et al . , 2004 ) .", "Rifampicin is a potent inducer of drug metabolism due to elevation of hepatic cytochrome P450 through increased gene expression ( Bolt , 2004 ) .", "This combination did indeed lower the levels of FLZ ( Panomvana Na Ayudhya et al . , 2004 ) , resulting in relapse of cryptococcal meningitis ( Coker et al . , 1990 ) .", "We hypothesize that an analogous process occurs when nafcillin is combined with FLZ , with nafcillin increasing ergosterol biosynthesis enzymes , counteracting FLZ’s activity .", "In a recent autopsy study , 10 or 16 patients who died of cryptococcosis were administered either a penicillin or a cephalosporin ( Hurtado et al . , 2019 ) .", "We recommend that these patients receive linezolid or vancomycin instead , since these drugs are used for similar bacterial targets but do not antagonize fluconazole activity ( Figure 5N ) .", "Our data demonstrate that O2M identifies promising new antifungal treatments that can rapidly move into the clinic .", "Our new combination of dicyclomine + FLZ almost doubled the median time-to-endpoint of mice treated with human dosages of dicyclomine ( Figure 7 ) , which is lower than dicyclomine’s fungal MIC .", "Additionally , dicyclomine + FLZ appear to kill C . neoformans cells , not just inhibit C . neoformans cell growth , which increases this combination’s therapeutic potential ( Figure 6M and Figure 6—figure supplement 2 ) .", "Dicyclomine’s serum concentration in mice administered 60 µg was 0 . 2–0 . 6 µg/mL at 18 hr post injection ( Karaka et al . , 2004 ) .", "While this is lower than dicyclomine’s antifungal MIC , dicyclomine improves mouse survival when combined with FLZ even at these low doses ( Figure 7 ) .", "Dicyclomine is orally bioavailable and able to cross the blood brain barrier ( Das et al . , 2013; Koerselman et al . , 1999 ) , which makes it particularly promising for fungal meningitis treatment .", "Since dicyclomine , like many of our new FLZ synergizers , is approved by the FDA for other indications , it could rapidly move into the clinic .", "In sum , O2M considerably streamlined the identification of important drug interactions affecting C . neoformans growth .", "These interactions are both synergistic and antagonistic among multiple fungal species capable of causing disease in humans .", "We focused on FDA-approved molecules to bypass the time and considerable expense it takes to develop a new drug ( Pushpakom et al . , 2019 ) .", "However , our method would work equally well on any library of small molecules or biologic drugs to discover new antifungals .", "We showed that identifying these drug interactions can quickly lead to additional interacting pairs by examining structure ( Figure 3 ) or by investigating underlying mechanism ( Wambaugh et al . , 2017 ) .", "Finally , our newly discovered interaction of dicyclomine and FLZ exhibited therapeutic potential in vivo , demonstrating the clinical potential of fluconazole-containing synergistic pairs in the clinic ." ], [ "Screening , validation , and structurally similar assays were performed with CM18 lab strain of C . neoformans .", "Screening with synergy prediction mutants ( CNAG_00573Δ and CNAG_03917Δ ) was in the CM18 background .", "Mechanistic studies were tested using the KN99 lab strain of C . neoformans .", "Clinical and environmental isolates of C . neoformans tested were a gift from Dr . John R . Perfect .", "C . deuterogattii strain R265 was purchased from ATCC .", "Candida albicans reference strain SC5314 and Candida glabrata reference strain CBS138 were used .", "Candida auris strains AR0383 and AR0384 were from the CDC .", "We inoculated either CM18 wild-type or CNAG_00573Δ or CNAG_03917Δ cells at 1000 cells per well of YNB + 2% glucose , then added small molecule to a final concentration of 1 µM .", "Plates were incubated for 48 hr at 30°C .", "OD600 was measured on the BioTek plate reader model Synergy H1 .", "Small molecules that altered growth by absolute value 0 . 22 in both synergy prediction mutants but not the wild type strain was considered significant .", "We found altered growth by 0 . 22 to give us the lowest false discovery rate when testing known synergistic and non-synergistic molecules .", "All assays were performed in 1x YNB + 2% glucose .", "To determine MICs , an overnight culture was grown at 30°C with rotation , diluted to OD600 = 0 . 02925 and 1000 cells were added to each well ( 2 µL of culture into 100 µL of media per well ) .", "Plates were incubated at 30°C unless otherwise stated .", "Small molecules were dissolved in DMSO to their highest soluble concentration and gradients were diluted in 2-fold dilution series .", "MIC values were calculated after 48 hr of incubation below the inhibitory effects of DMSO alone .", "We followed previously published methods ( Hsieh et al . , 1993; Orhan et al . , 2005 ) .", "Starting inoculation of either fungal strain was 2 µL of an OD600 = 0 . 02925 ( about 1000 cells per well of 100 µL medium ) .", "This inoculum was used for all fungal species and strains .", "Standard 96-well plates were grown statically for 48 hr at 30°C with minor shaking/resuspension of cells at 24 and 48 hr .", "Checkerboards were read at 0 and 48 hr on a BioTek plate reader model Synergy H1 to measure the OD600 ( Candida albicans and Candida glabrata were read at 0 , 24 , and 48 hr ) .", "Growth inhibition was assessed and FICIs for 50% and 90% inhibition were determined using standard methods ( see Wambaugh and Brown , 2018 ) .", "Briefly , to determine the FICI for each tested drug combination , we use the following formula to calculate FICI for each well in a 96 well plate that exhibits an OD600 of ≤90% of the no drug control well: FICI = ( concentration of drug #1 in well ) / ( MIC90 of drug #1 ) + ( concentration of drug #2 in well ) / ( MIC90 of drug #2 ) .", "FICI ≤0 . 5 is considered synergistic and FICI ≥4 . 0 is considered antagonistic .", "When testing for a synergistic interaction , the FICI is determined by the lowest scoring well in the plate .", "The FICI-determining well must exhibit a 4-fold decrease in drug concentration for each drug compared to the MIC90 of each drug alone .", "For antagonistic interactions , the FICI is determined by the highest scoring well in a plate .", "Repeated results were averaged for the average FICI .", "Outliers with a different result ( e . g . All replicates were performed on different days from independent stating cultures and are independent biological replicates .", "FICI scores presented in the figures are the average of a minimum of two independent replicates .", "If two replicates do not yield the identical result ( i . e . both synergistic or neither synergistic ) , we repeat the assay and average the FICI scores to produce the final score .", "Outlier FICI scores are not included in the analysis because FICI scores are calculated on a log2 scale and a single outlier can skew the results .", "Outlier scores are defined as those that differ from the majority of scores ( e . g . if four replicates yielded FICI score of 0 . 25 , 0 . 31125 , 0 . 5 , and 1 . 0 , the 1 . 0 score is the only non-synergistic result of the four and would be considered an outlier ) .", "However , average these example scores would result in an FICI of 0 . 515 , which would not be considered synergistic .", "As this average differs from the result of the majority of the FICI scores , the outlier is excluded from the calculation .", "We created a gradient of fluconazole in a 96-well plate , then added small molecules at 10 µM or 100 nM final concentrations or vehicle .", "CM18 wild-type was added at 1000 cells per well of YNB+ 2% glucose .", "Percent growth was calculated for fluconazole , combinations , or small molecules alone .", "We then determined if growth inhibition caused by the combination was equal or greater than growth inhibition of the small molecules alone .", "Repeated results were averaged for the average Bliss score .", "All replicates were performed on different days from independent stating cultures and are independent biological replicates .", "Bliss scores presented in the figures are the average of a minimum of two independent replicates .", "If two replicates do not yield the identical result ( i . e . both negative indicated synergy or both positive indicating not interaction ) , we repeated the assay and average the Bliss scores to produce the final score .", "Outlier Bliss scores are not included in the analysis they can skew the results .", "Outlier scores are defined as those that differ from the majority of scores ( e . g . if four replicates yielded bliss scores < 0 and one score dramatically >0 , the >0 score is the only non-synergistic result and would be considered an outlier ) .", "A DMSO control was conducted each time a molecule was tested to ensure the assay was working correctly .", "All DMSO results were averaged for the final score .", "KN99 culture was grown overnight in YNB + 2% glucose .", "Cells were sub-cultured into various treatments ( vehicle control was YNB + 2% glucose ) .", "6 ODs of each culture were harvested and lyophilized overnight .", "Pellets were resuspended in 25% alcoholic potassium hydroxide , vortexed , and incubated at 85°C water bath for 1 hr .", "Water and n-heptane were added to each tube , vortexed , and the n-heptane layer was transferred to borosilicate glass tubes .", "Biological replicates were grown on separate days from independent starting cultures .", "Metabolomics analysis was performed at the Metabolomics Core Facility at the University of Utah which is supported by 1 S10 OD016232-01 , 1 S10 OD021505-01 , and 1 U54 DK110858-01 .", "YNB + 2% glucose agar plates with or without 1 . 65 mg/mL of dicyclomine were made .", "Deletion mutants in KN99 strain were grown in YNB + 2% glucose then pinned to YNB plates .", "Plates were assessed at 1 , 2 , and 3 days for resistance .", "An overnight culture of KN99 was grown in YNB + 2% glucose .", "This was then sub-cultured into the various treatments ( vehicle control was either 0 . 1% DMSO or only YNB + 2% glucose ) .", "Cultures were grown at 30°C with rotation for 24 hr .", "Cultures were washed twice and resuspended in PBS .", "3 µL of propidium iodide ( stock concentration = 1 mg/mL ) was added to the FACS tube .", "After 1 min , flow cytometry was performed .", "Voltage gates used were as follows: FSC: 500; SSC: 310; PE: 496 .", "100 , 000 cells were counted per sample .", "Experiments were repeated three times on separate days from a different starting culture for each experiment and thus represent biological replicates .", "An overnight culture of KN99 was grown in YNB + 2% glucose .", "This was sub-cultured into either dicyclomine ( 0 . 3 mg/mL ) , FLZ ( 3E-4 mg/mL ) , Synergy , or Vehicle ( YNB + 2% glucose ) and grown at 30°C with rotation for 24 hr .", "Cells were then sub-cultured again into honeycomb plates with those previous treatments ( dicylomine , FLZ , Synergy , or Vehicle ) with either 20 , 10 , 5 , 2 . 5 ug/mL of 5-FAA or 0 . 4 mg/mL 5-MT or vehicle ( 3 . 2% DMSO ) .", "Plates were incubated at at 30°C in a Bioscreen C ( Growth Curves USA ) which automatically takes OD600 measurements every 30 min .", "An overnight culture of KN99 was grown in YNB + 2% glucose then sub-cultured into either dicylomine , FLZ , Synergy , or Vehicle ( YNB + 2% glucose ) and grown at 30°C with rotation for 24 hr .", "Cells were washed with PBS and calcofluor white added to a final concentration of 50 µg/mL and stained for 5–15 min .", "Cells were washed once more with PBS and assessed by flow cytometry .", "Voltage gates as follows: FSC: 500; SSC: 317; BV421-A: 185 .", "Significance determined with Mann-Whitney test .", "This assay was adapted from McMullan et al . ( 2015 ) .", "KN99 was grown on YPAD agar plates and resuspended in YNB + 2% glucose .", "Cells were counted and transferred for a final concentration of 3 × 106 cells/mL cultures containing treatments .", "Vehicle was YNB + 2% glucose .", "Cultures were left grown at 30° C statically for 3 , 9 , or 24 hr .", "Heat killed cultures were placed in a water bath >65° C for at least one hour .", "Cultures were then spun down and resuspended in PBS containing BCECF-AM ( Invitrogen ) at a final concentration of 40 ug/mL .", "Cultures were stained for 15 min in the dark then washed and resuspended in PBS and assessed by flow cytometry .", "50000 cells were collected for each sample .", "Each treatment was a combination of at least three independent experiments .", "Voltage gates as follows: FSC: 500 , SSC: 250 , FITC-A: 230 .", "Cells were gated on single cells then for BCECF-AM staining .", "Significance was determined with either uncorrected Fisher’s Least Significant Distance test or Mann-Whitney test .", "Counts of CFU following heat killing of 3 × 106 cells/mL are shown in Figure 6—source data 1 .", "~8 week-old female CD-1 IGS outbred mice ( Charles River Laboratories ) were intranasally inoculated with C . neoformans strain KN99 and monitored for survival as follows .", "The inoculum was prepared by culturing C . neoformans overnight in liquid YNB+2% glucose medium .", "C . neoformans cells collected from the overnight culture were washed twice in 1XPBS , counted , and suspended at a concentration of 5 × 105 cells / mL .", "Mice were anesthetized with ketamine/dexmedetomidine hydrochloride ( Dexdomitor ) administered intraperitoneally ( i . p . ) and suspended by their front incisors on a line of thread .", "The inoculum was delivered intranasally by pipetting 50 μL ( 2 . 5 × 104 cells / mouse ) dropwise into the nostrils .", "After 10 min , mice were removed from the thread and were administered atipamezole ( Antisedan ) i . p . as a reversal agent .", "Mice were massed daily and euthanized by CO2 asphyxiation when they reached 80% of their initial mass .", "Beginning 8 days post-inoculation , they received daily i . p . injections of either 8 mg/kg FLZ , 1 . 15 mg/kg DIC , 2 . 30 mg/kg DIC , 8 mg/kg FLZ + 1 . 15 mg/kg DIC , 8 mg/kg FLZ + 2 . 30 mg/kg DIC , or PBS control ( vehicle ) .", "Dosages were determined from human doses ( Lexicomp , 2019a; Lexicomp , 2019b ) .", "All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Utah ." ] ]

[ "Invasive fungal infections cause 1 . 6 million deaths annually , primarily in immunocompromised individuals .", "Mortality rates are as high as 90% due to limited treatments .", "The azole class antifungal , fluconazole , is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells , necessitating long treatments .", "To improve treatment , we used our novel high-throughput method , the overlap2 method ( O2M ) to identify drugs that interact with fluconazole , either increasing or decreasing efficacy .", "We identified 40 molecules that act synergistically ( amplify activity ) and 19 molecules that act antagonistically ( decrease efficacy ) when combined with fluconazole .", "We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity .", "A promising fluconazole-synergizing anticholinergic drug , dicyclomine , increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole .", "In vivo , this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis .", "Thus , our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes ." ]

[ "Individuals with weakened immune systems – such as recipients of organ transplants – can fall prey to illnesses caused by fungi that are harmless to most people .", "These infections are difficult to manage because few treatments exist to fight fungi , and many have severe side effects .", "Antifungal drugs usually slow the growth of fungi cells rather than kill them , which means that patients must remain under treatment for a long time , or even for life .", "One way to boost efficiency and combat resistant infections is to combine antifungal treatments with drugs that work in complementary ways: the drugs strengthen each other’s actions , and together they can potentially kill the fungus rather than slow its progression .", "However , not all drug combinations are helpful .", "In fact , certain drugs may interact in ways that make treatment less effective .", "This is particularly concerning because people with weakened immune systems often take many types of medications .", "Here , Wambaugh et al . harnessed a new high-throughput system to screen how 2 , 000 drugs ( many of which already approved to treat other conditions ) affected the efficiency of a common antifungal called fluconazole .", "This highlighted 19 drugs that made fluconazole less effective , some being antibiotics routinely used to treat patients with weakened immune systems .", "On the other hand , 40 drugs boosted the efficiency of fluconazole , including dicyclomine , a compound currently used to treat inflammatory bowel syndrome .", "In fact , pairing dicyclomine and fluconazole more than doubled the survival rate of mice with severe fungal infections .", "The combined treatment could target many species of harmful fungi , even those that had become resistant to fluconazole alone .", "The results by Wambaugh et al . point towards better treatments for individuals with serious fungal infections .", "Drugs already in circulation for other conditions could be used to boost the efficiency of fluconazole , while antibiotics that do not decrease the efficiency of this medication should be selected to treat at-risk patients ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "microbiology and infectious disease" ]

[ [ "Small RNAs including small interfering RNAs ( siRNAs ) and microRNAs ( miRNAs ) are important regulators of gene expression via RNA interference ( RNAi ) pathways in many organisms ( Baulcombe , 2004; Wang et al . , 2011; Hauptmann and Meister , 2013; Meister , 2013 ) .", "The RNAi machinery contains many proteins involved in the biogenesis and function of specific types of small RNAs ( Chen , 2009; Wang et al . , 2011; Hauptmann and Meister , 2013; Meister , 2013 ) .", "Among these are two categories that act as the core of the RNA silencing machinery: DICER ( DICER-LIKE or DCL in plants ) and ARGONAUTE ( AGO ) .", "DICER and DCL proteins mainly function to process double-stranded or folded stem-loop precursor RNAs into small RNA duplexes .", "AGO proteins incorporate one strand of an siRNA duplex to form an effector complex called RNA-induced silencing complex ( RISC ) to regulate the stability/translatability of target RNAs or to mediate methylation of target DNA sequences ( Baulcombe , 2004; Wang et al . , 2011; Meister , 2013; Pfaff et al . , 2013; Li et al . , 2013a; Rogers and Chen , 2013 ) .", "In plants , the RNAi machinery has undergone extensive amplification and functional specialization during evolution .", "For instance , the model plant Arabidopsis thaliana encodes four DCLs that function indistinct and yet overlapping RNAi pathways to control diverse biological processes ranging from development , response to abiotic stresses , to defense against pathogens ( Deleris et al . , 2006; Chapman and Carrington , 2007; Garcia-Ruiz et al . , 2010; Vazquez et al . , 2010 ) .", "Arabidopsis encodes 10 AGOs whose functions are not all understood .", "Well-studied AGOs include AGO1 that mediates mRNA cleavage is critical for development , AGO4 that directs DNA methylation , and AGO2 that functions in DNA double strand break repair ( Baumberger and Baulcombe , 2005; Mallory and Vaucheret , 2010; Ye et al . , 2012; Wei et al . , 2012 ) .", "AGO1 is particularly notable in that its homeostasis is controlled at the transcriptional , post-transcriptional , and post-translational levels .", "At the post-transcriptional level , the AGO1 mRNA is a target of miR168 .", "Therefore , miR168-guided cleavage of AGO1 mRNA by AGO1 protein exerts auto-regulation .", "Moreover , AGO1 is co-expressed with miR168 and AGO1 protein can stabilize miR168 post-transcriptionally ( Vaucheret et al . , 2006; Vaucheret , 2008; Mallory and Vaucheret , 2010 ) .", "At the post-translational level , the accumulation of AGO1 can be reduced by F-box proteins in a proteasome-independent manner through the autophagy pathway ( Derrien et al . , 2012; Rogers and Chen , 2013 ) .", "The auto-regulation of AGO1 indicates that its level within a cell can be dynamic and this dynamics may significantly impact the biological activities of a plant .", "RNA-mediated immunity against viruses operates in plants , fungi , invertebrates , and mammals to specifically destroy viral RNAs through the cellular RNA silencing machinery ( Li et al . , 2013b; Maillard et al . , 2013 ) .", "In plants , it is well known that AGO1 is a major effector of antiviral RNAi; AGO1 associates with virus-derived siRNAs ( vsiRNAs ) and mediates the degradation of viral RNAs .", "Furthermore , AGO2 and AGO7 are induced during viral infection , and both proteins can bind viral siRNAs .", "The antiviral function of AGO2 and AGO7 requires their slicing activity ( Qu et al . , 2008; Wang et al . , 2011 ) .", "AGO2 is repressed by AGO1-associated miR403 , and AGO1 and AGO2 appear to exert antiviral functions in a non-redundant and cooperative manner .", "Specifically , AGO1 functions in the first layer of antiviral RNAi; when AGO1's antiviral function is inhibited , a second layer is activated involving AGO2 ( Harvey et al . , 2011; Jaubert et al . , 2011; Scholthof et al . , 2011; Wang et al . , 2011; Carbonell et al . , 2012; Xia et al . , 2014 ) .", "AGO2 also recruits miR393* to regulate plant immunity against bacterial infection ( Zhang et al . , 2011 ) .", "As a counter-defense strategy , some plant viruses have evolved silencing suppressors to target AGO1 ( Burgyán and Havelda , 2011 ) .", "Moreover , infection of many viruses can elevate the miR168 level to down-regulate AGO1 , thereby nullifying this layer of host defense ( Várallyay et al . , 2010 ) .", "Thus , regulation of AGO1 by both host and viral factors plays a critical role in determining host responses to viral infection .", "Whether a host has positive regulators to check the viral counter-defense activities is not understood .", "How different AGOs have evolved to regulate plant responses to pathogen infection also remains an outstanding question ( Ding and Voinnet , 2007; Ding , 2010; Garcia-Ruiz et al . , 2010 ) .", "Rice ( Oryza sativa ) is one of the most important food crops as well as an experimental model plant for monocotyledonous plants ( monocots ) .", "However , rice production and consequently food sustainability is under the constant threat of emerging and reemerging viral diseases .", "Rice viral pathogens are genetically diverse and many highly pathogenic viruses such as Rice stripe Tenuivirus ( RSV , with a genome comprising 4 negative-stranded RNAs ) and Rice dwarf Phytoreovirus ( RDV , with a genome comprising 12 double-stranded RNAs ) are transmitted persistently and solely by arthropod vectors ( Hibino , 1996; Ren et al . , 2010; Du et al . , 2011 ) .", "Because of the global circulation of these vectors and lack of virus resistance germplasms , the incidence and severity of rice viral diseases in many rice-growing regions are unpredictable .", "Infection by multiple viruses is also a common and severe challenge for other important crops .", "Therefore , developing new and effective strategies to control infection by multiple viruses for a crop , especially the prevalent food crops in the monocot group such as rice , maize , and wheat that have traditionally been under-investigated , is of paramount importance for human food sustainability .", "Successful development of such strategies requires a knowledge base of the mechanisms of viral infection and host defense responses .", "The rice genome encodes five DCLs and 19 AGOs .", "How different AGOs regulate antiviral RNAi in rice is not known .", "In this study , we report a critical role of RNAi in antiviral defense in rice under natural infection conditions , when the plants were inoculated , as in the field , by viruliferous insect vectors brown planthopper ( Laodelphax striatellus ) and leafhopper ( Nephotettix cincticeps ) that transmit RSV and RDV , respectively .", "Intriguingly , we found that AGO18 , a member of a new AGO clade that is conserved in monocots , is specifically induced by the infection of two taxonomically different viruses and is required for the antiviral function of AGO1 .", "Loss-of-function ago18 mutation abolishes , whereas over-expression of AGO18 increases , the AGO1 antiviral activity .", "We further demonstrated that AGO18 competes with AGO1 for binding miR168 , resulting in elevated levels of AGO1 in the infected plants to enable antiviral defense .", "Expression of an miR168-resistant AGO1a variant in the ago18 background rescues or increases rice antiviral activity .", "The antiviral function of AGO18 requires its small RNA-binding , not slicing activity .", "Our findings reveal a novel mechanism AGO1 homeostasis regulation by AGO18 for antiviral defense and have significant implications in understanding the evolutionary amplification of RNA silencing mechanisms and in developing novel antiviral strategies ." ], [ "Our understanding of RNAi as an antiviral defense mechanism in plants mainly comes from viral infection systems involving model plants Arabidopsis and Nicotiana benthamiana by mechanical inoculation with virions or in vitro viral RNA transcripts or by agro-infiltration with viral cDNAs .", "To investigate the importance of RNAi in antiviral immunity in economically important crops under natural infection conditions , we used the rice-RSV patho-system in which rice is infected by RSV through the transmission of insect vector brown planthopper .", "Given the established role of AGO1 in antiviral defense in Arabidopsis , we tested whether AGO1 functions similarly in rice by inoculating an ago1 RNAi line ( Wu et al . , 2009 ) by viruliferous brown planthopper carrying RSV to recapitulate the natural infection conditions .", "As shown in Figure 1A , the ago1 RNAi line , in which the expression of AGO1s is diminished ( Figure 1—figure supplement 1A ) , was much more susceptible to RSV infection and showed more severe symptoms .", "Northern blots showed a remarkable increase in the accumulation of RSV genomic RNAs in the ago1 RNAi line ( Figure 1B ) .", "Since AGO1a and 1b are expressed at much higher levels than AGO1c and 1d in both mock-inoculated and RSV-infected rice plants ( Figure 1—figure supplement 1B , C ) ( Kapoor et al . , 2008 ) , we presumed that AGO1a and 1b are more important for antiviral defense .", "Therefore , AGO1a and 1b were characterized further in subsequent studies .", "We immunoprecipitated AGO1a and 1b complexes from RSV-infected or mock-treated rice plants using specific antibodies .", "Northern blot analyses showed that RSV vsiRNAs were readily detected in the AGO1a and AGO1b complexes ( Figure 1C ) , suggesting that AGO1 is an effector of vsiRNA .", "Taking together , these data indicate a role for AGO1s in antiviral immunity under natural infection conditions in rice . 10 . 7554/eLife . 05733 . 003Figure 1 . AGO1 participates in antiviral immunity in rice .", "( A ) Symptoms of WT and ago1 RNAi rice plants infected with RSV , pictures were taken at 6 weeks post-inoculation .", "Scale bars , 15 cm .", "( B ) Detection of RSV genomic RNA segments in WT and ago1 RNAi rice plants by Northern blot .", "The blots were hybridized with radiolabeled riboprobes specific for each RNA segment .", "rRNAs were stained with ethidium bromide and served as loading controls .", "The RNA signals were quantified and normalized to rRNAs , and the relative values were calculated by comparison with those in RSV-infected WT ( arbitrarily set to 1 . 0 ) .", "( C ) Detection of vsiRNAs associated with AGO1a and AGO1b immunoprecipitates .", "The silver-stained gel shows that comparable amounts of AGO1 complexes were used for RNA preparation .", "The asterisks indicate the positions of AGO proteins .", "The position of a RNA size marker , electrophoresed in parallel , is shown to the right of the blots .", "U6 was also probed and served as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 00310 . 7554/eLife . 05733 . 004Figure 1—figure supplement 1 . Characterization of rice ago1 RNAi lines and expression profiles of rice AGO1a , 1b , 1c , and 1d .", "( A ) Detection of AGO1a , AGO1b , AGO1c , and AGO1d in WT and the ago1 RNAi line by semi-quantitative RT-PCR .", "Actin was detected and used as a control .", "( B ) Expression profiles of rice AGO1a , 1b , 1c , and 1d in vegetative tissues ( young leaf , mature leaf , and root ) , shoot apical meristem ( SAM ) , panicle at six different developmental stages ( P1–P6 ) , and seeds at five different developmental stages of seed development ( S1–S5 ) examined by microarray analysis .", "( C ) Detection of AGO1a , AGO1b , AGO1c , and AGO1d in mock- or RSV-inoculated rice plants by qRT-PCR .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 004 We have previously shown that rice AGO1 as well as AGO18 , a member of a new AGO clade that is conserved in monocots ( Figure 2—figure supplement 1 ) , is highly induced by viral infection ( Du et al . , 2011 ) .", "Consistent with mRNA expression patterns , AGO18 protein was hardly detectable in mock-inoculated rice plants but accumulated to a high level in RSV-infected plants ( Figure 2A ) .", "To further test whether RSV infection induces the transcription activity of AGO18 promoter , we generated transgenic rice plants that express β-glucuronidase ( GUS ) under the control of AGO18 promoter .", "RSV infection led to high levels of GUS expression in such plants as compared to the background levels in mock-inoculated plants ( Figure 2B , C ) .", "These data demonstrate that RSV infection , but not insect vector feeding , induced AGO18 expression at the transcriptional level . 10 . 7554/eLife . 05733 . 005Figure 2 . AGO18 is induced by viral infection and confers antiviral immunity in rice .", "( A ) Detection of AGO18 in mock- or RSV-inoculated rice plants by Western blot .", "Tubulin was probed and served as a loading control .", "( B ) Representative GUS staining images of mock- or RSV-inoculated AGO18p:GUS transgenic plants .", "Scale bars , 5 mm . ( C ) qRT-PCR analysis of the transcript level of GUS in the indicated plants .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown .", "( D ) Symptoms of wild-type ( WT ) and ago18 rice plants infected with RSV , pictures were taken at 6 weeks post-inoculation .", "Scale bars , 15 cm .", "( E ) Detection of RSV genomic RNA segments in WT and ago18 rice plants by Northern blot .", "The blots were hybridized with radiolabeled riboprobes specific for each RNA segment .", "rRNAs were stained with ethidium bromide and served as loading controls .", "The RNA signals were quantified and normalized to rRNAs , and the relative values were calculated by comparison with those in RSV-infected WT ( arbitrarily set to 1 . 0 ) .", "( F ) Detection of RSV CP in the RSV-infected WT and ago18 by qRT-PCR .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown .", "( G ) Symptoms of RSV-infected WT ( Non-transgenic ) , ago18 and transgenic lines overexpressing AGO18 , pictures were taken at 6 weeks post-inoculation .", "Scale bars , 15 cm ( upper panel ) and 5 cm ( lower panel ) .", "( H ) Detection of AGO18 in mock- or RSV-inoculated plants as indicated .", "Tubulin was probed and served as a loading control .", "( I ) Detection of RSV genomic RNA segments in the indicated plants by Northern blot .", "The blots were hybridized with radiolabeled riboprobes specific for each RNA segment .", "rRNAs were stained with ethidium bromide and served as loading controls .", "The RNA signals were quantified and normalized to rRNAs , and the relative values were calculated by comparison with those in RSV-infected WT ( Non-transgenic ) ( arbitrarily set to 1 . 0 ) .", "( J ) Detection of RSV CP in the indicated RSV-infected plants by qRT-PCR .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 00510 . 7554/eLife . 05733 . 006Figure 2—figure supplement 1 . Phylogenetic analysis of AGO family proteins in plants . Unrooted , neighbor-joining tree was constructed by alignments of AGO protein sequences from the indicated plants . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 00610 . 7554/eLife . 05733 . 007Figure 2—figure supplement 2 . RSV CP triggers AGO18 expression . Detection of CP , P2 , pC4 , P4 , and AGO18 in transgenic rice lines that overexpress RSV CP , P2 , pC4 , and P4 by Western blot analysis .", "Tubulin was also detected and served as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 00710 . 7554/eLife . 05733 . 008Figure 2—figure supplement 3 . Identification of the ago18 mutant .", "( A ) A diagram showing the site of Tos17 insertion in AGO18 locus in the ago18 mutant .", "( B ) Upper two panels: Genotyping of the ago18 mutant by PCR .", "LP: left primer; RP: right primer; LB: left T-DNA ( Tos17 ) border primer .", "Lower two panels: RT-PCR analysis of AGO18 expression in wild-type ( WT ) and ago18 plants .", "Actin was detected and served as a control . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 00810 . 7554/eLife . 05733 . 009Figure 2—figure supplement 4 . AGO18 is required for rice resistance to RDV infection .", "( A ) qRT-PCR analysis of AGO1 and AGO18 expression levels in mock- or RDV-inoculated WT and ago18 plants .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown .", "( B ) Detection of AGO1a , AGO1b , and AGO18 proteins in mock- or RDV-inoculated WT and ago18 plants .", "Tubulin was detected and served as a loading control .", "( C ) Symptoms of RDV-infected WT ( Non-trangenic ) , ago18 and transgenic lines overexpressing AGO18 , pictures were taken at 6 weeks post-inoculation .", "Scale bars , 15 cm ( upper panel ) and 5 cm ( lower panel ) .", "( D ) Detection of AGO18 in mock- or RDV-inoculated plants as indicated .", "Tubulin was probed and served as a loading control .", "( E ) Detection of RDV genomic RNA segments in the indicated plants by Northern blot .", "The blots were hybridized with radiolabeled riboprobes specific for each RNA segment .", "rRNAs were stained with ethidium bromide and served as loading controls .", "The RNA signals were quantified and normalized to rRNAs , and the relative values were calculated by comparison with those in RSV-infected WT ( arbitrarily set to 1 . 0 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 009 To investigate how RSV infection triggers the expression of AGO18 , we examined the possibility that viral proteins produced during infection induce AGO18 expression .", "To do this , we generated transgenic rice plants that over-express Myc-tagged RSV P2 , CP , pC4 , and P4 proteins ( Xiong et al . , 2008 ) , respectively ( Figure 2—figure supplement 2 ) .", "We found that AGO18 was induced only in the transgenic lines that over-expressed CP , but not in those that over-expressed three other viral proteins ( Figure 2—figure supplement 2 ) .", "This suggests that RSV CP is an effector to induce AGO18 expression during viral infection .", "The induction of AGO18 expression by RSV infection prompted us to investigate its function in antiviral defense .", "We obtained a Tos17 retrotransposon insertional mutant ago18 ( NF6013 ) ( Figure 2—figure supplement 3A ) from the Tos17 database and isolated homozygous plants ( Figure 2—figure supplement 3B ) .", "The ago18 mutant did not exhibit notable phenotypic differences from the WT plants in growth and development but were much more sensitive to RSV infection than the WT plants ( Figure 2D ) .", "Viral replication and infection rates also increased in ago18 mutant plants ( Figure 2E , F , and Supplementary file 1A ) .", "These results suggest that AGO18 may be dispensable for normal growth and development of rice , but is required for defense against RSV infection .", "Importantly , AGO1 is present in the ago18 mutants .", "Therefore , these data indicate that the antiviral function of AGO1 depends on the presence of AGO18 .", "We further over-expressed AGO18 under the control of the ACTIN promoter in the WT rice background .", "The antiviral activity of AGO18 in the three independent transgenic lines was directly correlated with the expression levels of AGO18 ( Figure 2G–J ) .", "AGO18 was not only induced by and functioned against RSV , but also was induced by and functioned against RDV , a double-stranded RNA Phytoreovirus ( Figure 2—figure supplement 4 ) .", "This indicates that AGO18 is effective in positively regulating defense against a broad spectrum of viruses with diverse genome structures .", "Given that AGO1 is present in the ago18 mutant and AGO18 is present in ago1 RNAi plant and that both ago18 mutant and ago1 RNAi rice plants were very susceptible to viral infection , AGO1 and AGO18 evidently depend on each other for their antiviral activities .", "A key question is how this mutual dependence operates .", "To investigate how AGO18 acts in antiviral defense , we first tested whether this protein may function as an effector of vsiRNAs , like AGO1 .", "We immunoprecipitated AGO18 complexes from RSV-infected WT rice plants using AGO18 antibodies ( Figure 3A ) .", "Northern blots using RSV RNA2 probes showed much weaker signals for vsiRNAs in AGO18 ( Figure 3A ) , compared to those in AGO1a and AGO1b ( Figure 1C ) .", "We further profiled small RNAs in AGO18 complexes prepared from RSV-infected plants using deep sequencing .", "For comparison , small RNAs in total extracts , AGO1a , and AGO1b complexes were also profiled in parallel .", "We found that vsiRNAs accounted for about 4 . 48% of the total small RNAs in RSV-infected plants and 17 . 37%–9 . 77% of those in the AGO1a and AGO1b complexes ( Figure 3B and Supplementary file 1B ) , indicating that vsiRNAs are highly enriched in the AGO1 complexes .", "In contrast , vsiRNAs accounted for only about 2 . 52% of the AGO18-bound small RNAs ( Figure 3B and Supplementary file 1B ) .", "These data suggest that AGO18 is not a major effector of vsiRNAs .", "Together with the finding that AGO18 is required for the antiviral activity of AGO1 ( see above ) , this observation suggests that AGO18 very likely uses a different strategy to regulate antiviral defense response in rice . 10 . 7554/eLife . 05733 . 010Figure 3 . AGO18 unlikely functions as an effector of vsiRNAs .", "( A ) Detection of vsiRNAs in total extracts ( Input ) or AGO18 immunoprecipitates prepared from mock- or RSV-inoculated plants by Northern blot .", "The silver-stained gel shows the quality of purified AGO18 complexes .", "The asterisk indicates the position of AGO18 .", "The position of an RNA size marker is shown on the right of the blot .", "U6 is probed and served as a loading control .", "( B ) Percentage of deep sequencing reads matching vsiRNAs in total reads obtained from total extracts , AGO1a , AGO1b , and AGO18-associated small RNAs .", "Samples for deep sequencing were prepared from RSV-infected rice plants . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 010 RSV infection increases the accumulation of miR168 as well as AGO18 ( Du et al . , 2011 ) ( Figure 2A–C and Figure 4A ) , suggesting that the increased miR168 is not efficiently loaded into AGO1 and hence not effective in targeting AGO1 .", "Intriguingly , from the small RNA deep sequencing analyses , we found that AGO18 recruited a large amount of miR168 in RSV-infected rice plants compared with AGO1a or AGO1b ( Figure 4A and Supplementary file 1C ) .", "Given that miR168 plays a critical role in AGO1 homeostasis in plants ( Mallory and Vaucheret , 2010 ) and that the antiviral function of AGO1 requires the presence of AGO18 , our observations suggest that AGO18 up-regulates AGO1 by competitively binding miR168 to enable antiviral defense . 10 . 7554/eLife . 05733 . 011Figure 4 . AGO18 competes with AGO1 for miR168 to up-regulate AGO1 upon viral infection .", "( A ) Percentage of deep sequencing reads matching the indicated miRNAs in total reads obtained from total extracts , AGO1a , AGO1b , and AGO18-associated small RNAs .", "Samples for deep sequencing were prepared from mock- or RSV-inoculated rice plants .", "( B ) Detection of the indicated miRNAs in total extract ( Input ) , AGO1a , AGO1b , and AGO18 complexes by Northern blot .", "The blots were stripped and reprobed for multiple times .", "The silver-stained gel shows that comparable amounts of different AGO complexes were used for RNA preparation .", "The asterisks indicate the positions of AGO proteins .", "The positions of RNA size markers are shown on the right of the blots .", "The RNA signals were quantified , and the relative values were calculated by comparison with those in total extracts or AGO1a complex prepared from mock-inoculated WT ( arbitrarily set to 1 . 0 ) .", "( C ) Northern blot analysis showing miR168 from AGO1a , AGO1b , and AGO18 complexes in RSV-infected WT and ago18 plants ( upper panel ) .", "Western blot gel shows that comparable amounts of different AGO complexes were used for RNA preparation ( lower panel ) .", "( D ) qRT-PCR analysis of the levels of AGO1a , AGO1b , and AGO18 in WT rice and ago18 mutants with or without RSV infection .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown .", "( E ) Western blot showing AGO1a , AGO1b , and AGO18 protein levels in WT and ago18 with or without RSV infection .", "Tubulin was probed and served as a loading control .", "( F ) qRT-PCR analysis of the levels of AGO1a , AGO1b , AGO2 , and AGO18 in the indicated plants .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 011 Immunoprecipitation ( IP ) -northern blot analyses further confirmed that RSV infection of WT rice plants increased the association of miR168 with AGO18 but decreased its association with AGO1a and 1b , whereas several control miRNAs ( including miR166 and miR156 ) showed no obvious changes ( Figure 4B ) .", "Consistent with these results , in RSV-infected ago18 mutants , where AGO18 is absent , more miR168 was now loaded into AGO1a and 1b ( Figure 4C ) , which was correlated with the reduced expression of AGO1 at mRNA ( Figure 4D ) and protein ( Figure 4E ) levels .", "Also in the AGO18OE#1 transgenic rice line , in which AGO18 was overexpressed , AGO1a and 1b both increased at the mRNA level ( Figure 4F ) , likely as a result of more miR168 being loaded into AGO18 .", "Consistent with our previous finding ( Du et al . , 2011 ) , AGO2 was induced by RSV infection in wild-type rice plants ( Figure 4F ) .", "Intriguingly , knockout or overexpression of AGO18 did not have an obvious effect on such induction , indicating that the induction of AGO2 by RSV infection is independent of AGO18 ( Figure 4F ) .", "Thus , AGO18 specifically up-regulates AGO1 in antiviral defense .", "To directly test whether AGO18 could indeed compete with AGO1 for binding miR168 , we transiently expressed a miR168 precursor together with Flag-AGO1a or Flag-AGO1b in the presence or absence of Myc-AGO18 in N . benthamiana leaves .", "MiR444 was used as a control , because it is a species-specific miRNA in monocots that can be loaded by AGO1 ( Wu et al . , 2009 ) , but not by AGO18 ( Supplementary file 1C ) .", "Results from IP-northern experiments showed that when AGO18 was co-expressed with AGO1a or AGO1b , there was a nearly five fold reduction in miR168 loading into AGO1a and AGO1b and concurrent increase in loading into AGO18 ( Figure 5 ) .", "The control miR444 was specifically loaded into AGO1a and 1b and co-expression of AGO18 did not decrease its loading into AGO1a and 1b ( Figure 5 ) .", "These data demonstrate that AGO18 can effectively compete with AGO1 for binding miR168 . 10 . 7554/eLife . 05733 . 012Figure 5 . AGO18 competes with AGO1 for miR168 in vitro . Specific AGO18–miR168 interaction was confirmed by in vitro assays in N . benthamiana .", "Constructs with the indicated combinations were introduced into N . benthamiana leaves for transient expression by agro-infiltration .", "Northern blots were conducted with total RNA ( Input ) and small RNAs recovered from immunoprecipitated AGO complexes ( IP ) .", "Western blot analyses were done with the crude extract and aliquots of the IP products using anti-Flag or anti-Myc antibodies .", "The positions of RNA size markers are shown on the left of the blots .", "U6 was probed and served as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 012 The above results show that AGO18 is unlikely an effector of vsiRNAs but has the novel activity of competing with AGO1 for binding miR168 .", "To further test this , we analyzed the functions of AGO18 domains in antiviral defense .", "AGO family proteins contain four characteristic domains: an N-terminal domain and conserved PAZ , MID , and PIWI domains ( Tolia and Joshua-Tor , 2007; Vaucheret , 2008 ) .", "The PAZ domain binds to the 3′ end of small RNAs ( Ma et al . , 2004 ) , whereas the MID domain recognizes the 5′ end ( Kidner and Martienssen , 2005; Mi et al . , 2008; Montgomery et al . , 2008; Frank et al . , 2010 ) .", "The PIWI domain adopts an RNaseH-like structure and exhibits endonuclease ( slicing ) activity when an Asp-Asp-His ( DDH ) catalytic triad is present ( Song et al . , 2004; Rivas et al . , 2005 ) .", "Our analysis indicates that AGO18 also contains all of the four AGO signature domains and the DDH catalytic triad ( Figure 6A ) .", "We investigated whether the small RNA binding and slicing activities of AGO18 are required for its role in antiviral defense by transgenic complementation experiments .", "We generated two AGO18 mutants , AGO18Y537A/F538A ( YF/AA ) and AGO18D833A ( D833A ) .", "YF/AA contains alanine substitutions at two conserved residues ( Y537F538 ) that are known to be required for small RNA binding ( Ma et al . , 2004; Guang et al . , 2008; Ye et al . , 2012 ) , whereas D833A contains an alanine substitution at the DDH catalytic triad ( Song et al . , 2004; Rivas et al . , 2005; Wee et al . , 2012 ) .", "We transgenically expressed WT AGO18 , YF/AA , and D833A mutants under the control of the native AGO18 promoter in the ago18 mutant background .", "We then inoculated these plants with RSV by insect vector transmission .", "Western blots confirmed the expression of WT and mutant AGO18 in the infected transgenic plants ( Figure 6B ) .", "Based on the disease symptoms ( Figure 6C ) and accumulation of viral genomic RNAs ( Figure 6D ) , both WT AGO18 and D833A could mostly complement the ago18 mutant for resistance to RSV infection , whereas the YF/AA mutant could not .", "These results suggest that AGO18 exerts its antiviral function mainly through small RNA binding , rather than slicing .", "We also measured the expression levels of AGO1 in these transgenic lines that were infected by RSV .", "We found that AGO1 expression was elevated by viral infection in the transgenic plants that express WT AGO18 or D833A mutant but not in those that express YF/AA ( Figure 6E ) , further suggesting that the small RNA binding but not slicing activity of AGO18 is required for its role in up-regulating AGO1 . 10 . 7554/eLife . 05733 . 013Figure 6 . Small RNA-binding but not slicing activity of AGO18 is required for its antiviral function .", "( A ) Domain structure of AGO18 protein .", "AGO18 consists of a variable N-terminal domain and conserved C-terminal PAZ , MID , and PIWI domains .", "The residues Y537 and F538 required for small RNA binding , and D833 , D906 , and H1045 required for slicing are indicated .", "( B ) Detection of AGO18 protein in mock- or RSV-inoculated WT ( Non-trangenic ) plants as well as ago18 mutants complemented with AGO18 and its derivatives by Western blot .", "Tubulin was probed and served as a loading control .", "( C ) Symptoms of mock- or RSV-inoculated WT ( Non-trangenic ) plants as well as ago18 mutants complemented with AGO18 or its derivatives , pictures were taken at 6 weeks post-inoculation .", "Scale bars , 15 cm ( upper panel ) and 5 cm ( lower panel ) .", "( D ) Detection of RSV genomic RNA segments in mock- or RSV-inoculated WT ( Non-trangenic ) plants as well as ago18 mutants complemented with AGO18 or its derivatives by Northern blot .", "The blots were hybridized with radiolabeled riboprobes specific for each RNA segment .", "rRNAs were stained with ethidium bromide and served as loading controls .", "The RNA signals were quantified and normalized to rRNAs , and the relative values were calculated by comparison with those in RSV-infected WT ( arbitrarily set to 1 . 0 ) .", "( E ) qRT-PCR analysis of the levels of AGO1a , AGO1b , and AGO18 in RSV-infected WT plants as well as ago18 mutants complemented with AGO18 or its derivatives .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 013 The above studies demonstrated that AGO18 regulates AGO1 homeostasis by sequestering miR168 during viral infection .", "To further test this , we generated transgenic rice plants expressing wild type ( AGO1a lines ) and miR168-resistant AGO1a ( AGO1a-Res lines ) in the ago18 background , under the control of the native AGO1a promoter ( Figure 7A ) .", "The steady-state AGO1a/AGO1a-Res mRNA levels of both AGO1a and AGO1a-Res transgenes accumulated to higher levels relative to the AGO1a levels in wild-type ( WT ) non-transgenic and ago18 plants ( Figure 7B ) .", "It is noteworthy that AGO1a mRNA level was reduced by RSV infection in the AGO1a transgenic line , whereas AGO1a-Res transgenic lines had no significant reduction in AGO1a-Res mRNA levels upon RSV infection ( Figure 7B ) , indicating that AGO1a is subject to regulation by miR168/AGO18 .", "Although the transcript levels of the miR168-resistant transgene AGO1a markedly increased , none of these lines showed notable phenotypic differences from WT or ago18 plants in growth and development .", "These transgenic lines , however , were much more resistant to RSV infection than ago18 and AGO1a plants ( Figure 7B , C ) .", "Viral replication significantly decreased in the AGO1a-Res plants ( Figure 7D ) .", "Thus , AGO1 over-expression could rescue the deficiency of ago18 for viral resistance .", "These data provided further compelling evidence that AGO18 functions through sequestering miR168 to up-regulate AGO1 against virus infections . 10 . 7554/eLife . 05733 . 014Figure 7 . Transgenic expression of miR168-resistant AGO1a rescued the deficiency of ago18 for viral resistance .", "( A ) Schematic drawing of AGO1a featuring the target site of miR168 .", "In AGO1a that is resistant to miR168 cleavage ( AGO1a-Res ) , seven synonymous nucleotide substitutions were introduced into the miR168 target site .", "The resistance of AGO1a-Res to miR168-directed cleavage was examined by in vitro cleavage assay using purified AGO1a complex .", "Positions of the cleavage products are indicated by arrows .", "( B ) qRT-PCR analysis of AGO1a and AGO1b expression levels in mock ( − ) or RSV-inoculated ( + ) WT , ago18 , and transgenic plants expressing AGO1a or AGO1a-Res in the ago18 mutant background .", "The expression levels were normalized using the signal from OsEF-1a .", "The average ( ± standard deviation ) values from three biological repeats of qRT-PCR are shown .", "( C ) Symptoms of mock- or RSV-inoculated WT ( Non-trangenic ) , ago18 , and transgenic plants expressing AGO1a or AGO1a-Res in the ago18 mutant background .", "Scale bars , 15 cm .", "( D ) Detection of RSV genomic RNA segments in the indicated plants by Northern blot .", "The blots were hybridized with radiolabeled riboprobes specific for each RNA segment .", "rRNAs were stained with ethidium bromide and served as loading controls .", "The RNA signals were quantified and normalized to rRNAs , and the relative values were calculated by comparison with those in RSV-infected WT ( Non-trangenic ) ( arbitrarily set to 1 . 0 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05733 . 014 Based on the above results , we conclude that AGO18 regulates AGO1 homeostasis by specifically loading miR168 during viral infection .", "Sequestering of miR168 by AGO18 leads to increased accumulation of AGO1 to embark on an effective antiviral defense response ." ], [ "AGO proteins are at the heart of RNAi machinery .", "Building upon the conserved core components of the RNA silencing machinery such as DCLs and AGOs , plants have evolved extensive variants of these components , especially AGOs .", "This evolutionary amplification of the machinery components implies amplification/diversification in regulatory mechanisms/functions .", "While the roles of some AGOs in specific functions of RNA silencing in gene regulation and innate immunity have been well studied , the roles of most AGOs remain unknown or poorly understood .", "Besides AGO1 , only the slicing function of AGO2 appears to be also important for antiviral defense in Arabidopsis ( Harvey et al . , 2011; Jaubert et al . , 2011; Scholthof et al . , 2011; Wang et al . , 2011; Carbonell et al . , 2012; Xia et al . , 2014 ) .", "Current studies on the roles of several Arabidopsis AGOs in antiviral defense are dictated by the target RNA-slicing paradigm .", "In the arms race between host defense and viral counter-defense , plant viruses have evolved various strategies , including encoding suppressors of RNA silencing , to interfere with different steps of the host RNA silencing defense pathways ( Ding and Voinnet , 2007; Ding , 2010 ) .", "In Arabidopsis and N . benthamiana , infection by many viruses leads to increased levels of AGO1 mRNA readying the plants to combat viruses , but the viruses also simultaneously increase the expression levels of miR168 to down-regulate AGO1 to defeat host defense ( Várallyay et al . , 2010 ) .", "RSV infection of rice also leads to an increase in miR168 ( Figure 4A , B ) ( Du et al . , 2011 ) .", "Thus , elevated miR168 expression appears to be a broad mechanism of viral counter-defense .", "In this study , we discovered a novel mechanism of positive regulation of AGO1 activity by AGO18 in antiviral RNAi .", "We showed that AGO18 does not have antiviral function by itself , but its presence is required for the antiviral function of AGO1 .", "AGO18 accomplishes this not via its slicing function but through its competition with AGO1 for binding miR168 .", "This binding inhibits miR168-mediated auto-regulation of AGO1 , thereby boosting the accumulation levels of AGO1 to enable antiviral function .", "Thus , AGO18 has evolved as a novel and dedicated positive regulator of the plant surveillance/defense system .", "Our findings suggest that AGO18-bound miR168 is not competent in cleaving AGO1 mRNA in RSV-infected rice plants , albeit AGO18 contains conserved DDH motif for slicing activity .", "Several possible mechanisms can be considered .", "First , the catalytic activity of AGO18 may not be as potent as that of AGO1 due to some sequence/structure differences between these two AGOs .", "Second , the slicing activity of AGO18 may be repressed by a yet-to-be-identified protein that specifically interacts with AGO18 .", "Third , the cellular compartmentation of AGO18/miR168 complex may be distinct from that of AGO1/miR168 and prevent its access to AGO1 mRNA .", "Finally , AGO18 binding of miR168 may subsequently induce the degradation of miR168 .", "It will be highly interesting to test these possibilities in future studies .", "There were previous examples of competitive binding of other miRNAs between other AGOs and AGO1 in Arabidopsis .", "miR168 can be incorporated into AGO10 to decrease the translation efficiency of AGO1 mRNA in Arabidopsis ( Mallory et al . , 2009 ) .", "The Arabidopsis miR166/165 are significantly enriched in AGO10-bound miRNAs , preventing them from being loaded into AGO1 to fulfill their normal roles in development ( Zhu et al . , 2011; Ji et al . , 2011; Manavella et al . , 2011 ) .", "MiR390 is associated with AGO7 to mediated trans-acting siRNA biogenesis ( Montgomery et al . , 2008 ) , through cooperative activity of AGO1 .", "In addition , miR408 associates with both AGO1 and AGO2 redundantly to regulate Plantacyanin mRNA levels ( Maunoury and Vaucheret , 2011 ) .", "This competition , however , does not affect AGO1 homeostasis .", "In contrast to these negative regulations of AGO1 , binding of miR168 by AGO18 in infected rice plants boosts AGO1 accumulation .", "Thus , among mechanisms of AGO-regulation of AGO1 homeostasis or activity , the up-regulation of AGO1 via AGO18 binding of miR168 represents a novel type of mechanism .", "Whether other AGOs function similarly in Arabidopsis , rice , and other plants to up-regulate AGO1 or another AGO to impact developmental processes or defense responses is an outstanding question to be addressed in future studies .", "It is also noteworthy that , in addition to miR168 , several miRNAs including miR528 , miR159a , and miR159b were also recruited by AGO18 in RSV-infected rice plants ( Supplementary file 1C ) .", "Thus , for AGO18 , we cannot rule out the possibility that AGO18 plays regulatory roles in other capacities or has its own independent biological functions .", "It is important to emphasize that most plant viruses are transmitted to plants by insect vectors under natural infection conditions in the field , and yet the vast majority of studies so far on the mechanisms of RNA silencing-mediated antiviral responses employed infection methods such as mechanical inoculation with in vitro viral RNA transcripts or virions and infiltration with agrobacteria carrying engineered viral DNAs .", "These studies often used viral delivery methods with much higher levels of inoculum than natural field conditions .", "How some of such manipulations would alter host responses remains an outstanding question .", "Here , we used insect vectors that carry the natural forms of viruses to inoculate plants , best reflecting what happens under field infection conditions with regard to the behavior of viruses and host responses .", "This is not only important to dissect the natural infection and defense mechanisms , but also important for developing effective technologies to combat viral infection under natural infection conditions .", "Recent studies indicated that natural infection in early stage of virus infection may trigger different host-defense reaction ( Garcia et al . , 2014 ) .", "AGO18 is of special interest because it has evolved as a conserved clade in monocots that encompass many important crop plants for foods and biofuels .", "Whether it plays a similar role in antiviral defense in other monocots warrants further investigations .", "From a broader perspective , we expect that further studies on the extensive RNA silencing machinery components evolved in crop plants may lead to new discoveries about their functions in shaping plant diversity with regard to phenotypes and innate immunity mechanisms .", "Finally , sequestering miR168 by AGO18 to regulate AGO1 homeostasis represents an evolutionary novelty .", "It raises the question of whether small RNA competition-based regulations between other types of proteins have also evolved to regulate different biological processes in plants and other organisms .", "This mechanism functions against infection by two evolutionarily distinct viruses , and likely has broader significance in resistance against more viruses in a wide range of monocots .", "We propose that engineering rice and other cereals to over-express AGO18 may provide a new strategy for the control of diverse viral pathogens ." ], [ "Plant growth and virus inoculation were essentially carried out as described ( Du et al . , 2011 ) .", "Briefly , rice ( O . sativa spp . japonica ) seedlings were grown in a greenhouse at 28–30°C and 60 ± 5% relative humidity under natural sunlight for 4 weeks .", "The viruliferous ( RSV and RDV-carrying ) insects of L . striatellus and N . cincticeps , as well as virus-free L . striatellus and N . cincticeps ( mock ) were used for inoculation .", "After feeding 3 days , the insects were removed , and the rice seedlings were returned to the greenhouse to grow under the greenhouse conditions above .", "3 weeks post-inoculation when the newly developed leaves started to exhibit viral symptoms , the whole seedlings were harvested .", "For each sample , at least 15–20 rice seedlings were pooled for RNA extraction .", "Non-preference tests were performed for all rice seedlings of the different genetic backgrounds with the two viral transmission insect vectors brown planthopper ( L . striatellus ) and leafhopper ( N . cincticeps ) .", "Details of the procedures were described previously ( Hiroshi et al . , 1994 ) .", "Plants were infiltrated with 50 mM sodium phosphate ( pH 7 . 0 ) , 10 mM EDTA , and 0 . 5 mg/ml X-gluc ( Apollo Scientific , UK ) , followed by incubation at 37°C in the dark overnight and then destained in 70% ethanol before photographing .", "Synthetic peptides AGO1aN ( KKKTEPRNAGEC ) , AGO1bN ( KKRTGSGSTGEC ) , and AGO18N ( YHGDGERGYGRC ) were used to raise rabbit polyclonal antibodies against AGO1a , AGO1b , and AGO18 , respectively , essentially as described ( Wu et al . , 2009; Mi et al . , 2008 ) .", "The antisera were affinity purified and used for immunoprecipitation ( IP ) ( 1:50 dilution ) .", "Gateway system ( Invitrogen , Carlsbad , CA ) was used to make binary constructs .", "Several destination vectors were created for transient expression in N . benthamiana and stable rice transformation .", "The binary gateway vector pMDC32 ( Karimi et al . , 2007 ) was modified to obtain rice AGO18 promoter ( p32:pAGO18 ) or AGO1a promoter ( p32:pAGO1a ) .", "Most cDNA and miRNA genes were cloned into pENTR/D vectors and pENTR/D-Flag-AGO18D833A , YF/AA and AGO1a:miR168 Res clones were prepared by using the QuikChange site-directed mutagenesis kit ( Stratagene , La Jolla , CA ) .", "All these clones were confirmed by sequencing and transferred to the appropriate destination vectors by recombination using the Gateway LR Clonase II Enzyme mix ( Invitrogen ) .", "pCam2300:Actin1::OCS and pCam2300:35S::OCS vectors ( Wu et al . , 2009 ) were used to generate pCam2300:Actin1::Flag-AGO18 , pCam2300:35S::Myc-AGO18 , Flag-AGO1a , Flag-AGO1b , miR168a , and miR444b , respectively .", "All PCR primers are listed in Supplementary file 1D .", "Assays of transient expression in leaves of N . benthamiana were performed as described ( Voinnet et al . , 2003 ) .", "A detailed protocol is available upon request .", "Protein samples were boiled with same volume of 2× protein loading buffer at 95°C for 5 min and separated by SDS-PAGE gel .", "Proteins were then transferred to PVDF membranes and detected with antibodies against AGO18 , AGO1a , AGO1b , MYC ( 11667203001 , Roche , Switzerland ) , FLAG ( F1804 , Sigma–Aldrich , St . Louis , MO ) , and tubulin ( T5168 , Sigma–Aldrich ) .", "Rice AGO-containing complexes were immunopurified from RSV-inoculated rice plants as previously described ( Qi et al . , 2005; Wu et al . , 2010 ) .", "The quality of purification was examined by SDS-PAGE followed by silver staining or IP-western blotting , and the bands of expected sizes were confirmed as AGO proteins by mass spectrometry .", "RNAs were isolated from total cell extracts and from the purified AGO complexes by Trizol reagent ( Invitrogen ) , resolved on a 15% denaturing PAGE gel , and visualized by SYBR-gold ( Invitrogen ) staining .", "Gel slices within the range of 18–28 nucleotides were excised , and the RNAs were eluted and purified for cloning .", "Small RNA cloning for Illumina sequencing was carried out essentially as described previously ( Mi et al . , 2008; Wu et al . , 2010 ) .", "A detailed protocol is available upon request .", "Northern blot analysis with total sRNAs or from purified AGO complexes was performed as described before ( Qi et al . , 2005 ) .", "32P-end labeled oligonucleotide probes complementary to sRNAs were used for Northern blots .", "The sequences of the probes are listed in Supplementary file 1D .", "Total RNAs were extracted from rice plants with Trizol ( Invitrogen ) .", "After removal of contaminating DNAs by digestion with RNase-free DNaseI ( Promega , Madison , Wisconsin , USA ) , the RNAs were reverse transcribed by SuperScript III Reverse Transcriptase ( Invitrogen ) using oligo ( dT ) .", "The cDNAs were then used as templates for quantitative PCR and RT-PCR .", "Quantitative PCR was performed using SYBR Green Real-time PCR Master Mix ( Toyobo , Osaka , Japan ) .", "The rice OsEF-1a gene was detected in parallel and used as the internal control .", "All the other primers used are listed in Supplementary file 1D .", "The adaptor sequences in Illumina 1G sequencing reads were removed by using ‘vectorstrip’ in the EMBOSS package .", "The sRNA reads with length of 19–27 nt were mapped to the rice nuclear , chloroplastic , and mitochondrial genomes ( http://rice . plantbiology . msu . edu/ , version 6 . 0 ) .", "The sRNAs with perfect genomic matches were used for further analysis .", "Rice miRNA annotations were from miRBase ( http://microrna . sanger . ac . uk/sequences , Release14 ) and our previous publication ( Wu et al . , 2009; Du et al . , 2011 ) .", "Statistical analysis of the sRNA data sets was done by using in-house-developed Perl scripts ( Supplementary file 2 ) .", "miRNA cleavage activity assay was performed essentially as described ( Qi et al . , 2005; Qi and Mi , 2010 ) , using immunopurified rice AGO1a complex and in vitro-transcribed AGO1a or AGO1a-Res transcripts .", "Small RNA data sets generated in this study are deposited in the NCBI sequence read archive ( SRA ) ( http://www . ncbi . nlm . nih . gov/sra ) under accession number PRJNA273330 ." ] ]

[ "Viral pathogens are a major threat to rice production worldwide .", "Although RNA interference ( RNAi ) is known to mediate antiviral immunity in plant and animal models , the mechanism of antiviral RNAi in rice and other economically important crops is poorly understood .", "Here , we report that rice resistance to evolutionarily diverse viruses requires Argonaute18 ( AGO18 ) .", "Genetic studies reveal that the antiviral function of AGO18 depends on its activity to sequester microRNA168 ( miR168 ) to alleviate repression of rice AGO1 essential for antiviral RNAi .", "Expression of miR168-resistant AGO1a in ago18 background rescues or increases rice antiviral activity .", "Notably , stable transgenic expression of AGO18 confers broad-spectrum virus resistance in rice .", "Our findings uncover a novel cooperative antiviral activity of two distinct AGO proteins and suggest a new strategy for the control of viral diseases in rice ." ]

[ "Rice is a major food crop , providing over a fifth of all calories consumed by people around the world .", "As such , it is important to find ways to prevent the diseases that affect rice plants .", "Many of the viruses that infect rice are transferred between plants by insects and many insects carry more than one virus at a time; this means it can be difficult to predict where a disease will next emerge .", "As a result , there is a pressing need to develop new and effective strategies that boost the ability of rice plants to fight off harmful viruses .", "One way that plants defend themselves from viruses involves using a system called RNA interference to identify and destroy the RNA molecules that viruses produce .", "This process depends on the Argonaute ( AGO ) family of proteins , although the roles of many of its members are not well understood .", "One of the better-studied AGO proteins is called AGO1 and is known to be important for defending plants against viruses .", "Unfortunately , a small RNA molecule called miR168 acts to limit the amount of AGO1 in a cell , and the levels of miR168 increase in virus-infected rice plants .", "Wu , Yang et al . exposed rice plants to two species of insect that each carried a different plant virus .", "Rice plants infected with these viruses increased their levels of both AGO1 and another AGO protein called AGO18 .", "Modifying the ability of rice plants to produce AGO18 revealed that the anti-viral activity of AGO1 is abolished in plants lacking AGO18 .", "However , plants that over-produce AGO18 are better able to fight off viral infections .", "Wu , Yang et al . further showed that AGO18 binds to miR168 and so prevents this small RNA from reducing AGO1 levels .", "Therefore , AGO1 and AGO18 work together to defend rice plants from viruses .", "Wu , Yang et al . suggest that engineering rice plants to make more AGO18 could make them more resistant to viruses .", "Further work will be needed to confirm whether AGO1 and AGO18 also work together to defend rice against viruses other than the two tested so far and to investigate whether these proteins also perform similar roles in other crops ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "When eukaryotic cells are deprived of amino acids , they activate autophagy , a conserved lysosomal degradation pathway that enables cells to survive short-term periods of starvation by recycling nutrients and maintaining energy homeostasis ( Levine and Klionsky , 2004; Mizushima and Komatsu , 2011 ) .", "The autophagy pathway is mediated by a set of evolutionary conserved proteins , the ATG proteins , that include proteins involved in the Atg1/ULK1 serine/threonine kinase protein complex , the Beclin 1/VPS34 class III phosphatidylinositol kinase ( PI3K ) complex , and the ATG5/ATG12/ATG16 and Atg8/LC3 protein conjugation complexes ( Levine and Klionsky , 2004 ) .", "The activity of these ATG protein complexes can be regulated by protein–protein interactions , post-translational modifications ( e . g . , ubiquitination , phosphorylation , acetylation ) , and at the transcriptional level ( Kroemer et al . , 2010; Abrahamsen et al . , 2012; Norris and Yao , 2012; Bánréti et al . , 2013; Webster et al . , 2014 ) .", "Despite extensive research over the past several years into the molecular mechanisms of autophagy , very little is understood about stress-responsive signals that regulate activation of autophagy protein complexes during amino acid deprivation .", "Inactivation of the autophagy repressor kinase mTOR ( mechanistic target of rapamycin ) in response to amino acid starvation contributes to autophagy through phosphorylation of ULK1/2 and ATG13 ( resulting in their dissociation ) and of ATG14 ( Meijer et al . , 2014 ) .", "The low-energy sensing kinase , AMPK , also acts directly on components of the autophagy machinery ( ULK1 , Beclin 1 , VPS34 ) to positively regulate glucose starvation-induced autophagy ( Egan et al . , 2011; Kim et al . , 2013 ) , but does not appear to function similarly in amino acid starvation-induced autophagy ( Kim et al . , 2013 ) .", "Thus , an important open question is whether any stress-responsive signals directly regulate autophagy proteins to promote autophagy during amino acid starvation .", "The autophagy protein , Beclin 1 , is a central regulator of autophagy and functions through its interaction with the Class III PI3K VPS34 , VPS15 , and the autophagy protein ATG14 in the initial stages of autophagosome formation ( Funderburk et al . , 2010; He and Levine , 2010; Abrahamsen et al . , 2012; Fu et al . , 2013 ) .", "It is a haploinsufficient tumor suppressor important for breast cancer and also acts in various other biological processes , including differentiation and development , innate immunity , lifespan extension , protein against neurodegeneration , and exercise-mediated effects on glucose metabolism ( Cao and Klionsky , 2007; Levine and Kroemer , 2008; He et al . , 2012 ) .", "During amino acid starvation , Beclin 1-associated VPS34 autophagy complex activity is negatively regulated by the direct binding of Beclin 1 to BCL2 or BCL2L1 ( Sinha and Levine , 2008 ) , and by inhibitory interactions with the oncogenic kinases , AKT and EGFR ( Wang et al . , 2012; Wei et al . , 2013 ) , the Golgi-associated protein , GLIPR2/GAPR-1 ( Shoji-Kawata et al . , 2013 ) , and the kinase MST1 ( Maejima et al . , 2013 ) .", "Several proteins positively regulate the Beclin 1-associated VPS34 autophagy complex , including those that disrupt BCL2/BCL2L1-Beclin 1 binding by either Beclin 1 phosphorylation ( e . g . , DAPK , ROCK [Zalckvar et al . , 2009; Gurkar et al . , 2013] ) or BCL2 phosphorylation ( e . g . , JNK1 [Wei et al . , 2008] ) and proteins that are part of the core autophagy machinery , such as ULK1 and AMBRA1 ( Fimia et al . , 2007; Di Bartolomeo et al . , 2010; Russell et al . , 2013 ) .", "These observations suggest that Beclin 1 may be a central target that is ‘turned on’ or ‘turned off’ in order to properly coordinate autophagosome formation with the needs of the cell to increase or decrease autophagy in a context-dependent manner .", "However , it remains unknown whether stress-activated kinases directly phosphorylate Beclin 1 ( or other ATG proteins ) to positively regulate autophagy in response to amino acid starvation .", "Here we describe a novel role for members of the stress-activated protein kinase ( p38 MAPK ) signaling pathway , MAPKAPK2 ( MK2 ) and MAPKAPK3 ( MK3 ) ( Cargnello and Roux , 2011 ) , in mediating Beclin 1 S90 phosphorylation , which we show is essential for its autophagy and tumor suppressor function .", "This MK2/3-dependent Beclin 1 S90 phosphorylation is inhibited by a mutant form of BCL2 that cannot be phosphorylated by the MAPK family member , JNK1 .", "Our findings reveal a crucial link between an evolutionarily conserved family of stress-activated protein kinases , MK2 and MK3 , and a component of the autophagy machinery , Beclin 1 , that plays an essential role in starvation-induced autophagy .", "Moreover , we demonstrate that blockade of MK2/MK3-dependent Beclin 1 phosphorylation is a mechanism by which BCL2 family proteins inhibit autophagy .", "These finding have important implications for understanding how cells respond to amino acid starvation and turn on autophagy ." ], [ "Activation of the Beclin 1/VPS34 complex is an essential event for starvation-induced autophagy ( Funderburk et al . , 2010 ) .", "To gain insights into potential mechanisms that regulate starvation-induced autophagy , we sought to identify post-translational modifications of Beclin 1 that occur specifically in response to the autophagy-inducing condition , amino acid starvation .", "Mass spectrometry analysis of SKNSH human neuroblastoma cells stably transfected with Flag epitope-tagged Beclin 1 grown in normal growth or starvation conditions identified only one phosphorylation event that increased specifically in response to amino acid deprivation—phosphorylation of the serine 90 residue of Beclin 1 ( data not shown ) .", "We confirmed starvation-induced phosphorylation of Beclin 1 S90 using HeLa cells transiently transfected with either Flag epitope-tagged wild-type Beclin 1 or Flag epitope-tagged mutant Beclin 1 containing a non-phosphorylatable alanine substitution at residue 90 of human Beclin 1 ( Beclin 1 S90A ) .", "Following metabolic labeling with 33P , immunoprecipitation with an anti-Flag antibody and autoradiography , minimal phosphorylation was detected during nutrient-rich conditions , whereas Beclin 1 phosphorylation increased in response to starvation ( Figure 1A , upper gel ) .", "Mutation of S90 to alanine almost completely abolished the 33P signal of Beclin 1 , indicating that S90 is a major starvation-induced phosphorylation site ( Figure 1A , upper gel ) .", "We confirmed that starvation induces Beclin 1 S90 phosphorylation by generating a phosphospecific antibody that recognizes Beclin 1 p-S90 .", "Beclin 1 p-S90 reactivity of wild-type Beclin 1 , but not Beclin 1 S90A , increased after nutrient starvation ( Figure 1A , middle gel ) . 10 . 7554/eLife . 05289 . 003Figure 1 . Beclin 1 S90 is phosphorylated in response to nutrient starvation .", "( A ) Detection of Beclin 1 S90 phosphorylation in HeLa cells transfected with indicated Flag-Beclin 1 construct by radiolabeling , immunoprecipitation with an anti-Flag antibody and autoradiography ( upper gel ) or by western blot analysis using an anti-Beclin 1 S90 phosphospecific antibody ( middle gel ) .", "Starvation ( − ) refers to growth in normal medium and starvation ( + ) refers to growth in HBSS for 2 hr . ( B ) Detection of endogenous Beclin 1 S90 phosphorylation in HeLa cells by immunoprecipitation with anti-Beclin 1 followed by immunoblot analysis with anti-Beclin 1 p-S90 phosphospecific antibody and detection of autophagy by p62 and LC3 immunoblot analysis in HeLa cells at indicated time points after starvation in HBSS .", "Actin is shown as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 003 We next evaluated whether endogenous Beclin 1 undergoes S90 phosphorylation in response to nutrient ( amino acid ) starvation using the Beclin 1 S90 phosphospecific antibody .", "Beclin 1 S90 phosphorylation was detected in HeLa cell lysates within 30 min after starvation and gradually increased over a 2 hr period ( Figure 1B ) .", "This increase in Beclin 1 S90 phosphorylation was accompanied by starvation-induced autophagy as measured by a time-dependent decrease in levels of the autophagy substrate , SQSTMI/p62 ( herein referred to as p62 ) , and increase in the conversion of the autophagy protein LC3-I to the lipidated , autophagosome-associated form , LC3-II ( Figure 1B ) .", "Thus , endogenous Beclin 1 undergoes phosphorylation at residue S90 in response to nutrient starvation in parallel with autophagy induction .", "Next , we asked whether Beclin 1 S90 phosphorylation is essential for starvation-induced autophagy .", "To evaluate this question , we used two different cell lines that are deficient in Beclin 1 expression and starvation-induced autophagy , including human MCF7 breast carcinoma cells ( Liang et al . , 1999 ) and U2OS cells that inducibly express shRNA targeted against beclin 1 ( Sun et al . , 2008 ) .", "MCF7 cells were derived from a patient with allelic loss of beclin 1 , have low levels of endogenous Beclin 1 expression , and are defective in starvation-induced autophagy in the absence of exogenous beclin 1 gene transfer ( Liang et al . , 1999 , 2001; Furuya et al . , 2005; Pattingre et al . , 2005; Wang et al . , 2012 ) .", "As reported , enforced expression of wild-type Beclin 1 rescued starvation-induced autophagy , as measured by decreased levels of p62 , increased LC3-II conversion and increased numbers of GFP-LC3 puncta ( a marker for autophagosomes ) ( Figure 2A–C ) .", "These readouts represented an increase in autophagic flux rather than a block in autophagosomal maturation , as treatment with the lysosomal inhibitor bafilomycin A1 blocked p62 degradation and further increased LC3-II accumulation and numbers of GFP-LC3 puncta ( Figure 2B , C ) .", "In contrast , enforced expression of the Beclin 1 S90A mutant failed to induce autophagy in response to starvation ( Figure 2A–C ) , indicating that the Beclin 1 S90 phosphorylation site is essential for autophagy induction in response to nutrient starvation .", "Moreover , a phosphomimetic mutant Beclin 1 S90E increased autophagy in basal conditions , suggesting that Beclin 1 S90 phosphorylation may be sufficient to induce autophagy ( Figure 2A–C ) . 10 . 7554/eLife . 05289 . 004Figure 2 . The Beclin 1 S90 phosphorylation site is required for autophagy induction in MCF7 and U2OS cells .", "( A ) Western blot results of MCF7 cells transiently transfected with empty vector , and Flag epitope-tagged wild-type Beclin 1 , Beclin 1 S90A , or Beclin 1 S90E .", "The cells were grown in normal medium ( starvation− ) or HBSS ( starvation+ ) for 3 hr in the presence or absence of 100 nM bafilomycin A1 .", "( B ) Representative images of GFP-LC3 puncta ( autophagosomes ) in MCF7 cells transiently co-transfected with indicated Flag-Beclin 1 constructs and a plasmid expressing GFP-LC3 and grown in normal medium or in HBSS for 3 hr ( starvation ) in the presence or absence of 100 nM bafilomycin A1 .", "( C ) Quantification of GFP-LC3 puncta in MCF7 cells in conditions shown in ( B ) .", "Bars are mean + SEM of triplicate samples ( >50 cells analyzed per sample ) .", "Similar results were observed in three independent experiments .", "***p < 0 . 001 , **p < 0 . 01 , NS , not significant; one-way ANOVA .", "( D ) Western blot detection of Beclin 1 , p62 and LC3 in U2OS cells expressing doxycycline-inducible shRNA against beclin 1 ( beclin 1 shRNA U2OS cells ) following treatment with 1 μg/ml doxycycline for 4 days in cells transduced with retroviral constructs expressing indicated shRNA-resistant Flag-Beclin 1 ( NTm , non-targetable mutant ) plasmids .", "Cells were either grown in normal medium ( starvation− ) or in HBSS for 3 hr ( starvation+ ) in the presence or absence of 100 nM bafilomycin A1 .", "See Figure 2—figure supplement 1 for comparison of Beclin 1 , p62 , and LC3 western blots in the presence and absence of doxycycline .", "( E ) Quantification of GFP-LC3 puncta ( autophagosomes ) in beclin 1 shRNA U2OS cells treated with 1 μg/ml doxycycline for 4 days and co-transfected with plasmids expressing GFP-LC3 and indicated shRNA-resistant Flag-Beclin 1 construct and grown in normal medium or in EBSS for 3 hr ( starvation ) in the presence or absence of 100 nM bafilomycin A1 .", "Bars are mean + SEM of triplicate samples ( >50 cells analyzed per sample ) .", "Similar results were observed in three independent experiments .", "**p < 0 . 01 , *p < 0 . 05 , NS , not significant; one-way ANOVA .", "( F ) Beclin 1-associated VPS34 in vitro lipid kinase assay and amounts of VPS34 and ATG14 in anti-Beclin 1 immunoprecipitates of beclin 1 shRNA U2OS cells following treatment with 1 μg/ml doxycycline for 4 days and transfection with indicated shRNA-resistant Flag-Beclin 1 ( NTm , non-targetable mutant ) plasmids .", "Cells were either grown in normal medium ( starvation− ) or in HBSS for 2 hr ( starvation+ ) .", "Dots shown in upper panel represent the amount of PI3P generated in an in vitro VPS34 lipid kinase assay using anti-Flag-Beclin 1 immunoprecipitates as input .", "( G ) Densometric quantitation of VPS34 in vitro lipid kinase activity in anti-Beclin 1 immunoprecipitates in conditions described in ( F ) .", "Results shown represent mean + SEM of values in three independent experiments .", "Similar results were observed in each independent experiment .", "Shown are the relative values of VPS34 lipid kinase activity compared to those observed in cells expressing WT Beclin 1 in normal media ( defined as 100% ) .", "To control for input in Beclin 1 anti-immunoprecipitates , values used to calculate VPS34 lipid kinase activity were normalized for levels of Beclin 1 determined by densitometric quantification of Beclin 1 western blot bands in anti-Beclin 1 immunoprecipitates .", "***p < 0 . 001 , **p < 0 . 01 , NS , not significant; one-way ANOVA .", "See also Figure 2—figure supplement 1 , Figure 1—figure supplement 2 , Figure 2—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 00410 . 7554/eLife . 05289 . 005Figure 2—figure supplement 1 . Doxycycline reduces Beclin 1 expression and starvation-induced autophagy in U2OS cells that express doxycycline-inducible beclin 1 shRNA . Detection of Beclin 1 and autophagy levels ( as measured by p62 and LC3 immunoblots ) in U2OS that express doxycycline-inducible beclin 1 shRNA .", "Cells were cultured in the presence or absence of 1 μg/ml doxycycline for 4 days and subjected to starvation in HBSS for the indicated time period . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 00510 . 7554/eLife . 05289 . 006Figure 2—figure supplement 2 . Bafilomycin A1-induced Beclin 1 S90 phosphorylation is reversed by the ROS scavenger , N-acetyl-L-cysteine . HeLa cells were either untreated , pre-treated or not pre-treated with 5 mM N-acetyl-L-cysteine for 1 hr , followed by culture either in the presence or absence 100 nM Bafilomycin A1 for 3 hr .", "Baf A1 , bafilomycin A1; NAC , N-acetyl-L-cysteine . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 00610 . 7554/eLife . 05289 . 007Figure 2—figure supplement 3 . Effects of Beclin 1 S90 phosphorylation o on subcellular localization of the Beclin 1/VPS34/ATG14 complex . Subcellular localization of Beclin 1 , VPS34 , and ATG14 in normal media or after starvation for 2 hr in HBSS in beclin 1 shRNA U2OS cells following treatment with 1 μg/ml doxycycline for 4 days and transfection with indicated shRNA-resistant Flag-Beclin 1 non-targetable wild-type ( WT ) or indicated mutant plasmids .", "Loading controls for the cytosol , mitochondria , and microscome fractions are GAPDH , TOM20 , and PDI , respectively .", "Asterix denotes non-specific signal due to membrane edge artifact . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 007 We observed similar results in U2OS cells with doxycycline inducible shRNA knockdown of endogenous Beclin 1 .", "Doxycycline treatment of these cells resulted in undetectable levels of Beclin 1 and lack of starvation-induced p62 degradation .", "Consistent with previous reports of Beclin 1 knockdown or knockout in other mammalian cells ( Matsui et al . , 2007; He et al . , 2013; Mandell et al . , 2014 ) and knockout of Atg6 in yeast ( Suzuki et al . , 2004 ) , knockdown of Beclin 1 did not block LC3 lipidation ( Figure 2—figure supplement", "1 ) but it did block the formation of GFP-LC3 puncta ( Figure 2D , data not shown ) ( Atg6/Beclin 1 are not invariably required for LC3 lipidation , but they are required for the localization of lipidated LC3 to the autophagosome [Mizushima et al . , 2010] ) .", "Expression of shRNA-resistant wild-type Beclin 1 , but not shRNA-resistant Beclin 1 S90A , rescued starvation-induced autophagic flux as measured by p62 degradation and quantification of GFP-LC3 puncta in the presence and absence of bafilomycin A1 ( Figure 2D , E ) .", "Expression of the phosphomimetic mutant Beclin 1 S90E increased autophagy in basal conditions to levels similar to those observed in starvation in cells expressing wild-type Beclin 1 ( Figure 2D , E ) .", "Taken together , the data in MCF7 cells and U2OS cells provide strong evidence that Beclin 1 S90 phosphorylation is both necessary and sufficient for autophagy induction .", "We note that there is not a complete block in autophagic flux in empty vector transfected MCF7 cells or in U2OS cells treated with doxycycline , presumably due to the presence of low levels of Beclin 1 expression .", "In both MCF7 cells and U2OS cells , we observed that bafilomycin A1 treatment resulted in Flag-Beclin 1 S90 phosphorylation in the absence of starvation .", "To determine whether bafilomycin A1 also induces phosphorylation of endogenous Beclin 1 S90 and whether such effects are due to reactive oxygen species ( ROS ) generation that occurs as a consequence of inhibition of vacuolar-type-H+-ATPases ( Zhdanov et al . , 2011; Yokomakura et al . , 2012 ) , we measured the effects of bafilomycin A1 treatment on Beclin 1 S90 phosphorylation in HeLa cells in the presence or absence of the ROS scavenger , N-acetyl-L-cysteine ( Figure 2—figure supplement 2 ) .", "Our results indicate that bafilomycin A1 results in endogenous Beclin 1 S90 phosphorylation which is blocked by N-acetyl-L-cysteine .", "Thus , ROS generation , as well as starvation , results in Beclin 1 S90 phosphorylation .", "We also observed Beclin 1 S90 phosphorylation in response to other stress stimuli ( data not shown ) , but chose to focus on starvation , given the crucial physiological importance of starvation in autophagy induction .", "To address the mechanism by which Beclin 1 S90 phosphorylation induces autophagy , we compared U2OS cells expressing wild-type Beclin 1 , the non-phosphorylatable Beclin 1 S90A mutant , and the phosphomimetic Beclin 1 S90E mutant with respect to ( 1 ) Beclin 1-associated VPS34 lipid kinase activity; ( 2 ) Beclin 1 immunoprecipitation of ATG14 and VPS34 , and ( 3 ) the membrane localization of Beclin 1 , ATG14 , and VPS34 .", "Our results show that the amounts of VPS34 and ATG14 that co-immunoprecipitate with Beclin 1 are not altered by starvation or by mutation of the Beclin 1 S90 site ( Figure 2F ) .", "In addition , no apparent differences were observed in the localization of Beclin 1 , ATG14 , or VPS34 to membrane fractions ( e . g . , mitochondrial-enriched , microsomal ) in cells expressing wild-type Beclin 1 , Beclin 1 S90A , or Beclin 1 S90E ( Figure 2—figure supplement 3 ) .", "However , despite similar levels of Beclin 1/ATG14 and Beclin 1/VPS34 binding , marked differences were observed in the VPS34 lipid kinase activity associated with wild-type and mutant forms of Beclin 1 ( Figure 2F–G ) .", "In cells expressing the phosphorylation-defective Beclin 1 S90A mutant , no increase was observed in Beclin 1-associated VPS34 lipid kinase activity in response to starvation .", "In cells expressing the Beclin 1 S90E phosphomimetic mutant , Beclin 1-associated VPS34 lipid kinase activity was higher in baseline and in starvation conditions than in cells expressing wild-type Beclin 1 .", "Thus , Beclin 1 S90 phopshorylation increases the lipid kinase activity of the Beclin 1/VPS34 complex .", "Beclin 1 is a tumor suppressor protein that inhibits the growth of MCF7 human breast carcinoma cells in immunodeficient mice ( Liang et al . , 1999 , 2001; Furuya et al . , 2005 ) .", "We investigated whether the Beclin 1 S90 phosphorylation site required for starvation-induced autophagy is also required for the tumor suppressor activity of Beclin 1 .", "MCF7 cells were transduced with retroviruses that express Flag epitope-tagged wild-type Beclin 1 or Beclin 1 S90A; injected into nu/nu mice implanted with slow release estrogen tablets; and monitored for their rate of tumor growth .", "Levels of Beclin 1 expression were comparable in MCF7 cells transduced with both Beclin 1-expressing viruses ( Figure 3A ) .", "Even in normal growth conditions , Beclin 1 S90 phosphorylation could be detected in MCF7 cells expressing wild-type Beclin 1 , presumably due to high levels of expression of the protein using a retroviral vector; such phosphorylation was absent in MCF7 cells transduced with a retrovirus expressing mutant Beclin 1 S90A . 10 . 7554/eLife . 05289 . 008Figure 3 . The Beclin 1 S90 phosphorylation site is required for tumor suppression function in MCF7 cells .", "( A ) Western blot detection of Beclin 1 p-S90 , Flag-Beclin 1 , p62 , and LC3 in MCF7 cells stably transduced with retroviruses expressing indicated Beclin 1 construct .", "( B ) Xenograft growth of cells in ( A ) in nu/nu mice .", "Tumor volumes shown represent mean + SEM for at least 11 mice per group .", "Differences in tumor growth among the different groups were analyzed using a linear mixed effect model; P = NS for Beclin 1 S90A vs empty vector; p < 0 . 001 for Beclin 1 WT vs empty vector .", "( C ) Representative images of p62 staining , Ki67 staining , and TUNEL-labeling of indicated MCF7 xenograft tumor genotype .", "( D–F )", "Quantification of relative reciprocal p62 intensity per high-power field ( D ) , percentage of Ki67-positive nuclei per high-power field ( E ) and number of TUNEL-positive nuclei per high power field ( F ) .", "At least 10 randomly selected fields per xenograft section were analyzed by an observer blinded to genotype .", "Bars are mean + SEM for 5–8 xenografts per MCF7 xenograft tumor genotype .", "***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 05 , NS , not significant; one-way ANOVA with Dunnett's test . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 008 Striking differences in the rate of MCF7 xenograft growth in nude mice were observed in cells expressing wild-type Beclin 1 as compared to Beclin 1 S90A; MCF7 cells expressing Beclin 1 S90A grew as rapidly as MCF7 control cells , whereas a marked reduction in tumor growth was observed in MCF7 cells expressing wild-type Beclin 1 ( Figure 3B ) .", "In xenografts expressing wild-type Beclin 1 , but not Beclin 1 S90A , there was a significant reduction in tumor p62 staining as compared with empty vector controls ( Figure 3C , D ) , suggesting increased levels of autophagy in the tumors .", "In parallel with decreased growth and decreased p62 staining , xenografts expressing wild-type Beclin 1 , but not Beclin 1 S90A , had decreased rates of cell proliferation ( Figure 3C , E ) , as measured by the percentage of cells with Ki67 staining .", "Xenografts expressing wild-type Beclin 1 also had decreased cell death ( Figure 3C , F ) , as measured by the percentage of TUNEL-positive cells .", "Together , these data confirm previous reports demonstrating that Beclin 1 simultaneously increases cell survival and decreases cell proliferation , resulting in a net decrease in tumor cell growth ( Wei et al . , 2013 ) , and provide new evidence that the S90 phosphorylation site is essential for the tumor suppressor function of Beclin 1 .", "Next , we sought to identify the upstream kinase ( s ) that mediate starvation-induced phosphorylation of Beclin 1 S90 .", "Therefore , we performed an in vitro kinase screen using a 15 amino acid peptide corresponding to Beclin 1 amino acid residues 83–97 as a substrate .", "Among a pool of 190 serine/threonine kinases , MAPKAPK3 , also known as and herein referred to as MK3 , was the only kinase with significant activity against the Beclin 1 peptide substrate ( Figure 4—figure supplement 1 , Supplementary file 1 ) .", "Since there are other serine and threonine residues in the Beclin 1 83–97 peptide , we repeated the in vitro kinase assay with MK3 using a Beclin 1 peptide spanning from amino acid residues 83–103 with either a wild-type sequence or an alanine substitution mutation at amino acid residue 90 .", "This mutation completely abrogated MK3-mediated phosphorylation of the Beclin 1 peptide , indicating that serine 90 is essential for it to serve as a substrate for MK3 ( Figure 4A ) . 10 . 7554/eLife . 05289 . 009Figure 4 . MK2 and MK3 mediate phosphorylation of Beclin 1 S90 . ( A ) In vitro kinase activity of MK3 using indicated Beclin 1 peptides as a substrate .", "( B ) In vitro kinase activity of MK2 , MK3 , and MK5 using the wild-type ( WT ) Beclin 1 peptide shown in A as a substrate .", "( C ) In vitro kinase activity of MK2 and MK3 using Flag epitope-tagged wild-type Beclin 1 or Beclin 1 S90 purified from HEK293T cells as a substrate .", "See also Figure 4—figure supplement 1 and Supplementary file 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 00910 . 7554/eLife . 05289 . 010Figure 4—figure supplement 1 . Results of in vitro kinase screen using the Beclin 1 83–97 peptide as a substrate . Kinases included in screen are listed in alphabetical order on the x-axis .", "See Supplementary file 1 for details . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 010 MK3 is highly homologous to MK2 , another MAP kinase-activated protein kinase , and shares ∼75% sequence identity and displays functional redundancy ( Gaestel , 2006 ) .", "We found that MK2 , but not MK5—another MAP kinase-activated protein kinase that is less structurally related to MK3 ( Cargnello and Roux , 2011 ) —also phosphorylated the Beclin 1 83–97 peptide in vitro ( Figure 4B ) .", "Furthermore , both MK2 and MK3 demonstrated in vitro kinase activity , resulting in Beclin 1 S90 phosphorylation , using Flag-Beclin 1 purified from HEK293T cells as a substrate ( Figure 4C ) .", "To investigate whether MK2 phosphorylates Beclin 1 S90 in cultured cells , we expressed either dominant-negative MK2 ( MK2 K/R ) ( containing an arginine substitution at a lysine position at amino acid residue 76 that is conserved between MK2 and MK3 ) , ( Winzen et al . , 1999 ) or constitutively active MK2 ( MK2 T205E/T317E; herein referred to as MK2 EE ) ( Engel et al . , 1995 ) in HeLa cells .", "Dominant-negative MK2 blocked starvation-induced Beclin 1 S90 phosphorylation , whereas constitutively active MK2 increased Beclin 1 S90 phosphorylation during nutrient-rich conditions ( Figure 5A ) .", "By measuring phosphorylation of the downstream MK2/MK3 substrate , p-HSP27 , we confirmed that starvation activated MK2/MK3 and that this was blocked by dominant negative MK2 expression .", "Taken together , these data indicate that MK2 and MK3 directly phosphorylate Beclin 1 S90 in vitro , and that MK2-like kinase activity also mediates starvation-induced Beclin 1 S90 phosphorylation in cultured cells . 10 . 7554/eLife . 05289 . 011Figure 5 . MK2 positively regulates autophagy .", "( A ) Effects of wild-type , dominant negative ( K/R ) and constitutively active ( EE ) MK2 on endogenous Beclin 1 S90 phosphorylation , MK2/MK3 activation ( levels of p-HSP27 ) and autophagy ( levels of p62 degradation and LC3-II conversion ) in HeLa cells grown in normal medium ( starvation− ) or HBSS for 2 hr ( starvation+ ) .", "Actin is shown as a loading control .", "( B ) Effects of wild-type , dominative negative ( K/R ) and constitutively active ( EE ) MK2 on autophagy ( GFP-LC3 puncta numbers in the presence or absence of 100 nM bafilomycin A1 ) in HeLa cells grown in normal medium ( starvation− ) or HBSS for 3 hr ( starvation+ ) .", "Bars are mean + SEM of triplicate samples ( >50 cells analyzed per sample ) .", "Similar results were observed in three independent experiments .", "**p < 0 . 01 , *p < 0 . 05 , NS , not significant; one-way ANOVA for indicated comparisons .", "( C ) Beclin 1 S90 phosphorylation and autophagy ( levels of p62 degradation and LC3-II conversion ) in MK2−/−/MK3−/− MEFs and MK2−/−/MK3−/− MEFs stably transformed with wild-type MK2 .", "Cells were grown in normal medium ( starvation− ) or HBSS for 2 hr ( starvation+ ) .", "( D ) Quantitation of GFP-LC3 puncta ( autophagosomes ) in MK2−/−/MK3−/− MEFs and MK2−/−/MK3−/− MEFs stably transformed with wild-type MK2 during growth in normal media or HBSS ( starvation ) for 3 hr in the presence or absence of 100 nM bafilomycin A1 .", "Bars are mean + SEM of triplicate samples ( >50 cells analyzed per sample ) .", "Similar results were observed in three independent experiments .", "***p < 0 . 001 , NS , not significant; one-way ANOVA .", "p < 0 . 001 for the magnitude of change between normal and starvation conditions in MK2−/−/MK3−/− MEFs vs that in in MK2−/−/MK3−/− + MK2 MEFs; two-way ANOVA .", "See also Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 01110 . 7554/eLife . 05289 . 012Figure 5—figure supplement 1 . Dominant p38α inhibits starvation-induced MK2 activation and Beclin 1 S90 phosphorylation .", "( A ) Effects of dominant-negative ( DN ) JNK1 and DN p38α on Beclin 1 S90 phosphorylation , MK2 activation ( MK2 band shift and p-HSP27 ) and autophagy ( p62 degradation and LC3-II conversion ) in HeLa cells grown in normal media ( starvation− ) or HBSS for 2 hr ( starvation+ ) .", "( B ) Constitutively active MK2 ( MK2 EE ) blocks DN p38α suppression of starvation-induced Beclin 1 S90 phosphorylation .", "HeLa cells were co-transfected with plasmids expressing Flag-DN p38α and either empty vector or MK2 EE and grown in normal media ( starvation− ) or HBSS for 2 hr ( starvation+ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 012 To study the effects of MK2 and MK3 on autophagy regulation , we first evaluated autophagy in HeLa cells transfected with dominant negative MK2 ( which blocks the kinase activity of both MK2 and MK3 ) or constitutively active MK2 .", "As measured by LC3-II conversion , p62 degradation , and numbers of GFP-LC3 puncta in the presence and absence of bafilomycin A1 , dominant-negative MK2 decreased starvation-induced autophagy ( Figure 5A , B ) .", "Conversely , constitutively active MK2 increased basal autophagy in HeLa cells ( Figure 5A , B ) .", "Thus , in parallel with regulation of starvation-induced Beclin 1 S90 phosphorylation ( Figure 5A ) , MK2 regulates starvation-induced autophagy .", "The best-characterized kinase upstream of MK2/MK3 is p38 MAPK ( Freshney et al . , 1994; Rouse et al . , 1994 ) .", "Therefore , we asked whether dominant negative p38α could block starvation-induced MK2 activation , Beclin 1 S90 phosphorylation , and autophagy .", "In HeLa cells transfected with a plasmid expressing dominant negative p38α , we failed to observe starvation-induced MK2 activation ( i . e . , an MK2 band shift or phosphorylation of its substrate HSP27 ) , Beclin 1 S90 phosphorylation , or autophagy ( Figure 5—figure supplement 1A ) .", "This block in Beclin 1 S90 phosphorylation in cells expressing dominant negative p38α was reversed by overexpression of constitutively active MK2 ( Figure 5—figure supplement 1B ) , providing additional proof that MK2 functions downstream of p38α to mediate Beclin 1 S90 phosphorylation in response to amino acid starvation .", "To determine whether endogenous MK2/MK3 function in the regulation of Beclin 1 S90 phosphorylation and autophagy , we evaluated Mapkapk2−/−/Mapkapk3−/− ( herein referred to as MK2−/−/MK3−/− ) MEFs retrovirally transduced with empty vector compared to MK2−/−/MK3−/− MEFs retrovirally transduced with MK2 ( Ronkina et al . , 2011 ) .", "In MK2−/−/MK3−/− MEFs , there was a marked decrease in starvation-induced Beclin 1 S90 phosphorylation which was rescued by MK2 expression ( Figure 5C ) .", "Low levels of Beclin 1 S90 phosphorylation were still detected in MK2−/−/MK3−/− MEFs following starvation , suggesting either that other members of the MAPKAPK family may partially compensate for the developmental loss of both MK2 and MK3 and/or that other unidentified kinase families may also play a minor role in the regulation of phosphorylation at this site of Beclin 1 .", "Nonetheless , loss of MK2 and MK3 was sufficient to decrease both basal and starvation-induced autophagy as assessed by p62 degradation , LC3-II conversion , and quantification of GFP-LC3 puncta in MK2−/−/MK3−/− MEFs as compared to MK2−/−/MK3−/− MEFs that stably express MK2 ( Figure 5C , D ) .", "The increase in GFP-LC3 puncta with MK2 reconstitution was not due to a block in autophagic maturation , as numbers further increased upon treatment with the lysosomal inhibitor , bafilomycin A1 .", "Also , we note that the decreased levels of total LC3 ( with an increased ratio of LC3-II to LC3-I ) in MK2 reconstituted cells during starvation conditions is consistent with a marked increase in autophagic flux , as LC3 ( similar to p62 ) is degraded by the autophagy pathway .", "Thus , these results in MK2−/−/MK3−/− MEFs demonstrate that endogenous MK2 and MK3 function in the positive regulation of Beclin 1 S90 phosphorylation and autophagy .", "To examine whether MK2/3 positively regulate autophagy through a mechanism that involves Beclin 1 S90 phosphorylation , we examined the effects of retroviral gene transfer of wild-type Beclin 1 , Beclin 1 S90A ( a non-phosphorylatable mutant ) , or Beclin 1 S90E ( a predicted phosphomimetic mutant ) on autophagy in MK2−/−/MK3−/− MEFs .", "Whereas expression of Beclin 1 S90A did not increase levels of basal or starvation-induced autophagy ( and expression of wild-type Beclin 1 had only minimal effects ) , Beclin 1 S90E expression was sufficient to markedly increase basal autophagy in cells lacking both MK2 and MK3 ( Figure 6A , B ) .", "The magnitude of this increase was almost comparable to that observed with enforced expression of MK2 .", "The observation that a Beclin 1 S90 phosphomimetic mutant can substitute for MK2/3 in MK2/3-deficient cells in regulating levels of autophagy is consistent with a model in which MK2/3 regulates autophagy through the phosphorylation of Beclin 1 S90 . 10 . 7554/eLife . 05289 . 013Figure 6 . MK2 positively regulates autophagy through the Beclin 1 S90 phosphorylation site .", "( A ) Western blot detection of Beclin 1 S90 phosphorylation and autophagy ( p62 degradation and LC3-II conversion ) in MK2−/−/MK3−/− MEFs retrovirally transduced with indicated expression constructs and grown in normal media or HBSS ( starvation ) for 4 hr . ( B ) Quantification of GFP-LC3 puncta ( autophagosomes ) in MK2−/−/MK3−/− MEFs retrovirally transduced with indicated expression constructs and grown in normal media or HBSS ( starvation ) for 4 hr .", "Bars are mean + SEM of triplicate samples ( >50 cells analyzed per sample ) .", "Similar results were obtained in two independent experiments .", "***p < 0 . 01 , NS , not-significant; one-way ANOVA with Dunnett's test .", "( C ) Effects of constitutively active MK2 ( MK2 EE ) on Beclin 1 S90 phosphorylation and autophagy ( as measured by p62 and LC3 western blot analysis ) in U2OS doxycycline-inducible beclin 1 shRNA knockdown cells expressing either shRNA-resistant wild-type Flag-Beclin 1 or mutant Flag-Beclin 1 S90A .", "Cells were treated with 1 μg/ml doxycycline for 4 days prior to western blot analyses with indicated antibodies and transfected with indicated shRNA-resistant plasmids after 2 days of doxycycline treatment .", "Cells were grown in normal media or HBSS ( starvation ) for 2 hr .", "NTm , non-targeting mutant .", "( D ) Effects of dominant-negative MK2 ( MK2 K/R ) on Beclin 1 S90 phosphorylation and autophagy ( as measured by p62 and LC3 western blot analysis ) in U2OS doxycycline-inducible beclin 1 shRNA knockdown cells expressing either shRNA-resistant wild-type Flag-Beclin 1 or mutant Flag-Beclin 1 S90A .", "Cells were treated with 1 μg/ml doxycycline for 4 days prior to western blot analyses with indicated antibodies , and transfected with indicated shRNA-resistant plasmids after 2 days of doxycycline treatment .", "Cells were grown in normal media or HBSS ( starvation ) for 2 hr .", "NTm , non-targeting mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 013 To more directly test the hypothesis that MK2/3 regulate autophagy through the Beclin 1 S90 phosphorylation site , we investigated whether active MK2 can induce autophagy in cells that lack detectable endogenous Beclin 1 and only express an shRNA-resistant mutant Beclin 1 S90A construct ( Figure 6C ) .", "U2OS cells with doxycycline treatment-induced knockdown of Beclin 1 fail to undergo increased autophagy ( as assessed by p62 degradation and LC3-II conversion ) in response to starvation , active MK2 expression , or both starvation and active MK2 expression .", "In cells that express an shRNA-resistant wild-type Beclin 1 , there is an increase in autophagy with starvation and active MK2 expression , and a further increase in autophagy with combined starvation and active MK2 expression .", "In contrast , in cells that express an shRNA resistant Beclin 1 S90A mutant construct , no increase in autophagy is observed in response to any of these treatment conditions .", "Thus , MK2-induced upregulation of autophagy requires the Beclin 1 S90 phosphorylation site .", "Conversely , we asked whether expression of a Beclin 1 S90E phosphomimetic mutant in cells lacking detectable endogenous Beclin 1 could bypass the inhibitory effects of dominant negative MK2 ( Figure 6D ) .", "In doxycline-treated beclin 1 shRNA U2OS cells that express shRNA-resistant wild-type Beclin 1 , dominant negative MK2 nearly completely suppressed starvation-induced autophagy , as assessed by p62 degradation and LC3-II conversion .", "However , in cells expressing shRNA-resistant Beclin 1 S90E , starvation induced a decrease in p62 levels and conversion of LC3-I to LC3-II even in the presence of dominant negative MK2 expression .", "This suggests that Beclin 1 S90 phosphorylation may be sufficient to partially bypass the inhibitory effects of MK2/3 inhibition on starvation-induced autophagy .", "Taken together , these data in MK2−/−/MK3−/− MEFs and in doxycycline-inducible beclin 1 shRNA U2OS cells ( reconstituted with wild-type Beclin 1 , the non-phosphorylatable Beclin 1 S90A mutant or the phosphomimetic Beclin 1 S90E mutant ) strongly suggest that MK2/3 regulate autophagy through a mechanism involving Beclin 1 S90 phosphorylation .", "BCL2 inhibits autophagy through a direct interaction with the BH3 domain of Beclin 1 , which consists of amino acid residues 105–128 ( Oberstein et al . , 2007 ) .", "Given the proximity of the BH3 domain to Beclin 1 S90 , we postulated that BCL2 binding to Beclin 1 might affect MK2-dependent Beclin 1 phosphorylation .", "In addition , the possibility that BCL2 binding to Beclin 1 ( a process disrupted by JNK1-mediated multisite phosphorylation of BCL2 during amino acid starvation [Wei et al . , 2008] ) might block MK2-dependent phosphorylation was suggested by two observations in this study , including ( 1 ) dominant-negative JNK1 blocked starvation-induced Beclin 1 S90 phosphorylation and autophagy but not starvation-induced MK2 activation ( Figure 5—figure supplement 1 ) ; and ( 2 ) the amount of active MK2-dependent Beclin 1 S90 phosphorylation was greater in starvation conditions than in normal nutrient conditions ( Figure 6C ) .", "To directly test the hypothesis that BCL2 binding to Beclin 1 blocks MK2-mediated Beclin 1 S90 phosphorylation , we performed an in vitro kinase assay to assess the effects of MK2 on Beclin 1 S90 phosphorylation in the presence of recombinant GST-BCL2 .", "We found that with increasing amounts of GST-BCL2 , there was decreased in vitro MK2-mediated phosphorylation of Beclin 1 S90 ( Figure 7A ) .", "These data provide direct evidence that BCL2 inhibits MK2-dependent Beclin 1 S90 phosphorylation . 10 . 7554/eLife . 05289 . 014Figure 7 . BCL2 inhibits MK2-dependent Beclin 1 S90 phosphorylation .", "( A ) In vitro kinase activity of MK2 using Flag-Beclin 1 purified from HEK293 cells as a substrate in the presence of the indicated amount of GST-BCL2 .", "( B ) Structural model showing that steric hindrance may prevent simultaneous binding of BCL2/BCL2L1 and MK2 to Beclin 1 .", "MK2 ( residues 44–216 , 236–284 and 288–345 ) , BCL2L1 ( residues 2–25 and 83–198 ) and Beclin 1 ( residues 79–181 ) are shown in cyan , pink and grey ribbon , respectively .", "MK2 residues important for catalysis and Beclin 1 residues important for binding to BCL2L1 , or for phosphorylation ( S90 and R87 ) are shown in stick , with atoms colored by element type: oxygen , red; nitrogen , blue; sulfur , green; and carbon , cyan or grey for MK2 or Beclin 1 respectively .", "Numbers indicate the last residue preceding or following regions missing from this model , that might cause further steric conflicts .", "( C ) Detection of Beclin 1 S90 phosphorylation and autophagy levels ( as measured by p62 and LC3 immunoblots ) in HeLa cells stably transfected with empty vector ( HeLa/control ) , wild-type BCL2 ( HeLa/BCL2 ) , or a BCL2 T69A/S70A/S87A mutant ( HeLa/BCL2 AAA ) and subjected to starvation in HBSS for the indicated time period .", "p-Hsp27 protein levels are shown as an indicator of MK2 activation .", "( D ) Detection of Beclin 1 S90 phosphorylation and autophagy levels ( as measured by p62 and LC3 immunoblots ) in HeLa/Control , HeLa/BCL2 , and HeLa/BCL2 AAA cells transiently transfected with a control empty vector or constitutively active MK2 ( MK2 EE ) and grown in normal medium ( starvation− ) or HBSS for 2 hr ( starvation+ ) .", "( E ) Starvation-induced Beclin 1 S90 phosphorylation and autophagy induction in vastus lateralis muscle of Bcl2AAA mice and control wild-type littermates .", "Mice were subjected to starvation for 48 hr prior to tissue collection and western blot analysis of muscle lysates with indicated antibodies .", "Each lane represents a muscle sample from an independent mouse . DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 014 To better understand how binding of BCL2 family proteins to Beclin 1 may inhibit MK2-mediated phosphorylation , we constructed a structure-based model ( Figure 7B ) to determine whether both BCL2 and MK2 could bind simultaneously to Beclin 1 .", "The structure of the BCL2-related anti-apoptotic protein , BCL2L1 ( which also inhibits autophagy through its interaction with Beclin 1 ) , bound to the helical BH3 domain of Beclin 1 has been solved ( Oberstein et al . , 2007 ) ( PDV ID: 2P1L ) , as has the structure of a region of MK2 bound to its auto-inhibitory peptide ( Meng et al . , 2002 ) ( PDB ID: 1KWP ) .", "Based on sequence analysis suggesting that Beclin 1 residues 80–104 that precede the BH3 domain are helical when bound to a suitable partner ( Mei et al . , 2014 ) , a model for this region was built by ‘in silico’ mutagenesis of the MK2-bound auto-inhibitory peptide to correspond to Beclin 1 residues 80–95 , such that S90 was positioned optimally for phosphorylation .", "This helix was then extended to include Beclin 1 residues 96–131 and BCL2L1 positioned by superposition of the bound Beclin 1 BH3 domain ( residues 105–128 ) on the modeled Beclin 1 extended helix .", "This model suggests that steric hindrance may prevent simultaneous binding of BCL2L1 ( or BCL2 ) and MK2 to Beclin 1 , thereby inhibiting MK2-dependent Beclin 1 S90 phosphorylation .", "Of note , residues 216–236 and the N- and C-terminal regions of MK2 and BCL2L1 residues 26–81 , were not present in the structures used to build this model , but in the full-length proteins these residues likely result in additional steric conflicts ( Figure 7B ) .", "Furthermore , the BCL2L1 C-terminal membrane-anchoring helix that was removed in the construct crystallized may provide cellular localization constraints that prevent MK2-mediated phosphorylation of Beclin 1 S90 .", "Next , we assessed whether BCL2 binding to Beclin 1 in cultured cells regulates Beclin 1 S90 phosphorylation ( Figure 7C , D ) .", "In HeLa cells stably transfected with wild-type BCL2 , the starvation-induced increase in Beclin 1 S90 phosphorylation was delayed and blunted compared to that observed in HeLa control cells; however , by 2 hr after starvation when there was complete disruption of BCL2 and Beclin 1 binding ( as measured by co-immunoprecipitation ) , an appreciable increase in Beclin 1 S90 phosphorylation was observed ( Figure 7C ) .", "In contrast , in HeLa cells stably transfected with the mutant BCL2 T69A/S70A/S87A ( BCL2 AAA ) , which lacks the JNK1 phosphorylation sites and fails to dissociate with Beclin 1 during starvation ( Wei et al . , 2008 ) , there was no increase in Beclin 1 S90 phosphorylation in response to starvation .", "The levels of starvation-induced autophagy , as assessed by p62 degradation and LC3-II conversion paralleled the levels of Beclin 1 S90 phosphorylation .", "Starvation-induced autophagy was delayed and attenuated in HeLa/BCL2 cells compared to HeLa/control cells , and completely blocked in HeLa/BCL2 AAA cells .", "These effects of BCL2 on starvation-induced Beclin 1 S90 phosphorylation and autophagy were not due to a blockade of MK2/MK3 activation , as levels of phospho-HSP27 increased similarly during starvation in HeLa/control , HeLa/BCL2 and HeLa/BCL2 AAA cells .", "Furthermore , even in the absence of starvation , active MK2 was able to increase Beclin 1 S90 phosphorylation and induce autophagy in HeLa/control cells but not in HeLa/BCL2 or HeLa/BCL2 AAA cells ( Figure 7D ) .", "Thus , BCL2 blocks both starvation- and active MK2-mediated upregulation of Beclin 1 S90 phosphorylation and autophagy .", "To confirm whether BCL2 regulates starvation-induced Beclin 1 S90 phosphorylation in vivo , we examined the levels of Beclin 1 S90 phosphorylation in Bcl2AAA mice that contain a T69A/S70A/S84A knock-in mutation in Bcl2 ( S84 is homologous to human S87 ) ( Figure 7E ) .", "Previously , we showed that this mutation blocks starvation-induced disruption of Bcl2/Beclin 1 binding and starvation-induced autophagy , including in cardiac and skeletal muscle of Bcl2AAA mice ( He et al . , 2012 ) .", "Following a 48 hr starvation period , we found the skeletal muscle of wild-type mice had activation of MK2 ( as evidenced by a band shift in MK2 and phosphorylation of the substrate , HSP27 ) , increased autophagy ( as detected by p62 degradation and LC3-II conversion ) , and increased Beclin 1 S90 phosphorylation .", "In contrast , in the muscles of Bcl2AAA mice , activation of MK2 appeared similar to that observed in wild-type muscles , but this was not accompanied by an increase in Beclin 1 S90 phosphorylation or in levels of autophagy .", "Thus , the constitutive binding of Bcl2 AAA to Beclin 1 during starvation in vivo is sufficient to block the effects of activated MK2 on Beclin 1 S90 phosphorylation .", "Based on our data above in cultured cells , we propose that this block in MK2-regulated Beclin 1 S90 phosphorylation may contribute to the mechanism by which Bcl2 inhibits autophagy in vivo ." ], [ "Autophagy is a fundamental cellular survival response during nutrient starvation , permitting cells to maintain nutrient and energy homeostasis when external food supply is limited ( Levine and Klionsky , 2004 ) .", "The negative regulation of this process by mTORC1 has been extensively studied , but less is known about stress-activated signals that turn on autophagy during starvation ( Kroemer et al . , 2010 ) .", "While AMPK activates components of the autophagy machinery during glucose starvation ( phosphorylating ULK1 , Beclin 1 , and VPS34 ) , it does not appear to regulate the Beclin 1/VPS34 complex in response to complete ( nitrogen and glucose ) starvation ( Kim et al . , 2013 ) .", "The signaling molecules that activate Beclin 1/VPS34 to rapidly initiate autophagosome formation during complete starvation have been heretofore unknown .", "Using unbiased approaches , we identified Beclin 1 S90 as a key phosphorylation site in starvation-induced autophagy and identified two stress-activated protein kinase signaling pathway family members , the p38 MAPK-activated protein kinases MK2 and MK3 , as crucial kinases that mediate Beclin 1 S90 phosphorylation .", "A previous study showed that Beclin 1 S90 is phosphorylated during starvation and may function in autophagy ( based on a ubiquitin-p62 degradation assay ) ( Fogel et al . , 2013 ) , but did not identify the kinase ( s ) responsible for this phosphorylation .", "In the present study , we found that alanine substitution of Beclin 1 serine 90 is sufficient to block detection of starvation-induced Beclin 1 phosphorylation , and sufficient to block Beclin 1-mediated rescue of starvation-induced autophagy in multiple different beclin 1-deficient cells using multiple different autophagic flux assays .", "The structurally and functionally-related p38 MAPK-activated protein kinase family members , MK2 and MK3 ( Cargnello and Roux , 2011 ) , but not the more distant family member , MK5 , are strong inducers of Beclin 1 S90 phosphorylation .", "MK2 and MK3 phosphorylate a Beclin 1 peptide spanning S90 in vitro; MK2 and MK3 phosphorylate Beclin 1 immunoprecipitated from cells in vitro; dominant negative MK2 blocks starvation-induced Beclin 1 S90 phosphorylation and constitutively active MK2 induces Beclin 1 S90 starvation during basal conditions; and MEFs from MK2/MK3 knockout mice are deficient in starvation-induced Beclin 1 S90 phosphorylation .", "Moreover , MK2 positively regulates autophagy through a mechanism that requires the Beclin 1 S90 phosphorylation site .", "These findings describe a previously unidentified direct link between specific members of the MAPK signaling pathway and the positive regulation of a core component of the autophagy machinery during starvation .", "MK2 was originally discovered as an ERK1/2 activated protein kinase that phosphorylates HSP25 and HSP27 ( Stokoe et al . , 1992 ) , and was later found to be stimulated by p38 in response to stress stimuli ( Freshney et al . , 1994; Rouse et al . , 1994 ) .", "MK3 was discovered independently through a yeast two-hybrid screen for p38-interacting proteins ( McLaughlin et al . , 1996 ) and through an analysis of genes commonly deleted in small-cell lung cancer ( Sithanandam et al . , 1996 ) .", "The substrate spectrum of MK2 and MK3 are virtually indistinguishable , and both enzymes modulate proteins involved in cytokine production , endocytosis , actin remodeling , cell migration , cell cycle control , chromatin remodeling , and transcriptional regulation ( Cargnello and Roux , 2011 ) .", "The role of MK2 and MK3 in LPS-induced inflammatory signaling is well-established in vivo; MK2 knockout mice show increased resistance to endotoxic shock and increased susceptibility to infections ( Kotlyarov et al . , 1999; Lehner et al . , 2002 ) and this phenotype is exacerbated by simultaneous deletion of MK3 ( Ronkina et al . , 2007 ) .", "To our knowledge , MK2 and MK3 have not been previously linked to starvation-induced stress responses or to phosphorylation of specific substrates involved in starvation-induced stress response pathways such as autophagy .", "The newly described role of these kinases in regulating starvation-induced autophagy raises the possibility that this arm of the MAPKAPK family ( sometimes referred to as the ‘p38 module’ ) may have evolved to integrate autophagy induction with diverse other cellular functions required for cellular adaption to stress .", "Our data also suggest that MK2/MK3 are activated during amino acid starvation by the well-characterized upstream kinase , p38α .", "Several reports have shown that p38 functions in the positive regulation of autophagy in response to other stress stimuli such as glucose starvation ( Moruno-Manchon et al . , 2013 ) , interferon-γ stimulation ( Matsuzawa et al . , 2012 ) reservatrol ( Chang et al . , 2014 ) or accumulation of mutant gilal fibrillary acidic protein in astrocytes ( Tang et al . , 2008 ) .", "However , Webber and Tooze found that p38α functions in the negative regulation of basal and starvation-induced autophagy in HEK293 cells ( Webber and Tooze , 2010 ) .", "The basis for the apparent discrepancy between this previous report and our current findings may reflect that p38α functions differently in different cell types or may reflect other differences in experimental design .", "For example , we did not directly examine the effects of p38α overexpression or p38α siRNA knockdown on MK2/MK3 activation and autophagy; instead , we used a dominant-negative p38α plasmid .", "One possibility is that there may be compensatory activation of different p38 isoforms that have different substrate signaling specificity in different experiments .", "In support of the concept that different p38 isoforms have different effects on starvation-induced MK2/MK3 activation , we found that dominant-negative p38β , but not dominant-negative p38γ , also blocks starvation-induced MK2/MK3 activation ( unpublished data ) .", "Another possibility is that p38α may function upstream of MK2/MK3 in amino acid starvation , but its effects on other substrates may , in some contexts , counterbalance such effects in terms of autophagy regulation .", "Given our findings that dominant negative p38α blocks starvation-induced MK2/MK3 activation , further studies are warranted to more clearly define the role of this signaling event in amino acid starvation-induced autophagy .", "Beclin 1 is a haploinsufficient tumor suppressor; it is frequently monoallelically deleted in human breast and ovarian cancers ( Aita et al . , 1999 ) ; heterozygous loss in mice results in an increased incidence of breast and other spontaneous tumors ( Qu et al . , 2003; Yue et al . , 2003; Cicchini et al . , 2014 ) ; decreased beclin 1 mRNA expression is associated with aggressive clinic-pathological features and poor prognosis in human breast cancer ( Tang et al . , 2015 ) ; and beclin 1 gene transfer impairs the ability of MCF7 human breast tumor cells to form tumor xenografts in immunodeficient mice ( Liang et al . , 1999 ) .", "Previous studies have shown that mutation of the nuclear export signal of Beclin 1 ( resulting in its nuclear retention ) or deletion of the evolutionary conserved domain of Beclin 1 ( which abrogates it binding to VPS34 and autophagy function ) blocks its tumor suppressor activity ( Liang et al . , 2001; Furuya et al . , 2005 ) .", "Our observation that a single point mutation in Beclin 1 that abrogates starvation-induced autophagy , Beclin 1 S90A , also eliminates it ability to suppress MCF7 mammary tumorigenesis provides additional support for the concept that Beclin 1 functions as a tumor suppressor through its autophagy activity .", "However , as this mutation impairs Beclin 1-associated VPS34 lipid kinase activity , we cannot rule out the possibility that the mutation blocks a VPS34-dependent , autophagy-independent function of the Beclin 1/VPS34 complex in tumor suppression .", "Regardless of the precise mechanisms by which the Beclin 1 S90A mutation blocks the tumor suppressor function of Beclin 1 , our data show a strong association between decreased autophagy , increased tumor growth , and increased cellular proliferation in human mammary tumor xenografts .", "This association occurs despite the presence of increased cell death in tumors with decreased autophagy , and is consistent with our previous findings showing that enhanced suppression of autophagy in non-small cell lung carcinomas is associated with enhanced tumor growth despite increased cell death ( Wei et al . , 2013 ) .", "Taken together , these findings suggest that the pro-survival function of autophagy in established tumors may not be the most important determinant of net tumor growth; other factors such as the effects of autophagy on cell growth control and genomic stability may be more important .", "Another implication of our finding relates to the potential relationship of MK3 deletion and Beclin 1 S90 phosphorylation in cancer development .", "Given the requirement of the Beclin 1 S90 site for the tumor suppressor activity of Beclin 1 , it will be interesting to determine whether the lack of MK3-dependent Beclin 1 S90 phosphorylation contributes to the pathogenesis of small cell lung carcinomas that commonly harbor deletions in MK3 ( Sithanandam et al . , 1996 ) or of other cancers with frequent heterozygous loss of MK3 such as invasive breast carcinomas , ovarian carcinomas , lung adenocarcinomas and lung squamous cell carcinomas ( www . cbioportal . org ) .", "PI3KC3-C1 consists of the lipid kinase , VPS34; the scaffolding protein , VPS15; and two autophagy proteins containing coiled-coil domains , ATG14 and Beclin 1 .", "Our data show that Beclin 1 S90 phosphorylation increases the VPS34 lipid kinase activity of this complex , without altering its composition or membrane localization .", "These data are consistent with predictions from a recent EM reconstitution of the ordered domains of the PI3KC3-C1 complex ( Baskaran et al . , 2014 ) .", "The architecture of the EM reconstituted complex predicts that signaling inputs at the N-terminus of Beclin 1 are likely to function in the allosteric regulation of its lipid kinase activity .", "There are no direct interactions between ATG14 and Beclin 1 and VPS34; however , the unstructured N-terminal region of Beclin 1 is predicted to be in proximity to the catalytic subunit of VPS34 , leading to the proposal that signals in the region of the Beclin 1 N-terminus regulate the activity of VPS34 lipid kinase domain .", "We speculate that MK2/MK3-dependent phosphorylation of Beclin 1 S90 may function to initiate autophagy precisely through the mechanism predicted by the recently solved structure of the PI3KC3-C1 complex , that is , through allosteric interactions that increase the activity of the VPS34 lipid kinase domain .", "Beclin 1 was originally identified as a BCL2-interacting protein ( Liang et al . , 1998 ) , and BCL2 and BCL2L1 inhibit autophagy by binding to the BH3 domain of Beclin 1 ( Pattingre et al . , 2005; Maiuri et al . , 2007 ) .", "Bcl2 plays a crucial role in the regulation of stimulus-induced autophagy in vivo , as mice that contain non-phosphorylatable mutations in Bcl2 that prevent disruption of its binding to Beclin 1 are deficient in both starvation- and exercise-induced induced autophagy ( He et al . , 2012 ) .", "Despite the importance of BCL2/BCL2L1 as negative regulators of autophagy , little is understood about the precise molecular mechanism by which their binding to Beclin 1 inhibits its autophagy function .", "The BH3 domain is in the N-terminal half of Beclin 1 and is not required for interaction with ATG14 or VPS34 or for the autophagy function of Beclin 1 .", "Homodimerization of Beclin 1 favors binding BCL2/BCL2L1 and disfavors binding ATG14 or UVRAG ( Noble et al . , 2008 ) , but the temporal order of Beclin 1 homodimerization and binding BCL2/BCL2L1 are not known .", "Even if BCL2/BCL2L1 binding to Beclin 1 precedes its homodimerization , it is not yet established that the promotion of Beclin 1 homodimerization and consequent prevention of binding to ATG14 is an important mechanism by which these proteins inhibit the autophagy function of Beclin 1 .", "Our data uncover a previously undescribed mechanism by which BCL2 inhibits the autophagy function of Beclin 1 .", "BCL2 binding to the BH3 domain of Beclin 1 blocks an essential step in the positive regulation of starvation-induced autophagy—MK2/MK3-dependent Beclin 1 S90 phosphorylation .", "Our structure-based model predicts that this may occur via steric hindrance of MK2/MK3 binding to Beclin 1 when BCL2 is bound to the BH3 domain of Beclin 1 , which is in close proximity to Beclin 1 serine 90 .", "While further studies will be required to test this structural model , our data provide strong functional evidence that BCL2 binding to Beclin 1 blocks Beclin 1 S90 phosphorylation , both in vitro and in vivo .", "BCL2 inhibits MK2-dependent in vitro phosphorylation of Beclin 1 S90 in a dose-dependent manner; BCL2 binding to Beclin 1 in cultured cells prevents starvation and active MK2-dependent Beclin 1 S90 phosphorylation; and Bcl2 AAA mice have deficient starvation-induced Beclin 1 S90 phosphorylation despite starvation-induced activation ( phosphorylation ) of MK2 .", "Taken together , our findings describe a central regulatory loop underlying starvation-induced activation of autophagy .", "This loop involves two distinct arms of the MAPK signaling pathway , which act in parallel to activate the autophagy function of Beclin 1 in response to starvation ( Figure 8 ) .", "Previously defined JNK1-mediated BCL2 multisite phosphorylation , leading to disruption of BCL2/Beclin 1 binding ( Wei et al . , 2008 ) , is a requisite event for subsequent for MK2/MK3-dependent Beclin 1 S90 phosphorylation and Beclin 1-dependent autophagy .", "The dual functions of two arms of the MAPK signaling pathway , JNK1 and MK2/MK3 , in mediating starvation-induced autophagy at the level of Beclin 1 S90 phosphorylation underscore the crucial importance of the interaction between the MAPK signaling pathway and Beclin 1 in starvation-induced autophagy .", "More broadly , these findings suggest that activation of autophagy is intricately coordinated with other MAPK signaling stress responses . 10 . 7554/eLife . 05289 . 015Figure 8 . Model of convergent functions of MAPK signaling molecules in amino acid starvation-induced autophagy and regulation of Beclin 1 S90 phosphorylation . In response to nutrient starvation , JNK1 results in multi-site phosphorylation of BCL2 and disruption of BCL2/Beclin 1 binding ( Wei et al . , 2008 ) .", "This permits active MK2/3 to phosphorylate Beclin 1 at residue serine 90 ( Figures 4–5 ) , which is essential for the autophagy and tumor suppressor function of Beclin 1 ( Figures 2–3 ) .", "Mutations in BCL2 that block multi-site phosphorylation by JNK1 ( and block starvation-induced disruption of BCL2/Beclin 1 binding [He et al . , 2012] ) block MK2-mediated Beclin 1 S90 phosphorylation both in vitro and in vivo ( Figure 7 ) .", "The MAPK signaling molecule , p38 , may function upstream to activate MK2/MK3 during amino acid starvation ( Figure 5—figure supplement 1 ) ; however , a definitive role for other kinases has not been excluded .", "Together , our data suggest a model in which autophagy activation during amino acid starvation requires two simultaneous functions of MAPK signaling molecules: ( 1 ) MK2/MK3-mediated Beclin 1 S90 phosphoryation; and ( 2 ) JNK1-mediated disruption of BCL2 binding to Beclin 1 ( which functions to block MK2/MK3-mediated Beclin 1 S90 phosphorylation ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05289 . 015" ], [ "HeLa , HEK293T , and MCF7 cells and SV40 immortalized murine embryonic fibroblasts ( MEFs ) retrovirally transduced with pMMP-IRES empty vector or pMMP-IRES MK2 ( Ronkina et al . , 2011 ) were grown in DMEM supplemented with 10% FBS , 100 U/ml penicillin and 100 μg/ml streptomycin .", "Doxycycline-inducible beclin 1 shRNA U2OS cells ( Sun et al . , 2008 ) were grown in DMEM supplemented with 10% tetracycline-free FBS .", "HeLa/Control , HeLa/BCL2 , HeLa/BCL2 AAA , and SKNSH/3xFlag-Beclin 1 cells were generated by transfection of the parental cells with pIRES . Neo , pIRES . Neo/Myc-BCL2 , pIRES . Neo/Myc-BCL2 AAA or pBICEP-CMV2-Beclin 1 , and selection with 0 . 5 μg/ml G418 .", "MCF7 cells retrovirally transduced with pBABE-puro empty vector or Flag-Beclin 1 ( WT or S90A ) were grown in DMEM supplemented with 10% FBS , 0 . 01 mg/ml human recombinant insulin and 1 μg/ml puromycin .", "MK2−/−/MK3−/− MEFs retrovirally transduced with pBABE-puro empty vector or Flag-Beclin 1 ( WT or S90 mutants ) were grown in DMEM supplemented with 10% FBS and 1 μg/ml puromycin .", "For autophagy assays , cells were grown in either the DMEM media described above for each cell line supplemented with 2× non-essential amino acids and 2 mM glutamine ( nutrient-rich conditions ) or in HBSS ( starvation conditions ) for the indicated time period .", "The HBSS used in starvation conditions contained glucose .", "The human beclin 1 gene was inserted into the EcoRI/BamH1 restriction site of the pBICEP-CMV2 vector .", "pBICEP-CMV2-Beclin 1 S90 mutants and pBABE-puro-Beclin 1 S90 mutants were constructed using a QuickChange site mutagenesis kit ( Agilent Technologies , Santa Clara , CA ) .", "The shRNA non-targetable Beclin 1 constructs harbor silent mutations ( mutated sequence: GGAAGCAAAACTAGTAACA ) in the region of beclin 1 targeted by shRNA ( GGGTCTAAGACGTCCAACA ) ( sense strand ) in order to rescue the effects of knockdown of endogenous Beclin 1 .", "All of the constructs were confirmed by sequencing .", "Other plasmids used in this study have been previously described , and include plasmids expressing GFP-LC3 ( Mizushima et al . , 2004 ) , dominant-negative MK2 ( MK2K76R ) ( Winzen et al . , 1999 ) , constitutively active MK2 ( T205E/T317E ) ( Engel et al . , 1995 ) , dominant-negative Flag-p38α ( Addgene plasmid #20352 [Enslen et al . , 1998] ) , and dominant-negative JNK1 ( Lei et al . , 2002 ) .", "Flag was detected using an anti-Flag M2 HRP antibody ( 1:2000 ) ( Sigma-Aldrich , St . Louis , MO ) .", "The phosphospecific Beclin 1 S90 antibody was produced by PhosphoSolutions ( Aurora , CO ) .", "Briefly , synthetic peptides corresponding to phosphorylated and dephosphorylated S90 of Beclin 1 were injected to two rabbits and sera were purified using a phosphopeptide affinity column followed by a dephosphopeptide affinity column .", "The phosphospecific Beclin 1 S90 antibody was used at a concentration of 1:500 .", "Beclin 1 , HSP27 , p-HSP27 , Actin , MK2 , p62 , LC3 , TOM20 , PDI , and GAPDH were detected using a rabbit or goat anti-Beclin 1 antibody ( Santa Cruz Biotechnology , Dallas , TX , 1:1000 dilution ) , a rabbit anti-HSP27 antibody ( Santa Cruz Biotechnology , 1:200 dilution ) , a rabbit anti-p-HSP27 antibody ( Santa Cruz Biotechnology , 1:200 dilution ) , an anti-β-Actin HRP antibody ( Santa Cruz Biotechnology , 1:2000 dilution ) , a rabbit anti-MK2 antibody ( Cell Signaling Technology , Beverly , MA; 1:1000 dilution ) , a mouse anti-p62 antibody ( Abnova , Walnut , CA; 1:2000 dilution ) a rabbit anti-LC3 antibody ( Novus Biologicals , Littleton , CO; 1:1000 dilution ) , a rabbit anti-TOM20 antibody ( Santa Cruz Biotechnology; 1:1000 dilution ) , a rabbit anti-PDI antibody ( Cell Signaling Techology; 1:1000 dilution ) , and a mouse anti-GAPDH antibody ( Chemicon International , Temecula , CA; 1:000 dilution ) .", "Lipofectamine 2000 ( Life Technologies , Grand Island , NY ) was used for cell transfection according to the manufacturer's protocol .", "For the GFP-LC3 assay , GFP-LC3 and Beclin 1 ( or Beclin 1 S90 mutants ) were co-transfected at a molecular ratio of 1:2 .", "For western blot analyses in beclin 1 shRNA U2OS cells , shRNA non-targetable Beclin 1 ( or Beclin 1 S90 mutants ) and MK2 mutants were co-transfected at a molecular ratio of 1:1 .", "Cells were lysed in lysis buffer ( 100 mM Tris pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 ) with protease inhibitors ( Roche Life Sciences , Indianapolis , IN ) and Pierce phosphatase inhibitors ( Thermo Fisher Scientific , Rockford , IL ) for 30 min .", "Cell lysates were separated on 4–15% TGX gels ( Bio-Rad Laboratories , Hercules , CA ) , transferred to PVDF membranes ( Bio-Rad Laboratories ) , and probed with the indicated antibodies .", "For immunoblotting levels of proteins in subcellular fractions , cytsolic , mitochondrial , and microsomal fractions were separated with a Qiagen Qproteome Mitochondrial Isolation Kit ( Qiagen , Valencia , CA ) , and resuspended in an equal volume of storage buffer .", "HeLa cells were plated on 6 cm dishes , and transfected the following day with pBICEP-CMV2 , pBICEP-CMV2-Beclin 1 , or pBICEP-CMV2-Beclin 1 S90A using lipofectamine 2000 .", "On the second day after transfection , cells were grown in phosphate-free medium ( phosphate free DMEM + 10% FBS ) for 3 hr .", "The cells were then washed with phosphate-free EBSS three times and were grown in phosphate-free EBSS or phosphate-free normal medium supplemented with 40 μCi 33P-orthophosphate for 2 hr .", "The cells were washed three times with PBS , and lysed with lysis buffer ( 100 mM Tris pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 with protease and phosphatase inhibitors ) for 30 min .", "Lysates were centrifuged for 10 min at 13 , 000 rpm at 4°C .", "The supernatants were collected and adjusted to equal protein concentrations using the Bradford assay .", "9 μl α-Flag ( M2 ) agarose ( Sigma-Aldrich ) was added to each sample and samples were rotated at 4°C overnight .", "The agarose was washed three times with PBS .", "30 μl SDS loading buffer was added to each sample and the samples were boiled at 95°C for 5 min . 10 μl of each sample were loaded on 4–15% TGX gels ( Biorad Laboratories ) .", "Peptides based on amino acid sequences of human Beclin 1 83–97 were synthesized ( JPT Peptide Technologies , Berlin , Germany ) and used as substrates for an in vitro protein kinase screen .", "The peptides were dissolved in 50 mM HEPES , pH 7 . 5 at a concentration of 200 μM .", "A radiometric Streptavidin-FlashPlate-based protein kinase assay was performed to measure the kinase activity of 190 serine/threonine kinases ( ProQinase , Freiburg , Germany ) .", "For mutational analysis , peptides ( wild-type and S90A ) based on amino acid sequences of human Beclin 1 83–103 were used .", "Immunoprecipitation of endogenous Beclin 1 was performed using a polyclonal goat anti-Beclin 1 antibody ( Santa Cruz Biotechnology; 1:50 dilution ) and immunoprecipitation of Flag-tagged Beclin 1 was performed using a monoclonal anti-Flag M2 antibody pre-conjugated to agarose ( Sigma–Aldrich; 1:20 dilution ) .", "For co-immunoprecipitation of Beclin 1 and BCL2 , immunoprecipitation was performed using a polyclonal rabbit anti-Beclin 1 antibody ( Santa Cruz Biotechnology; 1:50 dilution ) and BCL2 was probed with a monoclonal HRP antibody ( Santa Cruz Biotechnology; 1:200 dilution ) .", "For MK2/3 in vitro kinase assays , HEK293T cells were transfected with pBICEP-CMV2-Beclin 1 or pBICEP-CMV2-Beclin 1 S90A using lipofectamine 2000 ( Invitrogen; Grand Island , NY ) ; 6 μg DNA was transfected into each 10 cm dish .", "After 24 hr , each dish of cells was lysed with 500 μl lysis buffer ( 100 mM Tris pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 with protease inhibitors ) for 30 min at 4°C .", "Flag-Beclin 1 was immunoprecipitated using 75 μl anti-Flag ( M2 ) agarose ( Sigma-Aldrich ) for 3 hr at 4°C .", "The beads were washed twice with lysis buffer and another two times with phosphatase buffer .", "The samples were then treated with λ-protein phosphatase ( New England Biolabs , Ipswich , MA ) according to the manufacturer's protocol .", "For 60 μl Flag-agarose , 4 μl λ-protein phosphatase was used .", "The agarose was washed twice with lysis buffer with phosphatase inhibitors , and another two times with kinase reaction buffer ( 60 mM HEPES , pH 7 . 6 , 3 mM MgCl2 , 3 mM MnCl2 , 3 mM Na3VO4 , 1 . 2 mM DTT ) .", "For each in vitro kinase reaction , 15 μl Flag-agarose was mixed with 15 μl kinase buffer ( with 1 μM ATP ) .", "The kinases were added to a final concentration of 0 . 16 μM .", "The reactions were incubated at 30°C for 30 min . 30 μl SDS loading buffer was added to each reaction and the samples were incubated at 95°C for 2 min .", "After brief centrifugation , the supernatants were loaded onto 4–15% TGX gels ( Bio-Rad Laboratories ) , transferred to PVDF membranes and incubated with the indicated antibodies .", "To assess whether BCL2 blocks MK2-mediated phosphorylation of Beclin 1 in vitro , 5 μg GST ( negative control ) or various amounts of GST-BCL2 recombinant protein were added to each reaction tube .", "To measure Beclin 1-associated VPS34 in vitro lipid kinase activity , Beclin 1-VPS34 complexes were immunoprecipitated from U2OS cells with anti-Flag and used as substrates for a VPS34 in vitro lipid kinase assay as described ( Wang et al . , 2012; Wei et al . , 2013 ) .", "The thin layer chromatography spots and western blot band intensities were measured by densitometry ( Visionworks LS image acquisition and analysis software , UVP LLC ) .", "A 60-day slow-release 1 . 7 mg estrogen pellet ( Innovative Research of America , Sarasota , Florida ) was injected in the neck region of 7-week old female nu/nu mice .", "After 1 week , 5 × 106 MCF7 cells retrovirally transduced with Beclin 1 ( WT or S90 mutants ) were injected into the upper right mammary fat pad of each mouse .", "Tumor width and length were measured twice a week for a total experimental duration of 8 weeks .", "Each group contained 11 mice .", "All animal experiments were performed in accordance with institutional guidelines and approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committee .", "Mouse tumor xenografts were fixed in 4% PFA , embedded in paraffin , and sectioned ( ∼5 microns in thickness ) .", "Immunohistochemical staining of paraffin-embedded tumor tissues was performed using anti-p62 ( PROGEN Biotechnik , Heidelberg , Germany; 1:2000 dilution ) or anti-Ki67 ( Abcam , Cambridge , MA; 1:200 dilution ) primary antibodies and the ABC Elite immunoperoxidase kit ( Vector Laboratories , Burlingame , CA ) according to the manufacturer's instructions .", "TUNEL staining was performed according to the manufacturer's instructions , using Sigma FAST 3 , 3′-diaminobenzidine ( DAB ) tablets as the peroxidase substrate .", "Reciprocal intensity of p62 was quantified using the ImageJ 1 . 47v software ( http://imagej . nih . gov/ij ) .", "8-week-old Bcl2AAA knock-in and littermate control C57/BL6 mice ( He et al . , 2012 ) were starved for 48 hr and thigh muscles were snap-frozen in liquid nitrogen .", "The thigh muscles ( vastus lateralis ) were homogenized and protein was extracted in lysis buffer ( 100 mM Tris , pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 ) with protease ( Roche Life Sciences ) and Pierce phosphatase inhibitors cocktail ( Thermo Fisher Scientific ) prior to western blot analyses with indicated antibodies ." ] ]

[ "Autophagy is a fundamental adaptive response to amino acid starvation orchestrated by conserved gene products , the autophagy ( ATG ) proteins .", "However , the cellular cues that activate the function of ATG proteins during amino acid starvation are incompletely understood .", "Here we show that two related stress-responsive kinases , members of the p38 mitogen-activated protein kinase ( MAPK ) signaling pathway MAPKAPK2 ( MK2 ) and MAPKAPK3 ( MK3 ) , positively regulate starvation-induced autophagy by phosphorylating an essential ATG protein , Beclin 1 , at serine 90 , and that this phosphorylation site is essential for the tumor suppressor function of Beclin 1 .", "Moreover , MK2/MK3-dependent Beclin 1 phosphorylation ( and starvation-induced autophagy ) is blocked in vitro and in vivo by BCL2 , a negative regulator of Beclin 1 .", "Together , these findings reveal MK2/MK3 as crucial stress-responsive kinases that promote autophagy through Beclin 1 S90 phosphorylation , and identify the blockade of MK2/3-dependent Beclin 1 S90 phosphorylation as a mechanism by which BCL2 inhibits the autophagy function of Beclin 1 ." ]

[ "Cells keep themselves healthy by breaking down unneeded or damaged internal structures via a process called autophagy .", "This process also helps a cell to survive if it is starved of nutrients .", "For example , if a cell does not receive enough amino acids , it cannot make new proteins .", "Autophagy can break down existing non-essential proteins so that their amino acids can be re-used to build other proteins that the cell needs to survive .", "Autophagy is performed by a set of proteins that is found in many different species , ranging from yeast to humans and plants .", "How these proteins are activated when a cell is starved of amino acids is not fully understood .", "However , evidence suggests that activating one of these proteins , called Beclin 1 , by adding phosphate groups to it controls the extent to which autophagy occurs .", "It is also known from previous work that less autophagy occurs when Beclin 1 binds to another protein called BCL2 .", "Wei , An et al . identified two enzymes that attach a phosphate group to a specific site on Beclin 1 to activate it , and revealed that autophagy is defective in cells that lack these enzymes .", "Furthermore , Wei , An et al . found the BCL2 protein prevents autophagy by binding to Beclin 1 in such a way that stops these two enzymes from activating Beclin 1 .", "Beclin 1 is also known to prevent the growth of malignant tumors .", "Wei , An et al . found that to do so , Beclin 1 must have a phosphate group added to the same site that activates the protein during autophagy .", "This suggests that drugs that enhance the addition of this phosphate group to Beclin 1 could help activate autophagy and have anti-cancer effects ." ]

[ "Introduction", "Results", "Materials and methods" ]

[ "genetics and genomics" ]

[ [ "Hybridization between closely related species is remarkably common ( Mallet , 2005 ) .", "Many hybridizing populations and species remain genetically and ecologically distinct despite bouts of past admixture ( e . g . , Scascitelli et al . , 2010; Vonholdt et al . , 2010 ) .", "This has led to a surge of interest in identifying which and how many loci are important in maintaining species barriers .", "Recent work has focused on identifying so-called ‘genomic islands’ of high divergence between closely related species ( e . g . , Turner et al . , 2005; Nadeau et al . , 2012 ) .", "This approach assumes that the most diverged regions between species are most likely to be under divergent selection between species or important in reproductive isolation .", "However , divergence-based measures need to be interpreted with caution because they are susceptible to artifacts as a result of linked selection events ( including background selection and hitchhiking ) such that outlier regions might reflect low within-population polymorphism rather than unusually high divergence ( discussed in Charlesworth , 1998; Noor and Bennett , 2009; Renaut et al . , 2013 ) , and there are many possible causes of elevated divergence that are not linked to isolation between species .", "Investigating genome-wide patterns in naturally occurring or laboratory-generated hybrid populations is another approach to characterize the genetic architecture of reproductive isolation ( Payseur , 2010 ) .", "Hybridization leads to recombination between parental genomes that can uncover genetic incompatibilities between interacting genes .", "When genomes diverge in allopatry , substitutions that accumulate along a lineage can lead to reduced fitness when hybridization decouples them from the genomic background on which they arose .", "The best understood of these epistatic interactions , called ‘Bateson-Dobzhansky-Muller’ ( BDM ) incompatibilities ( Coyne and Orr , 2004 ) , can occur as a result of neutral substitution or adaptive evolution , and are thought to be common based on theoretical ( Orr , 1995; Turelli et al . , 2001 ) and empirical studies ( Presgraves , 2003; Presgraves et al . , 2003; Payseur and Hoekstra , 2005; Brideau et al . , 2006; Sweigart et al . , 2006 ) .", "One defining feature of BDM incompatibilities is that they are predicted to have asymmetric fitness effects in different parental backgrounds , such that only a subset of hybrid genotypes are under selection .", "Though the BDMI model is an important mechanism of selection against hybrids , other evolutionary mechanisms can contribute to hybrid incompatibility .", "For example , co-evolution between genes can result in selection on all hybrid genotype combinations due to the accumulation of multiple substitutions ( Seehausen et al . , 2014 ) .", "Similarly , natural or sexual selection against hybrid phenotypes can be considered a form of hybrid incompatibility; in this case the genotypes under selection will depend on their phenotypic effects .", "How common are hybrid incompatibilities and what is their genomic distribution ?", "Most studies to date have addressed this question by mapping hybrid incompatibilities that contribute to inviability or sterility ( Orr , 1989; Orr and Coyne , 1989; Barbash et al . , 2003; Presgraves , 2003; Presgraves et al . , 2003 ) , in part because these incompatibilities affect hybrids even in a lab environment .", "Initial genome-wide studies in Drosophila and other organisms suggest that the number of incompatibilities contributing to hybrid viability and sterility can range from a handful to hundreds accumulating between deeply diverged species ( Harushima et al . , 2001; Presgraves , 2003; Moyle and Graham , 2006; Masly and Presgraves , 2007; Ross et al . , 2011 ) ; recent work has also suggested that substantial numbers of incompatibilities segregate within species ( Cutter , 2012; Corbett-Detig et al . , 2013 ) .", "However , because research has focused primarily on postzygotic isolation , little is known about the total number of loci contributing to reproductive isolation between species .", "For example , research shows that selection against hybrid genotypes can be strong even in the absence of postzygotic isolation ( Fang et al . , 2012 ) .", "Thus , the focus on hybrid sterility and inviability is likely to substantially underestimate the true number of genetic incompatibilities distinguishing species .", "If negative epistatic interactions are important in maintaining reproductive isolation , specific patterns of genetic variation are predicted in hybrid genomes .", "In particular , selection against hybrid individuals that harbor unfavorable allele combinations in their genomes will lead to under-representation of these allelic combinations in a hybrid population .", "Thus , selection can generate non-random associations , or linkage disequilibrium ( LD ) , among unlinked loci in hybrid genomes ( Karlin , 1975; Hastings , 1981 ) .", "Patterns of LD in hybrid populations can therefore be used to identify genomic regions that are important in establishing and maintaining reproductive isolation between species .", "Only a handful of studies have investigated genome-wide patterns of LD in hybrid populations .", "Gardener et al . ( 2000 ) evaluated patterns of LD at 85 widely dispersed markers ( ∼0 . 03 markers/Mb ) in sunflowers and found significant associations among markers known to be related to infertility in hybrids .", "Similarly , Payseur and Hoekstra ( 2005 ) evaluated patterns of LD among 332 unlinked SNPs ( ∼0 . 12 markers/Mb ) in inbred lines of hybrid mice and identified a set of candidate loci with strong conspecific associations .", "More recently , Hohenlohe et al . ( 2012 ) investigated genome-wide patterns of LD at ∼2000 sites ( ∼4 . 5 markers/Mb ) in oceanic and freshwater sticklebacks and found two unlinked regions in strong LD that are highly differentiated between populations .", "These studies suggest hybridization can expose the genome to strong selection that leaves detectable signatures of LD in hybrids .", "In this study , we evaluate genome-wide patterns of LD in replicate hybrid populations of two species of swordtail fish , Xiphophorus birchmanni and X . malinche .", "These species are recently diverged ( 0 . 5% genomic divergence per site; 0 . 4% genomic divergence following polymorphism masking ) and form multiple independent hybrid zones in river systems in the Sierra Madre Oriental of Mexico ( Culumber et al . , 2011 ) .", "X . malinche is found at high elevations while X . birchmanni is common at low elevations; hybrids occur where the ranges of these two species overlap .", "The strength of selection on hybrids between these two species is unknown , but several lines of evidence have suggested that selection may be weak .", "Hybrids are abundant in hybrid zones , often greatly outnumbering parental individuals .", "Hybrids are tolerant of the thermal environments at the elevations in which they are found ( Culumber et al . , 2012 ) .", "Though there is some evidence of BDMIs between the species that cause lethal melanomas , these melanomas typically affect hybrids post-reproduction ( Schartl , 2008 ) and may constitute a weak or even favorable selective force ( Fernandez and Morris , 2008; Fernandez and Bowser , 2010 ) .", "Finally , recent behavioral studies show that once hybrids are formed , hybrid males actually have an advantage due to sexual selection compared to parental individuals ( Figure 1; and see Fisher et al . , 2009; Culumber et al . , in press ) .", "However , the genomes of adult hybrids sampled from hybrid populations have already been subject to multiple generations of selection ( at least 30; Rosenthal et al . , 2003 ) , making it difficult to evaluate the extent of selection on hybrids without genetic information .", "By surveying the genomes of hybrids from natural populations , we are able to identify interacting genomic regions under selection in hybrids , giving a powerful picture of the number of regions involved in reproductive isolation between this recently diverged species pair . 10 . 7554/eLife . 02535 . 003Figure 1 . Hybrids between X . malinche and X . birchmanni .", "( A ) Parental ( X . malinche top , X . birchmanni bottom ) and ( B ) hybrid phenotypes with sample MSG genotype plots for linkage groups 1–3 ( see Figure 1—figure supplement 1 for more examples ) for each population shown in the right panel .", "In MSG plots , solid blue indicates homozygous malinche , solid red indicates homozygous birchmanni and regions of no shading indicate heterozygosity .", "Hybrid individuals from Tlatemaco ( B top ) have malinche-biased ancestry ( solid blue regions ) while hybrids from Calnali ( B bottom ) have birchmanni-biased ancestry ( solid red regions ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 00310 . 7554/eLife . 02535 . 004Figure 1—figure supplement 1 . MSG ancestry plots for parental and hybrid individuals . Representative parental individuals of X . birchmanni ( A ) and X . malinche ( B ) in linkage groups 1–3 , shows that parental individuals are called as homozygous throughout the chromosome .", "Tick markers indicating calls to the other parent are the result of undetected polymorphism or error .", "More representatives from the parental panel are shown in ( C ) .", "Hybrids from Tlatemaco ( left ) and Calnali ( right ) are shown in panel ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 004 To evaluate genome-wide patterns of LD in hybrid populations , we further develop the multiplexed shotgun genotyping ( MSG ) protocol of Andolfatto et al . ( 2011 ) .", "Originally developed for QTL mapping in controlled genetic crosses , we describe modifications that make the technique applicable to population genetic samples from hybrid populations .", "With this approach , we assign ancestry to nearly 500 , 000 ancestry informative markers throughout the genome , allowing us to evaluate genome wide patterns of LD at unprecedented resolution ( ∼820 markers/Mb ) .", "The joint analysis of two independent hybrid zones allows us to distinguish the effects of selection against genetic incompatibilities from confounding effects due to population history ( Gardner et al . , 2000 ) .", "We further evaluate loci that are in significant LD to investigate the mechanisms of selection on these pairs .", "Our results support the conclusion that a large number of loci contribute to reproductive isolation between these two species , that these loci have higher divergence than the genomic background , and that selection against genetic incompatibilities maintains associations between loci derived from the same parental genome ." ], [ "We used a modified version of the MSG analysis pipeline , optimized for genotyping in natural hybrids ( ‘Materials and methods’ , Figure 1—figure supplement", "1 ) to genotype individual fish collected from two independently formed hybrid zones , Calnali and Tlatemaco ( Figure 1 , Culumber et al . , 2011 ) .", "We genotyped 143 hybrid individuals from Calnali , 170 hybrids from Tlatemaco , and 60 parents of each species , determining ancestry at 469 , 400 markers distinguishing X . birchmanni and X . malinche at a median density of 1 marker per 234 bp ( ‘Materials and methods’ ) .", "On average , hybrids from the Calnali hybrid zone derived only 20% of their genome from X . malinche , while hybrids from the Tlatemaco hybrid zone had 72% of their genomes originating from X . malinche .", "Most hybrid individuals were close to the average hybrid index in each group ( Tlatemaco: 95% of individuals 66–80% malinche ancestry; Calnali: 95% of individuals 14–35% malinche ancestry ) .", "We determined the time since hybridization based on the decay in linkage disequilibrium ( ‘Materials and methods’ ) , assuming an average genome-wide recombination rate of 1 cM/378 kb ( Walter et al . , 2004 ) .", "Estimates of hybrid zone age were similar for the two hybrid zones ( Tlatemaco 56 generations , CI: 55–57 , Calnali 35 generations CI: 34 . 5–35 . 6 ) .", "Interestingly , these estimates are remarkably consistent with available historical estimates , which suggest that hybridization began within the last ∼40 generations due to disruption of chemical cues by pollution ( Fisher et al . , 2006 ) .", "Ancestry calls at 469 , 400 markers genome-wide were thinned to 12 , 229 markers that are sufficient to describe all changes in ancestry ( ±10% ) across individuals in both populations ( ‘Materials and methods’ ) .", "Using this thinned data set , we analyzed patterns of LD among all pairs of sites .", "The physical distance over which R2 decayed to <0 . 5 was approximately 300 kb in both populations ( Figure 2—figure supplement 1 ) .", "Average genome-wide R2 between physically unlinked loci was 0 . 003 in Tlatemaco and 0 . 006 in Calnali ( Figure 2A ) , and did not significantly differ from null expectations ( 1/2n , where n is the sample size ) .", "A p-value for the R2 value for each pair of effectively unlinked sites was estimated using a Bayesian Ordered Logistic Regression t-test ( Figure 2B , ‘Materials and methods’ ) . 10 . 7554/eLife . 02535 . 005Figure 2 . R2 distribution and p-value distributions of the sites analyzed in this study .", "( A ) Genome-wide distribution of randomly sampled R2 values for markers on separate chromosomes ( see Figure 2—figure supplement 1 for R2 decay by distance; Figure 2—figure supplement 2 for a genome-wide plot ) .", "Blue indicates the distribution in Tlatemaco while yellow indicates the distribution in Calnali .", "Regions of overlapping density are indicated in green .", "The average genome-wide R2 in Tlatemaco is 0 . 003 and in Calnali is 0 . 006 .", "( B ) qq-plots of −log10 ( p-value ) for a randomly selected subset of unlinked sites analyzed in this study in each population; expected p-values are drawn from p-values of the permuted data . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 00510 . 7554/eLife . 02535 . 006Figure 2—figure supplement 1 . Decay in linkage disequilibrium . Average decay of R2 over distance in Tlatemaco ( black ) and Calnali ( red ) in 100 kb windows ( plot generated from 1000 randomly selected sites ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 00610 . 7554/eLife . 02535 . 007Figure 2—figure supplement 2 . Genome-wide linkage disequilibrium plot for Tlatemaco . This plot demonstrates that there are few regions in the X . birchmanni-malinche genomes that are inverted relative to the X . maculatus genome .", "Red indicates regions of R2 near 1 , while green indicates low R2 .", "Regions outlined in dark blue appear to be inverted based on the analysis in both Tlatemaco and Calnali , regions highlighted in gray appear to be inverted only in the Tlatemaco analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 007 The expected false discovery rates ( FDRs ) associated with given p-value thresholds applied to both populations were determined by simulation ( ‘Materials and methods’ , Figure 3 , Figure 3—figure supplement 1 ) .", "Using data from two hybrid populations to examine LD circumvents two problems: ( 1 ) within a single population cases of LD between unlinked sites are unlikely to be strong enough to survive false discovery corrections and ( 2 ) even interactions that are highly significant could be caused by population demographic history .", "The joint distribution of p-values in the two populations implies several hundreds of unlinked locus pairs are in significant LD in both populations ( Figure 3—figure supplement 1 ) ; 327 pairs of regions were significant at FDR = 5% , and 150 pairs were significant at a more stringent FDR = 2% ( Figure 3 ) .", "The genomic distribution of loci in significant LD at FDR = 5% is shown in Figure 4 .", "For simplicity , we focus on statistics for the less stringent data set ( Supplementary file 1A ) , but nearly identical results were found for the more stringent data set ( Tables 1 and 2 ) . 10 . 7554/eLife . 02535 . 008Figure 3 . Number of unlinked pairs in significant linkage disequilibrium and expected false discovery rates . Plot showing number of pairs of sites in significant LD in both populations in the stringent and relaxed data sets ( light blue ) .", "The expected number of false positives in each data set is shown in dark blue , and was determined by simulation ( see main text; Figure 3—figure supplement 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 00810 . 7554/eLife . 02535 . 009Figure 3—figure supplement 1 . False discovery rate ( FDR ) at different p-value thresholds .", "( A ) The number of pairs of loci in LD in both populations in black ( y-axis left ) vs the expected false discovery rate in red ( y-axis right ) at different p-value thresholds .", "( B ) Estimated number of true positives in the data set at different p-value thresholds for all pairs ( black ) , conspecific pairs ( blue ) , and heterospecific pairs ( red ) .", "Expected false discovery rate was determined by 1000 simulations randomly permuting markers from the real data in both populations . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 00910 . 7554/eLife . 02535 . 010Figure 4 . Distribution of sites in significant linkage disequilibrium throughout the Xiphophorus genome . Schematic of regions in significant LD in both populations at FDR 5% .", "Regions in blue indicate regions that are positively associated in both populations ( conspecific in association ) , regions in black indicate associations with different signs of R in the two populations , while regions in red indicate those that are negatively associated in both populations ( heterospecific in association ) .", "Chromosome lengths and position of LD regions are relative to the length of the assembled sequence for that linkage group; most identified LD regions are <50 kb ( Figure 4—figure supplement 1; Figure 4—figure supplement 2; Figure 4—figure supplement 3 ) .", "Analysis of local LD excludes mis-assemblies as the cause of these patterns ( Figure 4—figure supplement 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01010 . 7554/eLife . 02535 . 011Figure 4—figure supplement 1 . Log10 distribution of LD region length in base pairs . The dotted line indicates the median length of regions in cross-chromosomal LD ( 45 kb ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01110 . 7554/eLife . 02535 . 012Figure 4—figure supplement 2 . Plot of the number of recombination breakpoints detected along linkage group 2 . Number of breakpoints in Tlatemaco are indicated in blue and Calnali in red .", "( A ) Breakpoints counted in 1 Mb windows and ( B ) 100 kb windows .", "The high density of recombination events allows for the identification of narrow regions in linkage disequilibrium . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01210 . 7554/eLife . 02535 . 013Figure 4—figure supplement 3 . Example of the use of data from two populations to narrow candidate regions in cross-chromosomal LD . p-values for linkage disequilibrium between a marker on linkage group 2 and an interval on linkage group 16 ( blue: Calnali , black: Tlatemaco ) .", "Overlapping significant intervals from the two populations allows us to narrow candidate regions . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01310 . 7554/eLife . 02535 . 014Figure 4—figure supplement 4 . Regions in cross-chromosomal LD are also in LD with their neighbors . Decay in R2 of markers at the edge of LD blocks in both populations ( black lines ) compared to 95% confidence intervals of 1000 markers randomly selected from the genomic background ( blue ) in Tlatemaco ( A ) and Calnali ( B ) .", "Fewer than 5% of markers fall outside of the 95% confidence intervals in each 100 kb window in both populations .", "Average R2 and 95% CI for regions in significant cross-chromosomal LD are shown in purple .", "LD blocks without neighboring markers within 300 kb of the focal marker are excluded from this figure . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01410 . 7554/eLife . 02535 . 015Table 1 . Comparison of results for sites in significant LD at two different p-value thresholdsDOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 015Data setNumber of pairsProportion of pairs with conspecific associationsStringent ( FDR<2% ) 150Tlatemaco: 72% ( p<0 . 001 ) Calnali: 94% ( p<0 . 001 ) Relaxed ( FDR 5% ) 327Tlatemaco: 67% ( p<0 . 001 ) Calnali: 94% ( p<0 . 001 ) p-values were determined resampling the genomic background , see main text for details . 10 . 7554/eLife . 02535 . 016Table 2 . Sites in significant LD are more divergent per site than the genomic backgroundDOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 016Mutation typeMedian divergence genomic backgroundMedian divergence stringentMedian divergence relaxedAll sites0 . 00400 . 0045 ( p<0 . 001 ) 0 . 0044 ( p<0 . 001 ) Nonsynonymous0 . 000400 . 00065 ( p=0 . 001 ) 0 . 00040 ( p=0 . 6 ) Synonymous0 . 00400 . 0048 ( p<0 . 001 ) 0 . 0045 ( p=0 . 004 ) Results shown here are limited to regions that had conspecific associations in both populations ( stringent dataset: 200 regions , relaxed dataset: 414 regions ) .", "p-values were determined by resampling the genomic background .", "Average R2 for unlinked regions in LD is 0 . 08 in Tlatemaco and 0 . 12 in Calnali , both significantly higher than the genomic background ( p<0 . 001 , by bootstrapping ) .", "LD regions were non-randomly distributed in the genome ( χ2 = 87 , df = 23 , p<3e−9 ) .", "Contrary to findings of a large-X effect in other taxa ( ‘Discussion’ ) , we do not find a significant excess of LD pairs involving the X chromosome ( p=0 . 11 , Binomial test ) .", "Focusing on the overlap of significant regions in both populations allowed us to narrow candidate regions ( Figure 4—figure supplement 3 ) .", "Regions in significant LD in both populations have a median size of 45 kb ( Figure 4—figure supplement 1; Figure 4—figure supplement 2 ) ; 67% of regions contain 10 genes or fewer , and 13 pairs of regions are at single gene resolution ( Supplementary file 1B ) .", "Approximately , 10% of the genomic regions identified are very large and contain hundreds of genes ( >5 Mb , Supplementary file 1A ) , potentially as a result of reduced recombination or selection .", "Unlinked regions in significant LD were more strongly linked to neighboring loci ( Figure 4—figure supplement 4 ) , ruling out the possibility that mis-assemblies underlie the patterns we observe .", "Models of selection against hybrid genotypes ( Figure 5 , Figure 5—figure supplement", "1 ) predict that certain genotype combinations will be less common ( Karlin , 1975 ) .", "In particular , selection against hybrid incompatibilities is expected to generate positive R , or conspecific associations between loci .", "Among loci in significant LD , we found an excess of conspecific associations in both hybrid populations ( 94% of pairs in Calnali and 67% of pairs in Tlatemaco , p<0 . 001 for both populations relative to the genomic background by bootstrapping ) . 10 . 7554/eLife . 02535 . 017Figure 5 . Loci in significant conspecific linkage disequilibrium show patterns consistent with selection against hybrid incompatibilities .", "( A ) Posterior distributions of the selection coefficient and hybrid population size from ABC simulations for Tlatemaco and ( B ) Calnali .", "The range of the x-axis indicates the range of the prior distribution , maximum a posteriori estimates ( MAP ) and 95% CI are indicated in the inset .", "( C ) Departures from expectations under random mating in the actual data ( top—blue points indicate LD pairs , black points indicate random pairs from the genomic background ) and samples generated by posterior predictive simulations ( bottom , see ‘Materials and methods’ ) .", "The mean is indicated by a dark blue point; in the real data ( top ) smears denote the distribution of means for 1000 simulations while in the simulated data ( bottom ) smears indicate results of each simulation .", "Genotypes with the same predicted deviations on average under the BDM model have been collapsed ( Figure 5—figure supplement 1 , but see Figure 5—figure supplement 3 ) and are abbreviated in the format locus1_locus2 .", "These simulations show that the observed deviations are expected under the BDM model .", "The posterior distributions for s and hybrid population size are correlated at low population sizes ( Figure 5—figure supplement 2 ) .", "Deviations in Calnali also follow expectations under the BDM model ( Figure 5—figure supplement 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01710 . 7554/eLife . 02535 . 018Figure 5—figure supplement 1 . Different fitness matrices associated with selection against hybrid incompatibilities . In a classic BDMI model ( A and B ) , hybrid genotypes potentially under selection ( indicated in red ) are determined by the locus and order in which mutations occur .", "In a model of co-evolution between loci or extrinsic selection against hybrid phenotypes ( C ) , more genotype combinations are potentially under selection .", "( A ) Interaction between a mutation in locus 1 malinche and locus 2 birchmanni ( two-lineage model ) or first substitution occurring in locus 1 birchmanni or locus 2 malinche ( one-lineage model ) .", "( B ) Interaction between a mutation in locus 1 birchmanni and locus 2 malinche ( two-lineage model ) or first substitution occurring in locus 1 malinche or locus 2 birchmanni ( one-lineage model ) .", "Format of genotypes is as follows: haplotype1_locus1-haplotype1_locus2/haplotype2_locus1-haplotype2_locus2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01810 . 7554/eLife . 02535 . 019Figure 5—figure supplement 2 . Joint posterior distribution of hybrid population size and selection coefficient . Posterior distributions of hybrid population size and s indicate a relationship between these parameters in both populations ( A-simulations of Tlatemaco , B-simulations of Calnali ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 01910 . 7554/eLife . 02535 . 020Figure 5—figure supplement 3 . Deviations in genotype combinations compared to expected values under a two-locus selection model in both populations . Average deviation of genotype combinations ( dark blue point ) from expectations under random mating at conspecific LD pairs ( top ) compared to posterior predictive simulations ( bottom , see ‘Materials and methods’ ) in Tlatemaco ( A ) and Calnali ( B ) .", "The light blue smears indicate the distribution of means for 1000 bootstrap samples in the real data and result of individual simulations in the simulated data .", "Labels on the x-axis indicate the genotype in the format locus1_locus2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 020 We focus all subsequent analyses on the subset of significant LD pairs ( FDR = 0 . 05 ) with conspecific associations in both populations ( 207 locus pairs ) because these sites will be enriched for hybrid incompatibilities , but similar results are observed for the whole data set ( Supplementary file 1C ) .", "To estimate parameters under a classic BDM incompatibility model ( Figure 5—figure supplement 1 ) , we use an approximate Bayesian approach to simulate selection on two-locus interactions ( ‘Materials and methods’ ) .", "Though it is likely that other types of hybrid incompatibilities were identified in our analysis , estimates are similar using other models ( ‘Materials and methods’; Figure 5—figure supplement 1C ) .", "These simulations demonstrate that our results are well described by a model of selection against hybrid incompatibilities ( Figure 5 , Figure 5—figure supplement", "1 ) and that sites are on average under weak to moderate selection ( Figure 5 , maximum a posteriori estimates for Tlatemaco ŝ = 0 . 027 , and Calnali ŝ = 0 . 074 ) .", "Strikingly , the median divergence between X . birchmanni and X . malinche at loci in significant conspecific LD ( FDR = 0 . 05 ) was much higher than the genomic background ( p<0 . 001 by bootstrapping , Figure 6A ) .", "Elevated divergence could be caused by differences in selection on individual loci , differences in mutation rate , or reduced susceptibility of these genomic regions to homogenization by ongoing gene flow .", "To distinguish among these causes , we examined rates of synonymous substitution ( dS ) .", "Elevated divergence compared to the genomic background is also observed at synonymous sites ( p<0 . 01 by bootstrapping , Table 2 ) .", "We also examined the same regions in two swordtail species in an independent Xiphophorus lineage , X . hellerii and X . clemenciae and found that the level of genomic divergence in this species pair was not significantly different from the genomic background ( Figure 6B ) .", "Together , these results imply that variation in selective constraint or mutation rate do not explain elevated divergence between X . birchmanni and X . malinche at loci in conspecific LD . 10 . 7554/eLife . 02535 . 021Figure 6 . Divergence of LD pairs compared to the genomic background in two species comparisons .", "( A ) Regions identified in X . birchmanni and X . malinche and ( B ) orthologous regions in X . hellerii and X . clemenciae .", "The blue point shows the average divergence for genomic regions within significant LD pairs , and whiskers denote a 95% confidence interval estimated by resampling genomic regions with replacement .", "The histogram shows the distribution of the average divergence for 1000 null data sets generated by resampling the genomic background without replacement . DOI: http://dx . doi . org/10 . 7554/eLife . 02535 . 021 We performed gene ontology ( GO ) analysis on unlinked regions in significant conspecific LD and , surprisingly , found no significantly enriched GO categories ( ‘Materials and methods’ ) .", "This result holds when restricting the analysis to regions resolved to only a few genes ( ≤10 genes , 242 regions ) .", "This suggests that regions in significant LD contain genes with a broad range of functional roles .", "In this study , we assign ancestry to nearly 500 , 000 markers genome-wide in samples from two hybrid fish populations , providing a portrait of the genetic architecture of hybrid incompatibilities between two closely related species at unprecedented resolution .", "We discover significant LD between hundreds of pairs of unlinked genomic regions and show that a model of selection against hybrid incompatibilities describes the observed conspecific LD patterns .", "This implies that many negative epistatic interactions segregate in these hybrid fish populations despite the fact that intrinsic post-zygotic isolation is thought to be weak ( Rosenthal et al . , 2003 ) .", "Our analysis focuses on a high confidence set of unlinked loci in significant LD .", "Despite these findings , the true number of loci involved in incompatibilities may involve hundreds more interactions for several reasons .", "First , we conservatively exclude interactions within chromosomes due to the lack of detailed genetic map information .", "Second , we have only moderate power to detect incompatibilities ( e . g . , ∼30% using parameter estimates for Calnali ) .", "Third , relaxing the p-value threshold suggests the true number of pairs in significant LD could be much larger ( Figure 3—figure supplement 1 ) .", "Finally , our experimental design incorporates two populations with opposite trends in genome-wide ancestry and independent histories of hybridization .", "This conservative approach allows us to exclude effects of population history , but false negatives in our joint analysis may result from effects that are population-specific ( for example , different effects of extrinsic selection in the two populations ) .", "Investigating the role of such a large number of hybrid incompatibilities in determining the structure of hybrid genomes is an exciting area for future empirical and theoretical research .", "Using an approximate Bayesian approach , we estimate that the average selection coefficient on negative epistatic interactions is in the vicinity of s = 0 . 03 ( Tlatemaco ) to s = 0 . 07 ( Calnali ) ; scaling these by estimates of the effective population size ( 2 Ns∼10 and ∼45 , respectively ) suggests that selection is strong enough to be deterministic but of moderate strength relative to drift .", "Given the large number of putative incompatibilities , even weak selection would introduce substantial genetic load in hybrids .", "Depending on dominance effects , this could explain why hybrid populations are skewed in genome-wide ancestry .", "However , we may overestimate the potential for genetic load if epistatic interactions are complex .", "Though this study focuses on pairwise comparisons because statistically evaluating high-order interactions in a data set of this size is intractable , more complex interactions are predicted to be likely ( Orr , 1995 ) .", "The fact that many locus pairs ( ∼40% of regions localized to 1 Mb or less ) identified in this study interact with multiple partners provides indirect support for this prediction .", "Most work on hybrid incompatibilities has focused on characterizing specific incompatibilities at candidate genes , such as those associated with mapped QTL distinguishing species ( Ting et al . , 1998; Presgraves et al . , 2003; Lee et al . , 2008; Tang and Presgraves , 2009; Moyle and Nakazato , 2010 ) .", "The idea that negative epistatic interactions may be pervasive in closely related species or populations has come from multiple studies of potential candidate genes ( e . g . , Arabidopsis thaliana: Bomblies et al . , 2007; Smith et al . , 2011; Xiphophorus: Nairn et al . , 1996; Drosophila: Barbash et al . , 2000; Bayes and Malik , 2009; Tang and Presgraves , 2009; Caenorhabditis elegans: Seidel et al . , 2008 ) and several genome-wide studies ( Harushima et al . , 2001; Presgraves , 2003; Payseur and Hoekstra , 2005; Moyle and Graham , 2006; Masly and Presgraves , 2007; Matute et al . , 2010 ) .", "These studies suggest that on the order of 100 incompatibilities explain isolation between closely related species .", "In contrast , though X . malinche and X . birchmanni have a similar divergence time ( ∼2 Ne generations ) to a previously studied Drosophila species pair ( Masly and Presgraves , 2007 ) , we identify approximately fourfold more genetic incompatibilities at FDR = 0 . 05 .", "What accounts for this difference ?", "Previous studies on the genomic distribution of hybrid incompatibilities have focused almost entirely on incompatibilities involved in post-zygotic isolation ( in some studies , exclusively in males; Presgraves , 2003 ) .", "However , such studies can only provide a lower limit on the number of loci involved in reproductive isolation between lineages .", "A recent study in Drosophila simulans and D . sechellia investigated the effects of interspecific competition on the fitness of introgressed lines ( Fang et al . , 2012 ) .", "Though introgressed lines had no detectable differences in fertility or viability , the authors nonetheless detected strong selection on hybrids in competition experiments ( Fang et al . , 2012 ) , implying that the number of loci involved in reproductive isolation has been vastly underestimated in most studies .", "Our results lend support to this point of view .", "The hybrid genomes we analyze in this study have been exposed to over 30 generations of intrinsic and extrinsic selection .", "Given that post-zygotic isolation between X . birchmanni and X . malinche is weak ( Rosenthal et al . , 2003 ) , we propose that extrinsic selection is more likely the cause of the majority of hybrid incompatibilities detected in this study .", "Future research comparing hybrid incompatibilities in lab hybrids to natural hybrids will begin to elucidate how many incompatibilities are targets of extrinsic vs intrinsic selection .", "Many studies of hybrid incompatibilities have focused on organisms with clear species boundaries—those that do not frequently hybridize in nature .", "In species that do frequently hybridize , early research supported the conclusion that these species retain their identity through a few highly differentiated genomic regions , inferred to contain genes responsible for reproductive isolation ( Turner et al . , 2005; Ellegren et al . , 2012 ) .", "More recent studies have suggested that even hybridizing species remain genetically differentiated through much of the genome ( Lawniczak et al . , 2010; Michel et al . , 2010 ) .", "Our findings in X . birchmanni and X . malinche support the latter conclusion and suggest that hybrid incompatibilities may be a common mechanism of restricting gene flow genome-wide even in species with incomplete reproductive isolation .", "Theory predicts that more rapidly evolving genomic regions will be more likely to result in BDM incompatibilities ( Orr , 1995; Orr and Turelli , 2001 ) .", "However , an issue that arises in testing this prediction is that functionally diverged regions associated with hybrid incompatibilities may also resist homogenization due to gene flow , conflating the cause and the effect of elevated divergence ( but see below ) .", "While our study confirms that unlinked genomic regions in LD pairs are significantly more diverged between X . birchmanni and X . malinche compared to the genomic background ( Figure 6 ) , we also find that these regions do not show elevated divergence in an independent comparison of Xiphophorus fish ( Figure 6B ) .", "This supports the hypothesis that these regions are resistant to homogenization due to ongoing gene flow between X . birchmanni and X . malinche .", "This finding is interesting because theoretical work suggests BDM incompatibilities are ineffective barriers to gene flow , especially when migration rates are high ( Gavrilets , 1997; Gompert et al . , 2012 ) , but incompatibilities in which all hybrid genotypes are under selection more effectively limit gene flow ( Gavrilets , 1997 ) .", "Remarkably , we found not a single significantly enriched GO category in well-resolved pairs in significant LD .", "This is in stark contrast to previous studies , such as that of Payseur and Hoekstra ( 2005 ) , who found 17 over-represented GO categories in a data set of ∼180 pairs of loci ( at 2 Mb resolution ) detected in Mus .", "Our study suggests a much more equal representation of functional categories among genes involved in incompatibilities .", "One of the first putative BDM incompatibilities identified at the genetic level involves the Xmrk-2 gene in Xiphophorus hybrids .", "Hybrids between many Xiphophorus species develop lethal melanomas which have been hypothesized to reinforce species boundaries ( Orr and Presgraves , 2000 ) .", "Decoupling of Xmrk-2 from its repressor ( thought to be the gene cdkn2x ) through hybridization triggers melanoma development ( Nairn et al . , 1996 ) .", "Though melanomas can develop in X . malinche–X .", "birchmanni hybrids , we found no evidence of LD between Xmrk-2 and cdkn2x in either population .", "This may support previous hypotheses that melanoma is not under strong selection in hybrids because it affects older individuals ( Schartl , 2008 ) or provides an advantage in mate choice ( Fernandez and Morris , 2008; Fernandez and Bowser , 2010 ) .", "Alternatively , cdkn2x may not in fact be the repressor of Xmrk-2 .", "Theory predicts that the X-chromosome will play a major role in the establishment of reproductive isolation due to Haldane's rule , faster-X evolution or meiotic drive ( Presgraves , 2008 ) .", "Intriguingly , we do not see an excess of interactions involving group 21 , the putative X chromosome ( Schartl et al . , 2013 ) .", "This is in contrast to results in a large number of species that demonstrate that sex chromosomes play a disproportionate role in the evolution of reproductive isolation ( Sperling , 1994; Presgraves , 2002; Payseur et al . , 2004; Turner et al . , 2005; Pryke , 2010 ) , including studies on LD ( Payseur and Hoekstra , 2005 ) .", "However , the X chromosome in Xiphophorus is very young ( Schartl , 2004 ) and sex determination may also be influenced by autosomal factors ( Kallman , 1984 ) .", "Since the non-recombining portion of the Y chromosome is small in Xiphophorus , this will reduce the effects of recessive X-chromosome incompatibilities on male fitness .", "We detect an excess of conspecific associations among significant LD pairs , which we evaluate ( above ) in the context of selection on hybrid incompatibilities .", "However , the proportion of locus pairs in significant LD that are in conspecific association differs dramatically in the two populations .", "6% of locus pairs in Calnali and 33% in Tlatemaco have significant heterospecific associations ( i . e . , significantly negative R ) at FDR = 0 . 05 .", "Heterospecific associations may be the result of beneficial epistatic interactions that result in hybrid vigor .", "For example , hybrid males are better at buffering the locomotor costs of sexual ornamentation ( Johnson , 2013 ) and have an advantage compared to parental males from sexual selection ( Culumber et al . , in press ) , which could in part counteract the negative fitness effects of genetic incompatibilities .", "However , the fact that few loci are heterospecific in association in both populations ( 2% , Figure 3—figure supplement 1B ) suggests that these patterns are not repeatable across populations .", "One possible explanation for this is divergent effects of mate preferences in the two populations .", "Behavioral studies have shown that X . malinche females prefer unfamiliar male phenotypes ( Verzijden et al . , 2012 ) while X . birchmanni females prefer familiar male phenotypes ( Verzijden and Rosenthal , 2011; Verzijden et al . , 2012 ) .", "Given that Tlatemaco hybrids are primarily malinche and Calnali hybrids are primarily birchmanni , divergent effects of male phenotypes on mating preferences could produce the observed patterns .", "We find hundreds of pairs of unlinked regions in significant LD across the genomes of X . birchmanni–X .", "malinche hybrids in two independent hybrid populations .", "These associations are largely well described by a model of selection against hybrid incompatibilities , implying that reproductive isolation in these recently diverged species involves many loci .", "These regions were also more divergent between species than the genomic background , likely as a result of reduced permeability to ongoing gene flow between the species .", "By using samples from two populations with independent histories of hybridization , we are able to exclude population structure and drift as potential causes of these patterns .", "Our results suggest that past research has vastly underestimated the number of regions responsible for reproductive isolation between species by focusing on intrinsic postzygotic reproductive isolation .", "In addition , our results demonstrate that even in species without strong intrinsic post-zygotic isolation , hybrid incompatibilities are pervasive and play a major role in shaping the structure of hybrid genomes ." ], [ "We created ‘pseudogenomes’ of X . malinche and X . birchmanni based on the X . maculatus genome reference sequence .", "As raw materials , we used previously collected Illumina sequence data ( Acc # SRX201248; SRX246515 ) derived from a single wild-caught male for each species ( Cui et al . , 2013; Schumer et al . , 2013 ) and the current genome assembly for X . maculatus ( Schartl et al . , 2013 ) .", "We used a custom python script to trim reads to remove low quality bases ( Phred quality score <20 ) and reads with fewer than 30 nucleotides of contiguous high quality bases and aligned these reads to the X . maculatus reference using STAMPY v1 . 0 . 17 ( Lunter and Goodson , 2011 ) with default parameters except for expected divergence set to 0 . 03 .", "Between 98–99% of reads from both species mapped to the X . maculatus reference .", "Mapped reads were analyzed for variant sites using the samtools/bcftools pipeline ( Li and Durbin , 2009 ) .", "We used the VCF files and a custom python script to create a new version of the X . maculatus reference sequence for each species that incorporated variant sites and masked any sites that had depth <10 reads or were called as heterozygous .", "As an additional step to mask polymorphisms , we prepared multiplexed shotgun genotyping ( MSG ) libraries ( Andolfatto et al . , 2011 ) for 60 parental individuals of each species ( Figure 1—figure supplement 1 ) , generating 78 , 881 , 136 single end 101 nucleotide reads at MseI sites for X . malinche and 80 , 189 , 844 single end 101 nucleotide reads for X . birchmanni .", "We trimmed these reads as described above and mapped them to the X . malinche and X . birchmanni reference pseudogenomes , respectively , using bwa ( Li and Durbin , 2009 ) .", "We analyzed variant sites as described above and excluded all sites that were either polymorphic in the sampled parentals or had fixed differences between the sampled parentals and the reference ( excluding indels ) .", "After masking , 0 . 4% of sites genome-wide were ancestry informative markers ( AIMs ) between X . birchmanni and X . malinche .", "The total number of AIMs in the assembled 24 linkage groups was 2 , 189 , 807 .", "For the same 60 parental individuals of each species , we evaluated MSG output to determine any markers that did not perform well in genotyping the parental individuals ( average probability of matching same-parent <0 . 9 ) .", "We found that 1 . 7% of covered markers performed poorly in X . malinche and 0 . 3% of markers performed poorly in X . birchmanni; we excluded these 10 , 877 markers in downstream analysis , leaving 2 , 178 , 930 AIMs .", "The procedures used in this study were approved by the Institutional Animal Care and Use Committee at Texas A&M University ( Protocols # 2010-111 and 2012-164 ) .", "Individuals were collected from two independent hybrid zones ( Calnali-mid and Tlatemaco , Culumber et al . , 2011 ) in 2009 , and between 2012 and 2013 .", "Individuals were caught in the wild using baited minnow traps , and lightly anesthetized with tricaine methanesulfate .", "Fin clips were stored in 95% ethanol until extraction .", "Population turnover rate is high between years , and sites were sampled only once per year .", "We also performed relatedness analyses to ensure that individuals had not been resampled ( data not shown ) .", "DNA was extracted from fin clips using the Agencourt bead-based purification method ( Beckman Coulter Inc . , Brea , CA ) following manufacturer's instructions with slight modifications .", "Fin clips were incubated in a 55°C shaking incubator ( 100 rpm ) overnight in 94 μl of lysis buffer with 3 . 5 μl 40 mg/ml proteinase K and 2 . 5 DTT , followed by bead binding and purification .", "Genomic DNA was quantified using a Typhoon 9380 ( Amersham Biosciences , Pittsburgh , PA ) and evaluated for purity using a Nanodrop 1000 ( Thermo Scientific , Wilmington , DE ) ; samples were diluted to 10 ng/μl .", "MSG libraries were made as previously described ( Andolfatto et al . , 2011 ) .", "Briefly , 50 ng of DNA was digested with MseI; following digestion custom barcodes were ligated to each sample .", "5 μl of sodium acetate and 50 μl of isopropanol were added to each sample and samples were pooled ( in groups of 48 ) and precipitated overnight at −20°C .", "Following overnight precipitation , samples were extracted and resuspended in TE ( pH 8 . 0 ) and purified through a phenol–chloroform extraction and Agencourt bead purification .", "Pooled samples were run on a 2% agarose gel and fragments between 250 and 500 bp were selected and purified .", "2 ng of each pooled sample was amplified for 14–16 PCR cycles with custom indexed primers allowing us to pool ∼180 samples for sequencing on one Illumina lane .", "Due to multiplexing with other libraries , samples were sequenced on a total of four Illumina HiSeq 2000 lanes with v3 chemistry .", "All raw data are available through the NCBI Sequence Read Archive ( SRA Accession: SRX544941 ) .", "Raw reads were parsed by index and barcode; the number of reads per individual ranged from 0 . 4 to 2 . 8 million reads , with a median of 900 , 000 reads .", "After parsing , 101 bp reads were trimmed to remove low quality bases ( Phred quality score <20 ) and reads with fewer than 30 bp of contiguous high quality bases .", "If an individual had more than 2 million reads , reads in excess of 2 million were excluded to improve the speed of the MSG analysis pipeline .", "The following parameters were specified in the MSG configuration file: recombination rate recRate = 240 , rfac = 3 , X . birchmanni error ( deltapar1 ) = 0 . 05 , X . malinche error ( deltapar2 ) = 0 . 04 .", "See ’Materials and methods’ for details on parameters and parameter determination .", "All individuals were initially analyzed with naive priors ( probability of ancestry for parent 1 = 0 . 33 , parent 2 = 0 . 33 , and heterozygous = 0 . 33 ) with the MSG v0 . 2 pipeline ( https://github . com/JaneliaSciComp/msg ) .", "Based on genome-wide ancestry proportions given these priors , population-specific priors were calculated for Tlatemaco ( homozygous X . malinche = 0 . 49 , heterozygous = 0 . 42 , homozygous X . birchmanni = 0 . 09 ) and Calnali ( homozygous X . malinche = 0 . 04 , heterozygous = 0 . 32 , homozygous X . birchmanni = 0 . 64 ) .", "These estimates were used as new priors for a subsequent run of the MSG pipeline .", "This resulted in genotype information at 1 , 179 , 187 ancestry informative markers ( ∼50% of the total number of ancestry informative markers ) .", "MSG ancestry posterior probability files were thinned to exclude markers that were missing or ambiguous in >15% of individuals leaving 469 , 400 markers ( ∼820 markers/Mb ) .", "Following this initial culling , markers were further thinned using a custom Python script to exclude adjacent markers that did not differ in posterior probability values by ±0 . 1 .", "This resulted in 12 , 269 markers for linkage disequilibrium analysis; the median distance between thinned markers was 2 kb ( mean = 48 kb ) .", "Individuals were considered hybrids if at least 10% of their genome was contributed by each parent; using this definition , 100% of individuals collected from Tlatemaco and 55% of individuals collected from Calnali were hybrids .", "Three more individuals from Calnali were excluded because their hybrid index was not within the 99% CI of the mean hybrid index .", "Including them resulted in a significant deviation from the expected R2 between unlinked sites of 1/2n ( n: number of sampled individuals ) .", "The expected number of generations since initial hybridization was estimated using the LD decay with distance method described in Hellenthal et al . ( 2014 ) .", "First , we used genome sequences of 5 Xiphophorus outgroups ( X . maculatus , X . hellerii , X . clemenciae , X . variatus , and X . nezahualcoyotl ) to identify autapomorphic loci in X . malinche and X . birchmanni respectively .", "We limit the analyses to only the autapomorphic loci for the minor parental species in each hybrid zone .", "We then fit an exponential curve D = a*exp ( −T*x ) , where ‘D’ is disequilibrium , ‘a’ is a coefficient , ‘T’ is time since hybridization in generations and ‘x’ is the physical distance between markers in Morgans ( scripts are available in the Dryad data repository under DOI: 10 . 5061/dryad . q6qn0 , Schumer et al . , 2014 ) .", "Because we do not have access to a recombination map for our species , we assumed a uniform recombination rate of 1 cM/378 kb ( Walter et al . , 2004 ) .", "This assumption can underestimate the time since hybridization in some cases ( Sankararaman et al . , 2012 ) , but better estimates await more detailed genetic map information .", "For each pairwise combination of markers ( Figure 2—figure supplement 2 ) , we used a custom php script to calculate R , the correlation coefficient .", "R is typically defined as D , the disequilibrium coefficient , scaled by the square root of the product of the allele frequencies at the two loci ( Hartl and Clark , 2007 ) .", "We use the methods outlined in Rogers and Huff ( 2009 ) for calculation of R using unphased data .", "We recorded whether R was positive or negative , corresponding to conspecific vs heterospecific association .", "To assess significance of correlations , we used a Bayesian ordered logistic regression as implemented in the R package bayespolr ( http://rss . acs . unt . edu/Rdoc/library/arm/html/bayespolr . html ) to estimate Student's t; we used this estimate to determine the two-sided p-value for the correlation .", "We only considered pairs of markers belonging to different linkage groups; intrachromosomal comparisons were excluded due to concerns about false positives caused by recombination rate variation ( scripts are available in the Dryad data repository under DOI: 10 . 5061/dryad . q6qn0 ) .", "To determine our expected false discovery rates ( FDRs ) associated with given p-value significance thresholds , we used a simulation approach .", "For LD analysis , we surveyed 12 , 269 markers ( reduced from 1 . 2 million , see above ) , but many of these markers are tightly clustered .", "We used the Matrix Spectral Decomposition method described in Li and Ji ( 2005 ) as implemented in the program matSpDlite ( Nyholt , 2004 ) , to determine the effective number of markers .", "We used the correlation matrix for each pair-wise marker from Tlatemaco for these calculations; calculations based on the correlation matrix from Calnali resulted in a similar but slightly lower number of tests .", "We determined based on this analysis that the effective number of markers is 1087 .", "Based on these results , we randomly selected 1087 markers from our data set , randomly shuffled genotypes within columns , calculated R2 and p-values for the entire data set , and determined the expected number of false positives at different p-value thresholds .", "We repeated this procedure 1000 times .", "We compared the average number of false positives to the total number of positives in the actual data set at a number of p-value thresholds .", "Based on this analysis , we determined that p<0 . 013 in both populations resulted in an expected false discovery rate ( FDR ) of 0 . 05 for 1087 independent markers ( excluding within chromosome comparisons ) , while p<0 . 007 resulted in an expected FDR of 0 . 02 .", "Our analyses focused on the FDR = 0 . 05 data set , but we repeated these analyses with a more restricted data set ( FDR = 0 . 02 , Tables 1 and 2 ) .", "We also performed simulations to investigate the potential effects of demographic processes on p-value distributions ( see below ) .", "In most cases , dozens to hundreds of contiguous markers showed the same patterns of LD .", "To cluster these markers and delineate between independent and non-independent LD blocks , we used an approach designed to conservatively estimate the number of LD pairs .", "Within adjacent clusters on the same chromosome , we tested for independence between clusters of sites by determining the p-value for R2 between a focal site and the last site of the previous LD cluster .", "If p>0 . 013 ( our FDR = 0 . 05 significance threshold ) , we counted the focal site as the first site in a new cluster .", "If regions of the Xiphophorus genome are misassembled , incorrect assignment of contigs to different linkage groups could generate strong cross-chromosomal linkage disequilibrium ( see for e . g . , Andolfatto et al . , 2011 ) .", "To evaluate this possibility , we focused on markers at the edges of identified LD blocks and examine patterns of local LD in these regions ( Figure 4—figure supplement 4 ) .", "If markers had neighboring markers within 300 kb ( 86% ) , we evaluated whether the marker had stronger LD with neighboring markers than detected in any cross-chromosomal comparisons .", "Only 1 . 5% of markers in Calnali and 0 . 6% in Tlatemaco had stronger cross-chromosomal LD than local LD .", "Selection against hybrid incompatibilities is expected to generate an excess of conspecific associations .", "To investigate whether regions in significant LD were more likely to have conspecific associations , we determined the direction of association between markers in each population .", "We compared the sign of R in pairs in significant LD ( 327 pairs at FDR = 0 . 05 ) to the sign of R in 1000 data sets of the same size composed of randomly selected pairs from the genomic background ( p>0 . 013 in each population ) .", "To investigate what levels of selection might be required to generate the patterns we observe , we use a model of selection on locus pairs following Karlin ( 1975 ) and a regression approach to approximate Bayesian inference using summary statistics as implemented in the program ABCreg ( Beaumont et al . , 2002; Jensen et al . , 2008; Thornton 2009 ) .", "We focus only on sites that have positive R ( 207 pairs ) since these sites are expected to be enriched for hybrid incompatibilities .", "Because selection on two-locus interactions results in changes in the frequency of particular genotypes , we used the frequency of double homozygous genotypes for the major parent ( Tlatemaco: MM_MM , Calnali: BB_BB ) , frequency of homozygous–heterozygous genotypes for the minor parent ( Tlatemaco: MB_BB and BB_MB , Calnali: MM_MB and MB_MM ) , and average final ancestry proportion as summary statistics .", "Under the BDM incompatibility model , two distinct fitness matrices are possible ( Figure 5—figure supplement 1 ) .", "Because these models are equally likely ( Coyne and Orr , 2004 ) , we used the random binomial function in R to assign the 207 conspecific-associated locus pairs to each fitness matrix for each simulation .", "To simplify our simulations , we assume that selection is equal on all genotype combinations that have not previously been exposed to selection in ancestral populations ( see Figure 5—figure supplement 1 ) .", "For each simulation , we drew from uniform prior distributions for 4 parameters .", "Limits on the prior distribution for admixture proportions for the two populations were determined as 0 . 5 to A , where A is the 95% CI of 1000 bootstrap resamplings of population ancestry from the observed data .", "Each simulated replicate was generated as follows:Draw values for s ( 0–0 . 1 ) , initial admixture proportions ( Tlatemaco 0 . 5–0 . 72 , Calnali 0 . 18–0 . 5 ) , number of generations of selection ( Tlatemaco 40–70 , Calnali 20–50 ) , and hybrid population size ( 50–5000 ) .", "Random assignment of 207 pairs to each of two possible BDM incompatibility fitness matrices . Calculate expected frequencies of each two-locus genotype using these priors and the methods described by Karlin ( 1975 ) , introducing drift at each generation as sampling of 2N gametes . After iterating through step 3 for t generations , we multinomially sampled expected frequencies from step 3 for n individuals .", "To account for variation in sample size , we simulated the actual distribution of sample sizes in the observed data . Calculate the mean of each summary statistic . Repeat for 1 , 000 , 000 simulations . Run ABCreg with a tolerance of 0 . 5% .", "These simulations produced well-resolved estimates of the selection coefficient , s , and hybrid population size , N ( Figure 5 ) .", "We also repeated these simulations using a model of selection against all hybrid genotypes ( Figure 5—figure supplement 1C ) .", "These simulations also resulted in well-resolved posterior distributions of s and N and similar maximum a posteriori ( MAP ) estimates for both populations ( Tlatemaco s = 0 . 015 , N = 4360; Calnali s = 0 . 043 , N = 270 ) .", "This model may be more consistent with incompatibilities arising from co-evolving loci or selection against hybrid phenotypes .", "Scripts for this analysis are available in the Dryad data repository under DOI: 10 . 5061/dryad . q6qn0 .", "To check the consistency of our simulations with the observed data , we performed posterior predictive simulations by randomly drawing 100 values from the joint posterior ( of N , s , generations of selection , and admixture proportions ) with replacement for each population .", "For each draw , we then simulated selection using these parameters , applying the same significance threshold as we applied to the real data , until 207 pairs had been generated .", "Departures from expectations under random mating were compared to departures in the real data ( Figure 5 , Figure 5—figure supplement 3 ) .", "Regions involved in hybrid incompatibilities are predicted to be more divergent than other regions of the genome for a number of reasons ( see main text ) .", "To evaluate levels of divergence relative to the genomic background , we compared divergence ( calculated as number of divergent sites/length of region ) between X . malinche and X . birchmanni at 207 regions in significant conspecific LD ( FDR = 0 . 05 ) in both populations to 1000 data sets of the same size generated by randomly sampling regions throughout the genome that were not in significant LD ( p>0 . 013 with all unlinked regions ) using a custom perl script and the program fastahack ( https://github . com/ekg/fastahack ) .", "For LD regions that included only 1 marker ( n = 60 ) , we included the flanking region defined by the closest 5′ and 3′ marker .", "To analyze coding regions , we extracted exons from these regions and calculated dN , N , dS , and S for each gene using codeml in PAML with the F3x4 codon model ( Yang , 1997; scripts are available in the Dryad data repository under DOI: 10 . 5061/dryad . q6qn0 ) .", "For a phylogenetically independent comparison , we repeated this analysis using pseudogenomes for two swordtail species for which we previously collected genome sequence data , X . hellerii and X . clemenciae ( Schumer et al . , 2013 ) .", "Repeating all analyses for the full data set ( i . e . , including pairs in heterospecific LD in one or both populations ) did not substantially change the results ( Supplementary file 1C ) .", "To determine whether there is significant enrichment of certain gene classes in our data set , we annotated regions in significant LD .", "We only considered LD regions that contained 10 genes or fewer to limit our analysis to regions that are reasonably well-resolved .", "After excluding regions with no genes , this resulted in 242 regions for analysis containing 202 unique genes .", "We used the ensembl annotation of the X . maculatus genome ( http://www . ensembl . org/Xiphophorus_maculatus ) to identify the HUGO Genome Nomenclature Committee ( HGCN ) abbreviation for all the genes in each region .", "Using the GOstats package in R , we built a custom Xiphophorus database using the HGCN gene names listed in the genome and matching each of these to Gene Ontology ( GO ) categories as specified in the annotated human genome database ( in bioconductor ‘org . Hs . eg . db’ ) .", "This resulted in a total of 12 , 815 genes that could be matched to GO categories .", "We then tested for functional enrichment in GO categories , using the GOstats and GSEABase packages in R and a p-value threshold of <0 . 01 .", "Demographic processes such as bottlenecks and continued migration can affect LD measures and could potentially increase our false discovery rate .", "To explore how demographic changes might influence LD p-value distributions , we performed coalescent simulations using the MAP estimate of hybrid population size ( co-estimated with other parameters using ABC , see above ) .", "We used Hudson's ms ( Hudson , 1990 ) to simulate an unlinked pair of regions in two populations .", "We calculated time of admixture relative to the time of speciation using the relationship Tdiv4N= ( 1/2 ) ( ( Dxy/θ ) −1 ) , where Dxy is the average number of substitutions per site between species; we previously estimated ρ , the population recombination rate , θ , the population mutation rate ( ρ =θ = 0 . 0016 per site ) , and Ne , the effective population size ( Ne = 10 , 500 ) , for X . birchmanni based on the whole genome sequences ( Schumer et al . , 2013 ) .", "Because parameter estimates were similar for X . malinche , for simplicity , we used these estimates for both parental populations .", "For population 1 , we set the time of admixture ( tadmix/4N ) to 0 . 0014 generations , the proportion X . malinche to 0 . 7 ( derived from the average hybrid index in samples from Tlatemaco ) , and the sample size to 170 diploids .", "For population 2 , we set tadmix/4N to 0 . 000875 , the proportion X . malinche to 0 . 2 ( derived from the average hybrid index in samples from Calnali ) , and the sample size to 143 diploids .", "We specified a bottleneck at tadmix/4N reducing the population to 2% its original size in simulations of Calnali and 18% its original size in simulations of Tlatemaco ( based on results of ABC simulations , see ‘Results’ ) .", "In each simulated replicate , we selected one substitution from each unlinked region and accepted a pair of sites if they were fixed for different states between parents ( evaluated by generating 30 chromosomes of each species per simulation ) .", "Simulations were performed until 100 , 000 pairs had been simulated ( scripts are available in the Dryad data repository under DOI: 10 . 5061/dryad . q6qn0 ) ; we used the rate of false positives from these simulations to calculate the total expected number of false positives given our effective number of tests .", "Based on these simulations , our expected FDR at p<0 . 013 is ∼10% , slightly larger than our expected FDR based on permutation of the data .", "Because we do not have information about the migration history of these populations , we use simulations of migration only to explore how continued migration or multiple admixture events might affect our false discovery rate .", "We simulate unidirectional migration of individuals from each parental population to the hybrid population per generation ( 4Nm = 80 for each parental population ) .", "Under this migration scenario , our expected FDR at p<0 . 013 is 15% .", "In addition to scenarios of ongoing migration , we simulated migration bursts .", "For a short time interval that corresponds to ∼1 generation starting ∼10 generations ago ( tmig/4N = 0 . 00025–0 . 000275 ) , we set the migration rate to 4Nm = 4000 , or 10% of the population made up of migrants .", "We simulated three scenarios: ( 1 ) migration from the major parent ( expected FDR 15% ) , ( 2 ) migration from the minor parent ( expected FDR 11% ) , and ( 3 ) migration from both parents ( 4Nm = 2000 for each parental population , expected FDR 11% ) .", "None of these demographic scenarios increased expected FDR above 15% .", "Because MSG has not previously been used to analyze natural hybrids , we evaluated performance at a range of parameters and performed power simulations .", "To optimize the Hidden Markov Model ( HMM ) parameters of MSG for analysis of natural hybrids , we used a combination of empirical data and simulations .", "We set the error rate parameter ( deltapar ) for each parent based on the genome wide average rate of calls to the incorrect parent in the 60 parental individuals of each species analyzed ( deltapar = 0 . 05 and 0 . 04 for X . birchmanni and X . malinche , respectively ) .", "The transition probability of the HMM in MSG is determined by the mean genome-wide recombination rate multiplied by a scalar ( rfac ) .", "We set the recombination rate to 240 based on the estimate of approximately 1 recombination event per chromosome per meiosis ( 24 , 1 cM/378 kb , Walter et al . , 2004 ) , and an a prior expectation of at least 10 generations of hybridization .", "We then increased the recombination factor step-wise to the maximum value that did not induce false breakpoints in parental individuals; we determined that rfac could be set to 3 without leading to spurious ancestry calls for parental individuals .", "At some point , ancestry blocks will be too small for the HMM to detect given our density of ancestry informative markers .", "To determine the ancestry block size at which sensitivity decreases , we used the pseudogenomes to generate 25 Mb chromosomes with a homozygous block for the alternate parent randomly inserted ( 40 , 60 , 80 , 100 , 120 kb ) .", "We simulated 100 replicates of each size class and generated 1 million reads at MseI sites genome-wide .", "We then analyzed these simulated individuals using the MSG pipeline and determined whether the homozygous segment was detected .", "Based on our simulations , we determined that we had low power to detect ancestry blocks smaller than 80 kb ( probability of detection ≤80% ) and high power to detect blocks 120 kb or larger ( probability of detection ≥97% ) .", "To determine how much of the genome we are failing to detect in small ancestry segments , we fit an exponential distribution to the observed block sizes in the real data for each parent and for each population , generated samples from an exponential distribution with the lambda of the observed distribution , and determined what percent of bases pairs were found in ancestry blocks below our detection threshold .", "Based on this analysis , we determined that in both hybrid zones , less than 5% of base pairs in the genome are likely to fall into undetectable segments .", "As an additional evaluation of MSG's effectiveness in genotyping hybrid genomes with similar properties to ours , we simulated a 25 Mb admixed chromosome for 100 individuals , drawing ancestry size blocks from the block size distribution observed in the real data .", "We then generated MSG data in silico for each simulated individual ( 1 million reads ) , and compared MSG ancestry calls to true ancestry .", "We found that on average 91 . 4% of raw calls were made to the correct genotype; if ambiguous calls were excluded ( posterior probability ≤0 . 95 , 7% of sites ) , MSG's accuracy increased to >98% .", "The median size of regions for which incorrect calls were made was 29 kb , much smaller than the median block size in the whole data set ." ] ]

[ "Hybridization is increasingly being recognized as a common process in both animal and plant species .", "Negative epistatic interactions between genes from different parental genomes decrease the fitness of hybrids and can limit gene flow between species .", "However , little is known about the number and genome-wide distribution of genetic incompatibilities separating species .", "To detect interacting genes , we perform a high-resolution genome scan for linkage disequilibrium between unlinked genomic regions in naturally occurring hybrid populations of swordtail fish .", "We estimate that hundreds of pairs of genomic regions contribute to reproductive isolation between these species , despite them being recently diverged .", "Many of these incompatibilities are likely the result of natural or sexual selection on hybrids , since intrinsic isolation is known to be weak .", "Patterns of genomic divergence at these regions imply that genetic incompatibilities play a significant role in limiting gene flow even in young species ." ]

[ "In nature , closely related species often interbreed to produce hybrids .", "However , hybrids are often less fertile or unable to compete with parent species , making them less likely to thrive in the wild .", "When the genomes of two different species are mixed , versions of genes that are meant to work together can become separated , which means that these genes do not work as well as they should .", "This reduces the hybrids' chances of survival , and a poor survival rate of hybrids is one barrier that keeps different species distinct , even though the species can interbreed .", "Two species of swordtail fish live in the rivers in Mexico , and although they mostly live in different stretches of these rivers , the two species interbreed to produce hybrids in the regions where they overlap .", "These hybrids can outnumber the parental species in these ‘hybrid zones’ , but the two species have remained separate in other parts of the rivers .", "Though some genetic incompatibilities that might keep the species distinct have previously been suggested , it is not known how many incompatibilities there are in these fish's genomes .", "Schumer et al . have searched the genomes of wild hybrids between these species and found hundreds of genetic incompatibilities .", "These were identified by looking for species-specific pairs of genes that are found together more often than would be expected if there were no selection against hybrids .", "It is likely that many of these incompatibilities reduce the evolutionary fitness of the hybrid fish and Schumer et al . suggest that many could be the result of environmental pressures and the fish's mating preferences .", "Furthermore , Schumer et al . demonstrate that genes close to identified incompatibilities are more different between species , on average , than genes that are further away .", "When there is on-going interbreeding between two the species ( as is the case with the swordtails ) , this finding is expected only if these incompatibilities reduce the hybrids' chances of finding mates or surviving .", "The findings of Schumer et al . demonstrate how conflicts in the genomes of two species allow these species to remain distinct even when they live in overlapping environments and frequently interbreed .", "Future work will investigate how these genetic incompatibilities shape the hybrid populations that are found in the wild; and which incompatibilities are caused by poor survival of the hybrids or by the fish's mating preferences selecting against the hybrids ." ]

[ "Introduction", "Results", "Discussion", "Materials and" ]

[ "structural biology and molecular biophysics", "neuroscience" ]

[ [ "Neurotransmitter release is crucial for interneuronal communication .", "This exquisitely regulated process involves the docking of synaptic vesicles at presynaptic active zones , priming of the vesicles to a readily-releasable state and very fast ( <1 ms ) Ca2+-triggered fusion of the vesicle and plasma membranes ( Südhof , 2013 ) .", "The machinery that controls release contains core proteins that form part of a universal intracellular membrane fusion apparatus .", "These proteins include ( Rizo and Xu , 2015; Südhof and Rothman , 2009; Jahn and Fasshauer , 2012 ) :", "( i ) the synaptic vesicle SNAP receptor ( SNARE ) synaptobrevin and the plasma membrane SNAREs syntaxin-1 and SNAP-25 , which together form a tight four-helix bundle called the SNARE complex that brings the two membranes together and is key for membrane fusion ( Söllner et al . , 1993; Hanson et al . , 1997; Poirier et al . , 1998; Sutton et al . , 1998 ) ;", "( ii ) N-ethylmaleimide-sensitive factor ( NSF ) and soluble NSF attachment proteins ( SNAPs; no relation to SNAP-25 ) , which disassemble the SNARE complex to recycle the SNAREs ( Söllner et al . , 1993; Mayer et al . , 1996; Banerjee et al . , 1996 ) ; and", "( iii ) the Sec1/Munc18 ( SM ) protein Munc18-1/UNC-18 , which ( together with Munc13s/UNC13 ) orchestrates SNARE complex formation in an NSF-SNAP-resistant manner ( Ma et al . , 2011 , 2013 ) .", "In addition , the neuronal exocytotic machinery contains multiple additional proteins , such as the Ca2+ sensor Synaptotagmin-1 , RIMs , Rab3s , CAPS and complexins , which confer the tight regulation of neurotransmitter release ( Rizo and Xu , 2015 ) .", "This tight regulation also arises in part from the core components themselves .", "Thus , Munc13s contain a MUN domain ( Basu et al . , 2005 ) that has homology to tethering factors from diverse membrane compartments ( Pei et al . , 2009; Li et al . , 2011 ) and hence provides a core function , but also multiple domains with key regulatory roles ( e . g . Lu et al . [2006]; Deng et al . [2011]; Liu et al . [2016] ) .", "Syntaxin-1 also modulates neurotransmitter release by forming a self-inhibited ‘closed conformation’ that involves intramolecular binding of its N-terminal Habc domain to its SNARE motif , and that binds tightly to Munc18-1 ( Dulubova et al . , 1999; Misura et al . , 2000 ) .", "Opening of syntaxin-1 to form the SNARE complex is mediated by the Munc13 MUN domain ( Ma et al . , 2011; Yang et al . , 2015 ) .", "This transition constitutes a central step that can be regulated via Munc13s in presynaptic plasticity processes that mediate diverse forms of information processing in the brain ( Rizo and Rosenmund , 2008 ) .", "However , the mechanism of syntaxin-1 opening and the exact role of Munc18-1 in SNARE complex assembly have been enigmatic .", "Some evidence suggested that the Munc13-1 MUN domain opens syntaxin-1 through a weak interaction with its SNARE motif , providing a template for SNARE complex assembly ( Ma et al . , 2011 ) .", "However , an alternative model postulated that Munc18-1 provides a template for the formation of the SNARE complex .", "This model is based in part on the findings that Munc18-1 binds to synaptobrevin ( Xu et al . , 2010b ) and that a Munc18-1 mutation that disrupts this interaction ( L348R ) impairs the stimulation of liposome lipid mixing by Munc18-1 in reconstitution assays ( Parisotto et al . , 2014 ) .", "Tantalizing evidence supporting a general role for SM proteins as templates for SNARE complex assembly was provided by two crystal structures of Vps33 , the SM protein involved in yeast vacuolar fusion , bound either to Vam3 or to Nyv1 , the respective homologues of syntaxin-1 and synaptobrevin in this system ( Baker et al . , 2015 ) .", "Superposition of the two structures clearly shows that binding to the SM protein places the two SNARE motifs in close proximity and in the right register to form the SNARE complex ( Figure 1A ) .", "Munc18-1 is likely to template SNARE complex assembly through similar interactions: the crystal structure of closed syntaxin-1 bound to Munc18-1 ( Misura et al . , 2000 ) revealed an orientation of the SNARE motif similar to that observed for Vam3 bound to Vps33 ( compare Figure 1A and B ) .", "Furthermore , L348 side chain implicated in synaptobrevin binding ( Parisotto et al . , 2014 ) is located in a groove between two helices of domains 3a of Munc18-1 ( Figure 1C ) , which is involved in the binding of Vps33 to Nyv1 ( Figure 1A ) . 10 . 7554/eLife . 24278 . 003Figure 1 . Autoinhibition by a loop of Munc18-1 is likely to inhibit binding to synaptobrevin .", "( A ) Ribbon diagram of the structure of Vps33 ( blue ) bound to the Nyv1 SNARE motif ( red ) ( PDB code 5BV0 ) superimposed with the structure of Vps33 ( not shown ) bound to the Vam3 SNARE motif ( yellow ) ( PDB code 5BUZ ) ( Baker et al . , 2015 ) .", "The two helices connected by a loop in domain 3a of Vps33 , which are involved in Nyv1 binding , are shown in cyan .", "( B ) Crystal structure of Munc18-1 ( blue ) bound to closed syntaxin-1 ( SNARE motif in yellow; N-terminal region in orange ) ( PDB code 3C98 ) ( Burkhardt et al . , 2008 ) superimposed to the crystal structure of Munc18-1 ( gray ) bound to a syntaxin-4 N-terminal peptide ( pink ) ( PDB code 3PUJ ) ( Hu et al . , 2011 ) .", "The helix-loop-helix of domain 3a of Munc18-1 in the complex with syntaxin-1 is in cyan .", "( C ) Close-up of the helix-loop-helix of domain 3a of Munc18-1 bound to syntaxin-1 ( not shown ) illustrating how the loop forms a ‘furled conformation’ by folding back onto the groove between the two helices that is putatively involved in synaptobrevin binding .", "Side chains are shown as stick models and those that were mutated to disrupt synaptobrevin binding are labeled .", "( D ) Close-up of the helix-loop-helix region from the superposition shown in ( B ) but without showing syntaxin-1 .", "Note how in the complex with closed syntaxin-1 , the Munc18-1 loop is furled ( cyan ) .", "whereas in the complex with the syntaxin-4 N-terminal peptide , the loop is unfurled and forms a helical extension of one of the helices but with a bend ( gray ) .", "This bend has P335 in the corner , and the homologous residue of Vps33 ( P355 ) is also in the corner of the helix bend ( A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 00310 . 7554/eLife . 24278 . 004Figure 1—figure supplement 1 . Sequence alignment of the helix-loop-helix region of domain 3a of rat Munc18-1 ( residues 298–355 ) with the corresponding sequences of Munc18-1 from different species and of Vps33 from Chaetomium thermophilum . Residues D326 , M330 and L348 of rat Munc18-1 are marked by a * .", "The alignment was prepared using Clustal Omega ( http://www . ebi . ac . uk/Tools/msa/clustalo/ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 004 Despite this convergence of results , the nature of the synaptobrevin-Munc18-1 interaction remains unclear , as it is very weak and appears to be primarily mediated by the C-terminal region of the synaptobrevin SNARE motif ( Xu et al . , 2010b ) .", "This C-terminal region includes the sequence KLKRKYWWK ( residues 83–91 ) , which contains five basic and three aromatic residues and is thus likely to be promiscuous , as shown by the finding that this region also binds to the Munc13-1 MUN domain ( see below ) .", "Its homologous region in Nyv1 is not involved in Vps33 binding ( Baker et al . , 2015 ) .", "The weak affinity of Munc18-1 for synaptobrevin may arise because of autoinhibition by a loop that connects the two aforementioned helices of domain 3a of Munc18-1 , which occludes the putative synaptobrevin binding site in the crystal structure of Munc18-1 bound to closed syntaxin-1 , forming what is referred to as a 'furled loop conformation' ( Figure 1B , D ) ( Hu et al . , 2011 ) .", "Interestingly , this loops adopts an ‘unfurled conformation’ in the crystal structure of Munc18-1 bound to a syntaxin-4 N-terminal peptide ( Figure 1B , D ) ( Hu et al . , 2011 ) , forming an extension of one of these helices that resembles the conformation observed for the homologous sequence in the Vps33-Nyv1 structure ( Figure 1A ) .", "Thus , a conformational change in this loop may modulate synaptobrevin binding .", "Indeed , a P335A mutation designed to favor this helical extension enhanced Munc18-1 activity in lipid mixing assays ( Parisotto et al . , 2014 ) and caused a gain-of-function in secretory cells ( Han et al . , 2014; Munch et al . , 2016 ) .", "However , the basis for these results is unclear because the mutation did not appear to affect synaptobrevin binding ( Parisotto et al . , 2014 ) , and this region does not form a continuous helix in Vps33 bound to Nyv1; rather , it forms a bent helix with a proline in the corner ( P355 , homologous to Munc18-1 P335; see Figure 1A ) .", "Furthermore , we do not yet know how the Munc18-1-synaptobrevin interaction is related to the functional interplay between Munc18-1 and Munc13s in orchestrating SNARE complex assembly .", "In the study presented here , we addressed these questions using biophysical approaches and genetic experiments in C . elegans .", "NMR data show that the synaptobrevin SNARE motif binds to Munc18-1 through two types of interactions , one that involves the C-terminal region of the synaptobrevin SNARE motif and is likely non-specific , and another involving more central synaptobrevin sequences that is specifically impaired by the L348R mutation and enhanced by a mutation ( D326K ) designed to disrupt the furled loop conformation of Munc18-1 .", "Using reconstitution assays in which liposome fusion strictly requires Munc18-1 and a Munc13-1 C-terminal fragment ( Ma et al . , 2013; Liu et al . , 2016 ) , thus mirroring the total abrogation of neurotransmitter release observed in the absence of Munc18-1 or Munc13s ( Verhage et al . , 2000; Richmond et al . , 1999; Aravamudan et al . , 1999; Varoqueaux et al . , 2002 ) , we find that the L348R mutation strongly impairs membrane fusion whereas the D326K mutation leads to a dramatic gain-of-function that can partially overcome the requirement for Munc13-1 for membrane fusion .", "Furthermore , the Munc18-1 D326K mutant rescues physiological defects observed in C . elegans unc-18 nulls much more efficiently than WT Munc18-1 , thus demonstrating a gain-of-function in a live animal .", "Overall , our data support the proposal that Munc18-1 acts as a template for the assembly of the SNARE complex by binding not only to syntaxin-1 but also to synaptobrevin .", "Moreover , our results suggest that autoinhibition of Munc18-1 binding to synaptobrevin enables or facilitates the existence of Munc13-dependent modes of regulation of neurotransmitter release , adding to the notion that diverse intra- and intermolecular inhibitory interactions within the release machinery provide opportunities for multiple layers of regulation that are fundamental for brain function ." ], [ "In previous cross-linking experiments , we found that a sequence from the C-terminus of the synaptobrevin SNARE motif was cross-linked with a sequence from the loop in domain 3a of rat Munc18-1 ( Xu et al . , 2010b ) .", "Because rat Munc18-1 tends to aggregate , we used NMR spectroscopy in that study to analyze the binding of synaptobrevin to squid Munc18-1 , which is more soluble , and also found that binding was dominated by the C-terminus of the synaptobrevin SNARE motif ( Xu et al . , 2010b ) .", "In subsequent studies of interactions of neuronal SNAREs with Munc13-1 , we found that the C-terminus of the synaptobrevin SNARE motif also binds to the Munc13-1 MUN domain via its C-terminus ( Figure 2—figure supplement 1 ) .", "These findings raised the possibility that this C-terminal region of the synaptobrevin SNARE motif is promiscuous , which is not surprising because it includes five basic and three aromatic residues .", "This sequence is close to the transmembrane region , and is therefore likely to interact with the synaptic vesicle membrane through its abundant basic and hydrophobic residues in vivo; however , in the absence of membranes , this sequence is expected to be prone to non-specific protein interactions .", "These observations , and the finding that the homologous sequence at the C-terminus of the Nyv1 SNARE motif does not bind to Vps33 ( Baker et al . , 2015 ) , led us to examine the binding of synaptobrevin to Munc18-1 in more detail using NMR spectroscopy .", "For this purpose , we used a 15N-labeled fragment that spans the cytoplasmic region of synaptobrevin [synaptobrevin ( 1-96 ) ] and acquired 1H-15N HSQC spectra in the absence and presence of rat Munc18-1 at moderate concentrations ( 14 . 5 µM ) , which limited Munc18-1 aggregation while maintaining high sensitivity .", "Note that these spectra contain one cross-peak for each non-proline residue in a 15N-labeled protein , and that the intensity of the cross-peaks should decrease strongly upon binding to another protein ( Rizo et al . , 2012 ) , particularly considering that the synaptobrevin cytoplasmic region is unstructured in isolation ( Hazzard et al . , 1999 ) .", "Our 1H-15N HSQC spectra confirmed the binding of Munc18-1 to the C-terminus of synaptobrevin ( 1-96 ) , but showed that additional sequences from the SNARE motif also bind to Munc18-1 ( Figure 2 ) .", "The finding that the cross-peaks do not disappear completely indicates that the interaction is weak but , interestingly , examination of the cross-peak intensity ratios in the presence and absence of Munc18-1 shows clear intensity decreases not only for the C-terminal region ( beyond residue 82 ) but also for residues 60–80; even residues 42–55 exhibit some meaningful intensity decreases albeit smaller .", "Note that the absolute intensities and relative intensity patterns in these experiments are highly reproducible , as illustrated by comparison of absolute intensities from two 1H-15N HSQC spectra acquired for two different samples of 15N-synaptobrevin ( 1-96 ) plus Munc18-1 ( Figure 2—figure supplement 2 ) , and by the similarity of intensity ratio patterns observed in spectra acquired with WT and mutant Munc18-1s in regions where the mutations did not affect binding ( see below and Figure 3 ) . 10 . 7554/eLife . 24278 . 005Figure 2 . Munc18-1 binds to central sequences and the C-terminus of the synaptobrevin SNARE motif .", "( A ) 1H-15N HSQC spectra of 14 . 5 µM 15N-synaptobrevin ( 1-96 ) in the absence ( black contours ) and presence ( red contours ) of 14 . 5 µM Munc18-1 .", "Selected cross-peaks that were particularly broadened by Munc18-1 binding are labeled .", "( B ) Plots of the ratios of the intensities of the synaptobrevin ( 1-96 ) 1H-15N HSQC cross-peaks observed in the presence of Munc18-1 versus those observed in its absence , as a function of residue number .", "Intensities were measured only for well-resolved cross-peaks .", "Ratios were calculated from experiments performed with two separate samples and averaged .", "Note that the synaptobrevin SNARE motif spans approximately residues 29–91 ( Sutton et al . , 1998 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 00510 . 7554/eLife . 24278 . 006Figure 2—figure supplement 1 . The Munc13-1 MUN domain binds to the C-terminus of the synaptobrevin SNARE motif but not to more central sequences .", "( A ) 1H-15N TROSY-HSQC spectra of 20 µM 15N-synaptobrevin ( 29-93 ) in the absence ( black contours ) and presence ( red contours ) of 16 . 5 µM Munc13-1 MUN domain .", "Selected cross-peaks that were particularly broadened as a result of MUN domain binding are labeled .", "( B ) Plots of the ratios of the intensities of the synaptobrevin ( 29-93 ) 1H-15N TROSY-HSQC cross-peaks observed in the presence of Munc13-1 MUN domain versus those observed in its absence , as a function of residue number .", "Intensities were measured only for well-resolved cross-peaks . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 00610 . 7554/eLife . 24278 . 007Figure 2—figure supplement 2 . Reproducibility of 1H-15N HSQC cross-peak intensities . The plot shows the intensities ( arbitrary units ) from the 1H-15N HSQC cross-peaks of two separate samples of 14 . 5 µM 15N-synaptobrevin ( 1-96 ) in the presence of 14 . 5 µM Munc18-1 .", "Intensities were measured only for well-resolved cross-peaks .", "There is one black bar and one orange bar for each residue , representing the measurements made in the two separate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 00710 . 7554/eLife . 24278 . 008Figure 3 . Selective enhancement and disruption of synaptobrevin binding by Munc18-1 mutations .", "( A–C )", "Plots of the ratios of the intensities of the synaptobrevin ( 1-96 ) 1H-15N HSQC cross-peaks observed in the presence of WT or mutant Munc18-1 versus those observed in its absence , as a function of residue number .", "The plots compare the results obtained with WT Munc18-1 ( black bars ) with those obtained with the D326K ( red bars , [A] ) , M330E ( green bars , [B] ) or L348R ( blue bars , [C] ) Munc18-1 mutants .", "Intensities were measured only for well-resolved cross-peaks .", "Ratios were calculated from experiments performed with two separate samples and averaged . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 008 It is also worth noting that , based on the homology between synaptobrevin and Nyv1 and on the crystal structure of Vps33 bound to Nyv1 ( Figure 1A ) , an analogous binding mode for the neuronal proteins would predict that residues 41–55 of synaptobrevin bind to Munc18-1 at the loop region containing the helical extension , while residues 59–78 of synaptobrevin bind to the groove between the two helices of domains 3a .", "This prediction fits approximately with the decreases in cross-peak intensities observed in our 1H-15N HSQC spectra , particularly considering that the smaller intensity decreases observed for residues 42–55 ( Figure 2B ) may arise because the binding of this region may be hindered by competition with the C-terminus , which interacts with the same region of Munc18-1 according to the cross-linking experiments ( see above ) .", "In summary , these results suggest that the Vps33-Nyv1 binding mode is conserved for neuronal Munc18-1 and synaptobrevin , and that analysis of this interaction by methods that do not provide residue-specific information is complicated by the existence of non-specific binding involving the C-terminus of the synaptobrevin SNARE motif .", "We attempted to use 1H-15N HSQC spectra to analyze Munc18-1 binding to a 15N-labeled synaptobrevin fragment spanning residues 1–82 , which lacks the promiscuous C-terminal region , but no cross-peaks could be observed for the SNARE motif of this fragment because of aggregation .", "Previous biochemical data suggested that the L348R mutation disrupts binding of Munc18-1 to synaptobrevin and impairs the activity of Munc18-1 in liposome lipid mixing assays ( Parisotto et al . , 2014 ) .", "To design additional mutations that could potentially correlate synaptobrevin binding to Munc18-1 function , we examined which residues of Munc18-1 might be involved in binding based on homology with the Vps33-Nyv1 structure , and which side chains may stabilize the furled conformation of the loop in domain 3a of Munc18-1 ( Figure 1C ) , as destabilization of this conformation may enhance synaptobrevin binding .", "These analyses were hindered by the low sequence similarity between Munc18-1s and Vps33 ( Figure 1—figure supplement 1 ) , and because the furled conformation of the Munc18-1 loop is not very well defined in the crystal structure of its complex with syntaxin-1 .", "( Note that the conformation of the loop is different in the original published structure ( Misura et al . , 2000 ) and that refined later with the same data [Burkhardt et al . , 2008] ) .", "Nevertheless , D326 appeared to be a good candidate that may stabilize the furled loop because it may participate in hydrogen-bonding interactions ( Figure 1C ) and is highly conserved throughout evolution in Munc18-1s ( Figure 1—figure supplement 1 ) .", "Conversely , M330 may contribute to synaptobrevin binding because it is in a conserved hydrophobic position and its homologous residue of Vps33 participates in binding to Nyv1 .", "We used 1H-15N HSQC spectra to assess whether a mutation that reverses the charge of D326 ( D326K ) in Munc18-1 or a mutation that disrupts the hydrophobicity of M330 ( M330E ) could alter binding to 15N-synaptobrevin ( 1-96 ) , and also analyzed the effects of the L348R mutation .", "Importantly , the D326K Munc18-1 mutant caused stronger decreases in the intensities of cross-peaks from residues 45–83 than those caused by WT Munc18-1 ( Figure 3A ) .", "These effects seem moderate , but they do reflect a clear increase in the specific binding of the D326K mutant to this region .", "By contrast , the patterns of intensity ratios resulting from the addition of the D326K mutant or WT Munc18-1 are very similar for other regions of synaptobrevin , including the 40 N-terminal residues that are not involved in binding and the C-terminal region , which does bind to Munc18-1 but through interactions that are not affected by the D326K mutation .", "The M330E Munc18-1 mutant caused very similar intensity changes on the synaptobrevin 1H-15N HSQC cross-peaks to those caused by WT Munc18-1 ( Figure 3B ) .", "Hence , the M330 side chain does not contribute substantially to the affinity of Munc18-1 for synaptobrevin , but the M330E mutation provides a convenient negative control as a mutation in the loop region that is predicted to have no or very limited functional effects .", "Conversely , the L348R Munc18-1 mutant caused practically no decreases in the intensities of the synaptobrevin 1H-15N HSQC cross-peaks except for those from the C-terminus , where the changes exhibited a similar pattern to those caused by WT Munc18-1 .", "These data correlate with previous biochemical results showing impaired binding of the L348R Munc18-1 mutant to synaptobrevin ( Parisotto et al . , 2014 ) .", "Moreover , these results provide an emphatic illustration of the power of this method of analysis of protein–protein interactions , showing that the L348R mutation specifically impairs binding of Munc18-1 to the sequences of the synaptobrevin SNARE motif that are expected to bind according to the crystal structure of the Vps33–Nyv1 complex , and that the mutation has a clearly less marked effect on the non-specific binding involving the C-terminus .", "Overall , these results support the notion that there is a specific interaction between Munc18-1 and synaptobrevin that resembles that observed between Vps33 and Nyv1 , and that this interaction is autoinhibited by the furled conformation of the loop in domain 3a of Munc18-1 .", "We also analyzed whether the D326K , M330E and L348R mutations alter the binding of Munc18-1 to syntaxin-1 or to the SNARE complex using isothermal titration calorimetry ( ITC ) ( Table 1 ) .", "The KDs measured for the binding of WT and mutant Munc18-1s to syntaxin-1 were all very low , refining to less than 2 nM .", "The differences in the KDs calculated in duplicate experiments for each Munc18-1 protein and the confidence intervals derived from the fits ( Table", "1 ) indicate that none of the differences in KDs measured for WT Munc18-1 and the mutants can be considered significant .", "Hence , these data show that the three Munc18-1 mutants retain very high affinity for syntaxin-1 , although we cannot rule out the possibility that the mutations alter the affinity to some extent that is not measurable in these experiments .", "Similarly , there do not appear to be major differences in the affinities of WT and mutant Munc18-1s for the SNARE complex , although it is plausible that the D326K mutant has an increased affinity .", "Because Munc18-1 is relatively unstable ( Xu et al . , 2011 ) , we also measured the thermal denaturation curves of the three Munc18-1 mutants using circular dichroism .", "We observed no significant differences in the melting temperature compared to WT Munc18-1 , indicating that the mutations do not destabilize the protein .", "Therefore , the functional consequences of the D326K , M330E and L348R mutations in Munc18-1 are not expected to arise from misfolding . 10 . 7554/eLife . 24278 . 009Table 1 . Summary of the ITC data obtained to analyze the binding of WT and mutant Munc18-1s to syntaxin-1 and the SNARE complex* .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 009SyringeCellCell concentration correct factor†KD [nM]ΔH[kcal/Mol]Syx2-253WT-10 . 98 [0 . 97 , 0 . 98]0 . 10 [0 , 0 . 15]−22 . 80 [–23 . 13 , –22 . 46]WT-20 . 92 [0 . 92 , 0 . 93]0 . 34 [0 , 0 . 67]−22 . 03 [–22 . 36 , –21 . 71]D326K-10 . 89 [0 . 89 , 0 . 89]0 . 23 [0 . 11 , 0 . 37]−23 . 56 [–23 . 75 , –23 . 37]D326K-20 . 86 [0 . 85 , 0 . 87]0 . 30 [0 , 0 . 67]−25 . 45 [–26 . 30 , –24 . 61]M330E-10 . 92 [0 . 91 , 0 . 94]1 . 76 [0 . 82 , 3 . 23]−21 . 56 [–22 . 21 , –20 . 92]M330E-20 . 97 [0 . 94 , 1 . 00]1 . 52 [0 . 49 , 3 . 47]−25 . 67 [–27 . 43 , –24 . 04]L348R-10 . 86 [0 . 85 , 0 . 87]0 . 54 [0 . 23 , 0 . 98]−25 . 42 [–25 . 90 , –24 . 95]L348R-20 . 85 [0 . 85 , 0 . 85]0 . 71 [0 . 51 , 0 . 93]−25 . 12 [–25 . 31 , –24 . 92]SNARE complex‡WT-11 . 07 [0 . 90 , 1 . 18]1 , 382 [952 , 2172]−5 . 21 [–7 . 31 , –4 . 24]WT-21 . 01 [0 . 84 , 1 . 11]1 , 479 [1018 , 2355]−5 . 46 [–7 . 99 , –4 . 38]D326K-11 . 05 [0 . 98 , 1 . 12]442 [296 , 681]−4 . 29 [–4 . 94 , –3 . 84]D326K-20 . 98 [0 . 81 , 1 . 11]361 [179 , 834]−3 . 13 [–4 . 66 , –2 . 51]M330E-10 . 96 [0 . 79 , 1 . 08]730 [414 , 1459]−2 . 48 [–3 . 72 , –1 . 99]M330E-20 . 95 [0 . 82 , 1 . 12]1 , 205 [993 , 1462]−3 . 69 [–4 . 30 , –3 . 25]L348R-10 . 95 [0 . 89 , 1 . 00]677 [521 , 895]−5 . 06 [–5 . 74 , –4 . 56]L348R-20 . 89 [0 . 77 , 0 . 98]781 [527 , 1245]−5 . 20 [–7 . 00 , –4 . 30]*Two independent experiments were performed for WT Munc18-1 and for each mutant .", "For all parameters , 68 . 3% confidence intervals calculated using the error-surface projection method are indicated between square brackets .", "†Correction factor calculated as part of the fitting procedure to account for differences in the concentrations of active proteins ( Brautigam et al . , 2016 ) .", "‡The SNARE complex was formed with syntaxin-1 ( 2–253 ) , synaptobrevin ( 1–96 ) , SNAP-25 ( 11-82 ) and SNAP-25 ( 141–203 ) .", "Previous analyses of the effects of the L348R mutation on the activity of Munc18-1 in stimulating lipid mixing between reconstituted SNARE proteoliposomes were very insightful but did not include Munc13 , NSF or SNAPs ( Parisotto et al . , 2014 ) , and hence cannot connect these effects to the interplay between Munc18-1 , Munc13 , NSF and SNAPs in SNARE complex assembly ordisassembly .", "To study the functional consequences of the D326K , M330E and L348R mutations in Munc18-1 , we used assays that monitor both lipid and content mixing ( Zucchi and Zick , 2011 ) between liposomes containing synaptobrevin ( V-liposomes ) and liposomes containing syntaxin-1-SNAP-25 ( T-liposomes ) in the presence of different combinations of NSF , αSNAP , Munc18-1 , a fragment spanning the two C2 domains of Synaptotagmin-1 ( C2AB ) and a fragment spanning the conserved C-terminal region of Munc13-1 ( i . e . containing its C1 , C2B , MUN and C2C domains , referred to as C1C2BMUNC2C ) ( Liu et al . , 2016 ) .", "Monitoring of content mixing is necessary to ascertain that real membrane fusion occurs , as extensive lipid mixing can occur without content mixing ( Chan et al . , 2009; Zick and Wickner , 2014; Liu et al . , 2016 ) .", "The inclusion of NSF and αSNAP is critical to render membrane fusion strictly dependent on both Munc18-1 and Munc13 ( Ma et al . , 2013 ) , mirroring the total abrogation of neurotransmitter release observed in their absence ( Verhage et al . , 2000; Richmond et al . , 1999; Aravamudan et al . , 1999; Varoqueaux et al . , 2002 ) .", "All experiments were started in the absence of Ca2+ , and Ca2+ was added after 300 s to study how it affects membrane fusion .", "As expected , control experiments performed in the presence of NSF-αSNAP revealed very little fusion between V- and T-liposomes in the absence of Ca2+ , and highly efficient fusion upon Ca2+ addition when both WT Munc18-1 and Munc13-1 C1C2BMUNC2C were included , but not when either one or both of these proteins were absent ( Figure 4A , B ) .", "Interestingly , in analogous experiments that included Munc13-1 C1C2BMUNC2C and Munc18-1 mutants , we found that fusion was strongly impaired by the L348R mutation , which disrupts binding of Munc18-1 to synaptobrevin , whereas the D326K mutation that enhances synaptobrevin binding caused a dramatic enhancement of fusion before Ca2+ addition , and fusion was not affected by the M330E mutation that does not alter synaptobrevin binding ( Figure 4C , D; see quantification of content mixing after 150 s and 500 s in Figure 4—figure supplement 1A , B ) .", "These results establish a clear correlation between the activity of the Munc18-1 mutants in supporting membrane fusion in this reconstituted system and their ability to bind to synaptobrevin . 10 . 7554/eLife . 24278 . 010Figure 4 . Effects of Munc18-1 mutations that alter synaptobrevin binding on membrane fusion in reconstitution assays . Lipid mixing ( A , C , E ) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids .", "Content mixing ( B , D , F ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "In ( A , B ) , the assays were performed in the presence of NSF-αSNAP with or without WT Munc18-1 ( M18 ) and/or Munc13-1 C1C2BMUNC2C ( M13 ) as indicated .", "In ( C , D ) , assays were performed in the presence of NSF-αSNAP , Munc13-1 C1C2BMUNC2C and WT or mutant Munc18-1s as indicated .", "In ( E , F ) , assays were performed in the presence of NSF-αSNAP , synaptotagmin-1 C2AB fragment and WT or mutant Munc18-1s as indicated .", "Experiments were started in the presence of 100 μM EGTA and 5 µM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 01010 . 7554/eLife . 24278 . 011Figure 4—figure supplement 1 . Effects of Munc18-1 mutations that alter synaptobrevin binding on membrane fusion in reconstitution assays .", "( A , B )", "Quantification of content mixing in the experiments of Figure 4D .", "Bars represent averages of the normalized fluorescence observed after 150 s ( A ) and after 500 s ( 200 s after Ca2+ addition ) ( B ) in experiments performed at least in triplicate .", "Error bars represent standard deviations .", "( C , D )", "Lipid and content mixing assays performed under the same conditions as those shown in Figure 4C , D but containing in addition synaptotagmin-1 C2AB fragment . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 01110 . 7554/eLife . 24278 . 012Figure 4—figure supplement 2 . Effects of Munc18-1 mutations that alter synaptobrevin binding on membrane fusion in reconstitution assays .", "( A , B )", "Lipid and content mixing assays performed under the same conditions as those shown in Figure 4E , F but in the absence of the synaptotagmin-1 C2AB fragment .", "The gray curves show a reference experiment performed in the presence of NSF-αSNAP , Munc13-1 C1C2BMUNC2C and WT Munc18-1 .", "( C ) Quantification of content mixing in the experiments shown in Figure 4F .", "Bars represent averages of the normalized fluorescence observed after 800 s ( 500 s after Ca2+ addition ) in experiments performed at least in triplicate .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 012 As expected , the Synaptotagmin-1 C2AB fragment did not have major effects in these experiments ( Figure 4—figure supplement 1C , D ) because the Ca2+-dependence of fusion arises primarily from Ca2+ binding to the C2B domain of Munc13-1 C1C2BMUNC2C , although Synaptotagmin-1 C2AB may induce an acceleration of Ca2+-dependent fusion that can only be measured at faster time scales ( Liu et al . , 2016 ) .", "As the Munc13-1 C1C2BMUNC2C fragment bridges V- and T-liposomes in the absence of Ca2+ , and because this activity is key for its role in supporting membrane fusion ( Liu et al . , 2016 ) , Ca2+ binding to the Munc13-1 C2B domain probably helps to overcome an energy barrier to membrane fusion in these experiments .", "The finding that the D326K mutation designed to unfurl the Munc18-1 loop can support efficient fusion before Ca2+ addition suggests that the furled loop conformation imposes another energy barrier in WT Munc18-1 that is tightly coupled to the barrier associated with Munc13-1 C1C2BMUNC2C; thus , when the D326K mutation decreases the energy barrier to unfurl the Munc18-1 loop , the requirement for Ca2+ binding to the Munc13-1 C2B domain is relaxed .", "These observations led us to test the possibility that the D326K mutation may overcome , at least partially , the overall requirement for Munc13-1 C1C2BMUNC2C for membrane fusion .", "In experiments performed in the presence of NSF-αSNAP and in the absence of Munc13-1 C1C2BMUNC2C , neither the WT nor the Munc18-1 mutants were able to support fusion between V- and T-liposomes ( Figure 4—figure supplement 2A , B ) .", "However , when we performed analogous experiments in the presence of the Synaptotagmin-1 C2AB fragment , the D326K Munc18-1 mutant was able to stimulate fusion ( Figure 4E , F; Figure 4—figure supplement 2C ) .", "This fusion was slow but was dramatically more efficient than that observed with WT Munc18-1 or the M330E and L348R mutants .", "These results suggest that Munc13-1 is normally critical for membrane fusion because it is needed to overcome the energy barriers , imposed by the closed conformation of syntaxin-1 and by the furled conformation of the Munc18-1 loop , to SNARE complex assembly templated by Munc18-1 .", "Thus , the D326K mutation in Munc18-1 probably helps to overcome the need for Munc13-1 in these assays , much as a so-called LE mutation that disrupts the closed conformation of syntaxin-1 helps to overcome the requirement of Unc13 for neurotransmitter release in C . elegans ( Richmond et al . , 2001 ) .", "The gain-of-function associated with the D326K mutation is expected to reflect an enhanced ability to mediate SNARE complex formation .", "To test this notion , we used an assay that monitors SNARE complex assembly using native PAGE ( Yang et al . , 2015 ) .", "Binary complexes of syntaxin-1 and WT or mutant Munc18-1s containing the M330E , D326K or L348R substitutions were formed and then incubated with synaptobrevin and SNAP-25; they were then analyzed by native PAGE .", "SNARE complex assembly was reflected in the appearance of a characteristic band in the middle of the gel , which was concomitant with the depletion of the band corresponding to the syntaxin-1–Munc18-1 complex at the top of the gel ( Figure 5 ) .", "Only a small amount of SNARE complex was observed when WT Munc18-1 or the M330E mutant was used , and practically no SNARE complex assembly was observed for the L348R mutant .", "Conversely , the D326K mutation led to a clear increase in SNARE complex formation compared to that observed with WT Munc18-1 ( Figure 5 ) .", "These results show a clear correlation between SNARE complex assembly and the activities of the mutants in the reconstitutions assays , supporting the notion that these activities are a consequence of their ability to template SNARE complex assembly . 10 . 7554/eLife . 24278 . 013Figure 5 . Effects of Munc18-1 mutations on SNARE complex assembly starting with the binary syntaxin-1–Munc18-1 complex . Complexes of syntaxin-1 ( 2–253 ) with WT or mutant Munc18-1 were incubated with SNAP-25 and the synaptobrevin SNARE motif , and analyzed by native PAGE followed by Coomassie blue staining ( five lanes on the right ) .", "The four lanes on the left show controls with the same amounts of the syntaxin-1–Munc18-1 complexes .", "The middle lane shows a SNARE complex assembly reaction performed with syntaxin-1 ( 2–253 ) , the synaptobrevin SNARE motif and SNAP-25 in the absence of Munc18-1 .", "The positions of the different complexes are indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 013 The design of the D326K mutation was based on the furled conformation of the loop that connects two helices of domain 3a in the crystal structure of rat Munc18-1 bound to closed syntaxin-1 ( Figures 1B–D and 6A–B ) ( Burkhardt et al . , 2008 ) , but this loop is involved in crystal contacts that could influence the structure in this region .", "Similarly , the unfurled conformation of this loop in the crystal structure of Munc18-1 bound to the syntaxin-4 N-terminal peptide ( Figures 1B , D and 6C–D ) ( Hu et al . , 2011 ) might have been induced by dimerization within the crystals ( Figure 6C ) , and the crystal structure of squid Munc18-1 ( Bracher et al . , 2000 ) revealed another unfurled conformation of the loop ( Figure 6—figure supplement", "1 ) that could also be caused by crystal contacts .", "These findings show that the structure of this region of Munc18-1 is malleable and it is unclear whether the loop adopts a defined conformation in Munc18-1 alone and/or bound to syntaxin-1 .", "Moreover , it is uncertain whether the effects of the D326K mutation on synaptobrevin binding and Munc18-1 activity arise because of structural perturbations caused by the mutation , as predicted , or from alteration of direct interactions of D326 with the SNAREs . 10 . 7554/eLife . 24278 . 014Figure 6 . The D326K mutation destabilizes the structure of the Munc18-1 loop .", "( A ) Ribbon diagram of Munc18-1 ( violet ) bound to syntaxin-1 ( SNARE motif in yellow; N-terminal region in orange ) ( PDB code 3C98 ) ( Burkhardt et al . , 2008 ) .", "The red ellipse shows the location of the loop that connects two helices of domain 3a of Munc18-1 .", "( B ) Close-up view of the domain 3a loop region from panel A . Side chains from the loop and the two helices are shown as stick models , and the atoms of the side chains from D326 , M316 , M324 , M330 and M334 are color-coded ( carbon green; oxygen red; sulfur yellow ) .", "The diagram illustrates that these side chains are well packed within the furled structure of the loop , although they have different degrees of solvent accessibility .", "( C ) Ribbon diagram showing the dimeric structure observed in the crystals of Munc18-1 bound to a syntaxin-4 N-terminal peptide ( not shown ) ( PDB code 3PUJ ) ( Hu et al . , 2011 ) .", "One Munc18-1 molecules is shown in gray and the other in blue .", "Note that dimerization involves the two domain 3a helices but the loop is at the end of the dimerization interface .", "The red ellipse shows the location of the loop for the gray molecule .", "( D ) Close-up view of the loop in the gray Munc18-1 molecule of panel C . Atoms from the loop and the two helices are shown as stick models , and the atoms observed for M324 , D326 , M330 and M334 are color-coded ( carbon green; oxygen red; sulfur yellow ) .", "Note that residues 315–323 , as well as the side chains of M324 , D326 and M330 , were not observable , probably because they are not packed against other regions of Munc18-1 and they are thus dynamic .", "( E ) Expansions from the methionine methyl region of 1H-13C HMQC spectra of WT 50%-2H-ILMV-13CH3-Munc18-1 free ( black contours ) and bound to syntaxin-1 ( 2–253 ) ( orange contours ) .", "The arrows indicate potential shifts caused by syntaxin-1 ( 2–253 ) binding on the cross-peaks tentatively assigned to M330 and M334 ( Figure 6—figure supplement 2 ) .", "( F ) Expansions from the methionine methyl region of 1H-13C HMQC spectra of D326K 50%-2H-ILMV-13CH3-Munc18-1 free ( magenta contours ) and bound to syntaxin-1 ( 2–253 ) ( cyan contours ) .", "( G , H )", "Expansions of the 1H-13C HMQC spectra shown in panels E , F but superimposing the spectra of WT and D326K 50%-2H-ILMV-13CH3-Munc18-1 in isolation ( G ) or bound to syntaxin-1 ( 2–253 ) ( H ) .", "The spectra were plotted at higher contour levels to emphasize the spectral changes induced by the D326K mutation .", "Arrows indicate cross-peak shifts .", "Cross-peaks are labeled with residue assignments based on spectra acquired on methionine mutants ( Figure 6—figure supplement 2 ) ; the ?", "symbols after the residue numbers indicate the tentative nature of the assignments .", "Green boxes indicate regions of the spectra that were plotted at lower levels to show weak cross-peaks .", "New cross-peaks caused by the D326K mutation are indicated by ** .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 01410 . 7554/eLife . 24278 . 015Figure 6—figure supplement 1 . Structure of squid Munc18-1 . Ribbon diagram of the crystal structure of squid Munc18-1 ( PDB code 1EPU ) ( Bracher et al . , 2000 ) showing the unfurled structure of the loop connecting two helices of domain 3a . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 01510 . 7554/eLife . 24278 . 016Figure 6—figure supplement 2 . Assignment of methionine cross-peaks in 1H-13C HMQC spectra of 50%-2H-ILMV-13CH3-Munc18-1 . ( A–F ) Superpositions of the methionine methyl region of 1H-13C HMQC spectra of WT ( black contours ) and M316L ( A , B ) , M330E ( C , D ) or M334L ( E , F ) mutants ( red contours ) 50%-2H-ILMV-13CH3-Munc18-1 free ( A , C , E ) or bound to syntaxin-1 ( 2–253 ) ( B , D , F ) .", "The spectra were plotted at different contour levels to help with visualizing the most pronounced perturbations caused by the mutations .", "Arrows show cross-peak shifts .", "Tentative assignments are indicated for cross-peaks that shift or disappear due to the mutations .", "All assignments must be considered tentative ( hence the ? symbols ) because of the complexity of the perturbations caused by the mutations , but we feel more confident with the assignments of the syntaxin-1 ( 2–253 ) -bound form .", "Blue boxes indicate regions of the spectra that were plotted at lower levels to show weak cross-peaks .", "New cross-peaks caused by the mutations that do not arise from an obvious cross-peak shift are labeled with ** .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 01610 . 7554/eLife . 24278 . 017Figure 6—figure supplement 3 . The D326K mutation causes selective decreases in the intensities of the 1H-13C HMQC cross-peaks of methionines from the domain 3a loop of Munc18-1 . The plots show the ratios between the intensities of methionine cross-peaks observed for D326K 50%-2H-ILMV-13CH3-Munc18-1 and those observed for WT 50%-2H-ILMV-13CH3-Munc18-1 in the absence ( left panel ) and presence ( right panel ) of syntaxin-1 ( 2–253 ) .", "The data for the cross-peaks assigned to the four methionines from the domain 3a loop are grouped on the left of each plot , while those for other well-resolved ( unassigned ) cross-peaks are grouped on the right .", "To account for small differences in protein concentrations , all intensities in each spectrum were normalized with the average intensity of the unassigned cross-peaks . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 017 To shed light into these questions , we first investigated whether dimeric structures such as that observed in the Munc18-1-syntaxin-4 N-peptide crystals ( Figure 6C ) predominate in solution .", "Using static light scattering ( SLS ) , we measured the following weight-average molecular mass ( presented as the average of 30 determinations ± the standard deviation in kDa ) at 15 µM concentration: WT Munc18-1 77 . 2 ± 0 . 1; D326K Munc18-1 78 . 7 ± 0 . 9; WT Munc18-1 bound to an unlabeled fragment spanning most of the cytoplasmic region of syntaxin-1 [syntaxin-1 ( 2–253 ) ] 97 . 1 ± 0 . 6; D326K Munc18-1-syntaxin-1 ( 2–253 ) 102 . 1 ± 0 . 9 .", "The expected molecular weights are 68 . 6 and 98 . 0 for free and syntaxin-1 ( 2–253 ) -bound Munc18-1 , respectively .", "Hence , these data show that the free and syntaxin-1 ( 2–253 ) -bound Munc18-1 are largely monomeric under these conditions , and that the D326K mutation does not alter this behavior substantially .", "We next turned to NMR spectroscopy .", "Clearly , it is very difficult to obtain detailed structural information on a small region of a 68-kDa protein such as Munc18-1 by any available technique in solution , but methyl TROSY-based heteronuclear multiple quantum coherence ( HMQC ) spectra of perdeuterated proteins that are specifically 1H , 13C-labeled at Ile , Leu , Met and Val methyl groups ( 2H-ILMV-13CH3-labeling ) offers the possibility of observing cross-peaks from methyl groups of very large proteins with high sensitivity ( Ruschak and Kay , 2010 ) , thus providing a powerful tool to investigate structural perturbations around these methyl groups .", "Application of this approach to Munc18-1 was hindered by its limited solubility and stability , and attempts to obtain 2H-ILMV-13CH3-labeled Munc18-1 yielded very low amounts of soluble protein .", "We circumvented this problem by preparing ILMV-13CH3-labeled Munc18-1 with only 50% perdeuteration ( 50%-2H-ILMV-13CH3-Munc18-1 ) , which we could obtain with reasonable yields in stable form .", "The quality of the 1H-13C HMQC spectra obtained for 15 µM 50%-2H-ILMV-13CH3-Munc18-1 in isolation or bound to syntaxin-1 ( 2–253 ) was rather limited in the Ile and Leu-Val methyl regions but , fortunately , multiple well-resolved cross-peaks could be observed with sufficient sensitivity in the Met methyl region ( Figure 6E ) and the domain 3a loop contains four methionines ( M316 , M324 , M330 and M334; Figure 6B ) .", "Hence , the methyl cross-peaks from these methionines might provide information on the structural perturbations caused by the D326K mutation .", "Comparison of 1H-13C HMQC spectra of 50%-2H-ILMV-13CH3-Munc18-1 in a free state and bound to syntaxin-1 ( 2–253 ) shows that syntaxin-1 binding induces shifts in many methionine methyl cross-peaks ( Figure 6E ) , consistent with the extensive surface involved in the interaction ( Misura et al . , 2000 ) .", "The 1H-13C HMQC spectra acquired for D326K mutant 50%-2H-ILMV-13CH3-Munc18-1 free and bound to syntaxin-1 ( 2–253 ) revealed similar changes ( Figure 6F ) to those observed for WT , as expected because this mutant retains very high affinity for syntaxin-1 ( 2–253 ) ( Table", "1 ) and the mutation is not in the binding interface .", "Attempts to assign the cross-peaks from the four methionines in the loop by acquiring 1H-13C HMQC spectra of three mutants containing single methionine substitutions ( M316L , M330E and M334L ) did not yield unambiguous assignments because the mutations perturbed several cross-peaks for both free and syntaxin-1 ( 2–253 ) -bound 50%-2H-ILMV-13CH3-Munc18-1 ( Figure 6—figure supplement 2 ) .", "Nevertheless , the perturbations did identify four cross-peaks that are most perturbed by the mutations and hence are most likely to correspond to the M316 , M324 , M330 and M334 methyl groups for both the free and the syntaxin-1 ( 2–253 ) -bound 50%-2H-ILMV-13CH3-Munc18-1 ( Figure 6—figure supplement 2 ) .", "Reassuringly , superposition of the 1H-13C HMQC spectra obtained for WT and D326K mutant 50%-2H-ILMV-13CH3-Munc18-1 revealed that the D326K mutation caused shifts in the four cross-peaks corresponding to M316 , M324 , M330 and M334 for both the free and the syntaxin-1 ( 2–253 ) -bound forms ( Figure 6G , H ) .", "The observed cross-peak perturbations caused by the D326K mutation and the three methionine mutations show that the loop region adopts well-defined structures in both isolated Munc18-1 and its complex with syntaxin-1 ( 2–253 ) .", "The perturbations are not compatible with unfurled conformations such as that observed in the crystal structure of Munc18-1 bound to the syntaxin-4 N-terminal peptide ( Figure 6D ) because the D326 , M316 , M324 and M330 side chains are not well packed and the loop region is rather flexible in this dimeric structure ( residues 315–323 , were not observable , and the side chains of M324 , D326 and M330 were also not observable ) .", "As Munc18-1 and its complex with syntaxin-1 ( 2–253 ) are largely monomeric under our conditions , additional flexibility would be expected if an unfurled loop structure were present .", "Conversely , the observed cross-peak perturbations are compatible with the furled loop structure observed in the crystals of Mun18-1 bound to closed syntaxin-1 ( Figure 6B ) .", "Nevertheless , the extent to which the observed structure was influenced by crystal contacts is still unclear , and the large changes caused by syntaxin-1 ( 2–253 ) binding on the cross-peaks tentatively assigned to the M330 and M334 ( Figure 6E ) indicate that binding may substantially alter the structure of the loop .", "The shifts of the tentatively assigned Met cross-peaks caused by the D326K mutation ( Figure 6G , H ) show that the mutation causes some structural perturbations in the region , but these perturbations are unlikely to underlie the functional effects of the mutation because the M330E mutation induces more substantial cross-peak shifts without having functional effects .", "It is noteworthy that the D326K mutation caused not only shifts but also decreased intensities in the cross-peaks from the four methionines of the Munc18-1 loop in the 1H-13C HMQC spectra of the syntaxin-1 ( 2–253 ) -bound form , whereas other methionine cross-peaks generally had similar intensities in the WT and D326K spectra ( Figure 6—figure supplement 3 , right panel ) .", "Moreover , two new cross-peaks were observed in the spectrum of D326K 50%-2H-ILMV-13CH3-Munc18-1 bound to syntaxin-1 ( 2–253 ) that were not present in the spectrum of the WT complex ( indicated by ** in Figure 6H ) .", "These observations suggest that the D326K mutation destabilizes the furled loop structure , inducing the formation of an alternative ( perhaps unfurled ) structure in a population of the Munc18-1-syntaxin-1 ( 2–253 ) complexes , and resulting in an equilibrium between the two structures that is relatively slow in the NMR time scale .", "The observation of only two new cross-peaks for the alternative structure might arise because its population is low , because of overlap with other cross-peaks and/or because exchange broadening renders some new cross-peaks unobservable .", "Note that exchange broadening may also contribute to the decreased intensities of the cross-peaks from the four methionines in the furled loop structure .", "For isolated Munc18-1 , the D326K mutation also caused decreases in the intensities of the four methionine cross-peaks from the furled loop , but the decreases were less marked ( Figure 6—figure supplement 3 , left panel ) , and only one of the new cross-peaks was observed ( ** in Figure 6G ) , but with lower intensity than for the syntaxin-1 ( 2–253 ) -bound form .", "Thus , the D326K mutation also appears to induce the formation of an alternative structure in isolated Munc18-1 but with a lower population than in the complex with syntaxin-1 ( 2–253 ) .", "It is tempting to speculate from these findings that formation of the alternative structure underlies the increased activity of the Munc18-1 D326K mutant in the membrane-fusion and SNARE-complex-assembly assays , as well as its increased binding to synaptobrevin , and that this increased binding is less pronounced than the increases in activity because formation of the alternative structure is less favored in isolated Munc18-1 than in the Munc18-1–syntaxin-1 complex .", "Mammalian Munc18-1 rescues the paralysis phenotype of unc-18 nulls in C . elegans , but the rescue is only partial ( Gengyo-Ando et al . , 1996 ) and rat Munc18-1 bearing a P335A mutation yields a much more efficient rescue than the WT protein ( S . Park and S . Sugita , unpublished results ) .", "As the functional effects caused by the P335A mutation are probably related to those induced by the D326K mutation , we tested the ability of the D326K rat Munc18-1 mutant to rescue the paralysis phenotype observed in C . elegans unc-18 ( e81 ) nulls .", "Indeed , the D326K Munc18-1 mutant was much more efficient in rescuing paralysis than was WT Munc18-1 ( Figure 7A ) .", "In addition , the behavioral enhancement of unc-18 ( e81 ) nulls expressing the mutant Munc18-1 was explained by increased synaptic transmission , which was indirectly measured as the sensitivity of C . elegans to aldicarb , an inhibitor of acetylcholine esterase ( see Materials and methods ) .", "In the presence of aldicarb , C . elegans becomes paralyzed due to the persistent contraction of muscles arising from the accumulation of acetylcholine at the neuromuscular junction ( Miller et al . , 1996; Nonet et al . , 1998; Mahoney et al . , 2006 ) .", "Consistent with the enhancement in motility , unc18 ( e81 ) nulls expressing the D326K mutant Munc18-1 exhibited higher sensitivity to 1 mM aldicarb than those expressing WT Munc18-1 ( Figure 7B ) .", "Immunoblots showed comparable levels of expression for the WT and D326K mutant Munc18-1 proteins ( Figure 7—figure supplement 1 ) , indicating that the observed functional differences do not arise from differential expression levels .", "Importantly , the aldicarb sensitivity of unc-18 ( e81 ) nulls expressing the mutant Munc18-1 reached levels that were close to those of WT ( N2 ) animals ( Figure 7B ) .", "These experiments show that the D326K mutation in Munc18-1 , which enhances synaptobrevin binding and the activity of Munc18-1 in the in vitro fusion assays , also leads to an overt gain of function in live animals . 10 . 7554/eLife . 24278 . 018Figure 7 . The Munc18-1 D326K mutation leads to a gain-of-function in C . elegans .", "( A ) The motility of C . elegans was measured by counting the number of thrashings per min in liquid medium .", "unc-18 null mutants expressing multiple copies of D326K Munc18-1 swim with higher thrashing rates than those expressing multiple copies of WT Munc18-1 .", "Error bars indicate SEM ( number n of worms per strain = 16–21 ) .", "An asterisk indicates statistical significance at p<0 . 05 .", "( B ) Aldicarb assays were conducted to assess the sensitivity of rescued unc-18 null mutants to 1 mM aldicarb .", "unc-18 null mutants expressing multiple copies of D326K Munc18-1 were more sensitive to aldicarb than those expressing multiple copies of WT Munc18-1 .", "The sensitivity of unc-18 nulls expressing multiple copies of D326K Munc18-1 was similar to that of WT N2 worms .", "Error bars indicate SEM ( number n of experiments = 4 , where 10–20 worms per strain were analyzed in each experiment ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 01810 . 7554/eLife . 24278 . 019Figure 7—figure supplement 1 . Immunoblot analysis of unc-18 ( e81 ) mutants expressing WT and D326K mutant forms of rat Munc18-1 . Anti-Munc18-1 monoclonal antibody ( 1:1000 ) from BD Biosciences detected a similar expression level of WT and D326K mutant forms of Munc18-1 in transgenic animals , but the antibody did not cross-react with endogenous UNC-18 protein expressed in N2 worms . DOI: http://dx . doi . org/10 . 7554/eLife . 24278 . 019" ], [ "Munc18-1 is crucial for neurotransmitter release much as SM proteins are crucial for traffic at most intracellular membrane compartments .", "The function of these proteins has proven enigmatic , in part because of the diversity of their interactions with SNAREs ( Rizo and Südhof , 2012 ) , but recent progress has brought key insights to help solve the mystery .", "Research on yeast vacuolar fusion showed that the HOPS tethering complex containing the SM protein Vps33 is required for SNARE complex assembly in the presence of Sec17 and Sec18 , the yeast homologues of αSNAP and NSF ( Xu et al . , 2010a ) , and Munc18-1 ( together with Munc13s ) was later shown to orchestrate SNARE complex assembly in an NSF-αSNAP-resistant manner ( Ma et al . , 2013 ) .", "The findings that a loop in domain 3a of Munc18-1 adopts distinct conformations ( Hu et al . , 2011 ) , that Munc18-1 binds to synaptobrevin ( Xu et al . , 2010b ) , and that the L348R mutation that disrupts this interaction impairs Munc18-1 function , led to the notion that Munc18-1 provides a template for the formation of the SNARE complex ( Parisotto et al . , 2014 ) .", "This proposal and its generality for SM protein function were emphatically supported by the crystal structures of Vps33 with Vam3 and Nyv1 ( Baker et al . , 2015 ) .", "However , the dependence of Munc18-1 function on synaptobrevin binding was not clearly established , and the relation of this role of Munc18-1 to its tight functional interplay with Munc13s was unknown .", "Our results now reveal a clear correlation between Munc18-1 binding to synaptobrevin and its activity in stimulating membrane fusion in reconstitution assays that depend critically on both Munc18-1 and Munc13-1 .", "Moreover , the gain-of-function observed in these assays for the D326K mutation , which was designed to unfurl the Munc18-1 loop , is mirrored in a gain-of-function in C . elegans .", "These results strongly support the template model of SM-protein function and the notion that synaptobrevin binding is autoinhibited by the furled loop conformation in domain 3a .", "Our data also suggest that this autoinhibition of Munc18-1 function is important to enable control of release by Munc13s , thus allowing a wide range of Munc13-dependent regulatory processes that are vital for brain function .", "It may seem surprising that , although the molecular mechanism of neurotransmitter release has been studied intensely for 30 years , the importance of Munc18-1 binding to synaptobrevin has been recognized only recently .", "This delay arose in part because much attention was paid to the tight interaction of Munc18-1 with syntaxin-1 ( Hata et al . , 1993 ) and its implications in inhibiting or perhaps assisting in SNARE complex assembly ( Dulubova et al . , 1999; Misura et al . , 2000 ) .", "Munc18-1 was later found to bind to the SNARE complex and synaptobrevin binding was proposed to be important in this context ( Shen et al . , 2007; Dulubova et al . , 2007 ) .", "Binding of Munc18-1 to isolated synaptobrevin was probably missed in some early studies because it is weak and difficult to detect with the pulldown assays commonly used to identify protein–protein interactions .", "Recent pulldown assays did detect the interaction , but using a large synaptobrevin excess and antibody detection ( Parisotto et al . , 2014 ) .", "Munc18-1–synaptobrevin binding washas been readily observed by NMR spectroscopy , which is well suited to the analysis of weak interactions ( Rizo et al . , 2012 ) , but the interaction was dominated by the promiscuous C-terminus of the synaptobrevin SNARE motif ( Xu et al . , 2010b ) .", "Our NMR analyses now provide crucial clarification in this area .", "On one hand , the findings that the C-terminus of the synaptobrevin SNARE motif also binds to the Munc13-1 MUN domain ( Figure 2—figure supplement 1 ) and that its interaction with Munc18-1 is largely unaffected by the D326K or L348R mutations ( Figure", "3 ) strongly supports the conclusion that this interaction is not specific , although we cannot rule out its physiological relevance .", "On the other hand , our NMR results reveal an interaction of Munc18-1 with more central sequences of the synaptobrevin SNARE motif ( Figure", "2 ) that are not involved in binding to the Munc13-1 MUN domain and that , on the basis of the Vps33-Nyv1 crystal structure , are expected to be involved in binding to Munc18-1 .", "Moreover , Munc18-1 binding to these sequences is specifically altered by the D326K and L348R mutations ( Figure 3 ) .", "The D326K mutation designed to destabilize the furled loop conformation of domain 3a of Munc18-1 increases binding , whereas the L348R mutation impairs binding as shown earlier ( Parisotto et al . , 2014 ) and in agreement with the Vps33-Nyv1 binding mode ( Baker et al . , 2015 ) .", "In correlation with these findings , the L348R mutation impairs Munc18-1 function in reconstitution assays and the D326K mutation stimulates Munc18-1 activity ( Figure 4 ) , results that also correlate with the ability of these mutants to support SNARE complex assembly starting from their binary complex with syntaxin-1 ( Figure 5 ) .", "Moreover , the D326K mutation leads to a dramatic gain-of-function in rescue experiments in C . elegans ( Figure 7 ) .", "Together with previous studies ( Parisotto et al . , 2014; Baker et al . , 2015 ) , these results strongly support the notion that Munc18-1 templates SNARE complex assembly via interactions with synaptobrevin and syntaxin-1 that resemble those of Vps33 with Nyv1 and Vam3 , and that are likely to be conserved across the SM protein and SNARE families .", "Establishing definitively whether and how the D326K mutation in Munc18-1 alters the structure of the domain 3a loop is challenging , but our NMR data ( Figure 6 and Figure 6—figure supplement", "2 ) do show that the loop adopts defined structures that do not appear to be unfurled in both isolated and syntaxin-1 ( 2–253 ) -bound Munc18-1 .", "The data are consistent with the furled loop structure observed in the crystal structure of the Munc18-1-syntaxin-1 complex , but indicate that the structure differs considerably in the free and syntaxin-1-bound Munc18-1 .", "The decreases in the intensities of the methionine methyl cross-peaks from the loop region observed in the 1H-13C HMQC spectra ( Figure 6—figure supplement", "3 ) and the appearance of new cross-peaks caused by the D326K mutation ( Figure 6G , H ) suggest that the mutation destabilizes the structure of the loop both in the isolated Munc18-1 and in the Munc18-1-syntaxin-1 ( 2–253 ) complex , but more markedly in the latter .", "Thus , although these results are not definitive , they support the notion that the effects of the D326K mutation on synaptobrevin binding and on the activity of Munc18-1 in the membrane fusion and SNARE complex assembly assays arise from structural destabilization , and hence that Munc18-1 is autoinhibited both in isolation and when bound to syntaxin-1 .", "Such autoinhibition may underlie in part the weak affinity of the Munc18-1–synaptobrevin interaction , but it appears that the interaction is intrinsically weak , as binding is not saturated at 14 . 5 µM protein concentration even for the D326K mutant ( Figure 2 ) .", "However , when a synaptic vesicle docks at the active zone , or when V- and T-liposomes dock in our reconstitution assays , binding should be favored by the high local concentrations of not only synaptobrevin but also Munc18-1 , which initially is most likely to be bound to syntaxin-1 ( Ma et al . , 2013 ) .", "Note also that weak interactions can have dramatic effects in catalyzing the assembly of a protein complex , as a ten-fold acceleration in assembly rate requires a decrease in the energy barrier of just 1 . 4 kcal/mol , which can be provided by a single hydrogen bond .", "Hence , the Munc18-1–synaptobrevin interaction might play a catalytic role in promoting SNARE complex assembly by placing the synaptobrevin and syntaxin-1 SNARE motifs near each other , in the correct register and orientation ( as suggested by the Vps33 crystal structures with Nyv1 and Vam3 [Baker et al . , 2015] ) .", "At the same time , these interactions may prevent antiparallel interactions of synaptobrevin with syntaxin-1 ( see [Weninger et al . , 2003] ) that could inhibit synaptic vesicle fusion .", "This model does not rule out the notion that Munc18-1 may remain bound to the SNARE complex after assembly and might participate in downstream events that lead to membrane fusion .", "In this context , it is worth noting that the D326K mutation may increase the binding of Munc18-1 to the SNARE complex ( Table 1 ) , which might also underlie in part the gain-of-function caused by the mutation .", "The tight Ca2+-dependence of membrane fusion in our reconstitution assays depends on Ca2+ binding to the Munc13-1 C2B domain by a mechanism that is not understood but that may be related to a Ca2+-sensing role in release or promotion of an activated state of Munc13-1 during repetitive stimulation ( Liu et al . , 2016; Shin et al . , 2010 ) .", "Regardless of which of these two possibilities is correct , the facts that Ca2+-free Munc13-1 C1C2BMUNC2C can dock V-liposomes to T-liposomes and that there can be efficient lipid mixing with little content mixing before Ca2+ addition in our fusion assays indicate that Munc13-1 C1C2BMUNC2C helps in the fusion reaction by promoting such docking ( Liu et al . , 2016; Xu et al . , 2017 ) , but also imposes an energy barrier that is overcome by Ca2+ binding to its C2B domain .", "In reconstitution experiments with WT Munc18-1 , the additional energy barriers caused by the closed conformation of syntaxin-1 and the furled loop conformation of Munc18-1 probably hinder SNARE complex assembly to such an extent that full assembly and membrane fusion can only occur when Ca2+ binding to the Munc13-1 C2B domain lowers one of the existing energy barriers .", "Our reconstitution data show that this Ca2+ binding event becomes less critical when Munc18-1 bears the D326K mutation ( Figure 4C , D ) , probably because this mutation decreases the energy barrier caused by the furled loop conformation .", "In fact , some fusion can occur even in the absence of Munc13-1 C1C2BMUNC2C when using the Munc18-1 D326K mutant ( Figure 4E , F ) , suggesting that , without a furled loop conformation in Munc18-1 , SNARE complex formation can occur without an absolute requirement for the membrane bridging and syntaxin-1 opening activities of Munc13-1 C1C2BMUNC2C .", "This notion is reminiscent of the finding that syntaxin-1 bearing an LE mutation that disrupts its closed conformation ( Dulubova et al . , 1999 ) can partially rescue neurotransmitter release in unc-13 nulls in C . elegans ( Richmond et al . , 2001 ) , and suggests that there are at least two energy barriers within the Munc18-1–syntaxin-1 complex that hinder SNARE complex assembly and thus contribute to make Munc13-1 critical for release: one arises from autoinhibition of syntaxin-1 and the other from autoinhibition of Munc18-1 .", "Without these autoinibitory interactions , Munc13-1 would not be essential , but then the varied modes of regulation of release during presynaptic plasticity that depend on Munc13-1 would be lost .", "These ideas emphasize the importance of autoinhibitory interactions in achieving the exquisite regulation of neurotransmitter release and need to be explored with further research in the future ." ], [ "We previously described expression in BL21 Escherichia coli cells and purification of the following proteins: rat syntaxin 1A ( residues 2–253 ) ; rat synaptobrevin 2 ( residues 1–96 or 29–93 ) ; rat SNAP-25A full length ( with the four cysteines mutated to serines ) ; and fragments corresponding its SNARE motifs ( residues 11–82 and residues 141–203 ) ; full-length rat Munc18-1; rat synaptotagmin-1 C2AB fragment ( residues 131–421 ) ; full length Cricetulus griseus NSF V155M mutant; and full-length Bos taurus αSNAP ( Dulubova et al . , 1999; 2007; Chen et al . , 2002 , 2006; Xu et al . , 2013; Brewer et al . , 2015; Ma et al . , 2013 ) .", "Starting from the full-length rat Munc18-1 sequence , we used standard site-directed mutagenesis techniques and custom-designed primers to generate Munc18-1 mutants ( M316L , D326K , M330E , L348R , and M334L ) .", "Mutants were expressed and purified as WT Munc18-1 .", "Rat Munc13-1 C1C2BMUNC2C fragment ( residues 529–1735 , with the sequence from a flexible loops corresponding to residues 1408–1452 replaced by EF ) was expressed and purified from Sf9 cells as described previously ( Liu et al . , 2016 ) .", "To obtain uniformly 15N-labeled synaptobrevin , we used M9 minimal expression media with 15NH4Cl as the sole nitrogen source ( 1 g/L ) .", "WT and mutant 50%-2H-ILMV-13CH3-Munc18-1 proteins were produced using M9 expression media in 50% D2O with 2H-glucose as the sole carbon source ( 3 g/L ) and 15NH4Cl as the sole nitrogen source ( 1 g/L ) , and by adding [3 , 3-2H2] 13C-methyl alpha-ketobutyric acid ( 80 mg/L ) , [3-2H] 13C-dimethyl alpha-ketoisovaleric acid ( 80 mg/L ) , and 13C-methyl methionine ( 250 mg/L ) ( Cambridge Isotope Laboratories , Tewksbury , Massachussetts ) to the cell cultures 30 min prior to induction with 0 . 4 mM isopropyl β-D-1-thiogalactopyranoside overnight at 20°C .", "NMR spectra were acquired on Agilent DD2 spectrometers operating at 800 or 600 MHz .", "1H-15N HSQC spectra ( Zhang et al . , 1994 ) were acquired at 20°C .", "Samples contained 14 . 5 µM 15N-synaptobrevin ( 1–96 ) with or without 14 . 5 µM Munc18-1 ( WT or mutant versions ) dissolved in 20 mM HEPES ( pH 7 . 2 ) , 120 mM KCl an 1 mM TCEP , containing 5% D2O .", "1H-13C HMQC spectra ( Ruschak and Kay , 2010 ) were acquired at 25°C .", "Samples contained WT or mutant 50%-2H-ILMV-13CH3-Munc18-1 ( 15–20 µM ) in the absence or presence of excess syntaxin-1 ( 2–253 ) ( 20–22 µM ) , and were dissolved in 50 mM Tris pH 8 . 0 , 150 mM KCl , 2 . 5 mM CaCl2 and 1 mM TCEP containing 8% D2O .", "Total acquisition times ranged from 1 . 5 to 6 hr for 1H-15N HSQC spectra and from 48 to 60 hr for 1H-13C HMQC spectra .", "All data were processed with NMRpipe ( Delaglio et al . , 1995 ) and analyzed with NMRView ( Johnson and Blevins , 1994 ) .", "ITC experiments were performed using a VP-ITC system ( MicroCal , Westborough , Massachusetts ) at 20°C .", "Syntaxin-1 ( 2–253 ) ( 8–24 μM ) was titrated into the cell containing Munc18-1 or mutants ( 0 . 5–2 μM ) in buffer A ( 20 mM Hepes , pH 7 . 2 , 120 mM KCl and 1 mM TCEP ) .", "SNARE complex ( 30–50 μM ) was titrated into Munc18-1 or mutants ( 1 . 5–2 . 5 uM ) in buffer B ( 20 mM Hepes , pH 7 . 4 , 150 mM KCl and 1 mM TCEP ) .", "SNARE complexes were formed with SNAP-25 ( 11–82 ) , SNAP-25 ( 141–203 ) , syntaxin-1 ( 2–253 ) and synaptobrevin ( 1–96 ) .", "Complex assembly was accomplished by incubating a mixture of the purified fragments overnight and then removing remaining unassembled fragments by repeated rounds of size-exclusion gel filtration to efficiently eliminate unassembled syntaxin-1 ( 2–253 ) , which binds very tightly to Munc18-1 and could thus perturb the measurements of SNARE complex binding .", "All proteins and the SNARE complex were purified by gel filtration and dialyzed in the same buffer before the experiments .", "The data were baseline corrected with NITPIC , fitted with a nonlinear least squares routine using a single-site binding model with ITCsy , and plotted with GUSSI ( Brautigam et al . , 2016 ) .", "The ‘A + B <-> AB’ model was used for the fitting , and apparent concentration errors for the cell contents were compensated for by refining a single correction factor for this parameter .", "The 68 . 3% confidence intervals were obtained using the error surface projection method .", "Global analysis with SEDPHAT or ITCsy was not carried out in order to capture experiment-to-experiment variation in refined parameters .", "Assays that simultaneously measured lipid mixing from de-quenching of the fluorescence of Marina Blue-labeled lipids and content mixing from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes were performed as described previously ( Liu et al . , 2016 ) .", "V-liposomes with synaptobrevin ( protein-to-lipid ratio 1:500 ) contained 39% POPC , 19% DOPS , 19% POPE , 20% cholesterol , 1 . 5% NBD PE , and 1 . 5% Marine Blue PE .", "T-liposomes with syntaxin-1–SNAP-25 ( protein-to-lipid ratio 1:800 ) contained 38% POPC , 18% DOPS , 20% POPE , 20% cholesterol , 2% PIP2 and 2% DAG .", "Liposomes were in a buffer that included 25 mM HEPES , pH7 . 4 , 150 mM KCl , 0 . 5 mM TCEP and 10% glycerol ( v/v ) .", "V-liposomes ( 0 . 125 mM lipids ) were mixed with T-liposomes ( 0 . 25 mM lipids ) with various additions of the other components in a total volume of 200 µl at final a concentration of 2 . 5 mM MgCl2 , 2 mM ATP , 0 . 1 mM EGTA , 5 µM streptavidin , 0 . 8 µM NSF , 2 µM αSNAP , 1 µM Munc18-1 ( wild type or mutants ) , and 1 µM excess SNAP-25 , with or without 0 . 5 µM Munc13-1 C1C2BMUNC2C , and with or without 1 µM synaptotagmin-1 C2AB .", "T-liposomes were first incubated with NSF , MgCl2 , ATP , EGTA , streptavidin , NSF , αSNAP and Munc18-1 at 37°C for 25 min , and then mixed with V-liposomes and excess SNAP-25 with or without Munc13-1 C1C2BMUNC2C and/or synaptotagmin-1 C2AB as indicated in the figure legends .", "CaCl2 at final concentration of 0 . 6 mM was added after 300 s of the start of the reaction .", "The fluorescence signals were monitored with a PTI spectrofluorometer ( Edison , NJ ) .", "The emission fluorescence of Marina Blue-PE at 465 nm ( excitation at 370 nm ) was measured to monitor lipid mixing .", "The emission fluorescence of Cy5–streptavidin at 670 nm was measured with excitation of PhycoE-biotin at 565 nm to monitor content mixing .", "All experiments were performed at 30°C .", "At the end of each reaction , 1% w/v β-OG was added to solubilize the liposomes , and all the lipid mixing data were normalized to the maximum fluorescence signal achieved after addition of β-OG .", "Control experiments without streptavidin were performed to measure the maximum Cy5 fluorescence attainable upon detergent addition .", "WT or mutant Munc18-1 ( 6 µM ) was incubated with syntaxin-1 ( 2–253 ) ( 5 µM ) for 20 min at room temperature .", "Synaptobrevin ( 29–93 ) ( 10 µM ) and SNAP-25 ( 10 µM ) were then added and samples were incubated at room temperature for 3 hr in 25 mM Hepes , pH 7 . 4 , 150 mM KCl , 10% glycerol ( v/v ) .", "Samples were loaded onto 15% tris-glycine native gels and run at 80 V , 4°C for 6 hr .", "Gels were stained with Coomassie blue and imaged on a ChemiDoc MP Imaging System ( Bio-Rad Laboratories , Hercules , California ) .", "The monomeric/oligomeric state of WT and mutant Munc18-1 proteins alone or bound to syntaxin-1 ( 2–253 ) was investigated at 25°C by static light scattering using a Wyatt DynaPro NanoStar instrument ( Wyatt Technology , Santa Barbara , CA ) .", "Samples contained 15 µM protein concentrations dissolved in 50 mM Tris pH 8 . 0 , 150 mM KCl and 1 mM TCEP .", "The data were analyzed using the program Dynamics version 7 . 5 . 0 ( Wyatt Technology , Santa Barbara , CA ) .", "pMunc18-1 ( a kind gift from Dr Hitoshi Kitayama , Kyoto University ) described in Gengyo-Ando et al . ( 1996 ) was used as template to generate the rescue constructs that express WT or D326K-mutant forms of rat Munc18-1 .", "These plasmids contained ~3 kb of unc-18 promoter , ~1 . 8 kb cDNA of Munc18-1 , and ~1 kb of 3’ poly ( A ) signal of the unc-18 gene ( Gengyo-Ando et al . , 1996 ) .", "A mixture of each Munc18-1 expression plasmid and a co-injection maker ( pMyo3-RFP ) was prepared such that the final concentration of each DNA was ~50 ng/ul .", "This mixture was injected into the gonads of young adult worms of the CB81 strain ( genotype: unc-18 ( e81 ) ) .", "Three to 4 days after the injection , the F1 generation , which is the progeny of the injected worms , was screened for red fluorescence , and only RFP-positive F1 worms were singled out .", "The motility of C . elegans was evaluated by counting the number of thrashings per min in liquid medium .", "One-day-old adult worms free of OP50 bacteria were first transferred to a non-seeded plate containing 1 ml of M9 buffer .", "After a 2 min adjustment period in M9 buffer , the worms were video-recorded for 1 min .", "A single trashing was defined as a bending at the mid body , with both head and tail pointing to the opposite direction of mid body movement .", "For comparison of multiple groups , one-way ANOVA ( analysis of variance ) followed by Tukey’s range test was conducted , with a significance level of 0 . 05 .", "All statistical tests were performed with OriginPro 9 . 0 .", "The sensitivity of C . elegans to aldicarb was tested by examining one-day-old adult worms on non-seeded NGM plates containing 1 mM aldicarb .", "During the assays , animals were assessed for paralysis at multiple time points .", "They were considered paralyzed if their tail did not move after their head was tapped three times .", "Protein extract was prepared as described in Weimer et al . ( 2003 ) .", "Briefly , we plated ~50 RFP ( co-injected marker protein ) -positive unc-18 ( e81 ) mutant worms expressing WT and D326K mutant rat Munc18-1 on 6 cm nematode growth medium ( NGM ) plates , and cultured them while manually removing RFP-negative offspring worms .", "As controls , we also cultured unc-18 ( e81 ) mutant worms and N2 worms .", "When the plates were full of worms with little OP50 left , worms were harvested and rinsed three times with a buffer containing 360 mM sucrose , 12 mM HEPES , and a protease inhibitor cocktail ( 1 µg/ml pepstatin A , 1 µg/ml leupeptin , 1 µg/ml aprotinin and 0 . 1 mM PMSF ) .", "The worms were resuspended in around five times the volume of the buffer and frozen at −80°C until use .", "The defrosted worms were sonicated on ice ten times with a 5 s burst .", "The resulting lysate was centrifuged for 15 min to pellet the cuticle , nuclei , and other debris .", "After centrifugation , the supernatant ( final protein concentrations were ~3–5 mg/ml ) was transferred to a clean microcentrifuge tube with an equal volume of sample buffer 2X .", "50 µg of samples were subjected to SDS-PAGE electrophoresis followed by immunoblotting .", "The expression of transgenic Munc18-1 was detected by anti-Munc18-1 monoclonal antibody ( 1:1000 ) from BD Biosciences ." ] ]

[ "Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release .", "Munc18-1 binds to synaptobrevin , but the relevance of this interaction and its relation to Munc13 function are unclear .", "NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin .", "Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the ‘furled conformation’ of a Munc18-1 loop .", "Correspondingly , the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation .", "Moreover , the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls .", "Together with previous studies , our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly , and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1 ." ]

[ "Nerve cells communicate with other nerve cells by releasing small molecules called neurotransmitters .", "The neurotransmitters are first packaged inside bubble-like structures called vesicles , which fuse with the membrane of the nerve cell when it is stimulated .", "Once the vesicle and membrane have fused , the neurotransmitters are released outside the nerve cell and are detected when they bind to proteins on the surface of other nearby nerve cells .", "A machinery of different proteins controls membrane fusion .", "Amongst these proteins are five called Munc18-1 , Munc13-1 , syntaxin-1 , synaptobrevin and SNAP-25 .", "The last three form a tight bundle called SNARE complex that brings the vesicle and cell membrane together and is essential for the two to fuse .", "Munc18-1 and Munc13-1 orchestrate the assembly of the SNARE complex .", "Previous studies suggested that Munc18-1 binds to synaptobrevin , providing a template to bring syntaxin-1 and synaptobrevin together and thereby helping the SNARE complex to form .", "However , the importance of the interaction between Munc18-1 and synaptobrevin was not clearly established , and it was not known how Munc13-1 is involved .", "Sitarska , Xu et al . have now measured how mutated versions of Munc18-1 bind to synaptobrevin and tested how the mutations affect membrane fusion .", "A mutation in Munc18-1 that increased binding to synaptobrevin increased membrane fusion too , while a mutation that decreased binding had the opposite effect and reduced fusion .", "The results support the idea that Munc18-1 provides a template for the SNARE complex to form .", "One mutation stimulated Munc18-1 so that Munc13-1 was no longer needed for fusion when the mutant Munc18-1 was tested in fusion assays with artificial membranes .", "This mutation was designed to perturb the structure of a region of Munc18-1 protein that normally inhibits the binding of synaptobrevin .", "These results suggest that by adopting a state where it cannot bind synaptobrevin , Munc18-1 can only be stimulated to form the SNARE complex and trigger release of neurotransmitter when Munc13-1 is present .", "This provides a way for Munc13-1 , which is regulated by many factors , to fine-tune the release of neurotransmitter .", "Future work will test whether these proteins work in the same way in living animals .", "This will help us understand how communication between neurons is finely controlled to enable the brain to carry out its many different tasks ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "In daily life , reward contingencies are often unstable; an action that once produced a valued outcome may over time become less desirable .", "The ability to shift responses away from the previously valued action-outcome pair is a hallmark example of behavioral flexibility .", "Flexible responding ( i . e . , adapting behavior to reflect new reward contingencies or reward values ) has been evaluated extensively through the use of reinforcer devaluation tasks ( Málková et al . , 1997; Hatfield et al . , 1996 ) .", "In these tasks , the value of secondary reinforcers or operanda ( e . g . , objects ) , and actions that were once favored decrease following an experimental reduction in the value of the associated primary reinforcer .", "Processes required for optimal performance in the reinforcer devaluation task include: [1] registering a change in value of the primary reinforcer , [2] integrating the new value of the primary reinforcer with the cognitive representation of the secondary reinforcers/operanda ( e . g . , objects that signal the reward ) , and [3] guiding action selection to target the now optimal choice .", "In the standard version of this task used in macaques , animals are trained to associate sets of objects with one of two rewards ( primary reinforcers; e . g . , peanuts or fruit snacks ) .", "After the association between objects and particular rewards is established , subjects are presented with a forced choice between objects associated with each of the two foods .", "The proportion of choices between the objects rewarded with one type of food ( e . g . , peanuts ) versus the other ( e . g . , fruit snacks ) represents the baseline preference .", "An experimental reduction of reward value by selective satiation ( i . e . , providing one food to satiety ) produces a devaluation effect , i . e . , a decrease in the proportion of objects associated with the sated food that are selected .", "These processes critically rely on an interactive network including the orbitofrontal cortex ( OFC ) , the amygdala , and the mediodorsal thalamus ( MD ) .", "The amygdala projects directly to OFC , and indirectly to the OFC via the MD ( Timbie and Barbas , 2015 ) .", "MD is reciprocally interconnected with two critical subregions of the OFC , Areas 11 and 13 ( Ray and Price , 1993 ) .", "Lesions to each of these nodes impair the typical shift away from the objects that predict the sated food ( Málková et al . , 1997; Izquierdo et al . , 2004; Izquierdo and Murray , 2010; Browning et al . , 2015; Pickens , 2008; Mitchell et al . , 2007 ) .", "Moreover , crossed lesions of any two nodes of this circuit likewise impair performance .", "While lesions provide strong evidence that these brain regions are required for task performance , they do not have the temporal specificity needed to dissociate the contributions during discrete phases of task performance .", "By contrast , focal pharmacological manipulations , which can transiently suppress activity within a brain region , have revealed differential roles for the amygdala and the OFC in particular phases of this task ( Wellman et al . , 2005; West et al . , 2011 ) .", "While neither the amygdala , nor the OFC are needed to register a change in value of the primary reinforcer , both structures play critical and differential roles in the subsequent processes .", "The amygdala is necessary for adjusting the object representations to reflect the new value of the primary reinforcer , but not necessary for optimal action selection ( Wellman et al . , 2005 ) ; a similar profile has also been observed for the OFC Area 13 .", "By contrast , Area 11 is critical only for action selection ( Murray et al . , 2015 ) .", "Because MD receives input from amygdala and is reciprocally connected with both Areas 11 and 13 of the OFC ( Timbie and Barbas , 2015 ) , it suggests that this structure may be central to the devaluation circuit .", "However , the role of the MD in specific phases of devaluation is unknown .", "Here , we considered two competing hypotheses regarding the role of the MD in this network: [1] it mirrors the function of the amygdala and Area 13 of the OFC , serving primarily as a ‘relay’ to process information regarding value updating between the amygdala , Area 11 and Area 13 , and [2] it mirrors the function of Area 11 of the OFC , contributing primarily to action selection .", "To dissociate between these outcomes , we tested four adult male rhesus macaques on a reinforcer devaluation task while transiently inactivating the MD during various stages of the task ( Málková et al . , 1997 ) ." ], [ "Animals were first trained on a set of forty concurrent object discriminations; through repeated trials the animals learned the association between each object and a particular reward ( see Materials and methods ) .", "Next , they were tested for preference on a baseline object probe test ( baseline probe ) .", "In this test , two objects , each baited with one of the two foods , were pitted against each other .", "On another day , animals underwent selective satiation ( pre-feeding with one of the two foods ) and were again tested on an object probe test ( sated probe ) , administered in a similar way as the baseline probe .", "This weekly sequence ( baseline probe followed by sated probe ) was repeated such that each food was sated for each experimental condition ( see Figure 1 for experimental timeline ) .", "In the above probe tests , the animal selected between two objects , each baited with one of the two food rewards .", "Thus , the objects were the cue used to guide action .", "We also tested animals in a ‘consummatory’ probe , in which they chose between two competing food rewards in the absence of objects; this served as a control to ensure successful devaluation of the primary reinforcer .", "A shift in choices between the baseline and sated probe tests is reflected by a positive proportion shifted ( see Materials and methods ) and indicates successful devaluation .", "A value of 1 indicates a complete shift away from objects predicting the sated food , a value of 0 indicates no shift in preference .", "Thus , decreases in proportion shifted after experimental manipulations indicate impaired reinforcer devaluation .", "On separate testing weeks , we microinjected the GABAa receptor agonist muscimol ( MUS , 9 nmol ) , the glutamate receptor antagonist kynurenic acid ( KYNA , 450 nmol ) , or saline into the MD , either prior to selective satiation or prior to the sated probe test ( Figures 2A1–4 ) .", "MRI-guided stereotaxic targeting of the MD was performed and confirmed ( Figure 2B ) as we have described for other regions ( Wellman et al . , 2005; West et al . , 2011 ) .", "We microinjected drug or vehicle at one of these two points during the task ( see Figure 2A ) , to dissociate potential impairments in value updating ( infusion before satiation; Figure 2A1 and A3 ) from impairments in action selection ( infusion prior to probe; 2A2 , 2A4 ) .", "Under baseline conditions , animals displayed a slight , but significant , preference for one type of reward ( Figure 2F ) .", "This was evident in their choosing a larger proportion of objects predicting that reward ( baseline preference ratio ) .", "This pattern is similar to what has been previously reported ( Mitchell et al . , 2007; West et al . , 2011 ) .", "Under saline-infused condition , animals displayed robust devaluation following satiation; the devaluation effect was demonstrated by a shift away from choices of objects associated with the devalued food .", "Microinjection of MUS either before satiation or before the sated probe test significantly disrupted the devaluation effect compared to sham/saline infusion; this was manifest as a decrease in the proportion shifted ( Figure 2C ) .", "This disruption was evident in all four subjects , resulting in a significant main effect of treatment ( F1 . 1 , 3 . 4=13 . 3; p=0 . 029 ) .", "Pairwise comparisons revealed a significant impairment in performance when MUS was infused either before satiation ( p=0 . 032 ) or before the sated probe test ( p=0 . 035 ) .", "The magnitude of impairment did not differ between these conditions ( p=0 . 13 ) .", "In contrast to the object probe sessions , MUS injection failed to alter choices in the consummatory probe ( Figure 2D; F1 . 3 , 4 . 0=1 . 25; p=0 . 35 ) .", "Because animals still displayed a typical shift away from sated primary reinforcers in the consummatory probe , the deficits seen in Figure 2C cannot be explained by impairment in satiety or valuation of primary reinforcers .", "Thus , the disruption in the devaluation effect was specific to adjusting the value of the objects to reflect the new value of the primary reinforcer ( i . e . , the sated food ) .", "In prior studies ( Izquierdo and Murray , 2010; Mitchell et al . , 2007; Wellman et al . , 2005; West et al . , 2011 ) , it has been reported that after displacing objects and revealing a devalued food reward , monkeys will avoid consuming the devalued food .", "While we did not systematically record the consumption of food during the object probe tests , anecdotally , there were trials in which the animals did not eat the devalued food after displacing the object .", "To determine if deficits in devaluation were secondary to impaired object recognition , we tested animals ( on a separate test day ) on the concurrent visual discrimination task following drug infusion .", "MUS injection was associated with a small , but significant impairment in concurrent visual discrimination ( Figure 2E; t = 4 . 09 , df = 3 , p=0 . 026 ) .", "It is possible that this finding is due to a drug-related deficit in object recognition , reward associations , and/or appropriate action selection .", "Interestingly , this deficit also likely contributed to the significant decrease in baseline preference ratio ( see Materials and methods ) observed after MUS injection ( Figure 2F; t = 4 . 28 , df = 3 , p=0 . 023 ) .", "Indeed , unlike under baseline ( non-sated ) condition when animals typically choose significantly more objects associated with one type of reward over the other , MUS presence abolished this baseline preference ratio ( Figure 2F ) .", "MUS injection produces long lasting inhibition , with effects evident for hours after drug injection ( Dybdal et al . , 2013 ) .", "Thus , injection before satiation likely results in a suppression of the activity within MD both during satiation and the probe test .", "Because injections both before and after satiation disrupted devaluation , these data alone were unable to clarify what , if any , role the MD played during the period of selective satiation .", "To determine if the MD is required specifically during selective satiation , we next turned to microinjection of KYNA , which has a short duration of action ( ~30 min ) ( Forcelli et al . , 2014 ) and short half-life within the brain ( 8–30 min ) ( Vécsei and Beal , 1990; Turski and Schwarcz , 1988 ) .", "Based on the timing of our experiments ( the probe session was conducted 45 min after drug infusion , ~3 half-lives ) , less than 10% of KYNA is expected to remain during the probe test .", "Thus , injection before satiation is expected to only disrupt activity during the period of satiation and not during the sated probe test .", "We confirmed , in two monkeys , that lower doses of KYNA infused immediately prior to the sated probe ( Figure 2—figure supplement 1 ) , did not impact performance .", "Thus the trace amounts of KYNA remaining when infused prior to satiation are unlikely to be sufficient to impact behavior during the probe test .", "KYNA infusion either during satiation , or before the sated probe test , impaired the devaluation effect ( Figure 2G ) .", "This pattern was evident in all three subjects , resulting in a significant main effect ( F1 . 0 , 2 . 1=52 . 3; p=0 . 017 ) with both test conditions differing significantly from saline-infused control sessions ( Infusion before satiation: p=0 . 021 , Infusion before sated probe: p=0 . 021 ) .", "Moreover , the impairment was of greater magnitude when KYNA was infused before the sated probe test ( p=0 . 043 ) .", "Similar to MUS infusion , KYNA infusion did not disrupt performance in the consummatory task ( Figure 2H; F1 . 1 , 2 . 1=1 . 11; p=0 . 40 ) .", "In contrast to MUS infusion , KYNA infusion spared performance in concurrent visual discrimination ( Figure 2I; identical values for all animals under all conditions precluded inferential statistical analysis across treatments ) .", "Similarly , KYNA infusion was without effect on baseline preference ratio ( Figure 2J; t = 0 . 277 , d = 2 , p=0 . 81 ) .", "In conclusion , impaired devaluation , in the absence of other deficits , indicates that the MD is required both for adjusting the value of object representations and for appropriate action selection ." ], [ "Our present findings delineate the role of the MD in each of the cognitive processes needed for reinforcer devaluation .", "We conclude that: [1] Because the expected shift in primary reinforcer preference seen after satiation occurred under all experimental conditions , the MD is not necessary for registering a change in primary reinforcer value .", "This is consistent with prior studies in which the MD was lesioned .", "[2] Because inactivation of the MD during satiation impaired reinforcer devaluation , activity in the MD is necessary for adjusting the value of the objects to reflect the new value of primary reinforcers .", "This deficit is similar to that seen after inactivation of the amygdala or Area 13 of the OFC .", "[3] Because inactivation of the MD during the probe session ( i . e . , after satiation was completed ) also impaired reinforcer devaluation , activity in the MD is required for appropriate action selection , a role also attributed to Area 11 of the OFC .", "Thus , we have demonstrated that the MD is required for both reward valuation and action selection; this represents a unique profile within the circuit supporting this behavior , differing from both the amygdala and subregions of the orbitofrontal cortex .", "We found a dissociation between the effects of MUS and KYNA with respect to performance on concurrent visual discrimination .", "While in the case of MUS , at least a portion of the observed deficit in reinforcer devaluation may be due to impaired object discrimination , this is not so for KYNA .", "After KYNA infusion , concurrent visual discrimination performance was left intact , but deficits in reinforcer devaluation were still evident .", "In prior studies , large lesions to the MD resulted in deficits in visual recognition/concurrent visual discrimination ( Gaffan and Parker , 2000; Aggleton and Mishkin , 1983 ) whereas lesions that damaged only the magnocellular portion of the MD ( the region we targeted with our drug infusions ) , did not ( Mitchell et al . , 2007 ) .", "Because of the differences in duration of action and timing of experimental manipulations , MUS likely spread to a larger area of the MD than did KYNA .", "While gadolinium is a reasonable proxy of drug spread , and prior functional data from our labs ( Dybdal et al . , 2013; Malkova et al . , 2015 ) and others help to estimate the volume of drug spread ( Martin and Ghez , 1999; Martin , 1991; Allen et al . , 2008 ) , it is indeed a technical limitation of temporary pharmacological inactivation that we cannot directly document the spread of drug for each individual infusion .", "While speculative , broader inactivation of the MD with MUS may have caused the deficit in concurrent visual discrimination .", "The degree to which inactivation of the MD would impair performance in other tasks where lesions have produced deficits ( Browning et al . , 2015; Chakraborty et al . , 2016 ) remains to be explored .", "Inactivation of the MD during the probe test ( KYNA infusion after satiation ) resulted in a larger deficit than inactivation during satiation; we consider at least two explanations .", "[1] Amygdala neurons ( necessary during satiation ) project directly to the OFC ( Timbie and Barbas , 2015; Timbie and Barbas , 2014 ) , and indirectly to the OFC via the MD ( Timbie and Barbas , 2015; Ray and Price , 1993 ) .", "Thus , even in the absence of MD function , amygdalofrontal projections may partially support updating of the object values .", "[2] Because Areas 11 and 13 of the OFC support different components of this task , cross-talk between these regions may be critical .", "Both of these regions are reciprocally interconnected with the MD ( Ray and Price , 1993 ) , thus , the MD may modulate and facilitate transmission between these cortical regions ( see Figure 2—figure supplement 2 for a comparison across studies ) .", "Consistent with this notion , crossed lesions to the OFC and the MD impaired devaluation ( Browning et al . , 2015 ) .", "Moreover , recent studies in rodents have shown that optogenetic activation of the MD enhances cortico-cortical communication and improves performance on prefrontal cortex dependent tasks , whereas optogenetic silencing of the MD increases errors during prefrontal-dependent task performance ( Schmitt et al . , 2017; Bolkan et al . , 2017 ) .", "Our present findings , which provide novel temporal information regarding the role of MD in reinforcer devaluation , are in general agreement with prior studies .", "Lesions to the MD produced similar impairments to those that we observed .", "While the effect magnitude has varied slightly from study to study , the general pattern has been consistent .", "Given the similarity in the findings between lesion and pharmacological inactivation methodologies , neither compensatory circuit alterations after lesions , nor off-target effects after drug infusion are likely to account for the deficits observed .", "Thus , together , these data and those previously published underscore a critical role for processing within the MD in behavioral flexibility .", "The present data delineate the role of the MD in each of the processes integral to reinforcer devaluation: [1] The MD is not necessary for registering a change in primary reinforcer value .", "[2] Activity in the MD is necessary for adjusting the value of the objects to reflect the new value of primary reinforcers .", "[3] The MD is required for appropriate action selection .", "Together , this pattern of deficits seen with inactivation of the MD is unique; unlike the amygdala , or individual subregions of OFC , the MD is required for multiple components of task performance .", "Rather than serving as a parallel pathway for information transfer between the amygdala and the OFC , these data instead suggest that the MD functions as a privileged and critical interface between other nodes of the devaluation circuitry ." ], [ "Four adult , male , rhesus macaque ( Macaca mulatta ) were subjects in the present study .", "At the start of the first reinforcer devaluation study they weighed 8 . 2–9 . 8 kg .", "They were housed with visual access to conspecifics in standard home cages ( 61 × 74×76 cm each ) .", "Water was available ad libitum in the home cage .", "Meals ( LabDiet #5049 ) were provided twice daily and supplemented with fresh fruit .", "The first meal was always given after behavioral testing occurred .", "The study was conducted under a protocol approved by the Georgetown University Animal Care and Use Committee ( #2016–1115 ) and in accordance with the Guide for Care and Use of Laboratory Animals ( Committee for the Update of the Guide for the Care and Use of Laboratory Animals , Institute for Laboratory Animal Research , Division on Earth and Life Studies , National Research Council , 2010 ) .", "These animals were previously tested on a within-session concurrent discrimination learning task ( unpublished ) and a previous study using the current reinforcer devaluation task with systemic drug administration ( Waguespack et al . , 2018 ) .", "The monkeys were trained in a Wisconsin General Testing Apparatus located in a darkened sound-shielded room .", "The test tray , which was located at the level of the floor of the monkey transport cage , contained two food wells spaced 18 cm apart ( center to center ) on the midline of the tray .", "The wells were 25 mm in diameter and 5 mm deep .", "The stimuli were 80 junk objects that differed widely in shape , size , color , and texture .", "We selected two highly palatable foods that are commonly used as reinforcers across laboratories for this task: fruit snacks and peanuts ( Málková et al . , 1997; Izquierdo and Murray , 2010; Wellman et al . , 2005 ) .", "Food one was a fruit snack ( Sharkbites; General Mills ) and food two was half of a honey roasted peanut ( Planters; Kraft Foods ) .", "Animals did not receive these food reinforcers outside of the context of this task ( e . g . , as enrichment or reinforcers for other tasks ) .", "Monkeys were trained on the task , as described previously ( Málková et al . , 1997 ) .", "The animals were first trained on a set of 40 object-discrimination problems .", "The objects were placed over the food wells; the monkey could only see and retrieve the food by displacing an object .", "In each of the 40 object pairs , one object ( S+ ) was baited with a food reinforcer and the other was unbaited ( S− ) .", "Half of the S+ objects were baited with fruit snacks and the other half baited with peanuts , intermixed within a session .", "The S+ and S− assignment of the objects , the order of the object pairs , and the food reinforcer paired with particular objects remained constant across days; however , the left–right positions of the S+ object was pseudorandomized .", "The intertrial interval between the presentations of two consecutive pairs was 20 s .", "The monkeys were trained at a rate of one session per day , 5 d a week , until they reached criterion , which was set at a mean of 90% correct responses or better over five consecutive days ( i . e . , 180 or more correct responses of 200 ) .", "During the course of this training animals learned , incidentally , the food-object association .", "Upon reaching criterion on the concurrent visual discrimination , the animals’ choices of objects were assessed in probe sessions , in which only baited objects ( S+ ) were used .", "For each probe session , the 40 S+ objects were randomly assigned to create 20 pairs of probe trials , each offering a choice between an object baited with fruit snack and an object baited with peanut .", "The left-right position of the object-food pairs was randomized within a session and across days .", "On each probe trial , the animal was allowed to select one of the two objects and retrieve the food reward .", "By repeating this for each of the 20 pairs of probe trials , this allowed for the assessment of baseline preference ( Figure 2F and Figure 2J ) for object-food pairs .", "Animals typically choose more objects baited with one type of food reward ( e . g . , Food", "1 ) over those baited with the other type ( e . g . , Food 2 ) .", "Those chosen in higher numbers are considered ‘preferred’ .", "Baseline preference ratio was calculated with the following equation:Fpreferred/Ftotalwhere the number of objects chosen with preferred primary reinforcer were divided by the total number of objects chosen .", "Other probe sessions ( sated probe; see below ) were preceded by a devaluation procedure , in which the monkey was allowed to consume one of the two food rewards to satiation ( selective satiation ) .", "To experimentally induce ‘devaluation’ of one reinforcer , animals were subjected to selective satiation .", "Selective satiation sessions were conducted >14 hr after the last feeding .", "During the satiation procedure , a food box attached to the monkey’s home cage was filled with one of the two food reinforcers ( either food 1 or food", "2 ) of a measured quantity .", "The monkey was allowed to eat the food for 15 min , at which point the experimenter re-entered the room and checked the amount of food eaten .", "If the monkey consumed all of the initially offered food , the experimenter gave the monkey more food .", "This was repeated until the animal did not take additional food over a five minute period .", "At that time , the remaining food was removed and measured .", "In all cases , 30 min was a sufficient time to complete this procedure .", "After selective satiation , animals were tested in the probe sessions as described above .", "Typically-responding animals will shift behavior such that objects associated with the sated ( devalued ) food are rejected in favor of the objects associated with the non-sated ( non-devalued ) food .", "This results in a clear shift in the preference score toward the objects associated with the non-devalued food .", "Each object was presented only once during the probe session , so that the assessment was uncontaminated by new learning .", "Therefore , the rejection of an object associated with the sated food is an indication that the devaluation of the food was cognitively transferred to the object .", "In a counterbalanced manner , this procedure was repeated for each of the two foods for each test condition .", "Data across these two devaluation sessions for each condition were used to evaluate the magnitude of devaluation , which was assessed by measuring the proportion shifted , previously described ( Murray et al . , 2015 ) .", "Proportion shifted was calculated according to the formula:Proportionshifted=[ ( F1N−F1D ) +[ ( F2N−F2D ) ]/ ( F1N−F2N ) where F1N is the number of objects baited with fruit snacks chosen during the baseline probe , F1D is the number objects baited with fruit snack chosen during the sated probe , F2N is the number of objects baited with peanuts chosen during the baseline probe and F2D is the number of objects baited with peanuts chosen during the sated probe .", "The amount of food consumed during the selective satiation experiments is shown in Supplementary file 1a .", "Prior to training on the task , each monkey was implanted with a head post and a stereotaxically positioned infusion chamber .", "The chamber allowed a removable injector , fitted with an infusion cannula of adjustable length to be inserted into predetermined sites in the brain through the guiding channels of a grid .", "The rectangular chamber ( 38 mm length x 51 mm width ) was covered with a removable top that was secured with four screws .", "For drug infusions , the top was removed and a grid was inserted to provide guiding channels for the placement of the injector and cannula .", "The grid ( 25 mm length , 41 mm width ) contained 2 mm - length guiding channels set 1 mm apart ( center to center ) .", "Surgery was performed as previously described ( Murray et al . , 2015 ) .", "A custom-built telescoping injector made of polyethylene terephthalate polyester ( Elmeco Engineering ) was designed to fit snugly into the infusion grid and allowed for sub-millimeter adjustment of infusion cannula ( 27 ga stainless steel tubing ) length ( Wellman et al . , 2005; West et al . , 2011 ) .", "Thus , our final spatial resolution for infusion targeting was 2 mm in the anteroposterior and mediolateral planes , and 0 . 25 mm in the dorsal-ventral plane .", "Postoperatively , each monkey received one or more T1-weighted scans to determine and/or verify the coordinates for the infusion sites .", "Scans were conducted as previously described ( Wellman et al . , 2005; West et al . , 2011 ) with an effective resolution of 0 . 25 × 0 . 25 × 0 . 25 mm .", "To calculate infusion site coordinates , a dilute gadolinium solution was instilled into the chamber .", "Gadolinium contrast allowed for the detection of the position of each of the guide channels within the infusion grid .", "To verify the volume of diffusion of the infused solution , we infused 1 ul ( 5 nmol ) of an MRI contrast agent , gadolinium ( 5 mM solution diluted in sterile saline; Magnevist ) as previously described .", "The resolution of this scan was 1 mm x 1 mm x 1 mm .", "The range of diffusion visualized in MRI sections was limited to a diameter of 3 mm at 60 min after infusion , in agreement with previous gadolinium imaging in our laboratory and others ( Forcelli et al . , 2014; Krauze et al . , 2005; Fiandaca et al . , 2009 ) .", "For each drug , we evaluated performance in the: ( Málková et al . , 1997 ) Object Probe [baseline probe , sated probe] , ( 2 ) Consummatory Probe , ( Timbie and Barbas , 2015 ) Baseline Preference , and ( 4 ) Concurrent Visual Discrimination .", "For Object and Consummatory Probe tests , we compared performance after drug infusion with matched saline ( or sham ) infusions .", "For baseline preference and concurrent visual discrimination , performance was compared to non-injected baselines .", "In order to minimize penetrations of the brain , animals received one saline infusion and one sham infusion for each drug .", "Sham infusions were performed in the same manner as drug infusions except no cannula was inserted .", "A pair of tests , comprised of one sham infusion and one saline infusion , were performed for each drug ( i . e . , one pair was performed for MUS , one pair for KYNA ) .", "Each pair contained a manipulation performed before satiation and a manipulation performed before the probe session .", "Furthermore , each pair contained one control with each of the two foods .", "These tests were performed in a balanced manner , as shown in Supplementary file 1b .", "Animals AN , TA , EL , and LO were used for experiments with MUS .", "LO was not included for experiments with KYNA , as he became uncooperative after the completion of experiments with MUS .", "The GABAa receptor agonist muscimol ( MUS; Sigma Aldrich ) was dissolved in sterile saline at a concentration of 9 mM .", "The broad-spectrum ionotropic glutamate receptor antagonist , kynurenic acid ( KYNA; Sigma Aldrich ) was dissolved first in a small quantity of NaOH , neutralized with dilute HCl , and adjusted to the final concentration of 300 mM by addition of sterile water .", "All drug solutions were filter sterilized ( 0 . 20 µm pore size , Corning ) before being stored in 1 ml frozen aliquots ( −20C ) .", "Drug doses and concentrations were selected on the basis of our prior microinfusion studies in macaques .", "We have previously reported functional effects of muscimol within the range of 4 . 5 to 9 nmol per infusion ( i . e . , 0 . 5 to 1 ul of a 9 mM solution ) ( Wellman et al . , 2005; West et al . , 2011 ) .", "Similarly , we have reported functional effects of KYNA infusion in the range of 300–600 nmol per infusion ( Forcelli et al . , 2014; Malkova et al . , 2015 ) ( i . e . , 1–2 ul of a 300 mM solution ) .", "As shown in Figure 2A , for MUS , we matched the time between infusion and probe test to ensure an equal diffusion of drug between conditions .", "To accomplish this , we inserted a 30 min delay between infusion and probe test , when the drug was infused after satiation but before the probe .", "This strategy is similar to that used in the amygdala and orbitofrontal cortex ( Wellman et al . , 2005; West et al . , 2011 ) .", "The duration of action of MUS when microinjected into the brain easily exceeds 90 min; in a prior study , we found that effects of MUS infusion increased over the duration of a 90 min infusion period and observed behavioral responses that in some cases lasted for several hours ( Dybdal et al . , 2013 ) .", "Thus , it was not possible to allow sufficient time for clearance of MUS prior to the probe test .", "In order to determine , what , if any , effect the MD plays exclusively during the period of satiation , we required a compound that would be cleared by the time the probe test occurred .", "For this reason , we turned to KYNA , which is quickly cleared in the brain and has a relatively short duration of action ( Forcelli et al . , 2014; Vécsei and Beal , 1990; Turski and Schwarcz , 1988 ) .", "In rodent studies , the half-life of KYNA in brain has been reported to range from 5 to 30 min ( Turski and Schwarcz , 1988 ) .", "Consistent with this timecourse , in a prior study , we observed normalization of cognitive function within 45 min of infusion of KYNA ( Forcelli et al . , 2014 ) ( and unpublished observations ) in the primate brain .", "To maximize time for clearance of KYNA when infused prior to satiation , we inserted a 15 min delay following the satiation procedure; thus the total time from the end of the infusion to the start of the probe was ~45 min .", "Finally , we infused KYNA at a lower dose ( 150 nmol ) in two animals ( EL/LO ) .", "This dose of KYNA was without effect on the proportion shifted when infused before the probe test ( 0 . 71 and 0 . 72 after KYNA , as compared to 0 . 73 and 0 . 85 after control , respectively; see Figure 2—figure supplement 1 ) .", "Given the half-life of KYNA in brain , even if sparse amounts of KYNA remained when animals were infused before satiation , the concentration of KYNA remaining would not be sufficient to produce the significant deficits observed .", "Drug infusions were performed using an aseptic technique while the monkey was seated in a primate chair with its head posted .", "The chamber was cleaned immediately prior to each infusion .", "Coordinates for drug infusion were determined based on individual MRI scans for each monkey .", "Animals were infused , bilaterally , with either MUS , KYNA , or sterile saline ( 0 . 9% NaCl ) .", "Drugs were infused at a rate of 0 . 2 ul/min using the removable injector described above , connected by sterile tubing to Hamilton syringe driven by an injection pump ( New Era Pumps Systems ) .", "Infusion progress was monitored by the displacement of a small air bubble introduced into the tubing .", "Weekly testing occurred per the following schedule ( see Figure 1 ) .", "On Day 1 , animals were tested on concurrent visual discrimination .", "If animals correctly chose >90% baited S+ objects during concurrent visual discrimination , on Day two they were tested on a baseline probe .", "If animals performed below this criterion , they were re-tested on concurrent visual discrimination until they again met the criterion of >90% .", "On Day 3 , animals were again tested on concurrent visual discrimination .", "On Day 4 , animals were infused with drug or saline before satiation or after satiation period , then tested on the object probe session .", "To determine if drug manipulations impaired the typical change in value of primary reinforcers that occurs after satiation , we evaluated performance in a consummatory probe session .", "Immediately after the completion of both the baseline and sated object probe sessions ( described above ) , animals were presented with 20 pairs of primary reinforcers ( fruit snack and peanut ) in the absence of objects .", "The left-right positions of each reinforcer were pseudorandomized across the 20 trials .", "Animals typically display a small , but significant preference for objects associated with one of the two food reinforcers .", "To determine if inactivation of the MD impacted this baseline preference , animals were tested in a weekly sequence consisting of concurrent visual discrimination on Day 1 , baseline probe on Day 2 , and re-tested on the baseline probe following drug infusion on Day 3 .", "After infusion of KYNA , animals were immediately transferred into the WGTA; following MUS infusion , animals were returned to the home cage for 30 min prior to transfer to the WGTA and testing .", "This timing was selected to match the timing used for the sated object probe .", "To determine if drug manipulations impaired concurrent visual discrimination performance , animals were tested in a sequence consisting of the concurrent visual discrimination task on Day 1 .", "On Day 2 , animals were infused with either MUS or KYNA , transferred to the WGTA , and re-tested on the concurrent visual discrimination task .", "The timing of drug infusions and testing follows that described above for Baseline Preference testing .", "All statistical comparisons were made on a within-subject basis .", "Results of saline/sham infusions for each animal ( both before and after selective satiation ) were pooled to generate a cumulative preference shift; in this way , both foods were represented in the control condition .", "Devaluation magnitude ( proportion shifted ) for both the object and consummatory probes were analyzed by analysis of variance with treatment as a within subject condition .", "The Greenhouse-Geisser correction for violations of sphericity was applied .", "A priori determined pairwise comparisons ( Control >Drug Infused ) were analyzed using a one-tailed Holm-Sidak corrected post-test .", "A one-tailed analysis was selected for these comparisons because of our strong a priori hypothesis that drug infusion would impair not improve reinforcer devaluation .", "We had no a priori hypothesis regarding the magnitude of devaluation when drug was infused before satiation as compared to before the probe test , thus these data were analyzed using a two-tailed Holm-Sidak corrected post-test .", "Concurrent visual discrimination and baseline preference ratio were analyzed by paired t-test .", "Baseline preference ratios for MUS also analyzed using a one-sample t-test comparing preference to a neutral preference score ( 0 . 5 ) .", "This analysis was not performed for KYNA infusion , as there were insufficient data points to analyze due to the attrition of one subject prior to KYNA testing .", "All data were analyzed using GraphPad Prism ( ver 7 , Graph Pad , La Jolla , CA ) .", "In all cases , P values less than 0 . 05 were considered statistically significant .", "Full statistical results are shown in Supplementary file 1c .", "The data generated and analyzed during this study are all presented in summary form in the manuscript .", "Data for object selection during the testing sessions are shown in Supplementary file 1d ." ] ]

[ "Reward contingencies are dynamic: outcomes that were valued at one point may subsequently lose value .", "Action selection in the face of dynamic reward associations requires several cognitive processes: registering a change in value of the primary reinforcer , adjusting the value of secondary reinforcers to reflect the new value of the primary reinforcer , and guiding action selection to optimal choices .", "Flexible responding has been evaluated extensively using reinforcer devaluation tasks .", "Performance on this task relies upon amygdala , Areas 11 and 13 of orbitofrontal cortex ( OFC ) , and mediodorsal thalamus ( MD ) .", "Differential contributions of amygdala and Areas 11 and 13 of OFC to specific sub-processes have been established , but the role of MD in these sub-processes is unknown .", "Pharmacological inactivation of the macaque MD during specific phases of this task revealed that MD is required for reward valuation and action selection .", "This profile is unique , differing from both amygdala and subregions of the OFC ." ]

[ "Most of us have experienced feeling full after a main course , only to discover that we somehow still have room for dessert .", "Eating a particular foodstuff to the point of satiety makes that item temporarily less appealing .", "This is an example of reward devaluation .", "We typically respond to this phenomenon by adjusting our behavior .", "We give up on the main course , for example , and turn our attention instead to dessert .", "This ability to adjust our actions based on changes in the value of their outcomes is a form of behavioral flexibility .", "Several brain regions contribute to behavioral flexibility .", "These include the amygdala , parts of the orbitofrontal cortex , and the mediodorsal thalamus .", "Wicker et al . have now explored the role of the mediodorsal thalamus by temporarily inactivating it in monkeys performing a task involving reward devaluation .", "The monkeys learned to associate one set of objects with peanuts and another with fruit .", "They were then given unlimited access to either peanuts or fruit .", "Finally , they were offered a choice between the two sets of objects .", "Like people who opt for dessert rather than another helping of a main course , the monkeys that had received peanuts chose the objects associated with fruit , and vice versa .", "Temporarily inactivating the mediodorsal thalamus prevented this change in behavior .", "This occurred if the inactivation took place while the monkeys had unlimited access to the reward , or if it took place while they were choosing between the two objects .", "The mediodorsal thalamus is thus required both to update the value of a reward and to select the best course of action .", "This is in contrast to the amygdala and the orbitofrontal cortex , which each support only one of these processes .", "Impaired behavioral flexibility is a hallmark of neuropsychiatric disorders , including addiction .", "Understanding the brain networks that support flexible responding may help improve the treatment of such disorders .", "As therapies that involve electrically stimulating the brain become more common , knowing which regions to avoid will be just as important as identifying new targets ." ]

[ "Introduction", "Results", "Discussion", "Conclusions", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Ever since life became terrestrial ∼360 million years ago , animals have developed increasingly sophisticated methods to navigate their environments ( Dickinson et al . , 2000 ) .", "Locomotion is essential for animals to escape from predators , find mates , and search for food .", "One of the most common forms of terrestrial locomotion depends on the movement of multi-jointed legs .", "For this to occur , animal nervous systems face two main computational challenges .", "First , multiple leg joints must move rhythmically and in a precisely coordinated fashion to allow the stereotyped movements that occurs during the swing and stance phases of each step cycle .", "Second , these movements must be coordinated between legs , which number four in a typical tetrapod and six in a hexapod .", "Both challenges are met in part by interactions between central pattern generators ( CPGs ) , neural networks within the central nervous system that have the capacity to generate rhythmic outputs ( MacKay-Lyons , 2002 ) .", "When used for walking , individual CPGs result in the rhythmic and alternating activity of motor neurons that control the flexion and extension of single leg joints ( Bässler , 1977; Strauss , 2002; Borgmann et al . , 2009; Büschges et al . , 2011 ) .", "Leg movement coordination is also assisted by proprioceptive sensory inputs that report the load and position of leg joints ( Bässler , 1977; Borgmann et al . , 2009 ) .", "Other sensory modalities , such as visual , olfactory and gravitational , also modulate the activity of locomotor CPGs to allow animals to readily change their motor behavior in response to their environment ( Frye , 2010 ) .", "An understanding of locomotion requires the identification of the neurons that comprise locomotor neural circuits .", "Given the complexity of these circuits , insects provide an attractive model to achieve this goal due to their relative simplicity , approachable physiology and availability of genetic tools .", "Moreover , adult insects share with vertebrates the same general principles of locomotion ( Pearson , 1993 ) .", "Nevertheless , despite many anatomical similarities , it is less clear how similar the neural circuitries underlying locomotion are in vertebrates and invertebrates .", "Groundbreaking studies in cockroaches , locusts and stick insects have identified many components and fundamental rules that regulate the walking apparatus , such as the definition of multiple gaits ( Graham et al . , 1985; Burrows , 1992; Zill et al . , 2004; Ritzmann and Büschges , 2007; Büschges et al . , 2008 ) .", "However , unlike in vertebrates , where gait transitions are discontinuous , it is less clear in which situations insects use distinct gaits and how they transition between them .", "Although theoretical considerations suggested the possibility that gait transitions may be more gradual than they are in vertebrates , this question has not been fully resolved ( Graham et al . , 1985 ) .", "Walking speeds in cockroaches , for example , cluster into two groups , but both rely on the tripod gait ( Bender et al . , 2011 ) .", "The stick insect tends to use a tetrapod gait at slower speeds and a tripod gait at faster speeds ( Graham et al . , 1985 ) .", "Drosophila , on the other hand , are reported to primarily use the tripod gait ( Strauss and Heisenberg , 1990 ) .", "Similar questions arise with respect to the role of sensory feedback in coordinated walking .", "Although feedback is thought to be critical for coordination between legs ( Bässler , 1977; Borgmann et al . , 2009 ) most studies address this question using electrophysiology readouts with tethered animals .", "In addition , with the exception of surgical ablation experiments ( Usherwood et al . , 1968; Cruse et al . , 1984 ) , it has been difficult to dissect the contribution of individual sensory modalities .", "Gaining further insights into these questions may benefit by the use of systems that use freely walking animals with access to genetic tools to identify and manipulate the individual components of locomotor circuits .", "The fruit fly , D . melanogaster , is a powerful genetic model with a large collection of mutants and an increasingly sophisticated genetic toolkit ( Pfeiffer et al . , 2008; Venken et al . , 2011 ) .", "However , despite the availability of powerful genetic tools , there is a lack of quantitative and robust methods to analyze the consequences of manipulating locomotor circuits in the fruit fly .", "Consequently , many studies focus on relatively low-resolution locomotor assays such as monitoring the average speed of a population , walking trajectories , or the ability to fulfill simple motor tasks such as climbing a vertical surface ( Ganetzky and Flanagan , 1978; Branson et al . , 2009; Slawson et al . , 2009; Robie et al . , 2010 ) .", "To obtain kinematic data , researchers have relied on the manual frame-by-frame analysis of videos ( e . g . Strauss and Heisenberg , 1990 , 1993; Wosnitza et al . , 2012 ) .", "The lack of a high-resolution and accessible assay to monitor fruit fly walking has greatly limited the use of this model system to study locomotion .", "In order to quantitatively analyze locomotion in Drosophila , we developed an optical system to monitor walking that uses frustrated Total Internal Reflection ( fTIR ) coupled with high-speed video imaging .", "Our fTIR-based method is similar in principle to that used for analyzing the gaits of larger animals ( e . g . CatWalk ( Vrinten and Hamers , 2003 ) ; http://www . noldus . com/animal-behavior-research/products/catwalk/ ) , but differs significantly due to the small size and rapid walking speed of fruit flies .", "Using this approach , we are able to track the position of each footprint relative to the body at high spatial and temporal resolution as an untethered fly walks freely on a flat surface .", "Custom analysis software allows the extraction of many parameters of fly walking behavior , including gait , coordination between legs , and footprint positions .", "Using this method , we comprehensively characterized the walking behavior of wild type animals .", "Unlike walking in vertebrates , our data demonstrate that flies do not abruptly switch from one gait to another , but instead rely on a continuum of gait patterns that correlate with walking speed .", "However , several readouts suggest that flies may use distinct neural programs at slow , medium , and fast walking speeds .", "Genetic manipulations to specifically disrupt sensory feedback from the legs show that blocking proprioception results in altered step parameters and reduced walking precision , especially at slower speeds , but does not interfere with the ability of flies to execute a tripod gait .", "Together , these data reveal the underlying parameters of wild type walking in Drosophila and show that proprioceptive sensory feedback is important , but not absolutely required , for coordinated locomotion ." ], [ "The analysis of locomotion in large animals , notably mammals , often relies on the placement of visual marks in strategic positions , usually joints that can be readily detected and tracked ( Akay et al . , 2006 ) .", "However , in smaller insects such as Drosophila , such a strategy becomes not only technically challenging but is also likely to disturb walking behavior and generate artifacts .", "Such challenges have precluded a more detailed examination of the components that comprise the fly's walking behavior .", "To overcome these obstacles , and to measure the biomechanical features underlying walking in Drosophila we turned to an optical effect known as frustrated Total Internal Reflection ( fTIR ) ( Zhu et al . , 1986 ) .", "Total Internal Reflection occurs when light traveling through a medium—in this case optical glass—hits an interface with another medium with a lower refractive index , such as air .", "If the angle of incidence is above the so-called critical angle ( as compared to the normal of the surface ) , defined by Snell's Law ( Katz , 2002 ) , the light is no longer refracted but is internally reflected .", "For a glass-air interface this corresponds to ∼43° .", "However , if a denser material , such as the tarsus of an insect leg , contacts the surface of the glass , then the locally ‘frustrated’ total internal reflection will scatter the light , which can be recorded by a high-speed video camera ( Figure 1 and Figure 1—figure supplement 1 ) ( Sumriddetchkajorn and Amarit , 2006 ) .", "A sample video of the unprocessed fTIR effect can be seen in Video 1 . 10 . 7554/eLife . 00231 . 003Figure 1 . fTIR apparatus and FlyWalker software .", "( A ) .", "Schematic of the fTIR optical effect .", "LED light sources are located at the edges of an optical glass and light propagates within the glass via internal reflection .", "Tarsal contacts lead to light scattering detected by a high-speed camera .", "See Figure 1—figure supplement 1 for more details .", "( B ) .", "Single frame of a fTIR video .", "The fTIR effect can be seen for three legs in stance phase ( yellow arrows ) .", "Background light partially illuminates the fly's body ( orange dashed ellipse; the center of the body is indicated by an orange cross ) .", "( C ) .", "Image generated by the FlyWalker software .", "The fly's footprints and body center are tracked throughout the video .", "Present footprints are identified and labeled ( yellow arrows ) .", "The fly body and trajectory are visualized by a blue line ( white arrow ) .", "Past footprints can also be recorded ( red arrows ) .", "A scale bar can be introduced .", "Step length is defined as the distance between two consecutive footprints ( green arrows ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 00310 . 7554/eLife . 00231 . 004Figure 1—figure supplement 1 . Additional information on fTIR apparatus .", "( A ) .", "Detailed schematic of the fTIR apparatus .", "Red dashed line shows position of the cross-section image in panel ( B ) .", "( B ) Cross-section of schematic at red dashed line in panel ( A ) .", "( C ) Photograph of the fTIR apparatus . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 00410 . 7554/eLife . 00231 . 005Figure 1—figure supplement 2 . FlyWalker program .", "( A ) .", "Screenshot of the FlyWalker graphical user interface used to edit fTIR videos .", "( B ) .", "Screenshot of the FlyWalker parameters window . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 00510 . 7554/eLife . 00231 . 006Video 1 . Unprocessed fTIR video . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 006 To automate the tracking of the footprints and fly body revealed by the fTIR method , we created a program called FlyWalker that tracks and outputs several user-defined parameters ( Figure 1—figure supplement 2 ) .", "The program , which is freely available for download at http://biooptics . markalab . org/FlyWalker , evaluates the fTIR signals in each video and generates a set of graphs that describe the walking behavior ( Video 2 shows a FlyWalker-processed video; see ‘Materials and methods’ for a list of parameters , their definitions and a list of graphs generated by FlyWalker ) .", "In addition , the program also creates a spreadsheet that contains the full data set .", "From this , the user can analyze , compile and compare several samples . 10 . 7554/eLife . 00231 . 007Video 2 . Processed video by FlyWalker . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 007 As a proof-of-principle , we examined the walking behavior of freely walking upright wild-type adult flies on a flat horizontal surface .", "The distance recorded was on average 1 . 27 cm , a distance covered by the camera without compromising the fTIR signal .", "We collected 71 videos of animals that walked in a straight manner without any stops .", "Average speeds of each fly varied between 7 . 2 and 44 . 7 mm/s with 28 mm/s as the most representative speed ( Figure 2A ) , similar to previously reported values ( Robie et al . , 2010 ) .", "Interestingly , speeds ∼20 mm/s were underrepresented in this data set ( see ‘Materials and methods’ ) , reminiscent of underrepresented speeds at gait transitions in humans or ponies ( Hoyt and Taylor , 1981 ) . 10 . 7554/eLife . 00231 . 008Figure 2 . General walking parameters .", "( A ) .", "Speed histogram of 71 videos recorded for wild type flies , with 2 mm/s bins .", "Average speeds vary between 7 . 2 and 44 . 7 mm/s , with 28 mm/s the most represented speed .", "Speeds at ∼20 mm/s are underrepresented in this dataset .", "( B ) .", "Durations of stance ( blue ) and swing ( red ) phases as a function of speed .", "Swing phase duration remains mostly constant while stance phase duration is inversely proportional to speed .", "Graphical fits for swing and stance phases versus speed are represented in red and blue lines , respectively .", "( C ) .", "Step period is inversely proportional to average speed .", "The blue line is a graphical fit .", "( D ) .", "Duration of the metachronal lag as a function of the hindleg period for slow ( blue ) and fast ( red ) flies .", "metachronal lag closely matches hindleg period for fast flies ( HLagF ≈ Period ) .", "Regression line for the slow walking flies: HLagF = Period × 0 . 505 + 27 . 627 .", "A total of 220 metachronal waves were used , error bars correspond the standard error of the mean .", "( E ) , ( F ) .", "Step length ( E ) and swing speed ( F ) increase linearly with speed .", "A graphical fit is shown by the blue lines .", "Error bars in ( B–F ) correspond to standard error of the mean . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 00810 . 7554/eLife . 00231 . 009Figure 2—figure supplement 1 . Gait definitions . For each leg , swing phases are represented in black and stance phases are represented in white ( from top to bottom: right hind ( RH ) ; right mid ( RM ) ; right front ( RF ) ; left hind ( LH ) ; left middle ( LM ) ; left front ( LF ) ) .", "Step period is the time for one complete cycle ( initiation of consecutive stance phases ) and is divided into stance duration and swing duration .", "The metachronal lag is defined as the time between the start of sequential swing phases for the hind and forelegs on the ipsilateral side ( red arrows ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 00910 . 7554/eLife . 00231 . 010Figure 2—figure supplement 2 . Gait parameters by segment .", "( A ) – ( D ) .", "Each column corresponds to the legs in a particular segment .", "Graphical fits are represented by blue lines .", "( A ) Stance duration .", "( B ) Swing duration .", "( C ) Step length .", "( D ) Swing speed . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 010 To initially describe the walking behavior of wild type animals , we plotted several step parameters ( Figure 2B—F and Figure 2—figure supplement 1 ) .", "As previously noted in several insect species ( Wilson , 1966; Graham , 1972; Strauss and Heisenberg , 1990 ) as speed increases , stance phase duration becomes shorter while swing phase duration remains largely constant; at the fastest speeds the durations of both swing and stance phases equalize ( Figure 2B ) .", "Consequently , step period varies inversely with average speed up to approximately 30 mm/s , when step period reaches a steady state of about ∼60 ms , corresponding to a frequency of 16 cycles per s ( Figure 2C ) .", "Next we examined the relationship between step pattern and period for faster and slower walking animals .", "For this , we plotted the metachronal lag , ( i . e . , the time between sequential swing onsets from hind to forelegs on an ipsilateral side ) , as a function of hindleg period ( Graham , 1972 ) ( Figure 2D , Figure 2—figure supplement 1 ) .", "We compared data extracted from slow animals ( <20 mm/s ) with those from fast animals ( >34 mm/s ) .", "These speed groups were chosen and consistently used in this and all subsequent analyses because several of our readouts exhibit discontinuous or non-linear behavior at ∼20 and ∼34 mm/s ( see Figures 2A; 3C and 5B for examples ) .", "Data extracted from the fast set reveal that hindleg period equals the metachronal lag ( HLagF ≈ PH ) , typical of the tripod gait ( also called Gait I; ( Graham , 1972 ) ) .", "In contrast , flies walking slower than 20 mm/s showed a period-dependent metachronal lag with a regression curve of ( HLagF = 0 . 505* PH + 27 . 6 ) , typical of the tetrapod gait ( also called Gait II; ( Graham , 1972 ) ) , see also ‘Gait parameters’ , below ) ( Figure 2D ) .", "We also find that step length increased almost linearly with speed ( Figure 2E ) .", "As a consequence , swing speed also follows this trend ( Figure 2F ) . 10 . 7554/eLife . 00231 . 011Figure 3 . Spatial parameters .", "( A ) .", "Stance traces .", "Representative plot of an animal walking at 28 . 82 mm/s .", "Traces are generated by the position of the stance phase footprints relative to the body center ( set at 0 . 0 , 0 . 0 ) .", "For each leg , stance onset corresponds to the Anterior Extreme Position ( AEP ) while stance offset is termed Posterior Extreme Position ( PEP ) .", "( B ) .", "Method to quantify the stance linearity index .", "For each stance trace ( brown ) , a smoothed trace is generated ( using data from every five frames; yellow trace ) , and the average of the difference between these two lines ( orange arrows ) corresponds to the stance linearity index .", "( C ) .", "Stance linearity as a function of speed .", "Each data point corresponds to the average of all traces for all six legs for a single video greater than ∼34 mm/s , stance linearity becomes constant ( red box ) .", "( D ) .", "Method to quantify footprint clustering .", "For each set of AEP/PEP footprints ( red circles ) , an average ±STD xy point is created ( blue cross ) .", "The footprint clustering value is calculated as the vector sum of the two STD values ( orange arrow ) .", "( E ) .", "Quantification of footprint clustering .", "Data were grouped into slow ( <20 mm/s ) , medium ( between 20 and 34 mm/s ) and fast ( >34 mm/s ) speeds .", "Boxplots represent the median as the middle line , with the lower and upper edges of the boxes representing the 25% and 75% quartiles , respectively; the whiskers represent the range of the full data set .", "Values are normalized for body size .", "Asterisks indicate the significance of the decrease in footprint clustering between the AEP and the PEP ( using the paired parametric t test in the case of the slow speed group and the Wilcoxon non-parametric test for the remaining groups , **p<0 . 005 , ***p<0 . 001 ) .", "A comparison between the three speed groups also displays statistical significance ( Kruskal–Wallis-ANOVA , p values of 0 . 0015 and 0 . 0014 for AEP and PEP , respectively ) .", "Dunn's post hoc significance tests show: slow AEP vs medium AEP , not significant ( NS ) ; medium AEP vs fast AEP , **; slow AEP vs fast AEP , **; slow PEP vs medium PEP , NS; medium PEP vs fast PEP , NS; slow PEP vs fast PEP , *** .", "( F ) .", "Footprint position relative to the body center .", "Data were pooled as in the previous panel .", "AEP and PEP values for each leg are represented on the left and right sections of the plot , respectively .", "Values are normalized for body size . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 01110 . 7554/eLife . 00231 . 012Figure 3—figure supplement 1 . Representative examples of stance traces and corresponding stance linearity values . A less linear stance trace ( e . g . , ( A′ ) compared to ( A ) ) corresponds a higher stance linearity value . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 01210 . 7554/eLife . 00231 . 013Figure 3—figure supplement 2 . AEP and PEP clustering values for all segments as a function of speed . Linear trend lines for AEP and PEP versus average speed are shown as blue and red lines , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 01310 . 7554/eLife . 00231 . 014Figure 3—figure supplement 3 . AEP and PEP clustering values for each segment as a function of speed .", "( A ) .", "Linear trend lines for AEP and PEP are represented as blue and red lines , respectively .", "( A ) forelegs; ( A′ ) midlegs; ( A″ ) hindlegs .", "( B ) .", "Boxplots of the data presented in ( A ) .", "Data were grouped into slow ( <20 mm/s ) , medium ( between 20 and 34 mm/s ) and fast ( >34 mm/s ) animals .", "Boxplots represent the median as the middle line , with the lower and upper edges of the boxes representing the 25% and 75% quartiles , respectively , and the whiskers representing the range of the full data set .", "Values are normalized for body size .", "Asterisks indicate statistical significance between AEP and PEP .", "( Data analyzed by the Wilcoxon or paired-t test the , **p<0 . 005 , ***p<0 . 001 ) .", "Values are normalized for body size . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 014 The FlyWalker program also extracts data for each individual leg for each of the three thoracic segments ( Figure 2—figure supplement 2 ) .", "Although legs in different segments show very similar trends for most parameters , some distinctions can be made .", "For example , as average speed increases there is a small but noticeable decrease in swing phase duration in the forelegs when compared to other segments .", "In addition , compared to other leg segments , foreleg swing speed varies more linearly with average speed , as seen by a larger R value ( Figure 2—figure supplement 2 ) .", "These observations support the hypothesis of a leading role for forelegs in forward locomotion suggested by experiments in the stick insect ( Akay et al . , 2007; Bässler and Büschges , 1998 ) .", "In addition to following the six footprints , FlyWalker also monitors the position of the fly body .", "From these data we can reconstruct the stance positions relative to the center of the body ( Figure 3A ) .", "In order to account for variations in body size , these plots are normalized to body length .", "Stance traces are generated as each leg contacts the glass .", "The onset of these traces is termed the Anterior Extreme Position ( AEP ) , which corresponds to the position where the leg first contacts the glass after touchdown at the end protraction ( Cruse , 1976 ) .", "The position at the end of the stance phase , just before the tarsi enter swing phase , corresponds to the Posterior Extreme Position ( PEP ) ( Cruse , 1976 ) .", "Stance traces can be compared by their straightness , which is a measure of how much wobble there is in body position relative to each footprint , which are stationary .", "This parameter , the stance linearity index , is calculated by computing the average difference between an actual stance trace and a smoothed version of the trace ( Figure 3B , Figure 3—figure supplement 1 ) .", "Measuring this parameter , faster animals have straighter stance traces ( lower stance linearity indexes ) compared to slower animals .", "This trend is seen up to approximately 34 mm/s , after which this value stabilizes ( Figure 3C ) .", "Another informative parameter corresponds to the clustering of the AEPs and PEPs for each leg in a single video .", "This parameter—termed footprint clustering—corresponds to the vector sum of standard deviations ( STDs ) from the mean for all AEPs or PEPs calculated for each leg ( Figure 3D ) .", "For example , a small footprint clustering value for the foreleg AEP means that the AEP coordinates ( relative to the fly's body ) were similar for all of the foreleg steps in a video .", "Using this parameter , we asked if there was a relationship between footprint clustering and average speed .", "For the reasons given above , we binned the speed values into three groups: <20 mm/s; between 20 and 34 mm/s; and >34 mm/s .", "For both AEP and PEP , the footprint clustering values were smaller for faster flies ( Figure 3E and Figure 3—figure supplements 2 and 3 ) , suggesting that animals have more spatially restricted steps as they increase their speed .", "We also found that AEP clustering values are generally smaller than PEP clustering values ( Figure 3E ) , suggesting a tighter motor control at touchdown ( AEP ) compared to stance offset ( PEP ) .", "Pooling the data for all legs , AEP clustering was smaller than PEP clustering in 61 out of 71 ( 86% ) videos .", "This difference is largest for the forelegs and is very small for the hindlegs ( Figure 3—figure supplement 3 ) .", "Knowing that flies increase their speed by making longer strides ( Figure 2E ) , we next asked if there is a relationship between average speed and AEP or PEP .", "To address this question we plotted AEP and PEP for each leg for the three speed groups ( Figure 3F ) .", "At faster speeds , for all legs , AEP coordinates are shifted anteriorly , while PEP coordinates are shifted posteriorly .", "Interestingly , at faster speeds midleg AEP and PEP are also shifted laterally , perhaps to increase stability at higher speeds .", "In contrast , the hindlegs are positioned closer to the body at higher speeds , perhaps to allow for a stronger power stroke .", "Previous work described insect gaits as either tripod or tetrapod , depending on the speed and body load ( Graham , 1972 ) .", "The tripod gait is characterized by three legs in stance phase and three legs in swing phase at any one time ( Figure 4—figure supplement 1 ) .", "Each group of three legs is composed of the fore and hind legs on one side and the midleg on the contralateral side .", "In contrast , in an idealized tetrapod gait only two legs are in swing phase while the remaining four legs are in stance phase ( Figure 4—figure supplement 1 ) .", "The two legs in swing phase are on contralateral sides and are offset by one segment .", "We also observed many noncanonical stance combinations that do not fit either of the idealized gaits , which we analyze below ( Figure 4—figure supplement 1 and Table 1 ) . 10 . 7554/eLife . 00231 . 015Figure 4 . Gait parameters .", "( A ) , ( B ) .", "Upper panels show the step pattern for representative videos of animals walking at 44 . 7 ( A ) and at 14 . 6 mm/s ( B ) .", "For each leg swing phases are represented in black ( from top to bottom: right hind ( RH ) ; right mid ( RM ) ; right front ( RF ) ; left hind ( LH ) ; left middle ( LM ) ; left front ( LF ) ) .", "( A′ ) , ( B′ ) show the instantaneous speed for the same video .", "Thick and thin lines correspond to integration times of 25 or 12 . 5 ms , respectively .", "In faster animals ( A ) , peak speeds are observed halfway through the stance phase ( red dashed line ) , while minimum speeds are observed during gait transitions ( blue dashed line ) .", "( A″ ) , ( B″ ) , for each frame the corresponding gait was color coded as follows: green ( tripod ) , blue ( tetrapod ) , and gray ( non-canonical ) .", "Red brackets indicate the eight frame windows used to generate the gait index plots ( A‴ ) , ( B‴ ) .", "Tripod = +1; tetrapod = −1; and noncanonical = 0 .", "( C ) .", "Quantification of the average gait index for three speed groups .", "For each video the average gait index was calculated for all frames ( p<0 . 0001 for Kruskal–Wallis-ANOVA test . Dunn's post hoc significance test: **p<0 . 005 , ***p<0 . 001 ) .", "( D ) , ( E ) .", "Tripod ( D ) and tetrapod ( E ) indexes as a function of speed .", "Graphical fits are represented in blue .", "Data for points labeled ( A ) and ( B ) are shown in panels ( A ) and ( B ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 01510 . 7554/eLife . 00231 . 016Figure 4—figure supplement 1 . Gait features .", "( A ) – ( C ) .", "Walking gaits and leg combinations .", "Each leg can be either in a swing or stance phase , represented by white or black circles , respectively .", "Underneath each combination the corresponding numerical code ( ‘1’ and ‘0’ correspond to stance or swing , respectively .", "Digit order: left foreleg; left midleg; left hindleg; right foreleg; right midleg; right hindleg .", "( A ) Tripod gait combinations .", "( B ) Tetrapod gait combinations .", "Tetrapod gait can be either right or left handed depending on which side is swinging more anteriorly .", "( C ) Representative noncanonical combinations , which are not represented by tripod or tetrapod configurations . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 01610 . 7554/eLife . 00231 . 017Figure 4—figure supplement 2 . Gait index plot .", "( A ) .", "Average gait index as a function of speed .", "Each point corresponds to the average of all frames in a video ( Figure 4A‴ , B‴ ) .", "( B ) .", "Instantaneous speed , gait map and gait index plots for data points labeled in ( A ) by I . , II .", ", and III .", "are shown .", "Data for points labeled A* and B* are shown in Figure 4A , B , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 017 For each video , a step pattern can be generated while simultaneously plotting the instantaneous speed and gait characteristics with high temporal resolution ( Figure 4A , B ) .", "Notably , the instantaneous speed plot has a wave-like appearance ( Figure 4A′ , B′ ) , particularly in high speed animals , with maxima and minima that can differ up to 30 mm/s .", "Consistent with the observations of Graham in the first instar stick insect ( Graham , 1972 ) , peak speeds are observed midway through each stance phase when the retracting tripod reaches its maximum motor output .", "Conversely , minimum speeds are observed at the transition between phases , when the stance switches to a different set of legs .", "The difference between the maxima and minima in these instantaneous speed plots decreases at slower speeds ( Figure 4B ) , suggesting that power is more evenly distributed throughout each period at slower speeds .", "For each frame in a video we also classify whether the fly is in a tripod , tetrapod , or noncanonical stance .", "The resulting gait map graphically illustrates the gaits used over time ( yellow for tripod , blue for tetrapod , and grey for noncanonical ) ( Figure 4A″ , B″ ) .", "Visual inspection of the full data set shows that flies walk preferentially using the tripod gait ( data not shown ) ( Strauss and Heisenberg , 1990; Wosnitza et al . , 2012 ) , but also underscores that as flies decrease their speed they increasingly use tetrapod and noncanonical combinations .", "To quantify these gait maps we plot the gait index .", "To calculate the gait index each frame is assigned a value ( +1 for tripod , −1 for tetrapod and 0 for noncanonical ) and these values are averaged for a sliding window of n frames; empirically , we find that n = 8 is most effective at distinguishing flies primarily using the tetrapod and tripod gaits ( Figure 4A‴ , B‴ and Figure 4—figure supplement 2 ) .", "The average value for an entire video corresponds to the average gait index .", "We calculated the average gait index for all 71 videos in our data set and binned the results using the same three speed groups used previously ( Figure 4C ) .", "Each group ( slow , medium , and fast ) had statistically distinct average gait indexes: slower animals had more negative values while faster animals had increasingly positive values , reflecting their increasing use of the tripod gait .", "As a complementary method to quantify differences in gait , we analyzed our fTIR data to determine the fraction of time flies spend using either idealized tripod or tetrapod configurations , and if these fractions depend on average speed ( Figure 4D , E ) .", "We define the tripod or tetrapod indexes as the fraction of frames in a video in which a leg combination matches one of these two gaits .", "As expected , faster flies spend a larger fraction of time in a tripod configuration , which presumably allows flies to maximize leg thrust ( Figure 4D ) .", "The inverse relationship is seen for the tetrapod configuration , where slower animals display the highest proportion of this gait ( Figure 4E ) .", "Two additional conclusions can be derived from these data .", "First , there is not an abrupt transition between the tripod and tetrapod gaits at a particular speed .", "Instead , the proportion of time a fly spends using each gait varies gradually according to average speed .", "Second , the idealized tripod and tetrapod combinations only account for a subset of all stance configurations present in our data set .", "For example , the fastest flies in our data set had tripod and tetrapod indexes of ∼70% and <10% , respectively .", "Noncanonical leg configurations in which none or only one leg is in swing phase account for 48% to 21% of all combinations , depending on the average speed ( Table 1 ) .", "Inspection of these videos reveals that these noncanonical combinations generally occur at the transitions between stances ( data not shown and Figure 4A″ ) .", "Thus , slower animals spend less time using the tripod gait in part because they use the tetrapod gait more , but also because they spend a larger fraction of time in gait transitions , where noncanonical leg configurations play a dominant role .", "In addition , at slow speeds occasional short sequences of pentapod stances ( ≤3 steps ) fit the so-called ‘wave gait’ in which individual legs swing in a wave-like pattern from front to back ( Wosnitza et al . , 2012 ) . 10 . 7554/eLife . 00231 . 018Table 1 . Gait combinations in three speed classesaDOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 018Slow [≤19 . 9 mm/s]Medium [20–33 . 9 mm/s]Fast [≥34 mm/s]Tripod31 . 37Tripod51 . 63Tripod64 . 98Tetrapod25 . 45Tetrapod15 . 90Tetrapod7 . 26Total56 . 82Total67 . 54Total72 . 24Additional combinationsAdditional combinationsAdditional combinations1111108 . 271111114 . 761110114 . 891101117 . 601111104 . 611111114 . 251111116 . 380111114 . 300111114 . 071110115 . 621110114 . 260101113 . 360111115 . 030101113 . 841110102 . 800101112 . 471101113 . 791101112 . 321110102 . 451110102 . 911111101 . 291011111 . 941011110 . 710110111 . 251111011 . 690110110 . 520110100 . 69Pentapod30 . 15Pentapod14 . 37Pentapod13 . 333 gaits combinedb86 . 973 gaits combinedb81 . 913 gaits combinedb85 . 57Totalc98 . 3Totalc97 . 2Totalc97 . 15aValues are expressed as percentage ( % ) .", "Leg order in combination: LF LM LH RF RM RH .", "1 , footprint present; 0 , footprint is absent . bThe sum of tripod , tetrapod , and pentapod gaits . cThe sum of all gait patterns listed in the table .", "Typically for hexapods , leg touchdown occurs close to where the immediately anterior ipsilateral leg made contact , a behavior termed follow-the-leader ( Dean and Wendler , 1983; Cruse et al . , 1984; Song and Choi , 1989; Brunn and Dean , 1994; Sponberg and Full , 2008 ) .", "This behavior depends on sensory feedback and specialized intersegmental interneurons ( Brunn and Dean , 1994 ) .", "One possible advantage of this behavior is to ensure that the animal places its legs on safe ground , particularly on rough terrain .", "As a consequence , mid and hindleg footprints fall close to where the foreleg was placed ( Figure 1C ) .", "We quantified this tendency by measuring the footprint alignment parameter , which corresponds to the mean standard deviation of the projection of the fore , mid , and hind footprints along the body's displacement axis ( Figure 5A ) .", "Accordingly , footprints that are more aligned have a smaller footprint alignment value ( Figure 5—figure supplement 1 ) .", "Interestingly , plotting footprint alignment as a function of speed revealed a non-linear relationship in which the data points cluster into three groups ( Figure 5B ) .", "At slow speeds ( <20 mm/s ) , footprint alignment values were relatively large while at fast speeds ( >34 mm/s ) these values were much smaller , suggesting that footprint alignment is highly constrained .", "Notably , at intermediate speeds ( between 20 and 34 mm/s ) , there was no correlation between speed and footprint alignment .", "The more aligned footprints observed in faster flies could assist the maintenance of static stability during tripod transitions ( Song and Choi , 1989 ) .", "In support of this possibility , the tripod index increases with lower footprint alignment values ( data not shown ) .", "The abrupt difference seen in alignment values between slow , intermediate , and fast flies suggests that the motor circuits controlling walking in these three speed groups may differ .", "Interestingly , one of the transitions in footprint alignment values occurs at speeds ( ∼20 mm/s ) that are underrepresented in our data set , consistent with the existence of a possible gait transition at this speed .", "Coordinated walking requires that CPGs interact with other CPGs .", "Such coordination is seen when examining the phase differences between the contralateral and ipsilateral legs of the same segment .", "For example , during a tripod gait , contralateral legs within the same segment maintain a consistent phase value of 0 . 5 ± 0 . 05 ( Graham , 1972 ) .", "The FlyWalker software computes the phase values for contralateral legs within the same segment and between adjacent ipsilateral legs .", "We compared these values for the slow ( <20 mm/s ) and fast ( >34 mm/s ) groups of flies in our data set .", "In both groups , contralateral legs within the same segment display an average phase of approximately 0 . 5 ( Figure 5C–E ) .", "However , slower animals display a higher degree of variability as seen by a shorter r vector in these radial plots .", "Examining hindlegs vs midlegs and midlegs vs forelegs revealed a decrease in the phase values for slower animals ( Figure 5—figure supplement 2 ) , consistent with an increased step period without a proportional increase in the step lag .", "The mechanosensory system constantly reports the surface properties and the relative position of each of an animal's appendages ( Bässler , 1977 ) .", "Moreover , sensory feedback is thought to trigger inter-leg coupling of local circuits to allow stable and well-coordinated gaits ( Brunn and Dean , 1994 ) .", "Different types of proprioceptor organs , positioned in different regions of the legs , report different aspects of the posture and terrain .", "For example , the tibial campaniform sensilla ( or sensilla campaniformia ) are mostly responsible for measuring body load while the femoral chordotonal organ ( ChO ) is a stretch sensor , reporting joint angles as the animal walks ( Shanbhag et al . , 1992; Zill et al . , 2004 ) .", "To quantify the effect of inactivating sensory neurons in the Drosophila leg , we tested both inactivation of a small subset of neurons and broader sensory inactivation within the legs .", "First , we tested the inactivation of the leg ChO .", "The nanchung ( nan ) gene encodes for a cation channel subunit and is expressed exclusively in the sensory cilia of chordotonal organs ( Kim et al . , 2003 ) ( Figure 6A ) .", "Loss of function alleles for nan display loss of sound perception , hygrosensation and negative gravitaxis defects , in addition to an ‘uncoordinated’ phenotype ( Liu et al . , 2007; Kamikouchi et al . , 2009; Sun et al . , 2009 ) .", "Second , we tested neuronal inactivation induced by the expression of tetanus toxin ( TNT ) in a large subset of sensory neurons in the legs ( Figure 6B ) .", "To restrict TNT expression to leg sensory neurons we relied on a intersectional approach using a UAS-FRT-stop-FRT-TNT line ( Stockinger et al . , 2005 ) , the pan-sensory driver 5-40-Gal4 ( Hughes and Thomas , 2007 ) , and a 567 base pair cis-regulatory enhancer fragment of dachshund ( dac ) to drive the FLP recombinase in the leg imaginal disc ( Giorgianni and Mann , 2011 ) .", "For simplicity , we refer to this genotype as 5-40Leg>TNT .", "Video 3 shows an example of walking by these flies . 10 . 7554/eLife . 00231 . 022Figure 6 . Effects of sensory deprivation on walking .", "( A ) .", "Expression pattern driven by nanchung-Gal4 .", "Genotype: F-Gal4 , UAS-GFP .", "( B ) .", "GFP expression under combinatorial control of 5-40-Gal4 and dacRE-flp .", "Genotype: 5-40-Gal4 , dacRE-flp , UAS-FRT-stop-FRP-GFP .", "All classes of sensory neurons in the leg express GFP .", "tChO , tibia chordotonal organ; fChO , femur chordotonal organ; hp , hair plates; cs , campaniform sensilla; ms , mechanosensory brisles .", "Bar , 100 μm .", "( C ) .", "Average speed .", "Boxplots with the median as the middle line and the lower and upper edges of the boxes representing the 25% and 75% quartiles , respectively; the whiskers represent the range of the full data set .", "Statistical analysis with one-way-ANOVA ( p<0 . 0001 ) followed by Tukey's post hoc test , ***p<0 . 001 .", "( D ) .", "Tripod index .", "Lines represent graphical fits .", "See Table 2 for statistical analysis .", "( E ) .", "Gait patterns , instantaneous speeds , and gait maps for representative wild type and sensory deprived animals walking at similar speeds .", "See Figure 2—figure supplement 1 for details .", "Tripod gait properties remain unchanged . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 02210 . 7554/eLife . 00231 . 023Video 3 . Processed video of 5-40Leg>TNT fly . DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 023 Flies of both genotypes ( nan36a and 5-40Leg>TNT ) walked slower than wild type flies but , remarkably , maintained a typical tripod gait ( Figure 6C , D ) .", "These flies also exhibited normal left-right and intersegmental coordination ( Figure 6E ) .", "Strikingly , however , stance traces for nan36a and 5-40Leg>TNT flies highlight several locomotion defects ( Figure 7A ) , which were quantified ( Figure 7 and Table 2 ) .", "We observed an increase in the step length ( Figure 7B ) ; a more wobbly body placement ( reflected by an increase in stance linearity , Figure 7C ) ; and a larger variability in footprint clustering ( Figure7D ) .", "In addition , both AEP and PEP were altered , in part because of a longer stride , and midleg placement was farther from the body ( Figure 7A and data not shown ) .", "Both swing and stance duration increased ( Figure 7E , F ) resulting in a longer period .", "The increase in step length reflects both overreaching ( more anterior AEPs ) and delayed swing onset ( more posterior PEPs ) .", "However , swing speeds were minimally affected ( Figure 7G ) , suggesting that the motor neurons , themselves , were not compromised in proprioception deficient flies .", "Footprint alignment values also decreased in flies deprived of sensory feedback ( Figure 7H ) .", "This phenotype was partially a consequence of a longer stride but also because protraction was often completed when direct contact was made with the next anterior leg .", "Importantly , although both nan36a and 5-40Leg>TNT affect sensory structures in the antennae , only an increase in swing duration was observed upon surgical removal of the antenna , arguing that all other phenotypes are a result of knocking out sensory feedback specifically from the legs ( data not shown ) . 10 . 7554/eLife . 00231 . 024Figure 7 . Quantification of gait parameters in sensory deprived animals .", "( A ) .", "Stance traces of three representative animals walking at a similar speed .", "For simplicity , only left stance traces are shown .", "Traces for sensory deprived genotypes display a longer step length; higher jitter and a more variable AEP and PEP .", "( See panels ( B ) – ( D ) and ( H ) for quantification ) .", "In ( B ) , ( C ) and ( E ) – ( G ) , colored lines represent graphical fits .", "See Table 2 for statistical analysis of ( B ) , ( C ) , ( E ) , ( F ) and ( G ) .", "( B ) .", "Step length .", "Sensory deprived animals display an increased step length .", "( C ) .", "Stance linearity .", "Sensory deprived animals display a more jittery movement .", "( D ) .", "AEP and PEP footprint clustering .", "Wild type data correspond to the speed range of nan36a and 5-40Leg>TNT ( 12 . 6 to 27 . 7 mm/s ) .", "In ( D ) , ( H ) , boxplots represent the median as the middle line and the lower and upper edges of the boxes representing the 25% and 75% quartiles , respectively; the whiskers represent the range of the full data set .", "Statistical analysis with Kruskal–Wallis-ANOVA ( p<0 . 0001 ) followed by Dunn's post hoc test , ***p<0 . 001 .", "( E ) .", "Swing duration .", "( F ) .", "Stance duration .", "( G ) .", "Swing speed .", "( H ) .", "Footprint alignment .", "Slow-walking sensory deprived animals display more aligned footprints compared to wild type flies .", "Data were grouped into slow ( <20 mm/s ) and medium speeds ( between 20 and 34 mm/s ) .", "Kruskal–Wallis-ANOVA test: p=0 . 0003 and NS for the slow and medium speed groups , respectively .", "Asterisks indicate the significance of the decrease in footprint clustering between genotypes .", "( Data analyzed by the Dunn's post hoc significance test , **p<0 . 005 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 02410 . 7554/eLife . 00231 . 025Table 2 . Multiple regression models for wild type versus sensory-deprived fliesaDOI: http://dx . doi . org/10 . 7554/eLife . 00231 . 025Wild type vs nan36aStep length ( Figure 7B ) Stance linearity ( Figure 7C ) Swing duration ( Figure 7E ) Swing speed ( Figure 7G ) Stance duration ( Figure 7F ) Tripod index ( Figure 6D ) CoefSEp-valueCoefSEp-valueCoefSEp-valueCoefSEp-valueCoefSEp-valueCoefSEp-valueWT y intercept746 . 793 . 80 . 0291 . 632 . 60 . 03 . 6 × 10−21 . 9 × 10−30 . 019 . 12 . 10 . 02 . 3 × 10−11 . 2 × 10−20 . 0−9 . 2 × 10−44 . 2 × 10−20 . 982Δ y intercept587 . 4270 . 50 . 0349 . 896 . 40 . 02 . 2 × 10−25 . 2 × 10−30 . 0−4 . 55 . 90 . 4511 . 9 × 10−13 . 4 × 10−20 . 0−4 . 1 × 10−11 . 2 × 10−10 . 001WT slope47 . 23 . 30 . 0−70 . 99 . 90 . 0−1 . 3 × 10−46 . 4 × 10−50 . 0430 . 60 . 10 . 0−5 . 4 × 10−23 . 5 × 10−30 . 01 . 8 × 10−21 . 4 × 10−30 . 0Δ Slope−11 . 312 . 90 . 4−89 . 132 . 10 . 007−8 . 4 × 10−42 . 5 × 10−40 . 0010 . 50 . 30 . 115−1 . 6 × 10−24 . 8 × 10−30 . 0021 . 6 × 10−25 . 6 × 10−30 . 006Wild type versus 5-40Leg>TNTWT y intercept746 . 793 . 80 . 0291 . 634 . 00 . 03 . 6 × 10−21 . 9 × 10−3<0 . 00119 . 12 . 1<0 . 0012 . 3 × 10−11 . 2 × 10−20 . 0−9 . 2 × 10−44 . 3 × 10−20 . 983Δ y intercept622 . 8295 . 30 . 038242 . 2115 . 70 . 0391 . 7 × 10−21 . 0 × 10−3<0 . 00110 . 56 . 40 . 11 . 6 × 10−14 . 1 × 10−20 . 0−3 . 6 × 10−11 . 4 × 10−10 . 009WT slope47 . 23 . 20 . 0−70 . 910 . 30 . 0−1 . 0 × 10−46 . 0 × 10−50 . 040 . 70 . 1<0 . 001−5 . 4 × 10−23 . 7 × 10−30 . 01 . 8 × 10−21 . 5 × 10−30 . 0Δ Slope11 . 415 . 60 . 465−54 . 839 . 70 . 17−2 . 4 × 10−53 . 1 × 10−40 . 94−0 . 50 . 30 . 12−4 . 4 × 10−21 . 4 × 10−20 . 0031 . 6 × 10−27 . 1 × 10−30 . 032aCoef stands for the estimated regression coefficient , SE represents its standard error .", "In this model we log transformed average speed for parameters that were non-linear with respect to speed ( step linearity and stance duration ) .", "WT y intercept indicates the y intercept for wild type .", "Δ y intercept indicates the difference in the y intercept between the experimental condition and wild type .", "WT slope reports the slopes of the wild type regression lines .", "p-values >0 . 05 are in bold italics; those <0 . 05 are in italics underlined .", "If Δ slope is >0 . 05 ( bold italics ) the regression curves are considered non-interacting ( ∼parallel ) .", "If Δ y intercept is <0 . 05 ( italics underlined ) , the parameter is considered different from WT .", "Interestingly , several of the differences we observed in flies deprived of sensory feedback were more pronounced in animals that walked more slowly compared to faster flies ( e . g . stance duration and footprint alignment; Figure 7F , H ) .", "The tripod index also decreased in slower flies ( Figure 6D ) .", "It is also noteworthy that several of the trends observed in sensory-deprived flies ( such as AEP , PEP , and footprint alignment ) are the same as those observed in faster wild type flies .", "In addition , sensory deprived flies position their midlegs further from their bodies , similar to the trend seen in fast flies ( Figures 3F and 7A ) .", "These observations are consistent with the idea that flies walking at slow , medium , and fast speeds use distinct neural programs , and that flies walking at fast speeds are less dependent on sensory feedback ." ], [ "In larger insects such as the stick insect and locust , the role of sensory feedback in coordinated walking has been addressed by local surgical ablations coupled to electrophysiological measurements ( Usherwood et al . , 1968; Cruse et al . , 1984; Akay et al . , 2001; Borgmann et al . , 2009 ) .", "For example , using single leg preparations of the stick insect , it was found that for mid and foreleg steps to be out of phase ( as they are in wild type walking ) , stimulation of sensory afferents in the midleg was required; without this stimulation , the CPGs for these legs fired in phase ( Borgmann et al . , 2009 ) .", "These , as well as additional observations ( Büschges et al . , 2008 ) , argue that sensory feedback from legs in stance phase is required for interleg coordination .", "The use of Drosophila brings an additional set of tools to address these questions .", "In one set of experiments , we took advantage of a mutation that specifically disrupts the function of the ChOs while a second set of experiments used a combinatorial misexpression approach to inactivate the majority of sensory neurons in the leg , including the ChO , hair plates , campaniform sensilla and mechanosensory bristles .", "In both sets of experiments , gait parameters and interleg swing phases were largely normal when sensory feedback was impaired .", "These results suggest that in the fly interleg coordination is not dominated by sensory feedback .", "Instead , they suggest that communication between CPGs , perhaps by local interneurons , may be sufficient for coordination , a suggestion that is supported by recent studies in vertebrates ( ( Kiehn , 2011 ) for review ) .", "In insects , interneurons have been implicated in interjoint coordination within individual legs and in footfall placement , but their role in interleg coordination has not been established ( Brunn and Dean , 1994; Brunn , 1998; Matheson , 2002 ) .", "The different requirements for sensory feedback observed here compared to electrophysiology studies in the stick insect may in part be because in our experiments leg sensory neurons were inactivated in all six legs ( in the case of 5-40leg>TNT ) or all ChOs in the animal ( in the case of the nan mutant ) .", "We speculate that when all six legs are equally impaired , the flies resort to a CPG and interneuron-dominated system , which is sufficient to execute the tripod gait .", "Consistent with this notion , although interleg coordination was impaired upon amputation of a single hindleg in Drosophila , the flies were able to recover a partial tripod gait ( Wosnitza et al . , 2012 ) .", "The data derived from sensory-deprived flies also supports the idea that flies may be less dependent on sensory feedback when they walk fast because , for several parameters , the defects were significantly more pronounced in slow flies .", "This observation is consistent with the idea that when flies walk fast , they use a largely sensory-independent CPG-based system where individual CPGs communicate by local interneurons to achieve coordination .", "In contrast , in slower flies , sensory feedback would be invoked to allow animals to negotiate more complex terrains .", "These speculations are supported by our observations that several parameters measured here , most prominently footprint clustering , appear distinct at slow , medium , and fast walking speeds .", "Thus , although the tripod gait is used at all speeds , distinct neural programs may come into play that are more or less dependent on sensory feedback , depending on the speed ." ], [ "The fTIR method and FlyWalker software provides a robust suite of tools for analyzing walking in Drosophila .", "Using this approach , we present many parameters that comprehensively describe walking by wild type flies .", "Our initial analysis of sensory-deprived flies reveal that proprioception , at least from the legs , is largely dispensable for coordinated walking and the tripod gait .", "In the future , additional expression tools will allow the targeting of other subsets of sensory and interneurons and the ability to carry out gain-of-function experiments .", "Moreover , the fTIR apparatus can be used with other arthropods as long as they , like Drosophila , possess adhesive structures on their tarsi ( Gorb et al . , 2007 ) .", "However , although footprints and body position can be readily identified with this method , our current setup does not provide the ability to follow individual leg joints , which can be done in larger insects such as the stick insect ( Cruse and Bartling , 1995 ) .", "Nevertheless , the set of parameters measured here underscore the complex mechanisms and circuits regulating hexapod locomotion .", "Our approach , in combination with the growing collection of genetic tools available in Drosophila should open many additional opportunities to unravel the mechanism of locomotion in animals and improve bio-inspired machines ." ], [ "Oregon R flies were reared on standard cornmeal food at 25°C .", "nan-Gal4 ( F-Gal4 ) was obtained from the Bloomington Stock Center .", "5-40-Gal4 was a gift from Cynthia Hughes ( Hughes and Thomas , 2007 ) , UAS-FRT-stop-FRT-TNT was from Barry Dickson ( Stockinger et al . , 2005 ) and nan36a was kindly provided by Marco Gallio .", "dacRE-FLP was generated by TOPO cloning the Ring Enhancer ( RE ) fragment from the regulatory region of dac ( Giorgianni and Mann , 2011 ) into an entry clone followed by a Gateway reaction into a FLP destination plasmid .", "Transgenic lines were generated by standard procedures in a yw background .", "All experiments were carried out with 1 to 7 day old animals at room temperature .", "To select the correct genotype , flies were anesthetized on a cold plate and allowed to recover at least 24 h .", "Before imaging , flies were kept in clean glass vials for ∼15 min .", "Five Neutral White ( 4100K ) LEDs from Luxeonstar ( Brantford , Ontario , Canada ) , Pre-Mounted on a 10 mm Square Base ( 230 lm @ 700mA ) were wired in series and glued to a 60 mm CPU heat-sink and fan ( StarTech . com Lockbourne , Ohio ) .", "Three LED/heat-sink sets were wired in parallel and clamped to the edges of a 150 mm × 150 mm × 6 . 5 mm Borofloat Glass ( Advanced Optics , Pewaukee , WI ) .", "The edges of the glass were scribed and broken so they would be clear , but not polished .", "The fourth edge of the glass was fixed to a filter mount and the whole system was attached to a bench plate by steel mounting posts ( Edmund optics , Barrington , NJ ) .", "The LEDs were powered by a variable power supply ( Tekpower , Montclair , CA ) .", "In order to minimize deflected light , the edges of the glass and the LEDs are covered with black ink or black cardboard .", "Consequently , most of the light from the LEDs enters the glass below the critical angle .", "To recycle light that reaches the edges of the optical glass , the remaining edges of the glass are covered with aluminum foil .", "Movies were acquired using a Photron ( San Diego , CA ) Fastcam MC2 camera using a 55 mm Computar ( Commack , NY ) telecentric lens , which has very little optical distortion .", "By measuring 1 mm intervals at multiple positions within the field of view we determined the optical distortion to be less than 4% .", "The lens aperture ( f/2 . 8 ) was maximized in order to increase light sensitivity and minimize depth of field .", "If the glass–air interface becomes disrupted by the footprints of a fruit fly , the light becomes deflected and is detected by the high-speed video camera .", "Consequently , when a leg is in stance phase it is detected by fTIR and the absence of signal indicates a swing phase .", "In addition , a small amount of reflected light illuminates the fly's body ( Figure 1—figure supplement 1 ) .", "In the center of the fTIR apparatus , a plexiglass tunnel limits the mobility of the fly and increases the chance it will move in a straight line ( Figure 1—figure supplement 1 ) .", "This tunnel is slightly separated from the glass by a nylon string in order to minimize the contact between the plexiglass and optical glass , which would produce a fTIR signal .", "In order to trigger an optomotor response , two optical fibers connected to a UV light source are present at both ends of the tunnel ( Figure 1—figure supplement 1 ) .", "Images are detected within a ∼1 . 2 cm2 area by a high-speed camera at a rate of 250 frames per second at f/2 . 8 .", "This allows sufficient light to be detected per frame while permitting a high temporal resolution of 4 milliseconds per frame .", "For the fly tunnel , a rectangle measuring 7 by 40 mm was cut out of a transparent plexiglass plate measuring 4 . 76 × 46 × 23 mm .", "At both ends , a 1 mm diameter hole was made in order to accommodate an optical fiber .", "As a cover for the tunnel , a second piece plexiglass of equal size was glued on top .", "A 5 mm hole was drilled on the cover approximately 1 cm from the edge in order to insert the flies into the chamber .", "In order to raise the chamber slightly , two small holes 1 mm apart , were drilled at each of the four corners of the chamber .", "A nylon wire—0 . 48 mm diameter—was inserted through the two holes to create a loop that functions as spacers to prevent the chamber from contacting the optical glass .", "Finally , the chamber was painted in black on the outside and the interior walls coated with Fluon AD-1 ( www . entosupplies . com . au ) mixed with black India ink in order to encourage walking on the optical glass ( Figure 1—figure supplement 1 ) .", "FlyWalker was created in MATLAB , and analyzes the sequence of images from the videos by registering the position of the body and each footprint .", "Once the image sequence is loaded into the FlyWalker interface ( Figure 1—figure supplement 2 ) , its analysis becomes a two-step process .", "First , body and footprints are tracked automatically by the software based on sudden increases of brightness within preset thresholds , as well as information from preceding frames .", "Any fTIR signal present in frames prior to the appearance of the fly ( due for example to dust ) is subtracted from all subsequent frames to minimize false positive signals .", "Sequential frame comparison sets the displacement axis , which helps with the identification of each of the six footprints , for example the left and right fore ( LF , RF ) , left and right mid ( LM , RM ) , and left and right hind ( LH , RH ) legs .", "Second , the software allows the user to manually correct any mislabeled or missed footprints .", "A sample video of the processed fTIR effect can be seen in Video 2 .", "For each frame , the user can reject a mislabeled footprint or add a footprint that is visible , but was not included because it fell below the preset threshold .", "The script is optimized to minimize the time spent editing each video .", "A setup window allows the user to define auto-tracking parameters and which features to show on-screen ( Figure 1—figure supplement 2 ) .", "For example , depending on the user-defined settings , the body trace and/or past footprints can be visualized ( Figure 1C ) .", "Importantly , through the use of a calibration reticle , the user can input the pixel/μm ratio , which allows the program to introduce a scale bar ( Figure 1C ) , calculate distances and speeds .", "Speed ( instantaneous and average ) Frequency Period Metachronal lag Swing speed ( average and for individual steps ) Step length ( average and for individual steps ) Swing time ( average and for individual steps ) Stance time ( average and for individual steps ) Footprint alignment ( average and for individual footprint clusters ) Anterior Extreme Position ( AEP ) Posterior Extreme Position ( PEP ) Footprint clustering ( AEP and PEP ) Stance trace ( average and for individual segments ) Body trace Tripod index Tetrapod index Gait index Step period ( LH:RH; LM:RM; LF:RF; LH:LM; LM:LF; RH:RM; RM:RF ) Angle between footprint and displacement axis vs time Footprint distance to body center vs time Footprint parallel distance to body center vs time Footprint perpendicular distance to body center vs time Instantaneous speed vs time Gait vs time Combined gait and instantaneous speed vs time Anterior extreme position plus stance trace Posterior extreme position plus stance trace Geometric combinations generated by footprints Step size vs time Gait maps ( fixed and automatic time scale ) Gait index vs time ( fixed and automatic time scale ) Step velocity; instantaneous speed; color code and gait index plots combined vs time ( fixed and automatic time scale ) .", "Summary plots including combined gait and instantaneous speed and anterior extreme position plus stance trace; plus values for average speed , tripod index , tetrapod index , stance trace and average step distance .", "Excel file with all parameters Image sequence from the tracking program .", "For most plots , each data point comes from a single video , except for phase and metachronal lag plots where each video generated multiple data points .", "Because some parameters changed with speed we fit multiple regression models ( Table 2 ) .", "The interaction term corresponds to the slope variation ( Δ slope ) .", "When the relationship between the parameter and the average speed was non linear ( stance linearity and stance duration ) , we transformed the average speed by its natural log .", "Because of the different behaviors of the slow , medium , and fast speed groups , some parameters were compared between individual speed groups .", "In these cases , the data were presented as box and whisker plots .", "For independent observations , comparisons between speed groups ( Figures 3E and 4C ) or between mutant groups ( Figures 6C and 7D , H ) were done using Kruskal–Wallis test followed by Dunn's post hoc test ( for non-normal distributions ) or one-way-ANOVA followed by Tukey's post hoc tests ( for normal distributions ) .", "For paired observations ( AEP vs PEP in Figure 3E and Figure 3—figure supplement 3 ) with a normal distribution , a paired t-test was used .", "Otherwise a Wilcoxon signed rank test was used ( GraphPad Prism , San Diego , CA ) .", "The least square regression line and the R correlation coefficient are indicated in all scatter plots .", "Although average speeds of ∼20 mm/sec are underrepresented in our collection of wild type videos , the histogram shown in Figure 2A is not statistically different from a normal distribution ( Shapiro-Wilk test; p=0 . 31 ) .", "However , a normal probability plot shows non-linearity of the data at <20 mm/s and greater than ∼35 mm/s ( data not shown ) , suggesting that the distribution has significant non-Gaussian outliers in the tails .", "Terms and definitions for parameters used in this paper .", "§ indicates definitions described previously by Strauss and Heisenberg 1990 ( and references within ) ." ] ]

[ "Coordinated walking in vertebrates and multi-legged invertebrates such as Drosophila melanogaster requires a complex neural network coupled to sensory feedback .", "An understanding of this network will benefit from systems such as Drosophila that have the ability to genetically manipulate neural activities .", "However , the fly's small size makes it challenging to analyze walking in this system .", "In order to overcome this limitation , we developed an optical method coupled with high-speed imaging that allows the tracking and quantification of gait parameters in freely walking flies with high temporal and spatial resolution .", "Using this method , we present a comprehensive description of many locomotion parameters , such as gait , tarsal positioning , and intersegmental and left-right coordination for wild type fruit flies .", "Surprisingly , we find that inactivation of sensory neurons in the fly's legs , to block proprioceptive feedback , led to deficient step precision , but interleg coordination and the ability to execute a tripod gait were unaffected ." ]

[ "Most animals need to be able to move to survive .", "Animals without limbs , such as snakes , move by generating by wave-like contractions along their bodies , whereas limbed animals , such as vertebrates and arthropods , walk by coordinating the movements of multi-jointed arms and legs .", "Locomotion in limbed animals involves bending each joint within each arm or leg in a coordinated manner , while also ensuring that the movements of all the limbs are coordinated with each other .", "In bipeds such as humans , for example , it is critical that one leg is in the stance phase when the other leg is in the swing phase .", "The rules that govern the coordination of limbs also depend on the gait , so the rules for walking are not the same as the rules for running .", "The nervous systems of bipeds and other animals that walk solve these problems by using complex neural circuits that coordinate the firing of the relevant motor neurons .", "Two general mechanisms are used to coordinate the firing of motor neurons .", "In one mechanism , local interneurons within the central nervous system coordinate motor neuron activities: in vertebrates these interneurons are found in the spinal cord .", "A second mechanism , termed proprioception , relies on sensory neurons that report the load and joint angles from the arms and legs back to the central nervous system , and thereby influence the firing of the motor neurons .", "Remarkably , both of these mechanisms , and also the types of neurons that comprise motor neuron circuits , are conserved from arthropods to vertebrates .", "Mendes et al . describe a new approach that can be used to analyze how the fruit fly , D . melanogaster , walks on surfaces .", "They use a combination of an optical touch sensor and high-speed video imaging to follow the body of the fly as it walks , and also to record when and where it places each of its six feet on the surface as it moves .", "Then , using a software package called FlyWalker , they are able to extract a large of number of parameters that can be used to describe locomotion in adult fruit flies with high temporal and spatial resolution .", "Many of these parameters have never been measured or studied before .", "Mendes et al . show that fruit flies do not display the abrupt transitions in gait that are typically observed in vertebrates .", "However , they do modify their neural circuits depending on their speed: indeed it appears that flies use subtly different neural circuitry for walking at slow , medium and fast speeds .", "Moreover , when genetic methods are used to block sensory feedback , the fly is still able to walk , albeit with reduced coordination and precision .", "Further , the data suggest that proprioception is less important when flies walk faster compared to when they walk more slowly .", "The next step in this research will be to combine this new method for analyzing locomotion in flies with the wide range of genetic tools that are available for the study of Drosophila: this will allow researchers to explore in greater detail the components of the motor neuron circuitry and their role in coordinated walking ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "microbiology and infectious disease" ]

[ [ "Cis-acting regulatory elements play essential yet diverse roles in the life cycle of RNA viruses ( Liu et al . , 2009; Nicholson and White , 2014 ) .", "These elements have been found to regulate viral replicase binding and viral RNA ( vRNA ) synthesis ( Filomatori et al . , 2006; Vogt and Andino , 2010; Wu et al . , 2009 ) , nucleocapsid assembly ( Goto et al . , 2013; Keane et al . , 2015; Morales et al . , 2013 ) , and viral translation ( Brierley and Dos Ramos , 2006; Kieft , 2008; Liu et al . , 2009; Simon and Miller , 2013 ) .", "In particular , local RNA elements in viral genomes are often involved in long-range RNA-RNA interactions ( Alvarez et al . , 2005; Nicholson and White , 2014; Shetty et al . , 2013 ) , which are often referred to as genome cyclization and can bring regions separated by several thousand nucleotides into proximity .", "Such strategies have been observed in different classes of RNA viruses and play different roles in virus propagation ( Nicholson and White , 2014 ) .", "In fact , the entire genomes of some RNA viruses are highly structured and have complex global architecture ( Athavale et al . , 2013; Wu et al . , 2013 ) .", "The genus flavivirus includes important causative agents for human diseases , such as dengue virus ( DENV1-4 ) , West Nile virus ( WNV ) , Japanese encephalitis virus ( JEV ) , yellow fever virus ( YFV ) and the emerging Zika virus ( ZIKV ) ( Fauci and Morens , 2016; Mlakar et al . , 2016 ) .", "Major phylogenetic groups ( Blitvich and Firth , 2015; Moureau et al . , 2015 ) of flaviviruses include mosquito-borne flaviviruses ( MBFVs ) , tick-borne flaviviruses ( TBFVs ) , insect-specific flaviviruses ( ISFVs ) and flaviviruses with no known vectors ( NKVs ) .", "Leading to diseases with symptoms ranging from mild fever and rash to severe hemorrhagic fever and encephalitis , the emergence and re-emergence of flaviviruses always arouses global concern .", "The flaviviruses are single-stranded , positive-sense RNA viruses , and their genome is approximately 11 kb and contains a single ORF , which encodes a polyprotein precursor with more than 3000 residues .", "The precursor is further processed into 3 structural proteins ( capsid , pre-membrane/membrane and envelope ) and at least 7 nonstructural proteins ( NS1 , NS2A , NS2B , NS3 , NS4A , NS4B and NS5 ) .", "The largest nonstructural protein , NS5 , performs methyltransferase ( Egloff et al . , 2002; Zhou et al . , 2007 ) , guanylyltransferase ( Issur et al . , 2009 ) and RNA-dependent RNA polymerase ( RdRp ) activity ( Nomaguchi et al . , 2003; Uchil and Satchidanandam , 2003; Yap et al . , 2007 ) .", "NS5 interacts with vRNA , other viral nonstructural proteins and host factors to assemble to the viral replication complex essential for vRNA synthesis ( Klema et al . , 2015 ) .", "On the other hand , the highly structured 5′ and 3′ untranslated regions ( UTRs ) occupy the termini of the viral genome ( Friebe and Harris , 2010; Gebhard et al . , 2011; Villordo et al . , 2016 ) and contain cis-acting elements crucial for vRNA replication ( Clyde et al . , 2008; Filomatori et al . , 2006; Liu et al . , 2013; Lodeiro et al . , 2009; Rouha et al . , 2011; Villordo et al . , 2010 ) , translation ( Chiu et al . , 2005; Clyde and Harris , 2006 ) , viral pathogenesis ( Chapman et al . , 2014; Funk et al . , 2010; Roby et al . , 2014 ) and host adaptation ( Villordo et al . , 2015 ) .", "Hybridization of complementary sequences at the 5′ and 3′ termini , which include the 5′-3′ upstream AUG region ( UAR ) ( Alvarez et al . , 2005; Zhang et al . , 2008 ) , downstream AUG region ( DAR ) ( Friebe and Harris , 2010; Friebe et al . , 2011 ) and cyclization sequence ( CS ) ( Khromykh et al . , 2001 ) elements , circularizes the genome of MBFV .", "Genome cyclization , which is also modulated by the downstream of 5′ CS pseudoknot ( DCS-PK ) in the capsid-coding region ( de Borba et al . , 2015; Liu et al . , 2013 ) , is crucial for the translocation of NS5 from the 5′ stem-loop A ( SLA ) promoter to the RNA synthesis initiation site at the 3′ end ( Filomatori et al . , 2006 ) .", "However , the mechanistic details of this critical process are poorly understood .", "The 5′ SLA structure and genome cyclization strategy were also identified in other phylogenetic groups of the flavivirus genus , although the specific details differ .", "The 5′ UAR element in MBFV usually participates in the formation of a local hairpin , stem-loop B ( SLB ) , in the linear conformation of the vRNA .", "The SLA and SLB elements are separated by a short uracil-rich sequence , which has been shown to mediate vRNA replication of DENV ( Lodeiro et al . , 2009 ) .", "In the present study , we demonstrated that the U-rich region of MBFV is involved in the formation of a conserved RNA duplex that is designated as a 5′-UAR-flanking stem ( UFS ) because it locks the 5′ UAR/SLB element between its two strands .", "A combination of our results suggested that the UFS and its cousin elements regulate the binding of viral RdRp to vRNA dynamically by switching their conformations in response to the long-range interactions between the viral 5′ and 3′ ends .", "Furthermore , UFS switching is a general replication strategy in flaviviruses with vertebrate hosts , highlighting their role in the host adaptation and evolution of flaviviruses ." ], [ "Although the U-rich region in the 5′ end of the MBFV genome has been shown to enhance vRNA replication ( Friebe and Harris , 2010; Lodeiro et al . , 2009 ) , there were controversial opinions regarding the detailed structures downstream of SLA in flaviviruses ( Dong et al . , 2008; Gebhard et al . , 2011; Liu et al . , 2013; Villordo and Gamarnik , 2009; Zhang et al . , 2008 ) .", "To clarify the structural characteristics of the region downstream of SLA , representative sequences derived from various flavivirus species were subjected to the mfold RNA folding server ( Zuker , 2003; Zuker and Jacobson , 1998 ) .", "Because the region of interest is involved in genome cyclization , both the 5′ end sequences and the query sequences composed of the 5′ and 3′ ends were analyzed .", "Figure 1A shows the representative results of the RNA structure prediction .", "The local RNA structures in the 5′ and 3′ ends of DENV4 vRNA are demonstrated , and elements involved in long-range interactions are highlighted .", "Structure prediction of the 5′ end local structures showed that the U-rich region of most MBFVs ( except the YFV clade ) forms a duplex with complementary sequences in or near the viral translational starting region ( Figure 1B ) .", "Because this duplex confines the 5′ UAR/SLB element between its two strands , we designated it as the UFS .", "Furthermore , the UFS duplex was predicted to unwind due to the formation of the panhandle structure between the 5′ and 3′ ends when long-range interactions between the genome termini were considered ( Figure 1—figure supplement 1 and Supplementary file 1 ) . 10 . 7554/eLife . 17636 . 003Figure 1 . Identification and comparison of the 5′ UAR-UFS elements among flaviviruses .", "( A ) Terminal RNA structures of the DENV4 genome .", "The UAR , DAR and CS elements are highlighted in yellow , green and red , respectively .", "Pseudoknotted interactions are labeled .", "The UFS stem region is indicated by parentheses .", "( B ) Comparison of the 5′ UAR-UFS and UFS-like elements in MBFVs , NKVs and ISFVs .", "*: KEDV and ZIKV were supposed to belong to the same clade herein .", "The MBFV 5′ UAR sequences and NKV sequences involved in genome cyclization are colored in yellow .", "The nucleotides that participate in the DAR interactions of MBFVs are labeled in green .", "Translational start codons in ( A ) and ( B ) are shown in red .", "Virus name abbreviations are annotated in Figure 1—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 00310 . 7554/eLife . 17636 . 004Figure 1—figure supplement 1 . Circular conformation of the DENV4 genomic RNA . The UAR , DAR and CS elements are shown in yellow , green and red , respectively .", "The sequence of the unwound UFS duplex is shown in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 00410 . 7554/eLife . 17636 . 005Figure 1—figure supplement 2 . Molecular phylogenetic analysis of the flavivirus genus . The ORF sequences of 48 flavivirus species were obtained from Genbank and aligned by the Clustal W method .", "The evolutionary relationships of flaviviruses were inferred by using the Maximum Likelihood method based on the Tamura-Nei model .", "Analyses were performed with MEGA6 .", "Different evolutionary groups as well as various clades among the MBFV group are annotated . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 005 Because the local folding pattern of the 5′ end and the mode of genome cyclization in the YFV clade are different than those in other MBFVs ( Figure 1B ) , the corresponding hairpin including the U-rich region in the YFV clade was recognized as ψUFS .", "Local structures similar to UFS were also identified in ISFVs ( Figure 1B ) and Modoc virus ( MODV ) among the NKVs ( Figure 1B ) , whereas the non-vectored Rio Bravo virus ( RBV , Figure 1B ) was shown to contain a ψUFS structure .", "The UFS and ψUFS in NKVs were also involved in genome cyclization , similar to the corresponding structures in MBFVs .", "In contrast , the UFS-like structures were not predicted to be involved in genome cyclization in ISFVs , suggesting that the UFS in MBFVs and UFS-like structures in ISFVs function differently .", "Finally , the UFS/ψUFS structure was not identified in TBFVs or in the Yokose virus clade of NKVs; instead , a short hairpin with a large loop occupied the analogous location of the UFS in these viruses ( Supplementary file 2 ) .", "Taken together , the above results demonstrated the conservation of UFS elements among the flavivirus genus and suggested that the secondary structures of UFS elements are affected by genome conformation .", "To investigate whether the duplex conformation of the UFS can indeed exist locally under in vitro conditions , RNA molecules corresponding to the 5′ ends of DENV1-4 , JEV and ZIKV were analyzed by selective 2′-hydroxyl acylation analyzed by primer extension ( SHAPE ) .", "The SHAPE reactivity data were annotated on RNA structure models generated by mfold prediction and sequence comparison ( Figure 2 ) .", "The SHAPE results were shown to agree well with the predicted RNA structures .", "It was shown that the UFS element of JEV forms a conical duplex structure under in vitro conditions ( Figure 2A ) because all nucleotides of the UFS exhibited low SHAPE reactivity .", "Similar results were obtained for DENV serotypes 1 , 3 , 4 and ZIKV ( Figure 2B , D , E and F ) , except that several nucleotides in the ZIKV and DENV1/3 UFS regions had elevated SHAPE reactivity , which was likely due to the presence of internal loops .", "In contrast , the SHAPE results indicated that the UFS duplex of DENV2 is highly unstable because the DENV2 UFS region showed considerable SHAPE reactivity ( Figure 2C ) .", "This result was consistent with thermodynamic calculations and suggested that the UFS duplex is a highly dynamic structure , at least in some flaviviruses .", "The presence of the DENV UFS duplex conformation was further confirmed by SHAPE analysis of DENV3 5′ end RNA molecules containing mutations in the UFS ( Figure 2—figure supplement 1 ) .", "It was shown that the SHAPE reactivity of the UFS region significantly increased in UFS-disrupted mutants ( Figure 2—figure supplement 1 , D3-M13A and D3-M13B ) , and restoration of UFS base-pairing endowed the corresponding region with low SHAPE reactivity ( Figure 2—figure supplement 1 , D3-M13C ) .", "The above results demonstrated that the UFS duplex can exist locally in the vRNA 5′ end under in vitro conditions , suggesting that the UFS can assume the duplex conformation , at least under certain states of the flavivirus genome . 10 . 7554/eLife . 17636 . 006Figure 2 . SHAPE analysis of the 5′ end RNA of representative flaviviruses . The SHAPE reactivity results are labeled in the structure models of different flaviviruses .", "Highly reactive nucleotides ( SHAPE reactivity > 0 . 85 ) are labeled in red , whereas nucleotides with moderate SHAPE reactivity ( 0 . 4–0 . 85 ) are labeled in green .", "Black-colored nucleotides indicate low or no SHAPE reactivity ( <0 . 4 ) .", "Nucleotides lacking SHAPE data are labeled in gray .", "The primer-binding region in the reverse transcription reaction is shown in light blue .", "( A ) JEV , strain SA-14-14-2 ( AF315119 ) .", "( B ) DENV1; the sequence is based on strain WestPac ( U88535 ) .", "( C ) DENV2; the sequence is based on strain NGC ( KM204118 ) .", "( D ) DENV3; the sequence is based on strain 80–2 ( AF317645 ) .", "( E ) DENV4; strain 814669 ( AF326573 ) , with two artificial nonsense mutations that do not affect the 5′ end secondary structures or vRNA replication .", "The two mutations , located in the loop region of cHP and DCS-PK Loop 3 , respectively , are labeled in black-colored font .", "( F ) ZIKV; strain FSS13025 ( KU955593 ) .", "Results presented were from two biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 00610 . 7554/eLife . 17636 . 007Figure 2—source data 1 . Source data for Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 00710 . 7554/eLife . 17636 . 008Figure 2—figure supplement 1 . SHAPE analysis of the DENV3 UFS mutants . SHAPE analysis of DENV3 5′-300 nt RNA molecules containing UFS mutations .", "the SHAPE result of WT 5′-300 nt RNA is shown in parallel .", "SHAPE diagrams of 5′ 25–255 nt are shown , and the regions corresponding to various 5′ elements are indicated .", "Negative SHAPE reactivity is set to zero .", "The SHAPE reactivity of the 5′ UAR-UFS region is annotated on the corresponding secondary structures .", "Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , moderately reactive sites ( 0 . 4–0 . 85 ) are labeled in green .", "Unlabeled sites have low or no SHAPE reactivity ( <0 . 4 ) .", "Two biological replicates were performed for each RNA . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 008 The UFS is located just downstream of the SLA promoter element , and it interlocks with cyclization sequences in the vRNA 5′ end .", "This peculiar localization of the UFS suggests a unique role in vRNA replication .", "Functional mutagenesis of the UFS was performed using a DENV4 replicon with internal ribosome entry site ( IRES ) -controlled viral translation ( Liu et al . , 2013 ) .", "To eliminate the possibility that productive 5′-cap-directed translation is caused by mutations altering the start codon ( Clyde and Harris , 2006 ) , which is embedded in the DENV4 UFS duplex , a nonsense mutation was introduced into the variable loop 3 of DCS-PK in the background of p4-cHPstop-SP-IRES-Rluc-Rep to generate the p4-Dualstop-SP-IRES-Rluc-Rep replicon ( Figure 3A ) .", "The replication characteristics of the two replicons were basically the same ( Figure 3B ) .", "First , a panel of mutants was generated , and their replication efficiency was assessed in BHK-21 cells .", "Disrupting the base-pairing of the UFS ( Figure 3C , D . M1A , M1B , M3A and M3B ) greatly reduced vRNA replication , whereas reconstituting base-pairing by combining the corresponding mutations was able to restore vRNA replication ( Figure 3C , D . M1C and M3C ) , although the M3C mutant replicated moderately less efficiently than the wild-type ( WT ) replicon , possibly due to the apparent primary sequence changes in the U-rich region .", "Next , the impact of the M1 and M3 mutations on the circularized vRNA structure was assessed using the mfold online server .", "It was shown that the overall genome cyclization pattern was only slightly affected by the M1 and M3 mutations ( Supplementary file 3 ) , and the changes in the stability of the predicted circularized structures was considerable smaller than the changes in the local UFS structure .", "To further confirm the function of local UFS structure in vRNA replication , we designed a panel of mutations ( Figure 3—figure supplement 1 ) , which nether affected vRNA cyclization pattern nor the internal loop structure in the circular form , and the corresponding mutants were assessed for replication efficiency .", "It was shown that disruption of UFS base pairing ( mutant AGU and GAU ) reduced vRNA replication greatly , whereas substituting of a few base pairs in the UFS ( mutant AUA and ACG ) only have moderate effects on vRNA replication . 10 . 7554/eLife . 17636 . 009Figure 3 . The secondary structure of the UFS is required for flavivirus vRNA replication .", "( A ) The organization of the DENV4 replicon constructs .", "In p4-cHPstop-SP-IRES-Rluc-Rep , the translation of viral nonstructural proteins is controlled by the EMCV IRES , and artificial stop codons ( red inverted triangles ) were introduced into the cHP loop region as well as the end of the capsid-coding region to abolish translation directed by the 5′ cap structure .", "In the improved version , p4-Dualstop-SP-IRES-Rluc-Rep , one additional stop codon was introduced into loop 3 of the DCS-PK element to abolish the potential translational start from a cryptic AUG in the 5′ CS element .", "( B ) Replication characteristics of the above two replicons in BHK-21 cells .", "Relative luciferase units are defined as the ratio of luciferase units measured at different time points after transfection to the value measured at 6 hr post-transfection .", "( C ) Demonstration of DENV4 UFS mutants .", "Mutations are shown in purple .", "The 'A' mutants contain mutations in the downstream strand of the UFS , and the 'B' mutants contain mutations in the upstream strand of the UFS .", "The 'C' mutant in each group combines the 'A' and 'B' mutations to restore the secondary structure of the UFS .", "Note that the secondary structures shown here are for illustration only and do not represent the actual folding of these mutants .", "( D ) Replication efficiency of the UFS mutants .", "The results of one of two independent biological replicates were shown .", "One hundred nanograms per well of replicon RNA species were transfected into BHK-21 cells in triplicate .", "The results are expressed as the percentage of the relative luciferase units of a UFS mutant at 72 hr post-transfection to the value of the WT replicon ( relative replication efficiency ) .", "The error bar represents the standard deviation in all figures .", "The mean values of relative replication efficiency are listed below the names of the replicon constructs . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 00910 . 7554/eLife . 17636 . 010Figure 3—source data 1 . Source data for 3B , 3D and Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01010 . 7554/eLife . 17636 . 011Figure 3—figure supplement 1 . Replication of DENV4 UFS mutants targeting the AUG region .", "( A ) Demonstration of mutants targeting the AUG start codon .", "Mutation sites are shown in purple .", "These mutants were designed without affecting either genome cyclization or the terminal topology of circularized genome .", "For detailed information , please refer to Supplementary file 3 .", "( B ) Relative replication efficiency of UFS mutants shown above at 48 hr post-transfection .", "Results shown were obtained from three parallel technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 011 To confirm that the RNA conformation was changed as expected by the corresponding mutations , SHAPE analysis was performed for DENV4 5′-300 nt RNA containing the M1A , M1C , M3A or M3C mutation , and the SHAPE results were compared with WT RNA ( Figure 4 ) .", "In WT RNA , nucleotides composing the UFS only showed low or background SHAPE reactivity , whereas the SHAPE reactivity of the UFS region increased significantly in the M1A and M3A mutants , suggesting that the UFS duplex was indeed disrupted by the corresponding mutations .", "In contrast , the M1C and M3C mutations greatly reduced the SHAPE reactivity of the UFS region .", "RNA structure predictions using the RNAstructure ( Reuter and Mathews , 2010 ) software with SHAPE constraints confirmed that the UFS structure was disrupted/destabilized or reconstituted as aimed , and these mutations did not affect the overall secondary structure of the 5′ end RNA ( Supplementary file 4 ) .", "Taken together , the above results demonstrated that the presence of the UFS greatly enhances vRNA replication , and this function is directly correlated to its duplex conformation . 10 . 7554/eLife . 17636 . 012Figure 4 . SHAPE analysis of the DENV4 UFS mutants . SHAPE analysis was performed for DENV4 5′-300 nt RNA containing the UFS mutations M1A , M1C , M3A or M3C .", "The SHAPE result for WT 5′ end RNA is shown in parallel .", "SHAPE diagrams of 5′ 20–200 nt are shown , and the regions corresponding to various 5′ elements are indicated .", "Negative SHAPE reactivity is set to zero .", "The SHAPE reactivity of the 5′ UAR-UFS region is annotated on the corresponding secondary structures ( left ) .", "Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , and moderately reactive sites ( 0 . 4–0 . 85 ) are labeled in green .", "Unlabeled sites have low or no SHAPE reactivity ( <0 . 4 ) .", "Two biological replicates were performed for each RNA , and the error bars represent the standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01210 . 7554/eLife . 17636 . 013Figure 4—source data 1 . Source data for Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 013 We further attempted to investigate the influence of the UFS on the propagation of infectious virus .", "Various mutations targeting the UFS element ( Figures 3 and 5A ) were introduced into a DENV4 infectious clone .", "vRNA copies in BHK-21 cells were determined at different time points post-electroporation ( Figure 5B–D ) .", "It was shown that the vRNA copies in BHK-21 cells transfected with the M1A , M3A or M5A mutants were significantly lower than those transfected with WT vRNA at 48–72 hr post-transfection , whereas restoration of the UFS element by complementary mutations partially ( Figure 5C , M3C ) or fully ( Figure 5B , D . M1C and M5C ) recovered the vRNA replication levels , highlighting the importance of the UFS for vRNA replication of infection-competent viruses .", "An indirect immunofluorescence assay ( IFA , Figure 5E ) revealed that the DENV-positive rates were apparently lower in cells transfected with UFS-disrupted mutants than in those transfected with UFS-restored mutants or WT vRNA at the same time points .", "The virus titers in the culture supernatants of M1A-transfected BHK-21 cells were also significantly lower than those of M1C- or WT-transfected cells ( Figure 5F ) . 10 . 7554/eLife . 17636 . 014Figure 5 . The role of the UFS in viral propagation .", "( A ) DENV4 UFS M5 series mutants .", "Mutations are shown in purple .", "( B–D ) vRNA replication profiles of different UFS mutants in transfected BHK-21 cells .", "WT and NS5-inactive GVD mutant vRNA , measured in parallel , are shown as controls .", "( B ) M1 series , ( C ) M3 series and ( D ) M5 series .", "( E ) Indirect immunofluorescence assay of WT , GVD , and UFS-mutated vRNA-transfected BHK-21 cells .", "( F ) Determination of the virus titers in the supernatants of the M1 series mutant-transfected BHK-21 cells .", "The virus titers in the supernatants of WT vRNA-transfected cells were determined in parallel .", "The virus titers were normalized according to input vRNA copies determined at 4 hr post-electroporation .", "( G–I )", "The RNA stability of the UFS mutants was determined by qRT-PCR and are expressed as the fold change relative to the vRNA copies at 4 hr post-transfection .", "( G ) M1 series , ( H ) M3 series and ( I ) M5 series .", "The results were from one biological replicate .", "Experiments to determine vRNA copies and virus titers were performed in triplicate .", "Note that the time point '72 hr' for the GVD group in ( D ) contains data from only two parallel wells ( technical replicates ) , because the result of the third sample was unreliable due to an error in the RNA isolation process . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01410 . 7554/eLife . 17636 . 015Figure 5—source data 1 . Source data for 5B and 5C . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01510 . 7554/eLife . 17636 . 016Figure 5—source data 2 . Source data for 5DDOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01610 . 7554/eLife . 17636 . 017Figure 5—source data 3 . Source data for 5FDOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01710 . 7554/eLife . 17636 . 018Figure 5—source data 4 . Source data for 5G , 5H and 5I . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 01810 . 7554/eLife . 17636 . 019Figure 5—figure supplement 1 . Replication of ZIKV UFS mutants in BHK-21 cells . Indirect immunofluorescence assay of WT and UFS-mutated ZIKV vRNA-transfected BHK-21 cells were performed .", "The secondary structures of mutants were demonstrated and mutation sites are shown in purple . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 019 The effect of UFS mutations on vRNA stability was then assessed .", "To this end , DENV4 NS5-GVD-vRNAs containing different UFS mutations were transfected into BHK-21 cells by electroporation , and vRNA copies were determined at 4 , 24 , 48 and 72 hr post-transfection .", "Only negligible differences in RNA stability were found between WT RNA and the mutants of the M1 or M3 groups ( Figure 5G , H ) , and the stability of the M5C mutant was moderately reduced compared with that of the M5A mutant ( Figure 5I ) .", "Thus , the differences in the RNA stability of the UFS mutants did not account for their different replication characteristics .", "The above results demonstrated that the UFS element is required for virus propagation due to its function in vRNA replication .", "In agreement with the function of DENV4 UFS , we also showed that disrupting of the UFS greatly reduced viral propagation of ZIKV , whereas reconstitution of the UFS duplex restores ZIKV propagation by IFA tests performed in transfected BHK-21 cells ( Figure 5—figure supplement 1 ) .", "To understand the mechanism of how the UFS fulfills its role in vRNA replication , a series of in vitro assays were performed ( Figure 6 ) .", "Because NS5 encodes the RdRp activity required for vRNA synthesis and has specific interactions with the 5′ end of vRNA ( Dong et al . , 2008; Filomatori et al . , 2006; Lodeiro et al . , 2009 ) , whether the UFS is involved in the recruitment of NS5 was examined .", "WT and UFS-mutated DENV4 5′-300 nt RNA molecules were incubated with different concentrations of the NS5 RdRp domain ( NS5Pol ) , and the reaction mixtures were analyzed by native PAGE .", "The binding of NS5Pol to 5′-300 nt RNA containing the M1A or M1B mutation in UFS was much weaker than the binding to 5′-300 nt WT RNA , whereas the 5′-300 nt M1C and WT RNA bound to NS5Pol with comparable affinity ( Figure 6C , top and middle panels ) .", "The 5′-300 nt M3A and M3B RNA also interacted with NS5Pol weakly , whereas the combination of the two mutations apparently restored the interaction between NS5Pol and 5′-300 nt M3C RNA ( Figure 6C , bottom panel ) .", "The above results indicated that the UFS duplex is required for efficient NS5Pol binding in DENV4 .", "Moreover , EMSA assay demonstrated that the UFS duplex is also important for the efficient binding of JEV NS5Pol to its 5′ end RNA ( Figure 6—figure supplement 1 ) , suggesting that the function of UFS in viral RdRp recruitment is conserved among flaviviruses . 10 . 7554/eLife . 17636 . 020Figure 6 . UFS is crucial for RdRp recruitment and de novo RNA synthesis .", "( A ) Simplified diagrams of DENV4 virus 5′ end RNA constructs used for RdRp binding and/or in vitro RdRp activity assays .", "The region corresponding to the UFS element is shown in blue , and the red regions represent the genome cyclization elements .", "( B ) SDS-PAGE of purified recombinant NS5Pol of DENV4 .", "( C ) The binding of DENV4 NS5Pol to 5′-300 nt RNA molecules containing UFS mutations was analyzed by EMSA .", "No NS5Pol was present in the left first lane of each group .", "The NS5Pol concentrations in the reactions were approximately 2 . 0 , 3 . 5 , 5 . 0 and 7 . 5 μM ( from left to right ) .", "( D ) In vitro RdRp activity assay using 5′-160 nt RNA as a template .", "The reactions were incubated at 30°C for 20 min .", "Detection was based on the incorporation of biotin-11-CTP into the products .", "Left , the results of the M1 series; right , the results of the M3 series .", "Two bands were detectable in the blot: the upper template-sized band was generated by the terminal addition of biotin-11-CTP to the input template due to the terminal nucleotide transferase activity of NS5Pol in the presence of Mn2+ , and the lower band represented the dsRNA product of de novo RNA synthesis , which was confirmed by comparison of the electrophoretic patterns of the RdRp products and annealed dsRNA molecules ( Figure 6—figure supplement 2 ) .", "( E ) EMSA was performed for the 5′-160 nt RNA molecules containing M1 series mutations .", "The NS5Pol concentrations in the reactions were approximately 3 . 6 , 7 . 2 , 10 . 7 and 14 . 3 μM ( left to right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02010 . 7554/eLife . 17636 . 021Figure 6—figure supplement 1 . The UFS is required for efficient NS5 binding to 5′ RNA in JEV .", "( A ) JEV UFS mutants .", "Mutations are shown in purple .", "( B ) SDS-PAGE of purified recombinant JEV NS5Pol .", "( C ) The binding of JEV NS5Pol to 5′-320 nt RNA molecules containing UFS mutations was analyzed by EMSA .", "No NS5Pol was present in the left first lane of each group .", "The NS5Pol concentrations in the reactions were approximately 3 . 3 , 5 . 0 , 6 . 6 and 7 . 5 μM ( from left to right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02110 . 7554/eLife . 17636 . 022Figure 6—figure supplement 2 . Identification of the products of the RdRp reactions . Lane 1: dsRNA prepared by the annealing of the positive and negative strand of DENV4 5′-160 nt RNA .", "Lane 2–4: products of RdRp assays using different 5′-160 nt RNAs as templates .", "Lane 2: WT , Lane 3: M2A , Lane 4: M2C . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 022 Next , in vitro RdRp assays using DENV4 5′-160 nt RNA as a template were performed to investigate whether the role of the UFS in NS5Pol recruitment is required for de novo RNA synthesis ( Figure 6D ) .", "Apparently fewer products were generated in the reactions containing templates with disrupted UFS elements compared with the reaction containing the 5′-160 nt WT template , and restoration of the UFS duplex greatly increased the amounts of RdRp products in reactions using the corresponding 5′-160 nt mutants as templates .", "Moreover , an electrophoretic mobility shift assay ( EMSA ) experiment using 5′-160 nt RNA as a probe confirmed the function of UFS in NS5Pol recruitment , although the overall affinity of 5′-160 nt RNA for NS5Pol was lower than that of 5′-300 nt RNA ( Figure 6E ) .", "Taken together , the above results demonstrated that the duplex conformation of the UFS element is required for efficient NS5 recruitment and de novo initiation efficiency , which is closely correlated with its function in vRNA replication .", "The UFS elements were shown to be rich in UA/AU base pairs , which contribute less to the stability of RNA structures than CG/GC base pairs .", "Furthermore , internal loops and/or wobble base pairs are prevalent inside the UFS or at the junction connecting the UFS and SLB , which further decreases their structural stability .", "To investigate whether the low stability of the UFS duplex is associated with its function , UA base pairs of the DENV4 UFS were progressively substituted with GC base pairs , and the replication efficiency of the corresponding mutants was assessed using the above replicon system in BHK-21 cells ( Figure 7 ) .", "As expected , all mutants containing disrupted UFS elements showed only extremely low levels of replication ( Figure 7 , M2A , M2B , M4A and M4B ) .", "One single UA-to-GC substitution at different positions ( Figure 7 , M2C1 and M2C2 ) had little effect on vRNA replication , in agreement with the above results ( Figure 3 , M1C ) .", "Surprisingly , all UFS mutants containing 2 or more UA-to-GC base pair substitutions replicated poorly compared with the WT replicon ( Figure 7 , M2C and M4C ) .", "In contrast , the M5 mutant series , in which only one UA-to-GC substitution was involved , exhibited replication characteristics similar to the M1 and M3 series .", "The above results indicated that increasing the stability of the UFS duplex is harmful for vRNA replication . 10 . 7554/eLife . 17636 . 023Figure 7 . Effect of UFS stability on vRNA replication .", "( A ) Demonstration of UFS mutants .", "The mutations are indicated in purple .", "The M2C mutant contained two UA-to-GC base pair substitutions , whereas the M4C mutant contained four substitutions in the UFS secondary structure .", "It should be noted that the secondary structures are meant for illustration purposes only , and the structures displayed for UFS-disrupted mutants are not the most thermodynamically favorable ones .", "( B ) Relative replication efficiency of UFS mutants shown in ( A ) at 72 hr post-transfection .", "The results of one of two independent biological replicates were shown .", "Fifty nanograms per well of replicon RNA were transfected into 96-well plates .", "Experiments were performed in triplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02310 . 7554/eLife . 17636 . 024Figure 7—source data 1 . Source data for Figure 7 and Figure 7—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02410 . 7554/eLife . 17636 . 025Figure 7—figure supplement 1 . Effects of mutations in the SLB-UFS internal loop on vRNA replication .", "( A ) Demonstration of mutants targeting the internal loop between SLB and UFS .", "Mutation sites are shown in purple .", "( B ) Relative replication efficiency of UFS mutants shown above at 72 hr post-transfection .", "Results shown were obtained from two parallel technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02510 . 7554/eLife . 17636 . 026Figure 7—figure supplement 2 . NS5Pol binding assay of the DENV4 5′-300 nt M2A and M2C mutants . The binding of NS5Pol to 5′-300 nt WT , M2A and M2C RNA was analyzed by EMSA assay .", "The left first lane in each group contained no NS5Pol .", "The NS5Pol concentrations in the reactions were approximately 2 . 0 , 3 . 5 , 5 . 0 and 7 . 5 μM ( from left to right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 026 To further confirm this finding and rule out the possibility that the base pair composition itself affects UFS function , another panel of replicon mutants , which targeted the 3-nt internal loop region between the DENV4 UFS and SLB , was generated .", "Shortening the length of the internal loop to 1 nt or 2 nt did not affect vRNA replication greatly .", "In contrast , total deletion of the internal loop , which caused the SLB to stack onto the UFS , significantly reduced the vRNA replication efficiency ( Figure 7 and Figure 7—figure supplement 1 , L1D , L2D and L3D ) .", "Moreover , replication assays performed for various purine-to-purine and purine-to-pyrimidine substituting mutations targeting the internal loop ( Figure 7—figure supplement 1 ) suggested that the purine-rich properties of the internal loop are optimal for efficient DENV4 vRNA replication .", "Taken together , the above results demonstrated that the stability of the UFS duplex is a critical determinant for its function in vRNA replication .", "The defects in the vRNA replication of the over-stabilized UFS mutants were not caused by alternations in their affinity for NS5Pol , as an EMSA indicated that the 5′-300 nt M2C RNA bound to NS5Pol efficiently ( Figure 7—figure supplement 2 ) .", "RNA structure prediction ( Figure 1—figure supplement 1 and Supplementary file 1 ) and a previous report ( Sztuba-Solinska et al . , 2013 ) suggested that the UFS duplex is melted after genome cyclization , whereas the 5′ UAR/SLB element , which is locked by the UFS duplex , is important for flavivirus genome cyclization ( Alvarez et al . , 2005; Zhang et al . , 2008 ) .", "We speculated that an over-stabilized UFS duplex would be too difficult to unwind and would hinder 5′-3′ UAR hybridization .", "To confirm this probability , a DENV4 5′-3′ RNA binding assay was employed ( Figure 8A ) .", "The M1A , M1B and M1C mutations ( Figure 8B ) , as well as the M3 series mutations ( Figure 8—figure supplement 1 ) , had little effect on the formation of 5′-3′ complex .", "However , when the M2 series mutants were assayed , although the M2A and M2B mutations also did not influence the formation of 5′-3′ RNA complex apparently , the complex formed by the 3′ UTR and 5′-300 nt M2C RNA was greatly reduced compared to the amounts of complexes formed by the 3′ UTR and other 5′-300 nt RNA species ( Figure 8B ) .", "Estimation of dissociation constant ( Figure 8—figure supplement 2 ) revealed that the Kd of 5′-300 nt M2C binding with 3′ UTR is approximately ten fold higher than the Kds of the other 5′ RNAs .", "These results together indicated that increasing the stability of the UFS hinders vRNA terminus interactions .", "Furthermore , the 5′-300 nt L3D mutant , in which the internal loop was deleted , also interacted with the 3′ UTR poorly ( Figure 8—figure supplement 1 ) . 10 . 7554/eLife . 17636 . 027Figure 8 . Increasing the stability of the UFS hinders vRNA cyclization .", "( A ) Schematic diagram of experimental design .", "As 5′-300 nt RNA is incubated with 3′ UTR RNA , a 5′-3′ RNA bimolecular complex is formed due to the interactions that promote genome cyclization .", "Various RNA structural elements in the DENV4 genomic ends are shown .", "The regions involved in terminal interactions are colored in red , and the UFS element is labeled in blue .", "( B ) RNA binding assay of the UFS mutants .", "3′ UTR RNA ( 20 ng/μl ) was incubated with different amounts ( 16 , 40 , 80 and 160 ng/μl ) of various 5′-300 nt RNA mutants in 20 μl reactions .", "The formation of RNA complexes was then analyzed by native TBE PAGE gels .", "A gel image of the M1 series mutants is shown on the top , and the results for the M2 series mutants are shown below .", "The molar ratios between 5′ and 3′ RNA are indicated above the gel lanes . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02710 . 7554/eLife . 17636 . 028Figure 8—figure supplement 1 . RNA binding assay of the UFS L1D , L3D and M3 series mutants . 3′ UTR RNA ( 20 ng/μl ) was incubated with different amount ( 16 , 40 , 80 and 160 ng/μl ) of the corresponding 5′-300 nt RNA mutants in 20 μl reactions .", "The formation of RNA complexes was then analyzed by native TBE PAGE gels .", "The molar ratios between 5′ and 3′ RNA are indicated above the gel lanes . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 02810 . 7554/eLife . 17636 . 029Figure 8—figure supplement 2 . The calculation of binding affinity of 3′ UTR for the 5′-300 nt RNAs . Reactions containing a series of concentrations of different 5′-300 nt RNAs and fixed concentration ( approximately 36 . 9 nM ) of 3′ UTR RNA were resolved in native PAGE gels .", "The gels were analyzed by ImageJ software and the percentages of bound 3′ UTR RNA were calculated .", "We assumed that the band intensity has a positive linear correlation with the molecular weights of the corresponding molecules , and that was taken into account in the calculation .", "The plotted data was fitted with % Bound=[X]/ ( Kd+[X] ) using GraphPad Prism 6 ( GraphPad Software , La Jolla , California , USA ) , where % Bound is the percentage of bound 3′ UTR RNA , and [X] is the concentration of 5′-300 nt RNA .", "By this method , the Kd was estimated and showed along with the binding curve . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 029 SHAPE analysis was then performed to observe the structural changes to 5′ end RNA caused by different concentrations of 3′ UTR RNA ( Figure 9 ) .", "When the 5′:3′ ratio was 1:1 , the SHAPE reactivity of the UFS element in 5′-300 nt WT RNA exhibited a significant increase compared with the reactivity when the 3′ UTR was absent , and the SHAPE reactivity of the SLB loop and 5′ CS region was obviously reduced .", "When the 3′ UTR was in 5-fold excess of 5′-300 nt RNA , the SLB loop region of WT 5′ RNA showed essentially no SHAPE reactivity , indicating a complete transition from the SLB structure to a UAR duplex .", "In contrast , when the 5′-300 nt M2C RNA was incubated with an equal concentration of 3′ UTR RNA , there were only minor changes in the SHAPE reactivity of the UFS and SLB loop regions compared with those of 5′-300 nt M2C RNA alone .", "When the 5′:3′ ratio was 1:5 , the SLB loop region of 5′-300 nt M2C RNA still showed moderate SHAPE reactivity .", "The above results demonstrated that the UFS duplex is melted when the 3′ UTR binds to 5′ end RNA , and stabilization of the UFS renders it difficult to unwind in response to the 3′ UTR and thus hinders genome cyclization .", "Thus , although the UFS does not participate in 5′-3′ hybridization directly , the stability of its secondary structure must be maintained below a threshold to enable the necessary conformational changes required for genome cyclization .", "It can be speculated that the unwinding of the UFS during genome cyclization may be important for its function in viral RNA synthesis . 10 . 7554/eLife . 17636 . 030Figure 9 . SHAPE analysis of the interactions between DENV4 genomic ends .", "( A ) SHAPE analysis was performed for DENV4 5′-300 nt WT and M2C RNA in the presence of various amounts of 3′ UTR RNA .", "For convenience , only the results for nucleotides 60–150 , which include critical elements for genome cyclization , are shown .", "The regions corresponding to various 5′ elements are indicated .", "Negative SHAPE reactivity is set to zero for clear demonstration .", "( B ) The changes in the SHAPE reactivity of the 5′ 60–150 region were obtained by subtracting the SHAPE reactivity when 3′ UTR RNA was absent from the corresponding values when 3′ UTR RNA was present in a 1:1 ratio with 5′-300 nt RNA .", "The results were from two biological replicates , and the error bars represent the standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 03010 . 7554/eLife . 17636 . 031Figure 9—source data 1 . Source data for Figure 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 031 As the UFS duplex is unwound after genome cyclization , it should not functionalize on circularized genomes .", "To investigate this inference , artificial mini-genomes ( Figure 10A ) , which have been widely used in studies of flavivirus vRNA synthesis , RdRp activity and RNA structures ( Niyomrattanakit et al . , 2015; Potisopon et al . , 2014; Sztuba-Solinska et al . , 2013; You and Padmanabhan , 1999 ) , were employed .", "The DENV4 WT mini-genome and those containing the desired mutations were constructed ( Figure 10A ) .", "First , using native PAGE , we found that the WT mini-genome , as well as the M1A , M1B , M1C , M3A , M3B , M3C and M2A mini-genomes , existed mostly in a circularized conformation ( Figure 10B ) , whereas the CS-M mutant , in which the 5′ CS sequence was mutated , was confined to a linear conformation , as expected .", "The M2C mutant also mainly stayed in a linear conformation , agreeing with the results of the 5′-3′ RNA binding assay .", "The above results demonstrated that these corresponding mini-genomes are highly circularized; thus , it is unlikely that the UFS duplex structure exists in them .", "Next , the affinity of different mini-genomes for NS5Pol was assessed .", "The WT , M1A , M1B and M1C mini-genomes all showed low levels of binding to NS5Pol ( Figure 10C ) .", "In contrast , the CS-M mini-genome bound to NS5Pol with apparently higher affinity than the other mini-genomes tested ( Figure 10C ) . 10 . 7554/eLife . 17636 . 032Figure 10 . Genome cyclization disables the function of the UFS in NS5 recruitment .", "( A ) Schematic diagram showing the conformational changes of the DENV4 genome .", "Two major conformations of vRNA , the linear and circular form , exist in equilibrium .", "The regions involved in terminal interactions are colored in red , and the UFS element is labeled in blue .", "The locations and sequences of the ΔSLA , cHP-M and CS-M mutants used in this section are indicated .", "( B ) The conformational equilibrium of mini-genome RNA containing different mutations was analyzed using native PAGE .", "The bands of the linear and circular conformations were identified using the CS-M mutant as a control .", "The faint and much larger bands were likely to be formed by misfolded RNA molecules .", "( C ) The binding of NS5Pol to different mini-genome RNA molecules was analyzed by EMSA .", "Mini-genomes containing the M1 series mutations in the UFS were assayed , and the WT and CS-M mini-genomes served as control reactions .", "No NS5Pol was present in the left first lane of each group .", "The NS5Pol concentrations in the reactions were approximately 2 . 0 , 3 . 5 , 5 . 0 and 7 . 5 μM ( from left to right ) .", "( D ) In vitro RdRp activity assay using various mini-genome RNA molecules as templates .", "The reactions were performed at 30°C for 40 min .", "Note that the signal of the ΔSLA lane did not correspond to the dsRNA product generated by de novo initiation .", "The blot was analyzed by ImageJ software and RdRp activity was expressed as percentage of WT .", "Note that the template activity of M1C-mini was calculated by analyzing a longer exposed version of the same blot shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 032 Next , an in vitro RdRp assay was performed using mini-genomes as templates .", "The CS-M mini-genome , together with a mini-genome lacking the SLA element ( ΔSLA ) or with a disrupted cHP structure ( cHP-M ) , were assayed in parallel as controls , which showed non-detected or reduced RdRp activity for de novo initiation ( Figure 10D ) , in agreement with their known defects in vRNA replication .", "The de novo products in reactions using M1 and M3 series mini-genomes as templates were also all reduced compared to those produced using the WT mini-genome as a template suggesting that the UFS is disabled in mini-genomes because of the unwinding of its duplex ( Figure 10D ) .", "The differences in RdRp activity between WT mini-genome and the UFS mutants were likely caused by changes of primary sequence in these constructs , In supporting of this , the M1C and M3C mutants were not able to reach the same level of replication as WT in replicon assay ( Figure 3D ) , and it has been reported that the base composition of U-rich sequence in the UFS was actually able to affect vRNA replication of DENV2 ( Friebe and Harris , 2010 ) .", "Taken together , the above results demonstrated that genome cyclization disables the function of the UFS in NS5Pol recruitment , suggesting that the UFS has to function dynamically during vRNA replication .", "Moreover , the attenuation of the affinity of NS5Pol for vRNA by genome cyclization is potentially important for the translocation of NS5Pol from the 5′ end to the 3′ end of vRNA .", "It has been suggested that both the linear and circular conformations of the flavivirus genome are required for vRNA replication and exist in equilibrium ( Villordo et al . , 2010 ) .", "However , circular states should be dominant among its conformations .", "Although the highly circularized mini-genome is much shorter than the actual viral genome , it has been theoretically proposed that the generic properties of a large RNA molecule lead its two ends to stay close to each other ( Yoffe et al . , 2011 ) , which was supported by results from single-molecule FRET assays ( Leija-Martinez et al . , 2014 ) .", "Thus , the 5′ and 3′ ends of the flavivirus genome have great opportunities to contact each other , and due to the high affinity between them , most vRNA molecules would stay circularized in the equilibrium state .", "Moreover , protein factors have been shown to facilitate the cyclization of mRNA and vRNA in vivo .", "In fact , the PABP protein , which bridges mRNA ends through poly ( A ) -PABP-eIF4E-5′ cap interactions , has been shown to bind to the DENV 3′ UTR ( Polacek et al . , 2009 ) .", "However , if the linear conformation of vRNA , the only conformation under which the UFS can exist , is present in low abundance , the question remains of how the latter can function .", "Inspired by the co-transcriptional folding of RNA molecules ( Frieda and Block , 2012; Gong et al . , 2015; Meyer and Miklos , 2004; Perdrizet et al . , 2012 ) , we speculated that the UFS duplex is folded during the ongoing synthesis of nascent positive-strand RNA because the 5′ end of the parental ( + ) strand is released from the dsRNA replication form ( RF ) by a potential strand-displacement mechanism during the process , given that the flavivirus genus exhibits semi-conservative replication ( Chu and Westaway , 1985; Uchil and Satchidanandam , 2003 ) , whereas the 3′ end of the parental ( + ) strand RNA remains in double-stranded form with the ( − ) strand .", "Based on the above considerations , we proposed that the UFS functions as an RNA switch , and its 'ON' and 'OFF' states are controlled by genome cyclization ( Figure 11 ) to fulfill different needs in vRNA replication .", "A mechanistic model to explain the function of the UFS is described below .", "( i ) vRNA released in the cytoplasm is translated to generate a sufficient level of viral proteins , which leads to the eventual binding of NS5 to the SLA and the initiation of ( − ) strand synthesis , even without the presence of the UFS duplex .", "( ii ) Completion of ( − ) strand synthesis generates the dsRNA RF , which serves as the template for the synthesis of progeny vRNA in the presence of viral NS5/NS3 and other viral/host factors ( not shown in Figure 11 ) .", "( iii ) During the elongation of the nascent ( + ) strand , the 5′ terminus of the parental ( + ) strand RNA is displaced first , and the UFS duplex is folded on the protruding 5′ end; this 'ON' state of the UFS facilitates the recruitment of NS5 to the free 5′ end .", "( iv ) After the completion of the nascent chain synthesis , the NS5-bound vRNA undergoes genome cyclization , and the UFS element is set to 'OFF' by the hybridization between genome termini , which decreases the binding strength of NS5 to the 5′ terminus and promotes its translocation to the 3′ terminus to initiate next-round synthesis of negative-strand vRNA .", "Such dynamic modulation of NS5 binding should be of great significance to maintain an appropriate level of ( − ) vRNA synthesis to ensure the global balance of vRNA replication because the dsRNA RF is a highly active template for ( + ) vRNA synthesis . 10 . 7554/eLife . 17636 . 033Figure 11 . Model explaining the functional mechanism of the UFS switch . A proposed mechanistic model of flavivirus vRNA replication .", "( i ) After the viral genome is released into the cytoplasm , first , translation occurs on the circularized genome to generate a sufficient level of viral proteins for downstream vRNA synthesis .", "( ii ) Accumulation of viral replication proteins ( only NS5 and NS3 are shown ) to high concentrations results in the formation of an RNA-viral replicase complex and initiates ( − ) RNA synthesis to generate the double-stranded replication form ( RF ) .", "( iii ) Then , the synthesis of nascent ( + ) RNA using RF as the template is initiated by NS5/viral replicase .", "During the process , 5′ local structures including UFS are formed in the displaced , parental ( + ) strand RNA .", "A free NS5/viral replicase is recruited to bind the 5′ end of the displaced ( + ) RNA .", "( iv ) As soon as the nascent ( + ) strand RNA synthesis completes , the released ( + ) ssRNA genome is circularized by terminal interactions , and the next round of ( − ) strand synthesis is ready to start . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 033 Furthermore , this RNA-switching mechanism is likely to be universal among flaviviruses with vertebrate hosts given that when the terminal secondary structures of both genome conformations of different flaviviruses were examined , we found that the 5′ local structures immediately following the SLA elements were always melted in response to genome cyclization ( Figure 12 and Figure 12—figure supplements 1–4 ) , suggesting that these structures may possibly possess the same regulatory function as the UFS . 10 . 7554/eLife . 17636 . 034Figure 12 . Genome conformation models of major groups of flaviviruses . Sequences involved in terminal interactions are shown in red .", "The 5′ RNA structures that are immediately downstream of SLA elements and consistently involved in genome cyclization are indicated by red arrows .", "The typical UFS element in MBFVs is shown in blue .", "The demonstrated structures are based on DENV4 for the MBFVs; tick-borne encephalitis virus ( TBEV ) for TBFVs; Wesselsbron virus ( WESSV ) for the YFV clade of the MBFVs and RBV for the NKVs .", "The kissing loop interaction identified recently in the tick-borne group ( Tsetsarkin et al . , 2016 ) is labeled by green letters ( KL ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 03410 . 7554/eLife . 17636 . 035Figure 12—figure supplement 1 . Terminal genomic RNA structures of the MBFV group . The demonstrated sequence was based on DENV4 .", "Sequences involved in genome cyclization are highlighted in yellow .", "The gray regions are not included in the demonstration of Figure 12 .", "The mfold software and VARNA application were utilized to generate the structural models . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 03510 . 7554/eLife . 17636 . 036Figure 12—figure supplement 2 . Terminal genomic RNA structures of the YFV clade in the MBFV group . The demonstrated sequence was based on WESSV .", "Sequences involved in genome cyclization are highlighted in yellow .", "The gray regions are not included in the demonstration of Figure 12 .", "The mfold software and VARNA application were utilized to generate the structural models . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 03610 . 7554/eLife . 17636 . 037Figure 12—figure supplement 3 . Terminal genomic RNA structures of the TBFV group . The demonstrated sequence was based on TBEV .", "Sequences involved in genome cyclization are highlighted in yellow .", "The gray regions are not included in the demonstration of Figure 12 .", "The mfold software and VARNA application were utilized to generate the structural models . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 03710 . 7554/eLife . 17636 . 038Figure 12—figure supplement 4 . Terminal genomic RNA structures of the NKV group . The demonstrated sequence was based on RBV .", "Sequences involved in genome cyclization are highlighted in yellow .", "The gray regions are not included in the demonstration of Figure 12 .", "The mfold software and VARNA application were utilized to generate the structural models . DOI: http://dx . doi . org/10 . 7554/eLife . 17636 . 038" ], [ "Herein , via the combination of bioinformatics , biochemical and reverse genetics approaches , the UFS element was shown to be crucial for efficient flavivirus vRNA replication through dynamic modulation of viral RdRp recruitment .", "Our results demonstrated that the UFS links two events of vRNA replication , NS5 recruitment and genome cyclization , together , and the structural stability of the UFS is elaborately balanced to meet the requirements of both .", "Especially , the conformational change of the UFS caused by genome cyclization is coupled with its function in modulating dynamic RdRp recruitment .", "As the function of UFS in viral replication and RdRp recruitment was investigated using different flaviviruses ( DENV4 , JEV and ZIKV ) , it is plausible to consider that the UFS is a general regulatory strategy of viral replication among the flavivirus genus .", "Although a previous report ( Filomatori et al . , 2011 ) has suggested that the UFS may not be functional in DENV2 , this was more likely to be an exception , as our SHAPE data also indicated that the UFS in DENV2 is highly unstable in vitro , in contrast to the UFS elements from other viruses ( Figure 2 ) .", "By applying a systemic RNA structure prediction and comparison approach covering different members of the flavivirus genus , the structure of the downstream SLA region in the 5′ end of the flavivirus genome was unambiguously illuminated .", "The UA-base-pair-rich UFS and the UFS-like element have been identified in MBFVs , two clades of NKVs , and ISFVs , demonstrating that the UFS element is evolutionarily conserved .", "Interestingly , while the UFS duplex unwinds during genome cyclization in MBFVs and NKVs , neither the conserved mode of genome cyclization nor the unwinding of UFS-like elements during this process was found in ISFVs .", "The ISFVs have been indicated to be the most divergent outgroup among the genus flavivirus ( Blitvich and Firth , 2015; Cook and Holmes , 2006; Moureau et al . , 2015 ) , which led us to assume that modern flaviviruses are evolved from an insect virus ancestor .", "We speculated that primordial UFS-like structures are potential viral replication enhancers with RdRp-recruiting functions in ancient ISFVs .", "When the viruses were introduced into vertebrates , the intense selection pressure caused by shifting between two hosts promoted the emergence of a highly efficient genome cyclization strategy , and the UFS-like elements evolved into the RNA switches in modern flaviviruses .", "Moreover , although the UFS structure itself was lost when the ancestral ISFV adapted into ticks and some special vertebrates , its functional mechanism was inherited by the appearance of elements such as 5′-SL2/CS-A in TBFVs .", "Thus , by looking into the evolutionary history of flaviviruses and exploring the modes of genome cyclization among them , we propose that the UFS may play a critical role during the evolution of the genus flavivirus .", "UFS elements usually localize close to , or overlap with , the translation start region , suggesting a possible role for the UFS in viral translation regulation .", "Moreover , the replication of most positive-strand RNA viruses occurs in virus-induced membrane structures ( Gillespie et al . , 2010; Welsch et al . , 2009 ) .", "It is possible that during ( + ) vRNA synthesis of flaviviruses , the parental ( + ) RNA is anchored to the vesicle membranes and prevented from being transported out of the vesicle through interactions between the UFS-containing 5′ end and the membrane-bound viral replicase .", "Interestingly , DENV has been reported to conceal its dsRNA in membrane structures to escape from host immunity surveillance ( Uchida et al . , 2014 ) .", "Thus , other potential functions of the UFS need further investigation .", "Functional long-range interactions are likely to be a general mechanism in positive-strand RNA viruses ( Nicholson and White , 2014 ) .", "Genome cyclization is responsible for the delivering of viral replication factors that bind 5′-terminally or internally to the very 3′ end of the viral genome .", "Such tactics have been observed in positive-strand RNA viruses , which infect animals , plants and bacteria ( Nicholson and White , 2014 ) .", "However , to our knowledge , this is the first report describing that an RNA switch triggered by genome cyclization integrates the recruitment and translocation of the viral RdRp into the dynamic process of nascent RNA synthesis .", "This deliberate control mechanism used by flaviviruses could also be used by other positive-strand RNA viruses in similar forms , and further pursuit of these mechanisms will provide deeper insight into the replication and evolution of positive-strand RNA viruses ." ], [ "The p4 infectious clone ( Durbin et al . , 2001 ) of the DENV4 814669 strain and its host E . coli strain BD1528 were kindly provided by Professor Stephen S . Whitehead .", "All replicon constructs were also manipulated using BD1528 .", "The p4-Dualstop-SP-IRES-Rluc-Rep replicon was generated by introducing a nonsense mutation into the 26th codon of the capsid ORF ( CAA to TAA ) in the background of p4-cHPstop-SP-IRES-Rluc-Rep ( Liu et al . , 2013 ) .", "p4-Dualstop-SP-IRES-Rluc-Rep-GVD , in which the NS5 catalytic triad was mutated , was generated based on p4-cHPstop-SP-IRES-Rluc-Rep-GVD using similar strategies .", "For the convenience of introducing mutations into the UFS region , the Asc I-SnaB I fragment from p4-Dualstop-SP-IRES-Rluc-Rep , which contained the SP6 promoter region and the DENV4 5′ UTR-capsid ORF sequence , was engineered such that the sequence corresponding to the 70–107 nt region of the DENV4 genome was substituted by the following: 5′- CTCCTCACACATACGTAATGGGGAGGAG-3′ .", "This engineered fragment contained two inversely positioned BseR I sites ( underlined above ) and was cloned into pGEM-T easy ( Promega , Madison , Wisconsin ) to generate the cloning cassette vector , pBseRI-Dualstop-04 .", "DNA oligos containing the desired mutations of the UFS were annealed and ligated with BseR I-digested pBseRI-Dualstop-04 to acquire Asc I-SnaB I fragments with the desired mutations , which were then subcloned back into p4-Dualstop-SP-IRES-Rluc-Rep .", "To generate UFS mutants in the p4 infectious clone and p4-GVD , the Asc I-BstE II fragments with desired mutations were generated by overlapping PCR using the corresponding replicon mutants and p4 infectious clone DNA as templates for amplification , cloned into pGEM-T easy , and then subcloned into p4 and p4-GVD , respectively .", "Site-directed mutagenesis method was employed for the generation of ZIKV fragments containing the desired UFS mutations , the generated Not I-Avr II restriction fragments were then subcloned into the infectious clone of ZIKV strain FSS13025 ( Shan et al . , 2016 ) , which was kindly provided by Professor Pei-Yong Shi .", "The DENV4 mini-genomes were constructed by joining various 5′-300 nt fragments with the 3′ UTR sequence using overlapping PCR techniques .", "The mutations targeting cHP and 5′CS were also introduced by overlapping PCR .", "The DENV2 and DENV3 5′ end sequences were acquired by RT-PCR using the DENV2 strain NGC ( KM204118 ) and the DENV3 strain 80–2 ( AF317645 ) RNA as templates respectively , and cloned into pMD-19T-simple ( Takara , Dalian , China ) .", "The 5′ end sequence of DENV1 strain WestPac ( U88535 ) was chemically synthesized and cloned into pUC57 by Thermo Fisher Scientific .", "The SP6 promoter sequence was placed upstream of the DENV1 5′ end sequence .", "These constructs , together with the JEV infectious clone pAJE70 ( Li et al . , 2013 ) , which contains the 5′ UTR-C-coding region of strain SA-14-14-2 ( AF315119 ) , were utilized to amplify the DNA templates for in vitro transcription .", "The infectious clone of ZIKV strain FSS13025 was used to amplify the DNA template for in vitro transcription of ZIKV 5′ end RNA .", "JEV UFS mutants were generated by site-directed mutagenesis , the resultant plasmids were utilized to amplify the DNA templates for in vitro transcription of the corresponding JEV 5′ RNA mutants .", "Replicon and full-length viral RNA were in vitro transcribed as described previously ( Liu et al . , 2013 ) .", "To generate RNA species for in vitro SHAPE , EMSA and RdRp activity assays , high-fidelity PCR-amplified DNA fragments , which contained the SP6 or T7 promoter , were used as templates for in vitro transcription reactions .", "RNA was purified with an RNeasy mini kit ( Qiagen , Hilden , Germany ) , and the concentrations were determined spectrophotometrically .", "Agarose/TAE gel electrophoresis was routinely conducted to examine the integrity of the purified RNA .", "The RNA preparations were stored at −80°C before use .", "Note that all DENV4 5′ RNA and mini-genome RNA molecules contained two artificial nonsense mutations at the corresponding sites in the capsid coding region that have been shown not to affect the folding of the corresponding RNA or vRNA replication ( Clyde et al . , 2008; Liu et al . , 2013 ) .", "For the evaluation of the replication characteristics of the p4-cHPstop-SP-IRES-Rluc-Rep and p4-Dualstop-SP-IRES-Rluc-Rep replicons , a replicon assay was performed as described previously ( Liu et al . , 2013 ) .", "For the replicon assays of the UFS mutants , ten thousand BHK-21 cells were seeded into each well of Nunc MicroWell 96-Well Optical-Bottom Plates ( Thermo Fisher Scientific , Waltham , Massachusetts ) one day prior to transfection .", "Unless otherwise specified , 100 ng of replicon RNA was transfected per well using Lipofectamine 2000 reagent ( Thermo Fisher Scientific , Waltham , Massachusetts ) , and the plates were cultured at 37°C with 5% CO2 .", "At the indicated time points , the cells in the microplates were lysed with 1× Renilla lysis buffer ( Promega , Madison , Wisconsin ) and stored at -20°C until use .", "The Renilla luciferase activity of the collected samples was measured as described previously ( Liu et al . , 2013 ) , except that the microplates were directly subjected to the GloMax-96 luminometer .", "Four million BHK-21 cells suspended in 400 μl of Opti-MEM I medium were electroporated with 5 μg of vRNA .", "The details of the electroporation are described elsewhere ( Liu et al . , 2013 ) .", "The electroporated cells were resuspended in 14 ml of Dulbecco's modified minimal essential medium ( DMEM , Thermo Fisher Scientific , Waltham , Massachusetts ) with 5% fetal bovine serum ( FBS , PAA , Morningside , QLD , Australia ) and dispensed into 24-well plates with or without sterile cover slips .", "The cells were cultured at 37°C in 5% CO2 .", "At 4 hr post-electroporation , a media change was performed for the plates intended for vRNA quantification analysis .", "At the indicated time points , the total RNA was isolated from the transfected cells , and qRT-PCR was performed to quantify vRNA copies as described previously ( Liu et al . , 2013 ) .", "The culture supernatants from the transfected cells were collected at different time points post-transfection , and virus titers were determined by plaque-forming assays .", "BHK-21 cells electroporated with corresponding vRNA were seeded onto cover slips in 24-well plate-format and cultured at 37°C in 5% CO2 .", "At the indicated time points , the cover slips were washed once with PBS ( phosphate buffered saline , pH 7 . 4 ) and fixed with acetone/methanol ( v/v: 3/7 ) at 4°C .", "The cover slips were incubated with the anti-dengue envelope protein monoclonal antibody 2A10G6 at 37°C for 1 hr for the cells transfected with DENV4 RNA , and anti-ZIKV envelope protein mAb clone 0302156 ( 1:1000 diluted , BioFront Technologies , Tallahassee , Florida , USA ) was used for the cells transfected with ZIKV RNA .", "After the incubation with primary antibodies , the cover slips were washed with PBS for three times .", "AlexaFluor 488-labeled goat anti-mouse IgG ( 1:200 diluted , zsbio , Beijing , China ) was then added , and after a 1-h incubation , the cover slips were washed as described above .", "For cell nuclei staining , 4' , 6-diamidino-2-phenylindole ( DAPI , 0 . 5 ng/μl ) was added onto the cover slips and incubated for 5 min .", "An Olympus BX51 microscope under the control of DP72 software was utilized for image capture .", "RNA molecules ( approximately 9 . 66 pmol ) corresponding to the WT or mutated 5′ end sequence of different flaviviruses were diluted into 12-μl reactions containing 0 . 5× TE buffer , heated at 95°C for 2 min and snap-cooled on ice immediately .", "Then 6 μl of 3 . 3× RNA folding buffer ( 333 mM HEPES , pH 8 . 0 , 333 mM NaCl and 20 mM MgCl2 , Wilkinson et al . , 2006 ) was added and the RNA was refolded at 37°C for 20 min .", "The 18-μl refolded RNA was splitted equally into two tubes and then modified by N-methylisatoic anhydride ( NMIA , Sigma-Aldrich , St . Louis , Missouri ) .", "For the analysis of 5′ end RNA-3′ UTR complexes , the two RNA segments were co-folded at the indicated molar ratios and then subjected to NMIA modification .", "The SHAPE ( + ) reactions contained 1 μl of 130 mM NMIA freshly dissolved in dimethyl sulfoxide ( DMSO , Sigma-Aldrich , St . Louis , Missouri ) , and 1 μl of DMSO was added into the parallel SHAPE ( − ) reactions .", "The SHAPE ( + ) and ( − ) reactions were incubated at 37°C for 45 min , then the RNA in the reactions was purified using RNA Clean and Concentrator-5 ( Zymo Research , Irvine , California , USA ) .", "Primer extension reactions utilizing fluorophore-labeled primers were performed with Superscript II reverse transcriptase ( Thermo Fisher Scientific , Waltham , Massachusetts ) in a 20 μl volume according to the manufacturer’s instructions .", "ddTTP dideoxy-sequencing reactions were performed in parallel .", "We routinely used VIC-labeled primers for primer extension of the SHAPE reactions and NED-labeled primers for sequencing reactions .", "Otherwise , FAM- and HEX-labeled primers were utilized for primer extension of the SHAPE and sequencing reactions , respectively .", "After primer extension , one microliter of 4 M NaOH was added to each reaction , and treatment at 95°C for 3 min was performed to hydrolyze the RNA templates .", "Two microliters of 2 M HCl were added to adjust the pH to neutral .", "Then , the sequencing reactions were split and added into the SHAPE ( + ) and ( − ) reactions .", "The final mixtures were purified by ethanol/EDTA precipitation , dissolved in Hi-Di formamide and separated via denaturing PAGE capillary electrophoresis by the DNA sequencing department of Thermo Fisher Scientific .", "The SHAPE data were analyzed using QuShape software ( Karabiber et al . , 2013 ) .", "All the processing steps were performed using the default software settings .", "Negative SHAPE reactivity was set to zero except for the calculations of the Δ SHAPE reactivity of WT and M2C 5′-300 nt RNA in response to 3′ UTR RNA .", "The coding sequence of the DENV4 NS5 RdRp domain ( residues 270–900 , NS5Pol ) was cloned into a pET-28a ( + ) expression vector using Nhe I/Xho I restriction sites .", "The recombinant NS5Pol carrying an N-terminal His-tag was expressed in E . coli Rosetta ( λDE3 ) in the presence of 0 . 125 mM isopropyl-β-D-thiogalactopyranoside ( IPTG ) at 16°C for 20 hr .", "The cell pellets were harvested and washed once with Buffer A ( 25 mM Tris . Cl , pH 8 . 0 , 500 mM NaCl , 25 mM imidazole and 5% glycerol ) and resuspended in Buffer A containing cOmplete EDTA-free protease inhibitor cocktail tablets ( Roche , Penzberg , Germany ) .", "After ultra-sonication , the suspension was centrifuged at 30 , 000 g at 4°C for 20 min .", "The soluble recombinant NS5Pol protein in the supernatants was then purified using a HisTrap HP column ( GE Healthcare , Little Chalfont , UK ) .", "The fraction containing NS5Pol was eluted with Buffer A containing 300 mM imidazole .", "The eluted protein was exchanged into 50 mM Tris . Cl ( pH 8 . 0 ) , 300 mM NaCl and 20% glycerol using a HiTrap desalting column ( GE Healthcare , Little Chalfont , UK ) and concentrated to mg/ml grade by ultrafiltration using Amicon Ultra-15 Centrifugal Filter Units ( 30 kDa-cutoff , Merck Millipore , Billerica , Massachusetts , USA ) .", "Dithiothreitol ( DTT ) was added into the purified NS5Pol protein to a final concentration of 1 mM .", "The final product was dispensed into single-use aliquots and stored at -80°C before use .", "NS5Pol of JEV SA-14-14-2 strain was cloned , expressed and purified using the same strategy .", "An RNA-RNA binding EMSA was performed as described previously ( Liu et al . , 2013 ) with minor modifications .", "For the NS5Pol-RNA EMSA assays , the different DENV4 5′-300 nt RNA species were first diluted in 0 . 5× TE buffer and heated at 95°C for 2 min .", "The RNA samples were then placed on ice immediately .", "5× RNA folding buffer T ( 250 mM Tris . Cl , pH 8 . 0 , 500 mM NaCl and 25 mM MgCl2 ) was added to the samples , and the RNA was refolded at 37°C for 20 min .", "The concentration of the renatured RNA was set to 500 nM .", "The binding reactions contained 50 nM 5′-300 nt RNA , 4 μl of 5× EMSA buffer ( 200 mM Tris . Cl , pH 8 . 0 , 300 mM NaCl , 25 mM MgCl2 ) , 0 . 05 mg/ml heparin sodium salt , 7 . 5% glycerol and different amounts of NS5Pol ( 0 , 3 , 5 . 25 , 7 . 5 and 11 . 25 μg ) in a 20 μl volume .", "Due to the solvent components in RNA and protein preparations , the final reactions contained 64 . 15 mM Tris . Cl ( pH 8 . 0 ) , 182 . 5 mM NaCl , 5 . 5 mM MgCl2 , 40 nM EDTA and 375 nM DTT .", "The reactions were incubated at 30°C for 30 min , and then , 10× gel loading solution ( Thermo Fisher Scientific , Waltham , Massachusetts ) was added , and the mixtures were separated by electrophoresis on 6% native PAGE gels running in 0 . 5× TBE .", "The gel apparatus was placed in an ice-water bath to prevent the dissociation of RNA-protein complexes .", "After running at 60 V for 4 hr , the gels were stained with SYBR Gold nucleic acid gel stain ( Thermo Fisher Scientific , Waltham , Massachusetts ) for 30 min , and gel pictures were captured using a BioSpectrum Imaging System ( UVP , LLC , Upland , California ) with a UV transilluminator .", "EMSAs using 5′-160 nt RNA or mini-genome RNA were performed similarly , with a few exceptions: for the 5′-160 nt RNA-based EMSA , the NS5Pol concentrations were higher ( 0 , 5 . 4 , 10 . 8 , 16 . 2 and 21 . 6 μg , respectively ) ; for the mini-genome-based EMSA , the heparin concentration was set to 0 . 16 mg/ml , and the electrophoresis was performed with 4 . 5% PAGE gels at 75 V for 6 hr in an ice-water bath .", "EMSA assay using JEV NS5Pol and 5′-320 nt RNA was performed with similar conditions for DENV4 5′-300 nt RNA EMSA assay .", "In vitro RdRp initiation assays were performed using: ( I ) 5′-160 nt RNA of DENV4 , ( II ) DENV4 mini-genome RNA as templates .", "The RNA templates were diluted with RNase-free water to a final volume of 8 μl , then heated at 95°C for 2 min and immediately cooled on ice .", "Two microliters of RNA fold buffer T2 ( 250 mM Tris . Cl , pH 7 . 4 , 100 mM NaCl , 25 mM MgCl2 ) was added , and the RNA templates were folded at 37°C for 20 min .", "Unless otherwise specified , the 30-μl RdRp reactions contained 450 ng of template RNA ( 3 μl of 150 ng/μl folded RNA ) , 55 . 6 mM Tris . Cl ( pH 7 . 4 ) , 12 mM NaCl , 5 . 5 mM MgCl2 , 2 mM MnCl2 , 10 mM DTT , 40 U of recombinant RNase inhibitor ( 40 U/μl ) , approximately 5 . 4 μg NS5Pol ( in 1 μl ) and 0 . 5 mM ATP , GTP , and UTP; 0 . 1 mM CTP; and 0 . 25 mM biotin-11-CTP ( Roche , Penzberg , Germany ) .", "According to previous reports ( Ackermann and Padmanabhan , 2001; Filomatori et al . , 2006 ) , the RdRp reactions were incubated at 30°C for the desired length of time .", "The reaction products were purified using an RNAclean Kit ( Tiangen , Beijing , China ) , and 2× RNA loading dye ( New England Biolabs , Ipswich , Massachusetts ) was added into the purified RNA .", "The samples were preheated at 70°C for 5 min , snap-cooled on ice , and analyzed using 6% PAGE/8 M urea gels .", "The gel-separated RNA was electro-transferred onto a Hybond N+ Nylon membrane ( GE Healthcare , Little Chalfont , UK ) .", "After air-drying the membrane , the transferred RNA was UV-crosslinked by a UV transilluminator for 10 min .", "The membrane was blocked using 10% non-fat dry milk , and then streptavidin-conjugated HRP ( Thermo Fisher Scientific , Waltham , Massachusetts , USA ) was incubated with the membrane at room temperature for 2 hr .", "The membrane was then extensively washed using PBS with 0 . 05% Tween 20 , and chemiluminescence signals were detected using a Pro-light chemiluminescent kit ( Tiangen , Beijing , China ) and a Smartchemi II imaging system ( Sage Creation Science Co , Beijing , China ) .", "Statistical analysis was performed using the statistical functions of GraphPad Prism 6 ( GraphPad Software , La Jolla , California , USA ) .", "Briefly , Paired t-test was performed for Figure 3B .", "One-way ANOVA and Dunnett’s multiple comparison test were performed for Figure 2—figure supplement 1 , Figure 3D , Figure 4 , Figure 7 and Figure 7—figure supplement 1 .", "Two-way ANOVA and Tukey’s multiple comparison test were performed for Figure 5B , C , D , F , G , H and I .", "The detailed results of statistical analysis were provided within the corresponding source data files ." ] ]

[ "Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication , yet the mechanism of their interplay remains elusive .", "Flaviviruses include numerous important human pathogens , e . g . , dengue virus ( DENV ) and Zika virus ( ZIKV ) .", "Here , we revealed a highly conserved , conformation-tunable cis-acting element named 5′-UAR-flanking stem ( UFS ) in the flavivirus genomic 5′ terminus .", "We demonstrated that the UFS was critical for efficient NS5 recruitment and viral RNA synthesis in different flaviviruses .", "Interestingly , stabilization of the DENV UFS impaired both genome cyclization and vRNA replication .", "Moreover , the UFS unwound in response to genome cyclization , leading to the decreased affinity of NS5 for the viral 5′ end .", "Thus , we propose that the UFS is switched by genome cyclization to regulate dynamic RdRp binding for vRNA replication .", "This study demonstrates that the UFS enables communication between flavivirus genome cyclization and RdRp recruitment , highlighting the presence of switch-like mechanisms among RNA viruses ." ]

[ "Flaviviruses include a large family of viruses that are harmful to human health , such as dengue virus , West Nile virus and Zika virus .", "Understanding the details of the life cycle of these viruses is important for better controlling and treating the diseases that they cause .", "The genetic information of flaviviruses is stored in single-stranded molecules of RNA .", "To form new copies of a virus , the RNA must be replicated in a process that involves two critical steps .", "First , an enzyme called viral RNA polymerase NS5 must be recruited to a specific end of the RNA strand ( known as the 5′ end ) .", "Then , the ends of the RNA strand bind together to form a circular loop .", "However , little is known about whether these two processes are linked , or how they are regulated .", "Using bioinformatics , biochemical and reverse genetics approaches , Liu et al . have now identified a new section of RNA in the 5′ end of the flavivirus RNA , named the 5′-UAR-flanking stem ( or UFS for short ) , which is critical for viral replication .", "The UFS plays an important role in efficiently recruiting the NS5 viral RNA polymerase to the 5′ end of the flavivirus RNA .", "After the RNA forms a circle , the UFS unwinds .", "This makes the NS5 polymerase less likely to bind to the 5′ end of the RNA .", "Stabilizing the structure of the UFS impairs the ability of the RNA strand to form a circle , and hence reduces the ability of the RNA to replicate .", "Thus , the UFS links and enables communication between the processes that form the flavivirus RNA into a circle and that recruit the viral RNA polymerase to the RNA .", "The structural basis of the interaction between the flavivirus RNA 5′ end , including the UFS element , and the viral RNA polymerase now deserves further investigation .", "It will be also important to explore whether other types of viruses regulate their replication via a similar mechanism ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "The development of complex tissues requires the coordination of cell fate decisions with the generation of correct tissue architecture .", "Asymmetric cell divisions ( ACDs ) serve to harmonize these two processes by both generating cellular diversity and dictating tissue structure ( Betschinger and Knoblich , 2004; Poulson and Lechler , 2012 ) .", "ACDs are driven by robust positioning of the mitotic spindle , a tightly regulated process that is orchestrated by several conserved proteins .", "However , our incomplete understanding of the mechanism underlying spindle orientation , as well as the lack of genetic tools to specifically perturb this process in adult mammalian tissues have prevented us from appreciating its function in various organ systems .", "An LGN/NuMA/dynein-dynactin complex is a conserved regulator of spindle orientation in a number of cell types , including epidermal and neural progenitors in mammals , neuroblasts in Drosophila and zygotic divisions in Caenorhabditis elegans .", "Loss-of-function studies on these proteins have revealed their essential roles in spindle orientation and force generation ( Bowman et al . , 2006; Izumi et al . , 2006; Nguyen-Ngoc et al . , 2007; Siller et al . , 2006; Skop and White , 1998; Williams et al . , 2011 ) .", "Furthermore , targeting dynein/dynactin to the cell cortex was sufficient to induce spindle movements , consistent with it playing a direct role in force generation ( Kotak et al . , 2012 ) .", "These data have led to the proposition that LGN and NuMA form a passive tether , which provides a high level of regulatory control over dynein accumulation at the cell cortex , and that dynein-dependent forces position the spindle ( Kotak et al . , 2012; Kotak and Gonczy , 2013 ) .", "The regulation of microtubule ( MT ) dynamics is another important factor in driving proper spindle orientation .", "For example , local taxol treatment at the posterior end of the C . elegans zygote prevented spindle movement toward that pole ( Nguyen-Ngoc et al . , 2007 ) .", "Additionally , in contrast to the current model in metazoans , the spindle orientation process in yeast requires not only dynein-dependent pulling forces , but also MT depolymerization at the cell cortex ( ten Hoopen et al . , 2012 ) .", "Present models include a kinetochore-like mechanism of capturing the energy from MT depolymerization to power spindle movement .", "Therefore , there is precedence for MT dynamics playing an important role during spindle orientation .", "In addition to its ability to recruit dynein/dynactin to the cell cortex , NuMA contains a domain that directly interacts with MTs ( Du et al . , 2002; Haren and Merdes , 2002 ) .", "This MT-binding domain ( MTBD ) is conserved among flies , worms , and mammals and has been characterized to stabilize and bundle MTs .", "Whether the MT-binding activity of NuMA is important for mitotic spindle orientation has not been tested .", "The epidermis has emerged an as ideal tissue to understand the mechanism and functions of spindle orientation ( Lechler and Fuchs , 2005; Poulson and Lechler , 2010; Williams et al . , 2011 ) .", "The tissue architecture allows for robust and reproducible determination of spindle angles while genetic and cell culture systems have allowed further exploration of the mechanisms .", "In embryonic development , spindle orientation is required for proper stratification and differentiation of the epidermis .", "The role of spindle orientation in epidermal appendages like hair follicles , which are highly organized structures , has not yet been reported .", "That said , stereotypical spindle orientations have been reported at several stages of hair follicle morphogenesis , suggesting that they may play important roles ( Niessen et al . , 2013; Rompolas et al . , 2012 ) .", "Direct testing of this will require development of genetic tools that specifically disrupt spindle orientation .", "Here , we report that NuMA can specifically localize to the tips of MTs , consistent with a role in mediating cortex/astral MT interactions .", "Loss of NuMA’s MTBD resulted in spindle orientation defects both in intact epidermis and in the hair follicle , leading to perturbation of differentiation and loss of tissue function ." ], [ "Previous studies have defined NuMA’s minimal MTBD ( Figure 1A ) ( Du et al . , 2002; Haren and Merdes , 2002 ) .", "When expressed in cultured mouse keratinocytes , however , we observed only weak co-localization between GFP-MTBD and MTs ( Figure 1E ) .", "This was true at both high and low levels of expression , and including or excluding the adjacent LGN-binding domain of NuMA ( Figure 1—figure supplement 1 ) .", "By contrast , when constructs that encoded additional amino-terminal regions were transfected , we noted robust association with the MT lattice in cells that expressed high levels of the fusion proteins ( Figure 1—figure supplement 1 ) .", "This was true in constructs containing the linker region between the 4 . 1 and LGN binding domains of NuMA , but not those that lacked this region .", "When highly overexpressed , the fusion proteins appeared to stabilize and bundle MTs ( Figure 1—figure supplement 1 ) .", "At lower levels of expression , however , we observed punctate MT localization of the fusion proteins that contained the linker region , LGN binding domain and the MTBD ( Figure 1B , C ) .", "Zoomed-in views of these images show that these puncta localize along MTs and often accumulate at MT tips ( Figure 1B’ , C’ ) .", "The MT density is very high in many keratinocytes , which makes it challenging to see a precise co-localization .", "We therefore examined the localization of these fusion proteins in regions of the cell periphery where MTs were sparse .", "Co-staining with α-tubulin revealed discrete localization to MT plus tips near the cell periphery ( Figure 1G–G’’ ) .", "Thus , a region of NuMA spanning the linker domain ( which follows the 4 . 1-binding domain ) to the MTBD localizes to MT tips .", "We refer to this entire region as 'NuMA-TIP' ( Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 12504 . 003Figure 1 . NuMA localizes to microtubule tips .", "( A ) Diagram of NuMA showing interaction domains for dynein/dynactin , 4 . 1 family proteins , LGN , and MTs as well as the nuclear localization sequence ( NLS ) .", "( B–F )", "Visualization of GFP-tagged NuMA constructs ( as diagrammed ) and α-tubulin ( red ) in cultured mouse keratinocytes .", "All cells displayed expressed GFP constructs at low levels .", "( B’ and C’ ) are zoomed-in views displaying the punctate localization of these constructs along MTs and MT tips .", "( G–G’’ )", "Co-localization of GFP-NuMA-TIP with MT ends ( arrows ) at the periphery of a cultured keratinocyte .", "Arrowheads indicate MT tips that lack NuMA-TIP label ( scale bar , 5 μm ) .", "( H ) Keratinocytes were transfected with GFP-NuMA-TIP and then fixed and stained for Eb1 ( red ) .", "Note the lack of co-localization between NuMA-TIP and Eb1 puncta .", "( I ) Keratinocytes were treated with 10 μM nocodazole for 15 min and then fixed before visualizing GFP-NuMA-TIP and MTs ( red ) .", "Images on right show a higher magnification view of NuMA-TIP localizing to both ends of shortened MTs .", "( J ) Quantitation of the percent of cells showing GFP-NuMA-TIP localization to MT tips following drug treatments ( only low-expressing cells were analyzed ) .", "Keratinocytes were treated with either DMSO , 2 nM vinblastine , or 10 μM taxol ( n>100 cells each , p<0 . 05 for each treatment relative to control ) .", "( K–K’’ )", "Three-color staining for Eb1 ( red ) , GFP-NuMA-TIP ( green ) and MTs ( blue ) in either untreated keratinocytes ( top 2 panel rows ) or nocodazole treated keratinocytes ( bottom panels ) .", "Scale bar is 0 . 5 μm .", "Unless noted , all scale bars are 10 μm .", "MT , microtubule . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 00310 . 7554/eLife . 12504 . 004Figure 1—figure supplement 1 . Localization of NuMA fragments when highly expressed .", "( A ) Diagram of NuMA indicating protein-protein interaction domains .", "( B–D )", "GFP-tagged NuMA fragments , as diagrammed , were expressed in cultured mouse keratinocytes .", "Cells were fixed and stained with anti-α-tubulin antibodies ( red ) .", "All these images are representative for cells showing high levels of expression of the transgene ( scale bars , 10 μm ) .", "( E ) Diagram of NuMA-TIP as well as the amino acids that this region spans in both human and mouse NuMA . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 00410 . 7554/eLife . 12504 . 005Figure 1—figure supplement 2 . HeLa cells transfected with GFP-NuMA-TIP were then treated with either DMSO ( A ) or 10 μM nocodazole ( B ) , fixed and stained with anti-α-tubulin antibodies ( red ) .", "Insets present zoomed-in views of NuMA-TIP at MT tips .", "Scale bars are 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 005 Eb1 is a well-characterized plus tip-binding protein that interacts specifically with growing MT ends .", "However , co-staining revealed that NuMA-TIP and Eb1 only very rarely colocalized on the same MT tip ( Figure 1H , K , K’ <2% of MTs , n = 200 ) .", "This could be due to their mutually exclusive steric interactions and/or association with distinct MT end structures .", "To better characterize the properties of the NuMA-TIP protein fragment , we examined how alterations in MT dynamics affected its localization .", "Many +TIP tracking proteins bind exclusively to growing MT ends and are displaced under depolymerization conditions , which can be triggered using nocodazole treatment .", "In stark contrast , NuMA-TIP localized specifically to both MT plus and minus ends following treatment with nocodazole ( Figure 1I , K’ , K’’ ) .", "This was in contrast to Eb1 , which was displaced from the tip and labeled the MT lattice upon nocodazole addition ( Figure 1K’’ ) .", "Since nocodazole significantly decreased the density of MTs and helped resolve MT ends , this treatment revealed that NuMA-TIP clearly favors binding to MT ends over MT sides .", "This same localization pattern was also observed in transfected HeLa cells , demonstrating that this MT tip-binding behavior of NuMA is not unique to keratinocytes ( Figure 1—figure supplement 2 ) .", "While MT plus and minus ends are structurally distinct , both undergo depolymerization that involves protofilaments curling off of the tube ( Tran et al . , 1997 ) .", "NuMA-TIP localization to nocodazole-treated MT ends suggested that it might preferentially bind to depolymerizing MTs and/or to those exhibiting protofilament curling .", "To determine whether NuMA-TIP localization correlates with the status of MT tips , we treated cells with either low doses of vinblastine , which promotes protofilament curling , or taxol , which straightens MT ends ( Elie-Caille et al . , 2007 ) .", "Under the vinblastine conditions used , this drug did not significantly alter MT density or organization .", "We found that a higher percentage of vinblastine-treated cells showed MT tip localization of NuMA-TIP when compared with DMSO-treated cells , while taxol treatment resulted in a significant loss of MT tip localization ( Figure 1J ) .", "This suggests that NuMA preferentially recognizes flayed MT ends over straight ends and is consistent with a report that NuMA interacts preferentially with vinblastine-induced curled MTs over taxol-stabilized MTs ( Volkov et al . , 2015 ) .", "Nevertheless , while NuMA-TIP co-localized well with MT tips in the presence of low-dose vinblastine , we did not observe association with the tubulin aggregates formed in cells treated with higher doses of vinblastine ( data not shown ) .", "Endogenous NuMA also exhibited MT tip localization .", "In early prometaphase , before NuMA had strongly accumulated at both the spindle poles and the cortex , we noted discrete puncta at MT tips ( Figure 2A ) .", "Briefly treating cells with nocodazole resulted in enhanced tip localization ( Figure 2B ) .", "Thus , MT-tip binding is a property of full-length endogenous NuMA . 10 . 7554/eLife . 12504 . 006Figure 2 . NuMA-TIP is necessary and sufficient for microtubule tip localization .", "( A ) Localization of endogenous NuMA ( red ) at MT tips ( green ) in a keratinocyte in early prometaphase .", "( B ) Keratinocytes were treated with 500 nM nocodazole for 5 min before fixation and analysis of NuMA ( red ) and MT ( green ) localization .", "Insets in A and B show high magnification images of NuMA on MT tips .", "Scale bars are 10 μm .", "( C ) Diagrams of NuMA structure and the NuMA mutants generated .", "( D–F ) .", "Localization analysis of GFP-tagged NuMA mutants with MTs ( red ) as indicated .", "Scale bars are 10 μm .", "( G , H )", "Kymographs of time-lapse movies of GFP-NuMA-TIP and tdTomato-EMTB expressing keratinocytes , taken at the periphery of the cell ( scale bar , 1 μm ) .", "( I–K ) 6XHis-GFP-NuMA-TIP was purified from bacteria , mixed with polymerized MTs and pelleted onto coverslips .", "( I ) Punctate labeling of MTs ( red ) by 6X-GFP-NuMA-TIP , ( approximately 300 nM ) .", "( J , K )", "Tip binding of GFP-NuMA-TIP at lower concentrations of the protein ( approximately 30 nM ) .", "Scale bars are 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 00610 . 7554/eLife . 12504 . 007Figure 2—figure supplement 1 . Kymographs showing GFP-NuMA-TIP dynamics at the cell periphery ( A ) and at the centrosome ( B ) .", "Small arrow indicates the centrosomes; the large arrowhead indicates MT tip-localized GFP-NuMA-TIP ( I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 007 While the NuMA-TIP region was sufficient for NuMA’s interaction with MTs , NuMA could also potentially interact with MTs indirectly through dynein/dynactin .", "We therefore examined which regions of NuMA were necessary for MT tip localization .", "Analysis of full-length NuMA localization requires the examination of mitotic cells , since NuMA is sequestered in the nucleus in interphase .", "To circumvent this issue , we examined the localization of NuMA fusion constructs lacking the nuclear localization sequence ( NLS ) alone , or in combination with the loss of the MTBD or the entire NuMA-TIP domain ( Figure 2C ) .", "We observed association of GFP-NuMAΔNLS with MT tips when the fusion protein was expressed at low levels ( Figure 2D ) .", "Conversely , cells expressing a NuMA construct that lacked both the NLS and TIP region did not exhibit any MT tip localization ( Figure 2F ) .", "This region is therefore both necessary and sufficient for NuMA’s MT tip interactions .", "Importantly , deletion of the MTBD domain alone ( which keeps the LGN-binding domain intact ) compromises tip-localization ( Figure 2E ) .", "The LGN-binding domain is required for cortical targeting of NuMA , which is necessary for its role in spindle orientation .", "Therefore , a NuMA mutant harboring a MTBD domain deletion allows us to test the role of MT tip-binding activity during spindle orientation without disrupting other critical domains of NuMA that are known to be required for its cortical localization .", "The association of NuMA with MT ends after nocodazole treatment suggested that it might associate with stalled or depolymerizing MTs .", "To address this , we performed time-lapse analysis of GFP-NuMA-TIP dynamics in cultured keratinocytes either alone or in combination with ensconsin’s MT-binding domain ( EMTB ) fused to tdTomato to label MTs .", "Live-imaging of cells expressing low levels of the construct revealed puncta that corresponded to MT tips .", "Many of the brighter puncta were stationary , consistent with NuMA-TIP’s role in stabilizing MTs .", "In addition , we observed motile GFP puncta tracking toward the cell periphery along MT plus tips ( Figure 2G , H; Figure 2—figure supplement 1 ) .", "Furthermore , these puncta remained localized to the ends of depolymerizing MTs .", "This behavior was also evident at the centrosome ( which displayed NuMA-TIP localization as well ) ( Figure 2—figure supplement 1 ) .", "These data demonstrate that NuMA can remain attached to both growing and shrinking MT ends and suggests that this may be functionally important for facilitating the interactions of astral MT tips with the cell cortex during spindle orientation .", "To determine whether NuMA-TIP could directly interact with MTs and MT tips , we purified a GFP and 6XHis-tagged version of this protein from bacteria .", "We took advantage of the GFP tag to ask whether we could detect association of purified NuMA-TIP with MTs .", "As shown in Figure 2I , NuMA-TIP at high concentrations decorated the MT lattice , although we often observed enhanced accumulation at MT tips .", "At lower concentrations , however , we observed specific association with MT tips ( Figure 2J ) .", "On short MTs , we also observed localization of GFP-NuMA-TIP on both MT ends ( Figure 2K ) , consistent with our findings in cells .", "In all these experiments , MTs were treated briefly with nocodazole before fixation to promote their depolymerization .", "While these data cannot rule out roles for accessory proteins in modulating NuMA’s association with MTs , they do demonstrate that NuMA-TIP is sufficient for direct interaction with MT ends .", "To directly test whether NuMA’s MTBD is required for spindle orientation , we took advantage of the robust ability of cultured keratinocytes to align their mitotic spindle with the cortical NuMA crescent ( Seldin et al . , 2013 ) .", "We used NuMA-MTBDfl/fl mice ( which harbor a floxed allele of Numa1 Exon 22 ) ( Silk et al . , 2009 ) to generate NuMAΔMTBD keratinocytes .", "Cre-induced recombination in these cells results in the in-frame deletion of NuMA’s MTBD ( Figure 3—figure supplements 1 ) .", "As described above , this mutation causes loss of MT tip localization , but preserves the LGN-binding domain that is necessary for targeting NuMA to the cell cortex and promoting spindle orientation .", "To generate these cells , we treated primary keratinocytes isolated from NuMA-MTBDfl/fl mice with either adeno-GFP ( control ) or adeno-Cre virus ( mutant ) , picked clones , and validated recombination by PCR ( Figure 3—figure supplement 2 ) .", "Loss of the MTBD did not impair localization of NuMA to either the spindle poles or cell cortex; however , it did cause a defect in spindle orientation ( Figure 3A–E ) .", "More than 80% of control cells had spindles oriented within 30 degrees of the center of the cortical NuMA crescent ( Figure 3D ) .", "In contrast , NuMAΔMTBD cells showed a complete randomization of division orientation ( Figure 3E ) .", "Similar results were found by overexpressing NuMAΔMTBD-GFP in wild-type keratinocytes , demonstrating that this acts as a dominant-negative allele ( Figure 3—figure supplement 3 ) .", "These data demonstrate that NuMA-MT interactions are essential for proper mitotic spindle orientation establishment in cultured keratinocytes . 10 . 7554/eLife . 12504 . 008Figure 3 . NuMA’s MTBD is required for spindle orientation in cultured keratinocytes and intact skin . Localization of endogenous full-length NuMA ( A ) and NuMAΔMTBD ( B ) in mitotic keratinocytes .", "( C ) Diagram illustrating how spindle angles wre measured with respect to the polarized cortical NuMA crescent in cultured keratinocytes .", "( D , E )", "Radial histograms representing the distribution of spindle orientation angles in control ( D ) and NuMAΔMTBD ( E ) keratinocytes ( p<0 . 0001 ) .", "( F , G )", "Co-localization of both endogenous NuMA ( F ) and NuMAΔMTBD ( G ) ( green ) with the p150glued subunit of dynactin ( red ) in mitotic keratinocytes .", "( H , I )", "Localization of NuMA ( H ) and NuMAΔMTBD ( I ) ( red ) in mitotic basal cells from e17 . 5 mouse backskin cryosections .", "Arrows and insets indicate the apical cortical accumulation of NuMA .", "Dashed lines indicate the basement membrane .", "Scale bars , 20 μm .", "( J , K )", "Radial histograms showing the distribution of spindle angles relative to the underlying basement membrane in e17 . 5 control epidermis ( J ) and NuMAΔMTBD epidermis ( K ) ( n = 75 cells , p<0 . 0001 ) .", "Unless noted , scale bars are 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 00810 . 7554/eLife . 12504 . 009Figure 3—figure supplement 1 . Binding sites and exon structure of NuMA .", "( A ) Diagram of the exon-intron structure of NuMA relative to its C-terminal protein-binding domains . The MTBD deletion mice were generated from a mouse line harboring a floxed exon 22 .", "( B ) Diagram of mouse NuMA exons 19–22 and their corresponding protein-binding domains .", "( C ) Amino acid numbers of the protein-binding domains in human NuMA ( 2115 amino acid isoform ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 00910 . 7554/eLife . 12504 . 010Figure 3—figure supplement 2 . PCR genotyping analysis of NuMAΔMTBD mice and clonal cell lines . Note that a fraction of Krt14-Cre;NuMA-MTBDfl/fl mice did not show an overt phenotype .", "PCR analysis of keratinocytes isolated from their backskin revealed poor recombination when compared with mice that exhibited an overt phenotype .", "This penetrance variability is thus due to mosaic recombination and is not biologically mediated . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01010 . 7554/eLife . 12504 . 011Figure 3—figure supplement 3 . Radial histograms of spindle orientation in cultured mouse keratinocytes expressing either full-length NuMA-GFP or NuMAΔMTBD-GFP . n = 50 and p<0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01110 . 7554/eLife . 12504 . 012Figure 3—figure supplement 4 . Co-localization of p150glued ( a dynactin subunit ) ( red ) with both NuMA-GFP and NuMAΔMTBD-GFP . Scale bar is 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01210 . 7554/eLife . 12504 . 013Figure 3—figure supplement 5 . Polarity and apoptosis markers in control and NuMA ( delta symbol ) MTBD epidermis .", "( A , B ) .", "Immunofluorescence images of the Golgi protein GRASP65 ( red ) in control ( A ) and NuMAΔMTBD ( B ) backskin ( scale bar , 10 μm ) .", "Dashed lines indicate the basement membrane .", "( C , D ) .", "Immunofluorescence images of activated caspase-3 in control ( C ) and NuMAΔMTBD ( D ) backskin ( scale bar , 50 μm ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 013 Considering that NuMA’s canonical function in spindle orientation is to recruit dynein to the cell cortex , we tested whether dynein/dynactin’s localization was perturbed in NuMAΔMTBD cells .", "We found that p150glued of the dynactin complex co-localized with the cortical crescents of both NuMA and NuMAΔMTBD ( Figure 3F , G ) .", "This was true in 100% of cells examined ( n = 50 from two experiments ) .", "In contrast , we recently published that knockdown of NuMA resulted in an almost complete loss of cortical dynactin ( Seldin et al . , 2013 ) .", "Similar results were found upon overexpression of NuMAΔMTBD-GFP in wild-type cells ( Figure 3—figure supplement 4 ) .", "While the fusion protein localized normally and co-localized with dynein/dynactin , its overexpression resulted in spindle misorientation .", "These data demonstrate that cortical recruitment of dynein/dynactin is not sufficient for proper spindle orientation and that NuMA’s MTBD provides an essential additional function .", "In contrast to cultured keratinocytes , mitotic spindles in situ align either parallel to the basement membrane to generate two basal progenitors , or perpendicular to it to generate one basal progenitor and one suprabasal cell that will differentiate ( Lechler and Fuchs , 2005; Poulson and Lechler , 2010; Smart , 1970 ) .", "To determine whether NuMA-MT interactions were critical for spindle orientation in vivo , we crossed the NuMA-MTBDfl/fl mouse line described above with Keratin 14-Cre mice ( Vasioukhin et al . , 1999 ) , generating an in-frame deletion of the MTBD in epidermal progenitors .", "Due to the small change in protein size , we validated recombination by PCR analysis of isolated epidermis ( Figure 3—figure supplement 2 ) .", "We analyzed NuMA localization in embryonic tissue sections and confirmed that the mutant form localized normally to spindle poles and the cortex of mitotic cells , consistent with our findings in cultured cells ( Figure 3H , I ) .", "We then scored spindle orientation using cryosections from e17 . 5 backskin by measuring the angle between the anaphase spindle and the basement membrane .", "In agreement with previous studies , controls showed a bimodal distribution of orientations ( Figure 3J ) .", "Quantitation of spindle angles in mutant epidermis showed a dramatic decrease in perpendicular spindles coupled with an increase in oblique and parallel spindles ( Figure 3K ) .", "This defect was not an indirect effect of loss of polarity or cell death , as both the Golgi and centrosomes showed proper polarization and cleaved caspase 3-positive cells did not significantly increase in number ( Figure 3—figure supplement 5 ) .", "While previous work on spindle orientation in NuMA knockdown mice revealed a dramatic shift toward parallel spindles ( Williams et al . , 2011 ) , it is important to note that the basal cells of those mice were largely devoid of NuMA , thus preventing cortical dynein recruitment .", "The randomized spindle angles observed in NuMAΔMTBD mice are likely due to the maintenance of cortical dynein , which cannot efficiently orient the spindle in the absence of NuMA-MT interactions .", "Loss of NuMA does not cause mitotic spindle assembly defects in cultured keratinocytes , mammalian epidermis or fly neuroblasts ( Bowman et al . , 2006; Seldin et al . , 2013; Siller et al . , 2006; Williams et al . , 2011 ) .", "However , previous analysis of NuMAΔMTBD mice harboring a full body deletion suggested that this region was essential for development and that cultured mutant fibroblasts had mitotic spindle defects ( Silk et al . , 2009 ) .", "To reconcile these findings , we analyzed mitotic spindles in both NuMAΔMTBD cultured keratinocytes and mouse epidermis .", "We did not detect any defects in mitotic morphology , stage , or number in mutant keratinocytes ( Figure 4A–G ) .", "Metaphase and anaphase spindles exhibited normal morphology ( Figure 4A , B ) .", "The localization of NuMA to the cell cortex , the incidence of abnormal spindles and the extent of mis-aligned DNA were all similar between control and NuMAΔMTBD cells ( Figure 4C–F ) .", "This was true both in primary keratinocytes directly isolated from newborn backskin as well as the clonal lines described above .", "There were also no significant differences in the percentage of cells in different mitotic stages or their spindle lengths ( Figure 4F , G ) .", "Furthermore , flow cytometry analysis of mutant epidermal cells revealed no evidence of aneuploidy or polyploidy ( Figure 4I , J ) .", "Finally , the phenotype that we observed is distinct from those caused by mutations in mitotic regulators , arguing against an underlying mitotic defect ( Foijer et al . , 2013 ) .", "This stands in stark contrast to past findings in cultured fibroblasts and thus suggests the possibility of cell-type specific roles for NuMA .", "Nevertheless , it should be noted that our data are consistent with previous studies on cultured keratinocytes , mouse epidermis and fly neuroblasts ( Bowman et al . , 2006; Seldin et al . , 2013; Siller et al . , 2006; Williams et al . , 2011 ) . 10 . 7554/eLife . 12504 . 014Figure 4 . Keratinocytes show no signs of mitotic spindle assembly defects upon loss of NuMA’s MTBD .", "( A , B )", "Immunofluorescence images of metaphase and anaphase mitotic figures from control and NuMAΔMTBD keratinocytes .", "MTs ( red ) , DNA ( blue ) .", "( C ) Primary keratinocytes were isolated from control and NuMAΔMTBD mice and analyzed for cortical localization of NuMA , formation of bipolar spindles and the presence of aligned chromosomes in metaphase .", "n = 40 , p>0 . 05 .", "( D ) Analysis as described in ( C ) was performed on control and NuMAΔMTBD clonal cell lines .", "n = 100 , p>0 . 05 .", "( E ) Representative images of the phenotypes scored in ( C ) and ( D ) .", "( F ) Analysis of mitotic stages in primary cells isolated from control and NuMAΔMTBD epidermis .", "n = 40 , p>0 . 05 for all .", "( G ) Quantitation of the number of astral MTs/spindle pole in control and NuMAΔMTBD keratinocytes .", "n = 60 spindle poles , p>0 . 05 .", "( H ) Quantitation of metaphase spindle length in control and NuMAΔMTBD keratinocytes .", "n = 20 spindles , p>0 . 05 .", "( I ) Keratinocytes were isolated from the backskin of control and NuMAΔMTBD mice , stained with propidium iodide and their DNA content analyzed by flow cytometry .", "n = 2 .", "( J ) Bar graph of data from ( J ) representing the percentage of cells in each cell cycle stage .", "n = 2 mice/genotype , p>0 . 05 for all .", "All scale bars are 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 014 Considering that spindle orientation defects could result from compromised astral MT integrity , we also quantified the number of astral MTs in both control and NuMAΔMTBD mice and found no significant differences ( Figure 4H ) .", "These data are consistent with loss of NuMA’s MTBD impairing its interaction with astral MTs at the cortex and not spindle assembly .", "Having established that loss of NuMA’s MTBD causes spindle orientation defects but not spindle assembly defects , we sought to determine the resulting consequences on epidermal development .", "Mice with epidermal loss of NuMA’s MTBD were neonatal lethal ( Figure 5—figure supplement 1 ) .", "This lethality was associated with a mild barrier defect , as observed by an X-gal penetration assay ( Figure 5A ) .", "These observations led us to investigate whether underlying differentiation defects contributed to lethality .", "In control mice , keratin 5/14 marked a monolayer of proliferative basal cells , while keratin 10 was present in their differentiated progeny ( Figure 5B ) .", "In mutant mice , we observed an expansion of K5/14- positive cells into suprabasal layers and their mixing with K10-positive cells ( Figure 5C ) .", "These defects were noticed as early as e16 . 5 , before epidermal barrier activity is fully established ( Figure 5—figure supplement 2 ) .", "Later differentiation markers , such as loricrin and filaggrin , were present at much lower levels in mutant skin , explaining in part the compromised barrier and neonatal lethality ( Figure 5D–F ) .", "There was also a dramatic upregulation of the stress marker keratin 6 in mutant skin ( Figure 5G–I ) .", "Consistent with a differentiation defect , we observed increased numbers of cycling cells in the mutant suprabasal epidermis by both BrdU and phospho-histone H3 staining ( Figure 5J–P ) .", "In addition , we noted an increase in basal cell proliferation , which is likely a consequence of barrier dysfunction .", "In support of this , analysis of e16 . 5 epidermis ( a stage at which the barrier is just beginning to form ) revealed an increase in suprabasal cell divisions , but not in basal divisions ( Figure 5N ) .", "These studies demonstrate that high levels of spindle misorientation are not tolerated and that the oblique divisions observed cannot promote proper differentiation of the epidermis .", "Previous data suggested that low levels of spindle misorientation were not detrimental to epidermal development ( Williams et al . , 2014 ) .", "Therefore , there appears to be a critical threshold at which a decrease in functionally asymmetric divisions impairs development . 10 . 7554/eLife . 12504 . 015Figure 5 . Loss of NuMA’s MTBD in the embryonic epidermis results in differentiation defects and neonatal lethality .", "( A ) X-gal penetration assay of e18 . 5 NuMAΔMTBD and littermate control mice shows a mild barrier defect in the mutant .", "( B , C )", "Immunofluorescence analysis of the basal cell marker keratin 5/14 ( green ) and the differentiated cell marker keratin 10 ( red ) in control ( B ) and NuMAΔMTBD ( C ) P0 backskin .", "( D , E )", "Expression of the granular cell marker loricrin ( red ) in control ( D ) and NuMAΔMTBD ( E ) epidermis .", "( F ) Western blot of filaggrin levels in protein lysates prepared from control and NuMAΔMTBD epidermis .", "( G , H )", "Expression of the stress marker keratin 6 ( red ) in control ( G ) and NuMAΔMTBD ( H ) epidermis .", "( I ) Western blot of keratin 6 levels in lysates prepared from control and NuMAΔMTBD epidermis .", "( J , K )", "Dams with e18 . 5 embryos were given a pulse of BrdU one hour before sacrifice .", "Control ( J ) and NuMAΔMTBD ( K ) embryonic backskin cryosections were stained with anti-BrdU antibodies ( red ) to assess proliferation rates .", "( L ) Quantitation of BrdU incorporation in basal and suprabasal cells of the control and mutant epidermis ( n = 3 mice/genotype , >500 cells analyzed . basal , p = 0 . 04; suprabasal , p = 0 . 03 ) .", "( M , N )", "Quantitation of the percentage of phospho-histone H3 positive keratinocytes in basal and suprabasal cells layers at e18 . 5 ( M ) and e16 . 5 ( n = 3 mice/genotype , >500 cells analyzed . basal , p = 0 . 05; suprabasal , p = 0 . 07 ) .", "* denotes p values ≤0 . 05 .", "All scale bars are 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01510 . 7554/eLife . 12504 . 016Figure 5—figure supplement 1 . Photographs of neonatal control and NuMAΔMTBD mice . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01610 . 7554/eLife . 12504 . 017Figure 5—figure supplement 2 . Analysis of differentiation in e16 . 5 control and NuMAΔMTBD embryos . Keratin 5 ( red ) and keratin 10 ( green ) were costained in tissues as indicated .", "Asterisks indicate suprabasal cells that are either co-labeled for K5 an K10 ( middle panel ) or that are undergoing mitosis ( bottom panel ) .", "Scale bar is 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 017 Loss of NuMA’s MTBD results in spindle orientation defects without affecting spindle assembly or cell polarity .", "The NuMAΔMTBD mouse line therefore serves as an invaluable tool that allows us to specifically address the role of spindle orientation in a tissue context .", "Previously , it has not been possible to specifically perturb spindle orientation during postnatal hair morphogenesis .", "The hair follicle is a highly organized epidermal appendage that undergoes cycles of growth , regression and rest throughout an animal’s lifetime .", "The growth phase , called anagen , is responsible for producing the fully differentiated hair shaft .", "Three proliferative compartments of the hair follicle , the bulge stem cells , the outer root sheath ( ORS ) and the transit-amplifying matrix cells , are responsible for generating the cell types that make up the mature hair follicle .", "We generated inducible epidermal NuMAΔMTBD mice by crossing to a keratin 5-CreER mouse line ( Figure 6A ) ( Van Keymeulen et al . , 2011 ) .", "Mice were injected with tamoxifen at both P18 and P20 , which coincide with the first telogen ( the resting phase of the hair cycle ) , and a small region of backskin was shaved to observe hair regrowth in the subsequent anagen ( Figure 6B ) .", "In contrast to the robust hair regrowth seen in littermates , we observed an almost complete absence of hair regrowth in the mutant mice ( Figure 6C ) . 10 . 7554/eLife . 12504 . 018Figure 6 . NuMA’s MTBD is required in hair follicle matrix cells for proper spindle orientation , differentiation , and hair generation .", "( A ) Diagram illustrating the mouse mating used to generate conditional inducible deletion of NuMA’s MTBD in the adult interfollicular epidermis and hair follicles .", "( B ) Diagram illustrating the experimental time course .", "Mice were injected twice with tamoxifen in telogen of the hair cycle , shaved and then examined for subsequent hair growth in the following anagen .", "( C ) Images of control and NuMAΔMTBD mice treated as described in ( B ) and photographed at postnatal day 44 .", "( D ) Expression of keratin 14 promoter-driven NuMA-GFP in both the interfollicular epidermis and outer root sheath ( ORS ) of a P2 hair follicle .", "The hair shaft signal in the top images is due to autofluorescence .", "The dashed line indicates the basement membrane that separates the dermal papilla and the matrix cells .", "Note the lack of NuMA-GFP expression in the matrix cells ( arrows ) .", "( E , F )", "Photographs of control and matrixΔMTBD mice ( K14-Cre; NuMA MTBDfl/fl;K14-NuMA-GFP ) at postnatal day 7 ( E ) and 18 ( F ) .", "Note that the small patch of dorsal hair is likely due to incomplete recombination and is lost in subsequent hair cycles ( Figure 6—figure supplement 4 ) .", "( G , H )", "Radial histograms of spindle angles in the matrix cells of control ( G ) and matrixΔMTBD ( H ) mice at P2 ( n = 40 cells . p = 0 . 005 ) .", "( I–T )", "Immunofluorescence analysis of the indicated hair differentiation markers ( green ) in control and matrixΔMTBD backskin at P4 .", "Keratin 5/14 labels the ORS , keratin 6 labels the companion layer , P-cadherin labels matrix cells , AE13 labels the cuticle and cortex of the hair shaft , and GATA3 labels the inner root sheath .", "All scale bars are 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01810 . 7554/eLife . 12504 . 019Figure 6—figure supplement 1 . MatrixΔMTBD mice ( 4 months old ) showing loss of all hair as the animals age . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 01910 . 7554/eLife . 12504 . 020Figure 6—figure supplement 2 . Representative images of anaphase spindles in the matrix of the hair follicle . Examples of symmetric ( SCD ) , oblique , and asymmetric ( ACD ) divisions are shown .", "Bottom panels show dividing cell chromatin at higher magnification .", "Phospho-histone H3 and β4 integrin are in red , DNA is blue . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 02010 . 7554/eLife . 12504 . 021Figure 6—figure supplement 3 . Hair follicle histology at P18 ( A , B ) and P30 ( C , D ) in control and MatrixΔMTBD mice . Note the abnormal structures that are present in the first telogen ( P18 ) of mutant follicles and the resumption of anaphase at P30 .", "( E , F )", "Analysis of proliferation by short-term BrdU uptake in P35 control ( E ) and MatrixΔMTBD ( F ) mice .", "Note the aberrant localization of S phase cells in matrixΔMTBD follicles . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 02110 . 7554/eLife . 12504 . 022Figure 6—figure supplement 4 . Analysis of BrdU positive cells in control and matrixΔMTBD mice at postnatal day 2 . Cells incorporating BrdU are labeled in red . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 022 We next wanted to isolate a single proliferative compartment within the hair follicle to examine the specific effects of deleting NuMA’s MTBD .", "Matrix cells are rapidly dividing and give rise to several distinct lineages , making them ideal candidates for utilizing spindle orientation .", "While bulge and ORS cells are contiguous with the interfollicular basal layer and express keratin 14 , matrix cells are keratin 14-negative .", "Using previously generated mice in which NuMA-GFP is expressed under the control of the keratin 14 promoter ( Poulson and Lechler , 2010 ) , we could rescue NuMA expression in the bulge and ORS ( in Krt14-Cre;NuMA-MTBDfl/fl mice ) , resulting in mice with a matrix-specific MTBD deletion that we will refer to as 'matrixΔMTBD' ( Figure 6D ) .", "Krt14-NuMA-GFP expression was sufficient to fully rescue the neonatal lethality caused by embryonic loss of NuMA’s MTBD ( Figure 6E ) .", "However , these mice displayed severe hair growth defects , indicating a critical role for NuMA’s MTBD in matrix cells ( Figure 6F ) .", "The small tufts of backskin hair present at early stages in matrixΔMTBD mice was lost during subsequent hair cycles ( Figure 6—figure supplement 1 ) .", "While these findings implicate spindle orientation in matrix function , they do not rule out the possibility that NuMA’s MTBD plays a critical role within other hair cell types as well .", "To determine whether spindle orientation was disrupted in matrixΔMTBD mice , we analyzed cryosections from P2 control and mutant follicular epidermis before overt changes in follicle architecture became apparent .", "Spindle orientation was quantitated similar to that in the embryonic epidermis by measuring the angle formed by the anaphase spindle relative to the underlying basement membrane ( Figure 6—figure supplement 2 ) .", "Compared with controls , the spindles of matrix cells lacking NuMA’s MTBD exhibited a randomization of orientations ( similar to the orientation pattern seen in knockout interfollicular epidermis ) , demonstrating that the MTBD is critical for robust spindle positioning within this cellular compartment ( Figure 6G , H ) .", "To determine the impact of this defective spindle orientation , we examined markers of the distinct cell lineages of the hair follicle .", "Keratins 5 and 6 mark the ORS and companion layer of the hair follicle , respectively , and demonstrated similar staining patterns in control and mutant follicles .", "Additionally , the matrix marker P-cadherin showed normal staining in both control and mutants , indicating that the matrix population was properly specified ( Figure 6I–N ) .", "In contrast , AE13 and GATA3 , which mark the hair shaft and inner root sheath ( IRS ) cells ( all matrix descendants ) , respectively , were decreased and/or disorganized in the mutant follicles ( Figure 6O–R ) .", "Therefore , robust spindle orientation is necessary for the proper differentiation of matrix progeny .", "Notably , the matrix-derived hair shaft and IRS cell lineages are also lost when BMP signaling is inhibited ( Andl et al . , 2004; Kobielak et al . , 2003; Kulessa et al . , 2000 ) .", "In fact , BMP signaling is one of the earliest detectable steps in the differentiation of these lineages .", "We therefore analyzed BMP activation status to determine whether spindle orientation was required for BMP activity .", "Analysis of BMP activity using anti-pSMAD1/5 antibodies revealed a loss of signaling ( Figure 6S , T ) .", "These data are consistent with BMP signaling acting downstream of these asymmetric divisions .", "Examination of older mice revealed additional phenotypic similarities with mice in which BMP signaling was blocked , including cyst formation and aberrant spatial localization of follicular proliferation ( Figure 6—figure supplement 3 ) .", "Although matrix-derived cells in the mutant mice lacked markers for IRS and hair shaft , they appeared to be non-proliferative , as judged by lack of BrdU incorporation ( Figure 6—figure supplement 4 ) .", "One of the simplest models suggests that BMP inhibitors in the dermal papilla locally impede BMP signaling in matrix cells , while more distant cells undergo active signaling .", "Our data demonstrate that a spindle orientation-dependent event is also required to activate BMP signaling , which is therefore not simply driven by compartmentalization ." ], [ "While precise control of spindle orientation has been documented in an increasing number of developmental and physiological contexts , the underlying mechanisms are only partially understood .", "Here , we have documented a new role for NuMA , one of the core components of the spindle orientation machinery .", "In addition to being important for the recruitment of dynein/dynactin to the cortex , we have found that NuMA’s dynein-independent interactions with MTs are also essential for spindle orientation .", "We report an unusual and previously unreported co-localization of NuMA with MT tips and define a region within NuMA that is both necessary and sufficient for this localization .", "While it is likely that the direct interactions provided by the NuMA-TIP region contribute to the specific binding of NuMA to MT tips , we cannot rule out that additional proteins are required to increase NuMA’s MT tip-binding affinity .", "The properties of NuMA-TIP are distinct from many tip-binding proteins .", "Most notably , NuMA-TIP remains associated with stalled and or depolymerizing MTs .", "To date , there are very few proteins known to exhibit MT depolymerization-tracking behavior ( either directly or indirectly ) .", "The few known examples are kinetochore proteins of different species , including the Dam1 complex , CENP-E , CENP-F , and the Ndc-80 complex .", "Of these , only Dam1 has been shown to follow depolymerizing MTs in vivo , while the others can do this in vitro , yet remain at the kinetochore-MT interface in vivo ( Westermann et al . , 2006 ) .", "Depolymerizing kinesins have also been observed to localize to both plus and minus ends of MTs ( Desai et al . , 1999 ) .", "While a previous study demonstrated that artificially targeting dynein to the cortex was sufficient to induce spindle oscillations ( Kotak et al . , 2012 ) , our data suggest that cortical dynein is not sufficient to robustly position the spindle and that NuMA-MT interactions are also required ( Figure 7 ) .", "A simple model in which all forces during spindle orientation are generated by dynein-dependent pulling on astral MTs is still formally possible , with NuMA acting simply to provide MT stabilization , thus making astral MTs better substrates for dynein-directed forces .", "While a viable possibility , this is not consistent with local stabilization of MTs inhibiting spindle movements ( Nguyen-Ngoc et al . , 2007 ) .", "Alternatively , NuMA’s unique MT-binding qualities revealed in this study could allow it to interact with depolymerizing MTs and harness their energy to promote spindle rotation , thus collaborating with dynein pulling forces to robustly orient the spindle .", "This latter idea is similar to kinetochores harnessing MT depolymerization for chromosome segregation ( McIntosh et al . , 2010 ) .", "Both the association of MT dynamics with force generation observed during spindle positioning ( Labbe et al . , 2003 ) , as well as work showing the ability of depolymerizing MTs to generate force ( McIntosh et al . , 2010 ) are consistent with this second possibility .", "This is not without precedent , as positioning of the budding yeast spindle relies on regulated depolymerization at the cell cortex ( Gupta et al . , 2006 ) .", "If and how depolymerization activity is coordinated with dynein/dynactin will require further investigation .", "It is also possible that both stabilization and tracking depolymerizing MTs are important activities of NuMA that are used at distinct sites/times .", "For example , NuMA has been shown to collect at the minus ends of severed MTs in the mitotic spindle where it may act to stabilize the free ends ( Elting et al . , 2014 ) . 10 . 7554/eLife . 12504 . 023Figure 7 . Models for NuMA and ACD functions .", "( A ) Diagram of interactions required for spindle orientation .", "NuMA interacts with MTs via a carboxy-terminal domain , as well as with dynein/dynactin in its amino terminus .", "Both these interactions are likely required for robust spindle orientation .", "Only a single NuMA dimer is illustrated for simplicity , although it is also thought to multimerize .", "NuMA may act to stabilize the MT end or , alternatively , may couple its depolymerization to force generation for spindle positioning .", "( B ) Comparison of the repetitive uses of spindle orientation in the embryonic epidermis and adult hair follicle .", "In both cases , spindles align perpendicular to the underlying basement membrane in a significant portion of cells in a NuMA-dependent manner .", "Whereas cell fate decisions in embryonic epidermis are Notch-dependent , cell fate choices in the hair follicle are BMP-dependent .", "In neither case do we understand the molecular connection between spindle orientation and the activation of these pathways . DOI: http://dx . doi . org/10 . 7554/eLife . 12504 . 023 Our data further demonstrate the developmental roles for spindle orientation in two distinct cellular environments .", "Consistent with previous studies , loss of spindle orientation in the developing epidermis resulted in embryonic lethality that is likely due to epidermal differentiation defects .", "These data raise the question of whether the early embryonic lethality observed in the full mouse NuMA MTBD knockout was due to mitotic defects , defects in spindle positioning or a combination of these effects ( Silk et al . 2009 ) .", "While the phenotype that resulted from the epidermal-specific loss of NuMA’s MTBD in this study was less severe than that seen upon shRNA depletion of either LGN or NuMA , there are two plausible explanations to reconcile this discrepancy .", "First , the gene ablations in the previous study were initiated prior to epidermal stratification , whereas our knockout occured during the process of stratification .", "Second , loss of full-length NuMA or LGN resulted in a drastic conversion of ACDs ( perpendicular spindles ) to SCDs ( parallel ) , while loss of NuMA’s MTBD resulted in a conversion of ACDs to randomized division orientations .", "The finding that the NuMA MTBD deletion primarily affects spindle orientation without observable effects on either cell polarity or cell division establishes the NuMA-MTBD deletion mice as a valuable tool to study the in vivoroles of spindle orientation in intact tissues .", "We have taken advantage of this by being the first to demonstrate an essential requirement for spindle orientation in hair follicle morphogenesis .", "While prior reports have presented correlative data to suggest that spindle orientation contributes to hair follicle morphogenesis ( Niessen et al . , 2013; Rompolas et al . , 2012 ) , we have quantitated division orientations within the rapidly proliferating matrix compartment and demonstrated the importance of spindle orientation in these cells .", "Similar to the interfollicular epidermis in the embryo , divisions can be either parallel or perpendicular to the underlying basement membrane ( Figure 7 ) .", "The percentage of matrix cell perpendicular divisions decreased in the mutant and resulted in differentiation defects in matrix-derived lineages .", "One of the earliest known steps of differentiation along these pathways is BMP activation ( Andl et al . , 2004; Kobielak et al . , 2003; Kulessa et al . , 2000 ) , and loss of BMP signaling resulted in phenotypes similar to loss of spindle orientation .", "In both cases , IRS and hair shaft markers are decreased or absent .", "The matrix-derived cells in the mutant mice described here as well as the BMPRIA mutant are non-proliferative , suggesting that they may be segregated from pro-proliferative signals from the basement membrane and/or dermal papilla .", "Surprisingly , in the absence of proper spindle orientation , the activation of BMP signaling does not occur .", "While the compartmentalized activation of BMP could have been due solely to the presence of BMP inhibitors in the DP preventing signaling in adjacent matrix cells , our data demonstrate that a spindle orientation-dependent step is required for cells to become responsive to BMP signaling .", "Whether this acts directly on the BMP signaling pathway or lies upstream of it is not currently known .", "Understanding the molecular basis of this requirement is a clear future goal to understand how differentiation and morphogenesis are controlled .", "In summary , we reveal that a reiterative use of spindle orientation machinery generates very distinct structures ( stratified interfollicular epithelium versus hair follicles ) within the same tissue ." ], [ "NuMA-MTBDfl/wt mice , K14-Cre , K5-CreER and K14-NuMA-GFP mice were previously reported ( Poulson and Lechler , 2010; Silk et al . , 2009; Van Keymeulen et al . , 2011; Vasioukhin et al . , 1999 ) .", "All mouse experiments were performed with approval from the Duke Institutional Animal Care and Use Committee .", "For BrdU experiments , mice were injected with 10 mg/kg of BrdU ( Sigma-Aldrich , St . Louis , MO ) and were left for 1 hr before sacrifice .", "Adeno-GFP and Adeno-Cre-GFP viruses ( Baylor Vector Development Labs , Houston , TX ) were used separately to infect NuMA-MTBDfl/fl mouse keratinocytes .", "Single cells from each infection plate were then seeded into 96-well plates , grown to confluency and subsequently trypsinized , replated and genotyped to identify knockout clones .", "Mouse keratinocytes were isolated from the backskins of newborn or embryonic mice .", "Mouse keratinocytes were grown in E low calcium medium and maintained in a 37°C incubator with 7 . 5% CO2 .", "Primary cells were isolated from mouse backskin by incubating the backskin overnight in a mixture of 1X phosphate-buffered saline ( PBS ) and Dispase II ( Hoffman-La Roche , Basel , Switzerland ) at a 1:1 ratio .", "The epidermis was then removed from the dermis and placed in a mixture of trypsin and versene at a 1:1 ratio for 3 min .", "Cells were resuspended in fresh media , filtered , pelleted , and plated onto glass coverslips coated with 100 μM laminin ( Invitrogen , Waltham , MA ) , or onto fibroblast feeders for long-term passage .", "For drug treatments , 10 μM nocodazole ( Sigma-Aldrich , St . Louis , MO ) and the corresponding concentration of DMSO was incubated with cells for 30 min before fixation .", "In mitotic cells , in which MTs are much more dynamic , nocodazole treatment was with 0 . 5 μM for 5 min .", "Vinblastine ( 2 nM ) and taxol ( 10 μM ) were both from Sigma , and treatments were for 5–10 min .", "For spindle orientation experiments on cultured keratinocytes , cells were plated on glass coverslips coated with 100 μM laminin ( Invitrogen ) .", "HeLa cells were grown at 37°C with 7 . 5% CO2 in DMEM media containing 10% Fetal Bovine Serum with antibiotics .", "Primary control and MTBD knockout cells were isolated as described above .", "After filtering and centrifugation , cells were resuspended in a mixture of PBS and 70% ethanol ( 1:5 ratio ) and placed on ice for 2 hr .", "Cells were then centrifuged for 5 min at 1000 rpm and the ethanol solution was removed .", "After an additional wash in PBS , the cell pellet was suspended in a 1 ml solution containing 0 . 1% Triton X-100 ( EMD Millipore , Darmstadt , Germany ) in PBS , 2 mg DNase-free RNase A ( Qiagen , Venlo , Netherlands ) and 200 µl of 1 mg/ml Propidium Iodide ( Sigma-Aldrich ) and incubated at room temperature for 30 min .", "Cell cycle analysis was then performed on these samples using a FACSCalibur analyzer ( BD Biosciences , Franklin Lakes , NJ ) .", "Cells were fixed for 3 min in −20°C methanol or 8 min in 20°C 4% paraformaldehyde .", "Primary antibodies used in this study include rabbit α-NuMA and rat α-BrdU ( Abcam , Cambridge , MA ) , mouse α-p150glued and rat α-β4 integrin ( BD Biosciences , San Jose , CA ) , mouse α-pankeratin ( AE13 ) , mouse α-Gata 3 and rat α-α tubulin ( all from Santa Cruz Biotechnology , Dallas , TX ) , m α-β-tubulin ( Sigma ) , rabbit α-keratin 6 ( Covance ) , chicken α-keratin 5/14 ( lab generated ) , rabbit α-phospho histone H3 and rabbit anti-pSMAD1/5 ( Cell Signaling Technology , Danvers , MA ) , and rabbit α-activated caspase 3 ( R&D Systems , Minneapolis , MN ) .", "Images were collected using a Zeiss motorized Axio Imager Z1 fluorescence microscope with Apotome attachment , an AxioCam MRm camera , a 10x , 20x , 40x , and 63×/1 . 4 numerical aperture ( NA ) Plan Apochromat objective , Zeiss Immersol 518F oil , and AxioVision Digital Image Processing Software .", "Photoshop ( Adobe ) and ImageJ software were used for postacquisition processing .", "Keratinocytes were plated on MatTek glass-bottom dishes ( no . 1 . 5 ) coated with 100 μM laminin ( Invitrogen ) and transfected with GFP-tagged NuMA constructs .", "Time-lapse imaging was performed using a Leica DMI6000 inverted microscope , a 63×/1 . 4 NA Plan Apochromat objective , Leica immersion oil , and an OrcaER camera ( Hamamatsu Photonics ) .", "The imaging chamber was maintained at 37°C with 5% CO2 .", "Simple PCI software was used for image acquisition ( eCommerce Solutions ) , and Photoshop and ImageJ software were used for post-acquisition processing .", "The entire GFP-NuMA-TIP coding sequence was inserted in frame in the 6XHis tagging vector pet28 .", "The protein was expressed in bacteria and purified over Nickel-agarose ( Qiagen ) .", "Tubulin ( Cytoskeleton ) was pre-cleared by centrifugation at 90000 × g for 10 min and then induced to polymerize by addition of 5% glycerol and incubation at 37°C for 1 hr .", "Assembled MTs were diluted to 1 mg/ml tubulin , and then added to purified 6XHis-GFP-NuMA-TIP .", "The 6XHis-GFP-NuMA-TIP was also precleared , but aggregates still precipitated out in the buffer conditions used .", "We used concentrations of GFP-NuMA-TIP ranging from 300 nM down to 30 nM , but the actual concentrations in solution might be lower due to the aggregation of some of the protein .", "After 2 min , 1 μM nocodazole was added to promote MT depolymerization .", "After 1 min , reactions were fixed with 10 volumes of 1% glutaraldehyde in BRB80 for 10 min at 37°C , and then further diluted with PBS .", "Fixed mixtures were spun onto poly-L-lysine coated coverslips at 25 , 000 × g for 30 min in a swinging bucket rotor .", "Coverslips were then fixed and stained for β-tubulin ( Sigma ) .", "Embryos were isolated from pregnant dams at day e18 . 5 and incubated at 30°C overnight in X-gal solution ( 1 . 3 mM MgCl2 , 100 mM NaPO4 , 3 mM K3Fe ( CN ) 6 , 0 . 01% Na deoxycholate , 0 . 2% NP-40 , 1 mg/mL X-gal [Invitrogen] , pH 4 . 5 ) .", "Spindle orientation in cultured cells was measured by determining the angle between the two spindle poles and the center of the cortical NuMA crescent ( as illustrated in Figure 3C ) .", "Spindle orientation in both interfollicular basal cells and hair follicle matrix cells was measured by determining the angle between a line bisecting both anaphase chromosomes ( identified using pHH3 staining ) and the underlying basement membrane ( β4-integrin staining ) .", "Student’s t-tests were used for all statistical analyses , with the exception of the radial histograms of spindle orientation that were analyzed using the Kolmogorov–Smirnov test ." ] ]

[ "Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis .", "A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types .", "Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules ( MTs ) to orient the spindle , with NuMA acting as a passive tether .", "In this study , we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain , which targets MT tips , is also required .", "Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation , leading to neonatal lethality .", "In addition , we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix .", "Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages .", "Our results reveal an additional and direct function of NuMA during mitotic spindle positioning , as well as a reiterative use of spindle orientation in the skin to build diverse structures ." ]

[ "Before a cell divides , it must duplicate its DNA so that each new cell receives a complete set of genetic material .", "A structure called the mitotic spindle helps to ensure each new cell gets the correct amount of DNA .", "Cells often precisely position their mitotic spindle during division , and this spindle orientation is important for generating different types of cells and for establishing the three-dimensional structure of tissues .", "How cells rotate their spindles into the correct position is not well understood , but a protein called NuMA is important for this process .", "Seldin et al . developed genetic tools that could disrupt spindle orientation in specific types of cells to determine where this orientation is important for proper tissue development .", "This revealed that the correct placement of the mitotic spindle is important for the development of the skin of mouse embryos and the formation of the hair of adult mice .", "Seldin et al . also found that the NuMA protein binds to the tips of the microtubules that make up the mitotic spindle .", "This binding activity is important for NuMA to be able to position the mitotic spindle correctly in the cell .", "The findings suggest similarities between how cells orient mitotic spindles and how they segregate DNA during cell division .", "More work is now needed to better understand how NuMA collaborates with force-generating molecular motors to precisely orient the mitotic spindle in the cell .", "In addition , understanding how spindle orientation dictates the fate of cells in the skin is an important future goal ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology" ]

[ [ "Human activities cause global environmental changes with important consequences for biodiversity and the functioning of ecosystems .", "Understanding these consequences is crucial for better policy and conservation strategies , which will ultimately promote human well-being too ( IPBES , 2019 ) .", "A key question is to what extent changes in ecosystem functioning are mediated by changes at which dimensions of biodiversity .", "Extensive research has demonstrated that biodiversity is needed for the stable provenance and enhancement of ecosystem processes and functions ( Cardinale et al . , 2012; Schuldt et al . , 2018; Tilman et al . , 2012 ) .", "However , this body of evidence is mostly based on experiments comparing ecosystem functioning in artificial communities with varying number of species .", "Such experiments might not capture the complex ways by which shifts in biodiversity induced by global change ultimately affect ecosystem functioning ( De Laender et al . , 2016; Eisenhauer et al . , 2019b ) .", "Early biodiversity-ecosystem function ( BEF ) experiments typically controlled for environmental gradients , thus not accounting for the underlying drivers of biodiversity change ( De Laender et al . , 2016; Srivastava and Vellend , 2005; Wardle , 2016 ) .", "These early experiments also focused on species richness as the sole biodiversity index , and manipulated it directly and randomly .", "However , environmental change will often elicit non-random changes in several facets of biodiversity ( Eisenhauer et al . , 2016; Giling et al . , 2019; van der Plas , 2019 ) ( community composition and population densities ( Glassman et al . , 2018; Spaak et al . , 2017 ) , functional diversity ( Cadotte et al . , 2011; Craven et al . , 2018; Heemsbergen et al . , 2004 ) , trophic diversity ( Soliveres et al . , 2016; Wang and Brose , 2018; Zhao et al . , 2019 ) .", "The selective effects of environmental change emerge because organisms differ in their response to environmental change .", "For example , larger organisms and predators are often more negatively affected than smaller organisms at lower trophic levels ( Hines et al . , 2015; Sheridan and Bickford , 2011; Srivastava and Vellend , 2005; Voigt et al . , 2007 ) .", "Using realistic extinction scenarios , experiments found contrasting effects of non-random shifts in biodiversity on ecosystem functioning ( e . g . Cárdenas et al . , 2017; Jonsson et al . , 2002; Melguizo-Ruiz et al . , 2020; Oliveira et al . , 2019; Smith and Knapp , 2003 , Zavaleta and Hulvey , 2004 ) .", "In addition , several variables that are not directly related to biodiversity control ecosystem functions ( e . g . physiological rates [Dib et al . , 2020; Thakur et al . , 2018] and alterations of physical and chemical conditions [De Laender et al . , 2016; Giling et al . , 2019] ) .", "When environmental change affects these mechanisms , teasing out the relative importance of biodiversity-mediated effects is complicated even more .", "Given the number of different potential mechanisms , quantifying the extent to which shifts in biodiversity underpin the effect of environmental change on ecosystem functioning under real-world scenarios of global change is a key challenge for ecology ( De Laender et al . , 2016; Duffy et al . , 2017; Eisenhauer et al . , 2019b; Srivastava and Vellend , 2005; van der Plas , 2019; Wardle , 2016 ) .", "Incorporating the impacts of environmental change drivers into BEF studies and meta-analyses is an important step forward to address such questions ( De Laender et al . , 2016; Eisenhauer et al . , 2019b ) .", "The vast majority of BEF experiments has focused on plant richness and ecosystem functions such as biomass production ( van der Plas , 2019 ) .", "However , litter decomposition has a tremendous importance in ecosystems and biogeochemical cycles ( Follstad Shah et al . , 2017 ) .", "Small changes in the rate of this process can have important consequences for the overall carbon balance .", "Indeed , increases in decomposition rates could have positive feedback effects on climate warming by enhancing C losses ( Kirschbaum , 2000 ) .", "The diversity of decomposers ( invertebrates and microorganisms that fragment and decompose organic matter in both aquatic and terrestrial systems ) is crucial for litter decomposition ( Eisenhauer et al . , 2012; García-Palacios et al . , 2013; Gessner et al . , 2010; Handa et al . , 2014; Hättenschwiler et al . , 2005 ) and for other ecosystem functions as well ( Eisenhauer et al . , 2019a; Lefcheck et al . , 2015; Schuldt et al . , 2018 ) .", "Despite the importance of decomposers , BEF experiments focusing on litter decomposition more often addressed the influence of plant litter diversity than of decomposers ( Gessner et al . , 2010; Tonin et al . , 2018 ) .", "In a meta-analysis , decomposer diversity had a greater effect on decomposition than the diversity of plant litter ( Srivastava et al . , 2009 ) , although also weak and neutral effects have been reported ( van der Plas , 2019 ) .", "Facilitation and complementarity through niche partitioning are primary mechanisms underlying the positive relationship between decomposer diversity and decomposition ( Gessner et al . , 2010; Hättenschwiler et al . , 2005; Tonin et al . , 2018 ) .", "Experiments conducted in natural conditions and reflecting realistic extinction scenarios are still relatively scarce , and demonstrate contrasting effects of non-random shifts in decomposer diversity on decomposition ( Cárdenas et al . , 2017; Jonsson et al . , 2002; Melguizo-Ruiz et al . , 2020; Wenisch et al . , 2017 ) .", "The need to quantify environmental change effects on decomposer diversity , along with potential knock-on effects on litter decomposition , is therefore particularly pressing .", "There is a variety of environmental change drivers , and different types of drivers may have diverse effects on biodiversity and ecosystem functions ( De Laender et al . , 2016; Dib et al . , 2020 ) .", "We postulate that there are two main categories of environmental change: stressors and resource shifts .", "While stressors cannot be consumed , and act as conditions that alter growth rates ( e . g . , temperature , drought , chemical stressors ) , resources are by definition consumed ( e . g . , CO2 or mineral nutrients ) , which has important implications for how they should enter theory ( Chase and Leibold , 2003; De Laender , 2018 ) .", "Chemical stressors and nutrient enrichment are important case studies of environmental stressors and resource enrichment , because their presence is increasing rapidly ( Bernhardt et al . , 2017 ) and they are projected to have severe effects on biodiversity ( Mazor et al . , 2018 ) .", "They are also of particular relevance for decomposer communities .", "Chemical stressors such as metals and pesticides decrease the diversity , abundance , growth and activity of decomposers across terrestrial and aquatic systems ( e . g . Hogsden and Harding , 2012; Pelosi et al . , 2014; Schäfer , 2019 ) .", "In contrast , nutrient enrichment can have positive impacts on the abundance and physiological rates of decomposer organisms by reducing resource limitations ( Treseder , 2008 ) , but at the same time decrease decomposer diversity ( Lecerf and Chauvet , 2008; Woodward et al . , 2012 ) .", "Across ecosystems , stressors and nutrients can exert opposite impacts on litter decomposition rates , with decreases in response to chemical stressors but increases following nutrient enrichment ( Ferreira et al . , 2015; Ferreira et al . , 2016 ) .", "In addition , decomposition involves both microorganisms and invertebrates ( Bardgett and van der Putten , 2014; Gessner et al . , 2010; Hättenschwiler et al . , 2005 ) that may respond differently to stressors and nutrients with a higher sensitivity of invertebrates than microorganisms ( Peters et al . , 2013; Siebert et al . , 2019 ) .", "Although many published case studies report shifts in decomposer diversity and in rates of litter decomposition at sites impacted by stressors and nutrients , biodiversity-mediated effects have not yet been quantified across systems .", "Here we addressed the question if the effects of stressors and nutrient enrichment on decomposer diversity and abundance explain the response of litter decomposition to these two types of pervasive environmental change drivers ( Figure 1 ) .", "We synthesized 69 published case studies reporting the impact of stressors ( metals , pesticides ) and nutrients ( nitrogen or phosphorous additions ) on litter decomposition and on decomposer diversity ( taxa richness , Shannon diversity , evenness ) or abundance ( density , biomass ) at sites differing in stressor or nutrient levels .", "Our comprehensive global dataset of 660 observations encompasses studies across taxonomic groups ( animal ( soil micro- , meso- and macrofauna , stream macroinvertebrates ) and microbial ( fungi and bacteria ) decomposers ) , ecosystems ( aquatic and terrestrial ) , and study types ( experimental and observational ) ( Figure 2 ) .", "We quantified the effect size of environmental change on decomposer diversity or abundance and on litter decomposition within studies using correlation coefficients between stressor or nutrient levels and decomposer diversity , abundance , and litter decomposition .", "We also characterized stressor and nutrient intensities , and standardized their levels in water , soil , or sediment using environmental quality criteria issued by environmental authorities ( e . g . ECHA , USEPA , UKTAG ) .", "Using meta-analysis and structural equation modelling ( SEM ) , we first compared the overall effects of stressors and nutrients on decomposers and decomposition across systems and studies ( first meta-analysis ) , and second , addressed to what extent changes in decomposer diversity and abundance mediate the impacts of these two contrasting drivers of environmental change on decomposition ( second meta-analysis and SEM ) .", "Third , we explored the effects of three main moderators on decomposers diversity , abundance , and decomposition responses , as found in the second meta-analysis: stressor or nutrient intensity , taxonomic group ( animal vs . microbes ) and study type ( experimental vs . observational studies ) .", "We expected that chemical stressors and nutrients would have contrasting effects on decomposer diversity and abundance , and on litter decomposition across ecosystems and studies ( Figure 1 ) .", "We hypothesized that chemical stressors generally decrease decomposer diversity , abundance ( Hogsden and Harding , 2012; Petrin et al . , 2008 ) , and litter decomposition rates ( Ferreira et al . , 2016; Peters et al . , 2013 ) , and that nutrients generally decrease decomposer diversity ( Lecerf and Chauvet , 2008; Woodward et al . , 2012 ) but increase decomposer abundance and litter decomposition rates ( based on physiological effects and decreasing resource limitations ( Bergfur et al . , 2007; Ferreira et al . , 2015; Treseder , 2008; Woodward et al . , 2012 ) .", "We further hypothesized that litter decomposition responses to environmental change depend on changes in decomposer diversity and abundance , and expected an overall positive relationship independent of environmental change intensity ( Srivastava et al . , 2009 ) ." ], [ "The final dataset contained 69 ( case ) studies from 59 publications , representing 660 observations .", "Data were mostly from Europe ( 44 ; 443 ( studies; observations ) ) and North and South America ( 19; 168 ) , while Asia ( 2; 9 ) and Oceania ( 4; 40 ) were less well represented ( Figure 2A ) .", "The studies covered aquatic ( 55; 388 ) and terrestrial systems ( 14; 272 ) ( Figure 2C ) , and used observational ( 43; 336 ) or experimental approaches ( 26; 324 ) .", "Studies reported abundance ( 66; 463 ) or diversity responses ( 48; 197 ) ( Figure 2B ) of soil and benthic invertebrates ( 48; 509 ) and microbes ( fungi and bacteria ) associated with litter materials ( 36; 151 ) ( Figure 2C ) .", "Chemical stressors were mostly metals ( 13; 257 ) and pesticides ( 12; 66 ) associated with industrial activities , accidental spills , and agricultural practices .", "Nutrient enrichment studies addressed fertilization by various N and/or P forms ( 26; 175 ) , and eutrophication due to agricultural runoffs ( 10; 59 ) or wastewater effluents ( 4; 44 ) .", "There was no study reporting nutrient enrichment impacts on soil decomposer diversity in the dataset .", "Funnel plots and intercepts of Egger’s regression showed evidence for positive publication bias in nutrient enrichment studies reporting decomposer abundance ( Appendix 2—figure 1; Appendix 2—figure 2; Appendix 2—table 1 ) .", "No publication bias was detected in the other datasets .", "We found largely contrasting effects of stressors and nutrients on each of the three response variables in a first-level meta-analysis comparing the overall effects of the two drivers of environmental change ( Figure 3 , Appendix 2—table 2 ) .", "Chemical stressors overall decreased decomposer diversity , abundance and litter decomposition across studies ( Figure 3 ) .", "Nutrient enrichment tended to decrease decomposer diversity but to increase abundance , and decomposition , although these trends were not significant as indicated by confidence intervals of the grand mean effects overlapping with zero ( Figure 3 ) .", "The responses of decomposition and of decomposer diversity and abundance to chemical stressors were correlated: decreases in decomposition were associated with decreases in decomposer diversity and abundance ( Figure 4 upper panels ) .", "We did not find such a relationship for nutrients .", "Instead , a range of positive and negative responses of decomposer diversity , abundance , and decomposition to nutrients were found , without significant associations between them ( Figure 4 lower panels ) .", "In addition , when decomposer diversity and abundance responses to nutrients were close to zero , there was a wide range of decomposition responses ( intercepts from Figure 4 lower panels ) .", "According to our overarching hypothesis , the SEM indicated that the effects of stressors on litter decomposition were mediated by shifts in decomposer diversity and abundance .", "Including the direct paths from decomposer diversity or abundance to litter decomposition improved both the models according to mediation tests and AIC comparisons ( Figure 5 ) .", "In addition , the path coefficients from diversity and abundance to the decomposition response to stressors had ( standardized ) values higher than 0 . 1 ( Figure", "5 ) and were statistically different from zero ( Appendix 2—table 3 ) .", "However , in contrast to chemical stressors , the SEM did not support biodiversity-mediated effects of nutrient enrichment on litter decomposition .", "While the mediation test and AIC indicated that the decomposer diversity-mediated path improved the model ( Figure 5 ) , the path coefficient was not significantly different from 0 ( Appendix 2—table 3 ) .", "The decomposer abundance-mediated path of nutrients was not supported by the data: an SEM without the direct path from decomposer abundance to decomposition could not be rejected based on the mediation test ( Figure 5 ) , and including this path did not improve the model according to the AIC comparison .", "Besides , we found publication bias in this dataset ( Appendix 2—figure 2 , Appendix 2—table 1 ) , and model check indicated that the residuals of the nutrients-abundance model were non-independent from the fitted values .", "Thus , the results from this model are reported here for comparison purposes only .", "The magnitude of the biodiversity-mediated effects of chemical stressors on decomposition was stronger than that of the direct effects of stressor intensity on decomposition .", "The indirect effect of stressors on decomposition mediated by diversity ( i . e . mathematical product of the standardized paths from stressor intensity to decomposer diversity and from diversity to decomposition Figure", "5 ) was higher than the direct effect of stressors on decomposition , while the abundance-mediated effect of stressors was negligible ( Figure 5 ) .", "In the case of nutrient enrichment , however , decomposition responses were not explained by shifts in decomposer diversity and abundance , and the direct effects of nutrient intensity dominated the total effect ( Figure 5 ) .", "Finally , between-model comparisons ( based on unstandardized path coefficients [Grace , 2006] ) revealed that decomposer diversity was a stronger driver of decomposition response to stressors than decomposer abundance ( unstandardized paths were 0 . 42 and 0 . 24 for diversity and abundance , respectively , Appendix 2—table 3 ) .", "Sensitivity analyses revealed that the results were robust to the inclusion of approximated standard deviations ( Appendix 3—table 1; Appendix 3—table 2 ) , and extreme values of effect sizes ( Appendix 3—table 3; Appendix 3—table 4 ) .", "We found partially different results when using log-response ratios as effect sizes ( Appendix 3—table 5; Appendix 3—table 6 ) , due to lower sample sizes and emergence of extreme values in these datasets .", "In addition , the log-response ratio is probably sensitive to the various metrics of biodiversity , abundance , and decomposition covered by the individual studies that we included , while correlation coefficients better accommodate such discrepancies ( Koricheva et al . , 2013 ) .", "Despite the overall negative effects of stressors on decomposition , negative responses in decomposition were not associated with higher stressor intensity ( Figure 5 , Figure 6 ) .", "This result held for two complementary approaches: multivariate SEM ( Figure", "5 ) that relied on data resampling to account for replicated values of decomposition matching several decomposer responses ( e . g . for different taxa in the same litterbag ) , and meta-regressions ( Figure", "6 ) where data resampling was not necessary ( see Materials and methods ) .", "There was mixed support for a stressor intensity effect on decomposer diversity across the two approaches: decomposer diversity responses decreased with stressor intensity according to the SEM ( Figure 5 ) , but this trend was not significant according to the second level meta-analysis ( Figure 6 ) .", "Similar slopes were obtained both with the SEM relying on data resampling ( the slope of the relationship was −0 . 10 ± 0 . 04 , Appendix 2—table 3 ) and with the meta-regression ( the slope was −0 . 05 ± 0 . 03 ) .", "The differences between the two approaches can be explained by the different data included .", "Decomposer abundance responses were not associated to stressor intensity in both the SEM and meta-regression approaches ( Figure 5 , Figure 6 ) .", "We found different patterns for nutrient enrichment , where decomposition responses decreased with nutrient intensity ( Figure 5 , Figure 6 ) , from positive effects at low intensity to negative effects at higher intensity ( Figure 6 ) .", "A similar pattern was observed for decomposer diversity , where responses decreased with nutrient intensity from positive to neutral to negative responses at high nutrient levels ( Figure 6 ) .", "Nutrient intensity , however , did not explain the responses of decomposer abundance ( Figure 5 , Figure 6 ) , and both positive and negative responses were found at high nutrient levels .", "The meta-analysis further revealed clear discrepancies between the response of animal and microbial ( fungi and bacteria ) decomposers to stressors and nutrients .", "Animal decomposers responded more strongly to chemical stressors than microbial decomposers .", "The mean effects of chemical stressors on animal decomposer diversity and abundance were more negative than that on microbial decomposers , confirmed by Wald type tests of the second-level meta-analyses ( Figure 7 upper panels , Appendix 2—table 4 ) .", "Animal decomposers overall decreased in diversity but increased in abundance in response to nutrient enrichment ( Figure 7 , lower panels ) .", "On the other hand , the mean effects of nutrients on microbial decomposer diversity and abundance had lower magnitudes compared to animals ( Appendix 2—table 4 ) , with confidence intervals overlapping with zero ( Figure 7 lower left panel ) .", "Finally , there was no clear difference between observational and experimental studies ( Figure 7 , Appendix 2—table 4 ) , and between biodiversity responses in terms of taxa richness or of diversity indices ( Appendix 2—table 4 ) ." ], [ "Chemical stressors caused consistent reductions in decomposer diversity and abundance as well as in litter decomposition rates , in line with several previous case studies ( Beketov et al . , 2013; Malaj et al . , 2014 ) and meta-analyses ( Ferreira et al . , 2016; Peters et al . , 2013 ) .", "Adding to the previous knowledge , the present meta-analysis shows that changes in decomposer diversity and abundance explained the decomposition response to stressors , providing evidence for the expectation that shifts in biodiversity mediate the impact of chemical stressors on decomposition .", "We acknowledge that despite the SEM analysis , the approach conducted here remains correlative .", "However , our study builds on a body of experimental and observational evidence that already demonstrated that more diverse and abundant decomposer communities support higher decomposition rates , albeit not under the influence of environmental change ( e . g . García-Palacios et al . , 2013; Handa et al . , 2014 ) .", "We especially complement a previous meta-analysis showing the importance of decomposer diversity for decomposition across experiments manipulating the richness of invertebrate and microbial decomposer communities ( Srivastava et al . , 2009 ) .", "We extend on this and show that non-random biodiversity losses induced by stressors are closely associated with decreases in decomposition across a wide range of studies .", "A recent review pointed out that in naturally assembled terrestrial communities , studies more often found neutral and to a lesser extent positive relationships between decomposer diversity and decomposition ( van der Plas , 2019 ) .", "In that review , communities were not influenced by environmental change drivers , and the vote counting approach used is sensitive to the statistical power of individual studies and could have increased the probability of finding non-significant relationships ( Koricheva et al . , 2013 ) .", "In line with our findings , an experiment mimicking the sequence in which freshwater invertebrate decomposers are lost after disturbances showed that decreasing non-randomly the number of species decreased decomposition rates ( Jonsson et al . , 2002 ) .", "Biodiversity-ecosystem function experiments manipulating biodiversity directly are key to understand the mechanisms involved in this relationship ( Eisenhauer et al . , 2016 ) , especially because they control for the effects of environmental heterogeneity or abundance .", "However , in real-world scenarios , environmental change drivers affect both biodiversity and abundance simultaneously .", "As demonstrated here , this is especially the case for stressors that decrease decomposer diversity and abundance ( Hogsden and Harding , 2012 ) .", "The abundance or biomass of different decomposers is of critical importance for decomposition ( e . g . Bergfur et al . , 2007; Ebeling et al . , 2014; Manning and Cutler , 2018 ) .", "Even at constant richness and community composition , strong decreases in abundance can have important impacts on ecosystem functioning ( Spaak et al . , 2017; but see Dainese et al . , 2019 ) .", "It is beyond the scope of the present meta-analysis to disentangle the effects of biodiversity from the effects of abundance , and we found that both contributed to explain shifts in decomposition in separate analyses .", "It is interesting to note that the few cases where negative effect sizes of stressors on biodiversity were associated with positive effect sizes on decomposition were also cases where decomposer abundance was positively associated with stressors ( Figure 4 ) .", "Although we cannot specifically test this with the present data , it seems that in those particular cases ( Lucisine et al . , 2015 ) , increases in decomposer abundance counteracted the negative effects of decreases in decomposer diversity ( Dornelas et al . , 2019 ) .", "Those results could therefore be in line with the mass-ratio hypothesis ( Grime , 1998; Smith and Knapp , 2003 ) .", "Indeed , an exclusion experiment showed that dominant , small , detritivores can compensate reductions in litter decomposition caused by the removal of large detritivores ( Cárdenas et al . , 2017 ) .", "These concomitant shifts in both diversity and abundance further have important implications for our estimates of diversity responses , as studies mostly reported richness to estimate decomposer diversity , but rarely corrected for the sampling effort ( Gotelli and Colwell , 2001 ) .", "This means that lower abundances rather than a lower number of species per se might have directly caused some of the negative effects on biodiversity reported here ( Chase and Knight , 2013 ) .", "This common caveat in meta-analysis approaches that rely on how individual studies report biodiversity , also applies to the present study , and reinforces the importance of reporting raw data in future studies on the impacts of chemical stressors on biodiversity and ecosystem functioning .", "The effects of changes in decomposer diversity and abundance on decomposition found in the present study might also have channeled changes in community and food-web structure not captured by our biodiversity metrics .", "Changes in keystone species ( Hättenschwiler et al . , 2005 ) , functional diversity ( Cadotte et al . , 2011; Dangles et al . , 2012; Heemsbergen et al . , 2004 ) , vertical diversity ( Gessner et al . , 2010; Melguizo-Ruiz et al . , 2020; Wang and Brose , 2018; Zhao et al . , 2019 ) , or dominance patterns ( Dangles and Malmqvist , 2004 ) might have shifted concomitantly to taxonomic diversity and abundance .", "Moreover , these different components of diversity might act at different timings of decomposition ( Oliveira et al . , 2019 ) .", "Unfortunately , studies rarely reported such measurements together with decomposition .", "For example in our dataset , only seven studies reported evenness .", "Future studies need to explore shifts in decomposer community composition in more detail to better understand what particular aspect of biodiversity is responsible for changes in decomposition rates ( Giling et al . , 2019; Hättenschwiler et al . , 2005 ) .", "In particular , few of the included studies reported comparable functional groups allowing to address the effect of functional diversity across the multiple systems and taxonomic groups addressed by the present analysis .", "Future synthesis work could specifically address the effect of functional diversity , by focusing on a given system type .", "Indeed , there is ample evidence that shifts in functional diversity are crucial for decomposition ( Heemsbergen et al . , 2004 ) , and that facilitative interactions occur primarily between decomposers of contrasting body size ( Dangles et al . , 2012; Tonin et al . , 2018 ) .", "This is especially the case for interactions between animal and microbial decomposers , where fragmentation of litter by detritivores facilitates access for microbial decomposers ( Eisenhauer et al . , 2010; Hättenschwiler et al . , 2005; Yang et al . , 2012 ) .", "Here , we found that invertebrates were more affected by chemical stressors than microbes , across aquatic and terrestrial ecosystems .", "Invertebrate decomposers are particularly sensitive to the impacts of metals and pesticides ( Hogsden and Harding , 2012; Pelosi et al . , 2014; Peters et al . , 2013; Schäfer , 2019 ) .", "Microbial decomposers are known to be sensitive to metals ( Giller et al . , 2009 ) and pesticides as well ( DeLorenzo et al . , 2001 ) .", "Nevertheless , our result is consistent with the general expectation that larger organisms are more sensitive to environmental change due to longer generation time , higher energetic demands and lower population densities ( Hines et al . , 2015; Sheridan and Bickford , 2011; Woodward et al . , 2005; Yvon-Durocher et al . , 2011; Baas and Kooijman , 2015 ) .", "These different sensitivities between groups of decomposers could imply that the biodiversity-mediated effects of stressors on decomposition are more strongly linked to shifts in invertebrates than microbes , as reported in a previous review ( Peters et al . , 2013 ) .", "However , in another meta-analysis focusing on microbial-driven decomposition rates , changes in fungal biomass and richness explained shifts in decomposition under the impacts of chemical stressors , but also of nutrient enrichment ( Lecerf and Chauvet , 2008 ) .", "The impacts of nutrient enrichment on litter decomposition and decomposer diversity were different from those caused by stressors , confirming our expectations .", "These different biodiversity and function responses led to different emergent relationships between decomposer diversity and decomposition compared to stressors .", "We found that nutrients had a variety of effects ranging from positive to negative depending on the taxonomic group ( Figure 7 ) and nutrient intensity ( Figure 6 ) , and resulting in neutral overall mean effects ( Figure 3 ) .", "Previous syntheses also found positive ( Ferreira et al . , 2015 ) as well as inconsistent ( Knorr et al . , 2005 ) responses of decomposition rates to nutrient enrichment in streams .", "The relatively small mean effect of nutrient enrichment on decomposition in the present meta-analysis could be explained by the use of correlation as an effect size , which does not capture potentially non-monotonic responses of decomposition to nutrients ( Woodward et al . , 2012 ) .", "However , we noted that most of the studies included in the present meta-analysis did not individually span nutrient gradients sufficiently large to capture this potential non-monotonous response .", "Taken together , the studies show positive effects on decomposition at low nutrient intensities that shifted toward neutral to negative effects at higher intensities ( Figure 6 ) , which is consistent with previous findings ( Ferreira et al . , 2015; Woodward et al . , 2012 ) .", "Low-nutrient intensities might have enhanced microbial activity and biomass by alleviating resource limitation , resulting in enhanced decomposition .", "At higher intensities , however , negative impacts on invertebrates might have decreased decomposition rates ( Peters et al . , 2013; Woodward et al . , 2012 ) .", "These nutrient intensity patterns contrasted with the results for chemical stressors .", "The overall negative effects of stressors ( Figure 3 ) on decomposition were not explained by stressor intensity levels ( Figure 6 ) , and there was mixed support for a stressor intensity effect on decomposer diversity based on two complementary data analysis approaches ( SEM based on data resampling ( Figure 5 ) vs . second level meta-analysis Figure 6 ) .", "Thus , negative responses to chemical stressors happened across the range of stressor intensity .", "Such contrasting patterns between stressor and nutrient intensity effects may reflect the greater number of stressor types ( different metals , pesticides , mixtures ) covered by individual studies compared to the limited number of nutrients .", "In addition , due to the higher variability of stressor types , we relied on more variable sources to standardize stressor levels compared to nutrients in the diversity dataset ( Materials and methods , Appendix 1—table 1 ) .", "With the data at hand , it was not possible to test the influence of the environmental quality criteria used to standardize stressor and nutrient levels , because such an effect would be confounded with stressor or nutrient types .", "The datasets were all dominated by environmental quality criteria based on similar methodologies ( for 75% to 100% of observations , see Material and Methods ) .", "However , future studies focusing on stressor intensity effects across ecosystems would greatly benefit from coordinated efforts to derive quality criteria encompassing the vast and rapidly increasing number of chemical stressors ( Wang et al . , 2020 ) .", "Contrary to our expectation , nutrient-induced shifts in decomposer diversity and abundance were not associated with shifts in decomposition rates across studies .", "We found that increasing nutrient intensity decreased the effects on decomposition and on decomposer diversity , but not on decomposer abundance .", "Statistically controlling for the effect of nutrient intensity with SEM indicated no residual association between shifts in decomposer diversity or abundance and in decomposition rates , that is a non-significant BEF relationship .", "Changes in microbial abundance in response to nitrogen deposition explained the responses of different ecosystem functions in terrestrial systems in previous meta-analyses ( García-Palacios et al . , 2015; Treseder , 2008 ) .", "Here , we show that this pattern cannot be generalized across aquatic and terrestrial systems and across animal and microbial decomposers .", "Contrary to stressors , when the diversity and abundance of animal and microbial decomposers were not affected by nutrients , we observed large positive and negative shifts in decomposition ( intercepts of Figure 4 ) , that were explained by nutrient intensity ( Figure 4: negative effects on decomposition at invariant biodiversity are associated with high intensities and positive effects with lower intensities ) .", "Together , these results show that nutrient-induced shifts in decomposer diversity were not as strong drivers of decomposition changes as stressor-induced biodiversity shifts .", "These differences may be partly due to the different mechanisms underlying the effects of stressors and nutrients .", "Based on previous studies , we speculate that our results are due to the complex responses of animal and microbial decomposers at different nutrient intensities ( Ferreira et al . , 2015; Lecerf and Chauvet , 2008; Treseder , 2008; Woodward et al . , 2012 ) .", "Animal decomposers showed a stronger response to nutrients than microbes .", "Invertebrate decomposers overall decreased in diversity , but they increased in abundance under nutrient enrichment .", "These results could reflect a loss of sensitive taxa to the benefit of tolerant taxa that were able to use additional resources and would then increase in density ( Bergfur et al . , 2007 ) .", "Overall , microbial decomposers responded little to nutrient enrichment , probably reflecting a mixture of positive and negative effects that nutrients can have on microbial growth ( Lecerf and Chauvet , 2008; Treseder , 2008 ) , as well as on different microbial taxa .", "Indeed , nutrients can alleviate resource limitations at low intensities , but can also exert toxic effects at high intensities .", "The initial levels of nutrients thus condition subsequent responses in decomposers and decomposition to nutrient enrichment ( Ferreira et al . , 2015; Knorr et al . , 2005 ) .", "Furthermore , at high intensities , nutrients can be associated with other chemical stressors ( e . g . pesticides in agricultural runoffs ) ( Ferreira et al . , 2015; Woodward et al . , 2012 ) .", "The influence of interactive effects of stressors and nutrients was impossible to quantify with the data at hand , given that only a few experiments assessed the effects of both drivers independently , but many observational studies may have been confounded by such joint effects .", "Chemical stressors and nutrients are often co-occurring in e . g . agricultural landscapes , and the consequences of such combinations are still poorly understood ( Alexander et al . , 2013; Alexander et al . , 2016; Barmentlo et al . , 2018; Chará-Serna et al . , 2019; Chará-Serna and Richardson , 2018; Fernández et al . , 2016 ) .", "Furthermore , stressor and nutrient effects might be modulated by climatic and other environmental conditions , and studies on interaction effects are scarce ( Rillig et al . , 2019; Thakur et al . , 2018 ) .", "Finally , although our comparison of stressors versus resources allowed us to test a clear concept , any kind of grouping in ecological studies may mask some of the variation within the categories and future studies may be interested in different categories .", "Indeed , a given environmental change driver can represent a stressor for a given species , and a resource for another species ( Connell et al . , 2018 ) .", "As data availability improves , future work could include different environmental change drivers .", "This would also allow to test additional groupings of drivers and ecological concepts unifying stressors and resources ( De Laender , 2018; Harley et al . , 2017 ) .", "This study brings new insights into the real-world patterns relating ecosystem function to non-random changes in biodiversity induced by environmental change .", "We found that the consequences of changes in biodiversity for ecosystem functioning depend on the type of environmental change .", "Real-world scenarios do not necessarily involve concomitant changes in both biodiversity and function across terrestrial and aquatic systems .", "We further found that with the environmental quality criteria used in risk assessment , there were already significant positive and negative effects on decomposers and decomposition ( Figure 6 ) , highlighting the need to better incorporate biodiversity and ecosystem function into ecological risk assessment programs ( De Laender and Janssen , 2013 ) .", "Finally , we report overall negative effects of chemical stressors on biodiversity and ecosystem functioning across terrestrial and aquatic ecosystems that reinforce recent calls to consider chemical stressors as important global change drivers and address their impacts on biodiversity and ecosystems ( Bernhardt et al . , 2017; Mazor et al . , 2018; Steffen et al . , 2015 ) .", "Positive real-world BEF relationships may be particularly significant in cases where environmental changes decrease biodiversity , such as in the case of chemical stressors .", "Such information are crucial if we are to design policy and conservation strategies able to reconcile human development with biodiversity conservation ." ], [ "We searched the Web of Science for studies that addressed the impact of environmental drivers and recorded decomposer community responses and litter decomposition rates .", "The search strategy is fully reported in Supplementary Methods ( Appendix 1 ) .", "The search retrieved 2536 references .", "Abstracts and titles were screened to identify a final set of 61 records that met our inclusion criteria ( PRISMA plot , Appendix 1—figure 1 , and list of included references ( Appendix 4 ) .", "To be included in the meta-analysis , studies had to: When a reference reported different environmental change drivers or geographical areas with a specific reference site for each case , we considered these as individual ( case ) studies ( García-Palacios et al . , 2015 ) .", "We extracted means or sums , standard deviations , and sample sizes of litter decomposition , decomposer diversity , and abundance ( outcomes ) in non-impacted vs . impacted sites ( control-treatment studies ) , or at each site when gradients of chemical stressors or nutrients were investigated ( gradient studies ) .", "When response variables were reported at different time points , we kept only the last time point to capture long-term responses .", "For studies reporting decomposition , decomposer abundance or diversity for several litter types ( e . g . different litter species ) , several groups of organisms ( e . g . functional feeding groups for macroinvertebrates ) , and several diversity metrics ( e . g . Shannon indices and taxon richness ) , we created separate observations within case studies .", "We also extracted chemical stressor or nutrient levels at those sites ( water , soil , or sediment concentrations of chemical stressors or nutrients , or application rate of pesticides or fertilizers ) .", "The study type ( experimental vs . observational ) , taxonomic group ( animal decomposers or microbial decomposers ) and metric of diversity ( taxa richness or diversity indices ( Shannon diversity and evenness ) ) were also recorded .", "We used the online software Webplotdigitizer to extract data from figures ( Rohatgi , 2018 ) .", "We converted standard errors and confidence intervals into standard deviations using the equations in Lajeunesse , 2013 .", "When reported as mass loss , litter decomposition data were transformed into k rates using the exponential decay equation used in Ferreira et al . , 2015 .", "We used z-transformed correlation coefficients as effect sizes in order to cope with the heterogeneity of data and study types ( Koricheva et al . , 2013 ) .", "For control-treatment studies , we first calculated Hedge’s d , and then transformed Hedge’s d into correlation coefficients ( Lajeunesse , 2013 ) .", "For gradient studies ( four or more treatment levels ) , we calculated correlation coefficients between the mean values of abundance , diversity , or decomposition rate and the corresponding chemical stressor or nutrient concentrations .", "When means , standard deviations , or sample sizes were missing , we contacted the authors to retrieve the data .", "When the information could not be retrieved , standard deviations were approximated from the data , using the linear relationship between mean values and standard deviations across our datasets ( Lajeunesse , 2013 ) .", "Given the variability in the different stressors and nutrients combinations in the studies , stressor and nutrient levels were standardized into a common environmental change driver intensity ( E⁢C⁢Di⁢n⁢t⁢e⁢n⁢s⁢i⁢t⁢y ) as follows:E⁢C⁢Di⁢n⁢t⁢e⁢n⁢s⁢i⁢t⁢y=l⁢o⁢g⁢ ( [C⁢o⁢m⁢p⁢o⁢u⁢n⁢di]t⁢r⁢e⁢a⁢t⁢m⁢e⁢n⁢t/[C⁢o⁢m⁢p⁢o⁢u⁢n⁢di]c⁢r⁢i⁢t⁢e⁢r⁢i⁢a ) where [C⁢o⁢m⁢p⁢o⁢u⁢n⁢di]c⁢r⁢i⁢t⁢e⁢r⁢i⁢a were environmental quality criteria set by European or US environmental authorities for the chemical stressor or nutrient considered ( Appendix 1—table 1 ) , and [C⁢o⁢m⁢p⁢o⁢u⁢n⁢di]t⁢r⁢e⁢a⁢t⁢m⁢e⁢n⁢t were the concentrations of the chemical stressor or nutrient at the treatment or impacted sites .", "When multiple stressors or nutrients were reported , we used the standardized intensity of the stressor or nutrient corresponding to the highest standardized intensity for the rest of the analyses .", "We used consistent sources for the environmental quality criteria as much as possible .", "For chemicals , we relied primarily on quality criteria from the European Chemical Agency ( ECHA ) and United States Environmental Protection Agency ( USEPA ) that use standardized procedures across aquatic and terrestrial realms based on ecotoxicological data .", "For nutrients , we relied mostly on European Water Framework Directive ( WFD ) benchmarks .", "Using various sources for those quality criteria was inevitable due to the high number of chemicals and the various way the authors reported stressor or nutrient levels in individual studies .", "When we could not find quality criteria for the stressors or nutrients considered in the studies in our main sources , we relied on the authors’ statements and expert knowledge regarding their stressor or nutrient levels ( e . g . citation for ecotoxicological data , or synthesis studies , or recommended application rates of pesticides [Appendix 1—table 1] ) .", "Despite this , the final datasets were all dominated by similar sources for standardizing stressor and nutrient intensity levels: thresholds from ECHA or USEPA for 80% and 90% of observations in the stressor-diversity and stressor-abundance datasets , respectively , and for nutrients , thresholds from WFD for 100% and 75% of observations in the nutrient-diversity and nutrient-abundance datasets , respectively .", "We first tested the differences between the effects of chemical stressors and nutrient enrichment on decomposer diversity , abundance and litter decomposition responses by quantifying the grand mean effect sizes on the three response variables ( first level meta-analysis ) .", "Three separate meta-analyses were conducted , one for each response variable , and included the type of driver ( stressors or nutrients ) as a categorical moderator , and a random effect of the case study .", "We used a weighted meta-analysis giving more weight to effect sizes derived from studies with larger sample sizes .", "Weights were the inverse of the variance in z-transformed correlation coefficients ( Viechtbauer , 2010 ) .", "Publication bias was evaluated using funnel plots with environmental change driver type as covariate .", "The intercepts from Egger’s regressions ( standardized effect size vs . precision = 1/SE ) were inspected for significant deviation from zero that would indicate publication bias ( Koricheva et al . , 2013 ) .", "Residual plots were used to detect strong deviation from normality and outliers .", "We estimated the grand mean effect sizes and compared the effect of chemical stressors and of nutrients using Wald-type chi-square tests .", "The rma . mv ( ) function of the R package metafor was used ( R Development Core Team , 2018; Viechtbauer , 2010 ) .", "An SEM was fitted to estimate the relationship between decomposer diversity or abundance and litter decomposition responses to environmental change drivers while controlling for the joint influence of stressor or nutrient intensity and categorical covariates .", "We used piecewise SEM ( Lefcheck , 2001 ) estimating two linear mixed effect models , one for decomposition ( zL⁢D ) and one for decomposer diversity or abundance responses ( zB ) , with a random effect of the case study on the intercepts .", "These two sub-models embedded in the piecewise SEM were the second-level meta-analyses in our hierarchical approach .", "The random effect structure , weighting approach and variance structure were coded with the R package nlme ( Pinheiro et al . , 2018 ) in a way that fully reproduced the meta-analysis approach of weighting and of known residual variance ( Viechtbauer , 2017 ) :zLD∼zB+ECDintensity+studytype , random=∼1|Casestudy/IDzB∼ECDintensity+studytype+taxonomicgroup ( +diversitymetric ) , random=∼1|Casestudy∕ID This SEM was tested separately for each of four datasets: Stressors – Biodiversity; Stressors – Abundance; Nutrients – Biodiversity and Nutrients – Abundance datasets .", "The influence of the diversity metric ( diversity indices versus taxa richness ) was tested in the Biodiversity datasets only .", "We initially considered more complex model structures , but were unable to use them for analysis due to data limitations ( in particular the effect of the ecosystem type and of interactions between our covariates ) .", "Outliers , relationships between covariates , and non-linear patterns between continuous covariates were explored graphically .", "Studies often reported different decomposer diversity or abundance values for the same litter decomposition ( e . g . when several taxonomic or functional groups were reported in the same litterbag ) .", "This variability could have affected the model estimates .", "We thus used data resampling to account for duplicated effect sizes on litter decomposition in the analyses .", "A stratified resampling was conducted , where for each duplicated value of effect size on decomposition , one randomly selected effect size on biodiversity was kept at each out of 1000 iterations .", "The models were fitted for each data resampling iteration , and we averaged model estimates and statistics across iterations and used the means as final values ( path coefficients and standard error of the path and intercepts , Chi-square statistics and AICs ) .", "Goodness-of-fit of the SEMs was assessed using directed separation tests based on the Fisher’s C statistic .", "We used mediation tests to explore the significance of the path between decomposer diversity or abundance and litter decomposition based on the Fisher’s C statistic of SEM that did not include the biodiversity-mediated path ( Lefcheck , 2001; Shipley , 2009 ) .", "We calculated the p-value associated with the mean Fisher’s C statistic across data resampling iterations ( p-value<0 . 05 indicated poor model fit ) .", "The AICs of models with and without the biodiversity-mediated paths were further compared using averaged AICs across data resampling iterations .", "We considered the biodiversity ( or abundance ) path to be consistent with the data when the SEM without the biodiversity-path had p-value<0 . 05 ( poor fit ) and was not associated with a better AIC value ( i . e . lower than two units ) than the SEM including the biodiversity path .", "Residuals from the two sub-models of each SEM were graphically evaluated for strong departure to normality and relationship with the fitted values ( Duffy et al . , 2015 ) .", "For these analyses , we averaged the residuals across data resampling iterations for each observation .", "We finally compared the relative magnitude of the biodiversity-mediated path versus the direct path from stressor or nutrient intensity to litter decomposition based on the mathematical product of the standardized path coefficients ( Grace , 2006 ) .", "In order to quantify the influence of the categorical ( study type , taxonomic group and diversity metrics ) and continuous ( environmental change intensity ) moderators on the three response variables , we further analyzed the results of the second-level meta-analyses ( i . e . the sub-models embedded in the SEMs ) .", "The data resampling used in the SEM was no longer necessary , because there were no repeated values of decomposition matching different decomposer diversity or abundance measurements in this univariate approach .", "We quantified the effects of the different moderators based on the Wald-type chi-square tests derived with the R package metafor ( Viechtbauer , 2010 ) .", "We finally tested the robustness of the results to the approximation of standard deviations , the presence of extreme values , and the metric of effect size used .", "The analyses were re-run with datasets that did not include the effect sizes for which we approximated standard deviations , for datasets that did not include extreme values of effect sizes ( values beyond the whiskers of boxplots that is below quantile 1 minus 1 . 5 times the interquartile range or above quantile 3 plus 1 . 5 times the interquartile range ) .", "Finally , we calculated log-response ratios instead of correlation coefficients as effect sizes and re-run the analyses ." ] ]

[ "Understanding the consequences of ongoing biodiversity changes for ecosystems is a pressing challenge .", "Controlled biodiversity-ecosystem function experiments with random biodiversity loss scenarios have demonstrated that more diverse communities usually provide higher levels of ecosystem functioning .", "However , it is not clear if these results predict the ecosystem consequences of environmental changes that cause non-random alterations in biodiversity and community composition .", "We synthesized 69 independent studies reporting 660 observations of the impacts of two pervasive drivers of global change ( chemical stressors and nutrient enrichment ) on animal and microbial decomposer diversity and litter decomposition .", "Using meta-analysis and structural equation modeling , we show that declines in decomposer diversity and abundance explain reduced litter decomposition in response to stressors but not to nutrients .", "While chemical stressors generally reduced biodiversity and ecosystem functioning , detrimental effects of nutrients occurred only at high levels of nutrient inputs .", "Thus , more intense environmental change does not always result in stronger responses , illustrating the complexity of ecosystem consequences of biodiversity change .", "Overall , these findings provide strong evidence that the consequences of observed biodiversity change for ecosystems depend on the kind of environmental change , and are especially significant when human activities decrease biodiversity ." ]

[ "Ecosystems are at their healthiest when they have a high level of biodiversity – that is , a wide variety of different species all living together .", "But human activity is changing the environment and putting ecosystems under pressure .", "One of the places this is most evident is in the communities of organisms responsible for breaking down dead plants .", "These organisms – called decomposers – are highly sensitive to pesticides , metals and other chemical stressors , as well as excess nutrients , such as nitrogen , released by industry and farming .", "Exposing decomposers to these chemicals can change both the number of individuals of each species and the number of different species that are present .", "In other words , these chemicals can , respectively , alter both the abundance and diversity of decomposer communities .", "Controlled experiments in simplified conditions suggest that these changes in biodiversity affect ecosystem health .", "But , it remained unclear to what extent these results applied to real-world scenarios of environmental change .", "To test the findings of controlled experiments , Beaumelle et al . investigated how chemical stressors and excess nutrients affect the breakdown of leaf litter – the debris of decomposing leaves that forms on top of soil .", "Previous studies suggest that the reduced biodiversity caused by chemicals should result in leaf litter breaking down more slowly .", "Whereas the loss in biodiversity caused by nutrients will increase the number of some decomposer species , causing leaf litter to break down faster or slower , depending on local conditions .", "Beaumelle et al . tested these predictions by gathering the results from 69 independent studies conducted across the globe .", "The results showed that stressors caused the diversity and abundance of decomposers to decline , which reduced the breakdown of leaf litter , as expected .", "But , the outcomes of excess nutrients were more varied .", "Low levels of excess nutrients increased the breakdown of leaf litter , but at high levels slowed down the rate leaves decomposed .", "Furthermore , the effect excess nutrients had on biodiversity in decomposer communities changed according to the types of organisms in the ecosystem .", "This suggests that variations in biodiversity can impact ecosystems differently depending on the type of environmental change .", "The breakdown of leaf litter plays a critical role in carbon balance , and this has knock-on effects for the Earth's climate .", "This work suggests that improving biodiversity is crucial to maintain the health of ecosystems , but successful strategies will have to be adjusted depending on the type of human impact ( for example , chemical stressors or nutrient additions ) .", "These findings could help researchers design better approaches for boosting ecosystem health in the future ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "medicine", "neuroscience" ]

[ [ "Myotonia congenita is one of the non-dystrophic muscle channelopathies .", "It is caused by loss-of-function mutations affecting the muscle chloride channel ( ClC-1 ) ( Lipicky et al . , 1971; Steinmeyer et al . , 1991; Koch et al . , 1992 ) .", "Patients with recessive myotonia congenita ( Becker disease ) experience muscle stiffness due to hyperexcitability ( Lehmann-Horn et al . , 2008; Trivedi et al . , 2014; Cannon , 2015 ) as well as transient weakness due to unknown factors ( Ricker et al . , 1978; Rüdel et al . , 1988; Zwarts and van Weerden , 1989; Deymeer et al . , 1998; the CINCH Consortium et al . , 2013 ) .", "Some patients with Becker disease report transient weakness in arm muscles as a greater impediment than muscle stiffness ( Rüdel et al . , 1988 ) .", "This weakness can last up to 90 s and is brought on by exertion following rest ( Ricker et al . , 1978; Rüdel et al . , 1988; Zwarts and van Weerden , 1989; Deymeer et al . , 1998 ) .", "The mechanism underlying transient weakness in Becker disease has remained unknown since its initial description close to 50 years ago ( Ricker and Meinck , 1972 ) .", "There appears to be loss of muscle excitability , as weakness is accompanied by a drop in compound muscle action potential ( CMAP ) amplitude during repetitive stimulation ( Ricker and Meinck , 1972; Brown , 1974; Aminoff et al . , 1977; Deymeer et al . , 1998; Drost et al . , 2001; Modoni et al . , 2011 ) .", "This drop in CMAP is associated with reduction in muscle fiber conduction velocity , which has been proposed to progress to depolarization block ( Zwarts and van Weerden , 1989 ) .", "What has remained unclear , and perhaps counterintuitive , is why a loss-of-function mutation of the muscle ClC-1 channels in myotonia congenita ( Lipicky et al . , 1971; Steinmeyer et al . , 1991; Koch et al . , 1992 ) leads to transient loss of excitability .", "The primary defect caused by loss of ClC-1 current is hyperexcitability of muscle , which causes myotonia .", "We established that a ClC-1 homozygous null ( ClCadr ) mouse model of Becker disease has transient weakness in vivo , mimicking the condition in human patients .", "Intracellular recording in both ClCadr muscle and a pharmacologic model of Becker disease ( due to block of ClC-1 with 9-AC ) have elucidated a novel phenomenon: transient depolarizations to voltages between −25 and −35 mV , lasting many seconds , which we termed ‘plateau potentials . ’ Blocking Na+ persistent inward current ( NaPIC ) with ranolazine prevented both development of plateau potentials and transient weakness .", "We conclude that NaPIC plays a central role in the development of plateau potentials , which are the mechanism underlying transient weakness in Becker disease ." ], [ "To study in vivo isometric motor performance in the Clcn1adr-mto2J ( ClCadr ) mouse model of recessive myotonia congenita ( Becker disease ) , a muscle force preparation that we used previously was employed ( Dupont et al . , 2019; Wang et al . , 2020 ) .", "Mice were anesthetized via isoflurane inhalation and the distal tendon of the triceps surae ( gastrocnemius , plantaris , and soleus muscles ) was dissected free and attached to a force transduction motor; then the sciatic nerve was stimulated with 45 pulses delivered at 100 Hz .", "In unaffected littermates , there was no myotonia following 45 pulses at 100 Hz , such that relaxation was immediate ( Figure 1A ) .", "In ClCadr mice , stimulation with 45 pulses caused full fusion of force , but relaxation was slowed , due to the presence of myotonia ( Figure 1C ) .", "To determine whether transient weakness was present , the sciatic nerve was additionally stimulated with 15 pulses at 100 Hz every 4 s for 1 min .", "In unaffected littermates , this caused stable force production with a mild , gradual reduction that was likely due to fatigue ( Figure 1B ) .", "In myotonic mice , the same stimulation protocol revealed transient weakness , as force fell over the first 10–15 s and then recovered ( Figure 1D ) .", "To avoid inclusion of fatigue in measurement of transient weakness , we normalized to force at the end of the 1 min of intermittent stimulation .", "The plot of the mean normalized force revealed transient weakness , which peaked in severity 15–20 s after the initial stimulation and resolved within 1 min ( Figure 1E ) .", "These data indicate that transient weakness is present in ClCadr mice .", "In myotonic patients , transient weakness is paralleled by a drop in CMAP amplitude ( Ricker and Meinck , 1972; Modoni et al . , 2011 ) .", "This finding suggests that weakness is due to loss of excitability .", "To look for inexcitability of ClCadr muscle , intracellular current clamp recordings were performed .", "In both unaffected littermates and ClCadr mice , stimulation with a 200 ms injection of depolarizing current triggered repetitive firing of action potentials during the stimulus .", "In muscle from unaffected littermates , the firing ceased as soon as the stimulus was terminated ( Figure 2A ) .", "In muscle from ClCadr mice , there was myotonia ( continued firing of action potentials following termination of the stimulus ) in 100% of fibers ( Figure 2B ) .", "The myotonia often persisted for many seconds .", "While most runs of myotonia ended with repolarization to the resting membrane potential ( Figure 2B ) , in some instances , myotonia terminated with the development of depolarizations lasting 5 to >100 s to a membrane potential near −35 mV ( Figure 2C , D ) .", "During these prolonged depolarizations , there was a gradual repolarization of the membrane to near −45 mV , followed by sudden repolarization back to the resting potential .", "In some cases , the sudden repolarization was preceded by the development of oscillations in the membrane potential ( Figure 2D ) .", "The prolonged depolarizations occurred in 30% of ClCadr muscle fibers ( n = 36/119 fibers from 10 mice ) .", "Of the 36 fibers with plateau potentials , 26 repolarized to within 4 mV of their initial resting potential .", "The 10 fibers that did not fully repolarize may have become damaged and thus were not analyzed .", "Depolarizations lasting less than 1 s to a membrane potential close to −60 mV have been described in a toxin-induced model of hyperkalemic periodic paralysis and were termed plateau depolarizations ( Cannon and Corey , 1993b ) .", "While the depolarizations we identified could be due to similar mechanisms as those described in hyperkalemic periodic paralysis , they seemed more similar to prolonged depolarizations in spinal motor neurons , which can last many seconds , and have been termed plateau potentials ( Alaburda et al . , 2002; Heckman and Enoka , 2012; Hounsgaard , 2017 ) .", "We thus chose the term plateau potentials to describe them .", "To determine whether plateau potentials cause inexcitability , ClCadr muscle fibers were stimulated during the plateau phase .", "During and immediately following plateau potentials , action potential generation in response to current injection failed ( Figure 2E ) .", "At later times following repolarization , action potential generation was again possible .", "The inexcitability at earlier times following repolarization is likely due to slow inactivation of Na channels following the many-second depolarization ( Ruff , 1996; Ruff , 1999; Rich and Pinter , 2003 ) .", "These data are consistent with the possibility that plateau potentials and the resultant inexcitability of muscle are the mechanisms underlying transient weakness in myotonia congenita .", "In ClCadr muscle , ClC-1 chloride conductance has been absent throughout development such that plateau potentials could be a compensatory response to muscle hyperexcitability .", "To test this possibility , we acutely blocked ClC-1 chloride channels in muscle from unaffected littermates with 100 µM 9-anthracene carboxylic acid ( 9-AC ) .", "This dose of 9-AC blocks more than 95% of ClC-1 chloride channels in skeletal muscle ( Palade and Barchi , 1977 ) and has been used to model myotonia congenita both in vitro and in vivo ( van Lunteren et al . , 2011; Desaphy et al . , 2013; Desaphy et al . , 2014; Skov et al . , 2015 ) .", "Acute blocking of ClC-1 triggered myotonia and plateau potentials in 92% of fibers ( Figure 3A , 49/53 fibers from eight mice ) .", "Following acute block of ClC-1 channels with 9-AC , all plateau potentials terminated with sudden repolarization to within 4 mV of the previous resting potential ( 49/49 fibers ) .", "These data strongly suggest that the ion channels responsible for development of plateau potentials are present in wild-type skeletal muscle .", "In contrast to the relative consistency in voltage at onset and termination of plateau potentials , the duration in both the ClCadr and 9-AC treated models of myotonia was highly variable ( Figure 3B , Table 1 , variance = 3131 s for ClCadr and 623 s for 9-AC treated muscles ) .", "The reason for the high variance of duration was that the rate of repolarization during the plateau potential varied by more than 100-fold ( Figure 3B , Table 1 ) .", "It was generally not possible to record multiple plateau potentials in individual ClCadr fibers due to the long median duration .", "However , it was possible to record multiple plateau potentials within individual fibers of 9-AC treated muscle , as most lasted only a few seconds .", "Within individual fibers , the slope of repolarization of plateau potentials was less variable such that duration was relatively constant with a mean variance of 0 . 5 s ± 0 . 7 s ( n = 17 9-AC treated fibers in which four or more plateau potentials were recorded ) .", "In both ClCadr muscle and 9-AC treated muscle , plateau potentials did not occur following every run of myotonia ( Figure 3C ) .", "To determine why some runs of myotonia ended in plateau potentials while others did not , we compared the mean voltage at the end of runs of myotonia that produced plateau potentials vs . the mean voltage at the end of runs that did not produce plateau potentials , in 9-AC treated fibers .", "There was a strong correlation between the mean membrane potential during the final 500 ms of runs of myotonia and development of plateau potentials .", "In 22/22 fibers , the mean membrane potential was more depolarized in runs of myotonia that produced plateau potentials ( mean = −40 . 1 ± 3 . 1 mV vs −49 . 0 ± 4 . 5 mV , p=5 × 10−11 , paired t-test , Figure 3D ) .", "This suggested that a voltage-dependent current might be involved .", "However , another feature of myotonia that determines whether a plateau potential is triggered is the firing rate , which might correlate with changes such as build-up of K+ in t-tubules or elevation of intracellular Ca2+ .", "Thus , we examined and found a strong correlation between the firing rate of myotonia runs and subsequent development of plateau potentials: In 21/22 fibers , the mean firing rate was higher for runs of myotonia ending in plateau potentials ( mean = 31 . 2 ± 5 . 7 Hz vs 23 . 4 ± 2 . 9 , p=7 × 10−6 , paired t-test , Figure 3E ) .", "Thus , while voltage-dependent channels appear to be involved , changes in ion concentrations due to differences in firing rates remain a possible contributor .", "Another factor that might determine whether plateau potentials are triggered is the duration of the preceding myotonia .", "However , recordings of multiple plateau potentials in individual 9-AC treated muscle fibers did not reveal a consistent pattern of duration of myotonia prior to entry into plateau potentials ( Figure 4 ) .", "While this does not rule out a contribution of duration of myotonia , it suggests other factors predominate .", "To determine whether development of plateau potentials is due primarily to opening or closing of ion channels , we measured the membrane response to injection of hyperpolarizing or depolarizing square current pulses at baseline and during plateau potentials .", "Injection of 5 nA of either depolarizing or hyperpolarizing current during plateau potentials led to variable responses that early in the plateau potential appeared passive , but prior to repolarization triggered an overshoot of membrane potential after termination of current injection ( Figure 5 ) .", "The presence of an overshoot fits with the propensity of membrane potential to oscillate prior to termination of plateau potentials ( Figure 2D ) .", "Since the response appeared passive during the early phase of plateau potentials we estimated relative input resistance , which was reduced by 50% following initial depolarization versus baseline ( 0 . 43 ± 0 . 05 vs 0 . 90 ± 0 . 12 MΩ , p<0 . 01 , paired t-test , n = 14 fibers ) .", "While caution must be used in interpreting these data , they favor the possibility that there is a net increase in membrane conductance during the early phase of plateau potentials .", "One possible explanation for the overshoot in voltage following termination of current injection during the late phase of plateau potentials is voltage-dependent opening and closing of KV channels .", "The voltage near the end of plateau potentials is near the midpoint of activation of rodent skeletal muscle KV channels ( Beam and Donaldson , 1983 ) .", "With the injection of hyperpolarizing current , KV channels would be caused to close , resulting in a depolarizing overshoot following termination of current injection .", "With the injection of depolarizing current , the converse would happen .", "This may not occur during the initial phase of plateau potentials if KV channels are mostly open or inactivated ( DiFranco et al . , 2012 ) .", "The finding that membrane conductance is increased during the initial phase of plateau potentials suggests opening of either Ca2+ or Na+ channels .", "In spinal motor neurons , L-type Ca2+ channels play a central role in generation of plateau potentials ( Alaburda et al . , 2002; Heckman and Enoka , 2012; Hounsgaard , 2017 ) .", "To determine whether Ca2+ current through skeletal muscle L-type channel ( Cav1 . 1 ) triggers plateau potentials , we performed recordings on skeletal muscle fibers from a mouse model ( ncDHPR ) in which the pore region of Cav1 . 1 carries a point mutation leading to ablation of inward Ca2+ current ( Dayal et al . , 2017 ) .", "We used voltage clamp of FDB/IO fibers to verify the absence of inward currents in ncDHPR mice .", "In wild-type littermate controls , ramp depolarization following block of Na+ , K+ , and Cl− channels triggered a large , inward Ca2+ current , which began to activate at −15 . 1 ± 6 . 9 mV ( n = 4 fibers ) with a mean amplitude of 165 ± 46 nA ( Figure 6A , B ) .", "Ramp depolarization triggered no inward Ca2+ current in ncDHPR muscle fibers ( mean = 0 ± 0 nA , n = 10 fibers; Figure 6C ) .", "To determine whether current flow through Cav1 . 1 contributes to generation of plateau potentials , current clamp recordings were performed .", "Application of 100 µM 9-AC led to the development of plateau potentials in 41/49 fibers from four wild-type mice and in 47/49 fibers from three ncDHPR mice ( Figure 6D and E ) .", "Analysis of the characteristics of plateau potentials suggests that Ca2+ current flow through Cav1 . 1 channels influences the duration of the plateau .", "While there was no difference in the beginning or ending voltages of plateau potentials , the duration was shorter in ncDHPR muscle ( Figure 6D and E , Table 2 ) .", "The cause of the shorter plateau potential was an increase in the rate of repolarization ( Table 2 ) .", "These data suggest that Ca2+ influx through Cav1 . 1 does not play a role in the initiation of plateau potentials , but is involved in sustaining them .", "The Nav1 . 4-mediated Na+ current responsible for the generation of action potentials in skeletal muscle inactivates within ms of depolarization , making it highly unlikely that it contributes to development of plateau potentials .", "However , a Na+ current lacking fast inactivation ( NaPIC ) was found to be involved in the generation of plateau depolarizations in muscle in a toxin model of hyperkalemic periodic paralysis ( Cannon and Corey , 1993b ) .", "We recently determined that NaPIC contributes to repetitive firing occurring during myotonia ( Hawash et al . , 2017; Metzger et al . , 2020 ) .", "NaPIC is present in normal skeletal muscle and likely derives from modal gating in which a small subset of Nav1 . 4 channels reversibly enter a mode lacking fast-inactivation ( Patlak and Ortiz , 1986; Gage et al . , 1989 ) .", "We term the Na+ channels responsible for action potentials ‘fast-inactivating Na+ channels’ and Na+ channels lacking fast inactivation ‘NaPIC’ .", "To determine whether NaPIC might play a role in the generation of plateau potentials , we applied ranolazine to 9-AC treated muscle .", "Ranolazine has been found to preferentially block NaPIC in brain , heart , peripheral nerve , and skeletal muscle ( El-Bizri et al . , 2011; Kahlig et al . , 2014 ) .", "We previously found that ranolazine was effective in eliminating myotonia by blocking NaPIC while sparing enough fast-inactivating Na+ channels to allow for repetitive firing of action potentials triggered by current injection ( Novak et al . , 2015; Hawash et al . , 2017 ) .", "As shown in Figure 7A , when myotonia was triggered by the treatment of muscle with 9-AC , 94% of fibers ( n = 53 fibers from eight mice ) developed plateau potentials in response to a 200 ms injection of depolarizing current .", "Following treatment with 40 µM ranolazine , 0/22 fibers from three mice developed plateau potentials after 200 ms current injection ( Figure 7A , p<0 . 01 vs untreated ) .", "These data were consistent with NaPIC playing a role in the development of plateau potentials .", "However , as myotonia was greatly reduced by ranolazine ( Novak et al . , 2015; Hawash et al . , 2017 ) , it was possible that elimination of plateau potentials was secondary to a reduction in the number of myotonic action potentials .", "We thus changed our stimulation protocol to a 2 s train of 3 ms stimulus pulses delivered at 20 Hz .", "This firing rate and duration of firing mimics the duration and rate of firing during runs of myotonia ( Hawash et al . , 2017 ) .", "For trains of stimuli , the amplitude of 3 ms pulses of current were first adjusted to find the lowest current required to elicit an action potential .", "The current was then increased by 10 nA prior to delivering a train of stimuli .", "2 s of 20 Hz stimulation triggered plateau potentials in 46/55 fibers ( n = 5 mice , Figure 7B ) .", "Out of the 46 fibers with plateau potentials triggered by 20 Hz trains of stimuli , 11 fibers entered plateau potentials with limited ( three or less myotonic APs ) or no myotonia .", "When 40 µM ranolazine was applied , plateau potentials developed in 0/68 fibers ( n = 5 mice , p<0 . 01 vs untreated , Figure 7B ) .", "These data suggest that ranolazine is not eliminating plateau potentials via a secondary effect of prevention of repetitive firing .", "As shown in Figure 3C and D , the mean membrane potential at the end of a run of myotonia correlated with whether myotonia terminated in a plateau potential or with repolarization .", "Thus , we tested if ranolazine prevented plateau potentials by hyperpolarizing the mean membrane potential prior to the development of plateau potentials .", "Untreated fibers with additional action potentials and plateau potentials occurring during the 2 s stimulation ( myotonia ) were excluded from the analysis to ensure that the presence of myotonia or plateau potentials did not account for the difference in mean membrane potential .", "As all untreated fibers initially had plateau potentials or myotonia during repetitive stimulation , the analysis was not possible .", "To address this issue , the warm-up phenomenon was induced using 8 Hz 10 s trains , which lessens myotonia due to slow inactivation of Na channels ( Novak et al . , 2015 ) .", "It must thus be noted that the untreated muscle was not at baseline but had already undergone some Na channel inactivation .", "No induction of warm-up was necessary following treatment with ranolazine , as no fibers had myotonia or plateau potentials .", "In the absence of ranolazine , the mean membrane potential during the 500 ms prior to the plateau potential was −49 . 6 ± 2 . 5 mV ( n = 5 muscles , 28 fibers ) .", "In the presence of 40 µM ranolazine , the mean membrane potential was less depolarized ( −56 . 2 ± 2 . 9 mV , p<0 . 05 vs untreated , n = 5 muscles , Figure 7D ) .", "These data suggest that ranolazine prevents plateau potentials by lessening depolarization of the mean membrane potential during the 20 Hz stimulation .", "There are two contributors to the mean membrane potential during repetitive stimulation: ( 1 ) the membrane potential during action potentials , and ( 2 ) the membrane potential during the interspike interval .", "We analyzed the contribution of each of these to the hyperpolarization caused by ranolazine .", "By the 30th action potential of the 20 Hz stimulation , action potential duration had increased to close to 8 ms . This widening of the spike-form was likely due to failure of the membrane potential to fully repolarize between action potentials , given that depolarization has previously been found to cause widening of action potentials ( Renaud and Light , 1992; Yensen et al . , 2002; Miranda et al . , 2017 ) .", "We examined the mean membrane potential during the 8 ms encompassing the 30th action potential and found a trend ( not statistically significant ) toward lessening of depolarization following treatment with ranolazine ( Figure 7C , E: −28 . 2 ± 3 . 4 vs −32 . 9 ± 3 . 8 mV , p=0 . 07 ) .", "With 20 Hz stimulation , there is an action potential every 50 ms . After taking the mean membrane potential for the 8 ms encompassing the 30th action potential , there remained 42 ms in which there was no action potential .", "The membrane potential for this 42 ms interspike interval before the 30th action potential was less depolarized following treatment with ranolazine ( Figure 7C , F: −56 . 0 ± 2 . 3 vs −61 . 7 ± 3 . 0 mV , p<0 . 05 ) .", "As the interspike interval accounts for 84% ( 42/50ms ) of the time between spikes during 20 Hz stimulation , hyperpolarization of this interval is largely responsible for hyperpolarization of the mean membrane potential .", "The finding that ranolazine eliminates plateau potentials allowed us to explore whether plateau potentials are the mechanism underlying transient weakness in vivo .", "We recorded triceps surae force in 5 ClCadr myotonic mice before and 45 min after intraperitoneal ( i . p . ) injection of 50 mg/kg of ranolazine .", "As shown previously ( Novak et al . , 2015 ) , treatment with ranolazine decreased myotonia such that muscle was able to more rapidly relax following termination of stimulation ( Figure 8A ) .", "In addition to lessening myotonia , treatment with ranolazine eliminated transient weakness in all five mice ( Figure 8A and B , p<0 . 01 vs untreated at 16 s ) .", "The elimination of both plateau potentials and transient weakness by ranolazine supports the hypothesis that plateau potentials are the mechanism underlying transient weakness in vivo ." ], [ "Our data suggest that muscle from a mouse model of Becker disease undergoes a rapid transition between the states of hyperexcitability ( myotonia ) and inexcitability ( due to plateau potentials ) , shown in Figure 9 .", "Repeated firing of action potentials during voluntary contraction triggers myotonia , which often transitions to a depolarization that forms a plateau potential .", "During plateau potentials , fibers cannot generate action potentials in response to stimulation , providing an explanation for the drop in CMAP amplitude reported in patients ( Ricker and Meinck , 1972; Brown , 1974; Aminoff et al . , 1977; Deymeer et al . , 1998; Drost et al . , 2001; Modoni et al . , 2011 ) .", "The reason for the rapid transition between myotonia ( hyperexcitability ) and plateau potentials ( inexcitability ) is that both states are caused by depolarization secondary to loss of muscle Cl- current .", "The difference is one of degree: when depolarization is mild , Na+ channels are not inactivated , such that repetitive firing of action potentials is triggered .", "When depolarization worsens , Na+ channels inactivate , such that inexcitability and paralysis ensue .", "This proposal is similar to the current understanding of hyperkalemic periodic paralysis , in which there is often myotonia at the beginning of attacks ( during the initial , mild depolarization ) and weakness at the height of an attack ( when depolarization is maximal ) ( Cannon , 2015; Statland et al . , 2018 ) .", "Involvement of voltage-gated channels in the generation of plateau potentials is suggested by the strong correlation between membrane potential and the initiation and termination of plateau potentials .", "In runs of myotonia that terminated in a plateau potential , the mean membrane potentials averaged to include both action potentials and interspike intervals prior to development of a plateau potential was −40 mV , whereas , within the same fibers , runs of myotonia terminating with repolarization had a mean membrane potential prior to repolarization of −49 mV .", "The midpoint of the difference between these values is close to −44 mV .", "In ClCadr muscle , the mean membrane potential prior to termination of plateau potentials was −46 mV , and in 9-AC treated muscle it was −45 mV .", "Thus , a mean membrane potential of −44 mV appears to predict both entry into , as well as termination of , plateau potentials .", "Both Cav1 . 1 and Nav1 . 4 are voltage-gated channels that could depolarize muscle to potentials achieved during plateau potentials .", "In spinal motor neurons , CaV1 . 3 channels play a central role in the generation of plateau potentials that last many seconds ( Alaburda et al . , 2002; Heckman and Enoka , 2012; Hounsgaard , 2017 ) .", "Here , we show that the development of plateau potentials was not affected in ncDHPR muscle that lack Ca2+ current through Cav1 . 1 .", "However , there was a reduction in the duration of plateau potentials in ncDHPR muscle .", "These data suggest that Ca2+ flux through Cav1 . 1 channels contributes to sustaining plateau potentials .", "This was a surprise , as the membrane potential during plateau potentials is more negative than voltages at which there is significant current through CaV1 . 1 channels ( García and Beam , 1994; Bannister and Beam , 2013 ) .", "One explanation is that in intact , mature fibers , prolonged depolarization during plateau potentials allows for activation of CaV1 . 1 at more negative potentials than during the shorter step depolarizations typically used for voltage clamp studies .", "Our findings suggest that despite Ca2+ current through CaV1 . 1 having no essential role in healthy muscle ( Dayal et al . , 2017 ) , it may contribute to pathologic depolarization in muscle channelopathies .", "It is not clear at this point whether the primary role of Ca2+ flux through CaV1 . 1 channels in prolonging plateau potentials is a direct effect of increased Ca2+ conductance or whether it is due to effects of Ca2+ accumulation on other ion channels .", "Partial block of Nav1 . 4 channels with ranolazine eliminated plateau potentials , suggesting involvement of NaV1 . 4 .", "However , the majority of Nav1 . 4 channels inactivate within ms ( fast-inactivating NaV1 . 4 channels ) , such that they cannot contribute to prolonged depolarization during plateau potentials .", "There is a NaPIC in skeletal muscle that appears to arise from modal gating whereby , with very low frequency , any NaV1 . 4 channel may enter the non-inactivating mode ( Patlak and Ortiz , 1986; Gage et al . , 1989; Hawash et al . , 2017 ) .", "The subset of NaV1 . 4 channels in the non-activating mode appears to play a role in the development of plateau potentials .", "Ranolazine has been found to preferentially block NaPIC ( El-Bizri et al . , 2011; Kahlig et al . , 2014 ) .", "When myotonic muscle was treated with ranolazine to block NaPIC , depolarization of the mean membrane potential during repetitive firing was decreased and plateau potentials were prevented .", "An important contributor was lessening of depolarization of the membrane potential during the interspike interval .", "Previously , the only identified contributor to depolarization of the interspike membrane potential was K+ build-up in the transverse ( t ) -tubules ( invaginations of the sarcolemma ) , which depolarizes the K+ equilibrium potential ( Adrian and Bryant , 1974; Adrian and Marshall , 1976; Wallinga et al . , 1999; Fraser et al . , 2011 ) .", "The finding that block of NaPIC lessens depolarization of the interspike membrane potential suggests that NaPIC is activated during this interval .", "We hypothesize that activation of NaPIC combines with K+ build-up in t-tubules to depolarize muscle to a mean membrane potential of −44 mV during myotonia , such that a plateau potential is triggered .", "An additional contributor to this depolarization may be the lessening of inward rectifier potassium channel ( Kir ) conductance with depolarization ( Standen and Stanfield , 1980; Struyk and Cannon , 2008 ) .", "It is unclear whether NaPIC , K+ build-up , and decreased Kir conductance can fully account for depolarization during plateau potentials .", "Based on the studies of sustained depolarizations ( sometimes termed plateau potentials ) in neurons , a family of ion channels that might contribute is the transient receptor potential ( TRP ) ion channel family ( Yan et al . , 2009; Phelan et al . , 2012 ) .", "Members of the TRP ion channel family are expressed in skeletal muscle ( Brinkmeier , 2011; Gailly , 2012 ) , and we recently found that activation of TRPV4 plays a role in triggering percussion myotonia ( Dupont et al . , 2020 ) .", "While entry into plateau potentials was often gradual ( Figure 4 ) , it could also occur over two to four action potentials ( ~100–200 ms , Figure 2 , Figure 4 ) .", "Repolarization occurred over 200–300 ms . While we do not know all the channels involved , we hypothesize one contributor to the instances of rapid onset and termination of plateau potentials is activation and deactivation of NaPIC ( Figure 9 ) .", "NaPIC in muscle can activate and deactivate within 10 ms ( Gage et al . , 1989 ) .", "The impression that gating of NaPIC is slow comes from the slow ramp protocols often used to study NaPIC .", "These protocols enable inactivation of fast Na current such that NaPIC can be studied in isolation ( Hawash et al . , 2017 ) .", "Ion channels promoting repolarization are likely also involved in the sudden termination of plateau potentials .", "Oscillations in membrane potential often occurred at the beginning or end of plateau potentials .", "In motor neurons , oscillations in membrane potential are caused by a balance between two voltage-gated ion channels: one promoting depolarization and one promoting hyperpolarization ( Iglesias et al . , 2011; Sciamanna and Wilson , 2011; Nardelli et al . , 2017 ) .", "Kv channels could participate in oscillations occurring during plateau potentials given that Kv conductance in muscle is large and the channels begin to activate at voltages reached during plateau potentials ( Beam and Donaldson , 1983; DiFranco et al . , 2012 ) .", "Thus , it may be possible to prevent plateau potentials by combining partial block of channels promoting depolarization such as NaPIC with partial opening of channels promoting repolarization such as Kv channels .", "As an example of how such an approach might work , plateau potentials in 9AC treated muscle were significantly shorter than in ClCadr muscle .", "Two factors may explain the difference .", "The first is that ClC-1 conductance is not completely blocked following application of 100 µM 9-AC ( Palade and Barchi , 1977 ) .", "While small , the remaining current through ClC-1 channels promotes repolarization and shortens plateau potentials .", "The second is that NaPIC current is larger in ClCadr muscle compared to wild-type muscle ( used for the 9-AC model of myotonia ) ( Hawash et al . , 2017 ) .", "The combination of slightly higher ClC-1 conductance and slightly smaller NaPIC likely combine to shorten plateau potentials in the 9-AC model of myotonia .", "However , despite the shorter duration of plateau potentials in the 9-AC model of myotonia , they were more frequent ( 92% of fibers vs 30% of fibers ) .", "The reason for this difference is not known and raises the possibility that currents not yet identified play a role in development of plateau potentials .", "In a mouse model of myotonia congenita , ranolazine prevents both development of plateau potentials in vitro and transient weakness in vivo .", "In open label trials of ranolazine in both myotonia congenita and paramyotonia congenita , there were statistically significant reductions in the degree of self-reported weakness ( Arnold et al . , 2017; Lorusso et al . , 2019 ) .", "Taken together , these data suggest that the clinical benefit of blocking Na+ channels in some diseases with myotonia may result , in part , from prevention of transient weakness secondary to development of plateau potentials .", "Our data also raises the possibility that blocking of Cav1 . 1 channels might reduce transient weakness by shortening the duration of plateau potentials .", "Since blockers of L-type Ca2+ channels are used clinically to treat hypertension and have few side effects , a trial of their efficacy in reducing transient weakness may be worthwhile .", "Mutations of NaV1 . 4 responsible for hyperkalemic periodic paralysis increase NaPIC ( Cannon et al . , 1991; Cannon and Strittmatter , 1993a ) , which appears to play a central role in triggering the depolarization that underlies attacks of transient weakness ( Lehmann-Horn et al . , 1987; Jurkat-Rott et al . , 2010; Cannon , 2015 ) .", "This suggests that blocking NaPIC should be effective in treating hyperkalemic periodic paralysis .", "However , treating patients with Na+ channel blockers that are effective in treating myotonia , such as mexiletine , was previously found to be ineffective ( Ricker et al . , 1983; Ricker et al . , 1986 ) .", "The finding that blocking NaPIC with ranolazine lessens depolarization of the interspike membrane potential suggests it might be worth considering a trial of this FDA-approved drug’s efficacy in hyperkalemic periodic paralysis .", "We identified currents contributing to plateau potentials that are responsible for transient weakness in recessive myotonia congenita .", "We also determined that blocking a current contributing to plateau potentials provided effective amelioration of transient weakness .", "Currents contributing to plateau potentials are present in wild-type muscle; thus , they might contribute to depolarization in other muscle channelopathies with transient weakness , such as hyper- and hypokalemic periodic paralysis .", "Identification of channels involved in generation of plateau potentials in skeletal muscle may thus advance understanding of regulation of excitability in both healthy and diseased muscle ." ], [ "All animal procedures were performed in accordance with the policies of the Animal Care and Use Committee of Wright State University and were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals .", "The genetic mouse model of myotonia congenita used was Clcn1adr-mto/J ( ClCadr ) mice , which have a homozygous null mutation in the Clcn1 gene ( Jackson Laboratory Stock #000939 ) .", "The pharmacologic model of Becker disease involved treatment of muscle with 100 µM 9-anthracenecarboxylic acid ( 9-AC ) .", "The mouse model of Ca2+ non-conducting CaV1 . 1 used was ncDHPR , carrying a point mutation in the Cacna1S gene coding for N617D in pore loop II ( Dayal et al . , 2017 ) .", "Genotyping of ClCadr mice was performed as previously described to select heterozygous mice for breeding ( Dupont et al . , 2019 ) .", "Otherwise , homozygous myotonic mice were identified by appearance and behavior as previously described ( Novak et al . , 2015 ) .", "Unaffected littermates were used as controls .", "Genotyping for selection of homozygous ncDHPR was performed as previously described ( Dayal et al . , 2017 ) .", "Both male and female mice were used from 2 months to 6 months of age .", "As mice with myotonia have difficulty climbing to reach food , symptomatic mice were supplied with moistened chow paste ( Irradiated Rodent Diet; Harlan Teklad 2918 ) on the floor of the cage .", "Current and voltage clamp recordings were performed at 20–22°C .", "Mice were sacrificed using CO2 inhalation followed by cervical dislocation , and both extensor digitorum longus ( EDL ) muscles were dissected out tendon-to-tendon .", "Muscles were maintained and recorded at 22°C within 6 hr of sacrifice .", "The recording chamber was continuously perfused with Ringer solution containing ( in mM ) NaCl , 118; KCl , 3 . 5; CaCl2 , 1 . 5; MgSO4 , 0 . 7; NaHCO3 , 26 . 2; NaH2PO4 , 1 . 7; glucose , 5 . 5 ( pH 7 . 3–7 . 4 at 20–22°C ) , and equilibrated with 95% O2 and 5% CO2 .", "Intracellular recordings were performed as previously described ( Novak et al . , 2015; Hawash et al . , 2017; Dupont et al . , 2019 ) .", "Briefly , muscles were loaded with 50 μM N-benzyl-p-toluenesulfonamide ( BTS , Tokyo Chemical Industry ) for at least 30 min prior to recording to prevent contraction .", "BTS was dissolved in DMSO and added to the perfusate prior to bubbling with 95% O2 and 5% CO2 , as we have found that BTS is more soluble at a basic pH . Prior to recording , muscles were stained for 3 min with 10 µM 4- ( 4-diethylaminostyrl ) -N-methylpyridinium iodide ( 4-Di-2ASP , Molecular Probes ) to allow imaging muscle with an upright epifluorescence microscope ( Leica DMR , Bannockburn , IL ) .", "Micro-electrodes were filled with 3M KCl solution containing 1 mM sulforhodamine to visualize the electrodes with epifluorescence .", "Resistances were between 15 and 30 MΩ , and capacitance compensation was optimized prior to recording .", "Fibers with resting potentials more depolarized than –74 mV were excluded from analysis .", "In cases where there was failure of action potentials during trains of stimulation , we did not determine whether the failure was due to the presence of an absolute or relative refractory period by altering current injection during the train of stimuli .", "Successful repolarization following a plateau potential was defined as a return to within 4 mV of the resting potential prior to the plateau potential .", "Fibers that did not fully repolarize may have become damaged and were thus discarded from further analysis .", "Flexor digitorum brevis ( FDB ) and interosseous ( IO ) muscle fibers were isolated as previously described ( Waters et al . , 2013; Hawash et al . , 2017 ) .", "Briefly , muscles were surgically removed and enzymatically dissociated at 37°C under mild agitation for ∼1 hr using 1000 U/mL of collagenase type IV ( Worthington Biochemical ) .", "Mechanical dissociation was completed using mild trituration in buffer with no collagenase .", "The fibers were allowed to recover at 20–22°C for 1 hr before being used for electrical measurements .", "Both the current-passing and voltage-sensing electrodes were filled with internal solution ( see below ) and had resistances of ~15 MΩ .", "After impalement , 10 min of hyperpolarizing current injection was allowed for equilibration of the electrode solution .", "Data were acquired at 20 kHz and low-pass filtered with the internal Axoclamp 900A filters at 1 kHz .", "The voltage clamp command signal was low-pass filtered with an external Warner LFP-8 at 1 kHz .", "Internal solution ( in mM ) was as follows: 75 aspartate , 30 EGTA , 15 Ca ( OH ) 2 , 5 MgCl2 , 5 ATP di-Na , five phosphocreatine di-Na , five glutathione , 20 MOPS , and pH 7 . 2 with CsOH .", "Extracellular solution ( in mM ) was as follows: 144 NaCl , 4 CsCl , 1 . 2 CaCl2 , 0 . 6 MgCl2 , five glucose , 1 NaH2PO4 , 10 MOPS , 0 . 05 BaCl2 , 0 . 1 9-AC , 0 . 001 TTX , 0 . 01 Ouabain , 0 . 1 3 , 4-diaminopyridine ( 3 , 4-DAP ) , and pH 7 . 4 with NaOH .", "In vivo muscle force recordings were performed as previously described ( Dupont et al . , 2019; Wang et al . , 2020 ) .", "Briefly , mice were anesthetized via isoflurane inhalation; then the distal tendon of the triceps surae muscles was attached to a force transduction motor and the sciatic nerve was stimulated while isometric muscle force generation was measured .", "The sciatic nerve was stimulated with constant current injection .", "The amplitude of the current pulse was adjusted to 150% of the current required to trigger a single action potential .", "To induce myotonia , 45 pulses of 1 ms duration were delivered at 100 Hz .", "To follow the development of transient weakness , 15 pulses delivered at 100 Hz were delivered every 4 s .", "Muscle temperature was monitored with a laser probe and maintained between 29°C and 31°C with a heat lamp .", "The muscle was kept moist by applying mineral oil .", "Ranolazine was administered via intraperitoneal ( i . p . ) injection at a dose of 50 mg/kg dissolved in water .", "The typical volume of water injected was 100 µl .", "Sample size was determined by past practice , where we have found that an n of 5 muscles from five different mice , studied on different days , yields statistically significant differences in muscle action potential properties .", "At least five muscle fibers were recorded from each muscle .", "However , for studies of plateau potentials in ClCadr mice we obtained recordings with adequate preservation of resting membrane potential in only a subset of fibers , so fewer fibers per muscle were included in the final analysis .", "This was due to the prolonged duration of plateau potentials , which required maintaining prolonged impalement of individual fibers with two electrodes .", "No outlier data points were excluded .", "Intracellular recording data from different mice were analyzed using nested analysis of variance with n as the number of mice , with data presented as mean ± SD .", "p<0 . 05 was considered to be significant .", "The numbers of animals and fibers used are described in the corresponding figure legends and text .", "For parameters that were not normally distributed , such as duration of the plateau potential and slope of the repolarization , differences between two data sets were analyzed after applying a log transformation , which yielded normally distributed data .", "For comparisons of data within individual muscle fibers ( mean membrane potential and mean firing rate for runs of myotonia , without and with plateau potentials ) , the paired Student’s t-test was used with n as the number of fibers .", "For force recording comparisons before and after ranolazine treatment , the paired Student’s t-test was used with n as the number of mice .", "For comparisons of force recordings between myotonic mice and unaffected littermates , a two-sample Student’s t-test was used ." ] ]

[ "In addition to the hallmark muscle stiffness , patients with recessive myotonia congenita ( Becker disease ) experience debilitating bouts of transient weakness that remain poorly understood despite years of study .", "We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness .", "Our recordings reveal transient depolarizations ( plateau potentials ) of the membrane potential to −25 to −35 mV in the genetic and pharmacologic models of Becker disease .", "Both Na+ and Ca2+ currents contribute to plateau potentials .", "Na+ persistent inward current ( NaPIC ) through NaV1 . 4 channels is the key trigger of plateau potentials and current through CaV1 . 1 Ca2+ channels contributes to the duration of the plateau .", "Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo .", "These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita ." ]

[ "Myotonia is a neuromuscular condition that causes problems with the relaxation of muscles following voluntary movements .", "One type of myotonia is Becker disease , also called recessive myotonia congenita .", "This is a genetic condition that causes muscle stiffness as a result of involuntary muscle activity .", "Patients may also suffer transient weakness for a few seconds or as long as several minutes after initiating a movement .", "The cause of these bouts of temporary weakness is still unclear , but there are hints that it could be linked to the muscle losing its excitability , the ability to respond to the stimuli that make it contract .", "However , this is at odds with findings that show that muscles in Becker disease are hyperexcitable .", "Muscle excitability depends on the presence of different concentrations of charged ions ( positively charged sodium , calcium and potassium ions and negatively charged chloride ions ) inside and outside of each muscle cells .", "These different concentrations of ions create an electric potential across the cell membrane , also called the ‘membrane potential’ .", "When a muscle cell gets stimulated , proteins on the cell membrane known as ion channels open .", "This allows the flow of ions between the inside and the outside of the cell , which causes an electrical current that triggers muscle contraction .", "To better understand the causes behind this muscle weakness , Myers et al . used mice that had either been genetically manipulated or given drugs to mimic Becker disease .", "By measuring both muscle force and the electrical currents that drive contraction , Myers et al . found that the mechanism underlying post-movement weakness involved a transient change in the concentrations of positively charged ions inside and outside the cells .", "Further experiments showed that proteins that regulate the passage of both sodium and calcium in and out of the cell – called sodium and calcium channels – contributed to this change in concentration .", "In addition , Myers et al . discovered that using a drug called ranolazine to stop sodium ions from entering the cell eliminated transient weakness in live mice .", "These findings suggest that in Becker disease , muscles cycle rapidly between being hyperexcited or not able to be excited , and that targeting the flow of sodium ions into the cell could be an effective treatment to prevent transient weakness in myotonia congenita .", "This study paves the way towards the development of new therapies to treat Becker disease as well as other muscle ion channel diseases with transient weakness such as periodic paralysis ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "short report", "immunology and inflammation" ]

[ [ "Women live for longer than do men in most modern societies ( Regan and Partridge , 2013 ) but suffer from higher levels of morbidity later in life ( Abad-Díez et al . , 2014; Barnett et al . , 2012 ) .", "Sex differences in health during aging are underpinned by differences in patterns of decline in the structure and function of specific tissues .", "For example , the gut ages differently in men and women , such that many gastrointestinal diseases and cancers are gender-biased ( Chang and Heitkemper , 2002; Kim et al . , 2015; Jemal et al . , 2011 ) .", "However , the mechanisms underlying sex differences in intestinal pathology are not well understood .", "Females of the fruit fly Drosophila melanogaster show substantial gut pathology during aging , which limits female lifespan ( Biteau et al . , 2010; Rera et al . , 2013; Wang et al . , 2014 ) .", "Drosophila are mostly post-mitotic as adults , but the gut contains intestinal stem cells ( ISCs ) ( Micchelli et al . , 2006; Ohlstein et al . , 2006 ) , and their division drives age-related intestinal hyperplasia ( Biteau et al . , 2008; Choi et al . , 2008 ) .", "Epithelial barrier function declines during aging , and its failure is predictive of death ( Rera et al . , 2012; Clark et al . , 2015 ) .", "However , it is not known to what extent males suffer from intestinal pathology during aging; indeed , studies of aging in male Drosophila are generally less common ( Magwere et al . , 2004; Partridge et al . , 1985; Tu et al . , 2002 ) , and little is known about tissue-specific sexual dimorphisms in aging phenotypes ( Boyle et al . , 2007; Camus et al . , 2012; Mackenzie et al . , 2011 ) .", "Interestingly , Drosophila females show a much greater longevity response to dietary restriction ( DR ) than do males ( Magwere et al . , 2004 ) , although the role of the gut in this sex difference has not been investigated .", "We have uncovered substantial sexual dimorphism in the incidence of gut pathology during aging , with females showing widespread deterioration in epithelial structure and loss of gut barrier function , while males generally maintain both even at very late ages .", "However , males succumbed to oral bacterial infections to which females were resistant , with female guts containing a higher number of proliferating cells , suggesting a trade-off between gut plasticity and/or repair mechanisms , and old age pathology in females .", "Gut pathology in aging females was ameliorated by DR , suggesting that the greater response of female lifespan to DR may be a consequence of improved gut function .", "Taking advantage of cell autonomous sex determination in Drosophila , we produced males that had a region of the midgut genetically feminized and we found that this region alone showed the female pattern of aging-related pathology and increase in mitotic stem cells .", "Furthermore , these feminized males also showed a greater increase in lifespan in response to DR , comparable to that seen in females .", "Males demonstrated higher age-related , systemic inflammation compared to females , and sensitivity to oral bacterial infection , indicative of immune dysfunction , despite better maintenance of gut barrier function .", "Intriguingly , this points to further sex bias ( es ) in immunity that could contribute to male mortality ." ], [ "ISC division becomes dysregulated with age , leading to a hyperplastic intestinal pathology .", "ISC over-proliferation and a build-up of undifferentiated enteroblasts ( EBs ) lead to cell crowding and eventual tumor formation ( Biteau et al . , 2008; Choi et al . , 2008; Patel et al . , 2015 ) .", "In order to better understand the effect of these events for intestinal epithelial organization , we subjected guts from outbred , wild type females and males with labeled epithelia ( wDah;Resille-GFP ) to high-resolution imaging over the entire adult lifespan at weekly intervals .", "Several recent studies have shown the Drosophila gut to be highly regionalized in function , cell type and gene expression ( Buchon et al . , 2013; Dutta et al . , 2015; Veenstra et al . , 2008 ) .", "We therefore analyzed four gut regions , identifying them with high fidelity ( Figure 1A ) .", "These were the proventriculus ( PV ) , midgut region 2 ( R2 ) and midgut regions 4–5 ( R4/R5 ) , spanning most of the length and functional diversity of tissues in the adult midgut .", "Young females showed a well-organized PV , with a honeycomb arrangement of tightly packed cells forming the outer wall ( Figure 1B–D ) , arranged in a columnar epithelium with perfectly aligned nuclei ( Figure 1E–G ) .", "In all distal midgut regions , large , polyploid , absorptive , enterocytes ( ECs ) were aligned to form a single layer epithelium with evenly spaced nuclei ( Figure 1H–M and Figure 1—figure supplement 1 ) .", "ISCs were nested at intervals along the basal side of the gut ( Figure 1K–M ) and were mitotically active as visualized by phosphohistone H3 ( PH3 ) immunostaining ( Figure 2J ) . 10 . 7554/eLife . 10956 . 003Figure 1 . Intestinal stem cell activity produces severe epithelial pathology in females .", "( A ) Outline of the adult gut indicating specific regions and areas subjected to image analysis ( orange dashed boxes ) .", "( B-D’ )", "Surface ( B ) and corresponding zoom ( C-D’ ) of the proventriculus ( PV ) from 7-day ( B-D ) and 42-day ( C’ , D’ ) –old females .", "Zoom panels show the green ( epithelium; Resille-GFP ) and blue ( nuclei; DAPI ) channels separately .", "Yellow arrowheads denote wound rosettes ( C’ ) and yellow asterisks denote multinucleated cells ( C’ , D’ ) .", "( E-G’ )", "Central section of the PV ( E ) and corresponding zoom panels ( F-G’ ) in 7-day ( E-G ) and 42-day ( F’ , G’ ) –old females .", "Yellow arrowheads denote extra , tumor-like cells in the epithelium ( I’ ) and yellow asterisks denote their corresponding nuclei ( J’ ) .", "( H-J’ )", "Surface of the gut at R2 ( H ) and zoom panels ( I-J’ ) , at 7days ( H-J ) and 42 days ( I’ , J’ ) .", "Yellow arrowheads denote small , tumor-like cell clusters .", "( K-M’ )", "Luminal section at R2 ( K ) and zoom panels ( L-M’ ) .", "Yellow arrowhead denotes basal ISC ( L , M ) .", "( N-R’ ) pathology in very old ( 70 day-old ) females: PV surface ( N ) and corresponding zoom ( O ) ; R2 surface ( P ) and corresponding zoom ( O’ ) .", "( Q-R’ )", "R4 section and corresponding zoom .", "Zoom panels ( R , R’ ) have had their colors inverted to better visualize tumor nuclei . DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 00310 . 7554/eLife . 10956 . 004Figure 1—figure supplement 1 . Epithelial pathology in females . PV at 21 days , section and surface , an example of intermediate pathology ( cat III ) .", "R4 at 7 days and 42 days for comparison .", "R4 pathologies here include epithelial holes and tumors .", "PV , proventriculus . DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 00410 . 7554/eLife . 10956 . 005Figure 2 . Females have more severe age-related intestinal pathologies than males .", "( A-D )", "Young ( 7 day-old ) male and female flies had comparable epithelial organization in the PV ( A , B ) , but at old age ( 42 days ) only females showed epithelial pathology ( C , D ) .", "For R2 region , see Figure 2—figure supplement 1 .", "( E-H )", "Females raised on a high-yeast diet developed a more severe pathology than males by 42 days ( G , H ) .", "( I ) PV pathologies were binned into scaled categories , where I = WT undisrupted honeycomb , II = loss of regularity in epithelial cell size and pattern; few ( <5 ) rounded unpolarized cells on apical side .", "III = sporadic wound healing rosettes and/or 5–10 apical cells; IV = widespread rosettes and/or >10 apical cells; V = severe pathology including holes , scars and tumors .", "Low yeast females tended to have less pathology than high yeast females ( n=12 guts per condition , ordinal logistic regression , OLR; p=0 . 07 ) ( J-L ) Female flies had more actively dividing ISCs than males , visualized by anti-pH3+ immunostaining .", "Images from R5 in 35-day-old flies are presented ( J , K ) ; quantification of pH3+ cells per gut demonstrated that females had more mitoses than males at 35 days ( n=20 guts per sex; student’s t test , p < 0 . 001 ) ( L ) .", "( M-N )", "Barrier function was compromised in old females but not males .", "‘Smurf’ flies with leaky intestines ( M ) were present in female , but not male cohorts of wD;Resille flies at 42 days ( n≥150 flies per condition , representative of three repeated experiments , Fisher’s exact , p = 0 . 008 ) ( N ) .", "( O-P )", "Males succumbed to oral infection with the gram-negative bacterium Erwinia carotovora ( Ecc ) at 35 days , whereas females were resistant ( O ) .", "PH3+ cell number per gut was increased in females ( n≥10 per condition; student’s t test , p = 0 . 0042 ) and males ( p = 0 . 0003 ) upon Ecc oral infection .", "More mitoses were induced in female compared to male guts ( p = 3 . 6E-05 ) ( P ) .", "( Q-T )", "AMPs and ROS were higher in challenged and unchallenged males compared to females .", "Diptericin expression was higher in both sham- and Ecc-infected males , compared to females at 35 days ( n≥3 samples per condition , 10 individuals pooled per sample , 2 technical repeats; t test with Welch’s correction , p = 0 . 0135 for sham , p = 0 . 0012 for infected ) and was upregulated significantly upon infection in males ( p = 0 . 0132 ) , and tended to be higher after infection in females ( p = 0 . 0571 ) ( Q ) .", "Duox expression was not upregulated after infection in 35-day-old males or females ( n≥3 samples per condition , 10 individuals pooled per sample , 2 technical repeats; t test with Welch’s correction , p = 0 . 8639 for females , p = 0 . 2303 for males ) , but was higher in males than females overall ( p = 0 . 0060 for sham , p= 0 . 0793 for infected ) ( R ) .", "Systemic diptericin was higher in males than females and increased with age in males ( n≥3 samples per condition , 10 individuals pooled per sample , 2 technical repeats; t test with Welch’s correction , p = 0 . 0062 for 7-day-old females vs males; p = 0 . 0158 for 42-day-old females vs males; p = 0 . 2435 for 7-day-old vs 42-day-old females; p = 0 . 0003 for 7-day-old vs 42-day-old males ) ( S ) .", "Duox expression did not increase with aging in either sex , but expression was higher in males than females at both 7 and 42 days ( n≥3 samples per condition , 10 individuals pooled per sample , 2 technical repeats; t test with Welch’s correction , p = 0 . 0029 for 7-day-old females vs males; p = 0 . 0206 for 42-day-old females vs males; p = 0 . 4531 for 7-day-old vs 42-day-old females; p = 0 . 4857 for 7-day-old vs 42-day-old males ( T ) .", "Males had a lower aerobic bacterial load than females at 21 days , ( n≥8 samples per condition , 5 individuals pooled per sample; Wilcoxon test , p = 0 . 05 ) ( U ) .", "A similar result was obtained for anaerobic load . DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 00510 . 7554/eLife . 10956 . 006Figure 2—figure supplement 1 . Females have more severe age-related intestinal pathologies than males . Examples of the R2 region in females and males on low- and high- yeast diets at 7 days and 35 days for comparison .", "Both surface and luminal section images are presented .", "In females at 35 days , groups of stem cells can be seen on the surface which are associated with multiple layered nuclei in luminal sections .", "Females on high-yeast food often presented a more severe pathology than those on low-yeast diets . DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 00610 . 7554/eLife . 10956 . 007Figure 2—figure supplement 2 . Sex differences in pathology , barrier dysfunction and response to intestinal challenge .", "( A ) Pathology at 64 days in wD;Resille males and females .", "Categories are described in Figure 2 legend .", "Males show significantly less pathology than females in all regions ( n≥16 per group; ordinal logistical regression analysis , p<0 . 00000001 for PV , p<0 . 01 for R2 , p<0 . 001 for R4 ) .", "( B-C )", "Analysis of barrier dysfuction; at 80 days in wDah did not identify Smurf phenotype in males ( n≥80 per group , Fisher’s exact , p<0 . 01 ) ( B ) ; at 42 days in w1118 identified the Smurf phenotype in a small proportion of males , but significantly more females ( n≥200 per group , Fisher’s exact , p<0 . 01 ) ( C ) .", "( D ) Lifespan analysis of female ( red ) and male ( blue ) wDah flies on two different yeast dilutions .", "Females live longer than males on both diets ( mean lifespans: female 1SYA = 68 . 2 , male 1SYA = 51 . 6 , female 2SYA = 63 . 7 , male 2SYA = 50 . 7 . Log rank; 1SYA , p=1 . 3E-26; 2SYA , p=1 . 34–20 ) .", "Females live longer on 1SYA than 2SYA ( Log rank , p=0 . 0088 ) , but male lifespan is not extended on 1SYA compared to 2SYA ( p=0 . 37 ) ( E ) 30-day-old males and females were exposed to DDT by feeding for 18 hr and total deaths were scored .", "By 18 hr , the majority of males had died ( 83%; red ) .", "Compare to females where most survived ( 2 . 7% dead; Fisher’s exact , p<0 . 0001 ) ( G ) .", "PV , proventriculusDOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 007 In aging females , disruptions to the PV epithelium began to appear at between 2 and 3 weeks of age , with rounded , unpolarized cells clustering apically ( Figure 1F’-G’ ) .", "This abnormality was progressive ( Figure 1—figure supplement 1 ) , and eventually led to tumor formation .", "The effect of this hyperplasia on the basal side of the PV epithelium was marked; cells were pulled away from the basal edge due to tumor formation or apoptosis , leading to the appearance of rosettes characteristic of epithelial wound healing ( Figure 1C’ and Figure 1—figure supplement 1 ) .", "These wounds became more numerous until , at very late ages , the epithelium did not heal properly , resulting in the formation of large scars , holes and multinucleated cells ( Figure 1C’-D’ , N-O and Figure 1—figure supplement 1 ) .", "R2 and R4 regions also showed dramatic aging phenotypes driven by an accumulation of small nuclei cells on the basal side - ISCs and EBs - which disrupted the single layer and eventually led to tumor formation and loss of epithelial organization ( Figure 1I’ , J’ , L’ , M’ , O’-R’ and Figure 1—figure supplement 1 ) .", "Strikingly , aged males showed only a low incidence of pathology in both the PV and midgut ( Figure 2A–H , Figure 2—figure supplement 1 ) .", "Indeed , many males in the oldest cohorts examined ( up to 64 days post-eclosion ) had well-maintained intestinal epithelia ( Figure 2—figure supplement 2A ) .", "For example , outer wall cells of the PV were maintained as a single layer columnar epithelium with well-aligned nuclei ( Figure 2B , D , F , H ) .", "The R2 midgut region retained regular spacing of ECs with few ISCs nested between them , similar to young guts ( Figure 2—figure supplement 1 ) .", "Aged males had far fewer PH3-positive cells in the midgut when compared to females ( Figure 2J–L ) , suggesting that ISCs were largely quiescent .", "To determine whether this sex difference in ISC activity and epithelial integrity was reflected in barrier function , we fed aging wDah females and males a blue dye that normally does not traverse the gut ( Rera et al . , 2012 ) , at 42 days and 80 days , and scored flies that leached dye into the body cavity ( ‘Smurfs’ ) .", "A low , but consistent , proportion of Smurfs was found in old females , while males never produced Smurfs , even in the oldest ( 80 day-old ) cohorts ( Figure 2M–N , Figure 2—figure supplement 2B ) .", "This is striking given the maximum lifespan for males ( final surviving 10% of the population ) is 64 days ( Figure 2—figure supplement 2D ) .", "A recent study , describing the Smurf technique , found that the inbred laboratory strain w1118 does produce male smurfs , but at a lower rate than females ( Rera et al . , 2012 ) .", "We confirmed this , finding a small proportion of w1118 male smurfs ( <2% ) at 42 days ( Figure 2—figure supplement 2C ) .", "While dysregulated ISC division may be detrimental at older ages ( e . g . Biteau et al . , 2010 ) , ISC responsiveness to gut damage maintains intestinal homeostasis and promotes survival in young females ( Buchon et al . , 2009; Chatterjee et al . , 2009 ) .", "We hypothesized that males , which have fewer ISCs and with a lower basal rate of division ( Jiang et al . , 2009; this study ) , would be more susceptible to intestinal stress such as oral infection , compared to females .", "The bacterium Erwinia carotovora ( Ecc ) induces antimicrobial peptide ( AMP ) expression and ISC division in the midgut when ingested ( Basset et al . , 2000; Ayyaz et al . , 2015 ) but is not usually lethal to healthy adult females ( Ha et al . , 2005 ) .", "When we challenged aged females and males with oral infection by Ecc , we found that females were resistant to the infection .", "However , a high proportion of males succumbed after around 72 hr ( Figure 2O ) .", "Upon analysis of the ISC response to Ecc infection , we found that infected female midguts had a significantly higher number of actively dividing ( PH3+ ) cells at 18-hr post-infection compared to controls .", "Although male midguts also responded to infection , the overall number of mitotic cells was significantly lower than in infected females ( Figure 2P ) .", "In addition , when we exposed flies to a xenobiotic agent in their food ( DDT; Slack et al , 2011 ) , female flies were significantly more resistant to this challenge ( Figure 2—figure supplement 2E ) .", "These findings suggest that , despite the patent hyperplastic pathology , older females are more resilient to intestinal challenge than are males .", "This sex bias may , in part , be promoted by higher numbers of dividing ISCs in females , despite this being detrimental to the maintenance of epithelial integrity at older ages .", "Although repair of the epithelium is an important trait for survival during intestinal stress , other factors will also contribute .", "We therefore assessed the systemic response to Ecc oral infection in aged males and females .", "Diptericin ( dipt , an antimicrobial peptide [AMP] responsive to gram-negative infection sensed by the Imd pathway ) , was expressed at higher levels in unchallenged males compared to females , and was strongly upregulated upon infection in males ( Figure 2Q ) .", "This high systemic dipt did not induce a high survival rate for males , many of which later succumbed to the infection , and may instead be indicative of an infection not effectively contained by the gut and/or a sepsis-like response .", "To better understand inflammatory status during aging in females and males , we measured expression of dipt during aging .", "Only males showed increased expression of dipt as they aged and , at both ages , males expressed dipt at a significantly higher level than did females ( Figure 2S ) .", "We also analyzed expression of the ROS-producer dual oxidase ( duox ) at young and old ages .", "Duox is expressed in immune-active epithelia , especially the gut , and is necessary for survival to foodborne pathogens ( Ha et al . , 2005 ) .", "Duox levels did not change significantly during aging , but males had higher expression than did females at both ages ( Figure 2T ) .", "This suggests that , even in the absence of acute infection , males suffer from a higher level of systemic inflammation than do females , despite better maintenance of gut barrier function .", "The sex differences in gut pathology and systemic inflammation could affect the bacterial load in the intestinal microbiota , which could in turn affect pathology and inflammation .", "We therefore analysed internal bacterial load in females and males at 21 days , and found that females had a higher load than did males ( Figure 2U ) .", "Higher bacterial load in the gut correlated with the higher levels of dysfunction and pathology observed in females compared to males ( Rera et al . , 2012; Broderick et al . , 2014; Clark et al . , 2015 ) , and suggests that factors apart from total load , such as specific composition of the microbiota ( Broderick et al . , 2014; Clark et al . , 2015 ) , or infection through other routes ( Gendrin et al . , 2009 ) , may explain the increased systemic dipt in males .", "ISC division is regulated by both diet and nutrient sensing IIS ( Choi et al . , 2011; Biteau et al . , 2010; O’Brien et al . , 2011 ) .", "DR of dietary protein can extend lifespan in a wide range of animals ( Lee et al . , 2008; Skorupa et al . , 2008; Fontana and Partridge , 2015; Solon-Biet et al . , 2015 ) .", "When we analyzed the guts of flies fed on two different yeast dilutions , we found that pathology was lower in old females that had been exposed to the low-yeast diet ( Figure 2C , G , I; Figure 2—figure supplement 1 ) , in line with studies showing that ISC division ( Choi et al . , 2011; O’Brien et al . , 2011 ) and intestinal barrier dysfunction ( Rera et al . , 2012 ) are reduced in females on restricted diets .", "Male flies , however , derived no obvious benefit to intestinal morphology from DR , largely because very little pathology was evident in males , even on a high-yeast diet ( Figure 2D , H , I; Figure 2—figure supplement 1 ) .", "Sex determination in fruit flies is cell autonomous , with X-chromosome counting leading to a cascade of splicing events and expression of sex-specific transcription factors ( Salz et al . , 2011 ) .", "We exploited this cell autonomy , by mis-expressing the female-specific spliceform of transformer ( UAS-traF ) in male flies , using the midgut driver NP1-Gal4 ( Zaidman-Rémy et al . , 2006 ) , to achieve midgut-specific feminization .", "We then analyzed the effect on intestinal pathology during aging .", "Cell size is larger in female flies than in males , in all tissues ( Alpatov et al . , 1930; French et al . , 1998 ) .", "Accordingly , males with feminized midguts had larger ECs compared to control males ( Figure 3—figure supplement 1A ) .", "Quantitative PCR on dissected guts demonstrated that doublesex ( dsxF ) , the direct downstream target of traF ( Lee et al . , 2002 ) , was expressed in feminized male guts at high levels ( Figure 3—figure supplement 1B ) .", "As further proof-of-principle for sex reversal , we expressed UAS-traFin whole flies using the ubiquitous driver da-Gal4 , producing transgendered males that were feminized in all sexually dimorphic structures ( Figure 3—figure supplement 1C ) . 10 . 7554/eLife . 10956 . 008Figure 3 . Feminized male guts develop female-like intestinal pathologies .", "( A-G )", "Mis-expression of traF feminizes male midguts .", "( A-F” )", "PV ( A-B” ) and R2 ( C-F” ) morphology in +/traF females ( A-F ) , +/traF males ( A’-F’ ) and NP1>traF males ( A”-F” ) at 7 and 35 days , reveal female-like pathology in the R2 region of NP1>traF males at 35 days ( F” ) .", "The NP1 driver is not expressed in the majority of the PV and accordingly , the PV is well-maintained at 35 days ( B” ) .", "Control females and feminized males increased ISC proliferation over age , but control males did not ( n=10–20 guts per condition , student’s t test , p=0 . 0366 for 3 vs 35 day-old +/traFfemales , p=0 . 0015 for 3 vs 35 day-old NP1>traF females , p=0 . 7057 for 3 d vs 35 d +/traFmales , p=0 . 00022 for 3 vs 35 day-old NP1>traF feminized males ) .", "Feminized male guts ( NP1>traF ) had more mitoses at 35 days than control ( +/traF ) male guts ( p=0 . 00018 ) ( G ) .", "( H-J ) barrier dysfunction and systemic AMP expression were increased in feminized males .", "Barrier dysfunction was significantly higher in feminized males than control ( +/traF ) males at 42 days ( n≥150 per group , Fisher’s exact , p=0 . 0001 ) and control ( +/traF ) females ( p=0 . 0002 ) ( H ) .", "Diptericin expression was increased over aging in all genotypes ( n≥3 samples per condition , 10 individuals pooled per sample , 2 technical repeats; 2-way ANOVA , age p=0 . 0487 , condition p=0 . 1031 , interaction p=0 . 3485 ) and was increased in feminized males relative to control males at 7 days only ( t test with Welch’s correction , p=0 . 0018 for NP1>traFvs +/NP1 at 7 days; p=0 . 5152 for NP1>traFvs +/NP1 at 42 days; p=0 . 0011 for NP1>traFvs +/traF at 7 days; p=0 . 8907 for NP1>traFvs +/traF at 42 days ) ( I ) .", "Doux expression did not increase over aging in any genotype , but was higher in males than females overall ( J ) .", "( K ) Aerobic bacterial load tended to increase between 7 and 21 days for both sexes and genotypes ( n≥8 samples per condition , 5 individuals pooled per sample; Monte Carlo Markov Chain Generalised Linear Model with Poisson Error Family , where pMCMC=0 . 040 for males and pMCMC=0 . 064 for females ) .", "Feminized males had a significantly higher load than control males ( pMCMC<0 . 001 ) .", "In addition , the direction of bias compared to females was switched in feminized males , such that control males had lower load than females , but feminized males had a higher load .", "A similar result was obtained for anaerobic load .", "( L-N )", "Pathologies in feminized males are responsive to diet and rapamycin treatment .", "Pathologies were binned into scaled categories and quantified , n≥12 per condition .", "PV categories as described in Figure 2 legend ( see Figure 3—figure supplement 2 for PV scoring ) .", "R2 and R4 categories were defined as follows: I = WT , single layer epithelium with low number of basal ISCs .", "II = sporadic pathology of small nuclei ‘nests’ without significant disruption to the epithelium; III = widespread pathology , majority of epithelium has several layers of nuclei; IV = widespread pathology plus clear tumor formation .", "Gut feminized males have significantly worse pathology than control males on both diets in R2 ( OLR , low-yeast , z=-3 . 916 , p=0 . 0000899; high-yeast z=-4 . 339 , p=0 . 0000143 ) and R4 ( low-yeast , z=-4 . 012 , p=0 . 0000602; high-yeast z=-4 . 520 , p=0 . 0000617 ) .", "The incidence of severe pathology and tumors ( cat IV ) in R2 was greater in feminized males than control females on high yeast diet ( p=0 . 04 ) but not low yeast diet ( p=0 . 48 ) , suggesting that there was a cost of feminization that was partly alleviated by DR ( L-M ) .", "Rapamycin treatment decreased mitoses in females and feminized males at 16 days ( n≥10 guts per condition , students t test; control ( +/traF ) females , p=0 . 0079; control ( +/traF ) males , p=0 . 1; control females ( NP1/traF ) , p=0 . 22; feminized males ( NP1/traF ) , p=0 . 0001 ) .", "( N ) O-R Feminized males were more sensitive to oral infection , but acquired a lifespan response to dietary restriction .", "At 42 days males succumbed to Ecc oral infection while females did not .", "Feminized males died significantly sooner than controls ( O ) .", "After Ecc oral infection at 7 days , males and females of all genotypes increased gut mitoses compared to sham infected ( n≥10 guts per condition , students t test; control ( +/NP1 ) females , p=2 . 082E-06; control ( +/NP1 ) males , p=0 . 0011; control females ( NP1/traF ) , p=0 . 00017; feminized males ( NP1/traF ) , p=0 . 00045 ) .", "However , females and feminized males lost the response to infection against a background of high proliferation in unchallenged individuals at 42 days ( n≥10 guts per condition , students t test; control ( +/NP1 ) females , p=0 . 2; control ( +/NP1 ) males , p=0 . 0088; control females ( NP1/traF ) , p=0 . 1478; feminized males ( NP1/traF ) , p=0 . 2344 ) ( P ) .", "Systemic dipt expression was increased after 18 hr continuous infection in all genotypes at 42 days ( n≥10 guts per condition , t test with Welch’s correction; +/NP1 females , p=0 . 0571; +/NP1 males , p=0 . 0132; +/traF females , p=0 . 0376; +/traF males , p=0 . 0282; NP1/traF females , p=0 . 0110; NP1/traF feminized males p=0 . 0331 ) , but at a higher level in males than females in both sham and infected conditions ( sham: +/NP1 females vs males , p=0 . 0135; +/traF females vs males , p=0 . 0428; NP1/traF females vs males , p=0 . 0022 . Infected: NP1/+ females vs males , p=0 . 0012; +/traF females vs males , p=0 . 0964; NP1/traF females vs males , p=0 . 0237 . ) ( Q ) .", "Lifespan analysis of NP1>traF males and +/NP1 control males and females on two yeast dilutions .", "NP1>traF males were significantly shorter lived than control males on both standard ( low yeast; log rank , p=0 . 0023 ) and double ( high yeast; log rank , p=2 . 06E-11 ) yeast dilutions , whereas +/NP1 control males did not differ between food conditions ( log rank , p=0 . 34 ) .", "This is a representative lifespan of three with similar outcomes .", "Cox proportional hazards analysis of the lifespan demonstrated a significantly increased risk of dying on high-yeast vs low-yeast food overall ( p=2 x 10–16 ) , and a significant difference in the response to food between control male genotypes and NP1>traF ( gut feminized ) males ( p=0 . 0298 ) .", "For full analysis , see Figure 3—source data 1 .", "PV , proventriculus . DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 00810 . 7554/eLife . 10956 . 009Figure 3—source data 1 . Output table for Cox Proportional Hazards analysis of the NP1>traF ( feminized gut ) lifespan ( Figure 3Q ) , showing hazard ratios , z and p values , and significance for all interactions . DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 00910 . 7554/eLife . 10956 . 010Figure 3—figure supplement 1 . Feminization by misexpression of traF .", "( A-C )", "Misexpression of traF feminized males .", "Enterocyte cell size was greater in control females ( wD;+/UAS-traF , Resille-GFP; traF/+ ) than control males ( student’s t test , p=0 . 003 ) , and was increased in 7-day-old feminized male ( wD;NP1-Gal4/UAS-traF , Resille-GFP; NP1>traF ) guts , compared to control male guts ( student’s t test , p=0 . 005 ) ( A ) .", "The female-specific form of doublesex ( dsxF ) , the direct downstream target of traF , was expressed at higher levels in the midguts of NP1>traF compared to control traF/+ male guts ( student’s t test , p=0 . 037 ) by quantitative PCR .", "Expression in NP1>traF feminized males was approx .", "Two fold higher than in control traF/+ females ( student’s t test , p=0 . 05 ) and NP1>traF females ( student’s t test , p=0 . 04 ) .", "NB traF/+ and NP1>traF females expressed comparable levels of dsxF ( student’s t test , p=0 . 263 ) , suggesting feedback regulation of dsxF expression in females ( B ) .", "Males flies were feminized in sexually dimorphic structures by ubiquitous mis-expression of the female-specific isoform of transformer ( UAS-traF ) , using the daughterless-Gal4 ( da-Gal4 ) driver .", "Feminized males ( da/traF ) partially regained female stripe pattern on the posterior dorsal cuticle , had feminized genitalia and loss ( red asterisk ) of sex combs ( red arrow ) ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 01010 . 7554/eLife . 10956 . 011Figure 3—figure supplement 2 . Feminized males increase mitoses but do not resist oral Ecc infection .", "( A ) The driver NP1-Gal4 was not expressed at high levels in the PV .", "Accordingly , pathologies did not appear in the PV in NP1>traF males , where control females developed worse pathologies than feminized males ( z=-2 . 182 , p=0 . 0291 ) .", "( B ) Analysis of midgut mitoses .", "pH3+ cell number was increased in NP1>traF midguts at 35 days , compared to control traF/+ males ( student’s t test , p=1 . 52E-05 ) .", "There was a higher number of mitoses in high-yeast traF/+ females ( student’s t test , p=0 . 033 ) compared to low yeast .", "NP1>traF males did not increase mitoses on high yeast ( means; low yeast = 18 . 7 , high yeast = 20 . 7; p=0 . 6 ) .", "( C ) Control males and females ( NP1/+ and traF/+ ) do not differ in mitoses per gut at 35 days ( n=10 , students t test , p=0 . 956 for females and p=0 . 601 for males ) .", "( D-E )", "Survival to oral infection at 7 days ( D ) .", "Survival to oral infection at 42 days showing all controls ( E ) ( F-G ) Average feeding on normal and infected food .", "Feminized males ( NP1>traF ) do not feed differently from control males on normal food ( n=8 groups of five individuals , students t test , NP1>traF males vs control males: NP1/+ 1SYA ( low yeast ) p=0 . 352 , 2SYA ( high yeast ) p=1; traF/+ 1SYA p=0 . 272 , 2SYA p=0 . 852 ) ( F ) .", "No significant differences between sexes , or between sham and infected ( with students t test ) ( G ) .", "( H ) Full NP1>traF lifespan with all controls ( see Figure 3 legend and Figure 3—source data 1 for statistical analyses ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10956 . 011 We analyzed intestinal aging in gut-feminized males ( wD;NP1-Gal4/UAS-traF , Resille-GFP; NP1>traF ) and found that age-related pathologies were apparent at 35 days post-eclosion , whereas male control flies ( wD;+/UAS-traF , Resille-GFP; +/traF ) generally had well-maintained guts at this age .", "Pathology in the feminized guts was comparable to that seen in females , or more severe ( Figure 3A–F” ) .", "The NP1-Gal4 driver is expressed throughout the midgut from R1 , but only in a small number of cells in the PV ( Zaidman-Rémy et al . , 2006 ) , serving as a useful internal control .", "Accordingly , the PV in NP1>traF males did not develop age-related pathologies ( Figure 3A–B” , Figure 3—figure supplement 2A ) .", "PH3-positive cell number was dramatically increased in aged NP1>traF males compared to control males , as predicted by the observed pathologies ( Figure 3G ) .", "When we analyzed barrier dysfunction in these flies , we found that at 42 days , gut-feminized males produced significantly more flies with a Smurf phenotype than control males ( who produced none ) and control females ( Figure 3H ) .", "Feminized males presented a high level of systemic dipt expression ( Figure 3I ) and Duox expression ( Figure 3J ) at young and old ages .", "In addition , when we analyzed aerobic and anaerobic bacterial load at 7 and 21 days , we found that load increased with age as previously reported ( e . g . Broderick et al . , 2014; Clark et al . , 2015 ) .", "Importantly , while load in control females was higher than in males , this trend was switched in feminized males , who had a higher load than did females of the same genotype ( Figure 3K ) .", "To investigate whether midgut homeostasis in feminized males was responsive to diet , we raised NP1>traF flies on two different yeast dilutions .", "Similar to females , feminized males on the high-yeast diet appeared to show a more severe pathology at 35 days than did those on low yeast ( Figure 3L–M ) .", "In line with this , when we analyzed the rate of tumor formation ( category IV ) in a specific region of the gut ( R2 ) , we found that DR significantly decreased the risk of tumor formation in feminized males ( Figure 2L ) .", "The observed pathologies demonstrate that female and feminized ISCs respond to DR , however , when we quantified PH3+ cell number in 35 day-old guts , although female high-yeast guts had more mitoses , this difference was not significant in feminized males ( Figure 3—figure supplement 2B ) .", "One possible explanation is that the observed pathology is an accumulation of ISC activity , which could expose subtle differences in mitotic rate over a lifetime .", "Another possibility is that ISC division and pathology are linked but pathology is driven by other factors that are responsive to diet , such as changes to the microbiota ( Clark et al . , 2015; Petkau et al . , 2014 ) .", "The TOR pathway inhibitor rapamycin extends lifespan in females , and to a much lesser extent in males ( Bjedov et al . , 2010 ) , and a recent study showed that it decreases ISC division and slows barrier dysfunction in females ( Fan et al . , 2015 ) .", "When we treated males , females and feminized males with rapamycin , we found that ISC proliferation and gut pathology were reduced in females and feminized males ( Figure 3N ) .", "This shows that , similarly to DR , females derive a greater benefit from rapamycin than do males , possibly explaining , at least in part , the sex bias in the magnitude of lifespan extension by the drug .", "To assess whether the acquired ISC activity in gut-feminized males could contribute to a more robust survival to intestinal stress , we challenged them to Ecc oral infection at 7 and 42 days of age .", "At 5-days post-infection , 7-day-old females and males survived , but approximately 50% of gut-feminized males had died ( Figure 3—figure supplement 2D ) .", "Analysis of ISCs post-infection showed that feminized males induced significantly higher proliferation than control males , but this was clearly not protective ( Figure 3P ) .", "In aged ( 42 day-old ) flies , as before , we found that only males succumbed to Ecc oral infection .", "Feminized males were most susceptible , dying more quickly than did control males ( Figure 3O , Figure 3—figure supplement 2E ) , and this was not related to a change in feeding on either normal food ( Figure 3—figure supplement 2F ) or the infection pellet ( Figure 3—figure supplement 2G ) .", "At this age , female guts had a high number of mitoses , but these did not significantly increase on infection , and nor did they in feminized males ( Figure 3P ) .", "One interpretation of this result is that , in aged females and feminized males , the pool of ISCs has been exhausted by continued mitotic activity through the lifespan , or that due to age-related triggers such as inflammation or dysbiosis , ISC activity is already at a maximum .", "Markedly different to females , however , was the high induction of systemic dipt in control and feminized males ( Figure 3Q ) , in line with their reduced survival .", "Altogether , these data show that age-related sensitivity of males to intestinal infection was not rescued by feminization of the midgut .", "One probable contributing factor to the high death rate of feminized males is the barrier dysfunction observed at 42 days ( Figure 3H ) .", "However , their sensitivity to Ecc at young ages points to other , compromised immune responses .", "We hypothesize that a sex mis-match between the gut and other immune tissues such as the fat body and hemocytes , may be detrimental .", "Females show a greater lifespan extension than do males when subjected to DR ( Magwere et al . , 2004 ) .", "Males from the outbred line wDah used in this study have a mean lifespan that is 22 days shorter than that of their female siblings’ ( Figure 2—figure supplement 2D ) , and only females showed a lifespan extension when raised on food with a 50% reduction in yeast ( Figure 2—figure supplement 2D ) .", "Comparison of the lifespans of gut-feminized males with control female and male flies raised on a standard diet showed that gut-feminized males were significantly shorter lived than control males ( Figure 3R , Figure 3—figure supplement 2H ) , suggesting that their acquired intestinal hyperplasia was detrimental to their lifespan .", "Interestingly , when we measured the responses of lifespan to two different yeast dilutions , we found that midgut-feminized males had acquired a response to DR similar in magnitude to that seen in control females ( Figure 3R , Figure 3—figure supplement 2H and Figure 3—source data 1 ) .", "Taken together with our data on tissue sex and gut pathology , this finding suggests that lowered levels of intestinal pathology contribute to increased female longevity upon DR , and offers one explanation for the difference between males and females in the response of their lifespans to diet ." ], [ "In marked contrast to the gut pathology previously reported in aging females ( Biteau et al . , 2008; Choi et al . , 2008; Patel et al . , 2015 ) , and the extensive deterioration seen in multiple regions of the gut in the present study , we found that males generally have well-maintained guts into old age , even at ages close to the maximum male lifespan .", "Female intestinal pathology is driven by stem cell activity ( Patel et al . , 2015 ) , which is responsive to diet ( Choi et al . , 2011; O’Brien et al . , 2011 ) .", "Gut hyperplasia is limiting for female lifespan: when ISC division is genetically reduced , female lifespan is extended ( Biteau et al . , 2010; Hur et al . , 2013; Rera et al . , 2011; Ulgherait et al . , 2014 ) .", "We showed that , by switching the sexual identity of male midgut cells , ISC activity was increased , pathologies appeared and lifespan was shortened .", "In addition , the lifespan of males with feminized guts became responsive to diet , with DR both reducing the risk for developing severe pathology and increasing lifespan .", "The marked sexual dimorphism in gut pathology during aging provides a potential explanation for why males do not increase lifespan as much as do females in response to DR . Because males do not suffer from age-related hyperplasia , they do not derive the same benefit from DR-induced stem cell quiescence .", "Limited hyperplasia could also at least partly explain the much greater increase in lifespan seen in females than in males upon reduced IIS ( Clancy et al . , 2001; Tatar et al . , 2001 ) ; given that ISC activity in females is responsive to insulin signaling ( Biteau et al . , 2010; O’Brien et al . , 2011; Hur et al . , 2013 ) .", "Intestinal barrier function decreases during aging in females , and this decline is associated with an increase in immune activation , a decrease in nutrient absorption and death ( Rera et al . , 2012 ) .", "We showed that sustained ISC division has drastic consequences for the female gut epithelium , causing scars and holes to appear on the basal side .", "Interestingly , in transcriptomic analysis of aging guts of flies raised in both natural and axenic conditions , genes involved in wound healing were increased during aging ( Guo et al . , 2014 ) .", "In line with the absence of severe pathology in males , barrier function was maintained into old age .", "Despite this , males succumbed to oral infection and xenobiotic stress to which females were resistant , and this sensitivity was not rescued by feminization .", "We found that the gut ISC response to infection , a repair process , was significantly lower in male flies and increased in feminized males .", "Regulation of ISC activity is therefore not the only difference between males and females affecting resistance: other epithelial immune responses such as ROS and antimicrobial peptide production or tolerance to Ecc-derived toxins could also be sexually dimorphic ( Vincent and Sharp , 2014 ) .", "The male sensitivity and sepsis-like response to Ecc oral infection , age-related systemic inflammation and high levels of ROS production shown in this study are striking and present a starting point for the analysis of sex differences in intestinal immunity , which will be relevant for resistance to opportunistic infection and shaping the microbiome .", "Intestinal plasticity is important not only to resist infection ( Buchon et al . , 2009 ) , but also to respond predictively to the increased energy demands of egg production upon mating ( Reiff et al . , 2015 ) , which could explain why females and males invest so differently in ISC activity .", "Male reproduction does not require the same amount of nutritional input and , therefore , large-scale intestinal remodeling would probably not increase reproductive rate , which is promoted instead by increased detection and courtship of females ( Bretman et al . , 2011 ) .", "This sex difference is evolutionarily conserved , in that female mammals extensively grow and remodel their guts , increasing digestive and absorptive capacity in accordance with the increased nutritional demands of lactation ( Hammond et al . , 1997; Speakman et al . , 2008 ) .", "Possibly related to this greater gut plasticity , women have higher rates of region-specific colon cancer than do men ( Kim et al . , 2015; Jemal et al . , 2011 ) , although the mechanisms underlying this difference are not well understood .", "Female-specific intestinal remodeling during reproduction could be a driving factor .", "Intriguingly , higher parity and earlier childbearing are protective against colon cancer , whereas later child-bearing is a significant risk factor ( Wernli et al . , 2009 ) , suggesting that the timing of reproductive events may affect ISC ( mis- ) regulation .", "Further studies linking early gut plasticity , nutrition and fecundity with severity of hyperplastic pathology during aging in Drosophila may help elucidate conserved mechanisms underlying sex differences in cancer rates ." ], [ "All mutants were backcrossed for six generations into the wild type , outbred strain , wDahomey ( wDah ) , maintained in population cages .", "The following fly stocks were obtained from the Bloomington Stock Centre: P{UAS-tra . F}20J7 , P{GawB}Myo31DFNP0001 ( referred to as NP1-Gal4 ) , and daughterless-Gal4 .", "P{PTT-un1}CG8668117-2 ( Resille-GFP ) was originally obtained from the Flytrap project and was a gift from A Jacinto .", "Stocks were maintained and experiments conducted at 25°C on a 12 hr:12 hr light:dark cycle at 60% humidity , on food containing 10% ( w/v ) brewer’s yeast , 5% ( w/v ) sucrose , and 1 . 5% ( w/v ) agar unless otherwise noted ( referred to as ‘low yeast’ food denoting an arbitrary 1:1 sugar to yeast ratio , which is changed to 1:2 ( 20% yeast , 5% sucrose ) for ‘high yeast’ food ) .", "For measurement of gut histology and lifespans , flies were reared at standard larval density and eclosing adults were collected over a 12-hr period .", "Flies were mated for 48 hr before being sorted into experimental vials at a density of 10 flies per vial .", "Flies were transferred to fresh vials thrice weekly , and for lifespan , deaths/censors were scored during transferral .", "Guts were dissected from live flies in ice-cold PBS and immediately fixed in 4% formaldehyde for 15 min .", "Guts were mounted in mounting medium containing DAPI ( Vectastain ) and endogenous GFP was imaged immediately .", "Between 6 and 14 guts were analyzed per condition , per time point .", "Images were captured with a Zeiss ( UK ) LSM 700 confocal laser scanning microscope using a 40x oil-immersion objective .", "The following antibodies were used for cell division analyses; primary antibodies: rabbit anti-PH3 ( Cell Signaling ( Danvers , MA ) 9701 ) 1:500; mouse anti-GFP ( Cell Signaling 2955 ) 1:1000 .", "Secondary antibodies: Alexa Fluor 594 donkey anti-rabbit ( ( A21207 ) Thermo Fisher Scientific , Waltham , MA ) 1:1000; Alexa Fluor 488 donkey anti-mouse ( A21202 ) 1:1000 .", "Guts were dissected in ice cold PBS and immediately fixed in 4% formaldehyde for 15 min , serially dehydrated in MeOH , stored at -20°C , and subsequently stained .", "Guts were washed in 0 . 2% Triton-X / PBS , blocked in 5% bovine serum albumin / PBS , incubated in primary antibody overnight at 4°C and in secondary for 2 hr at RT .", "At least 10 guts per condition were mounted , scored and imaged as described above .", "Gut barrier efficiency was analyzed by placing flies on blue food ( minimum 110 flies per condition , except at 80 d , min . 80 flies ) prepared using using 2 . 5% ( w/v ) FD&C blue dye no . 1 ( Fastcolors ) as previously described ( Rera et al . , 2012 ) , except flies were kept on the blue food for 24 hr before the Smurf phenotype was scored .", "At least 3 x colonies of Ecc15 were grown in separate overnight cultures in Luria-Bertani medium , pooled , pelleted and adjusted to OD600 = 200 with fresh LB .", "Bacteria were mixed 1:1 with 5% sucrose ( final OD600 = 100 ) ; LB / sucrose was used for sham infection .", "Flies were starved for 5 hr in empty vials .", "Experimental vials were lined with 10 pieces of Whatman filter paper to which 1 ml Ecc15 / sucrose ( or LB / sucrose ) was added .", "Starved flies were added at a density of 20/vial; a minimum of 80 flies/condition .", "Filter paper was refreshed each day with newly grown Ecc15 / sucrose ( or LB / sucrose ) solutions , using a Pasteur pipette .", "Deaths were scored thrice daily .", "Ten flies per vial , at least 110 flies per condition , were fed with the organochloride dichlorodiphenyltrichloroethane ( DDT; Supelco , Sigma-Aldrich , UK ) for 18 hr .", "To standard SYA food , 0 . 03% ( w/v ) DDT , made from a stock solution of 2% ( w/v ) DDT in ethanol , was added .", "Deaths were scored thrice daily .", "Flies were transferred to vials at a density of 5 per vial on the evening before the assay .", "Vials were coded and placed in a randomized order on viewing racks at 25°C overnight .", "The assay occurred with minimal noise and physical disturbance .", "Thirty minutes was allowed between the arrival of the observer and commencement of the assay .", "Observations were performed blind for 90 min , 1 hr after lights-on .", "Vials were observed for approximately 3 s during which the number of flies feeding was noted .", "A feeding event was scored when a fly extended its proboscis and made contact with food .", "Successive rounds of observations were carried out giving an observation rate of once / 5 min .", "Feeding data are expressed as a proportion by experimental group ( sum of scored feeding events divided by total number of feeding opportunities , where total number of feeding opportunities = number of flies in vial×number of vials in the group×number of observations ) .", "For statistical analyses , comparisons between experimental groups were made on total feeding events by all flies within a vial , to avoid pseudoreplication .", "As described for guts ( Broderick et al . , 2014 ) , whole flies were surface sterilized for 1 min in 95% ethanol , rinsed and pooled into groups of 5 , n≥8 per condition , on ice for homogenization .", "Three 1/5 serial dilutions were plated on Man , Rogosa and Sharpe ( MRS ) plates and cultured at 29°c both aerobically and anaerobically , and scored at 48 and 72 hr .", "Nearest-neighbour internuclear distance in the R2 region was measured from raw LSM files ( Zeiss ) using the Measure function in Fiji ( Image J ) ; 20 distances per gut , n ≥ 6 guts per condition .", "RNA was isolated from n ≥ 14 guts per sample with TRIzol ( Invitrogen , Thermo Fisher Scientific , Waltham , MA ) and cDNA was synthesized by using SuperScript II Reverse Transcriptase ( Invitrogen ) according to manufacturers instructions .", "qPCR was carried out by using Power SYBR Green PCR Master Mix ( Thermo Fisher Scientific ) on QuantStudio6 Flex real-time PCR ( Applied Biosystems , Thermo Fisher Scientific , Waltham , MA ) .", "The following primer sequences ( Eurofins , UK ) were used in the analysis: dsxF-F ( TCAACACGTTCGCATCACAAA ) ; dsxF-R ( TAGACTGTGATTAGCCCAATAACTGA ) , act5C-F ( CACACCAAATCTTACAAAATGTGTGA ) ; act5C-R ( AATCCGGCCTTGCACATG ) ; dipericin-F ( GCGGCGATGGTTTTGG ) ; dipericin-R ( CGCTGGTCCACACCTTCTG ) ; Duox-F ( TAGCAAGCCGGTGTCGCAATCAAT ) ; Duox-R: ACGGCCAGAGCACTTGCACATAG .", "Statistical analyses were performed in Excel ( Microsoft ) or Prism ( Graphpad , La Jolla , CA ) , except for Ordinal Logistical Regression ( OLR ) , Cox Proportional Hazards and Monte Carlo Markov Chain Generalised Linear Model with Poisson Error Family analyses , which were performed in R ( using the clm function from the 'ordinal' library for OLR ) .", "Statistical tests used are indicated in the figure captions .", "Outliers were rarely excluded from the data , but when excluded conformed to the rule of lying more than two standard deviations away from the mean .", "They are clearly marked as data points on graphical displays ." ] ]

[ "Women live on average longer than men but have greater levels of late-life morbidity .", "We have uncovered a substantial sex difference in the pathology of the aging gut in Drosophila .", "The intestinal epithelium of the aging female undergoes major deterioration , driven by intestinal stem cell ( ISC ) division , while lower ISC activity in males associates with delay or absence of pathology , and better barrier function , even at old ages .", "Males succumb to intestinal challenges to which females are resistant , associated with fewer proliferating ISCs , suggesting a trade-off between highly active repair mechanisms and late-life pathology in females .", "Dietary restriction reduces gut pathology in aging females , and extends female lifespan more than male .", "By genetic sex reversal of a specific gut region , we induced female-like aging pathologies in males , associated with decreased lifespan , but also with a greater increase in longevity in response to dietary restriction ." ]

[ "Women live longer than men , and many age-related diseases are more common in one sex than the other .", "In addition , some treatments that extend the healthy lifespan of laboratory animals are more effective in females than in males .", "These include dietary restrictions , where food or specific dietary constituants are kept in short supply .", "Stem cells can help to repair old and damaged tissue because , when they divide , they can form a cell that can specialize into one of several mature cell types .", "Previous studies of the fruit fly Drosophila melanogaster have shown that stem cell activity in the gut affects how long female flies live .", "Now , Regan et al . have looked in detail at the guts of male and female fruit flies as they age .", "This revealed that female guts deteriorate as the flies grow old because the stem cells in the gut divide more often and form small tumours .", "These stem cells help young females to grow and repair their guts , but start to turn against them as they age .", "In contrast , male guts stay well maintained and do not show the same signs of ageing .", "Females fed less food had guts that aged more slowly , suggesting they might live longer on a restricted diet because it improves their gut health .", "Regan et al . then used a genetic trick to make male flies with female guts .", "These feminized males had more gut tumours than normal males , but they also showed a greater increase in lifespan when placed on a restricted diet , because the poorer condition of their ageing gut meant there was more scope for the diet to improve their health .", "So if gut deterioration does not limit male lifespan , what do males die of ?", "Pursuing this question may ultimately help us to understand how human lifespans are affected by sex differences and develop treatments for ageing and age-related diseases that everyone can benefit from ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "computational and systems biology" ]

[ [ "Multicellularity arose multiple times across evolution ( Kaiser , 2001; Knoll , 2011 ) , yet how selective pressures shaped and optimized the cellular configurations of these complex assemblies remains poorly understood ( Ollé-Vila et al . , 2016 ) .", "Multicellular organs are more than the sum of their cells , and the collective interactions between cells on a global scale confer higher order functionality to the system through a structure-function relationship ( Thompson , 1942 ) .", "Cellular functionality therefore emerges from cellular associations and synergies , and is not cell autonomous .", "Understanding the emergent properties of complex multicellular assemblies , and the structure-function relationship between cell organization and organ function , remains an open challenge in both developmental and systems biology .", "This question has been examined previously in the field of neuroscience in the investigation of the relationship between cellular organization and nervous system function ( Cajal , 1911; White et al . , 1986 ) .", "This was first systematically applied to the simple nervous system of C . elegans ( White et al . , 1986 ) , and more recently the field of ‘connectomics’ has extended this approach to more complex nervous systems ( Bullmore and Sporns , 2009 ) .", "Here a distinction between structural and functional networks is drawn , the former being the physical associations between cells representing all possible routes of information flow , and the latter the paths which information is observed to follow ( Bullmore and Sporns , 2009 ) .", "Uncovering the organizational properties of complex multicellular assemblies has not been performed previously at a whole organ or organism level .", "In plants , cells are glued together through shared cell walls and do not migrate with respect to one another , as in animal systems ( Green , 1969 ) .", "This invariance between adjacent cells provides a simplified opportunity to examine multicellular complexity by looking at whole organ cellular interaction networks that remain topologically invariant following their formation .", "By viewing plant organs as a complex system of interacting cells , a systems-based approach to understanding organ construction and optimization at a cellular level can be undertaken .", "Cellular interactions play a key role during plant development ( Benitez-Alfonso et al . , 2013; Lucas and Lee , 2004 ) .", "Mobile information in the form of proteins , RNAs and small molecules move locally through physical cell-to-cell interactions .", "These local interactions mediate patterning , self-organization and underlie cell identity in plants ( Leyser , 2011; Sabatini et al . , 1999; Sena et al . , 2009; Sugimoto et al . , 2010 ) .", "Genetically encoded patterning mechanisms mediate the self-organization process that leads to the creation of functional cellular interactions and patterns that constitute plant organs ( Besson and Dumais , 2011; Di Laurenzio et al . , 1996; Yoshida et al . , 2014 ) .", "While the importance of intercellular interactions is well established , much less is known about the global properties of these assemblies , and how they come together to form coherent organs .", "Previous efforts to understand local interactions between cells in two-dimensional cellular sheets have been explored using the developing Drosophila wing disk .", "Here a network consisting of nodes representing cell-cell junctions was generated , and cell shape based on polygon classes ( or local connectivity ) was generated ( Gibson et al . , 2006 , 2011; Heller et al . , 2016 ) .", "In plants , local cellular interactions and their role in mediating cell division plane placement in neighbors has also been investigated ( Gibson et al . , 2011; Sahlin and Jönsson , 2010; Willis et al . , 2016 ) .", "While these approaches are informative , they are limited to the local topological analysis within the immediate vicinity of a cell , and are also restricted to cells on the surface of organs .", "Advances in whole organ 3D imaging at cellular resolution and computational image analysis enable organ-wide cellular interaction networks to be extracted and annotated by cell type ( de Reuille et al . , 2015; Montenegro-Johnson et al . , 2015 ) ( Figure 1A ) .", "This multidimensional topological phenotyping pipeline captures global cellular interactions in whole organs and enables the simultaneous analysis of both higher-order and local properties of these complex cellular configurations .", "These structural networks provide cellular roadmaps of possible information flux through organs , and understanding the structure of these cellular communities provides insight into the function of the system as a whole , and individual cells within organs . 10 . 7554/eLife . 26023 . 003Figure 1 . Computational workflow .", "( A ) Pipeline for imaging , annotating , quantitatively analyzing , and visualizing global cellular interaction networks .", "( B ) 3D layout of an annotated cellular connectivity network for the Arabidopsis Colombia hypocotyl .", "( C ) Explanation of the network measures employed in this study .", "Nodes are colored to indicate values . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 00310 . 7554/eLife . 26023 . 004Figure 1—figure supplement 1 . Filtering small edges in connectivity networks . Distribution of absolute shared cell wall area ( µm² ) in connected cells across different cell types in the A . thaliana Col hypocotyl .", "The black arrow indicates where a cutoff was applied , below 2 μm2 .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent ± standard deviation within a bin . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 00410 . 7554/eLife . 26023 . 005Figure 1—figure supplement 2 . Topological buffering of sample boundaries .", "( A ) In situ false color degree measurements in the A . thaliana Col hypocotyl with and without a buffer region ( in white ) .", "Pink arrows indicate a change in degree when using a cellular buffer region .", "( B ) In situ false color betweenness centrality measurements in the A . thaliana Col hypocotyl with and without a buffer region ( in white ) .", "Arrows indicate a change in betweenness centrality when using a buffer region . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 005 We examined the embryonic plant stem ( hypocotyl ) to understand the principles with which cells come together to create this organ .", "This radially symmetric multicellular assembly elongates exclusively through cell expansion during early seedling establishment , rendering cellular topology invariant across development .", "The hypocotyl also serves as a conduit linking the above and below ground portions of the seedling during early growth , serving both a transport and structural function ." ], [ "In order to understand both the local and higher-order properties of complex multicellular organization in whole organs , an image analysis and analytical framework was employed ( Figure 1A ) .", "To achieve robust topological analyses of cellular patterning in plant organs , we sought to generate fully represented and accurate cellular interaction networks .", "Every cell within the unexpanded embryonic hypocotyl was digitally captured using whole mount high resolution confocal microscopy and 3D segmentation ( de Reuille et al . , 2015; Montenegro-Johnson et al . , 2015 ) ( Figure 1A ) .", "Cell types within these samples were identified and annotated using 3DCellAtlas ( Montenegro-Johnson et al . , 2015 ) .", "Cellular connectivity networks were extracted by identifying shared surfaces between adjacent cells using 3D cellular meshes , representing intercellular associations .", "Organ-wide cellular connectivity networks that are annotated by cell type were generated ( Figure 1A–B , Video 1 ) .", "Towards the achievement of accurate cellular interactomes , segments representing air spaces between cells were computationally removed ( Montenegro-Johnson et al . , 2015 ) .", "Relatively small interfaces ( below 2 μm2 ) were filtered out due to their likely limited role in transport processes , and potential generation due to image processing artefacts ( Figure 1—figure supplement 1 ) ( Bassel , 2015 ) .", "Cells in the periphery of imaged organs were included in analyses , but only cells from the central regions of organs were reported in figures ( Figure 1—figure supplement 2 ) .", "This provided a topological buffer to the boundaries introduced following image acquisition .", "Collectively , these processes yielded precise global cellular interaction networks annotated by cell type , enabling cell type specific topological analyses of organ patterning to be performed . 10 . 7554/eLife . 26023 . 006Video 1 . Hypocotyl cell connectivity network of Arabidopsis thaliana Col . Cell types are gradually removed to allow internal visualisation of the network . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 006 A persistent issue with analyses using biological network datasets is their completeness and accuracy .", "The networks presented in this work are both fully represented ( no missing nodes or edges ) and highly accurate .", "In all instances , biological triplicates are used for the analysis of organ-wide patterning in this study .", "A remaining challenge is to identify an appropriate analytical framework to extract biologically meaningful information from these connectivity datasets .", "Considering the importance of intercellular interactions within plant organ function , these networks may be viewed as a complex system ( Newman , 2010 ) .", "The spatial constraints of plant cells and their embedding in space mean these networks resemble 3D lattices ( Barthelemy , 2011 ) .", "The properties of these undirected cell interaction networks can therefore be analyzed using quantitative network analyses to reveal the organizational properties of these multicellular assemblies ( Barabási , 2016; Bullmore and Sporns , 2009 ) .", "Following topological analyses , node data can be visualized by importing cell properties back into MorphoGraphX ( de Reuille et al . , 2015 ) as a heatmap for in situ 3D visualization within the cellular context of the segmented organ ( Figure 1A ) .", "Edge information can be graphically explored by importing these data into the BioLayout3D visualization tool and using the edge heatmap functions ( Figure 1B ) ( Theocharidis et al . , 2009 ) .", "Finally , statistical analyses of data exported from network analysis software can be performed using standard statistical software ( Hagberg et al . , 2008 ) .", "This approach enables the capture , discretization and quantification of local and global cellular organization , or patterning , in whole organs .", "A simple measure of the local properties of a cell ( node ) within an organ ( network ) is to count the number of neighbours it has ( Figure 1C ) .", "This is termed the degree of a node , and is a local measure of how a cell fits within its immediate context ( Newman , 2010 ) .", "While informative , degree is unable to capture the higher-order properties of multicellular systems ( Gibson et al . , 2006 , 2011; Heller et al . , 2016; Newman , 2010; Willis et al . , 2016 ) .", "In light of the 3D spatial constraints of immobilized plant cells within organs , and the central role of intracellular communication in plant development , path length represents an important and biologically relevant topological property of these networks ( Barthelemy , 2011 ) .", "A well-established measure of path length in networks is betweenness centrality ( BC ) ( Barabási , 2016; Freeman , 1977; Newman , 2010 ) ( Figure 1C ) .", "BC uses knowledge of the complete network to count the number of times a nodes lies on a shortest path between all other nodes ( Barabási , 2016; Newman , 2010 ) .", "A node with a high BC therefore lies on many shortest paths between other nodes .", "In this study we normalized BC by network size in order to make data comparable between different samples ( van Wijk et al . , 2010 ) .", "The use of BC enables the identification of short paths through multicellular networks , representing topologically poised optimized routes for information movement across the system .", "This combination of acquiring complete cellular interactomes and their analysis using path-length-based measures enables the higher-order principles of global cellular patterning to be revealed .", "The properties of three wild-type Arabidopsis hypocotyls of the ecotype Colombia ( Col ) were topologically analyzed at cell type specific resolution ( Figure 2A ) .", "To illustrate key findings , we focus the reporting of results on epidermal cell patterning , with additional cell type analyses presented as corresponding figure supplements . 10 . 7554/eLife . 26023 . 007Figure 2 . Cell type-specific quantification of the topological features of hypocotyl interaction networks in three Arabidopsis ecotypes: Colombia ( Col ) , Landsberg erecta ( Ler ) and Cape Verdi Islands ( Cvi ) .", "( A ) Surface and longitudinal-section meshes of the three ecotypes , with false color indicating cell type ( dark green – trichoblast , light green – atrichoblast , blue – outer cortex , yellow – inner cortex , pink – endodermis , cyan – vasculature ) .", "( B–C )", "Hypocotyl meshes with false color heat maps of ( B ) degree and ( C ) betweenness centrality .", "( D–E )", "Average normalized frequency distributions of ( D ) degree and ( E ) BC across trichoblast and atrichoblast cell types .", "( F ) Edge BC false colored onto edges of virtual cross and longitudinal sections of the hypocotyl networks of each ecotype .", "( G ) Normalized frequency distributions of edge BC in different cell interfaces within different cell types ( T – trichoblast , A – atrichoblast ) .", "( H ) Schematic showing cells lying upon short path lengths within the Arabidopsis hypocotyl .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent ± standard deviation within a frequency bin .", "Asterisks ( * ) indicate significant differences between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 00710 . 7554/eLife . 26023 . 008Figure 2—figure supplement 1 . Comparisons of topological features of Arabidopsis hypocotyl cellular arrangements between Arabidopsis ecotypes . Average normalized frequency distributions of ( A ) degree and ( B ) betweenness centrality ( BC ) in trichoblasts , atrichoblasts , the outer cortex , the inner cortex and the endodermis , in three A . thaliana ecotypes; Col , Ler and Cvi .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent the standard deviation within a bin .", "An asterisk ( * ) represents significant difference between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons used in this study ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 00810 . 7554/eLife . 26023 . 009Figure 2—figure supplement 2 . Cell interface sizes in unexpanded embryo hypocotyls .", "( A ) Shared cell wall areas between epidermal cells ( T – trichoblast , A – atrichoblast ) in Arabidopsis ecotypes , ( B ) within cells in the outer cortex ( OC ) and inner cortical layers ( IC – inner cortex ) , ( C ) between epidermal cell types and the cortex , and ( D ) between cells in the cortical cell layers and with the endodermis .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent ± the standard deviation within a bin . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 00910 . 7554/eLife . 26023 . 010Figure 2—figure supplement 3 . Cell surface area and weighted BC measurements .", "( A ) The average cell wall area ( µm² ) of specific cell types in the hypocotyl , comparing three Arabidopsis ecotypes , Col , Ler , and Cvi .", "Cells from biological replicates were grouped and data represent the mean across all cells within the triplicate samples .", "Error bars represent ± standard deviation of the mean .", "( B ) In situ false color weighted betweenness centrality in the Arabidopsis ecotypes Col , Ler , and Cvi .", "( C ) Distributions of weighted node betweenness centrality ( BC ) in different cell types of three Arabidopsis ecotypes .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent ± the standard deviation within a bin .", "An asterisk ( * ) represents significant difference between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons used in this study ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 010 The spatial distribution of cell ( node ) topological properties can be visualized by false coloring cells of segmented organs using centrality data ( Figure 2B–C ) ( de Reuille et al . , 2015 ) .", "This provides an in situ view of the distribution of node properties across the segmented organ within individual cells .", "Cell degree ( number of immediate neighbors ) is greater in cortical cells than in the epidermis ( Figure 2B and Figure 2—figure supplement 1 ) as this latter cell type lacks exterior neighbors .", "Degree is also high in elongated vascular cells representing xylem vessels ( Figure 2B ) .", "Node BC is low in most cells of the Arabidopsis Col hypocotyl , and cells lying upon shorter paths ( higher BC ) are observed in a small fraction of the network , as observed by the presence of a tail in this distribution ( Figure 2E and Figure 2—figure supplement 1 ) .", "The highest node BC , representing the shortest paths across the organ , is observed in the vascular system ( Figure 1C ) , consistent with the role of this cell type in facilitating long distance transport .", "High BC nodes are also observed in epidermal cell files belonging to the non-hair forming atrichoblast cell type ( Duckett et al . , 1994 ) ( Figure 2C and E ) .", "These cells lie on significantly shorter paths than their adjacent cell type , the hair forming trichoblast cells ( Figure 2E ) .", "This topological analysis identified a previously undescribed coherent conduit of interconnected cells providing a short path length across the atrichoblast cell type of the Arabidopsis Col hypocotyl epidermis ( Figure 2C and E ) .", "These data demonstrate that in addition to the vasculature , atrichoblast cell files are topologically poised to mediate the optimized movement of information along the longitudinal length of the hypocotyl axis .", "While the degree of trichoblast cells is significantly greater than that of atrichoblast cells ( Figure 2D ) , it is these latter cells with fewer connections that lie upon shorter paths .", "This represents a non-intuitive global topological property of this multicellular system .", "The higher order properties of cells within the hypocotyl are therefore not dictated by their number of local interactions , but rather their context within the wider system .", "We next examined each the geometric and topological properties of the interfaces , as opposed to the nodes , between adjacent cells .", "These are represented as edges within cellular connectivity networks .", "These intracellular interfaces provide the physical conduits by which information moves through these multicellular systems ( Figure 2F and Video 1 ) .", "The geometric size of these shared interfaces between cells was computationally measured using 3DCellAtlas ( de Reuille et al . , 2015; Montenegro-Johnson et al . , 2015 ) , and examined across the unexpanded embryonic Col hypocotyl ( Figure 2—figure supplement 2 ) .", "In absolute terms , cellular interactions between the large cortical cells had the greatest interface sizes , reflecting differences in cell size , and cells smaller in size had reduced contact areas with their neighbors ( Figure 2—figure supplement 2 ) .", "Topological analysis of these cell interfaces is provided using edge BC , which measures the participation of edges in the creation of short paths between all pairs of nodes ( Newman , 2010 ) .", "Atrichoblast-atrichoblast edge BC was greatest of all cellular junctions between different cell types within the epidermis ( Figure 2G and Video 2 ) .", "This demonstrates that intercellular interactions between atrichoblast cells contribute most towards the creation of short path lengths across this cell type . 10 . 7554/eLife . 26023 . 011Video 2 . Hypocotyl cell connectivity network of the epidermis of Arabidopsis thaliana Col . Edge colour represents the edge random walk betweenness centrality ( scale 0–0 . 0015 , blue to red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 011 Both cellular connectivity networks and cell interaction sizes can be extracted from whole organs using 3DCellAtlas .", "This enables the integration of these two pieces of information by weighting edges in the network with the geometric size of their associated cellular interfaces .", "In this case , weighted node betweenness centrality was calculated using the reciprocal of the connecting cell wall area ( edge weight ) , so that large cell-cell interactions generate small weights , which contributes more to the creation of short paths ( Newman , 2010 ) .", "The topological analysis of weighted hypocotyl networks generated a bias based on cell size whereby weighted BC identified large cortical cells as having the lowest path length ( Figure 2—figure supplement 3 ) .", "The superior size of cortical cells in the hypocotyl endow these cells with a geometrically facilitated reduction in path length in these weighted networks .", "Information flow which is limited by the size of cellular interfaces may therefore prefer to move through this cell type , however considering the scale of cellular interface sizes included in networks which is in the order of microns ( Figure 2—figure supplement 2 ) , this does not likely represent a limiting factor in this context .", "Increased surface area has biological relevance to mediating intercellular trafficking through molecular transport components , which occupy physical space .", "The role of cell interface size towards mediating topologically distinguishing features however appears to be limited in the context of the unexpanded embryonic hypocotyl in Arabidopsis Col due to large differences in size across cell types .", "Wild Arabidopsis plants have been collected from diverse locations across the planet , facilitating the study of naturally occurring variation within this species ( Koornneef et al . , 2004 ) .", "Cellular organization in the Cape Verdi Islands ( Cvi ) and Landsberg erecta ( Ler ) ecotypes was compared with that of Col to explore topological principles of patterning across diverse genetic backgrounds .", "Local epidermal patterning was similar in all ecotypes , with the trichoblasts ( hair forming cells ) having a significantly higher local connectivity than the atrichoblast cells ( Figure 2D ) .", "The degree of Cvi trichoblast cells was greater than the other ecotypes , and the atrichoblast cells of Ler also had a greater number of neighbors ( Figure 2D , Figure 2—figure supplement 1 ) .", "The distributions of node BC were similar in the trichoblast cells of all three ecotypes , however path length distributions varied in atrichoblast cells across these different genetic backgrounds ( Figure 2—figure supplement 1 ) .", "Comparing these two cell types within each ecotype , significantly atrichoblast cells lay upon significantly shorter paths than trichoblasts in Col and Ler , while no significant difference was present in Cvi ( Figure 2E ) .", "These observations indicate the presence of natural variation in path length , specifically within the atrichoblast cell type of the Arabidopsis hypocotyl .", "This topological plasticity in turn leads to contrasting path lengths between each of the two epidermal cell types in Col and Ler , but not the Cvi accession .", "The optimized movement of information through networks follows shortest paths ( Barabási , 2016; Newman , 2010 ) .", "Topological analyses of Arabidopsis hypocotyl cellular interaction networks revealed the presence of reduced path lengths in the epidermis specifically within the atrichoblast cell files of the Col and Ler ecotypes , but not that of Cvi ( Figures 2E and 3A ) .", "We examined these topological predictions of preferential cell type-specific flux of molecular information by tracking the movement of the small fluorescent molecule fluorescein .", "Seedlings were transferred to media containing fluorescein , and following an incubation period , hypocotyls were live imaged using confocal microscopy .", "Epidermal cells in the hypocotyl typically do not produce root hairs , despite having each trichoblast and atrichoblast cell identities .", "The observation of the passage of fluorescein along the hypocotyl occurs following molecular uptake in roots , where hairs are produced , and represents long distance transport along the length of the plant rather than local uptake . 10 . 7554/eLife . 26023 . 012Figure 3 . Transport of the small fluorescent molecule fluorescein in the hypocotyl epidermis of different Arabidopsis ecotypes .", "( A ) Illustration of the cells having reduced path length in the hypocotyls of Landsberg erecta ( Ler ) , Colombia ( Col ) and Cape Verdi Islands ( Cvi ) ecotypes indicated in green .", "( B ) Confocal image of the Ler hypocotyl ( white ) and fluorescein ( red ) imaged within the epidermis .", "( C ) Same as ( B ) with Col . ( D ) Same and ( B ) with Cvi .", "( E ) Quantification of fluorescein concentration within individual epidermal cells of the Ler hypocotyl epidermis .", "( F ) Same as ( E ) with Col . ( G ) Same as ( E ) with Cvi .", "Atrichoblast and trichoblast cell types are indicated by an A or T above each cell .", "The scale bar in ( E ) indicates mean normalized values for the quantification of fluorescein concentration visualization in ( E ) - ( G ) .", "( H ) Violin plot of fluorescein concentration in each atrichoblast and trichoblast cells .", "The mean is indicated by a black bar .", "An asterisk ( * ) indicates a significant difference in fluorescein concentration between atrichoblasts and trichoblasts within an ecotype ( t-test , p≤0 . 001 ) .", "Normalized fluorescence concentration is indicated on the y-axis .", "Data from three biological replicates for each ecotype were pooled and mean normalized for comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 012 Z-stacks were obtained and epidermal cells were segmented in 3D to quantify the concentration of fluorescein within each epidermal cell type ( de Reuille et al . , 2015 ) .", "This enabled the transport of this molecule through these multicellular networks to be captured at single cell resolution .", "Considering plants do not produce fluorescein , no specific transporter for this molecule is likely to be present ( Barclay et al . , 1982 ) .", "These measurements may therefore capture the non-specific bulk flow of molecules from the site of uptake in the roots , up through the hypocotyl .", "In all ecotypes , fluorescein was observed to be transported along the hypocotyl of seedlings examined ( Figure 3B–D ) .", "In each Ler and Col , a qualitatively greater amount of fluorescein was observed in atrichoblast than trichoblast cells ( Figure 3B–C ) , and this distinction was less clear in Cvi ( Figure 3D ) .", "Quantification of fluorescein concentration within each of these cell types across ecotypes ( Figure 3E–G ) revealed a significantly greater abundance of this molecule in atrichoblast cells over trichoblast cells in Ler and Col , and no significant difference between the cell types was observed in Cvi ( Figure 3H ) .", "These measurements support the link between path length analyses and the bulk movement of molecules along the epidermis in hypocotyls .", "The reduced path length in the atrichoblast cells of Ler and Col predict the preferential movement of small molecules through this epidermal cell type , while in Cvi where path length is equivalent between both epidermal cell types , no bias in molecular movement is observed .", "This suggests that BC can be used to predict the long range movement of molecules in this multicellular plant organ at single cell resolution .", "The construction of these conduits through the emergent property of global patterning therefore plays a functional role in optimized molecular transport across this organ .", "In the absence of active transport processes , molecules will preferentially move following these paths of reduced path length .", "Components participating in the active transport of molecules may also exploit these optimized routes by populating these cells with transport machinery .", "This higher order property of multicellular organization may also have a functional role within the context of epidermal cell patterning ( Dolan et al . , 1993 ) .", "The acquisition of nutrients in the soil by root hairs ( trichoblasts ) is dependent upon the ability to import these into cells .", "The accumulation of imported solutes within a root hair would impede this process owing to increased concentration within a cell as compared with that in the soil .", "A solution to this could be to acquire nutrients in root hairs and to move these into the adjacent non-hair atrichoblast cell which enables the hair cell to acquire further solutes .", "The atrichoblast is in turn topologically poised to facilitate the optimized longitudinal movement of these nutrients along the root and up through the hypocotyl .", "This provides a functional division of labour in the two epidermal cell types , and may bridge the structure-function relationship in epidermal cell patterning owing to the global topological properties of this system .", "Collectively , this experiment connects the topological analysis of global cellular connectivity using BC to molecular transport processes across the Arabidopsis hypocotyl .", "Plasticity in differential path length between epidermal cell types observed across Arabidopsis ecotypes suggests this system is not an intrinsic property of all hypocotyls .", "To determine the extent to which this higher-order property of global cellular organization is present in different contexts , two additional plants were selected for comparison with the Rosid species Arabidopsis belonging to the Brassicacea family .", "These were foxglove ( Digitalis purpurea ) , an Asterid from the Plantaginaceae family , and the common poppy ( Papaver rhoeas ) , a basal eudicot from the Papaveraceae family ( Figure 4A and Video 3 ) .", "These three species were selected based on their representation of diverse lineages of dicot angiosperms , enabling the topological investigation of multicellular complexity across distinct taxa .", "They are also all present in Western Europe and have annual , or in the case of foxglove biennial , life cycles .", "This enables comparisons within similar geographic distributions and life histories . 10 . 7554/eLife . 26023 . 013Figure 4 . Cell type-specific topological characterization of hypocotyl cellular interaction networks from three plant species: Arabidopsis thaliana ( Col ) , poppy and foxglove .", "( A ) Surface and longitudinal section meshes of poppy and foxglove hypocotyls , with false color denoting cell type ( dark green – trichoblast , light green – atrichoblast , blue – outer cortex , yellow – inner cortex second layer , navy blue – inner cortex third layer , pink – endodermis , cyan – vasculature ) .", "( B–C )", "Hypocotyl meshes with false color heat maps of ( B ) degree and ( C ) betweenness centrality ( BC ) .", "( D ) Average normalized frequency distributions of degree and ( E ) BC in epidermal cell types .", "( F ) Edges describing hypocotyl cellular interactions false colored by edge BC .", "( G ) Normalized frequency distribution of edge BC in the interfaces between different epidermal cell types ( T – trichoblast , A – atrichoblast ) .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent the standard deviation within a bin .", "An asterisk ( * ) represents significant difference between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 01310 . 7554/eLife . 26023 . 014Figure 4—figure supplement 1 . Cell type specific topological analysis of Arabidopsis , poppy and foxglove hypocotyl cellular arrangements . Average normalized frequency distributions of ( A ) degree and ( B ) betweenness centrality ( BC ) in trichoblasts , atrichoblasts , the outer cortex , the inner cortex and the endodermis , A . thaliana Col , poppy and foxglove .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent ± the standard deviation within a bin .", "An asterisk ( * ) represents significant difference between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons used in this study ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 01410 . 7554/eLife . 26023 . 015Video 3 . Comparison of the hypocotyl cell connectivity networks of Arabidopsis thaliana Col ( left ) , poppy ( centre ) and foxglove ( right ) .", "Edge colour represents the edge random walk betweenness centrality ( scale 0–0 . 0015 , blue to red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 015 Both poppy and foxglove showed a significantly higher degree distribution in trichoblast cells than atrichoblasts ( Figure 4D ) , consistent with all Arabidopsis ecotypes examined ( Figure 2D ) .", "This property of local cell connectivity is therefore conserved across most wild-type genetic backgrounds examined ( Figure 4—figure supplement 1 ) .", "The path length over atrichoblast cells is different in all species examined ( Figures 2E and 4E , Figure 4—figure supplement 1 ) .", "Poppy epidermal cells lie upon significantly longer paths than both other species , while path length is shortest in Arabidopsis in these same cell types ( Figure 4—figure supplement 1 ) .", "The quantitative distribution of BC is structurally and qualitatively similar between both epidermal cell types of poppy , yet a significantly shorter path length is present in trichoblasts ( Figure 4E ) .", "This observation contrasts the presence of shorter atrichoblast path lengths observed in the Col and Ler ecotypes of Arabidopsis ( Figure 2E ) .", "While a significant difference in path length is detected between epidermal cell types in poppy , their relative contrast is less pronounced than between epidermal cell types in Arabidopsis , in addition to their path lengths being significantly longer diminishing the optimization of potential transport through these conduits ( Figure 2E , Figure 4—figure supplement 1 ) .", "No significant difference in path length was detected between the two epidermal cell types of foxglove .", "Topologically this species is therefore similar to that of the Arabidopsis Cvi ecotype which also does not have a BC differential in the epidermis ( Figure 2E ) .", "Epidermal edge betweenness was greatest in Arabidopsis relative to each poppy and foxglove ( Figure 5F–G ) , reflecting the shorter node path length present in this Rosid species relative to the others . 10 . 7554/eLife . 26023 . 016Figure 5 . Comparisons of cell type-specific patterning and path length in the hypocotyls of wild-type A . thaliana ( Col ) and three A . thaliana mutants: cdka1;1 , monopteros and laterne .", "( A ) Surface and longitudinal section meshes of the three mutants , with false color cell type annotation as in Figure 2 .", "( B–C )", "Mutant hypocotyl meshes with false color heat maps of ( B ) degree and ( C ) betweenness centrality ( BC ) .", "( D ) Degree and BC in the cdka1;1 mutant and corresponding Col wild-type control .", "( E ) Degree and BC in the mp mutant and corresponding Col wild-type control .", "( F ) Degree and BC in the laterne mutant and corresponding Ler wild-type control .", "( G ) Edges describing cellular interactions false colored by edge BC in mutant hypocotyls .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent the standard deviation within a bin .", "An asterisk ( * ) represents significant difference between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 01610 . 7554/eLife . 26023 . 017Figure 5—figure supplement 1 . Cell type specific topological analysis of mutant Arabidopsis hypocotyl cellular arrangements .", "( A–B )", "Average normalized frequency distributions of ( A ) degree and ( B ) betweenness centrality ( BC ) in trichoblasts , atrichoblasts , the outer cortex , the inner cortex and the endodermis , in three Arabidopsis genetic backgrounds: wild type Col , cdka1;1 and monopteros .", "( C–D )", "Average normalized frequency distributions of ( C ) degree and ( D ) betweenness centrality in trichoblasts , atrichoblasts , the outer cortex , the inner cortex and the endodermis , in two Arabidopsis genetic backgrounds: Ler and laterne .", "Biological replicates were treated as individual samples and data represent the mean frequency of the bins across the triplicate samples .", "Error bars represent ± the standard deviation within a bin .", "An asterisk ( * ) represents significant difference between distributions using the chi-squared test for degree and the Kolmogorov–Smirnov test for BC , at the p≤1 . 56×10−5 level ( p≤0 . 05 after Bonferroni correction for 3200 distribution comparisons used in this study ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 017 Path length between epidermal cell types is therefore plastic both within Arabidopsis ecotypes , and across different species .", "A progressive decrease in epidermal path length is observed from poppy , to foxglove , then Arabidopsis , and the contrast in path length between epidermal cell types is greatest in the Col and Ler ecotypes of this latter species .", "This emergent property of reduced atrichoblast path length may represent a recent evolutionary innovation with regards to the optimization of transport along the exterior of the plant hypocotyl .", "The diversity in morphological complexity which is observed in biological systems results from the intersection between design principles which have been selected for , and constraints which are imposed upon possible configurations ( Avena-Koenigsberger et al . , 2015; Thompson , 1942 ) .", "Geometric , topological , mechanical and functional properties all act to limit the possible arrangements observed ( McGhee , 2006 ) .", "Differences in the topological properties of epidermal patterning suggest the global organization of this cell type is subject to modulation by genetically-encoded factors .", "In plants , different cellular configurations are also possible within the same species ( Di Laurenzio et al . , 1996 ) .", "The loss of genes mediating cellular patterning can yield viable offspring with alternative morphologies , and typically , compromised fitness .", "Characterizing the topology of cells in mutants where the causal genetic agent is known enables the contribution of gene activity towards the creation of topological properties to be investigated .", "Here we examined the properties of organ-wide hypocotyl cellular organization within three different cellular patterning mutants in Arabidopsis , selected based on previous reports of defects in the configuration of their cells .", "CYCLIN DEPENDENT KINASEA1;1 ( CDKA1;1 ) encodes a regulator of the cell cycle and mediates cellular patterning including asymmetric cell divisions ( Figure 5A and Video", "4 ) ( Nowack et al . , 2012 ) .", "The MONOPTEROS ( MP ) gene encodes a transcription factor that regulates auxin-mediated gene expression and cellular patterning ( Hardtke and Berleth , 1998 ) .", "The mp mutant embryo lacks vasculature tissue at maturity and provides the opportunity to examine the impact these cell types play in controlling organ-level topology ( Figure 5A and Video", "5 ) ( Schlereth et al . , 2010 ) .", "The laterne phenotype lacks embryonic leaves ( cotyledons ) in the mature embryo due to defects in localized auxin signaling ( Treml et al . , 2005 ) ( Figure 5A and Video 6 ) .", "The topological analysis of this mutant can link the activity of these mutations to patterning while exploring the role of non-cell autonomous signaling from the cotyledons in pattern formation within the hypocotyl during embryogenesis .", "The contribution of these genes towards the construction of the previously observed higher-order properties in epidermal patterning was examined by comparing their topological properties with their corresponding wild-type controls . 10 . 7554/eLife . 26023 . 018Video 4 . Comparison of the hypocotyl cell connectivity networks of wild-type Arabidopsis thaliana Col ( left ) , and the mutant cdka1;1 . Edge colour represents the edge random walk betweenness centrality ( scale 0–0 . 0015 , blue to red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 01810 . 7554/eLife . 26023 . 019Video 5 . Comparison of the hypocotyl cell connectivity networks of wild-type Arabidopsis thaliana Col ( left ) , and the mutant monopteros . Edge colour represents the edge random walk betweenness centrality ( scale 0–0 . 0015 , blue to red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 01910 . 7554/eLife . 26023 . 020Video 6 . Comparison of the hypocotyl cell connectivity networks of wild-type Arabidopsis thaliana Col ( left ) , and the mutant laterne . Edge colour represents the edge random walk betweenness centrality ( scale 0–0 . 0015 , blue to red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 020 The cdka1;1 mutant hypocotyl is qualitatively similar in appearance to the wild-type ( Figure 5A and Video 4 ) , and shows a similar spatial distribution and frequency of degree to its control accession Col ( Figure 5B , D and Figure 5—figure supplement 1 ) ( Dissmeyer et al . , 2009 ) .", "The number of local connections in each trichoblast and atrichoblast cells did not show any significant differences between this mutant and wild type , with trichoblast cells being more highly connected .", "The difference in path length between the two epidermal cell types is however lost in the cdka1;1 mutant ( Figure 5D ) , while a significant reduction in endodermal path length is also observed ( Figure 5—figure supplement 1 ) .", "This demonstrates that the patterning mediated by the CDKA1;1 gene product is responsible for the generation of reduced path length within the atrichoblast cell files of the epidermis , and also limits the construction of short paths in the endodermis .", "This further links the activity of this gene to both local and global patterning consequences within a specific cell type in this alternate cellular configuration .", "The trichoblast degree distribution of mature mp embryos is similar to its Col wild-type equivalent while atrichoblasts have more local connections ( Figure 5B , E ) .", "Node BC is however much higher in both epidermal cell types of the mutant , and these cells lie upon significantly shorter paths ( Figure 5E ) .", "This demonstrates that MP-mediated patterning acts to generate longer paths in the hypocotyl epidermis during embryogenesis , but is not required for the path length asymmetry between the two constituent cell types .", "The consequences of lacking a vascular system in this mutant can be observed through the visualization of the spatial distribution of edge betweenness ( Figure 5G ) .", "Here a radial route of short path length interfaces between cells is observed through the center of this cellular assembly , a property qualitatively absent in other hypocotyl cellular interfaces .", "The topological analysis of the laterne mutant revealed no significant difference in degree across all cell types , with the exception of the endodermis ( Figure 5F , Figure 5—figure supplement 1 ) .", "Each epidermal cell type lies upon longer paths than in its Ler wild-type equivalent , yet the relationship in path length between the epidermal cell types is the same as the control with atrichoblasts lying upon shorter paths than trichoblasts ( Figure 5F , Figure 5—figure supplement 1 ) .", "The laterne phenotype therefore limits global path length in the hypocotyl epidermis and may be controlled by non-cell autonomous signaling from the cotyledons , or be a local consequence of the cellular patterning activity of these gene products during embryogenesis ( Treml et al . , 2005 ) .", "The topological analysis of global cellular patterning in genetic mutant backgrounds allows the quantitative contribution of gene activity to be the linked to the formation of complex cellular assemblies , and their global organizational properties at cell type specific resolution .", "These analyses also enable the exploration of extant topological morphospace within plant organs at cellular resolution ( Avena-Koenigsberger et al . , 2015; Ollé-Vila et al . , 2016 ) .", "These alternative cellular topologies represent additional cellular configurations which are both geometrically and topologically possible , yet most likely compromised in their fitness , and thus function .", "Understanding the properties of these alternative configurations and their associated functional fitness is key to uncovering the structure-function relationship at the level of global cellular organization , and how these properties are genetically encoded .", "To understand how cell geometry is related to topology , mutual information measured in Shannon entropy was calculated within all samples used in this study .", "This determines the extent to which two variables are related to one another ( Kraskov et al . , 2004 ) .", "A high Shannon entropy between two entities demonstrates a high level of relatedness , and the ability of one variable to predict the value of the other .", "A similar pattern of mutual information was observed across all cell types and samples ( Figure 6 , Figure 6—figure supplements 1–2 ) .", "Each cell area and volume share a high degree of similarity , while mutual information between each area and degree , and degree and BC , show much less in common ( Figure 6 , and Figure 6—figure supplements 1–2 ) .", "Each area and BC , and volume and BC , have minimal mutual information , demonstrating that the global property of path length is not related to cell size .", "Likewise , the number of neighbours a cell has is weakly associated with the length of the path it lies upon .", "The control of path length in the hypocotyl is therefore largely independent of each cell size and the number of neighbours it has , and represents a property emerging from the global context within which a cell is situated in an organ .", "This conclusion is supported by the lack of significant differences in degree distributions which in turn show significant differences in path length distribution ( Figure 2—figure supplement 1 , Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 26023 . 021Figure 6 . Mutual information between geometric and topological cell features in the hypocotyl epidermis .", "( A–C )", "Estimated mutual information between area , volume , degree and betweenness centrality of epidermal cells in ( A ) three A . thaliana ecotypes , Col , Ler and Cvi , ( B ) poppy and foxglove , and ( C ) three A . thaliana mutants , cdka1;1 , monopteros and laterne .", "Mutual information was measured using bits of Shannon entropy and is plotted along the y-axis . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 02110 . 7554/eLife . 26023 . 022Figure 6—figure supplement 1 . Mutual information between geometric and topological hypocotyl cell features in all cell types , atrichoblasts , and trichoblasts . Estimated mutual information between area ( µm2 ) , volume ( µm3 ) , degree and betweenness centrality in hypocotyls of ( A ) A . thaliana Col , ( B ) A . thaliana Ler , ( C ) , A . thaliana Cvi , ( D ) poppy , ( E ) foxglove , ( F ) A . thaliana cdka1;1 , ( G ) A . thaliana monopteros and ( H ) A . thaliana laterne . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 02210 . 7554/eLife . 26023 . 023Figure 6—figure supplement 2 . Mutual information between geometric and topological hypocotyl cell features in the outer cortex , inner cortex and endodermis . Estimated mutual information between area ( µm2 ) , volume ( µm3 ) , degree and betweenness centrality in hypocotyls of ( A ) A . thaliana Col , ( B ) A . thaliana Ler , ( C ) , A . thaliana Cvi , ( D ) poppy , ( E ) foxglove , ( F ) A . thaliana cdka1;1 , ( G ) A . thaliana monopteros and ( H ) A . thaliana laterne . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 02310 . 7554/eLife . 26023 . 024Figure 6—figure supplement 3 . Mutual information between edge features across the hypocotyl cell connectivity network . Estimated mutual information between shared cell wall surface area and edge betweenness centrality in ( A ) three A . thaliana ecotypes: Col , Ler and Cvi , ( B ) other plant species: poppy and foxglove , and ( C ) three A . thaliana mutants: cdka1;1 , monopteros and laterne . DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 024 The spatial distribution of degree and BC is unevenly distributed within individual cells of a given cell type in all samples examined ( Figures 2 and 4–5 ) .", "This highlights the presence of topological variability in cell organization within these organs .", "We explored the extent of the variability present in BC in individuals across the Arabidopsis ecotypes , different species and cellular patterning mutants .", "The coefficient of variation for BC for each cell type was plotted to examine the extent of the variability in the creation of path lengths between individual hypocotyls ( Figure 7A–B and Figure 7—figure supplement 1 ) .", "Both epidermal cell types across all samples examined showed no significant differences in their robustness to generating consistent path lengths relative to one another , with the exception of the mp mutant which had a greater extent of variability ( Figure 7A–B ) .", "Similar results were observed in the cortical cell layers of this mutant relative to other genotypes examined ( Figure 7—figure supplement 1 ) .", "The MONOPTEROS gene product therefore promotes patterning robustness and the generation of consistent path lengths in Arabidopsis hypocotyls with regards to organ-level cell topology . 10 . 7554/eLife . 26023 . 025Figure 7 . Robustness in cellular patterning and topological properties in the plant hypocotyl . Comparisons of the coefficient of variation ( CV ) of betweenness centrality in different cell types of hypocotyls from A . thaliana ecotypes ( Col , Ler and Cvi ) , mutants ( cdka1;1 , monopteros and laterne ) and other plant species ( poppy and foxglove ) .", "( A ) Mean CV in trichoblast cells .", "( B ) Mean CV in atrichoblast cells .", "Data represented are the means and standard deviation of three biological replicates .", "ANOVA tests suggested significant differences between groups ( p≤0 . 01 ) and an asterisk ( * ) indicates significant difference to all other groups ( Tukey’s test , p≤0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 02510 . 7554/eLife . 26023 . 026Figure 7—figure supplement 1 . Variability in hypocotyl betweenness centrality . Relative variability is measured using the coefficient of variation ( CV ) , which is the standard deviation of a group divided by the mean .", "( A ) The mean coefficient of variability of log10betweenness centrality in all hypocotyl connectivity network samples , in outer cortical cells .", "( B ) The mean coefficient of variability of log10betweenness centrality in all hypocotyl connectivity network samples , in inner cortical cells .", "( C ) The mean coefficient of variability of log10betweenness centrality in all hypocotyl connectivity network samples , in the endodermis .", "Error bars represent ± the standard deviation in CV measurements ( n = 3 for each ecotype , species or mutant hypocotyl ) .", "ANOVA tests suggested significant differences between groups ( outer cortex , inner cortex: p≤0 . 01 , endodermis p≤0 . 05 ) and different lowercase letters represent statistically significant differences between means ( Tukey’s HSD , p≤0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 026 Beyond the epidermis , cell type-specific data describing local connectivity and path length in other cell types were generated .", "Their analysis provides the opportunity to understand the overarching principles underlying the assembly of cells in this organ across diverse genetic backgrounds and species .", "Across Arabidopsis genotypes , degree distributions show no significant differences in either cortical cell layers or the endodermis ( Figure 2—figure supplement 1 ) , indicating a robust control in local connectivity within the internal cell layers of this species .", "Fewer significant differences in BC distribution are seen from the outer to inner cell layers , with no significant differences being observed in the endodermis across ecotypes ( Figure 2—figure supplement 1 ) .", "Degree distribution also shows no significant difference across all cell types in foxglove , poppy and Arabidopsis Col hypocotyls , with the exception of the endodermis ( Figure 4—figure supplement 1 ) .", "BC distribution is the same in all species within in the inner cortex , however the endodermis shows some significant differences highlighting the presence of plasticity in path length generation ( Figure 4—figure supplement 1 ) .", "In contrast , the outer cortex showed similar path lengths across all species investigated .", "To better understand the relationship between the topological properties of different cell types and how both degree and BC change across the radial arrangement of the hypocotyl , scatterplots of each mean and median average value , respectively , were generated to visualize these relationships ( Figure 8A–E ) .", "Path length remains relatively constant within both epidermal cell types across species and genetic backgrounds , however degree shows a relatively broader range ( Figure 8A–B ) .", "The degree to BC relationship in the inner cortex contrasts this with mean degree being relatively constant across genetic backgrounds , and path length showing a greater relative spread in values ( Figure 8D ) .", "Each the outer cortex and endodermis have a relatively broad range of degree and BC across the samples analysed .", "These observations suggest that the epidermal cell types are topologically distinct , and that the outer cortex and endodermis are more similar to one another than to the inner cortex .", "In all instances monopteros samples showed a broad distribution of degree and BC values , highlighting the topologically unique nature of this mutant and a lack of robustness in its global pattern formation ( Figure 7 ) . 10 . 7554/eLife . 26023 . 027Figure 8 . Cell type-specific topological principles of hypocotyl multicellular architecture .", "( A–E )", "Scatterplots of mean degree and median betweenness centrality in ( A ) trichoblasts , ( B ) atrichoblasts , ( C ) , the outer cortex , ( D ) the inner cortex and ( E ) the endodermis for each sample used in this study .", "( F–G )", "Analysis of degree distribution relationships using the inverse of the chi-squared test statistic as a measure of distance .", "( F ) pairwise significance tests for degree distribution and ( G ) a dendrogram of the data from ( F ) .", "( H–I )", "Analysis of betweenness centrality distributions using the inverse of the Kolmogorov–Smirnov test statistic as a measure of distance , with ( H ) pairwise significance tests and ( I ) a dendrogram of the data from ( H ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26023 . 027 While informative , the use of mean values as a summary statistic in scatterplots fails to capture the breadth of the data represented within a distribution .", "To address this we performed pairwise statistical tests for each degree and BC distribution , respectively , for all cell types to determine whether their topological properties were significantly similar or not .", "The test statistic obtained by comparing two distributions ( chi-squared value for the discrete variable degree , Kolmogorov–Smirnov test value for the continuous variable betweenness centrality ) was used as a measure of the distance between the distributions .", "These distances were used to generate a distance matrix , enabling their clustering .", "Significant relationships between degree distributions were calculated and were placed into three clusters ( Figure 8F ) .", "Each wild-type atrichoblast and trichoblast cells clustered independently ( Figure 8G ) .", "All other cell types were present in the third group , with the exception of the poppy endodermis .", "This revealed that each epidermal cell type has a distinct local connectivity , while all other internal cell types share similar properties in this regard .", "Degree alone may therefore distinguish different epidermal cell types , and cells internal to the epidermis .", "The clustering of significant similarities between BC distributions enabled the relationships between the higher-order properties of diverse cell types across genetic backgrounds to be established .", "Three clusters were selected to categorize these pairwise similarities ( Figure 8H ) .", "All wild-type trichoblasts , with exception of poppy , cluster together ( Figure 8I ) .", "This is likely a consequence of the stringent regulation of path length we previously identified to be present within this cell type , and increased path length within the poppy epidermis ( Figure 2—figure supplement 1 , Figure 4—figure supplement 1 ) .", "Conversely , atrichoblasts have greater plasticity in their path length across genetic backgrounds ( Figure 2—figure supplement 1 , Figure 4—figure supplement 1 ) and are more broadly distributed across the clustering diagram .", "Arabidopsis Cvi and Foxglove atrichoblasts remained in the trichoblast cluster as these do not show a significant path length differential between them ( Figures 2E and 4E ) .", "These relationships between epidermal cell path lengths reflect the previously observed higher-order properties of this cell type .", "All wild-type outer cortical cells clustered together along with Arabidopsis endodermal cells , which have similar path lengths ( Figure 8I ) , supporting earlier conclusions relating to the topological relationship between these two cell types .", "Inner cortical cells also clustered together highlighting their topological uniqueness within the hypocotyl .", "Collectively these results suggest that each epidermal cell type is topologically distinct from all other cell types , as is the inner cortex .", "The outer cortex and endodermis share global organizational similarities .", "These observations reveal general principles of multicellular architectural design within the wild-type plant hypocotyl .", "It is through these higher-order path length principles that the movement of molecules occurs , bridging a structure-function relationship between cellular patterning and the molecular processes which unfold within complex plant organs .", "Understanding these principles and the ability to calculate them at single cell resolution enables the mathematical prediction of intercellular and optimized molecular flux and intercellular communication which underlies plant growth and development ." ], [ "Extensive research into cellular patterning has been undertaken previously in diverse biological systems .", "In both plants and animals , rules governing the placement of cell division planes in a default state have been described ( Besson and Dumais , 2011; Hofmeister , 1867 ) and are sufficient to explain pattern formation in contexts including the shoot apical meristem of plants ( Sahlin and Jönsson , 2010; Shapiro et al . , 2015 ) and Drosophila development ( Gibson et al . , 2011 ) .", "Deviations from this default rule have been considered to be actively regulated cell divisions that lead to the generation of novel patterns .", "In the context of plant embryo development , this has been reported to be mediated by the hormone auxin ( Yoshida et al . , 2014 ) and downstream factors controlling auxin-mediated patterning have been described to be involved in diverse aspects of this formation ( Schlereth et al . , 2010 ) .", "In addition to genetically controlled events , a role for mechanical feedbacks in the control of cellular patterning have also been reported in plants ( Hamant et al . , 2008; Louveaux et al . , 2016 ) .", "While the mechanisms underlying the creation of cellular patterns have been studied widely , bridging their structure-function relationship , and understanding of the functional role of these patterns in organ biology remains less clear .", "The network-based analysis of cellular patterning can bridge this gap by enabling cellular patterning to be captured and quantified .", "This approach was originally applied in the field of neuroscience where global neuronal connectivity was mapped in the worm C . elegans ( White et al . , 1986 ) .", "This approach has since been employed to more complex nervous systems in the growing field of connectomics ( Bullmore and Sporns , 2009 ) .", "Methodological imaging and computational advances are now leading to the generation of cellular resolution connectomes in complex brains ( Economo et al . , 2016 ) , though that of C . elegans remains the only complete connectome to date .", "The immobility of cells within plant organs simplifies the topological analysis of multicellular arrangements as cellular associations are maintained throughout development .", "The application of this approach to other biological systems where cell migration occurs is also possible , and would involve the analysis of 3D cellular interaction dynamics ( Heller et al . , 2016 ) .", "The transient nature of edges in these systems will dynamically alter the topology of these cellular interaction networks .", "Analytical frameworks to analyze temporal network datasets such as these have been developed ( Holme and Saramäki , 2012 ) .", "The analysis of animal epithelium development has also been studied using a network-based topological approach .", "Local epithelium cellular connectivity was sufficient to distinguish different species and organs ( Escudero et al . , 2011 ) .", "A separate study in Drosophila imaginal discs revealed cell degree to be tightly regulated during the development of this tissue ( Gibson et al . , 2006 ) .", "These same authors then extended this approach to identify a bias in the placement of the cleavage plane following the degree of adjacent cells during pattern formation , a phenomenon also present in the shoot apex of plants ( Gibson et al . , 2011 ) .", "In each of these studies , local connectivity was examined , with the global higher-order principles of organ design remaining unexplored .", "By viewing organs as a complex system of interacting cells , this study applied a systems-based approach to understand the quantitative principles of multicellularity and uncover their higher order properties .", "The capture and analysis of organism-wide cellular connectivity enabled both the local and global topological features of complex organism design to be investigated , revealing how multicellular assemblies operate as an integrated system and their emergent properties .", "The application of cell-type specific topological analysis using BC identified the presence of a previously undescribed optimized transportation route in the atrichoblast epidermal cell files of the Arabidopsis hypocotyl in the Col and Ler ecotypes ( Figure 2E ) .", "This cell type is uniquely poised to regulate information flow along the hypocotyl by lying on shorter paths , despite having fewer local neighbours ( Figure 2D–E ) .", "This property was not present in Arabidopsis Cvi , nor in other species studied including poppy and foxglove ( Figure 4E ) .", "This previously undescribed higher order property of epidermal patterning is therefore plastic across diverse genetic backgrounds .", "The functional significance of these reduced path lengths was demonstrated by tracking the movement of the fluorescent small molecule fluorescein through Arabidopsis seedlings ( Figure 3 ) .", "BC predicted the bulk flow of these small molecules through diverse epidermis topologies at single cell resolution .", "These patterns therefore provide optimized routes for molecular trafficking through organs , though they are not obligatory for organ function in light of the plastic nature of their presence .", "The reduction of epidermal path length may support rapid plant growth , or represent a recent evolutionary innovation of higher-order multicellular optimization that has not yet been widely adopted across diverse genetic backgrounds .", "The topological analysis of patterning mutant hypocotyls revealed the quantitative contribution of genetically-encoded factors towards the creation of higher-order features at cell type-specific resolution ( Figure 5 ) .", "The construction of reduced atrichoblast path length was established to be mediated by the patterning activity of the CDKA1;1 gene , and overall epidermal path length is limited by MONOPTEROS and promoted in laterne ( Figure 5D–F ) .", "This approach provides a step change beyond qualitative descriptions of altered local cellular organization , and enables quantitative links between the molecular and cellular scales of complexity to be bridged .", "The genetic contribution towards the regulation of consistent global path length in the hypocotyl epidermis also revealed an additional link between the activity of patterning genes and control over robustness in the generation of higher order path length ( Figure 7 ) .", "This provided novel insight into the role of MONOPTEROS in the control of topological robustness in cellular patterning during embryogenesis .", "These observations further demonstrated that while epidermal path length is plastic between genetic backgrounds , it is not variable across individuals .", "The structural networks generated in this study may be considered the roadmaps upon which molecular events occur within the hypocotyl .", "The annotation of these structural templates with additional molecular information will lead to the creation of functional networks , which will provide multidimensional insight into the molecular complexity which unfolds in the context of multicellular organ development .", "Uncovering the organizing principles ( Sena and Birnbaum , 2010 ) of multicellular assemblies in extant organs provides insight into the cellular configurations which are each selected for in wild-type plants , and which are possible in the case of patterning mutants ( Avena-Koenigsberger et al . , 2015 ) .", "These cellular interaction networks result from pattern formation , and represent the topological output of the self-organizing processes which underlie plant organ formation .", "Uncovering nature’s multicellular structural design principles and the functions they encode will provide fundamental insight into the evolutionary forces driving increased complexity and optimization following the transition to multicellularity ( Ollé-Vila et al . , 2016 ) .", "This enhanced understanding of these properties establishes a framework for the rational re-design of multicellular configurations and organ functionality ." ], [ "Arabidopsis wild-type plants of ecotypes Colombia , Cape Verdi Islands ( Cvi ) and Landsberg erecta ( Ler ) and associated mutants laterne ( Treml et al . , 2005 ) , monopteros ( mp B4149 ) ( Schlereth et al . , 2010 ) and cdka1;1 ( ProCDKA;1::CDKA;1-T14D;Y15E ) ( Dissmeyer et al . , 2009 ) were grown in environmentally controlled cabinets using 16 hr light ( light intensity 150–175 μmol m2s−1 at 23°C and 8 hr dark at 18°C ) .", "Plants with dry siliques were harvested and seeds cleaned through a 500 μm mesh .", "Poppy ( Papaver rhoeas ) seeds were grown under field conditions , and foxglove ( Digitalis purpurea ) was collected from the Birmingham Botanical Gardens .", "Embryos were dissected from germinating seeds with a scalpel and forceps using a binocular microscope at 3 hr following their imbibition .", "Samples were prepared for confocal microscopy as described previously ( Bassel et al . , 2014; Truernit et al . , 2008 ) and imaged using a Zeiss LSM 710 confocal microscope .", "This was performed as described previously ( Montenegro-Johnson et al . , 2015 ) .", "Cell type designation was achieved by using CellAtlas3D , followed by manual corrections .", "Epidermal cell types were assigned based on their positions above underlying cortical cell layers , with trichoblasts bridging multiple underlying cortical cell files .", "After using 3DCellAtlas , further manual annotation was based on position in the hypocotyl and cell shape .", "Shape became particularly important in assigning cell types in cdka1;1 and monopteros mutant samples , where endodermal cells are not present throughout the entire hypocotyl .", "Cellular interaction networks were exported from MorphoGraphX as text files with shared associations between cells as edge weights in μm2 .", "Edge lists were imported into the Python package NetworkX ( Hagberg et al . , 2008 ) where analyses were performed .", "Text files were in turn imported into MorphoGraphX as heat maps to visualize node properties in situ by false coloring segmented cells .", "Edge effects in topological analysis due to imaging boundaries were minimized by including all cells in topological analyses , but only examining the properties of those in the central regions ( Figure 1—figure supplement 1 ) .", "Vascular cells were captured and included in topological analyses , though quantitative data were not reported due to the presence of sporadic cellular segmentation errors .", "Biological triplicates were used across this study .", "To account for imaging artifacts affecting cellular connectivity , edges representing the connection of two adjacent cells which shared less than 2 µm² cell wall area were removed between all cell types except the vasculature ( Figure 1—figure supplement 1 ) .", "BC for mean and standard deviation calculations was log10 transformed to generate normal distributions .", "3D networks were visualized using BioLayout3D ( Theocharidis et al . , 2009 ) .", "All network data are available in Source Dataset .", "To account for differences in network size across samples , node betweenness centrality was normalized by 2/ ( ( N−1 ) ( N−2 ) ) , and edge betweenness centrality was normalized by 2/N ( N−1 ) ) where N is the number of nodes in a network .", "Due to inherent constraints of physical Euclidean space , centrality was not normalized by network density in the hypocotyl samples .", "Where networks were weighted , the inverse of the absolute connecting cell wall area ( µm² ) was used in the calculation of weighted betweenness centrality ( Newman , 2010 ) .", "Estimated mutual information ( Shannon entropy ) was calculated using the Non-Parametric Entropy Estimation Toolbox ( NPEET ) , a Python package that incorporates functions for both continuous ( betweenness centrality , area , volume ) and discrete ( degree ) variables ( Kraskov et al . , 2004 ) .", "Two-sample t-tests , chi-squared tests and two-sample Kolmogorov-Smirnov tests were performed using Python and the SciPy library ( Jones et al . , 2001 ) .", "ANOVA with Tukey’s HSD post-hoc tests were carried out using SPSS .", "Hierarchical clustering was performed using Python and the scikit-learn package ( Pedregosa et al . , 2011 ) .", "The Bonferroni correction for multiple comparisons was used to correct the p-value in every chi-sqaured test and Kolmogorov-Smirnov test , based on the 3200 pairwise comparisons used throughout the study .", "The three shown ecotypes were germinated in 1/2 MS plates in complete darkness for 4 days , then exposed for 2 . 5 hr to 0 . 8% agar plates containing 50 μM fluorescein ( Sigma Aldrich ) .", "Before imaging by fluorescence confocal microscopy , samples were treated with ( 10 mg/mL ) propidium iodide to visualize cell walls .", "3D cell segmentation and quantification of fluorescence density within each cell was performed using FIJI ( Schindelin et al . , 2012 ) and MorphoGraphX ( de Reuille et al . , 2015 ) .", "Cells were meshed using a cube size of 2 and no smooth passes were used .", "Fluorescein concentration within individual cells was calculated using the Heat Map function with Volume and Internal Signal selected .", "Data presented consist of at least 40 segmented cells from three independent biological replicates for each ecotype ." ] ]

[ "Multicellularity arose as a result of adaptive advantages conferred to complex cellular assemblies .", "The arrangement of cells within organs endows higher-order functionality through a structure-function relationship , though the organizational properties of these multicellular configurations remain poorly understood .", "We investigated the topological properties of complex organ architecture by digitally capturing global cellular interactions in the plant embryonic stem ( hypocotyl ) , and analyzing these using quantitative network analysis .", "This revealed the presence of coherent conduits of reduced path length across epidermal atrichoblast cell files .", "The preferential movement of small molecules along this cell type was demonstrated using fluorescence transport assays .", "Both robustness and plasticity in this higher order property of atrichoblast patterning was observed across diverse genetic backgrounds , and the analysis of genetic patterning mutants identified the contribution of gene activity towards their construction .", "This topological analysis of multicellular structural organization reveals higher order functions for patterning and principles of complex organ construction ." ]

[ "In plants and other multicellular organisms , cells work together in tissues and organs to achieve outcomes that they would not be able to accomplish on their own .", "For example , the cells that make up the stem of a plant hold the leaves , flowers and other organs in place , and provide a transport network that allows molecules to move around the plant .", "Mapping the locations of cells within tissues and analysing the connections between them will help researchers to understand how the organisation of tissues influences the tasks cells perform .", "There are several different layers of tissue within a plant’s stem .", "The surface of the stem has a protective layer of tissue called epidermis .", "The epidermis contains two different types of cells known as trichoblasts and atrichoblasts , but it was not clear why these cells are organised the way they are .", "Arabidopsis thaliana is a small plant that is often used in studies of how plants grow and develop .", "Jackson et al . combined microscopy with computational techniques to study the stems of young A . thaliana seedlings .", "The experiments reveal that the two types of epidermal cells appear to adopt distinct roles .", "The trichoblasts form hair-like structures and acquire nutrients from the external environment , while their neighbours the atrichoblasts provide shortcut routes for these nutrients to be unloaded and moved up the stem .", "This pattern was not present in several other plant species including foxglove or poppy , suggesting it may be an adaptation in A . thaliana plants that helps them grow in the particular environments this plant faces .", "The findings of Jackson et al . show that cells are carefully arranged in plant stems and suggest that there is an optimal way for a plant to make a stem depending on its environment .", "Further work is now needed to understand how different molecules use the shortcuts provided by the atrichoblasts during plant development , and whether alternative configurations are possible .", "In the future , such studies may help provide a framework to genetically engineer plants that are better adapted to grow in different environments ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "Viruses can cause massive and rapid changes in a cell’s transcriptome as they churn out viral mRNAs and hijack cellular machinery .", "For instance , cells infected with influenza virus at high multiplicity of infection ( MOI ) express an average of 50 , 000 to 100 , 000 viral mRNAs per cell , corresponding to 5% to 25% of all cellular mRNA ( Hatada et al . , 1989 ) .", "Infection can also trigger innate-immune sensors that induce the expression of cellular anti-viral genes ( Killip et al . , 2015; Iwasaki and Pillai , 2014; Crotta et al . , 2013 ) .", "This anti-viral response is another prominent transcriptional signature of high-MOI influenza virus infection in bulk cells ( Geiss et al . , 2002 ) .", "However , initiation of an actual influenza infection typically involves just a few virions infecting a few cells ( Varble et al . , 2014; Poon et al . , 2016; Sobel Leonard et al . , 2017; McCrone et al . , 2017 ) .", "The dynamics of viral infection in these individual cells may not mirror bulk measurements made on many cells infected at high MOI .", "Over 70 years ago , Max Delbruck showed that there was a ∼100-fold range in the number of progeny virions produced per cell by clonal bacteria infected with clonal bacteriophage ( Delbrück , 1945 ) .", "Subsequent work has shown similar heterogeneity during infection with other viruses ( Zhu et al . , 2009; Schulte and Andino , 2014; Combe et al . , 2015; Akpinar et al . , 2015 ) , including influenza virus ( Heldt et al . , 2015 ) .", "In the case of influenza virus infection , targeted measurements of specific proteins or RNAs have shed light on some factors that contribute to cell-to-cell heterogeneity .", "The influenza virus genome consists of eight negative-sense RNA segments , and many infected cells fail to express one more of these RNAs ( Heldt et al . , 2015; Dou et al . , 2017 ) or their encoded proteins ( Brooke et al . , 2013 ) .", "In addition , activation of innate-immune responses is inherently stochastic ( Shalek et al . , 2013; Shalek et al . , 2014; Bhushal et al . , 2017; Hagai et al . , 2017 ) , and only some influenza-infected cells express anti-viral interferon genes ( Pérez-Cidoncha et al . , 2014; Killip et al . , 2017 ) .", "However , the extent of cell-to-cell variation in these and other host and viral factors remains unclear , as does the association among them in individual infected cells .", "Here we use single-cell mRNA sequencing to quantify the levels of all cellular and viral mRNAs in cells infected with influenza virus at low MOI .", "We find extremely large variation in the amount of viral mRNA expressed in individual cells .", "Both co-infection and activation of innate-immune pathways are rare in our low-MOI infections , and do not appear to be the major drivers of cell-to-cell heterogeneity in viral transcriptional load .", "Individual infected cells often fail to express specific viral genes , and such gene absence explains some but certainly not all of the cell-to-cell heterogeneity .", "A variety of cellular genes , including ones involved in the oxidative-stress response , co-vary with viral transcriptional load .", "Overall , our work demonstrates remarkable heterogeneity in the transcriptional outcome of influenza virus infection among nominally identical cells infected with a relatively pure population of virions ." ], [ "We performed single-cell mRNA sequencing using a droplet-based system that physically isolates individual cells prior to reverse transcription ( Zheng et al . , 2017; Macosko et al . , 2015; Klein et al . , 2015 ) .", "Each droplet contains primers with a unique cell barcode that tags all mRNAs from that droplet during reverse-transcription .", "Each primer also contains a unique molecular identifier ( UMI ) that is appended to each mRNA molecule during reverse transcription .", "The 3’ ends of the mRNAs are sequenced and mapped to the human and influenza virus transcriptomes to determine transcript identities .", "This information is combined with that provided by the UMIs and cell barcodes to quantify the number of molecules of each mRNA species that have been captured for each cell .", "Infected cells will express viral as well as cellular mRNAs – however the cell barcodes and UMIs cannot distinguish whether a cell was initially infected by one or multiple viral particles .", "We therefore engineered an influenza virus ( strain A/WSN/1933 ) that additionally carried viral barcodes consisting of synonymous mutations near the 3’ end of each transcript ( Figure 1A ) .", "Critically , these synonymous mutations did not greatly impact viral growth kinetics ( Figure 1B ) .", "We infected A549 human lung carcinoma cells with an equal mix of the wild-type and synonymously barcoded viruses .", "Cells infected by a single virion will exclusively express mRNAs from either wild-type or synonymously barcoded virus , whereas cells that are co-infected with multiple virions will often express mRNAs from both the wild-type and synonymously barcoded viruses ( Figure 1C ) .", "We took care to generate stocks of virus that were relatively ‘pure’ of defective particles .", "Stocks of viruses typically contain an array of biologically active viral particles , some of which are defective for replication owing to mutations or deletions in essential viral genes ( von Magnus , 1954; Huang and Baltimore , 1970; Brooke , 2014; Fonville et al . , 2015; Lauring and Andino , 2010; Dimmock et al . , 2014; Saira et al . , 2013 ) .", "These defective particles become prevalent when a virus is grown at high MOI , where complementation permits the growth of otherwise deleterious genotypes .", "To minimize the levels of defective particles , we propagated our viral stocks at low MOI for a relatively brief period of time ( Xue et al . , 2016 ) .", "We validated that our stocks exhibited greater purity of infectious particles than a stock propagated at high MOI by verifying that they had a higher ratio of infectious particles to virion RNA ( Figure 2A ) and to particles capable of inducing expression of a single viral protein ( Figure 2B ) .", "In addition , viral stocks with many defective particles are more immunostimulatory per infectious unit ( e . g . , TCID50 ) than low-defective stocks ( Tapia et al . , 2013; López , 2014 ) , in part simply because there are more physical virions per infectious unit ( Figure 2A , B ) .", "We confirmed that our viral stocks induced less interferon per infectious unit than a stock propagated at higher MOI ( Figure 2C ) .", "We infected A549 cells at low MOI with a mixture of the wild-type and synonymously barcoded viruses , and collected cells for sequencing at 6 , 8 , and 10 hr post-infection , performing two slightly different variants of the experiment for the 8 hr timepoint .", "For most of the samples , we replaced the infection inoculum with fresh media at one-hour post-infection , thereby ensuring that most infection was initiated during a narrow time window .", "However , for the second 8 hr sample ( which we denote as ‘8 hr-2’ in the figures ) , we did not perform this media change and instead left the cells in the original infection inoculum .", "The rationale for including a sample without a media change was to determine the importance of synchronicity of the timing of infection as discussed later in this subsection .", "We recovered between 3000 and 4 , 000 cells for each sample ( Figure 3A ) .", "As expected for a low-MOI infection , most cells expressed little or no viral mRNA ( Figure 3B , Figure 3—figure supplement 1 ) .", "Also as expected , the amount of viral mRNA per cell among infected cells increased over time ( Figure 3B , Figure 3—figure supplement 1 ) .", "But what was most notable was how widely the number of viral mRNA molecules varied among infected cells .", "While the fraction of mRNA derived from virus was <0 . 1% for most cells , viral mRNA constituted half the transcriptome in a few cells at 8 and 10 hr ( Figure 3B , Figure 3—figure supplement 1 ) .", "A complicating factor is that uninfected cells could have small amounts of viral mRNA due to leakage of transcripts from lysed cells .", "It is therefore important to establish a threshold for identifying truly infected cells .", "We can do this by taking advantage of the fact that roughly half the infecting virions bear synonymous barcodes .", "Reads derived from lysed cells will be drawn from both wild-type and synonymously barcoded viral transcripts .", "However , most cells are infected by at most one virion , and so the reads from truly infected cells will usually derive almost entirely from one of the two viral variants .", "Figure 4A shows the fraction of viral reads in individual cells from each viral variant , and Figure 4B indicates the fraction of viral reads from the most abundant variant in that cell .", "Most cells with large amounts of viral mRNA have viral transcripts exclusively derived from one viral variant – indicating non-random partitioning as expected from viral infection .", "However , cells with a small amount of viral mRNA often have viral transcripts from both variants , as expected from the random partitioning associated with simple mRNA leakage .", "Finally , a few cells with large amounts of viral mRNA have viral transcripts from both variants , likely reflecting co-infection .", "We determined the threshold amount of viral mRNA per cell for each sample at which the barcode partitioning clearly resulted from infection rather than leakage ( Figure 4C , Figure 4—figure supplement 2 ) , and used these thresholds to annotate cells that we were confident were truly infected .", "We also annotated as co-infected cells above this threshold that had mRNA from both viral variants .", "Figure 4D shows the number of cells annotated as infected and co-infected for each sample – these cells are just a small fraction of the number of cells with any viral read .", "These annotation thresholds are conservative , and may miss some true low-level infections .", "However , it is important that the analyses below are restricted to cells that are truly infected with virus , so we accepted the possible loss of some low-level infections in order to avoid false positives .", "In addition , the synonymous viral barcodes only identify co-infections by viruses with different barcodes – since the barcodes are at roughly equal proportion , we expect to miss about half of the co-infections .", "Since we annotate about ∼10% of the infected cells as co-infected by viruses with different barcodes ( Figure 4D ) , we expect another ∼10% of the infected cells to also be co-infected but not annotated as so by our approach .", "Because most cells are not infected , we subsampled the uninfected cells to the numbers shown in Figure 4D to balance the proportions of infected and uninfected cells for all subsequent analyses .", "Strikingly , the extreme variation in the number of viral transcripts per cell remains even after we apply these rigorous criteria for annotating infected cells ( Figure 4E ) .", "The fraction of viral mRNA per infected cell follows a roughly exponential distribution , with many cells having few viral transcripts and a few cells having many .", "At 6 and 8 hr <10% of infected cells are responsible for over half the viral transcripts , while at 10 hr <15% of infected cells produce over half the viral transcripts ( Figure 4—figure supplement 3 ) .", "One way to quantify the heterogeneity of a distribution is to calculate the Gini coefficient ( Gini , 1921 ) , which ranges from 0 for a completely uniform distribution , to one for a maximally skewed distribution .", "Figure 4—figure supplement 3 shows the Gini coefficients for the distribution of viral mRNA across infected cells for each sample .", "The Gini coefficients are ≥0 . 64 for all samples .", "As a fun point of comparison , these Gini coefficients indicate that the distribution of viral mRNA across infected cells is more uneven than the distribution of income in the United States ( Alvaredo , 2011 ) .", "One possible source of heterogeneity in the amount of viral mRNA per cell is variability in the timing of infection .", "If some cells are infected earlier in the experiment than others , then they might have substantially more viral mRNA .", "However , several lines of evidence indicate that this is not the major cause of heterogeneity across cells .", "First , the sample for which the infection inoculum was never removed ( 8 hr-2 ) only shows slightly more heterogeneity than samples for which the inoculum was washed away after one hour ( Figure 4E , Figure 4—figure supplement 3 ) , despite the fact that the potential time window for infection is much longer in the former sample .", "Second , in an independent experiment , we performed completely synchronized infections by pre-binding virus to cells on ice and then washing away unbound virus before bringing the cells to 37∘C ( Dapat et al . , 2014 ) .", "As shown in Figure 4—figure supplement 4 , flow cytometry staining found that the heterogeneity in the levels of individual viral proteins was not markedly different for these synchronized infections than in the absence of pre-binding and washing .", "Finally , viral mRNA expression from the secondary spread of virus from infected cells does not appreciably occur during the timeframes of our experiments , since Figure 4B does not show the pervasive presence of mixed barcodes that would occur in this case .", "Therefore , variability in the timing of infection is not the dominant cause of the cell-to-cell heterogeneity in our experiments .", "Notably , Figure 4E shows that there are co-infected cells with both low and high amounts of viral mRNA , suggesting that the initial infectious dose does not drive a simple continuous increase in viral transcript production .", "In support of this view , we used flow cytometry to quantify the levels of individual viral proteins in cells infected at various MOIs or for which we could delineate co-infection status ( Figure 4—figure supplement 5 ) .", "This analysis shows that sub-populations of cells that express similarly low and high levels of viral proteins persist across a wide range of infectious doses , although co-infection can influence the relative proportion of infected cells that fall into these sub-populations ( Figure 4—figure supplement 5 ) .", "The influenza genome is segmented , and cells can fail to express a viral mRNA if the encoding gene segment is not packaged in the infecting virion or fails to initiate transcription after infection .", "Indeed , several groups have reported that the majority of infected cells fail to express at least one viral gene ( Brooke et al . , 2013; Heldt et al . , 2015; Dou et al . , 2017 ) .", "We wondered if the absence of specific viral genes might be associated with reduced amounts of viral mRNA within single infected cells .", "In particular , transcription of influenza virus mRNAs is performed by the viral ribonucleoprotein ( RNP ) complex , which consists of the three proteins that encode the tripartite polymerase ( PB2 , PB1 , and PA ) as well as nucleoprotein ( NP ) ( Huang et al . , 1990 ) .", "Each viral gene segment is associated with one RNP in incoming infecting virions , but secondary transcription by newly synthesized RNPs requires the presence of the viral genes encoding each of the four RNP proteins ( Vreede et al . , 2004; Eisfeld et al . , 2015 ) .", "This secondary transcription is a major source of viral mRNAs , as evidenced by the fact that blocking synthesis of the RNP proteins reduces the amount of viral mRNA by several orders of magnitude in bulk cells ( Figure 5—figure supplement 1 ) .", "We examined the total amount of viral mRNA versus the expression of the genes from each viral segment ( Figure 5A , Figure 5—figure supplement 2 , Figure 5—figure supplement 3 ) .", "Note that influenza virus expresses ten major gene transcripts from its eight gene segments , as the M and NS segments are alternatively spliced to produce the M1/M2 and NS1/NEP transcript , respectively ( Dubois et al . , 2014 ) .", "However , an inherent limitation of current established single-cell mRNA sequencing techniques is that they only sequence the 3’ end of the transcript ( Zheng et al . , 2017; Macosko et al . , 2015; Klein et al . , 2015; Cao et al . , 2017 ) .", "Since the alternative spliceoforms M1/M2 and NS1/NEP share the same 3’ ends , we cannot distinguish them and therefore will refer simply to the combined counts of transcripts from each of these alternatively spliced segments as the M and NS genes .", "Cells that lack an RNP gene never derive more than a few percent of their mRNAs from virus , confirming the expected result that all four RNP genes are essential for high levels of viral transcription ( Figure 5A , Figure 5—figure supplement 2 , Figure 5—figure supplement 3 ) .", "However , we observe cells that lack each of the other non-RNP genes but still derive ≈40% of their mRNAs from virus , suggesting that none of the other genes are important for high levels of viral transcription .", "These results are statistically supported by Figure 5B , which shows that absence of any RNP gene but not any other viral gene is associated with reduced amounts of viral mRNA .", "However , gene absence clearly does not explain all of the variability in viral gene expression , since even cells expressing all viral genes exhibit a very wide distribution in the amount of viral mRNA that they express .", "Specifically , at both 8 and 10 hr , the amount of viral mRNA in individual cells expressing all eight viral genes still ranges from <1% to >50% ( Figure 5A , Figure 5—figure supplement 2 , Figure 5—figure supplement 3 ) .", "Furthermore , the actual distribution of viral mRNA per infected cell ( Figure 4E ) does not match the mostly bi-modal shape expected under a simple model where RNP gene absence and Poisson co-infection are the only factors ( Figure 5—source data 2 ) , indicating that there are additional sources of variability beyond whether cells have full complement of RNP genes .", "We also quantified the fraction of infected cells that completely failed to express a given gene .", "We limited this analysis to examining the presence/absence of the non-RNP genes in cells expressing all four RNP genes , since we might fail to detect viral transcripts that are actually present at low levels in RNP-deficient cells due to the lower viral burden in these cells .", "At the 8- and 10 hr time points , between 5% and 17% of cells fail to express any one of the four non-RNP genes ( Figure 5C , Figure 5—source data 1 ) .", "The absence of a given gene appears to be an independent event , as the probability of observing all four non-RNP genes in a cell is well predicted by simply multiplying the probabilities of observing each gene individually ( Figure 5C and Figure 5—source data 1 ) .", "If we extrapolate the frequencies at which cells lack non-RNP genes to the RNP genes , then we would predict that 35–50% of infected cells express mRNAs from all eight genes .", "This estimate of the frequency at which infected cells express mRNAs from all eight gene segments is slightly higher than previous estimates of 13% ( Brooke et al . , 2013 ) and 20% ( Dou et al . , 2017 ) .", "At least one difference is that Brooke et al . ( Brooke et al . , 2013 ) stained for proteins whereas we examined the expression of mRNAs – it is likely that some cells contain mutated viral genes that fail to produce stable protein even when mRNA is expressed .", "The results above show that the amount of viral mRNA in infected cells varies over several orders of magnitude .", "Does the relative expression of viral genes exhibit similar cell-to-cell variability ?", "To address this question , we focused on cells that derived >5% of their mRNA from virus , since estimates of relative viral gene expression will be less noisy in cells with more viral mRNAs .", "In contrast to the extreme variability in the total viral mRNA per cell , the fraction of this viral mRNA derived from each gene is much more consistent across cells ( Figure 6A ) .", "Total viral mRNA varies by orders of magnitude , but the fraction from any given viral gene is fairly tightly clustered around the median value for all cells ( Figure 6B ) .", "The relative levels of each viral mRNA in our cells are similar to prior bulk measurements made by Northern blots ( Hatada et al . , 1989 ) , which also found an expression hierarchy of M > NS ≫ NP > NA > HA ≫ PB2 ∼ PB1 ∼ PA .", "The cell-to-cell consistency in the relative expression of different viral genes is even tighter if we limit the analysis only to cells that express all eight viral genes ( Figure 6C ) .", "Therefore , with the exception of complete gene absence , the factors that drive the dramatic cell-to-cell variability in the amount of viral mRNA have roughly similar effects on all viral genes in a given cell .", "This finding is consistent with prior work showing positive correlations among the abundance of several viral genome segments in individual cells ( Heldt et al . , 2015 ) .", "Our sequencing enables us to identify the rare cells that were co-infected with both wild-type and synonymously barcoded viral variants .", "Overall , we captured 10 such co-infected cells that had >5% of their mRNA derived from virus ( Figure 7 ) .", "Seven of these 10 cells expressed all eight viral genes .", "The majority ( 4 of", "7 ) of these cells would not have expressed all the viral genes in the absence of co-infection , since they have at least one gene exclusively derived from each viral variant .", "For instance , the cell with 11 . 2% of its mRNA from virus in the upper right of Figure 7 expresses M only from the wildtype viral variant , and NP and HA only from the synonymously barcoded variant .", "Our data therefore provide the first direct single-cell observation of the fact that co-infection can rescue missing viral genes ( Brooke et al . , 2013; Brooke et al . , 2014; Fonville et al . , 2015; Aguilera et al . , 2017 ) .", "Another observation from Figure 7 is that co-infected cells usually express roughly equal amounts of transcripts from each of the two viral variants .", "This observation is consistent with the finding by Doud et al . , 2017 and Huang et al . ( 2008 ) that the temporal window for co-infection is short – if both viral variants infect a cell at about the same time , then neither will have a headstart and so each will have a roughly equal opportunity to transcribe its genes .", "To support this idea with a larger dataset albeit at lower resolution , we generated a virus in which the HA coding sequence was replaced by GFP .", "We then co-infected cells with a mix of wildtype and ΔHA-GFP virus and used flow cytometry to score cells for the presence of HA only ( infection by wildtype virus ) , GFP only ( infection by ΔHA-GFP virus ) , or both ( co-infection ) as shown in Figure 7—figure supplement 1 .", "As in our single-cell sequencing data , we found that expression of HA and GFP were highly correlated , indicating that co-infected cells typically expressed roughly equal amounts of transcript from each viral variant .", "Because our sequencing captured all polyadenylated transcripts , we can examine whether there are prominent changes in the host-cell transcriptome in sub-populations of infected cells .", "Influenza virus infection can trigger innate-immune sensors that lead to the transcriptional induction of type I and III interferons , and subsequently of anti-viral interferon-stimulated genes ( Killip et al . , 2015; Iwasaki and Pillai , 2014; Crotta et al . , 2013 ) .", "However , activation of the interferon response is stochastic and bi-modal at the level of single cells ( Chen et al . , 2010; Shalek et al . , 2013 , Shalek et al . , 2014; Pérez-Cidoncha et al . , 2014; Bhushal et al . , 2017; Hagai et al . , 2017 ) .", "We therefore hypothesized that we might see two sub-populations of infected cells: one in which the interferon response inhibited viral transcription , and another in which the virus was able to express high levels of its mRNA by evading or blocking this response .", "To examine whether there were distinct sub-populations of virus-infected cells , we used a semi-supervised t-SNE approach ( Van der Maaten and Hinton , 2008 ) to cluster cells by genes that co-varied with viral infection status .", "As shown in Figure 8A , B , this approach effectively grouped cells by the amount of viral mRNA that they expressed .", "Sample-to-sample variation was regressed away during the clustering , as cells did not obviously group by time-point , with expected exception that the uninfected and 6 hr samples had few cells in the region of the plot corresponding to large amounts of viral mRNA ( Figure 8C ) .", "But to our surprise , we did not see a prominent clustering of infected cells into sub-populations as expected if the interferon response was strongly activated in some cells .", "To investigate further , we annotated each cell by the total number of type I and III interferon transcripts detected .", "Remarkably , only a single cell expressed detectable interferon ( Figure 8D ) .", "We also examined interferon-stimulated genes , which are induced by autocrine and paracrine interferon signaling .", "Figure 8E shows expression of one such gene , IFIT1 ( 31 ) .", "As with interferon itself , expression of IFIT1 was rare and most prominent in the single interferon-positive cell , presumably due to the higher efficiency of autocrine versus paracrine signaling .", "Notably , interferon and interferon-stimulated genes were also relatively ineffective at blocking viral transcription in the single cell in which they were potently induced , since >10% of the mRNA in this cell was derived from virus ( Figure 8A , B , D , E ) .", "We posited that the paucity of interferon induction might be due to the activity of influenza virus’s major interferon antagonist , the NS1 protein ( García-Sastre et al . , 1998; Hale et al . , 2008 ) .", "We therefore identified cells that expressed substantial amounts of viral mRNA but lacked the NS gene ( Figure 8F ) .", "Consistent with the idea that NS1 is important for suppressing interferon , the one interferon-positive cell lacked detectable expression of the NS gene .", "But other cells that lacked NS expression still failed to induce a detectable interferon response , despite often having a substantial amount of their mRNA derived from virus ( Figure 8 ) .", "This result is in line with other work showing that NS1-deficient influenza virus does not deterministically induce interferon ( Killip et al . , 2017; Kallfass et al . , 2013 ) .", "Therefore , many individual infected cells fail to activate innate-immune responses even when the virus lacks its major interferon antagonist .", "We examined whether any host genes were differentially expressed in cells with more viral mRNA .", "We restricted this analysis to infected cells with all eight viral genes in order to focus on cellular genes that were associated with viral mRNA burden independent of effects due to the presence or absence of particular viral transcripts .", "We identified 43 cellular genes that co-varied with viral mRNA expression at a false discovery rate of 0 . 1 ( Figure 9 , Figure 9—source data 1 ) .", "A gene-set analysis shows that many cellular genes that are associated with the amount of viral mRNA are involved in the response to reactive oxygen species or hypoxia ( Figure 9—source data 2 ) .", "Genes known or suspected to be regulated by the Nrf2 master regulator in response to oxidative stress are often expressed at higher levels in cells with more viral mRNA ( Figure 9 ) .", "These genes produce proteins that are involved in detoxification of reactive oxygen species or resultant products , the management of misfolded proteins , the electron transport chain , or a general stress response ( Figure 9—figure supplement 1 ) .", "We additionally see reduced expression of the nitric oxide synthase interacting protein ( NOSIP ) .", "Transient oxidative stress is known to occur during viral infection , and may act in a proviral fashion via MAPK activation driving vRNP export ( Amatore et al . , 2015 ) .", "The antioxidant response is thought to be largely antiviral , potentially through inhibition of MAPK activity ( Lin et al . , 2016; Sgarbanti et al . , 2014 ) .", "To directly test the effect of transient oxidative stress , we compared the fraction of cells that expressed detectable viral protein when infected either with or without pre-treatment to suppress oxidative stress .", "Figure 9—figure supplement 2 shows that the cells pre-treated with an antioxidant exhibited less frequent detectable expression of viral protein .", "These results , in conjunction with the differential expression test in Figure 9 and the prior work mentioned above , suggest that oxidative stress acts in a proviral fashion .", "The gene-set analysis also found that the amount of viral mRNA was associated with the expression of genes involved in the G2-M cell-cycle checkpoint ( Figure 9—source data 2 ) .", "The cell-cycle associated genes CCND3 , MKI67 , UBE2S , and CENPF are all expressed at significantly lower levels in cells with more viral mRNA ( Figure 9 ) .", "However , our data are not sufficient to determine whether the lower expression of these genes is a cause or effect of the reduction in viral mRNA .", "Interestingly , none of the cellular genes that are significantly associated with the amount of viral mRNA in our study are among the 128 genes that Watanabe et al . ( 2010 ) report as having been identified multiple times in genome-wide screens for factors affecting influenza virus replication .", "One possible explanation is that most of the cell-to-cell heterogeneity in our experiments might arise from viral segment absence or mutations , pure stochasticity , or more subtle alterations in host-cell state – not due to changes in expression of the type of single large-effect genes that are usually identified in genome-wide knockdown/knockout studies ." ], [ "We have quantified the total transcriptome composition of single cells infected with influenza virus .", "While we observe a general increase in the amount of viral mRNA over time as expected from bulk measurements ( Hatada et al . , 1989; Shapiro et al . , 1987 ) , there is wide variation in viral gene expression among individual infected cells .", "The most obvious form of heterogeneity is the complete failure of some infected cells to express one or more viral genes , which we estimate occurs in about half the infected cells in our experiments .", "The absence of some viral genes in some infected cells has been noted previously ( Brooke et al . , 2013; Heldt et al . , 2015; Dou et al . , 2017 ) , and our work provides a holistic view by quantifying the total viral transcriptional load as a function of the level of each mRNA .", "We find that cells lacking expression of any of the four genes that encode the viral RNP express much less total viral mRNA , consistent with prior bulk studies ( Vreede et al . , 2004; Eisfeld et al . , 2015 ) .", "Interestingly , the reason some cells fail to express some viral genes remains unclear .", "The prototypical influenza virion packages one copy of each of the eight gene segments ( Noda et al . , 2006; Hutchinson et al . , 2010 ) , but some virions surely package fewer ( Brooke et al . , 2014 ) .", "However , it is also possible that much of the viral gene absence is due to stochastic loss of viral RNPs after infection but prior to the initiation of viral transcription in the nucleus .", "The absence of viral genes only partially explains the cell-to-cell variation in amount of viral mRNA , which still varies from <1% to >50% among cells expressing all the viral genes .", "It is likely that other viral genetic factors explain some of this remaining heterogeneity .", "The 3’-end sequencing strategy used in our experiments detects the presence of a viral gene , but does not identify whether that gene contains a mutation that might hinder viral replication .", "However , viral mutations are also unlikely to explain all the observed heterogeneity , since current consensus estimates of influenza virus’s mutation rate suggest that the typical virion in a stock such as the one used in our experiment should contain less than one mutation per genome ( Parvin et al . , 1986; Suárez et al . , 1992; Suárez-López and Ortín , 1994; Nobusawa and Sato , 2006; Bloom , 2014; Pauly et al . , 2017 ) .", "The rest of the heterogeneity must be due to some combination of cellular factors and inherent stochasticity .", "Some features of the cellular transcriptome co-vary with the amount of influenza mRNA .", "In particular , the viral load in individual cells is associated with the expression of genes involved in response to cellular stresses , including oxidative stress .", "It will be interesting to determine if these cellular transcriptional signatures are simply a consequence of the stress imposed by viral replication , or if their stronger activation in some cells is a causative factor that promotes viral transcription .", "However , it also would not be surprising if a substantial amount of the cell-to-cell heterogeneity cannot be ascribed to pre-existing features of either the viral genome or cellular state .", "Apparently stochastic heterogeneity is a common feature of many processes at a single-cell level ( Cai et al . , 2006; Raj et al . , 2006; Buganim et al . , 2012; Shalek et al . , 2013; Avraham et al . , 2015 ) – especially when those processes are initiated by very small numbers of initial molecules ( Elowitz et al . , 2002 ) , as is the case for low-MOI viral infection .", "Our data do suggest that the factors driving the heterogeneity in viral transcriptional load exert relatively concordant effects on all viral genes in a given cell .", "Specifically , despite the extreme heterogeneity in total viral mRNA per cell , the relative levels of the viral mRNAs are reasonably consistent across cells , and generally reflective of classical bulk measurements ( Hatada et al . , 1989 ) .", "Therefore , despite the stochasticity inherent in initiating transcription and replication of each gene from a single copy carried by the incoming virion , as long as a gene is not completely lost then the virus possesses mechanisms to control its relative expression ( Shapiro et al . , 1987; Hatada et al . , 1989; Perez et al . , 2010; Heldt et al . , 2012; Chua et al . , 2013 ) .", "One factor that surprisingly does not appreciably contribute to the heterogeneity in our experiments is activation of innate-immune interferon pathways .", "Only one of the hundreds of virus-infected cells expresses any detectable interferon , despite the fact that a number of cells fail to express the influenza-virus interferon antagonist NS1 .", "It is known that interferon activation is stochastic at the level of single cells in response to both synthetic ligands ( Shalek et al . , 2013 , Shalek et al . , 2014; Bhushal et al . , 2017; Hagai et al . , 2017 ) and actual infection ( Rand et al . , 2012; Pérez-Cidoncha et al . , 2014; Avraham et al . , 2015; Killip et al . , 2017 ) .", "But interferon expression is a prominent transcriptional signature of high-MOI influenza virus infection of bulk cells , including in the epithelial cell line and at the time-points used in our experiments ( Geiss et al . , 2002; Sutejo et al . , 2012 ) .", "So it is notable how rarely single cells express interferon .", "Interferon expression would surely be more common at later times or with a viral stock passaged at higher MOI , since paracrine interferon signaling ( Crotta et al . , 2013 ) and accumulation of defective viral particles enhance innate-immune detection ( Tapia et al . , 2013; López , 2014 ) .", "However , the early events of physiological influenza infection involve just a few virions ( Varble et al . , 2014; McCrone et al . , 2017 ) , and so it is interesting to speculate whether rare events such as interferon activation during the first few cycles of viral replication could contribute to heterogeneity in the eventual outcome of infection .", "Overall , our work shows the power and importance of characterizing cellular infection at the level of single cells ( Avraham et al . , 2015 ) .", "Viral infection can involve heterogeneity in the genetic composition of the incoming virion , the host-cell state , the bi-modality of innate-immune activation , and the inherent stochasticity of molecular processes initiated by a single copy of each viral gene .", "In our experiments with short-timeframe and low-MOI infections with a relatively pure stock of influenza virus , we find only a minor role for innate-immune activation , but a substantial role for heterogeneity in the complement of viral genes that are expressed in individual cells and at least some contribution of host-cell state .", "Our current experiments are not able to quantify the role of other possibly important factors such as mutations in viral genes , but we suspect that they may also contribute .", "Future extensions of the approaches described here should enable further deconstruction of the sources of cell-to-cell heterogeneity during viral infection , thereby enabling a deeper understanding of how the bulk features of infection emerge from processes within individual virus-infected cells ." ], [ "The following cell lines were used in this study: the human lung epithelial carcinoma line A549 ( ATCC CCL-185 ) , the MDCK-SIAT1 variant of the Madin Darby canine kidney cell line overexpressing human SIAT1 ( Sigma-Aldrich 05071502 ) , and the human embryonic kidney cell line 293T ( ATCC CRL-3216 ) .", "The A549 cells were tested as negative for mycoplasma contamination by the Fred Hutch Genomics Core , and authenticated using the ATCC STR profiling service .", "All cells were maintained in D10 media ( DMEM supplemented with 10% heat-inactivated fetal bovine serum , 2 mM L-glutamine , 100 U of penicillin/ml , and 100 μg of streptomycin/ml ) at 37 at 5% CO2 .", "Wildtype A/WSN/1933 ( H1N1 ) influenza virus was generated by reverse genetics using the plasmids pHW181-PB2 , pHW182-PB1 , pHW183-PA , pHW184-HA , pHW185-NP , pHW186-NA , pHW187-M , and pHW188-NS ( Hoffmann et al . , 2000 ) .", "The sequences of the viral RNAs encoded in these plasmids are in Figure 1—source data", "1 . Reverse-genetics plasmids encoding the synonymously barcoded WSN virus were created by using site-directed mutagenesis to introduce two synonymous mutations near the 3’ end of the mRNA for each viral gene .", "The sequences of the synonymously barcoded viral RNAs are in Figure 1—source data", "1 . To generate viruses from these plasmids , we transfected an equimolar mix of all eight plasmids into cocultures of 293T and MDCK-SIAT1 cells seeded at a ratio of 8:1 .", "At 24 hr post-transfection , we changed media from D10 to influenza growth media ( Opti-MEM supplemented with 0 . 01% heat-inactivated FBS , 0 . 3% BSA , 100 U of penicillin/ml , 100 μg of streptomycin/ml , and 100 μg of calcium chloride/ml ) .", "At 48 hr post-transfection we harvested the virus-containing supernatant , pelletted cellular material by centrifugation at 300 x g’s for 4 min , and stored aliquots of the clarified viral supernatant at −80 .", "We then titered thawed aliquots of viral by TCID50 on MDCK-SIAT1 cells , computing titers via the formula of Reed and Muench ( 1938 ) .", "To generate our ‘high-purity’ stocks of viruses for the single-cell sequencing experiments , we then infected MDCK-SIAT1 cells at an MOI of 0 . 01 , and let the virus grow for 36 hr prior to harvesting aliquots that were again clarified by low-speed centrifugation , aliquoted , stored at −80 , and titered by TCID50 .", "The high-MOI passage ( high-defective particle ) stock used in Figure 2 was generated by instead passaging in MDCK-SIAT1 cells twice at an MOI of 1 for 48 hr .", "For the experiments in Figure 7—figure supplement 1 , we created a virus that carried an HA gene segment in which GFP replaced most of the HA coding sequence , following a scheme first described by Marsh et al . ( Marsh et al . , 2007 ) .", "Briefly , we created a plasmid encoding a viral RNA with GFP in place of the HA coding sequence in the context of the pHH21 ( Neumann et al . , 1999 ) reverse-genetics plasmid , removing potential start codons upstream of the GFP ( see Figure 7—source data 1 for the sequence of the viral RNA ) .", "We then generated GFP-carrying virus by reverse-genetics in cells constitutively expressing HA ( Doud and Bloom , 2016 ) .", "To obtain sufficient titers , this HA-eGFP virus was expanded for 44 rather than 36 hr after initiating infection at an MOI of 0 . 01 .", "For the qPCR in Figure 2 and Figure 5—figure supplement 1 , A549 cells were seeded at 3×105 cells per well in a 6-well tissue culture plate in D10 the day prior to infection .", "On the day of infection , a single well was trypsinized and the cells were counted in order to determine the appropriate amount of virus to use to achieve the intended MOI .", "Immediately before infection , D10 was replaced with influenza growth media .", "For cells incubated with cyclohexamide , the compound was added to a final concentration of 50 μg/ml at the time of infection – previously confirmed to be sufficient to halt viral protein production ( Killip et al . , 2014 ) .", "RNA was purified using the QIAGEN RNeasy plus mini kit following manufacturer’s instructions .", "cDNA was synthesized using an oligoDT primer and the SuperScript III first-strand synthesis supermix from ThermoFisher using the manufacturer’s protocol .", "Transcript abundance was measured using SYBR green PCR master mix , using a combined anneal/extension step of 60 for one minute with the following primers: HA: 5’-GGCCCAACCACACATTCAAC-3’ , 5’-GCTCATCACTGCTAGACGGG-3’ , IFNB1: 5’-AAACTCATGAGCAGTCTGCA-3’ , 5’-AGGAGATCTTCAGTTTCGGAGG-3’ , L32: 5’-AGCTCCCAAAAATAGACGCAC-3’ , 5’-TTCATAGCAGTAGGCACAAAGG-3’ .", "Biological triplicates were performed for all samples .", "For the measurements of viral genomic HA content in Figure 2 , vRNA was harvested from 80 μl of viral supernatant by the addition of 600 μl of RLT plus before proceeding with the standard QIAGEN RNeasy Plus Mini kit protocol .", "The cDNA was generated using SuperScript III first-strand synthesis supermix using the manufacturer’s protocol , and using the universal vRNA primers of Hoffmann et al . ( Hoffmann et al . , 2001 ) with the modifications described in Xue et al . ( Xue et al . , 2017 ) .", "The qPCR was then performed as for mRNA measurements .", "A standard curve was generated from three independent dilutions of the HA-encoding reverse genetics plasmid .", "All vRNA values represent three independent RNA extractions with two replicate qPCR measurements .", "To determine viral titers in terms of HA-expressing units and for the flow cytometry , A549 cells were seeded in a 6-well plate and infected as described above for the qPCR analyses .", "Cells were harvested by trypsinization , resuspended in phosphate-buffered saline supplemented with 2% heat-inactivated FBS , and stained with 10 μg/ml of H17-L19 , a mouse monoclonal antibody confirmed to bind to WSN HA in a prior study ( Doud et al . , 2017 ) .", "After washing in PBS supplemented with 2% FBS , the cells were stained with a goat anti-mouse IgG antibody conjugated to APC .", "Cells were then washed , fixed in 1% formaldehyde , and washed further before a final resuspension and analysis .", "We then determined the fraction of cells that were HA positive and calculated the HA-expressing units .", "For NS1 staining , cells stained for HA as described above were permeabilized using BD Cytofix/Cytoperm following manufacturer’s instructions , stained with anti-NS1 ( GTX125990 , Genetex ) at 4 . 4 μg/ml , washed , stained with a goat anti-rabbit IgG antibody conjugated to Alexa Fluor 405 , washed , and analyzed .", "To analyze the effect of N-acetylcysteine , the compound was added to cells in D10 9 hr prior to media change and infection , and included in infection media .", "Stocks of N-acetylcysteine were reconstituted immediately prior to use , and the pH of growth media supplemented with this compound was adjusted using sodium hydroxide .", "After channels were compensated and cells gated to exclude multiplets and debris in FlowJo , data were extracted using the R package flowCore ( Le Meur et al . , 2007 ) and analyzed using a custom Python script .", "Guassian kernel density estimates were obtained using the scipy stats package method , guassian_kde , using automatic bandwidth determination ( van der Walt et al . , 2017 ) .", "For gating on NS1 positive cells , the percentage of influenza-infected cells was determined by HA staining alone , and the top quantile of NS1-stained cells matching that percentage were taken as the NS1 positive population .", "Single-cell sequencing libraries were generated using the 10x Chromium Single Cell 3’ platform ( Zheng et al . , 2017 ) using the V1 reagents .", "All time points except for the second 8 hr sample ( 8 hr-2 ) were prepared on the same day .", "For the infections , A549 cells were seeded in a 6-well plate , with two wells per time point .", "A single well of cells was trypsinized and counted prior to initiation of the experiment for the purposes of calculating MOI .", "Wild-type and synonymously barcoded virus were mixed to an estimated ratio of 1:1 based on prior , exploratory , single-cell analyses ( data not shown ) .", "At the initiation of our experiment , the wells for all time points were changed from D10 to influenza growth media .", "Cells were then infected with 0 . 3 HA-expressing units of virus per cell ( as determined by flow cytometry ) .", "The infections were performed in order of time point: first the 10 hr time point , then the 8 hr , and then the 6 hr time point .", "At one hour after infection , the media for each time point was changed to fresh influenza growth media .", "Note that the HA-expressing units were calculated without this additional washing step , and so likely represent an overestimate of our final infectious dose ( consistent with the fact that fewer than 30% of cells appear infected in the single-cell sequencing data ) .", "All cells were then harvested for single-cell analysis concurrently – ensuring all had spent equivalent time in changed media .", "For 8 hr-2 sample , cells were infected as above except that the cells were infected at 0 . 1 HA-expressing units of virus per cell but no wash step was performed , and the sample was prepared on a different day .", "After harvest , cells were counted using disposable hemocytometers and diluted to equivalent concentrations with an intended capture of 3000 cells/sample following the manufacturer’s provided by 10x Genomics for the Chromium Single Cell platform .", "All subsequent steps through library preparation followed the manufacturer’s protocol .", "Samples were sequenced on an Illumina HiSeq .", "Jupyter notebooks that perform all of the computational analyses are available in Supplementary file 1 and at https://github . com/jbloomlab/flu_single_cell ( Russell et al . , 2018 ) copy archived at https://github . com/elifesciences-publications/flu_single_cell ) .", "Briefly , the raw deep sequencing data were processed using the 10X Genomics software package CellRanger ( version 2 . 0 . 0 ) .", "The reads were aligned to a concatenation of the human and influenza virus transcriptomes .", "The human transcriptome was generated by filtering genome assembly GRCh38 for protein coding genes defined in the GTF file GRCh38 . 87 .", "The influenza virus transcriptome was generated from the reverse-complement of the wildtype WSN viral RNA sequences as encoded in the reverse-genetics plasmids ( Figure 1—source data 1 ) .", "The aligned deep sequencing data are available on the GEO repository under accession GSE108041 ( https://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? acc=GSE108041 ) .", "CellRanger calls cells based on the number of observed cell barcodes , and creates a cell-gene matrix .", "We used custom Python code to annotate the cells in this matrix by the number of viral reads that could be assigned to the wildtype and synonymously barcoded virus .", "Only about half of the viral reads overlapped the barcoded regions of the genes ( Figure 1A ) and could therefore be assigned to a viral barcode ( Figure 4—figure supplement 1 ) .", "So for calculations of the number of reads in a cell derived from each viral barcode for each viral gene , the total number of detected molecules of that gene are multiplied by the fraction of those molecules with assignable barcodes that are assigned to that barcode .", "This annotated cell-gene matrix is in Supplementary file", "2 . A Jupyter notebook that performs these analyses is in Supplementary file", "1 . The annotated cell-gene matrix was analyzed in R , primarily using the Monocle package ( version 2 . 4 . 0 ) ( Qiu et al . , 2017; Trapnell et al . , 2014 ) .", "A Jupyter notebook that performs these analyses is in Supplementary file", "1 . For each sample , cell barcodes that had >2 . 5-fold fewer or more UMI counts mapping to cellular transcripts than the sample mean were excluded from downstream analyses ( see red vertical lines in Figure 3B ) .", "In order to determine an appropriate cutoff for how many reads a cell needed to contain in order to be classified as infected , we calculated the mean viral barcode purity across all cells that contained at least a given fraction of viral mRNA and had multiple viral reads that could be assigned a barcode ( Figure 4B and Figure 4—figure supplement 2 ) .", "We then determined the threshold fraction of viral mRNA at which the mean purity no longer increased as a function of the amount of viral mRNA .", "This threshold represents the point at which we have effectively eliminated cells that have low barcode purity simply due to lysis-acquired reads sampled randomly from both viral barcodes .", "As is apparent from Figure 4B , only the 10 hr sample and the 8 hr-2 sample have the excess of mixed barcodes among cells with low amounts of viral mRNA .", "The likely reason is that these samples have more total viral mRNA ( and so there is more available mRNA to be acquired from lysed cells ) ; in addition , there is always some experimental variability in the amount of cell lysis during the 10X sequencing process , and these samples may simply have the most .", "So the above threshold procedure is appropriate for those two samples .", "For the other samples , we simply set a minimum threshold of requiring at least a fraction two×10-4 reads to come from viral mRNA as explained in the legend to Figure 4—figure supplement", "2 . The thresholds for each sample are shown in Figure 4C and Figure 4—figure supplement", "2 . This procedure is expected to be conservative , and may miss some truly infected cells with very low amounts of viral mRNA .", "For subsequent analyses , we retained all infected cells and a subsample of uninfected cells ( the greater of 50 or the number of infected cells for that sample ) .", "The rationale for subsampling the uninfected cell is that the vast majority of cells are uninfected , and we did not want these cells to completely dominate the downstream analyses .", "Cells were classified as co-infected if both viral variants had an RNA level that exceeded the threshold , and if the minor variant contributed at least 5% of the viral mRNA .", "For the semi-supervised t-SNE clustering , we used Monocle’s cell hierarchy function to bin cells into those with no viral mRNA , <2% viral mRNA , between 2% and 20% viral mRNA , and >20% .", "Candidate marker genes for t-SNE dimensionality reduction were then determined using the Monocle function markerDiffTable , excluding the effects of sample variation and the number of genes identified in a given cell , using a q-value cutoff of 0 . 01 .", "The specificity of these markers was determined using the function calculateMarkerSpecificity – the top 50 markers were retained , and used to place populations in a two-dimensional plane based on tSNE dimensionality reduction .", "For the analyses of cellular genes that differed in expression as a function of the amount of viral mRNA , we only considered cells that expressed all eight viral mRNAs to avoid effects driven simply by viral gene absence .", "We also only considered cellular genes in the differential gene analysis , since viral gene expression will tautologically co-vary with the amount of viral mRNA .", "Additionally , because influenza virus has the capacity to degrade or prevent the synthesis of host mRNAs ( Bercovich-Kinori et al . , 2016 ) and contributes significantly to the total number UMIs in some cells , we calculate size factors ( a scalar value representing efficiency of UMI capture ) based on cellular transcripts alone .", "Finally , we assigned all cells a ceiling fraction of mRNA from virus of 25% so that a few extremely high-expressing cells did not dominate .", "Cellular genes with expression that co-varied with the fraction of viral mRNAs in a cell were then determined using the Monocle differentialGeneTest , after removing variance explained by sample to sample variation .", "Figure 9 shows all genes that were significantly associated with the fraction of mRNA from virus at a false discovery rate of 0 . 1 .", "We performed the gene set analysis using the P -alues from the Monocle differentialGeneTest with piano ( Väremo et al . , 2013 ) using the hallmark gene set from GSEA v6 ( Subramanian et al . , 2005 ) and Fisher’s method ." ] ]

[ "Viral infection can dramatically alter a cell’s transcriptome .", "However , these changes have mostly been studied by bulk measurements on many cells .", "Here we use single-cell mRNA sequencing to examine the transcriptional consequences of influenza virus infection .", "We find extremely wide cell-to-cell variation in the productivity of viral transcription – viral transcripts comprise less than a percent of total mRNA in many infected cells , but a few cells derive over half their mRNA from virus .", "Some infected cells fail to express at least one viral gene , but this gene absence only partially explains variation in viral transcriptional load .", "Despite variation in viral load , the relative abundances of viral mRNAs are fairly consistent across infected cells .", "Activation of innate immune pathways is rare , but some cellular genes co-vary in abundance with the amount of viral mRNA .", "Overall , our results highlight the complexity of viral infection at the level of single cells ." ]

[ "When viruses infect cells , they take over the cell’s machinery and use it to express their own genes .", "This process has mostly been studied by looking at the average outcome of infection when many viruses infect many cells .", "However , it is less clear what happens in individual cells .", "For example , does the virus take over every cell to make lots of viral gene products , or do some cells produce far more viral gene products than others ?", "Russell et al . have now used a new technique called single-cell RNA sequencing to look at how well influenza virus genes were expressed in hundreds of individual mammalian cells .", "The goal was to work out how the outcome of infection varied between different cells .", "One way to quantify variability – also known as heterogeneity – is by using a statistical measure called the Gini coefficient .", "This statistic is often used to assess the inequality in incomes across a nation . In the hypothetical situation where everyone earned the same income , the Gini coefficient would equal zero; while if only one person had all the income and all others had none , the value would be very close to one .", "In reality , countries fall somewhere in between these two extremes .", "In the United States for instance , the Gini coefficient for income is 0 . 47 .", "When Russell et al . worked out the Gini coefficient for the amount of viral genes expressed in different cells , the value was at least 0 . 64 .", "This indicates that there is more unevenness in viral gene expression for influenza than there is income inequality in the United States .", "So , what characterizes the “Bill Gates” cells and viruses that have the highest viral gene expression ?", "Influenza viruses sometimes fail to express some of their genes .", "Russell et al . found that this failure often led to “poor” viruses that were less productive than “rich” viruses that expressed all the critical genes .", "However , the results suggest that there are also other factors that contribute a lot to the heterogeneity .", "Real influenza virus infections are usually started by very few viruses , so this new understanding of the variability that occurs when individual viruses infect individual cells might prove important for understanding the properties of infections at larger scales too ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "Periodic patterning of iterative forms is one of the most common features observed in developmental processes across the living kingdom .", "Unraveling the rules governing pattern formation helps to elucidate the genetic basis of cell differentiation .", "A key contribution in theoretical biology was made by Alan Turing in 1952 who demonstrated that the reaction of two molecules with different diffusion rates generates regular patterns ( Turing , 1952 ) .", "Later on , Turing equations were adapted in a new mathematical model that emphasized conspicuous features of biological development , in particular local activation linked to autocatalysis and long range inhibition ( Gierer and Meinhardt , 1972; Meinhardt and Gierer , 1974 ) .", "Valuable experimental approaches showed the recurrence of this mechanism in various developmental behaviors , such as: spacing of leaves , tissue regeneration in Hydra , epidermis of insects , pigmentation in Zebra fish , embryogenesis in Drosophila , and also differentiation in some prokaryotes ( for a review see Schweisguth and Corson , 2019 ) .", "In all these developmental situations several aspects of the molecular interactions involved to generate patterning remain to be elucidated .", "Among prokaryotes , several members of the Cyanobacteria phylum are capable of cell differentiation .", "The molecular basis of differentiation has been well documented for the cyanobacterium Anabaena/Nostoc PCC 7120 ( referred herein as Nostoc ) .", "Nostoc is a diazotrophic strain which can differentiate into specific type of cells responsible for fixing atmospheric nitrogen .", "When combined nitrogen is abundant Nostoc forms long filaments consisting of a single cell type .", "When the filaments of Nostoc are deprived of combined nitrogen , around 10% of the vegetative cells differentiate into heterocysts .", "These micro-oxic cells , which provide a suitable environment for N2-fixation , are non-dividing and are distributed semi-regularly along the filaments .", "Nostoc differentiation follows therefore a one-dimensional pattern of heterocysts separated by 10 vegetative cells .", "Heterocysts are unable to undergo cell division , but as vegetative cells continue dividing , the filaments grow and new heterocysts form in the middle of the intervals between pre-existing heterocysts .", "Consequently , the pattern is dynamic and persists throughout the growth ( Kumar et al . , 2010; Flores and Herrero , 2010 ) .", "The molecular signal inducing heterocyst differentiation is 2-oxoglutarate ( 2-OG ) , which accumulates in response to combined nitrogen starvation ( Laurent et al . , 2005 ) .", "Among the various genes involved in the regulation of heterocyst formation and patterning ( Herrero et al . , 2016 ) , the global regulator NtcA and the specific master regulator HetR are key transcriptional factors in the cascade resulting in heterocyst development .", "Upon combined nitrogen starvation , NtcA interacts with 2-OG and induces heterocyst differentiation by controlling , directly or indirectly , the expression of several genes , including hetR ( Herrero et al . , 2004; Valladares et al . , 2008 ) .", "HetR is essential for cell differentiation: its deletion abolishes differentiation , and its overexpression induces differentiation of multiple contiguous heterocysts under combined nitrogen-starvation and allows differentiation even under non-permissive conditions ( Buikema and Haselkorn , 1991 ) .", "It exists in different oligomeric states among which dimer and tetramer have been proposed to interact with DNA ( Huang et al . , 2004; Valladares et al . , 2016 ) .", "The HetR regulon includes hundreds of genes , which are either activated in response to nitrogen starvation or repressed in nitrogen-replete conditions ( Flaherty et al . , 2014; Mitschke et al . , 2011; Videau et al . , 2014 ) .", "The structure of HetR shows a unique fold comprising three domains: helix-turn-helix , flap and hood; with the latter encompassing the binding site of PatS ( Kim et al . , 2011; Hu et al . , 2015 ) .", "A key event in pattern formation in Nostoc is the positive autoregulation of hetR occurring specifically in the differentiating cell ( Black et al . , 1993; Rajagopalan and Callahan , 2010 ) and the inhibition of HetR in neighboring cells by the product of patS ( Golden and Yoon , 2003 ) .", "The deletion of the patS gene leads to the formation of multiple contiguous heterocysts , and its overexpression inhibits the differentiation process and hence induces a lethal phenotype under combined nitrogen starvation ( Yoon and Golden , 1998 ) .", "patS encodes a 13–17 amino acid peptide containing at its carboxy-terminal end a RGSGR pentapeptide ( PatS-5 ) that interacts with HetR , which inhibits its DNA-binding activity ( Huang et al . , 2004; Feldmann et al . , 2011 ) and blocks cell differentiation when added to culture medium ( Yoon and Golden , 1998 ) .", "The hexapeptide ERGSGR ( PatS-6 ) derived from PatS is also able to interact with HetR and to inhibit its activity ( Hu et al . , 2015 ) .", "A mutant strain producing a variant of HetR , R223W , that is no longer able to interact with PatS , forms multiple contiguous heterocysts ( Khudyakov and Golden , 2004; Hu et al . , 2015 ) .", "PatS-dependent signals are considered to diffuse along the filament; inhibiting HetR in the cells adjacent to the heterocyst , so preventing them from differentiating ( Risser and Callahan , 2009; Corrales-Guerrero et al . , 2013 ) .", "The RGSGR pentapeptide is also present in HetN; a protein required for the maintenance of the pattern ( Black and Wolk , 1994; Higa et al . , 2012; Corrales-Guerrero et al . , 2014 ) , and recently a third protein ( PatX ) containing a RGTGR motif , has been reported to inhibit heterocyst differentiation when overproduced in Nostoc ( Elhai and Khudyakov , 2018 ) .", "The transcription of patX is induced in the heterocyst and is under the control of NtcA ( Elhai and Khudyakov , 2018 ) .", "The HetR/PatS regulatory loop fits the local activation/long range inhibition scheme that has been adapted from the Turing model explaining pattern formation ( Brown and Rutenberg , 2014; Turing , 1952; Turing , 1990; Gierer and Meinhardt , 1972; Figure 1A ) .", "Furthermore , the action of HetN and PatX as inhibitory factors , the stochastically noisy expression of regulatory proteins ( HetR and NtcA ) among other features specific to cell differentiation of Nostoc , allow the emergence of more elaborated mathematical models that outline the principles governing pattern formation in cyanobacteria ( Di Patti et al . , 2018; Muñoz-García and Ares , 2016 ) .", "Because Nostoc is the simplest model to address development in a one-dimensional self-organizing system , valuable genetics and biochemical studies have provided an accurate picture of heterocyst patterning .", "Nevertheless , several questions are still unanswered and deserve investigation .", "In particular , how the differentiating cell , where patS is expressed at its highest level , becomes immune to self-inhibition is not fully understood yet .", "A genetic analysis of patS suggested that the seven amino acids at the N-terminus of PatS protect the producing cell from inhibition likely concomitantly with the export of the active form of PatS ( Corrales-Guerrero et al . , 2013 ) .", "Intriguingly , if patS or patS-5 are expressed specifically in the vegetative cells , or when PatS-5 is added to the culture medium , cell differentiation is abolished .", "However when produced in cells that have already initiated development ( proheterocysts ) , PatS-5 is not able to inhibit differentiation ( Yoon and Golden , 2001; Wu et al . , 2004 ) .", "These observations suggest that proheterocysts must acquire additional protection post-PatS export/processing .", "The hetL gene ( all3740 ) was unearthed in a genetic screen aiming to identify factors involved in PatS signaling ( Liu and Golden , 2002 ) .", "HetL is a single domain protein composed of 40 pentapeptides ( A ( D/N ) LXX ) , adopting a right-handed quadrilateral beta helix typical of an Rfr-fold common to all pentapeptide repeats containing proteins ( PRPs ) ( Ni et al . , 2009 ) .", "The ectopic expression of hetL in a background of patS overexpression restores the ability of the strain to differentiate heterocysts ( Liu and Golden , 2002 ) .", "hetL overexpression stimulates differentiation also when PatS-5 is added to the culture medium ( Liu and Golden , 2002 ) .", "Henceforth , HetL interferes with PatS inhibition but the molecular mechanism involved is unknown .", "This study aims to further explore the function of HetL in PatS signaling .", "We show that HetL interacts with HetR at the same interface as PatS .", "This interaction is needed for HetR to escape PatS inhibition , and does not inhibit its DNA-binding activity .", "Analyzing hetL transcription , we found that this gene is induced shortly after initiation of cell differentiation and that HetR is required for its expression .", "Finally , we show that the expression of hetL in heterocysts , but not in vegetative cells , is necessary to counteract PatS inhibitory effect .", "We conclude that , by interacting with HetR , HetL interferes with PatS fixation and therefore provides immunity to the developing cell ." ], [ "To get further insights into HetL function , we wondered whether its activity would be mediated by its direct interaction with HetR .", "To test this , we used the bacterial two hybrid assay ( BACTH ) , which is based on the reconstitution of adenylate cyclase ( CyA ) activity by two interacting proteins that bring the T18 and T25 domains of CyA into close proximity ( Karimova et al . , 1998 ) .", "T18 and T25 domains were fused to HetR and HetL proteins at their N-terminal extremities , and the dimerization ability of HetR was used as an internal control for this assay .", "The data in Figure 1B show that HetL displays a strong interaction with HetR .", "Interestingly , it seems that the HetR-hood domain is sufficient to mediate the interaction of HetR to HetL .", "Furthermore , this experiment indicated that HetL is able to form dimers ( or oligomers ) ( Figure 1B ) .", "To confirm this interaction , we developed a BioLayer interferometry ( BLi ) assay .", "For this purpose , HetR and HetL proteins were produced and purified using affinity chromatography ( Figure 1—figure supplement 1 ) .", "HetR was biotinylated and immobilized on streptavidin biosensors as the ligand , while HetL was used as the analyte .", "Upon addition of HetL , a concentration-dependent association was recorded and decreased during the dissociation step corresponding to the washing of the sensor , indicating a direct interaction between HetL and HetR ( Figure 1C ) .", "The estimated dissociation constant ( KD ) of the HetR-HetL interaction was 6 µM .", "The interaction between HetR and HetL observed in the BACTH assay was thus confirmed by BLi .", "As HetR acts by directly binding to promoters of a subset of its target genes , we tested if the interaction with HetL would impact its DNA binding activity .", "To this end , we conducted an electrophoretic mobility shift assay ( EMSA ) using the hetP promoter as a target ( Huang et al . , 2004; Hu et al . , 2015 ) .", "The previously reported ability of PatS-5 to inhibit HetR DNA-binding activity was used as a control ( Huang et al . , 2004; Hu et al . , 2015 ) .", "In the presence of HetL , HetR was still able to interact with the hetP promoter and the complex formed was higher than the one formed by HetR alone ( Figure 1D ) .", "This result indicates that , contrary to PatS-5 binding , the interaction between the two proteins does not inhibit the DNA-binding activity of HetR .", "Since HetL has been identified on the basis of counteracting PatS inhibition and as both PatS and HetL interact with HetR at its hood domain , we hypothesized that HetL could interact with HetR at the same interface as PatS , which would therefore interfere with its inhibiting action .", "To test this hypothesis , we took advantage of the fact that HetR-PatS interaction involves the Hood domain of HetR and that the residue R223 of HetR is required for this interaction .", "Interestingly , a variant of HetR bearing a R223W substitution lost the ability to interact with HetL ( Figure 2A ) .", "HetR ( R223W ) was still able to form dimers ( Figure 2A ) , which indicates that this variant is correctly folded .", "We conclude that the absence of interaction between HetR ( R223W ) and HetL indicates that this residue , in addition to being involved in the interaction with PatS-5/PatS-6 , is also required for the interaction with HetL .", "For a deeper investigation of HetR-HetL interaction , we exploited the available structures of HetL and of the Hood domain of HetR to build interaction models ( Ni et al . , 2009; Hu et al . , 2015 ) .", "Interestingly , a group of four models among the top 10 clusters share similar orientations of HetR on HetL ( Figure 2—figure supplement 1 ) .", "Importantly , these models present interesting properties regarding the binding interface of HetR-Hood to HetL:", "( i ) the residue R223 of HetR was involved in the binding interface with HetL ( Figure 2B , Figure 2—figure supplement 1 ) ,", "( ii ) the HetR interaction interface with HetL matches the one involved in PatS-5 interaction ( Figure 2B ) .", "In the retained models , the HetR-Hood:HetL interaction is maintained by a large network of electrostatic interactions and hydrogen bonds .", "For HetR , the binding interface is composed of the surface exposed residues from strand β3 and α-helix α12’ ( Figure 2B ) .", "In HetL , the large proportion of the binding interface includes the pentapeptides repeat localized in face three encompassed E84ADLT , K104ASLC , 124QADLR , 149YADLR , 169RANFG and 197YANLE .", "A small second binding interface is localized within face 2 and includes 40ADLRQ and 145ADLSY pentapeptide repeats .", "To gain further insight about the HetR:HetL interaction , we performed a site-directed mutagenesis to substitute the residue D151 from HetL to Alanine .", "Interestingly , this substitution impaired the binding of HetL to HetR as revealed by BACTH assay ( Figure 2A ) .", "Furthermore , HetL D151A variant was still able to form dimers which is an important indication to rule out the possibility that the mutation impacted the fold of this variant ( Figure 2A ) .", "Taken together , these results indicate that HetL and PatS-5 interact with HetR at the same interface , which implies that HetL and PatS must compete for the interaction with HetR .", "To check this assumption , the interaction between HetR and HetL was analyzed by BLi in the presence of increasing amounts of PatS-5 .", "The data obtained clearly indicated that the association between HetR and HetL is impaired with the addition of PatS-5 in a dose-response manner , with a total inhibition effect obtained at a concentration of 5 µM ( Figure 2C ) .", "Similar results were obtained with PatS-6 ( Figure 2—figure supplement 2 ) .", "The effect of PatS on HetR-HetL interaction was further analyzed by two hybrid assays .", "For this , a synthetic operon constituted of T18-hetL and patS-6 was constructed and used to question the interaction with HetR .", "While adenylate cyclase activity was restored in bacteria producing T25-HetR and T18-HetL fusions , the interaction between HetR and HetL was abolished when PatS-6 was produced along with HetL from the synthetic operon ( Figure 2—figure supplement 3 ) , which is a further demonstration that PatS and HetL share the same interaction site within HetR .", "The interference of PatS with the HetR-HetL complex was also observed in EMSA experiments where the addition of increasing amounts of PatS-5 abolished the binding of HetR-HetL to the hetP promoter ( Figure 2D ) .", "Altogether , these data support the hypothesis of a competition between PatS and HetL for the interaction with HetR .", "Based on the data presented above , HetR acquired immunity against PatS inhibition can be explained by an exclusion of the inhibitor as a consequence of HetR-HetL interaction .", "However , an additional titration-based mechanism through a direct interaction between HetL and PatS cannot be ruled out .", "To test this possibility , Isothermal titration calorimetry ( ITC ) was used to analyze the possible interaction between HetL and PatS-5 .", "This technique has been used previously to uncover the binding of PatS-5 to HetR ( Feldmann et al . , 2011 ) .", "We have confirmed that HetR does indeed interact with PatS-5 with a dissociation constant ( KD ) of 600 nM similar to that of 227 nM described in the literature with a stoichiomerty of 1:1 .", "The data in Figure 3 show that HetR ( left panel ) displays a reducing heat exchange when titrated with increasing amounts of Pats-5 , indicating a saturation of the HetR sites with PatS-5 .", "This reaction is an exothermic favorable reaction with an enthalpy of −7 . 43 ± 0 . 67 ΔH kcal/mol .", "On the contrary , HetL ( right panel ) does not show a relative heat exchange upon binding to PatS-5 .", "The interaction between HetR and PatS-5 observed in Figure 3 , validates our technical experiments and revokes the possibility that HetL could titrate PatS-5 by a direct interaction .", "As hetL was discovered on the basis of its capacity , when overexpressed , to suppress the inhibitory effect of PatS , we used this approach to evaluate the impact of the interaction between HetR and HetL on the differentiation process .", "The wild-type version of hetL or the mutated gene encoding HetL ( D151A ) were expressed in Nostoc cells under the control of the petE promoter , and quantitative RT-PCR analyses were carried out to check the accurate overexpression of these genes upon induction .", "Results revealed a 30-fold higher expression of the hetL and the hetL ( D151A ) genes in the recombinant strains compared to the wild type ( Figure 4A ) , indicating that the two versions of hetL are actually overexpressed .", "PatS effect was analyzed either by the overexpression of patS gene from the petE promoter or by the addition in the medium of PatS-5 ( or PatS-6 ) .", "The cultures were transferred into a nitrate-depleted medium during 48 hr to assess heterocyst development .", "In agreement with published data , the overproduction of PatS or the addition of PatS-5 inhibited the differentiation process , while the overexpression of hetL restored the ability of the PatS overproducing strain to form heterocysts ( Figure 4B ) .", "HetL overproduction also allowed the ability of the strain to form heterocysts when PatS-5 was added to the culture ( Figure 4—figure supplement 1 ) .", "The percentage of the heterocysts formed in the hetL overexpressing strain was equal to that of the wild-type strain ( Table 1 ) .", "Interestingly , the recombinant strain overproducing HetL ( D151A ) was not able to form heterocysts when PatS was overproduced or upon addition of PatS-5 in the growth medium ( Figure 4B , Figure 4—figure supplement 1 ) .", "The interaction of HetL with HetR is therefore needed to counteract PatS inhibition .", "hetL expression in the heterocyst is required for providing HetR immunity against PatS inhibition hetL expression level was too low to be detected by Northern blot or fluorescent gene fusions ( Liu and Golden , 2002 ) .", "We therefore chose quantitative RT-PCR approach to analyze hetL transcription during the differentiation process .", "The hetP gene whose transcription is activated by HetR 8 hr after nitrogen step-down was used as an internal control for this experiment ( Mitschke et al . , 2011 ) .", "RNAs were collected from the wild-type strain and the ΔhetR mutant at different times after nitrogen starvation and the transcript levels of the two genes were expressed relative to their amount at time zero .", "Results reveal that hetP expression was , as expected , strongly induced from 8 hr after nitrate depletion .", "Contrary to the 10-fold induction in the wild-type strain , the expression of hetP did not significantly increase in the ΔhetR strain which is consistent with the activation of this gene by HetR ( Figure 5A ) .", "The hetL gene showed a similar transcription profile to that of hetP , yet its expression level was much lower .", "In the wild-type strain , a 3 . 5-fold increase of hetL transcripts was observed 8 hr after nitrogen step-down and was maintained up to 24 hr .", "In the ΔhetR mutant , no induction of hetL transcription was observed .", "The region including 500 nucleotides upstream of the start codon of hetL , which must include the promoter of this gene , was analyzed to probe for the two binding site consensus reported for HetR: the high-affinity consensus ( GTAGGCGAGGGGTCTAACCCCTCATTACC ) , and the low-affinity one ( GCTTATGGTGGGCAATGCCCACCATAATA ) ( Videau et al . , 2014 ) .", "None of these sequences are present in the hetL promoter .", "We concluded that , even if low , the transcription of hetL gene is induced early during the differentiation program and that HetR is required for hetL activation .", "The action of HetR on the promoter of hetL may be either indirect and mediated by another factor , or direct through a degenerated consensus which might explain the low transcription level of this gene .", "From the results presented above , it can be deduced that HetL acts in the heterocyst .", "To further confirm this assumption , we expressed hetL either from the rbcL promoter , which is specific to vegetative cells , or from the patS promoter which is expressed in the heterocysts early after nitrogen stepdown .", "The ability of HetL to suppress heterocyst inhibition triggered by the addition of PatS-5 was analyzed .", "Results show that the strain expressing the PpatS-hetL gene was able to form heterocysts even in the presence of PatS-5 , but when expressed from the rbcL promoter , hetL was unable to prevent the inhibitory effect of PatS-6 ( Figure 5B ) .", "This result is in favor of HetL acting specifically in the heterocyst .", "In addition to the RGSGR pentapeptide , heterocyst pattern formation has been recently shown to involve the HRGTGR peptide derived from the PatX protein ( Elhai and Khudyakov , 2018 ) .", "To further characterize HetL function , we wondered whether it would be involved in PatX signaling as well .", "Bli experiments showed that the HRGTGR peptide , like PatS-5 , inhibited the interaction between HetR and HetL ( Figure 6A ) .", "In addition , the strain expressing the PpetE-hetL gene was able to form heterocysts even in the presence of the HRGTGR peptide ( Figure 6B ) , while the overproduction of HetL ( D151A ) did not bypass PatX-6 inhibition since heterocysts were not observed ( Figure 6B ) .", "From these results , we conclude that HetL provides immunity for the developing cells against the two inhibitory peptides involved in pattern formation .", "In addition to HetL , the genome of Nostoc contains 31 genes potentially coding for PRPs of various sizes .", "This large family includes proteins predicted to be located either in the cytoplasm , in the membrane or in the periplasm ( Ni et al . , 2009 ) .", "Five of them display PRs among other domains , while the others , such HetL , are integrally formed by PR domains ( Supplementary file 1 ) .", "Given the considerable sequence identity shared by these PRPs ( 32% in average ) , predicting functional specificity based on sequence similarity is not possible .", "Because hetL mutant does not show any specific differentiation phenotype ( Liu and Golden , 2002 ) , we wondered if this could be due to a cross-complementation with another PRP .", "In this regard , we analyzed the capacity of HetR to interact with some HetL homologs .", "For this , we chose All3256 and All4303 because they share the closest features with HetL .", "They have a similar size ( 237 , 268 , 213 amino acids , respectively ) , a similar organization of the PRs domains , and the three are predicted to be cytosolic ( Figure 7A ) .", "Figure 7B shows the results of a BACTH assay questioning the putative interaction of these proteins with HetR .", "Only All4303 displayed interaction with HetR , and even if this interaction is two-fold weaker than that of HetR-HetL it is significant compared to the negative control .", "This experiment indicates that at least one among the 31 PRPs is able to interact with HetR , which makes a cross-complementation of the hetL mutation with all4303 , or another PRP coding gene a possible scenario .", "An important perspective of this work is to study the impact of the hetL-all4303 double mutation on the differentiation process ." ], [ "Eighteen years ago , the hetL gene was discovered on the basis of its ability , when overexpressed , to bypass the inhibitory effect of patS overexpression on cell differentiation ( Liu and Golden , 2002 ) .", "In their conclusion , the authors of this study speculated that PatS and HetL might act by modulating HetR activity .", "This speculation reveals to be an accurate prediction as demonstrated by the results of the experiments presented in this manuscript , added to all the knowledge accumulated on HetR and PatS functions from previous valuable studies that are fundamental to our investigation .", "In particular , as the unique structural fold of HetR with its two exposed domains ( flap and hood ) was proposed to favor protein-protein interactions , we questioned the possible interaction of HetR with proteins involved in patterning .", "In this context , HetL was found to interact strongly with HetR without abolishing its DNA-binding activity , which suggests that HetR-HetL complex may be active regarding gene regulation in vivo ( Figure 1 ) .", "A possible mechanism to explain the role of HetL in PatS signaling is the titration by HetL of the inhibiting peptide .", "This hypothesis was ruled out since ITC assay did not show any interaction between HetL and PatS ( Figure 3 ) .", "Alternatively , a site-exclusion mechanism can be proposed for HetL .", "The observation that HetL and PatS-5 ( or PatS-6 ) interact with HetR at the same interface is in agreement with this suggestion ( Figure 2B ) .", "In Bli assays , increasing concentrations of PatS-5 interfered with HetR-HetL interaction , and in EMSA experiments addition of PatS-5 abolished the formation of HetR-HetL complex in a concentration-dependent manner ( Figure 2C–D ) .", "In addition to confirming the in silico docking model predicting a same HetR-interaction interface for PatS and HetL , these data imply that the concentration of HetL in the ( pro ) heterocyst must be higher than the PatS peptide , which is plausible because", "( i ) PatS peptide is diffusible ( Wu et al . , 2004; Yoon and Golden , 2001 ) ,", "( ii ) HetL features do not include any motif or domain that predicts a putative translocation/secretion or association with membranes that could decrease its amount in the producing cell .", "Moreover , as hetL transcription is activated by HetR ( Figure 4 ) , HetL protein is likely to accumulate in the cytoplasm of differentiating cells .", "The diffusion of the inhibiting peptides ( derived from PatS and HetN ) along the filament has been shown to occur through septal junctions ( SJ ) connecting adjacent cells ( Mariscal et al . , 2016 ) .", "Electron cryotomography analysis showed that these SJ consist of tubes traversing the septal peptidoglycan ( Weiss et al . , 2019 ) .", "Interestingly , the lumen of the tubes measures around 7 nm which is compatible with the diffusion of the inhibiting peptides but probably not with the transfer of HetL between cells .", "HetL protein is 3 . 5 nm wide and 6 . 5 nm length ( Ni et al . , 2009 ) , which might suggest that a monomer could be transferred via the SJ .", "However , it is important to note that that HetL is capable of forming dimers ( or multimers ) ( Figure 1B ) , the size of which is not consistent with transfer via the SJ .", "In addition , it is also possible that HetL surface charge might not be compatible with its transfer across the SJ tubes .", "Alternatively , it can be assumed that in the ( pro ) heterocyst the affinity of HetR for HetL must be higher than that for PatS .", "Bli and ITC experiments showed that in vitro , the affinity of HetR for PatS is 10-fold higher than for HetL ( compare data of Figure 2C and Figure 3 ) .", "One might speculate that in vivo , and especially in the ( pro ) heterocyst , the affinity of HetR for HetL increases either due to a modification of HetR or to its interaction with another factor .", "HetR has been shown to be regulated by phosphorylation ( Valladares et al . , 2016; Roumezi et al . , 2019 ) , if this posttranslational modification occurs only in the developing cell it could explain a high affinity of HetR for HetL specifically in the heterocyst .", "In addition , the observation that the overexpression of hetL in vegetative cells , from petE or rbcL promoters , did not lead to their differentiation into heterocsyts is rather in favor of a mechanism promoting the association between HetR and HetL specifically in the heterocyst .", "Resolving the structure of HetR-HetL complex and following the behavior of HetL protein and PatS in vivo will provide more information about the dynamics and the nature of HetR complexes in the two cell types through the differentiation process .", "A deletion mutant of hetL differentiates heterocysts as well as the wild-type strain ( Liu and Golden , 2002 ) .", "The possibility that hetL is not essential for heterocyst differentiation and that only its overexpression affects the differentiation process was one of the hypotheses proposed to explain the phenotype of the hetL mutant .", "The second hypothesis was functional redundancy based on a crosstalk between HetL and another PRP ( Liu and Golden , 2002 ) .", "The observation that at least one homolog of HetL ( All4303 ) interacted with HetR , is rather in favor of functional redundancy .", "A global transcription study using RNA sequencing has shown that all4303 transcription is induced in response to nitrogen starvation ( Flaherty et al . , 2011 ) .", "The hetL gene has also been reported in this study to be induced after nitrogen starvation , which is consistent with our data .", "The other PRPs coding genes whose transcription has also been reported to be regulated in this condition are reported in Supplementary file 1 .", "At this stage of our investigation , we cannot rule out that homologs of HetL may also be part of the PatS signaling , but the fact that the genetic suppressor screen for suppression of PatS inhibition selected only hetL would rather point to it as the principal factor involved in the differentiation process ( Liu and Golden , 2002 ) .", "Because of the presence of multiple homologs , cross-complementation might occur when hetL happens to be mutated .", "Genes encoding proteins containing tandem pentapeptide repeats are abundant in all cyanobacterial genomes , which render the study of their function by gene-deletion approaches unjustified .", "Interestingly , despite their high amino acid homology , their structures possess distinctive features that can help to investigate their function .", "Exploring these structural features and searching for potential binding-partners can be a successful strategy in their study .", "The gyrase-binding PRP is the first protein of this family whose partner was identified ( Tran et al . , 2005 ) .", "PRPs from this family are largely conserved among bacteria , where number of them have been , based on their ability , proposed to provide resistance to quinolone-type antibiotics .", "Structural studies revealed the distribution of large contiguous patches of negative electrostatic potential resembling DNA which gave insights into their function as mimicking DNA structure for binding to gyrase ( Xiong et al . , 2011 ) .", "The structure of HetL does not show such a large distribution of negative charges , but instead contiguous patches of positively and negatively charged surfaces are distributed at the surface of the structure .", "These patches have been suggested to mediate the binding to unknown potential partners , other than DNA ( Ni et al . , 2009 ) .", "In docking simulation analyses reported in this study , these charged surfaces especially those localized in face 2 and face 3 mediate the interaction with the HetR-Hood domain .", "To the best of our knowledge , the interaction of HetL with HetR constitutes the second case of protein-protein complex identified for a PRP member .", "In Nostoc two other integral PRPs ( PatL [Liu and Wolk , 2011] and FraF [Merino-Puerto et al . , 2013] ) and a pentapeptide repeat domain-containing protein ( HglK , Black et al . , 1995 ) are involved in cell differentiation , but their mechanism of action is not fully uncovered .", "Analyzing their interaction with their physiological partners will provide a better understanding of the role played by PRPs in the developmental program of Nostoc .", "Genetic experiments showed that HetL interaction with HetR allows the differentiating cell to escape from PatS overproduction and from the addition to the culture medium of the ( E ) RGSGR , ( H ) RGTGR inhibiting peptides ( Figures 4 and 6 ) .", "These data suggest that HetL provides immunity for the developing cells all along the differentiation process .", "HetL might therefore be important for pattern initiation and establishment , exerted by PatX and PatS , and also for pattern maintenance mediated by HetN and PatS .", "Therefore , we propose that HetR can be engaged in two different networks depending on cell types: a HetR-HetL-positive network in the ( pro ) heterocyt as a consequence of hetL expression , and a negative HetR-inhibiting peptide complex taking place in the HetL-free vegetative cells .", "The HetR/HetL complex therefore enriches the ‘local-activation and long-range inhibition model’ with a third module that we propose to call ‘local protection’ since this complex allows the differentiating cell to become immune to self-inhibition ( Figure 7C ) .", "An interesting particularity of patterning in Nostoc is that future heterocysts do not emerge as isolated cells .", "Rather , strings of 3–4 developing cells appear before resolving into a single proheterocyst .", "( Wilcox et al . , 1973 ) .", "These clusters of cells ‘competent’ for differentiation were suggested to be correlated to the stochastic fluctuation in hetR expression ( Corrales-Guerrero et al . , 2015 ) .", "In addition , we propose that a stochastic variation of hetL expression among the string of competent cells might also be part of the resolving mechanism fixing the decision to differentiate in the cell that inherits enough HetL to be protected from self-inhibition .", "Microfluidic approaches probing multiple and integrated aspects of heterocyst formation along with mathematical models should shed light on how patterning emerges , resolves and persists in Nostoc .", "An interesting evolutionary aspect resulting from the conservation of PRPs coding-genes in cyanobacterial genomes is that the function of HetL described here might be accurate for other heterocyst-forming cyanobacteria .", "It is also likely that understanding all the parameters and factors governing heterocyst-pattern formation will be useful in uncovering the molecular mechanisms underlying patterning in more complex biological systems ." ], [ "The strains , plasmids and primers used in this study are listed in Supplementary file 1 .", "All the cyanobacterial strains are derivatives of Nostoc PCC 7120 ( Pasteur Cyanobacterial Collection , https://www . pasteur . fr/fr/sante-publique/crbip/les-collections/collection-cyanobacteries-pcc ) .", "Nostoc and derivatives were grown in BG11 medium ( Rippka R et al . , 1979 ) at 30°C under continuous illumination ( 40 µE m−2s−1 ) .", "When appropriate , media were supplemented with antibiotics at the following concentrations neomycin ( 50 μg mL−1 ) , erythromycin ( 200 μg mL−1 ) .", "Heterocyst formation was induced by transferring the exponentially growing cultures ( OD 750 = 0 . 8 ) to BG110 ( BG11 without sodium nitrate ) medium .", "The presence of heterocysts was confirmed by microscopy .", "Conjugation of Nostoc was performed as described in Cai and Wolk , 1990 .", "Briefly , E . coli strains ( bearing the replicative plasmid and the RP-4 conjugative plasmid ) grown to exponential growth phase , were mixed to an exponentially grown Nostoc culture .", "The mixture was plated on BG11 plates and antibiotics were injected under the agar 24 hr later for plasmid selection .", "All the plasmids used in this study are listed in Supplementary file 1 .", "The strategy used for plasmid construction is briefly described below .", "All the recombinant plasmids were analyzed by sequencing .", "For HetL purification , the BL21DE3 strain containing the pXX12 plasmid was grown until an optical density ( OD 600 nm ) of 0 . 6 .", "The expression of hetL was induced by the addition of Isopropyl β-D-1-thiogalactopyranoside ( IPTG , SIGMA ) of 0 . 4 mM over night at 16°C .", "Cells were harvested at 8000 rpm at 4°C during 2 min .", "The pellet was re-suspended in 25 mL of lysis buffer ( 50 mM Tris HCl ( pH 8 ) , 0 . 3 M NaCl ) , and cells were disrupted using French press .", "After centrifugation at 8000 rpm for 30 min at 4 °C , the supernatant was loaded onto a HisTrap Excel column ( GE healthcare ) pre-equilibrated with lysis buffer containing 10 mM Imidazole .", "The column was rinsed with 10 mM and 35 mM Imidazole , both prepared in lysis buffer .", "Fractions were collected ( in 200 mM Imidazole ) .", "The Imidazole was eliminated using disposable PD 10 desalting columns ( GE Healthcare ) .", "The proteins were concentrated using Vivaspin columns ( SIGMA ) and quantified using the Bradford assay ( SIGMA ) .", "HetR purification was undergone as previously described ( Hu et al . , 2015 ) .", "The promoter region of the hetP gene ( alr2818 ) was obtained by PCR using PhetP fw and PhetP rv primers .", "The forward primer was modified at its 5’ end by adding the 6-carboxyfluorescein ( 6-FAM ) dye .", "Purified HetR ( 1 µM ) and HetL ( 2–4 µM ) proteins , were incubated with the hetP promoter ( 50 nM ) in a buffer containing 10 mM Tris ( pH 8 ) , 150 mM potassium chloride , 500 nM EDTA , 0 . 1% Triton X-100 , 12 . 5% glycerol , 1 mM dithiothreitol and 1 µg DiDC competitor ( poly ( 2′-deoxyinosinic-2′-deoxycytidylic acid ) sodium salt ) , at 4°C in dark for 30 min .", "The electrophoresis was performed at 250 volts for 60 min .", "The DNA was revealed using Typhoon FLA 9500 ( GE Healthcare Life Sciences ) .", "The experiment was repeated three times with independent protein purifications and one representative result is shown .", "RNAs were prepared using the Qiagen RNA extraction kit ( Qiagen ) following the manufacturer’s instructions .", "An extra TURBO DNase ( Invitrogen ) digestion step was performed to eliminate the contaminating DNA .", "The RNA quality was assessed by tape station system ( Agilent ) .", "RNAs were quantified spectrophotometrically at 260 nm ( NanoDrop 1000; Thermo Fisher Scientific ) .", "For cDNA synthesis , 1 µg total RNA and 0 . 5 μg random primers ( Promega ) were used with the GoScript Reverse transcriptase ( Promega ) according to the manufacturer instructions .", "Quantitative real-time PCR ( qPCR ) analyses were performed on a CFX96 Real-Time System ( Bio-Rad ) .", "The reaction volume was 15 μL and the final concentration of each primer was 0 . 5 μM .", "The qPCR cycling parameters were 95°C for 2 min , followed by 45 cycles of 95°C for 5 s , 55°C for 60 s .", "A final melting curve from 65°C to 95°C was added to determine the specificity of the amplification .", "To determine the amplification kinetics of each product , the fluorescence derived from the incorporation of BRYT Green Dye into the double-stranded PCR products was measured at the end of each cycle using the GoTaq qPCR Master Mix 2X Kit ( Promega ) .", "The results were analysed using Bio-Rad CFX Maestro software , version 1 . 1 ( Bio-Rad , France ) .", "The RNA 16S gene was used as a reference for normalization .", "All measurements were carried out in triplicate and a biological duplicate was performed for each point .", "The amplification efficiencies of each primer pairs were 80% to 100% .", "All the primer pairs used for qPCR are reported in Supplementary file 1 .", "For each construct , the studied proteins were fused to the carboxy terminus of the T25 or T18 domain of CyA , except that HetRhood was fused to the N-terminus of the T18 domain .", "Bacterial two-hybrid assays were performed following the procedure described by Karimova et al . , 1998 .", "Briefly , after co-transforming the BTH101 strain with the two plasmids expressing the T18- and T25- fusions , LB plates containing ampicillin and kanamycin were incubated at 30° C for 2 days .", "For each assay , 10 independent colonies were inoculated in 3 ml of LB medium supplemented with ampicillin , kanamycin and 0 . 5 mM IPTG , and incubated at 30°C overnight .", "ß-galactosidase activity was determined as previously described ( Zubay et al . , 1972 ) .", "The values presented are means of three independent assays .", "Proteins were fractionated by performing SDS-PAGE ( 4–20% ) stained with Coomassie blue ( Euromedex , Souffelweyrshim , France ) .", "For immunoblot analysis , the proteins were transferred to nitrocellulose membranes before being revealed with anti-Histidine monoclonal antibodies ( Qiagen ) .", "Immune complexes were detected with anti-rabbit peroxidase-conjugated secondary antibodies ( Promega ) and enhanced chemiluminescence reagents ( Pierce , Illkich , France ) .", "PatS-5 ( RGSGR ) , Pat-6 ( ERGSGR ) and PatX-6 ( HRGTGR ) peptides were synthesized by Genecust ( https://www . genecust . com/en/ ) .", "ITC was performed to demonstrate the interaction of HetR and HetL with PatS peptides .", "The working buffer for both proteins and peptides was PBS pH 7 . 4 to avoid buffer mismatch .", "The experiments were performed at 25°C using the MicroCal PEAQ-ITC ( Malvern UK ) with 19 injections , first with an initial injection of 0 . 4 µL followed by 18 injections of 2 µL .", "The protein ligands were in the cell and the peptide analytes were in the syringe .", "The reaction was performed with a constant stirring speed of 750 rpm , each injection lasted for 4 s with a 150 s space between each injection .", "A constant heat control ( offset ) was removed from the raw data to account for heat dilution before integration .", "The data were fitted using a ‘One Set of Sites’ model in the PEAQ-ITC Analysis Software .", "The experiment was repeated two times with independent protein purifications and one representative result is shown .", "The BLi machine ( BLItz ) from FortéBio was used to perform biolayer interferometry to determine the interaction between HetR and HetL .", "2 . 2 µM of biotinylated HetR was loaded onto streptavidin biosensors in PBS .", "A 30 s baseline in PBS was performed before a 120 s association step with various concentrations of HetL at 2 . 5 , 5 , 10 and 20 µM followed by a 120 s dissociation step .", "To determine if the PatS-5 and PatS-6 peptides compete with HetL for HetR binding , 10 µM of HetL was incubated five mins with different concentrations of PatS at 0 . 05 , 0 . 1 , 0 . 5 and 2 . 5 µM before adding HetL to the bound HetR .", "All bindings were performed in triplicate , the dissociation constant was obtained using the BLItz Pro Data Analysis software using a global 1:1 fit model .", "The available atomic coordinates of the hood domain of Nostoc HetR ( PDB ID: 4YNL ) and HetL ( PDB ID: 3DU1 ) were used as templates for molecular docking simulations .", "Molecular docking study of the HetR with HetL was performed using the HADDOCK2 . 2 webserver ( http://milou . science . uu . nl/services/HADDOCK2 . 2/ ) ( van Zundert et al . , 2016 ) .", "The goal was to identify critical residues involved in HetR-HetL complex formation .", "To define Haddock run restraints , the surface exposed residues of both HetR and HetL were considered as active residues directly involved in the interaction .", "For both structures , the surface exposed residues were selected manually using PyMol ( Supplementary file 1 ) .", "Two independent docking simulations were performed .", "For each run , 10 clusters were generated , and classified based on their HADDOCK score .", "The best model of each cluster was analyzed by PDBePISA to explore their binding interfaces ." ] ]

[ "Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns .", "A number of them involve the production by the developing cell of an inhibitory morphogen , but how this cell becomes immune to self-inhibition is rather unknown .", "Under combined nitrogen starvation , the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10–12 vegetative cells .", "Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens , diffusion of which establishes the differentiation pattern .", "Here , we show that HetR interacts with HetL at the same interface as PatS , and that this interaction is necessary to suppress inhibition and to differentiate heterocysts .", "hetL expression is induced under nitrogen-starvation and is activated by HetR , suggesting that HetL provides immunity to the heterocyst .", "This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum ." ]

[ "Cyanobacteria are the only bacteria on Earth able to draw their energy directly from the sun in the same way that plants do .", "In addition , some strains are able to ‘fix’ the nitrogen present in the atmosphere: they can extract this gas and transform it into nitrogen-based compounds necessary for life .", "However , both processes cannot happen in a given cell at the same time .", "A strain of cyanobacteria called Nostoc PCC 7120 can organise itself into long filaments of interconnected cells .", "Under certain conditions , one in every ten cells stops drawing its energy from the sun , and starts fixing atmospheric nitrogen instead .", "Exactly how the bacteria are able to ‘count to ten’ and organize themselves in such a precise pattern is still unclear .", "Cells can communicate and establish patterns by exchanging molecular signals that switch on and off certain cell programs .", "For instance , a protein called HetR turns on the genetic program that allows cyanobacteria to fix nitrogen; on the other hand , a signal known as PatS binds to HetR and turns it off .", "Cells starting to specialise in fixing nitrogen produce both HetR and PatS , with the latter diffusing in surrounding cells and preventing them from extracting nitrogen .", "However , it remained unclear how the nitrogen-fixing cell could ignore its own PatS signal and keep its HetR signal active .", "HetL – another protein produced by the future nitrogen-fixing cell – could potentially play this role , but how it acts was unknown .", "Here , Xu et al . show that HetL cannot diffuse from one cell to the other , and that it binds to HetR at the same place than PatS does .", "When both PatS and HetL are present , they compete to attach to HetR , which stops PatS from turning off HetR and deactivating the nitrogen-fixing program .", "Understanding how cyanobacteria fix nitrogen could help to develop new types of natural fertiliser .", "More generally , dissecting how these simple organisms can create patterns could help to grasp how patterning emerges in more complex creatures ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "microbiology and infectious disease" ]

[ [ "Inordinately high genetic variation is a hallmark of RNA viruses .", "The rapid evolution underlying this variation can occur as a result of mutation , recombination , or reassortment , with major consequences for human disease ( Andino and Domingo , 2015 ) .", "In the case of influenza virus , these consequences include poor vaccine efficacy rates , immune escape , antiviral resistance , and the emergence of novel strains ( Lyons and Lauring , 2018 ) .", "Within the past century , influenza A virus ( IAV ) pandemics occurred in 1918 ( H1N1 ) , 1957 ( H2N2 ) , 1968 ( H3N2 ) , and 2009 ( H1N1 ) ( Neumann et al . , 2009; Paules and Subbarao , 2017; Short et al . , 2018 ) .", "Each of the last three influenza pandemics was attributable to a reassortant strain composed of a novel combination of the eight viral RNA ( vRNA ) segments of the influenza virus genome ( Neumann et al . , 2009 ) .", "Thus , the emergence of pandemic strains is marked by a concomitant alteration in the influenza virus genome .", "Public health measures to limit the impact of influenza virus outbreaks prioritize emerging viruses based on perceived risk factors such as the potential for reassortment between circulating influenza viruses .", "Reassortment of vRNA segments must occur during selective assembly of all eight genomic segments , which occurs after export of vRNA segments from the nucleus ( Lakdawala et al . , 2014 ) .", "Genomic assembly contributes to heterogeneity in progeny viruses and could determine the fitness of reassortant strains after coinfection ( Brooke , 2017; Lowen , 2017 ) .", "Selective assembly is thought to be facilitated by intersegmental RNA-RNA interactions .", "Each vRNA segment encodes packaging signals that must be compatible for reassortment to occur ( Lowen , 2017; Richard et al . , 2018 ) .", "Although much remains unknown about the role of RNA-RNA interactions in genomic assembly , it is evident that disruption of interactions between two vRNA segments can alter interactions with other segments , leading to a model in which hierarchical interactions between vRNA segments ensure selective assembly ( Dadonaite et al . , 2019; Le Sage et al . , 2020; Marsh et al . , 2008 ) .", "Such complexity among vRNA interactions poses a significant hurdle to reassortment ( Gavazzi et al . , 2013; Noda et al . , 2006 ) .", "It is consequently imperative to identify the evolutionary constraints imposed by intersegmental vRNA interactions , as this may improve risk assessment efforts for emerging influenza viruses .", "Complex intersegmental RNA-RNA interactions could be governed by epistasis , the phenomenon by which a mutation in one gene is impacted by the presence or absence of mutations in other genes ( Sardi and Gasch , 2018 ) .", "A number of tools exist to examine the shared evolutionary trajectories resulting from epistatic interactions between genes , yet the current focus surrounds constraints on indirect interactions between proteins that may function together rather than on interactions between viral RNA ( Escalera-Zamudio et al . , 2020 ) .", "Previous work with probabilistic models revealed that several mutations in the influenza virus nucleoprotein ( NP ) that are destabilizing on their own became fixed as a result of counterbalancing compensatory mutations that improve the overall protein stability of NP ( Gong et al . , 2013 ) .", "These destabilizing mutations occur within T cell epitopes of NP that may be important for immune escape ( Gong et al . , 2013 ) .", "Stabilizing epistasis was similarly instrumental to the emergence of oseltamivir resistance mutations in the influenza neuraminidase ( NA ) ( Bloom et al . , 2010 ) .", "The rise of oseltamivir resistance mutations in NA spurred investigation of shared evolutionary trajectories , or parallel evolution , between NA and hemagglutinin ( HA ) , demonstrating that mutations in HA may have facilitated acquisition of oseltamivir resistance mutations in NA ( Jang and Bae , 2018; Kryazhimskiy et al . , 2011; Neverov et al . , 2015 ) .", "We propose that phylogenetics could be further employed to investigate epistasis arising from direct RNA-RNA interactions between IAV segments .", "Therefore , shared evolutionary trajectories , or parallel evolution , between vRNA segments could reveal epistatic constraints on genomic assembly and reassortment .", "In this study , we set out to combine phylogenetics and molecular biology to examine parallel evolution across vRNA segments genome-wide in seasonal human influenza viruses to identify potential epistatic relationships .", "Unlike previous studies , our objective was to identify relationships between vRNA segments that might play key roles specifically in genomic assembly .", "To evaluate phylogenetic relationships among vRNA segments , we relied upon the Robinson-Foulds distance ( RF ) , a widely used measure of topological distance between trees ( Robinson and Foulds , 1981 ) .", "This method determines the number of branch partitions that are not shared between two trees ( Robinson and Foulds , 1981 ) and is therefore a quantitative measure of the topological distance between trees .", "We combined the conventional RF with the clustering information distance ( CID ) , a recently described measure of tree similarity with greater sensitivity for distinguishing between trees ( Smith , 2020 ) .", "Lower RF/CID corresponds with greater tree similarity , with a tree distance of 0 indicating that two trees are topologically equivalent .", "Our approach relies upon the assumption that tree distance would be inversely correlated with the degree of parallel evolution between genome segments arising from either RNA-RNA or protein-protein interactions .", "Incompatible polymerase subunits exhibit replication deficiencies and are known restriction factors in reassortment ( Li et al . , 2008 ) .", "Accordingly , we would predict that trees built from PB2 , PB1 , and PA would have high similarity , reflective of a shared evolutionary trajectory .", "Likewise , mounting evidence from our group and others suggests that direct intermolecular interactions between vRNA segments coordinate selective assembly ( Dadonaite et al . , 2019; Le Sage et al . , 2020 ) .", "Highly similar trees could therefore also be reflective of direct interactions between vRNA segments that may facilitate selective packaging .", "To distinguish between the roles of RNA and protein , we further examine tree similarity in viral proteins , choosing gene segments with high gene tree similarity , but not high protein tree similarity , to probe for RNA-RNA interactions .", "Since genomic assembly occurs in the cytoplasm after nuclear export ( Lakdawala et al . , 2014 ) , we reasoned that assembly intermediates found in close proximity to the nucleus could serve as scaffolds for genomic assembly and sought to visualize this by confocal microscopy .", "Therefore , our approach systematically identifies putative epistatic relationships between vRNA segments to elucidate mechanisms of selective vRNA assembly ." ], [ "H1N1 and H3N2 viruses have cocirculated in the human population since 1977 ( Neumann et al . , 2009 ) .", "In order to identify shared evolutionary trajectories between vRNA segments in seasonal human IAV strains over time , we examined parallel evolution between vRNA segments in viruses representative of each subtype from multiple time periods ( Table 1 ) .", "Bracketing H3N2 viruses into two time intervals permitted investigation of conserved vRNA relationships over time in antigenically drifted H3N2 viruses .", "We took a similar approach with human H1N1 viruses , bracketing instead on the antigenic shift event in 2009 and the emergence of the pandemic swine-origin H1N1 virus in the human population .", "Comparison of vRNA relationships in pre-pandemic ( 2000–2008 ) and post-pandemic ( 2010–2018 ) human H1N1 viruses could reveal distinct vRNA relationships from viruses of two distinct lineages or alternatively , uncover vRNA relationships that remain conserved despite swapping of vRNA segments across multiple host species .", "Our approach outlined in Figure 1 examines evolutionary relationships between vRNA segments .", "We began our investigation with all seasonal human H3N2 viruses for which full-length sequence information was available in the Influenza Research Database ( IRD ) , yielding 1026 H3N2 viruses from 1995 to 2004 and 3879 H3N2 viruses from 2005 to 2014 ( Table 1 ) .", "Reconstructing phylogenetic trees from all available sequences was disadvantageous , as a preliminary analysis of 300 sequences suggested that a great deal of phylogenetic variation could not be statistically supported by bootstrapping ( branch support less than 70 ) .", "This lack of bootstrap support was problematic for our downstream analysis of tree similarity , since topological distance can result from misleading phylogenetic signal when branches are poorly supported .", "Thus , reliance upon larger , poorly resolved trees would lead to uninterpretable tree distances .", "To address this , we used a clustering approach to select representative strains that would produce more statistically robust trees .", "We first concatenated sequences from all strains into full-length genomes from which we built alignments ( Figure 1A ) and clustered into operational taxonomic units on a neighbor-joining species tree ( Figure 1B ) .", "Despite the fact that fewer full-length influenza virus genomic sequences were available prior to the 2000s , our approach resulted in a similar number of clusters within a subtype ( Table 1 ) , consistent with the notion that increased sequencing has led to more closely related sequences in public databases .", "The primary objective behind clustering was to minimize variation between trees that was not statistically supported by bootstrapping .", "The cutoff for sequence identity during clustering of the species tree was therefore an important consideration because it controlled how much unsupported variation remained in our trees .", "Higher cutoffs ( 98–99% sequence identity ) yielded species trees with more clusters containing fewer members while lower cutoffs ( 95–96% sequence identity ) contained increasingly fewer clusters with more members grouped in each cluster .", "We selected a cutoff of 97% sequence identity based on the observation that it produced vRNA trees with an intermediate number of clusters ( 16–17 clusters in each species tree ) .", "We selected several high-quality sequences from each cluster to build replicate vRNA trees for comparison ( Supplementary files 1 and 2 ) .", "Using this approach , more than half of all branches were consistently supported in PB2 , PB1 , and HA trees; however , NS trees remained largely unsupported regardless of the sequence identity cutoff selected .", "Branch support varied between replicate trees of PA , NP , and NA , with no single replicate yielding consistently high branch support across vRNA trees .", "Therefore , we analyzed all seven replicate trees for each of the eight vRNA segments , for a total of 56 trees analyzed from each set of H3N2 viruses ( Figure 1C , Figure 1—figure supplement 1 , and Supplementary files 1 and 2 ) .", "Tree similarity among different vRNA segments is expected to be highest when there are strong epistatic interactions between encoded protein and/or RNA complexes ( Kryazhimskiy et al . , 2011; Neverov et al . , 2015; Nshogozabahizi et al . , 2017 ) .", "We expected to observe such epistasis between protein subunits of the heterotrimeric polymerase complex such as the PB1 and PA segments ( Fodor , 2013 ) , whereas we did not expect to observe epistasis between PB1 and HA , which do not share any known protein function .", "Therefore , we examined the extent of similarity between the PB1 tree and the PA and HA trees in H3N2 viruses from 2005 to 2014 .", "Trees built from the PB1 and PA segments had low tree distances ( RF = 6 and CID = 0 . 25 ) ( Figure 2A ) , suggesting that these genes evolve in parallel .", "PB1 and HA trees from the same set of H3N2 strains had higher tree distances ( RF = 14 and CID = 0 . 44 ) ( Figure 2B ) , suggesting that parallel evolution between PB1 and HA is weaker than that of PB1 and PA .", "These data are consistent with known protein interactions between PB1 and PA and suggest that tree similarity can be used to identify direct intermolecular interactions that constrain evolution , leading to converging evolutionary trajectories in the trees .", "Thus , pairwise tree distances recapitulate anticipated protein-driven parallel evolution between two influenza proteins .", "Genome-wide inferences of tree similarity can distinguish the relative degree of parallel evolution of all eight genomic segments to each other and capture the strongest overall relationships between segments .", "To examine the extent of parallel evolution between all vRNA segments , we measured tree distances in all sets of vRNA trees from H3N2 viruses from 2005 to 2014 .", "Figure 2C shows the overall mean tree distances between each pair of vRNA segments as determined by RF ( refer to Figure 2—figure supplement 1 for the standard error of the mean , or SEM , between sets of trees ) .", "To establish a threshold for significance of tree distances , we determined a 95% confidence interval for RF using a null dataset of randomly generated trees with an equivalent number of leaves ( 12 in this case , Figure 2—figure supplement 3A , D ) .", "Low tree distances rarely occurred by chance , with the vast majority of tree distances being greater than 15 in null trees .", "By comparison , the mean RF of vRNA trees ranged from 6 . 5 ( PB1 and PA ) to 15 ( PA and NS ) .", "Surprisingly , the PB2 tree shared the highest similarity with the NA tree rather than the PB1 or PA trees , suggesting that the relationship between PB2 and NA may supercede the essential role of the PB2 protein in the polymerase complex .", "In contrast , the mean RF of the NS trees with most other vRNA trees were 14–15 , approaching the 95% confidence threshold of 15 . 3 .", "However , distances between the NS tree and the other vRNA trees were difficult to interpret , owing to a lack of branch support in NS trees ( Figure 1—figure supplement 1B ) .", "Pairwise intersegmental relationships determined by RF were remarkably reproducible when compared to CID ( Figure 2—figure supplement 2 ) .", "To further visualize relationships between all eight vRNA segments , we assembled networks of the pairwise tree distances ( Figure 2—figure supplement 4 ) .", "These networks reveal robust parallel evolution between PB1 , PA , NP , and NA in H3N2 viruses .", "Recent studies have identified a highly plastic and redundant network of interactions between vRNA segments in influenza virus particles produced during productive infection , many of which may be transient ( Dadonaite et al . , 2019; Le Sage et al . , 2020 ) .", "Based on these observations , it is plausible that vRNA relationships identified using our methods might change over time .", "To examine whether the shared evolutionary trajectories we observed in H3N2 viruses are conserved , we estimated tree distances between all pairs of vRNA trees in H3N2 viruses from an earlier time period ( 1995–2004 ) ( mean RF: Figure 3A; SEM of RF: Figure 3—figure supplement 1; mean CID: Figure 3—figure supplement 2A; SEM of CID: Figure 3—figure supplement 2B ) .", "As was seen in H3N2 viruses from 2005 to 2014 , tree distances ranged widely , with the highest tree similarity found between PB1 , PA , NP , and NA trees of this time period .", "Networks constructed from pairwise distances that visualize the overall relatedness of vRNA segments confirm that PB1 , PA , NA , and NP share the closest distances overall ( Figure 3—figure supplement 3 ) .", "Statistical differences between RF from each time period were only found for the NS segment ( Figure 3C; refer to Supplementary file 6 for exact p-values ) .", "However , NS trees had consistently low bootstrap support ( Figure 1—figure supplement 1A , B ) , so these differences may be attributable to insufficient resolution in the underlying trees .", "Finally , tree distances for H3N2 viruses from 1995 to 2004 were positively correlated with those from 2005 to 2014 ( Figure 3B and Figure 3—figure supplement 4 ) .", "Taken together , we conclude that phylogenetic relationships between vRNA segments in H3N2 viruses are largely conserved across these two time periods .", "Our results suggest that vRNA relationships are remarkably consistent across H3N2 viruses from a period spanning two decades .", "To examine whether our approach captures anticipated changes in vRNA relationships in seasonal human influenza viruses of other subtypes and lineages , we assessed these relationships in H1N1 viruses from 2000 to 2008 and 2010 to 2018 .", "Human H1N1 viruses from these time periods represent distinct lineages before and after the 2009 pandemic .", "This pandemic was caused by an antigenically shifted H1N1 virus that emerged from reassortment of two swine-origin viruses , the North American triple reassortant swine H1N1 virus and Eurasian swine H1N1 virus ( Garten et al . , 2009 ) .", "Therefore , different evolutionary relationships between vRNA segments would be expected for each lineage .", "Species trees comprising full-length concatenated H1N1 virus genomes from 2000 to 2008 or 2010 to 2018 were constructed and clusters were defined using the same approach described for H3N2 viruses ( Figure 1A–B ) .", "While this method produced a similar number of clusters for both sets of H1N1 viruses ( Table 1 ) , there were fewer clusters than in H3N2 viruses , owing to the higher rate of evolution observed in H3N2 viruses ( Bedford et al . , 2015 ) .", "Seven strains were selected from each cluster ( Supplementary files 3 and", "4 ) and replicate vRNA trees were built as in Figure 1C , Figure 1—figure supplement 1C , D .", "Figures 4A and 5A show the mean RF for each pair of vRNA segments in H1N1 viruses from 2000 to 2008 and 2010 to 2018 , respectively ( SEM: Figure 4—figure supplement 1 and Figure 5—figure supplement 1 ) .", "These heatmaps suggested that tree distances were very different for H1N1 viruses when compared to H3N2 viruses .", "Using linear regression , we found that tree distances from H1N1 viruses shared either a modest negative correlation or none at all with H3N2 viruses from either time period ( Figure 4B and C , and Figure 5—figure supplement 3 ) .", "Tree distances determined by CID ( 2000–2008 mean and SEM: Figure 4—figure supplement 2A , B , respectively; 2010–2018 mean and SEM: Figure 5—figure supplement 2A , B , respectively ) likewise indicated similar trends ( Figure 4—figure supplement 3 and Figure 5—figure supplement 4A , B ) .", "This is further supported by networks constructed from the pairwise distances for H1N1 viruses as compared to those from H3N2 viruses .", "The distance networks from H3N2 viruses suggest highest overall tree similarity between PB1 , NP , PA , and NA ( Figure 2—figure supplement 4 , Figure 3—figure supplement 3 ) .", "In contrast , the networks from pre-pandemic H1N1 viruses indicate highest tree similarity between PB1 , NP , M , and NS ( Figure 4—figure supplement 4 ) .", "Networks from post-pandemic H1N1 viruses likewise reflect a different pattern in tree relatedness from that seen in H3N2 virus networks ( Figure 5—figure supplement 5 ) .", "Therefore , our data suggest that parallel evolution between vRNA segments overall have significantly diverged between seasonal human influenza H1N1 and H3N2 viruses from similar time scales .", "Heatmaps comparing tree distances between vRNA pairs further suggested that vRNA relationships are not conserved across H1N1 viruses of different lineages ( Figure 4A vs . Figure 5A ) .", "Linear regression comparing tree distances between vRNA segments from pre-pandemic and post-pandemic H1N1 viruses confirmed no correlation between these trees ( Figure 5B and Figure 5—figure supplement 4C ) .", "To examine individual differences between pairs of vRNA trees in H1N1 viruses of different lineages , we plotted RF from pre-pandemic H1N1 viruses alongside RF from post-pandemic H1N1 viruses ( Figure 5C; refer to Supplementary file 6 for exact p-values ) .", "The 95% confidence interval cutoff for RF corresponding to trees with nine leaves was 8 . 6 ( Figure 2—figure supplement 3C ) and is the threshold used for statistical comparison of parallel evolution in vRNA segments from pre-pandemic and post-pandemic H1N1 strains .", "In stark contrast to the relatively conserved vRNA relationships observed in H3N2 viruses over time , many relationships between vRNA segments were disrupted in post-pandemic H1N1 viruses .", "Parallel evolution between PB1 and NP observed in pre-pandemic H1N1 viruses ( mean RF increased from 1 to", "5 ) was notably displaced by stronger coevolution of PB1 with NA in post-pandemic H1N1 viruses ( mean RF decreased from 9 to 3 ) .", "The M and NS trees shared similar topologies across H1N1 lineages , but each one was significantly more coevolved with the HA and NA trees in post-pandemic viruses .", "The PB2 trees diverged significantly from the PA trees in favor of greater parallel evolution with the NP and NS trees in post-pandemic H1N1 viruses .", "Some of these data can be explained by weaker bootstrap support in H1N1 trees , particularly those from H1N1 viruses from 2010 to 2018 ( Figure 1—figure supplement 1C , D ) .", "However , these data imply that shared evolutionary trajectories have significantly diverged between H1N1 lineages .", "The genomic segments of the swine-origin 2009 pandemic H1N1 virus were contributed by human , avian , and swine hosts ( Garten et al . , 2009 ) .", "Therefore , our data suggest that host origin may impact the evolutionary trajectory of emerging reassortant viruses from different lineages and the resultant relationships between genomic segments of contemporary H1N1 viruses in humans .", "As discussed previously , shared evolutionary trajectories could arise from either protein-protein or RNA-RNA interactions .", "We have already shown that known protein relationships between PB1 and PA , two members of the polymerase complex , are captured by our approach ( Figure 2A ) .", "However , the observation that PB2 , another member of the polymerase complex , is more coevolved with NA than with either PB1 or PA ( Figure 2C ) suggests that our method also reveals protein-independent parallel evolution , since these proteins are not known to function together during infection .", "Using H3N2 viruses from 2005 to 2014 , which yielded vRNA trees with the highest overall bootstrap support ( Figure 1—figure supplement 1B ) , we explored the extent to which parallel evolution between vRNA segments is driven by protein-coding mutations .", "To do so , we converted the vRNA sequence alignments , which are negative-sense , into positive-sense RNA ( i . e . coding sense ) and translated the coding sequences into amino acid alignments .", "For the M and NS sequence alignments that encode two splice variants each , the M1/M2 and NS1/NS2 amino acid alignments were both translated .", "Neighbor-joining trees were reconstructed from the amino acid alignments and the evolutionary relationships between H3N2 proteins were analyzed by RF .", "We constructed a network from the resultant RF between all pairs of protein trees as was previously done with vRNA trees ( Figure 6—figure supplement 1 ) .", "This network appears distinct from networks built from the corresponding gene ( vRNA ) trees ( Figure 2—figure supplement 4 ) .", "As might be expected , the greatest degree of parallel evolution lying at the core of this network was between HA and NA , two viral glycoproteins with coordinated functions in attachment , motility , and entry ( Bloom et al . , 2010; Sakai et al . , 2017 ) .", "To compare parallel evolution between influenza proteins to that of the parent vRNA segments , the mean RF from the gene trees were plotted against the mean RF from the protein trees ( Figure 6 ) .", "In the case of the M and NS segments , the mean RF of all protein trees derived from the same gene ( i . e . M1/M2 or NS1/NS2 ) were plotted against the mean RF of the corresponding gene trees .", "Many vRNA pairs , such as the polymerase subunits PB2 and PB1 , lie along the identity line , indicating that protein interactions are more likely to drive parallel evolution in those vRNA segments .", "Interestingly , HA and NA were the only pair of vRNA segments that lay significantly above the identity line , strongly supporting the observation made by others that epistatic interactions between these proteins constrain their evolution ( Jang and Bae , 2018; Kryazhimskiy et al . , 2011; Neverov et al . , 2015 ) .", "Of particular interest was that several vRNA segments , such as PB2 and NA ( Figure 6 , open diamond ) , lay significantly below the identity line .", "This could be indicative of either purifying selection or of greater parallel evolution between the vRNA segments than the proteins encoded .", "While this is not altogether unexpected , considering that the mutation rate of a protein is unlikely to be as high as the mutation rate of the corresponding gene , we would expect conserved RNA interactions to also have this effect .", "These observations suggest that parallel evolution may identify putative RNA interactions between vRNA segments that could facilitate selective assembly and packaging .", "To address whether parallel evolution between the PB2 and NA segments corresponds with their behavior during influenza virus infection , we examined whether these vRNA segments preferentially colocalize in infected cells ( Figure 1D ) .", "During influenza virus infection , vRNA are synthesized in the nucleus , bound by NP in viral ribonucleoprotein ( vRNP ) complexes , and then transported to the plasma membrane for packaging on endocytic vesicles ( Lakdawala et al . , 2016 ) .", "Direct RNA-RNA interactions are thought to drive selective assembly of all eight vRNA segments into virus particles , with a hierarchy existing between interactions ( Dadonaite et al . , 2019; Le Sage et al . , 2020; Le Sage et al . , 2018; Lee et al . , 2017; Marsh et al . , 2008 ) .", "Previous studies examining the intracellular localization of vRNA segments demonstrated that after genomic replication , vRNA segments are exported from the nucleus as incomplete subcomplexes , or assembly intermediates ( Lakdawala et al . , 2014 ) .", "The formation of complete complexes containing all eight segments occurs en route to the plasma membrane through dynamic fusion or fission of vRNA segments ( Bhagwat et al . , 2020; Lakdawala et al . , 2014 ) .", "Taken together , these data suggest that interactions between some vRNA segments may serve as a scaffold that facilitates formation of complete complexes of all eight vRNA segments .", "Our network analyses suggest a putative hierarchy that could in part reflect the proposed hierarchical nature of genomic assembly ( Figure 2—figure supplement 4 ) .", "We theorized that those vRNA segments that exhibit high gene tree similarity might preferentially form subcomplexes soon after nuclear synthesis .", "Using our extensive expertise in visualizing intracellular localization of vRNA segments ( Lakdawala et al . , 2014; Nturibi et al . , 2017 ) , we examined the localization of three vRNA segments ( PB2 , NA , and NS ) in the context of H3N2 virus infection .", "These segments encompass a pair with high gene-based parallel evolution ( PB2-NA ) as well as pairs with less evidence of parallel evolution ( PB2-NS; NA-NS ) ( Figures 2C and 6 , open diamonds ) .", "Quantification of colocalized vRNA segments was performed using fluorescence in situ hybridization ( FISH ) and immunofluorescence ( IF ) in productively infected cells ( Lakdawala et al . , 2014; Nturibi et al . , 2017 ) .", "Lung epithelial A549 cells were infected for 8 hr with a seasonal human H3N2 virus representative of the time period analyzed ( A/Perth/16/2009 ) and stained for three vRNA segments , NP , and nuclei .", "The NP antibody stain was used to normalize pairwise colocalization data to the total number of vRNP foci present in cells .", "Entire cell volumes were captured and the nucleus was masked to analyze colocalization of vRNA segments specifically within the cytoplasm .", "A representative image of an infected cell from one of three independently performed experiments is shown after processing ( Figure 7A ) and at various stages of image analysis ( Figure 7B ) .", "Whole cytoplasmic analysis of vRNP colocalization in 15 individually analyzed cells revealed that the majority of cytoplasmic foci contained all three vRNA segments ( Figure 7C ) .", "These data may represent heterogeneity in genomic assembly: whole cytoplasmic analysis is likely to capture vRNP subcomplexes at various stages of assembly , regardless of whether direct RNA-RNA interactions underlie colocalization ( Lakdawala et al . , 2014 ) .", "In contrast , perinuclear assembly intermediates are more likely to reflect essential RNA-RNA interactions ( Majarian et al . , 2018 ) .", "Therefore , we assessed the potential for PB2 , NA , and NS to colocalize at the nuclear periphery , where assembly intermediates first begin to form .", "We defined localization at the nuclear periphery to within 300 nm , the limit of resolution in this system .", "Examination of newly exported vRNP complexes within 300 nm of the nuclear periphery revealed an enrichment of PB2-NA vRNP complexes over either NA-NS or PB2-NS vRNP complexes ( Figure 7D ) .", "These data indicate that PB2 and NA preferentially colocalize with each other after nuclear synthesis and support the hypothesis that parallel evolution between segments can reveal putative RNA-RNA interactions .", "Moreover , these data implicate RNA-RNA interactions , in addition to protein interactions , as novel drivers of parallel evolution between vRNA segments in seasonal human influenza viruses ." ], [ "In this study , we used phylogenetics and molecular biology methods to investigate genome-wide relationships between vRNA segments in seasonal human IAV .", "We found that parallel evolution varies considerably between vRNA segments , with distinct relationships forming in different influenza virus subtypes ( H1N1 vs . H3N2 ) and between H1N1 virus lineages arising from distinct evolutionary paths .", "We further demonstrate that evolutionary relatedness between vRNA segments in H3N2 viruses is largely conserved over time .", "Importantly , our data suggest that parallel evolution cannot be attributed solely to protein interactions , and we successfully predicted intracellular colocalization between two coevolved vRNA segments during infection with an H3N2 virus .", "Thus , we present a phylogenetic approach for interrogating putative RNA associations that could be broadly applied toward the study of genomic assembly and reassortment in segmented viruses .", "Selective assembly of all eight genomic segments is fundamental to the production of fully infectious virus particles .", "We and others have used a variety of biochemical approaches to investigate the mechanisms that promote selective assembly ( Dadonaite et al . , 2019; Le Sage et al . , 2020 ) .", "We previously demonstrated that binding of vRNA segments by NP is non-uniform and non-random ( Le Sage et al . , 2018; Lee et al . , 2017 ) , supporting the model that intersegmental RNA interactions facilitate selective assembly .", "Biochemical approaches to define bona fide intersegmental RNA-RNA interactions demonstrated that the interaction network is highly flexible and varies between H1N1 and H3N2 viruses ( Dadonaite et al . , 2019; Le Sage et al . , 2020 ) .", "These observations are consistent with our conclusion that RNA interactions constrain parallel evolution between vRNA segments in a manner sensitive to the genetic context studied .", "The approach we present here differs from other experimental approaches in that we identify a novel , conserved RNA-driven relationship between vRNA segments in H3N2 viruses .", "For example , we found that relationships between PB1 , PA , NP , and NA are enriched over other segments in H3N2 viruses and conserved over time .", "One might expect PB1 , PA , and NP to coevolve because of the functions of the proteins they encode: the polymerase subunits PB2 , PB1 , and PA form a supramolecular complex around each vRNA segment with NP ( Fodor , 2013 ) .", "However , this explanation does not account for the parallel evolution observed between vRNP components and NA , and our microscopy data demonstrates that the NA segment preferentially colocalizes with the vRNA of one such vRNP component , supporting the possibility that parallel evolution of NA with PB1 , PA , and NP could also be driven by RNA-RNA interactions .", "These observations suggest that RNA relationships with the NA segment may facilitate selective assembly of vRNA segments .", "Further work should be directed at determining the underlying nature driving the novel relationship between these segments and whether similar assembly intermediates can be identified in H1N1 viruses .", "Previous pandemic influenza viruses emerged through reassortment ( Neumann et al . , 2009 ) .", "Risk assessment for future influenza pandemics relies on understanding assembly of vRNA segments within a cell .", "As we have discussed , experimental investigations of intersegmental RNA interactions indicate that the vRNA interactome is distinct among virus strains and highly plastic ( Dadonaite et al . , 2019; Le Sage et al . , 2020 ) .", "Therefore , experimental approaches are unlikely to provide the holistic view necessary to assess reassortment outcomes of two circulating influenza strains .", "In contrast , we identified several conserved relationships between vRNA segments in H3N2 viruses that could impose constraints on reassortment .", "In addition , we identified several key differences between the evolutionary trajectories of vRNA segments in pre-pandemic and post-pandemic H1N1 viruses of different lineages .", "Experimental investigation of the differences we present here may reveal key vRNA relationships that dictate reassortment and pandemic potential of influenza viruses .", "Thus , investigation of epistatic relationships between vRNA segments through phylogenetics could inform sequence-based implementation of barriers to reassortment in emerging influenza viruses ." ], [ "FASTA files of each genomic segment of human IAV sequences of H1N1 and H3N2 viruses were downloaded from the IRD ( http://www . fludb . org ) ( Zhang et al . , 2017 ) on June 22 , 2018 , and July 3 , 2018 , respectively .", "Strains lacking full-length genomic sequence data were excluded .", "Sequences were read into R ( version 3 . 5 . 2 ) using the DECIPHER ( version 2 . 18 . 1 ) package ( Wright , 2015 ) and subset into the time periods 1995–2004 and 2005–2014 ( H3N2 strains ) or 2000–2008 and 2010–2018 ( H1N1 strains ) .", "Time periods were selected in part to ensure a similar level of genetic diversity between strains .", "In each strain , all eight vRNA segments were concatenated into a full-length genome from which alignments were constructed ( Figure 1A ) .", "A neighbor-joining species tree was built by clustering strains into operational taxonomic units with sequence identity cutoffs ranging from 95% to 99% ( Figure 1B ) .", "In H3N2 viruses from 1995 to 2004 , there were 3 , 7 , 16 , 53 , and 259 clusters corresponding to cutoffs of 95% , 96% , 97% , 98% , and 99 % sequence identity , respectively .", "The 95–96% sequence identity cutoffs were discarded , as these produced trees with an insufficient number of branches for comparison by RF .", "However , as the cutoff for sequence identity was increased from 97% to 99% , we observed a corresponding decrease in bootstrap support for trees built from representative sequences .", "A sequence identity cutoff of 97% was therefore selected to ensure the greatest degree of robustness in tree topologies .", "Small clusters occurred infrequently and were either omitted or collapsed into a single cluster .", "Seven replicate strains were randomly chosen from each cluster for further study and visually inspected for sequencing ambiguities .", "A list of all strains analyzed and the corresponding accession numbers can be found in Supplementary files 1-4 .", "Maximum-likelihood trees were reconstructed under the Hasegawa et al . , 1985 model for either full-length genomes or individual vRNA segments with 100 or 1000 bootstrap replicates , as indicated , using the DECIPHER package in R ( Figure 1C ) .", "The phangorn package ( version 2 . 5 . 5 ) ( Schliep , 2011 ) was used to identify an appropriate model of evolution for phylogenetic reconstruction .", "Strain names are coded by cluster number in all trees .", "Phylogenetic trees of full-length concatenated genomes are shown in Figure 1—figure supplement 2 .", "Neighbor-joining protein trees were built from amino acid alignments after translation of the corresponding coding sequence alignments .", "Networks visualizing overall vRNA relatedness with mean tree distances between vRNA segments were built using the UPGMA method .", "Tanglegrams , or back-to-back trees with intersecting lines matching tips from the left tree to the right tree , were built from pairs of vRNA phylogenies within replicates using the phytools package ( version 0 . 7–70 ) ( Revell , 2012 ) .", "RF was calculated for each pair of trees using the ape package ( version 5 . 4–1 ) ( Paradis and Schliep , 2019 ) .", "CID was calculated with the TreeDist package ( version 2 . 0 . 3 ) ( Smith , 2020 ) .", "Linear regression was used to determine the overall association between tree distances from different sets of viruses ( RF or CID ) .", "A set of 1000 randomly sampled , unrooted trees with 8 , 9 , or 12 tips were built using the ape package to determine confidence intervals for the RF between phylogenetic trees .", "RF was calculated for all pairs of trees and these were fit to a linear regression model .", "For visualization purposes , null RF values were either log-transformed or transformed by the Yeo-Johnson method ( Yeo and Johnson , 2000 ) , as indicated .", "Mean RF calculated for pairs of vRNA trees were considered significant if they fell within the first five percentiles as compared to null RF from random trees with the same number of tips ( i . e . 95% of null RF were greater than the mean RF for a given pair of vRNA trees ) .", "A Mann-Whitney U test with a Benjamini-Hochberg post hoc correction was used to identify statistically significant differences between RF from two time periods .", "Human adenocarcinoma alveolar basal epithelial cells ( A549 , ATCC ) were maintained in high-glucose Dulbecco’s modified Eagle medium ( Sigma ) supplemented with 10% fetal bovine serum ( HyClone ) , 2% L-glutamine , and 1% penicillin/streptomycin .", "Recombinant virus was rescued as previously described ( Lakdawala et al . , 2011 ) .", "Virus titers were determined by 50% tissue culture infectious dose ( TCID50 ) using the endpoint titration method ( Reed and Muench , 1938 ) .", "Validation of cell lines was performed on a routine basis for mycoplasma contamination and cell line purity and identity .", "Mycoplasma screening was performed by the vendor and annually thereafter .", "Cell lines tested negative for mycoplasma at time of purchase ( Hoechst stain , agar culture , and PCR-based assays ) and mycoplasma status is confirmed negative annually using the MycoAlert Mycoplasma Detection Kit ( Lonza ) .", "The identity and purity of cell lines were verified at the time of purchase ( ATCC ) and annually thereafter by short tandem repeat profiling ( University of Arizona Genetics Core ) .", "Documentation of the A549 cell line purity and identity in these studies is available upon request .", "Low-passage stocks were maintained for no more than 20 passages after thawing to ensure maintenance of a pure cell population .", "Custom Stellaris RNA FISH oligonucleotide probes specific for the H3N2 virus NS , NA , and PB2 vRNA segments were purchased from BioSearch Technologies ( refer to Supplementary file 5 for FISH probe sequences ) .", "Each custom probe mix comprises 2040 20-mers that span the length of the vRNA segment of interest .", "Probes with high complementarity against other vRNA segments or positive-sense RNA were excluded during the design process .", "The NS probe was purchased with a terminal amine group and manually conjugated to the Alexa Fluor 488 fluorophore using the Alexa Fluor 488 Oligonucleotide Amine Labeling Kit ( Invitrogen ) .", "The NA and PB2 probes were labeled by the manufacturer with the Quasar 570 and Quasar 670 fluorophores , respectively .", "Three independent FISH-IF experiments were performed ( Figure 1D ) .", "A549 cells were seeded directly onto 1 . 5 mm circular coverslips ( Fisher Scientific ) in tissue culture dishes .", "The next day , cells were infected at a multiplicity of infection of 2 with A/Perth/16/2009 ( H3N2 ) or mock-infected in diluent .", "Cells were fixed at 8 hr post-infection with 4% paraformaldehyde and permeabilized overnight in ice cold 70% ethanol .", "Prior to hybridization , cells were rehydrated in wash buffer ( 10% formamide and 2× saline sodium citrate [SSC] in DEPC-treated H2O ) and then incubated at 28°C overnight in hybridization buffer ( 10% dextran sulfate , 2 mM vanadyl-ribonucleoside complex , 0 . 02% RNA-free BSA , 1 mg/ml Escherichia coli tRNA , 2× SSC , and 10% formamide in DEPC-treated H2O ) with anti-IAV NP antibody ( Millipore , 1:2000 ) and FISH probes .", "After hybridization , cells were washed and incubated with Alexa Fluor 594 goat anti-mouse ( Invitrogen , 1:2000 ) and DAPI ( Sigma , 1:5000 ) in wash buffer .", "Coverslips were mounted on slides in ProLong Diamond antifade mountant ( Thermo Fisher ) .", "Microscope slides were imaged on a Leica SP8 confocal microscope equipped with a pulsed white light laser as an excitation source and an acousto-optical beam splitter and Leica Hybrid Detectors .", "All imaging was performed with a 100× oil immersion objective with a numerical aperture of 1 . 4 .", "Sequential scanning with a line averaging of 3 between frames was used .", "To obtain Nyquist sampling , z-stacks of each cell were taken with a step size of 170 nm to achieve a pixel size of 45 nm × 45 nm × 170 nm .", "The following custom parameters were established using single-color infected controls for sensitive detection of all five fluorophores: 405 nm excitation wavelength ( λex ) with 0 . 5% laser power and a detection range of 415–470 nm ( PMT1; DAPI ) , 488 nm λex with 10% laser power and a detection range of 493–540 nm with time gating of 1–6 nanoseconds ( ns ) ( HyD4; Alexa Fluor 488 ) , 582 λex with 15% laser power and a detection range of 590–635 nm with time gating of 1 . 5–6 ns ( HyD4; Cal Fluor Red 590 ) , 545 nm λex with 5% laser power and a detection range of 545–568 nm with time gating of 1 . 5–6 ns ( HyD4; Quasar 570 ) , 647 nm λex with 5% laser power and a detection range of 670–730 nm with time gating of 1 . 5–6 ns ( HyD5; Quasar 670 ) .", "In each experiment , five volumetric z-stacks were imaged of infected cells and one z-stack was imaged of mock-infected cells .", "Background subtraction and deconvolution of confocal images were performed manually for each channel using Huygens Essential software ( version 19 . 04 , Scientific Volume Imaging B . V . ) .", "In each experiment , images taken of mock-infected cells were deconvolved using the same parameters as those of infected cells .", "3D reconstruction and colocalization analysis of the resulting images were performed using Imaris software ( version 8 . 4 . 2 , Bitplane AG ) as previously described ( Lakdawala et al . , 2014; Nturibi et al . , 2017 ) .", "Briefly , the cell of interest in each image was segmented using the ‘Surfaces’ and ‘Cell’ tools in Imaris software .", "DAPI signal was used to mask nuclear signal from the remaining channels .", "The ‘Spots’ tool was then used to populate the reconstructed cell with four different sets of Spots corresponding to foci from each of the remaining channels .", "In each experiment , the mock infected cell was analyzed in an identical manner and the fluorescence intensity for each channel of the mock-infected cell was used to establish fluorescence intensity thresholds at which 97% or more of the background signal was removed prior to Spot generation .", "A modified Matlab extension was then used to quantify spot colocalization using a distance threshold of 300 nm as previously described ( Nturibi et al . , 2017 ) .", "Colocalization data was imported into the Cell and all data was exported and analyzed in R . A Mann-Whitney U test was used to determine statistical significance of FISH-IF colocalization data .", "Custom code for analysis of parallel evolution in concatenated , full-length genomic influenza virus sequences is available on GitHub ( https://github . com/Lakdawala-Lab/Parallel-Evolution-Of-Influenza-Viral-RNA/ ( copy Jones , 2020 archived at swh:1:rev:27dc83b8eec1f461bbf9ef3f1dbeba61f0514fb3 ) ) ." ] ]

[ "The influenza A virus ( IAV ) genome consists of eight negative-sense viral RNA ( vRNA ) segments that are selectively assembled into progeny virus particles through RNA-RNA interactions .", "To explore putative intersegmental RNA-RNA relationships , we quantified similarity between phylogenetic trees comprising each vRNA segment from seasonal human IAV .", "Intersegmental tree similarity differed between subtype and lineage .", "While intersegmental relationships were largely conserved over time in H3N2 viruses , they diverged in H1N1 strains isolated before and after the 2009 pandemic .", "Surprisingly , intersegmental relationships were not driven solely by protein sequence , suggesting that IAV evolution could also be driven by RNA-RNA interactions .", "Finally , we used confocal microscopy to determine that colocalization of highly coevolved vRNA segments is enriched over other assembly intermediates at the nuclear periphery during productive viral infection .", "This study illustrates how putative RNA interactions underlying selective assembly of IAV can be interrogated with phylogenetics ." ]

[ "The viruses responsible for influenza evolve rapidly during infection .", "Changes typically emerge in two key ways: through random mutations in the genetic sequence of the virus , or by reassortment .", "Reassortment can occur when two or more strains infect the same cell .", "Once in a cell , viral particles ‘open up’ to release their genetic material so it can make copies of itself using the cell’s machinery .", "The new copies of the genetic material of the virus are used to make new viral particles , which then envelop the genetic material and are released from the cell to infect other cells .", "If several strains of a virus infect the same cell , a new viral particle may pick up genetic segments from each of the infecting strains , creating a new strain via reassortment .", "Several factors are known to affect the success of the reassortment process .", "For example , if the new strain acquires a genetic defect that hinders its replication cycle , it is likely to die out quickly .", "Other times , this trading of genetic information can create a strain that is more resistant to the human immune system , allowing it to sweep across the globe and cause a deadly pandemic .", "However , a key part of the reassortment process that still remains unclear is how genome segments from two different influenza strains recognize each other before merging together to create hybrid daughter viruses .", "To explore this further , Jones et al . used a technique called fluorescence microscopy .", "They found that genome segments that evolved along similar paths were more likely to cluster in the same area inside infected cells , and therefore , more likely to be reassorted together into a new strain during assembly of daughter viruses .", "This suggests that assembly may guide the evolutionary path taken by individual genomic segments .", "Jones et al . also looked at the evolution of different genome segments collected from patients suffering from seasonal influenza , and found that these segments had a distinct evolutionary path to those in pandemic-causing strains .", "This research provides new insights into the role of reassortment in the evolution of influenza viruses during infection .", "In particular , it suggests that how the genome segments interact with one another may have a previously unknown and important role in guiding this evolution .", "These insights could be used to predict future reassortment events based on evolutionary relationships between influenza virus genomic segments , and may in the future be used as part of risk assessment tools to predict the emergence of new pandemic strains ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "computational and systems biology" ]

[ [ "Cellular variability is likely a biological trait with significant phenotypic consequences .", "Technological advances in single-cell measurement methodologies reveal substantial cellular variability .", "For instance , single-cell quantification of protein concentration variability between cells shows that the concentration of many signaling molecules can vary by ∼25% ( coefficient of variation ) ( Sigal et al . , 2006; Bar-Even et al . , 2006; Spencer et al . , 2009 ) .", "Furthermore , a large and rapidly growing body of single-cell transcriptomics experiments further demonstrates that cells homogeneous in 'type' have substantially heterogeneous gene expression patterns ( Junker and Van Oudenaarden , 2014 ) .", "The origin of this cellular variability has been traced to fundamental properties of gene expression .", "Notably , single-molecule kinetics regulates gene expression and , as a result , is an inherently stochastic process ( Sanchez and Golding , 2013 ) .", "While the costs and benefits of cellular variability are likely dependent on the specific physiological context , the functional significance of cellular variability suggests that cellular variability magnitude is regulated .", "Functional analysis of cellular response variability demonstrates that the observed cellular variability affects the core function of signaling networks .", "Despite a homogenous environment , cells respond in a heterogeneous manner due to biological variability .", "Response variability potentially degrades transmitted information and decreases downstream effector ability to reliably respond to environmental changes ( Selimkhanov et al . , 2014; Cheong et al . , 2011; Voliotis et al . , 2014; Hansen and O'Shea , 2015 ) .", "The abundance of cellular variability throughout biological processes and the potential consequences of information degradation suggest that biological systems have developed mechanisms to regulate cellular variability .", "However , cellular variability is not necessarily detrimental to cellular function .", "In fact , cellular heterogeneity often plays a critical role in ensuring proper cellular response by mechanistically increasing the cellular response range to a constantly changing environment ( Altschuler and Wu , 2010 ) .", "For example , single-cell noise in NFκB dynamics creates robust population level responses to a wider range of inputs ( Hughey et al . , 2014; Kellogg and Tay , 2015 ) .", "Cells share information with each other via paracrine signaling , which effectively averages variable cellular responses and therefore reduces cellular variability .", "Overall population-level averaging decreases variability by following the statistical laws of the central limit theorem and the law of large numbers ( Piras and Selvarajoo , 2014 ) .", "Paracrine signaling averaging can decrease variability in a similar manner , but functions on a local population level .", "Specifically , paracrine signaling averaging functions such that the local concentration of the paracrine ligand , or the concentration of ligand a cell is exposed to , is the average ligand concentration secreted by local cells ( Figure 1A ) .", "Indeed , the benefits from paracrine communication were previously demonstrated to increase post-paracrine cellular response fidelity ( Rand et al . , 2012; Shalek et al . , 2014 ) .", "The process of local population 'information averaging' by each cell enables increased accuracy of inherently single cell decisions such as proliferation and differentiation . 10 . 7554/eLife . 09652 . 003Figure 1 . Local averaging using paracrine signaling reduces response variability in a communication distance dependent manner .", "( A ) A hypothesis for local averaging based reduction of response variability using paracrine signaling for ERK activation by P2Y receptors .", "ATP binds to P2Y receptors to increase cytosolic Ca2+ levels with high variability between cells despite equivalent ATP dosage per cell ( pink shading ) .", "EGF release from each cell is proportional to the primary response to ATP ( green arrows ) .", "Due to diffusion of EGF , the local concentration will be the average of EGF released from nearby cells and subject cells in a local neighborhood to the same level of EGF ( blue arrows ) to result in similar ERK activation .", "( B ) ATP activates ERK in a paracrine fashion in MCF-10A cells .", "ERK response to 10 μM ATP addition with ( green dashed ) and without ( green solid ) 1 μM of EGFR inhibitor tryphosphitin AG1478 .", "0 μM ATP addition with ( blue dashed ) and without ( blue solid ) 1 μM AG1478 shown as controls .", "( C ) MCF-10A cells were clustered based on their spatial proximity so that cells within a cluster were within a specific maximal communication distance ( MCD ) and cells in other clusters were farther than the MCD .", "Cluster denisty was calcuated by dividing the number of cells per cluster by the circular area inhabitated by each cluster .", "( D ) Standard deviation of ERK response per cluster to 10 μM ATP .", "Standard deviation decreases with increasing cluster density ( p-value <0 . 05 , Pearson correlation ) .", "( E ) Average maximum Ca2+ response with increasing ATP dosage .", "The standard deviation , that is noise , of the Ca2+ response to each ATP dosage is large when compared to the increase in average response with increasing ATP dose , that is signal ( SNR = 0 . 9 , gray error bars [standard deviation] ) .", "Noise decreases when Ca2+ response is locally averaged with a PCD of 100 μm ( SNR = 20 . 2 , black error bars [standard deviation] ) .", "x-axis shown in log-scale .", "F . Single-cell maximum Ca2+ response is locally averaged across an area specified by the PCD to produce a predicted single-cell ERK response .", "Variability between cells in the predicted ERK response decreases with increasing PCD .", "Response magnitude of Ca2+ and ERK response indicated by pink to yellow and blue to yellow colorbars , respectively .", "G . The SNR of the predicted ERK response from locally averaged Ca2+ data continually increases with increasing PCD shown for a model with rapid diffusion ( green ) or limited by the diffusion rates and integration time of paracrine signals ( blue , Materials and methods ) .", "SNR calculated in same manner as panel E with increasing PCD .", "Shaded area is SEM ( N = 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00310 . 7554/eLife . 09652 . 004Figure 1—figure supplement 1 . MCF-10A cells can be separated and analyzed in cell clusters when plated at low densities . Cells were grouped into clusters based on distance between cells .", "First , the distance between all cells was calculated based on the distance from the center of the cell .", "A hierarchical clustering based on the shortest distance linkage between cell groups was used to group cells .", "Cells linked within 200 μm ( maximum PCD , or MCD ) of each other were assigned as a cell cluster .", "To calculate the local cell density ( ρ ) within the cluster , the longest cell-to-cell distance within the cluster was measured ( d ) .", "Clusters with a d greater than 150 μm were removed since the PCD was calculated to be 99 . 5 μm .", "Effective area ( EA ) of the cluster was calculated by finding the area of the circle encompassing the cluster using d as the diameter of the circle .", "ρ was estimated by dividing the number of cells within the cluster by the EA .", "This process was repeated for all clusters . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00410 . 7554/eLife . 09652 . 005Figure 1—figure supplement 2 . Cluster standard deviation and cluster average as a function of cluster density show significant trends for ERK activation but not Ca2+ activation . Cells were plated at low density to form groups of cells and clustered according to Figure 1—figure supplement 1 .", "ERK and Ca2+ activation were monitored following the addition of 10 μm ATP .", "All p-values obtained through Pearson correlation .", "( A ) Standard deviation of ERK expression for each cluster significantly decreases with increasing cluster density ( p-value <0 . 05 ) indicating that ERK expression becomes more reliable as cells coordinate with more cells .", "The average ERK expression per cluster , however , does not significantly decrease ( p-value >0 . 05 ) verifying that the decrease in standard deviation of ERK expression is not related to changes in the average ERK expression .", "( B ) The standard deviation of Ca2+ activation does not significantly decrease with increasing cluster density ( p-value >0 . 05 ) assuring that decreases in ERK cluster standard deviation represent communication via paracrine signaling and not due to similarities in cellular expression between sister cells in a cluster .", "Similarly to A . , the average Ca2+ response for each cluster does not significantly decrease with increasing cluster density ( p-value >0 . 05 ) DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00510 . 7554/eLife . 09652 . 006Figure 1—figure supplement 3 . Paracrine ERK activation depends on Src prior to MMP activation . MCF-10A cells expressing EKAREV FRET ERK reporter were incubated with 10 µM of the selective Src kinase inhibitor PP2 ( Tocris ) for 30 min at normal cell culture conditions before addition of 10 µM ATP or 5ng/mL EGF ( A = AP , E = EGF , P = PP2 ) .", "Shaded region indicates SEM ( N = 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00610 . 7554/eLife . 09652 . 007Figure 1—figure supplement 4 . Inhibiting paracrine communication does not allow decreased cellular response variability . Cells were plated in a 96 well plate at low density to form groups and clustered according to Figure 1—figure supplement 1 .", "All p-values obtained through Pearson correlation .", "( A ) Cells were incubated with varying concentrations of PP2 ( 0 – 10 μM , red dashed X ) for 30 min in normal cell culture conditions to inhibit Src to varying degrees and therefore limit the concentration of secreted EGF ( black broken lines ) .", "Additional EGF ( 0 – 1 ng/mL ) was added to compensate for the lack of secreted EGF to ensure that the average ERK response of the well remained similar to controls .", "Cells were then perturbed with 10 μM ATP to activate ERK response in a paracrine fashion .", "PP2 , EGF , and ATP were added such that cells were perturbed with every combination of PP2 , EGF , and ATP .", "( B ) Wells that showed statistically similar average ERK activation ( cyan ) to the control well ( blue , 10 μM ATP ) were selected for analysis ( blue * , p-value >0 . 05 , t-test ) .", "Complete inhibition by PP2 shown as negative control ( yellow ) .", "The cells within wells labeled with a blue * were clustered in the same manner as Figure 1—figure supplement 1 .", "( C ) The standard deviation of ERK response per cluster is not significantly correlated with cluster density ( p-value >0 . 05 , Pearson correlation ) indicating that ERK variability does not decrease when paracrine communication is inhibited . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00710 . 7554/eLife . 09652 . 008Figure 1—figure supplement 5 . Mutual information and SNR both continue to increase with increasing PCD . A mutual information analysis using established methods on a Ca2+ dose response to ATP as a secondary and more sophisticated approach to our SNR analysis ( Selimkhanov et al . , 2014 ) .", "As expected , the mutual information exhibits a similar increase with increasing PCD with no upper bound , similar to the SNR analysis .", "Shaded area represents SEM ( N = 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00810 . 7554/eLife . 09652 . 009Figure 1—figure supplement 6 . Scaling of Paracrine Communication Distance . The scaling of Paracrine Communication Distance with the strength of the paracrine signal expressed as the ratio of released molecules to the number of molecules needed for signal detection .", "Cell radius is 10 μm , height 15 μm and chamber height is 60 μm .", "These values were chosen to approximate the analysis in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 00910 . 7554/eLife . 09652 . 010Figure 1—figure supplement 7 . Required Integration time . The required integration time for selected Paracrine Communication Distance values shown in linear ( left ) and log ( right ) sclaes .", "Geometry is the same as in Figure 1—figure supplement", "6 . Cell radius is 10 μm , height 15 μm and chamber height is 60 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 01010 . 7554/eLife . 09652 . 011Figure 1—figure supplement 8 . The effect of fluid flow on paracrine communication .", "( A ) Color map of Péclet number ( shown in Log10 scale ) .", "The red ( cyan ) region shows the combination of signal strength ( S ) and diffusion coefficient ( D ) where advection ( diffusion ) is dominating over diffusion ( advection ) .", "The dotted dark line shows the regime above which advection will have little effect on positional accuracy .", "( B ) The expected distance of advection based spread as a function of D for a signal strength of 1000 .", "The geometry and parameters here are the same as in Figure 1—figure supplement 6 and Figure 1—figure supplement", "7 . Interstitial flow rate used was 0 . 3 μm/s based on ( Polacheck et al 2011 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 01110 . 7554/eLife . 09652 . 012Figure 1—figure supplement 9 . The effect of cellular decoding schemes on paracrine communication . Comparison of Paracrine averaging weights between the Gaussian model used in this work to represent paracrine communication and an alternative model that is based on an assumption of instant release temporal averaging of paracrine signal . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 012 Despite promises of noise mitigation from paracrine averaging , parameters set by biological systems can limit these potential benefits .", "For example , population averaging due to paracrine communication may cause loss of information in a similar manner to the information loss of single-cell dynamics due to ‘population average’ bulk measurements ( Elowitz et al . , 2002; Newman et al . , 2006; Bar-Even et al . , 2006 ) .", "The potential information loss due to ‘over-averaging’ of variable single-cell responses demonstrates a limitation to paracrine communication .", "Limitations to paracrine communication are also observed in post-paracrine single-cell responses that remain highly variable despite paracrine averaging .", "These limitations suggest an overall functional constraint to the potential benefits of paracrine communication .", "However , the identity and source of these limitations on paracrine communication benefits are unknown .", "The initial paracrine signaling pathways that are activated in response to Damage Associated Molecular Patterns ( DAMPs ) are a good model system for investigating the influence and limits of paracrine communication on cellular response fidelity .", "Paracrine communication is pervasive during initial wound response .", "Wound healing begins as soon as the wound occurs and the initial cellular wound response provides the foundation for proper downstream healing .", "The initial cellular wound response relies on external environmental cues as well as programs inherent to the cell , including DAMPs as primary danger signals ( Enyedi and Niethammer , 2015 ) .", "DAMPs are released from necrotic cells and bind to extracellular receptors on surrounding cells .", "This binding initiates a signal in the surrounding cells to secrete a secondary set of cytokines and growth factors required to coordinate the wound healing process .", "Many DAMP signals , such as extracellular ATP , are transient and released in limited quantities .", "As a result , the initial wound response to such DAMPs shows high cellular variability and low fidelity .", "Despite the limited fidelity of the initial wound response , the wounded epithelium is able to establish a robust healing response .", "The complicated and multi-step wound healing process utilizes several paracrine communication mechanisms to share cellular information and coordinate the overall healing program .", "Here we use the paracrine release of epidermal growth factor ( EGF ) ligands initiated by ATP binding to P2Y receptors as a model to investigate the limits of cellular information sharing through paracrine communication to mitigate biochemical noise ( Figure 1A ) .", "We show that paracrine communication increases extracellular signal-regulated kinase ( ERK ) response fidelity using live single-cell quantitative fluorescent imaging of primary Ca2+ and secondary ERK responses downstream of P2YR and EGFR , respectively .", "Statistical analysis of the primary response signal-to-noise ratio ( SNR ) demonstrates that the increase in response fidelity is limited by paracrine communication distance ( PCD ) .", "To analyze this pathway in the physiological context of wound response we developed a new microfluidics device to monitor the spatial propagation of initial wound response signaling .", "Our results demonstrate that the interplay between the wound induced spatial signaling gradient and the cellular noise pattern produces an optimal PCD .", "The optimal PCD balances the benefits of decreased noise from local averaging with the cost of reduced signal of the spatial signaling gradient due to over-averaging .", "Empirical measurements of the PCD reveal that cellular communication occurs at a distance to maximize cellular response fidelity ." ], [ "Here we establish that the paracrine activation of ERK by ATP provides a suitable system to investigate signaling response fidelity changes due to paracrine communication .", "Extracellular ATP binding to P2YR results in EGF family ligand release to bind EGFR and activate ERK response as monitored by ERK activity following ATP addition .", "In the mammary epithelial cell line MCF-10A , addition of extracellular ATP increases ERK kinase activity in an EGFR dependent manner ( Figure 1B ) similar to results reported in other in vitro epithelial models ( Yin et al . , 2007 ) .", "ERK , as measured by the genetically encoded FRET sensor EKAREV ( Albeck et al . , 2013; Komatsu et al . , 2011 ) , increases when stimulated with ATP .", "Inhibiting EGFR with tryphostin AG1478 prevents ERK activation upon ATP addition showing that ERK activation depends on secreted EGF binding to EGFR ( Wetzker and Böhmer , 2003 ) .", "With our paracrine communication system established , we next confirmed the influence of paracrine communication on cellular response variability .", "Under conditions of low cell density we used a spatial clustering analysis to group cells such that the distance between groups effectively constrained communication to cells within groups ( Figure 1C , D , Figure 1—figure supplement 1 , 2 ) .", "In the case that cellular coordination is beneficial , we anticipated that the ERK response within groups of higher cellular density , that is cells have increased communication ability , would have reduced response variability than cell clusters with decreased communication ability .", "ERK response variability within clusters decreases with increasing cluster density indicating increased intercellular communication ability .", "Increased intercellular communication ability is not observed in the absence of paracrine communication such as with the primary Ca2+ response to ATP ( Figure 1D , Figure 1—figure supplement 2 ) .", "Furthermore , disrupting paracrine communication by partially inhibiting Src results in the loss of the observed benefit of paracrine communication in higher density clusters ( Figure 1D , Figure 1—figure supplements 3 , 4 ) .", "Together these observations support the hypothesis that paracrine communication decreases cellular variability by increasing cellular coordination .", "Next we developed a computational model that mimics the coordination-effects of paracrine communication ( see Materials and methods for details ) .", "This computational model quantifies the overall observed benefit of paracrine coordination and predicts the potential reduction in variability .", "Experimental single-cell dose response data of primary Ca2+ ( prior to paracrine communication ) response to ATP is used as an input to predict the secondary ERK response .", "We quantify cellular response fidelity by using a simple signal-to-noise analysis ( SNR ) .", "In this analysis the cellular response magnitude of the input ligand ( signal ) is divided by the cellular response variability ( noise ) .", "The signal is estimated by calculating the spread between the average cellular Ca2+ responses from multiple ATP concentrations using multi-well dose response data .", "Noise is calculated from the average variability between cellular responses to a single input ligand concentration .", "SNR is simply the ratio of these two estimates ( Figure 1E ) .", "To mimic the benefit of paracrine communication our computational model performs a local , spatially weighted average ( convolution ) of the primary Ca2+ response to predict the variability of response post paracrine communication ( ERK ) ( Figure 1F ) .", "In short , the convolution averages the signal for every cell with its associated surrounding cells by weighting the surrounding cells based on a Gaussian function parameterized with varying PCDs .", "The PCD represents how far the paracrine molecule travels from a single-cell to activate its associated surrounding cells .", "Local spatial averaging provides an upper bound of the possible benefit resulting from cellular communication in conditions where no additional noise exists in the paracrine pathway .", "This analysis indicates that paracrine averaging using a PCD of 100 μm increases response SNR from 0 . 9 to 20 . 2 by decreasing noise , or response variability , of the predicted ERK response ( Figure 1E , gray/black ) .", "To investigate the limits of paracrine averaging , we repeated this analysis for multiple PCDs .", "Interestingly , our analysis estimates that the overall response SNR can increase up to eightyfold at PCDs of 500 μm when paracrine diffusion is not limiting , and up to twenty fivefold when diffusion of the paracrine ligand is limiting ( Figure 1G ) .", "More sophisticated statistical measures , such as mutual information , produce similar results ( Figure 1G , Figure 1—figure supplement 5 ) .", "The large maximal SNR benefit suggests a potentially noise-free ERK response .", "However , experimental measurements of ERK response fidelity shows substantial ERK variability indicating potential factors that limit the benefit gained from paracrine communication ( data not shown ) .", "Extracellular ATP released from necrotic cells act as DAMPs to activate healthy cells proximal to the wound ( Cordeiro and Jacinto , 2013 ) .", "Given this role , the spatial component produced by the ATP concentration gradient and the resulting cellular positional information relative to the wound may be important in the analysis of paracrine communication that occurs over hundreds of microns from the wound .", "Our previous SNR analysis demonstrating an increasing SNR with increasing communication distance was done based on multi-well experiment data .", "However , the bolus addition of ATP creates a spatially uniform ligand concentration in the well and does not represent a physiologically relevant spatial component .", "To examine whether ATP spatial patterns influence the paracrine communication benefit we repeated the SNR analysis using single-cell wound response data .", "In order to measure the spatial wound response for epithelial cells , we first developed a convection-free , small-volume wounding device .", "Scratch-assays , where a monolayer of cells is mechanically wounded using a pipet tip , are traditionally used for epithelial cell wounding ( Sholley et al . , 1977 ) .", "Although the scratch-assay is useful for studying cell-migration following wounding , scratch-assays lack the ability to study paracrine signaling .", "The large volume above the cells and convection caused by the scratch present challenges to examine paracrine signaling due to the dilution and inadvertent mixing of any paracrine molecules released from a cell into the surrounding media .", "To circumvent these technical issues we developed a microfluidics based wounding device ( Figure 2A , B ) .", "Our device has two components: an air channel ( black ) and a cell chamber with a ∼2 . 5 μL volume ( orange ) .", "The ceiling of the cell chamber has a PDMS pillar that , when air pressure is increased in the upper air channel , lowers down on to the cells , thereby wounding the cells in the cell chamber in a highly controlled and reproducible manner ( Figure 2B , C , Figure 2—figure supplement 1 , Video 1 ) . 10 . 7554/eLife . 09652 . 013Figure 2 . Paracrine communication reduces response variability during wounding .", "( A ) Dual layer microfluidic-based wounding device with a top air channel ( black ) and bottom cell chamber ( orange ) .", "( B ) Schematic of wounding in the device .", "Cells are first loaded into the cell chamber ( top ) .", "Increasing the air pressure in the air channel lowers a pillar in the ceiling of the cell chamber until cells below the pillar are mechanically crushed ( middle ) .", "The pillar returns to the original height when air pressure is released ( bottom ) .", "( C ) Ca2+ response visualized by the Fluo-4 Ca2+ indicator dye over a period of 5 min following a 300 μm diameter wound ( black circle ) .", "( D ) Top: Maximum single-cell ( dots ) Ca2+ response to a 300 μm wound .", "Inset shows maximum Ca2+ response to 300 μm wound in the presence of the ATP scavenger apyrase .", "Bottom: Maximum single-cell ( dots ) of Ca2+ response to wound according to distance from the wound .", "( E ) Same as D but for maximum ERK response .", "( F ) Top: Cells are binned according to distance from the wound ( Figure 3A ) and the average and standard deviation ( error bars ) are found for each bin .", "Middle: Coefficient of variation ( CV ) calcuated by dividing the standard deviation of each bin by the mean of that bin .", "Bottom: Ca2+ has higher variability than ERK response for the wound according to the CV of every bin for all wounds ( Black bar = average CV , p-value by t-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 01310 . 7554/eLife . 09652 . 014Figure 2—figure supplement 1 . Microfluidic wounding device characterization demonstrates cell viability , isotropic wounding , wounding control , and reproducibility .", "( A ) Top ( top panel ) and side ( bottom panel ) view of the microfluidic wounding device .", "The wounding device is constructed of 2 layers , a top air layer ( black ) and bottom cell chamber ( orange ) .", "The air layer has an air hole at one end where air pressure is increased by flowing air through a small tube connected to the hole .", "The ceiling of the cell chamber has a PDMS pillar that is lowered down on to the cells in the cell chamber when air pressure is increased in the air layer .", "( B ) Cell chamber heights were measured by loading fluorescent beads into the device and allowing them to settle on the surfaces within the device ( bottom , pillar , ceiling ) .", "Height was measured by scanning the cell chamber through the Z-axis and seeing where the beads had settled based on where they were in focus ( bead sharpness ) .", "Cell chamber height within the device as measured by bead sharpness revealed a chamber of approximately 60 μm in height and a distance of approximately 15 μm between the bottom of the pillar and the bottom of the cell chamber .", "These distances provide ample room for cell growth while still limiting the z-space in which extracellular signals can diffuse .", "( C ) The change in height of the ceiling of the cell chamber ( orange ) and the pillar ( black ) with increasing air pressure as measured by bead sharpness shows controlled movement of the pillar with increasing air pressure .", "Although the ceiling of the cell chamber also moves with the pillar , this does not interfere with cellular wounding .", "( D ) MCF-10A cells were loaded in to the wounding device cell chamber and allowed to adhere in the presence of propidium iodide ( PI ) to measure cell viability within the device over time in static media conditions .", "Cells were able to adhere within 5 hr after which the media began to dry up and cells began to die indicating that cellular response following wounding can be measured for up to 5 hr in a static setting before cell death .", "( E ) Wounding in non-static ( with flow ) and static ( no flow ) media conditions .", "Static media conditions were accomplished by placing a piece of tape over one of the ports of the wounding device during imaging .", "When flow was present , the signal went in the direction of the flow .", "However , when flow was not present , the signal propagated isotropically from the wound indicating that the response was a measurement of signal transfer and not simply due to the flow in the environment .", "( F ) Ca2+ response according to distance away from the center of a 300 μm diameter wound shows reproducible wounding between 3 experiments as represented by 3 different colors .", "Cellular response is measured as the percentage of responding cells according to distance away from the wound .", "( G ) Due to the layer-by-layer photolithography manufacture of the molds used to make the wounding device , the size of the wound can be controlled and reproducible .", "We were able to measure the single-cell spatiotemporal Ca2+ response in cells wounded by a 450 μm , 300 μm , and 150 μm wound .", "The single-cell responses for 4 different cells at varying distances from the wound are highlighted for the 450 μm wound with colorbar indicating fold maximum increase . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 014 We used our wounding device to monitor Ca2+ and ERK response to a 300 μm diameter wound using a stable , dual reporter MCF-10A cell line expressing the genetically encoded Ca2+ indicator RGECO ( Akerboom et al . , 2013; Zhao et al . , 2011 ) and the EKAREV FRET reporter for ERK ( Albeck et al . , 2013; Komatsu et al . , 2011 ) ( Figure 2D , E; Video 2 ) .", "We verified the key role of ATP in initial wound response by wounding in the presence of apyrase , an enzyme that rapidly hydrolyzes ATP .", "Wounding in the presence of apyrase inhibits both Ca2+ and ERK response ( Figure 2D , E , insets ) .", "From each wound we quantified single-cell time traces for over 3000 cells .", "Notably , the maximum activity per cell shows a larger response in cells closer to the wound compared to cells farther away from the wound for both Ca2+ and ERK .", "These response gradients demonstrate the importance of the cellular position to determine the cellular response , or positional information ( Figure 2D , E ) .", "We used coefficient of variation ( CV ) to measure the variability of the post-paracrine ERK response and the pre-paracrine Ca2+ response in the wound ( Figure 2F ) .", "Indeed , the CV for Ca2+ wound response shows statistically higher variability than ERK wound response indicating that paracrine communication reduces response variability during initial wound response .", "We adapted the computational SNR analysis to wound response data to determine the influence of spatial patterns on response fidelity .", "As opposed to the dose-response data , the wound response data uses the distance of each cell from the wound as the input rather than the concentration of activating ligand ( Figure 3A ) .", "Similar to the dose-response data , noise is estimated by averaging the cellular response variability over all distances .", "The variability between the average response magnitude of each distance constituted the signal ( Figure 3B ) .", "Other statistical measures of response fidelity such as mutual information were also adapted for the wound context ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 09652 . 017Figure 3 . Signal to noise analysis of initial wound response shows limits to paracrine communication .", "( A ) Representative single-cell time traces of Ca2+ response to wounding , grouped according to distance from the wound ( concentric circle colors ) .", "( B ) SNR calculation method for Ca2+ response adpated to the wound .", "Horizontal bars represent bin average and error bars represent bin variance .", "( C ) Maximum single-cell ( dots ) Ca2+ response to wound with respect to distance away from the wound ( pink ) .", "Predicted ERK cellular response after paracrine communication as determined by local averaging ( gray ) .", "Local averaging done in same manner as Figure 1F .", "( D ) Predicted ERK response according to distance from the wound using PCDs of 0 to 600 μm ( colorbar ) .", "Predicted ERK response determined through local averaging using increasing PCDs results in decreased response magnitude over space .", "( E ) Signal ( blue ) , Noise ( green ) and SNR ( orange ) as function of PCD of locally averaged Ca2+ response trends in panel D . ( F ) SNR analysis of locally averaged Ca2+ response to a wound with increasing PCD shows a maximum SNR at PCD of 91 . 0 μm+/-6 . 3 μm indicated by the asterick ( blue , SEM indicated by shaded region , N = 5 ) .", "The maximum SNR for conditions controlled for biologically relevant integration times show the same maximum SNR ( green , Materials and methods ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 01710 . 7554/eLife . 09652 . 018Figure 3—figure supplement 1 . Mutual information analysis of locally averaged Ca2+ response to wounding shows similar peak to SNR analysis .", "( A ) Mutual information was calucated by binning the cells according to their distance away from the wound .", "The maximum response per cell ( gray circles ) is divided into bins such that there are the same number of cells in each bin ( blue plots ) .", "The conditional response ( H[R S] ) is calculated for each bin using the probability density ( PD ) of each cell .", "The PD of each cell is calculated by finding the 10th nearest neighbor for each cell , counting the number of cells within that space , and then dividing by the distance covering that space .", "The non-conditional response ( H[R] ) is calcuated by measuring the PD of all of the cells .", "The cells are divided into multiple sets of bins and the mutual information ( I ) is calculated for each set of bins .", "Each I is then plotted according to the number of bins after which the final MI is found by extrapolating back to zero to ensure no dependence on bin number .", "B . Mutual information was used as a measurement tool to further confirm the SNR analysis of locally averaged Ca2+ response .", "Indeed , the mutual information analysis found an optimal coordination length-scale of 114 . 33 μm+/-6 . 7 μm ( SEM , N = 5 ) , matching the optimal coordintation length-scale found in the SNR analysis ( shaded region indicates SEM , N = 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 018 The maximum primary Ca2+ response shows highly variable cellular response when plotted according to distance ( Figure 3C , pink ) .", "This variability complicates the ability for a cell to distinguish its respective position to the wound based on its response We again mimicked paracrine communication to predict the post-paracrine ERK response by locally averaging the single-cell Ca2+ wound response using a Gaussian kernel ( Figure 1F ) .", "Locally averaging the cellular Ca2+ response creates a smoother predicted ERK response pattern versus distance from the wound ( Figure 3C , gray ) .", "However , the reduction in variability also decreases the overall response pattern trend .", "Locally averaging the Ca2+ signal using increasing PCDs decreases the magnitude of change of the average predicted ERK response between cells closest to the wound and farthest from the wound ( Figure 3D ) .", "In other words , the response gradient becomes less obvious when cells are averaged over larger distances .", "Therefore , although the increase in PCD decreases response noise , the corresponding decrease in signal demonstrates the limit of PCD on the SNR benefit ( Figure 3E ) .", "The difference in rates at which the signal and noise decrease results in a maximum SNR at a PCD of 91 . 0+/-6 . 3 μm ( SEM , N = 5 ) ( Figure 3F ) .", "This peak corresponds to a PCD where the amount of noise is decreased to the lowest amount possible without reducing the response gradient due to ‘over-averaging’ .", "The predicted PCD with maximal benefit did not change when we expand the model to consider limitations due to diffusion ( Figure 3F green curve , Materials and methods ) .", "Similar analysis using mutual information statistics shows a similar PCD with the maximal mutual information at the distance that showed maximal SNR ( Figure 3E , F , Figure 3—figure supplement 1B ) .", "This analysis shows that the benefits from paracrine communication depend on how far a paracrine molecule travels which , in this specific case , has a maximal benefit at ∼100 μm , or approximately three cell diameters .", "We next empirically measured the PCD in our experimental system to compare to the PCD predicted to maximize the SNR in the wound context .", "To measure the PCD of ERK activation we first established a co-culture system that allows us to separate the effects of autocrine and paracrine signaling .", "Our assay utilizes a synthetic GPCR: Designer Receptors Exclusively Activated by Designer Drugs ( DREADD ) .", "The Gq human muscarinic derived GPCR DREADD is activated by a synthetic small molecule , clozapine-N-oxide ( CNO ) , that has no known endogenous receptors ( Armbruster et al . , 2007 ) .", "In addition , DREADD activates the Gq pathway similar to purinergic ATP receptors ( Dong et al . , 2010 ) .", "Using a co-culture of DREADD expressing ( activated by CNO ) and non-expressing cells ( not activated by CNO ) , we can determine which cells release EGF ( DREADD expressing-red ) and which cells accept EGF ( non-expressing-gray ) ( Figure 4A ) .", "Using a synthetic system allows us to directly measure the average communication distance of EGF .", "CNO addition selectively activates Ca2+ response in DREADD expressing cells while the surrounding non-expressing cells show no response indicating a lack of paracrine activation of Ca2+ response in cells ( Figure 4A ) .", "Although some systems show that Ca2+ response can propagate from cell-to-cell through gap junctions ( Ross 2012 ) , this does not appear to be the case in MCF-10A cells as non-expressing cells showed no cytosolic Ca2+ increase upon activation of DREADD cells .", "Alternatively , ERK response was found in both DREADD expressing and the surrounding non-expressing cells upon CNO addition but was inhibited in both cell types in the presence of the EGFR inhibitor tryphosphitin AG1478 , confirming paracrine activation of ERK in the DREADD system ( Figure 4B ) .", "The Ca2+ and ERK responses in the DREADD system suggest that local averaging takes place only at the EGF level between Ca2+ and ERK response .", "Additionally , increasing the ratio of DREADD cells to non-expressing cells shows an increasing ERK response magnitude in non-expressing cells , further supporting that paracrine communication locally activates ERK ( Figure 4C ) . 10 . 7554/eLife . 09652 . 019Figure 4 . Empirical PCD measurement using DREADD synthetic GPCR show that cells use an optimal level of paracrine communication to maximize cellular response fidelity .", "( A ) The addition of 5 μM CNO to a co-culture of DREADD expressing ( red ) and non-expressing ( gray ) MCF-10A cells shows increased fold Ca2+ response in DREADD expressing cells but not non-expressing cells ( SEM indicated by error bars , N = 3; *p-value<0 . 005 , t-test ) .", "( B ) Fold ERK increase in DREADD co-culture assay .", "Both DREADD and non-expressing cells show significant ERK increase when both DREADD cells and 5 μM CNO are present .", "ERK activation inhibited by 1 μM AG1478 ( SEM indicated by error bars , N = 3; *p-value <0 . 005 , t-test ) .", "( C ) The average maximum ERK response of non-expressing cells in a given well increases linearly with an increasing percentage of DREADD cells per well .", "( D ) Representative images from a timelapse experiment showing ERK activation ( cyan ) in non-expressing cells surrounding a single activated DREADD cell ( yellow ) over a 40 min time period .", "ERK activation level indicated by black to cyan colorbar .", "( E ) Average maximum ERK activation in non-expressing cells surrounding single DREADD cell according to distance from the DREADD cell .", "PCD was calcuated as the spread , or sigma , of the fitted Gaussian curve ( dashed line ) and measured to be 99 . 5 μm+/-19 . 6 μm ( blue * ) ( SEM indicated by error bars , N = 12 DREADD cells ) .", "Scale bar represents average length of a single-cell .", "F . Comparison between calculated optimal PCD per wound ( pink , Figure 3F ) and experimentally measured PCD found using the DREADD co-culture assay per DREADD cell ( blue , Figure 4E ) ( p-value>0 . 5 , t-test ) .", "Horizontal bars represent average . DOI: http://dx . doi . org/10 . 7554/eLife . 09652 . 019 We measured the PCD by monitoring local paracrine ERK activation with our DREADD co-culture assay .", "We co-cultured DREADD cells at a low concentration compared to non-expressing cells to ensure that neighboring non-expressing cells were activated only by a single DREADD cell .", "We then analyzed the ERK response of ∼1500 non-expressing cells neighboring a DREADD cell .", "( Figure 4D , E ) .", "Local ERK activation of non-expressing cells surrounding a DREADD cell show decreasing response with increasing distance from the DREADD cell .", "ERK response as a function of distance follows a Gaussian fit , consistent with how the concentration of diffusing molecules , like in paracrine signaling , changes over distance ( Figure 4E ) ( Berg , 1993 ) .", "The PCD was determined by calculating the spread , or sigma , of this Gaussian curve .", "According to our fit , the paracrine activation of ERK has a communication distance of 99 . 5+/-19 . 6 μm ( SEM , N = 12 DREADD cells ) .", "This empirically measured value is statistically similar to the predicted communication distance value that maximizes the SNR of wound response ( Figure 4F ) .", "In other words , the cellular communication distance is tuned to maximize the overall response fidelity during wound response signaling ." ], [ "Multicellular organisms utilize cellular diversity for specialization and division of labor .", "However , the variability between cells can be detrimental due to the potential loss of response fidelity ( Uda et al . , 2013; Hansen and O'Shea , 2015; Voliotis et al . , 2014; Cheong et al . , 2011; Selimkhanov et al . , 2014 ) .", "Paracrine communication can serve to share information between cells to regulate cellular variability .", "In this study we analyzed the benefits and limitations of paracrine communication based information sharing between cells as a mechanism to control cellular response variability .", "We analyzed the limits of paracrine communication on cellular response fidelity in two cases .", "First we analyzed the response to a spatially uniform ligand .", "Our analysis reveals that , under these conditions , the magnitude of single-cell response fidelity increases as a function of the PCD with no upper bound .", "In order for cells to facilitate larger PCDs , cells would need to synthesize larger amounts of paracrine signaling molecules or utilize fast diffusing paracrine ligands like H2O2 ( Enyedi and Niethammer , 2015 ) .", "The increased energy required to synthesize the additional molecules is likely to be minor in comparison to the overall energetic demand of a cell .", "Therefore cells could potentially take advantage of large PCDs to substantially mitigate biochemical noise .", "However , a spatially uniform input , while common in cell culture experiments , is likely an inadequate representation of physiological conditions .", "In the second case we analyzed the response of cells to spatially defined inputs in the form of a mechanical epithelial wound .", "We analyzed the cellular response to extracellular ATP gradients , a damage associated molecule , following a controlled wounding of an epithelial monolayer in vitro .", "Similar to developmental systems , an extracellular input ligand conveys positional information in wound response ( Dubuis et al . , 2013; Sonnemann and Bement , 2011 ) .", "Analysis of the paracrine communication benefit in our novel quantitative wound response assay with defined spatial perturbations demonstrates that paracrine communication increased cellular response fidelity , but with limitations .", "Unlike the spatially uniform ligand in the first case , the magnitude of the response fidelity benefit varies with increasing PCD .", "The maximal increase of cellular response fidelity occurrs at a PCD of ∼100 μm , or approximately 3 cell diameters .", "In vivo work measuring ERK propagation using the same EKAREV FRET sensor also showed propagation extending ∼100 μm ( Hiratsuka et al . , 2015 ) .", "Our results demonstrate that the paracrine information sharing benefit depends on the input ligand spatial scale , or PCD .", "Furthermore , empirical measurements of paracrine communication match the physiologically relevant spatial wound response maximum communication distance .", "The process of wound healing is a complex multi-stage program that coordinates the action of multiple cell types over multiple timescales , from minutes to weeks , to address an acute need .", "The initial steps of wound healing programs propagate information concerning the wound in a manner that is appropriate to the magnitude of damage .", "Both inflammatory and fibrotic processes , critical steps in wound response signaling , are damaging when they go awry .", "Therefore , the initial cellular responses and the establishment of signaling gradients are key steps in wound healing .", "The mechanistic details underlying how tissues robustly match the wound response magnitude to the extent of wound-induced damage remain unknown .", "Our results demonstrate that intercellular communication during the initial wound response is optimized to increase overall response fidelity and provides the initial evidence that matching the wound response to wound damage is a critical aspect to wound healing programs .", "Future work is needed to further investigate tissue level response fidelity during wound healing programs .", "Paracrine communication increases the fidelity of response at the single-cell level by mitigating biological noise at the single-cell level .", "Each cell integrates information from its local neighborhood to increase its individual response fidelity .", "Local averaging at the cellular level is a distinct mechanism compared to the benefit of global averaging at the population level .", "Without any paracrine communication , the reliability of the average of a cell population response can only increase with the size of the population .", "This is a consequence of the central limit theorem where the uncertainty of a sample average decreases with sample size .", "However , this increase in reliability is only true for the population average and not for individual cells in the population .", "Therefore , in cases where the biologically significant output is the collective action of the population , for example the secretion of a cytokine , intercellular information sharing is not required .", "However , when biologically significant output requires single-cell action , local information sharing via paracrine communication increases cellular response fidelity .", "Therefore , whether paracrine communication is required remains context dependent .", "It is possible that paracrine information sharing is more prevalent in signaling networks that support individual cellular decisions and less prevalent in cases where biologically meaningful outcomes result from population averages .", "In cases where paracrine information sharing is used as a method to mitigate biological noise , the breakdown of this system could be detrimental .", "In vivo studies of ERK response in mammary tumor cells using the same EKAREV FRET sensor utilized here show highly variable ERK response that may lead to the survival and propagation of cancer cells ( Kumagai et al . , 2014 ) .", "Although the cause for this heterogeneity is unknown , one possible mechanism may be the breakdown of paracrine communication between cells similar to how our partial inhibition of paracrine communication showed no decrease in ERK variability ( Figure 1—figure supplement 4 ) .", "The abundance of paracrine communication in mammals , that is the activation of a receptor by a ligand synthesized by another cell , demonstrates the heavy utilization of intercellular communication ( Ben-Shlomo et al . , 2003; 2007 ) .", "Paracrine averaging demonstrates how intercellular communication enables cellular collective decision making where the 'wisdom of the crowd' is greater than the individual cell .", "Theoretical and empirical work in humans and animal collectives has shown that the benefit of collective decision making depends on the size of the group; big crowds are not always better than small crowds ( Sasaki et al . , 2013; Kao et al . , 2014; Hoare et al . , 2004; Sueur et al . , 2011 ) .", "Therefore , it is likely that the extent of secretion of each paracrine ligand is adjusted to the level of cellular information sharing to ensure an effective collective decision .", "The optimal PCD we identified is not universal .", "Rather , the optimal distance depends on the specific shape of the spatial pattern of the initial activating ligand and the noise pattern of the primary response .", "Additionally , propagation patterns of the same activating ligand can depend on the physiological signaling context as demonstrated by differences found during in vivo ERK propagations under wound and normal conditions ( Hiratsuka et al . , 2015 ) .", "The effective PCD can be regulated at the cellular level by several possible factors to optimize the benefit of paracrine communication to the specific noise and spatial patterns characteristic to each signaling system ( Batsilas et al . , 2003; Muratov and Shvartsman , 2003a; 2003b ) .", "PCD also depends on the effective diffusion coefficient of the secreted molecule , transmitted signal strength ( e . g . number of secreted molecules ) , and receiver cell sensitivity ( e . g . receptor Kd ) .", "The diffusion coefficients of paracrine signaling molecules can vary by two orders of magnitude , let alone differences in signal strength and receptor sensitivity in individual paracrine signaling pathways ( Kreuz et al . , 1965; Gregor et al . , 2007 ) .", "Fine-tuning each of these factors provides a possible mechanism for cells to regulate the PCD and thereby the extent a cell locally communicates .", "The ability to specifically tune PCD raises the possibility that evolutionary pressures can tune paracrine communication to provide the optimal benefit in many other paracrine communication systems ." ], [ "MCF-10A cells were cultured following established protocols ( Debnath et al . , 2003 ) .", "Before plating cells , each surface was first treated with a collagen ( Life Technologies , Carlsbad , CA ) , BSA ( New England Biolabs ) , and fibronectin ( Sigma-Aldrich ) solution in order for cells to completely adhere , according to established methods .", "In order to maintain a viable environment , cells were imaged at 32°C and 5% CO2 .", "All EGF ( PeproTech ) titrations and DREADD experiments were conducted in 96-well plates using extracellular hepes buffer ( ECB ) to reduce background fluorescence ( 5 mM KCl , 125 mM NaCl , 20 mM Hepes , 1 . 5 mM MgCl2 , and 1 . 5 mM CaCl2 , pH 7 . 4 ) .", "All imaging for wounding was done in MCF-10A assay media ( Debnath et al . , 2003 ) .", "Cells were plated at a density of 2 , 000 , 000 cells/100mm plate and allowed to adhere overnight .", "Cells were transfected with the Gq-coupled DREADD HA-tagged hM3D with an mCherry tag using a 3:1 ratio of FuGene HD ( Promega ) to DNA and allowed to incubate overnight ( Dong et al . , 2010 ) .", "In order to measure the paracrine signal from a single-cell , non-transfected cells were mixed with DREADD-transfected cells at ratios of 1:0 , 1:1 , 1:2 , 1:5 , 1:7 , and 0:1 ( non-transfected:DREADD ) and plated in 96-well plates at a density of 30 , 000 cells/well .", "The following day , cells were loaded with 1 μM Hoechst dye for nuclear imaging for 30 min for cell segmentation purposes .", "5 μM clozapine-N-oxide ( CNO ) ( Enzo Life Sciences ) was added to each well to specifically activate DREADD cells .", "ERK activation was monitored using the EKAREV FRET reporter ( Albeck et al . , 2013; Komatsu et al . , 2011 ) and Ca2+ activation was monitored using the Ca2+ indicator dye Fluo-4 using the published protocol ( Invitrogen ) .", "In order to measure the standard deviation of Ca2+ and ERK activity within a small group of cells , MCF-10A cells were plated at densities of 1000 , 2000 , and 3000 cells per well in a 96-well plate , taking advantage of the natural tendency for MCF-10A cells to cluster together .", "Cells were stimulated with 10 μM or 100 μM ATP and imaged for 5 min every 3 s ( Ca2+ ) or 30 min every minute ( ERK ) .", "Standard deviation and average expression of Ca2+ and ERK were analyzed by grouping cells in to clusters based on the distances between cells and clusters ( Figure 1—figure supplement 1 ) .", "Following the cluster analysis , the average and standard deviation of Ca2+ and ERK activation were calculated for each cluster .", "ERK activation was measured using the ERK FRET reporter ( Albeck et al . , 2013; Komatsu et al . , 2011 ) and Ca2+ activation was monitored using the genetically encoded sensor R-GECO ( Zhao et al . , 2011 ) .", "Master molds for the microfluidics based wounding device were created using silicon wafers and layer-by-layer photolithography using established methods ( Ferry et al . , 2011 ) .", "A separate mold for both the air layer and cell layer were made using negative photoresists and masks .", "Chips were made by pouring uncured polydimethylsiloxane ( PDMS ) onto each mold , allowing the PDMS to harden , and bonding the layers together and subsequently to a glass slide .", "Cells were loaded into the devices through the inlet port using a 20G needle .", "During wounding the outlet port was plugged using tape and the inlet port held a reservoir of media to prevent evaporation in the chamber .", "Wounding was accomplished by increasing the air pressure in the top layer of the device until the pillar made contact with the bottom of the device after which the air pressure was released to raise the pillar back up .", "Cells were loaded in to the wounding device at a density of 15 , 000 , 000 cells/mL using a 20G needle .", "Following trypsinization and resuspension , cells were put on ice to prevent aggregation .", "Two o-rings were attached to the device surrounding both the inlet and outlet ports for media reservoirs .", "Each o-ring was attached using a thin film of vacuum grease .", "Wounding devices were kept in an empty pipet box filled with water to prevent media evaporation .", "Cells were allowed to adhere for 18–24 hr before wounding .", "Imaging was accomplished using a Nikon Plan Apo λ 10X/0 . 45objective with a 0 . 7x demagnifier and Nikon Eclipse Ti microscope with a sCMOS Zyla camera .", "All imaging was accomplished using custom automated software written using MATLAB and Micro-Manager ( Edelstein et al . , 2010 ) .", "Image analysis was accomplished using a custom MATLAB code published previously ( Selimkhanov et al . , 2014 ) and is available through GitHub repository https://github . com/rwollman/CellSegmentation . git .", "The paracrine ligand concentration ( P ) for a cell at position ( x , y ) observed by ( P[x , y] ) is the local average of the concentration of ligand released by cells in the local neighborhood ( Figure 1A ) .", "We modeled this paracrine ligand local average using a convolution of two functions: S ( x , y ) that represents the amount of ligand secreted by each cell and D ( x , y ) that represents the expected diffusion of the paracrine ligand during the timescale of paracrine signal integration: ( 1 ) P ( x , y ) =∫∫dudvS x , y D x−u , y−v The function S ( x , y ) was estimated using experimental cellular Ca2+ response data according to: ( 2 ) S ( x , y ) ={max ( Cai2+ ( t ) ) x , y ∈ cell i 0 x , y ∈ background The function D ( x , y ) was approximated to follow Gaussian weights with a length-scale we named the Paracrine Communication Distance ( PCD ) : ( 3 ) D ( x , y ) =1PCD2πe− ( x2+y2 ) PCD2 Detailed analysis of how PCD depends on diffusion , number of secreted paracrine molecules , sensitivity of detection , physiological levels of fluid flow ( Polacheck et al . , 2011 ) , and cellular decoding of time varying paracrine signal are presented in Materials and methods .", "In cases where biologically relevant integrations times may influence the predicted paracrine communication response , the PCD did not exceed the approximate distance EGF could travel before the first ERK response ( Figure 1G , Figure 3F , Materials and methods ) .", "Based on single-cell ERK data to ATP stimulation , this time was found to be ∼5 min which resulted in a maximum PCD of ∼300 μm ( data not shown ) based on EGF diffusion coefficient of 50 μm2/s .", "Signal to Noise ratio analysis on Ca2+ response to ATP titration data was estimated as was done previously ( Selimkhanov , 2014 ) .", "Briefely , the signal S was calculated using: ( 4 ) S = varbins ( avgcells ( maxt ( Ca2 + ( t ) ) ) ) The noise N was calculated by: ( 5 ) N = avgbins ( varcells ( maxt ( Ca2 + ( t ) ) ) ) Where Ca2+ ( t ) is the temporal time series of Ca2+ response measured experimentally .", "Cells are separated into bins according to either different dosages of ATP added to multiple wells ( Figure 1 ) or different distances from the wound source ( Figure 3 ) .", "SNR was then simply: SNR = S/N .", "In this section we analyze how the Paracrine Communication Distance ( PCD ) , the characteristic length-scale of paracrine communication , depends on factors related to the paracrine signal .", "Specifically we look into how the PCD depends on the diffusion coefficient D , the number of molecules released from a cell Nr , the number of molecules needed for detection Nd , and the total integration time T . To understand how PCD depends on the factors mentioned above , we considered the diffusion of a paracrine ligand from a single cell to its surrounding neighbors .", "We considered a 2D-like geometry where cylindrical cells , each of height hc and radius ρ , grow in a chamber of total hf height .", "We simplify the below analysis by approximating the cell monolayer geometry to a series of 'cell cylinders' .", "The key results of the scaling of PCD and required integration time are similar for other comparable geometries ( data not shown ) .", "Under these conditions one could write the analytical solution of the diffusion equations: ( 6 ) Cr , t=Nrhf·4Dπte-r24Dt Where C ( r , t ) is the concentration of paracrine ligand for distance r and time t .", "For a neighboring cell to respond to this paracrine signal , a critical number of molecules Nd needs to reach the volume surrounding the cell .", "We assume that a cell ‘senses’ a volume comparable to the volume of a cell itself .", "For a cylindrical cell of area πρ2 and height hcthe critical concentration required for cellular response will be: ( 7 ) Cdetect = Ndhcπρ2 This is simply the required number of molecules divided by the cell volume .", "Combining equations 6 , 7 we can solve for the distance and time of where the critical concentration will be reached .", "Solving for distance we get that ( 8 ) rdetect=2Dt lnρ 2hcNr4DtNdhf The concentration of the paracrine ligand is diluted as it diffuses from the source .", "Therefore , there is a point in space which is the maximal distance from the source that the critical detection concentration Cdetect will be reached at some point in time .", "Distances that are greater than the critical distance will only experience concentrations lower than the critical detection concentration Cdetect .", "The existence of such maximum can also be seen by the non-monotonous dependency of rdetect on t in equation", "8 . To find the maximal distance we can simply find the maximum of 1 . 3 in respect to t .", "Doing so we get that: ( 9 ) PCD=e-12ρhcNrhfNd We can simplify the analysis by the introduction of two dimensionless variables: 1 ) S = NrNd represents the strength of the signal and is defined as the ratio of released molecules Nr and the number of molecules needed to detect the signal Nd .", "2 ) η = hchf represents the fraction of the height of the flow chamber that cells occupy .", "When we substitute the new variables into Equation 1 , 4 we get that: ( 10 ) PCD = 12e−12ρηS Interestingly this shows that the value of PCD does not depend on the diffusion coefficient .", "Rather , PCD scales as a function of the square root of the strength of the signal S with a multiplicative constant that depends on the specific cell geometry .", "PCD also depends on cell geometry with the cell radius ρ and the relative height of a cell in the effective environment η .", "Figure 1—figure supplement 6 shows equation 10 graphically .", "While the analysis above shows that the diffusion coefficient has no influence on the overall PCD , the time required to reach this maximal distance has important biological implications .", "'Paracrine averaging' requires cells to integrate the signal .", "However , the time required for signal integration must be biologically feasible given the cellular response time and diffusion coefficient .", "From equation 8 we can identify the time by which the PCD is maximal to be: ( 11 ) Tint=ρ2ηS4eD The integration time grows linearly with signal strength S . This is because the PCD itself scales as a square root of S and the diffusion time grows with the square of the distance .", "The integration time decreases with increasing diffusion coefficient as expected .", "Figure 1—figure supplement 7 shows the scaling of the integration time with the diffusion coefficient for a few PCD values .", "For diffusion coefficient values of ∼10–100 μm2/s and a PCD of 100 μm ( similar to the distance measured in Figure 3 ) integration times ranged between 0 . 5 to 5 min .", "Given that ERK activation is observed only after 5 min post-activation , the required integration time does not pose an issue .", "However , larger PCDs will require higher diffusion coefficients to allow proper integration of the paracrine signal .", "Interestingly , H2O2 , another key paracrine signaling molecule critical to initial wound response signaling , has a diffusion coefficient of ∼2000 μm2/s .", "A larger diffusion coefficient could allow for a much longer PCD with reasonable biological integration times .", "All the analysis above assumed static conditions , that is no fluid mixing or advection of any kind .", "In this section we analyze the degree to which the principles of paracrine communication are applicable in non-static conditions .", "Non-static fluid conditions potentially have two effects on mass transport .", "1 ) Non-static fluid conditions can create mixing due to turbulence and 2 ) Laminar advection can transport secreted molecules away from the secretion source .", "Since the extracellular environment is characterized by a low Reynold’s number there is effectively no turbulent mixing in biologically relevant parameters .", "To analyze the relative contribution of advection and diffusive transport we utilize a dimensionless number , the Péclet number ( P ) , that represents the ratio between the contribution of advection and diffusion: ( 12 ) P = vLD Where v is the interstitial flow rate , D is the diffusion coefficient and L is the characteristic length scale .", "In our case , the characteristic length scale is the PCD , which depends on the signal strength as described above ( equation 10 and Figure 1—figure supplement 6 ) .", "Therefore , the P number can be expressed as a function of the signal strength S and diffusion coefficient: ( 13 ) P = SηρvDe Graphical representation of this expression is shown in Figure 1—figure supplement 8 where the map of D and S is color coded by the Péclet number with three highlighted regions: A red region where flow will dominate , a cyan region where diffusion will dominate , and the region in between where both advection and diffusion contribute to paracrine communication .", "To gain further insight into the relative contribution of advection and diffusion we looked at the distance molecules will travel via advection for a specific signal strength ( S = 1000 ) .", "As can be seen in Figure 1—figure supplement 8b , for diffusion coefficients of small protein ligands advection will contribute minimally .", "When considering positional accuracy of cellular response , an important consideration is that advection can potentially 'shift' the effects of paracrine signaling downstream of the flow .", "Even if the shift is characterized by low Péclet number , advection can interfere with positional information accuracy ( as analyzed in Figure 3 ) .", "To estimate the potentially degrading effects of flow we calculate the expected level of positional accuracy error induced by flow .", "We estimate that the wound induced signaling gradient ( Figure 3C ) to be >500 μm .", "Therefore the effect on positional accuracy will be minimal ( <10% ) at advection distances up to 50 μm , or for a PCD of 100 μm , a Péclet number up to 0 . 5 .", "The isocline of a Péclet number of 0 . 5 is shown in Figure 1—figure supplement 8a as a dotted black line .", "This shows that for paracrine ligands with a diffusion coefficient >40 μm2/sec , advection will have little effect on positional accuracy of initial wound response signaling .", "The analysis in the previous two sections assumes that the concentration of the paracrine ligand decreases over increasing distance from the source of secretion according to a Gaussian fit where the diffusion length-scale represents the PCD .", "The cellular response to a paracrine ligand depends on cellular decoding of the temporal paracrine concentration profile a cell observes .", "As both the temporal profile of the secreted paracrine molecule and the temporal cellular decoding are unknown , we consider the simple assumption of a Gaussian profile reasonable .", "To quantitatively test this assumption we compared the Gaussian profile to an alternative model that could be addressed analytically .", "In the alternative model , we assume that all paracrine molecules are released at T = 0 and that cellular decoding of the paracrine signal is simple temporal averaging .", "Under these assumptions one can write an expression of the temporal average of the paracrine concentration at a distance r from the source as: ( 14 ) Cavgr =∫0t0C r , t = ∫0t0Nrhf · 4Dπte-r24 Dt = -Nr4Dπhf t0ei-r24Dt0· Where all symbols follow Equation 6 and ei represent the exponential integral: ei ( x ) = ∫−∞xettdt Comparison of the two models can be seen in Figure 1—figure supplement", "9 . Overall , the two models generate very similar Paracrine Averaging Weights ( the effect of cellular decoding of paracrine signal ) .", "There is a small discrepancy between the two models at very low distances ( <50 μm ) .", "However , this discrepancy is most likely a result of the assumption in the alternative model that all the paracrine molecules are released at once .", "Under the more realistic assumption where paracrine molecule release duration is not much smaller than the time to diffuse 50 μm ( 12 . 5 s at D = 50 μm2/s ) we anticipate that the similarity between these two profiles will further increase ." ] ]

[ "Population averaging due to paracrine communication can arbitrarily reduce cellular response variability .", "Yet , variability is ubiquitously observed , suggesting limits to paracrine averaging .", "It remains unclear whether and how biological systems may be affected by such limits of paracrine signaling .", "To address this question , we quantify the signal and noise of Ca2+ and ERK spatial gradients in response to an in vitro wound within a novel microfluidics-based device .", "We find that while paracrine communication reduces gradient noise , it also reduces the gradient magnitude .", "Accordingly we predict the existence of a maximum gradient signal to noise ratio .", "Direct in vitro measurement of paracrine communication verifies these predictions and reveals that cells utilize optimal levels of paracrine signaling to maximize the accuracy of gradient-based positional information .", "Our results demonstrate the limits of population averaging and show the inherent tradeoff in utilizing paracrine communication to regulate cellular response fidelity ." ]

[ "The human body is made up of many different types of cell that are each specialized to perform particular roles .", "Although each cell type has the same set of genes , the level of activity ( or “expression” ) of these genes varies between each type .", "Additionally , gene expression in cells of the same type can vary due to randomness in the regulation of genes .", "Although variation in gene expression between cells can allow populations of cells to adapt to a changing environment , variability can also cause problems when many different cells need to work together .", "A system called “paracrine signaling” allows cells to communicate with each other by releasing signaling molecules that bind to and activate surrounding cells .", "The distance that this molecule travels , or the “paracrine communication distance” , determines how many surrounding cells each cell can communicate with to coordinate their responses .", "However , it is not clear what impact paracrine signaling has on the variability between cells , or what limitations there are on the size of the paracrine communication distance .", "Cells that are damaged during wounding immediately release a molecule called ATP , which acts as a danger signal to activate the wound healing process in the surrounding cells .", "The release of ATP from wounded cells forms a spatial gradient in the surrounding healthy cells and stimulates the release of molecules called growth factors that are required for the healing process .", "Here , Handly et al . developed a new device to study the responses of human cells to a wound and used it in combination with a computational model to measure the impact of paracrine communication on these responses .", "The experiments show that paracrine signaling by the growth factor EGF reduces the variability in the responses of cells to the ATP signal .", "However , this reduction is limited by the size of the paracrine communication distance .", "Paracrine communication distances that are too small or too large either do not provide adequate reduction in variability or result in “over-averaging” .", "Handly et al . ’s findings show that there is an optimal level of paracrine signaling during wounding that helps to coordinate the response in nearby cells without inappropriately over-averaging the signal ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "evolutionary biology", "microbiology and infectious disease" ]

[ [ "In addition to two subtypes of influenza A virus ( H1N1 and H3N2 ) , two lineages of influenza B viruses co-circulate in humans and cause seasonal influenza epidemics ( Klimov et al . , 2012 ) .", "Influenza B causes a significant proportion of influenza-associated morbidity and mortality , and in some years is responsible for the major disease burden ( Burnham et al . , 2013; Paul Glezen et al . , 2013 ) .", "Although type A and B influenza viruses are closely related and have similarities in genome organization and protein structure ( McCauley et al . , 2012 ) , they exhibit important differences in their ecology and evolution ( Chen and Holmes , 2008; Tan et al . , 2013 ) .", "While new influenza A viruses periodically emerge from animal reservoirs to become endemic in humans ( Neumann et al . , 2009; Smith et al . , 2009 ) , influenza B viruses , first recognized in 1940 , have circulated continuously in humans alongside influenza A viruses and are presumably derived from a single , as yet unknown , source ( Francis , 1940; Chen and Holmes , 2008 ) .", "Unlike influenza A viruses that can infect a wide range of species , influenza B infections are almost exclusively restricted to humans with only sporadic infections reported in wildlife ( Osterhaus et al . , 2000; Bodewes et al . , 2013 ) .", "While the evolutionary and epidemiological dynamics of human influenza A H1N1 and H3N2 viruses have been well documented at the global scale ( Rambaut et al . , 2008; Russell et al . , 2008; Bedford et al . , 2010; Bahl et al . , 2011 ) , the equivalent dynamics of the two influenza B virus lineages—B/Yamagata/16/88-like and B/Victoria/2/87-like , henceforth termed the Yamagata and Victoria viruses—are poorly understood .", "Human influenza A H3N2 viruses exhibit limited genetic diversity at individual time-points due to periodic bottlenecks caused by strong selection—known as ‘antigenic drift’—in the hemagglutinin ( HA ) and neuraminidase ( NA ) genes .", "This results in an HA phylogenetic tree with a characteristic slender ‘trunk’ ( Fitch et al . , 1997 ) appearance ( Figure 1A ) .", "H3N2 viruses also exhibit strong seasonal fluctuations in genetic diversity in temperate climate regions ( such as Australia and New Zealand ) ( Rambaut et al . , 2008 ) , mainly due to the local extinction of viral lineages at the end of each influenza season ( Rambaut et al . , 2008 ) .", "A similar but weaker evolutionary pattern is observed in the seasonal H1N1 viruses that have circulated in humans for the majority of the previous century ( 1918–1957 and 1977–2009 ) , with short-term co-circulation of diverging virus populations ( Nelson et al . , 2008b ) ( Figure 1B ) .", "The pandemic H1N1 ( H1N1pdm09 ) viruses have to date also only exhibited limited antigenic evolution since they emerged in 2009 ( Figure 1C ) .", "In contrast , influenza B viruses are currently composed of two distinct lineages ( Victoria and Yamagata ) ( Kanegae et al . , 1990; Rota et al . , 1990 ) ( Figure 1D ) that diverged approximately 40 years ago and which have since co-circulated on a global scale , despite frequent reassortment among them ( Chen and Holmes , 2008 ) .", "Although the HA genes of influenza B viruses are thought to exhibit lower rates of evolutionary change ( nucleotide substitution ) than both influenza A viruses ( Ferguson et al . , 2003; Chen and Holmes , 2008; Bedford et al . , 2014 ) , their antigenic characteristics are not well understood . 10 . 7554/eLife . 05055 . 003Figure 1 . Evolutionary dynamics of human influenza A and influenza B Victoria and Yamagata viruses . Evolution of the HA genes of influenza A H3N2 virus , 2002–2013 , ( A ) , H1N1 virus , 1998–2009 ( B ) , H1N1pdm09 virus , 2009–2013 ( C ) , and influenza B Yamagata ( red ) and Victoria ( black ) lineage viruses , 2002–2013 ( D ) .", "All phylogenetic trees were generated using approximately 1200 randomly selected full-length gene sequences sampled during 12 years . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 003 The advent of global influenza surveillance and full genome sequencing over the past decade has shown that seasonal epidemic outbreaks of each influenza type are caused by the stochastic introduction of multiple virus lineages ( Nelson et al . , 2008a ) and that the patterns of seasonal oscillation vary between temperate and tropical regions ( Rambaut et al . , 2008 ) .", "Population genetic analysis ( Rambaut et al . , 2008 ) , consistent with epidemiological data ( Goldstein et al . , 2011 ) , suggests that the H3N2 and H1N1 subtypes of influenza A virus compete with each other resulting in the epidemic dominance of a single subtype .", "However , it is unclear whether the same dynamic patterns can be extended to influenza B viruses , or why the Victoria and Yamagata lineages have co-circulated for such an extended time period .", "To understand the evolutionary and epidemiological dynamics of influenza B virus , we generated the full genomes of 908 influenza B viruses selected from over 26 , 000 laboratory confirmed influenza B cases in children and adults aged from birth to 102 years sampled during 2002–2013 in eastern Australia ( Queensland , n = 275; New South Wales , n = 210; and Victoria , n = 207 ) and New Zealand ( n = 216 ) ( Figure 2 ) .", "These regions were selected because influenza surveillance was well established and continuous during the sampling period and included the co-circulation and periodic dominance of influenza A and both influenza B virus lineages .", "Of note is that the influenza B virus strain used for vaccination in the region did not match the dominant circulating strain during 7 of the 13 years studied ( Figure 2B ) .", "Our overall aim was to integrate , for the first time , data obtained from genetic , epidemiological , and immunological sources to better understand the evolution and interaction of these two lineages of influenza B virus . 10 . 7554/eLife . 05055 . 004Figure 2 . Influenza B virus lineages in Australia and New Zealand , 2001–2013 and source of full genomes . Percentage prevalence of influenza B viruses isolated from the three eastern Australian states and New Zealand ( A ) .", "Coloured lines represent the proportion of influenza viruses typed as influenza B in each country ( blue ) and each of the eastern Australian states; Queensland ( yellow ) , New South Wales ( orange ) , and Victoria ( pink ) .", "Bars represent the percentage prevalence of Victoria ( black ) and Yamagata ( red ) .", "Data based on National Notifiable Diseases Surveillance system ( NNDSS ) for Australia and Environmental Science and Research ( ESR ) for New Zealand .", "The lineage of representative influenza B virus strains used in the trivalent influenza vaccine during these years in both countries ( B ) .", "Excluding the years 2003 and 2009 , influenza B viruses represented on average 24 . 6% ( range 9 . 5–53 . 7% ) and 31 . 5% ( range 0 . 5–86 . 9% ) of laboratory confirmed influenza viruses from Australia and New Zealand , respectively .", "The percentage of circulating influenza viruses that were influenza B was significantly lower in 2003 ( AUS , 3 . 4% ) and 2009 ( AUS , 0 . 8% ) than in other years , due to the dominance of a new H3N2 variant ( A/Fujian/412/2002-like ) in 2003 and the emergence of the H1N1 pandemic in 2009 .", "Source of full genomes of Victoria and Yamagata viruses ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 004" ], [ "We used the HA segment of both lineages to contrast their phylodynamics .", "First , to assess the changing patterns of genetic diversity of the two influenza B virus lineages in relation to their evolutionary histories , we used a flexible coalescent-based demographic model ( Minin et al . , 2008 ) , which revealed stark differences in the epidemiological dynamics of the Victoria and Yamagata lineages ( Figure 3A , B ) .", "Whereas the Victoria lineage experienced strong seasonal fluctuations in relative genetic diversity , little change was observed over the same time period for the Yamagata lineage , and these observations were not heavily affected by differences in sampling density ( Figure 3—figure supplement 1 ) .", "While the almost invariant relative genetic diversity of the Yamagata lineage resembled that of seasonal H1N1 viruses ( Figure 3D ) , the stark and almost annual changes of diversity in the Victoria lineage were similar to those observed for H3N2 virus ( Figure 3C ) ; although H3N2 viruses exhibited a greater frequency of oscillations than those estimated for Victoria lineage viruses .", "The strong seasonal fluctuations in diversity observed for Victoria lineage suggest that this lineage experiences strong bottlenecks between seasons similar to those described for H3N2 viruses ( Bedford et al . , 2011; Zinder et al . , 2013 ) , whereas the almost invariant relative genetic diversity for Yamagata suggests the continuous co-circulation of multiple lineages . 10 . 7554/eLife . 05055 . 005Figure 3 . Population dynamics of genetic diversity in Australia and New Zealand . The relative genetic diversity of the HA segments of influenza B Victoria ( A ) , Yamagata ( B ) and influenza A H3N2 ( C ) , and H1N1 2003–2008 and H1N1pdm09 2009–2013 viruses ( D ) , isolated in Australia and New Zealand using the Gaussian Markov Random Field ( GMRF ) model . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 00510 . 7554/eLife . 05055 . 006Figure 3—figure supplement 1 . Effect of sampling on the population dynamics of Influenza B virus . Relative genetic diversity of the Victoria ( black ) and Yamagata ( red ) lineages estimated using the Gaussian Markov Random Fields ( GMRF ) Skyride model ( as in Figure 3 ) , using a subsampled Victoria data set , in which , the number of Victoria lineage viruses was randomly reduced to match the size of Yamagata for that year . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 006 Marked differences between the Victoria and Yamagata lineages were apparent in phylogenetic trees of the HA ( Figure 4 ) .", "The phylogenetic analysis of the HA genes showed that the Victoria lineage was characterized by a single prominent tree ‘trunk’ , with side branches that circulated for short periods of time ( 1–3 years ) ( Figure 4 ) .", "This evolutionary pattern parallels that observed for seasonal H3N2 viruses and is indicative of frequent selective bottlenecks due to the serial replacement of circulating strains , as would be expected under continual antigenic drift ( Grenfell et al . , 2004 ) .", "In contrast , greater diversification was observed for the Yamagata lineage , with multiple clades co-circulating for extensive periods of time ( Figure 4 ) .", "For example , the three clades of Yamagata viruses circulating in 2013 diverged approximately 10 years ago , again paralleling the evolutionary pattern seen in seasonal H1N1 viruses .", "These patterns are clearly identifiable in the genealogical diversity skyline ( Figure 4 ) in which the average time to common ancestor between contemporaneous samples fluctuated from 0 to <5 years for Victoria lineage , except during 2010 and 2011 where the genealogical diversity marginally increased to 7 years .", "In contrast , the genealogical diversity of Yamagata was consistently greater and gradually increased during the sampling period .", "The maintenance of genetic diversity through epidemic peaks and troughs as described for Yamagata ( Figure 3B ) is expected to result in the gradual increase of divergence times of contemporaneous samples . 10 . 7554/eLife . 05055 . 007Figure 4 . Evolution of the hemagglutinin genes of influenza B viruses . Phylogenetic relationship of the HA genes of influenza B Victoria ( black ) and Yamagata ( red ) lineage viruses inferred using the uncorrelated lognormal relaxed clock model .", "Genetic diversity through time was estimated by averaging the pairwise distance in time between random contemporaneous samples with a 1-month window on the same dated Maximum clade credibility ( MCC ) trees . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 007 As each seasonal influenza epidemic provides important information on the epidemiological characteristics of both influenza B virus lineages , we utilized a birth–death susceptible-infected-removed ( BDSIR ) ( Kühnert et al . , 2014 ) phylodynamic model that simultaneously co-estimates seasonal phylogenies along with the basic reproductive number , R0 , for each lineage .", "However , because the infected population contains both susceptible and non-susceptible hosts we report the effective reproductive number , Re .", "This analysis showed a greater variation in Re ( median values , 1 . 1–1 . 3 ) between epidemics caused by the Victoria lineage , whereas the Re of Yamagata epidemics , were generally lower , varied only slightly , around 1 . 1 ( 1 . 08–1 . 14 ) ( Figure 5A ) , indicating greater heterogeneity in transmission between seasons for Victoria viruses .", "Years in which both influenza viruses co-circulated in sufficient numbers ( 2005 and 2008 ) offer a chance for direct comparison of their phylodynamics .", "Both lineages transmitted with nearly equal force in 2005 , whereas in 2008 the median Re of 1 . 27 ( 95% highest posterior density [HPD] of 1 . 18–1 . 37 ) estimated for the Victoria lineage was significantly greater than that of Yamagata at 1 . 11 ( 95% HPD 1 . 05–1 . 17 ) .", "Analysis of the cumulative number of all influenza B positive cases through time for each season ( Figure 5B ) reveals significant differences in the exponential growth phase between the lineages , where Victoria lineage exhibited significantly higher initial growth rate resulting in a faster epidemic with larger number of infections .", "These results also show that in 2008 the Victoria lineage exhibited a significantly faster growth rate , in correlation with the high Re , coinciding with the year in which a new antigenic variant of the Victoria lineage was first detected ( B/Brisbane/60/2008-like viruses ) in Australia and New Zealand .", "This antigenic variant emerged as the globally dominant influenza B strain in the following years and has been continuously recommended ( 2009–2015 ) as the influenza B vaccine component since that period in both the Northern and Southern Hemispheres ( Klimov et al . , 2012 ) . 10 . 7554/eLife . 05055 . 008Figure 5 . Phylodynamics and cumulative cases of influenza B viruses . Effective reproductive number ( Re ) of influenza B Victoria ( black ) and Yamagata ( red ) viruses ( of the HA data set ) estimated for single epidemics ( median and 95% highest posterior density ( HPD ) values ) during years with sufficient number of sequences estimated using the BDSIR model ( A ) .", "The cumulative number of cases from all influenza B virus positive samples for each of these years ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 00810 . 7554/eLife . 05055 . 009Figure 5—figure supplement 1 . Estimates of Re with various S0 values . Estimates of effective population size , Re , using various S0 values for all Victoria ( A ) and Yamagata ( C ) lineage viruses isolated in Australia and for the largest monophyletic group of Victoria ( B ) viruses in Australia that clearly represent a single introduction . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 009 The BDSIR model assumes a closed epidemic , but the large-scale phylogenies generated using all available global data indicated that each of the annual epidemics were caused by the introduction of multiple viral lineages that went extinct locally by the end of the seasonal epidemic ( data not shown ) .", "We therefore investigated the effect of virus migration on the estimates of Re .", "First , we identified lineages that conformed to the assumption of a closed epidemic ( i . e . , lineages resulting from a single introduction into Australia and New Zealand ) and with a sufficiently large local transmission for analysis ( i . e . , Victoria lineage viruses in 2005 , 2006 and 2008 ) .", "An independent estimation of Re for each of these lineages produced a minor but non-significant variation to those observed for the entire epidemic ( Figure 5—figure supplement 1B ) , indicating that , on average , the Re estimates for lineages resulting from multiple introductions were similar .", "Next , we used a continuous-time Markov chain ( CTMC ) phylogeographic process ( Minin and Suchard , 2008 ) to estimate the number of migration events into and from Australia and New Zealand during the same period ( Figure 6 ) .", "This indicated that the number of introductions per year was greater for the Yamagata lineage ( 15–22 , mean state transition count in all years ) than for Victoria ( 3–8 , except 16 and 14 during 2010 and 2011 , respectively ) ( Figure 6 ) , further suggesting an inverse relationship between Re ( Figure 5 ) and the number of introduction events .", "Indeed , our results show that introductions of viruses with greater transmission efficiency ( i . e . , high Re ) , such as Victoria/2008 , resulted in the epidemic dominance of such single strains , whereas epidemics of the Yamagata lineage with lower Re values likely resulted in slower and shorter transmission chains with reduced competition , in turn allowing the co-circulation ( and detection ) of multiple introduced lineages .", "Additionally , we identified that , combined , Australia and New Zealand were net importers of influenza viruses , except during 2002 and 2008 when the net export of the Victoria lineage was similar to the import observed during the same years ( Figure 6 ) .", "The higher transmission rate for Victoria/2008 viruses ( i . e . , B/Brisbane/60/2008-like viruses ) may have also caused the successful seeding of these viruses globally ( as described above ) .", "Taken together , our results support the concept of a global metapopulation seeding subsequent epidemics elsewhere ( Bedford et al . , 2010; Bahl et al . , 2011 ) , provided the virus is transmitted efficiently as observed during 2008 in this study . 10 . 7554/eLife . 05055 . 010Figure 6 . Estimation of migration of influenza B viruses into and out of Australia and New Zealand . Estimated counts of import and export of Victoria ( black ) and Yamagata ( red ) between Australia and New Zealand and rest of the world , using the HA gene data set .", "Error bars represent the 95% highest posterior density ( HPD ) values of each point . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 010 To understand the genome-wide evolutionary dynamics of the two influenza B virus lineages , we inferred temporal changes in genetic diversity for all remaining gene segments ( Figure 7 ) .", "These analyses showed that the patterns observed for the NA and internal gene segments were similar to those observed for the HA genes described above .", "The single exception was the NP genes of both lineages where substantial differences occurred throughout their history .", "During 2002–2007 , the peaks of relative genetic diversity of the Victoria NP was higher than all remaining gene segments following which this lineage was not identified in our surveillance , whereas the Yamagata NP showed additional peaks during 2010 and 2011 that corresponded to the NP peaks observed for the Victoria genes . 10 . 7554/eLife . 05055 . 011Figure 7 . Genome wide evolutionary dynamics—relative genetic diversity . Relative genetic diversity of each gene segments of Victoria ( black ) and Yamagata ( red ) lineages estimated using the Gaussian Markov Random Fields ( GMRF ) Skyride model ( as in Figure 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 011 As genomic reassortment impacts levels of genetic diversity , we conducted phylogenetic analyses of all eight genome segments of the 908 viruses .", "Comparison of these phylogenies revealed frequent reassortment within the two lineages of influenza B virus ( data not shown ) and a few instances of reassortment between them ( Figure 8 ) .", "During the sampling period , the Victoria lineage HA gene repeatedly acquired internal gene segments from Yamagata lineage viruses to form novel reassortants .", "In particular , during 2004 a subpopulation ( approximately 15% ) of Victoria-like viruses acquired all internal gene segments ( PB2 , PB1 , PA , NP , MP , and NS ) from the Yamagata lineage viruses .", "Interestingly , all remaining inter-lineage reassortment events of the Victoria HA lineages involved the acquisition of the Yamagata NP gene during 2007 and 2008 ( Figure 8E ) , which resulted in the extinction of the previously circulating Victoria lineage NP gene .", "These patterns were consistent with the reconstruction of the population genetic history for the NP gene where we observed additional peaks in genetic diversity following 2007/2008 when the Yamagata NP was acquired by Victoria viruses ( Figure 7 ) , indicating a major genome-level transition for Victoria lineage viruses .", "In contrast , the only inter-lineage reassortment events for the virus carrying the Yamagata HA occurred during 2002 and 2004 ( red arrows in Figure 8A , F ) , when the NA and MP genes were derived from the Victoria lineage viruses , but these viruses went extinct within the same influenza season .", "In sum , these results show that the HA gene of Victoria viruses is placed in different genetic backgrounds at a higher rate and this is likely to have important fitness consequences . 10 . 7554/eLife . 05055 . 012Figure 8 . Genome wide evolutionary dynamics—reassortment . Evolutionary relationships of neuraminidase ( A ) , polymerase basic 2 ( B ) , polymerase basic 1 ( C ) , polymerase acidic ( D ) , nucleoprotein ( E ) , matrix ( F ) , and non-structural ( G ) genes of Victoria and Yamagata lineage viruses inferred using the maximum likelihood analysis of 908 full genome sequences .", "Lineages are coloured based on the HA lineage: Victoria ( black ) and Yamagata ( red ) and arrows highlight inter-lineage reassortment . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 012 Phylogenies also suggest that the PB2 and PB1 gene trees ( Figure 8B , C ) exhibit deep divergence , similar to the HA gene where co-circulating viruses contain distinct Victoria and Yamagata genes .", "In contrast , the other gene segments exhibit relatively recent divergence indicating that the prevailing diversity of these genes originates from a single lineage .", "These results are consistent with a detailed investigation of long term reassortment patterns of influenza B virus lineages that revealed genetic linkage between the PB2 , PB1 and HA protein genes ( Dudas et al . , 2015 ) .", "Specifically , we observe that the PB2 , PB1 and HA genes were consistently derived from a single lineage , except for the short-lived subpopulation in 2004 .", "Despite the marked differences in their epidemiological and evolutionary dynamics , the HA genes of the two influenza B lineages both evolved at a rate of approximately 2 . 0 × 10−3 subs/site/year ( Table 1 ) , comparable to those previously estimated for a smaller ( n = 102 ) global sample of influenza B viruses collected during 1989–2006 ( Chen and Holmes , 2008 ) ( mean nucleotide substitution rate of 2 . 15 × 10−3 subs/site/year ) .", "These rates were considerably lower than those estimated for influenza A H3N2 and H1N1 viruses ( 5 . 5 × 10−3 subs/site/year , 4 . 0 × 10−3 subs/site/year , respectively ) ( Rambaut et al . , 2008 ) .", "In contrast , analysis of the ratio of the number of nonsynonymous and synonymous substitutions per site ( dN/dS ) revealed significant differences between the influenza B virus lineages , with the Victoria lineage viruses having accumulated more nonsynonymous substitutions ( dN/dS = 0 . 19 ) than the Yamagata lineage ( dN/dS = 0 . 13 ) ( p-value , <0 . 05 ) .", "In addition , two amino acid residues in the Victoria HA ( positions 212 and 214 ) were revealed to have experienced positive selection ( p < 0 . 05 ) , whereas no positively selected sites were observed in the Yamagata lineage over the time period studied .", "Similarly , the Victoria lineage exhibited a greater dN/dS ( ratio = 1 . 37 ) on internal vs external branches of the HA phylogeny compared to the Yamagata lineage ( ratio = 0 . 98 ) , indicating that amino acid changes have been fixed more frequently in Victoria than Yamagata lineage viruses ( Table 1 ) .", "Taken together , these results indicate that the Victoria lineage is under greater positive selection pressure , and hence likely to experience greater antigenic drift , than the more conserved Yamagata lineage . 10 . 7554/eLife . 05055 . 013Table 1 . Nucleotide substitution rates ( nucleotide substitutions/site/year ) and selection pressures ( dN/dS ) of influenza B viruses from Australia and New Zealand during 2002–2013DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 013Mean substitution ratesBranch dN/dSSite dN/dSSegment* ( 95% HPD ) Global dN/dSInternalExternalInternal/ExternalNo .", "+ve ( sites ) No .", "−veVictoria PB21 . 49 ( 1 . 28–1 . 69 ) 0 . 08 ( 0 . 07–0 . 09 ) 0 . 020 . 030 . 550373 PB10 . 14 ( 0 . 12–0 . 16 ) 0 . 08 ( 0 . 07–0 . 09 ) 0 . 060 . 051 . 081 ( 474 ) 402 PA1 . 65 ( 1 . 44–1 . 88 ) 0 . 13 ( 0 . 11–0 . 15 ) 0 . 080 . 081 . 031 ( 700 ) 334 HA2 . 00 ( 1 . 74–2 . 57 ) 0 . 19 ( 0 . 17–0 . 22 ) 0 . 120 . 091 . 372 ( 212 , 214 ) 239 NP1 . 04 ( 0 . 76–1 . 34 ) 0 . 09 ( 0 . 07–0 . 12 ) 0 . 070 . 051 . 22049 NA2 . 04 ( 1 . 72–2 . 36 ) 0 . 31 ( 0 . 28–0 . 35 ) 0 . 250 . 241 . 026 ( 46 , 73 , 106 , 145 , 146 , 395 ) 129 MP1 . 44 ( 1 . 17–1 . 70 ) 0 . 06 ( 0 . 04–0 . 09 ) 0 . 000 . 020 . 01087 NS1 . 71 ( 1 . 38–2 . 06 ) 0 . 45 ( 0 . 38–0 . 53 ) 0 . 110 . 300 . 373 ( 116 , 120 , 249 ) 13Yamagata PB22 . 00 ( 1 . 74–2 . 25 ) 0 . 06 ( 0 . 05–0 . 07 ) 0 . 030 . 021 . 440443 PB11 . 78 ( 1 . 56–2 . 00 ) 0 . 07 ( 0 . 05–0 . 08 ) 0 . 020 . 030 . 821 ( 357 ) 392 PA1 . 60 ( 1 . 35–1 . 84 ) 0 . 10 ( 0 . 08–0 . 12 ) 0 . 030 . 050 . 570204 HA2 . 01 ( 1 . 73–2 . 29 ) 0 . 13 ( 0 . 11–0 . 16 ) 0 . 070 . 070 . 980245 NP1 . 87 ( 1 . 65–2 . 10 ) 0 . 10 ( 0 . 08–0 . 11 ) 0 . 080 . 071 . 160308 NA2 . 25 ( 1 . 90–2 . 60 ) 0 . 20 ( 0 . 17–0 . 24 ) 0 . 300 . 181 . 701 ( 295 ) 124 MP2 . 20 ( 1 . 85–2 . 55 ) 0 . 05 ( 0 . 03–0 . 07 ) 0 . 050 . 022 . 080102 NS2 . 00 ( 1 . 66–2 . 39 ) 0 . 33 ( 1 . 66–2 . 39 ) 0 . 420 . 321 . 32030*Analysis was restricted to the non-overlapping regions of M1 and NS1 , for the MP and NS segments , respectively .", "We constructed antigenic maps ( Smith et al . , 2004 ) using hemagglutination inhibition ( HI ) assay measurements for 87 Victoria and Yamagata viruses isolated during 2002–2013 and using 20 reference antigens and antisera ( Figure 9A ) .", "These revealed that Victoria lineage viruses exhibited antigenic variation that generally clustered according to the year of isolation and phylogenetic distance , indicative of ongoing antigenic drift , and similar to that previously reported for H3N2 viruses ( Smith et al . , 2004; Bedford et al . , 2014 ) .", "In contrast , the antigenic distances for the Yamagata viruses had no correlation with time or phylogenetic distance and showed greater levels of antigenic cross-reactivity between antisera raised to both earlier and more recent viruses .", "Structural modeling showed that the degree of antigenic distance between strains of Victoria viruses was often linked to the proximity of single amino acid substitutions to the receptor binding pocket ( RBP ) of the HA ( Figure 9B; see structural differences section below ) , in agreement with recent work on H3N2 ( Koel et al . , 2013 ) .", "Importantly , the closer the amino acid change between two strains was to the RBP , the greater the antigenic difference between them . 10 . 7554/eLife . 05055 . 014Figure 9 . Antigenicity of influenza B viruses . Antigenic map showing relative antigenic differences of Victoria and Yamagata lineage viruses ( circles ) measured using the hemagglutinin inhibition ( HI ) assay for each strain and coloured by year of isolation ( A ) .", "Residues contributing to HI titer changes ( B ) .", "Among the nine amino acid changes that we detected between antigenically different Victoria viruses , three changes produced strong HI titer change ( >100 ) ( red ) , 3 medium ( ≈50 ) ( orange ) and 3 low ( <20 ) ( yellow ) .", "Changes that produced the strongest HI titer change were the closest to the receptor binding pocket ( blue arrow ) , highlighting the significance of their proximity to HI titer change .", "Amino acids were mapped on previously resolved influenza B virus structure ( PDB:4FQM ) .", "Detailed HI titer values and reference antigens used are provided in the Dryad source data ( Vijaykrishna et al . , 2015 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 014 In addition to genetic , antigenic , and evolutionary differences , we found a notable difference in the age distribution of infected cases for the two influenza B virus lineages ( Figure 10 ) that was generally consistent throughout our sampling period ( Figure 10—figure supplement 1 ) .", "On average , Victoria viruses infected a younger population ( mean 16 . 8 years , median 11 years ) compared to Yamagata viruses ( mean 26 . 6 years , median 18 years ) .", "Although the proportion of cases under 6 years were similar in both lineages ( 28 . 8% of Victoria and 26 . 8% of Yamagata ) , there were 1 . 7 times more cases aged 6–17 years infected with a Victoria lineage virus ( 39 . 0% Victoria vs 22 . 7% Yamagata ) , while this ratio was almost reversed for those aged 18 years and over ( 32 . 2% Victoria vs 50 . 0% Yamagata; χ2 , p < 0 . 0001 ) ( Table 2 ) .", "Thus , nearly 70% of Victoria lineage viruses were identified in children <18 years , whereas the Yamagata lineage exhibited a bimodal age distribution with a significant shift toward infections in individuals aged >25 years ( Figure 10 ) .", "These differences in age distribution are significant and unlikely to be explained by systematic bias because the same pattern was observed in both countries , and are consistent with data from Guangdong , China ( Tan et al . , 2013 ) , and Slovenia ( Sočan et al . , 2014 ) during the 2009–2010 and 2010–2013 epidemic seasons , respectively . 10 . 7554/eLife . 05055 . 015Figure 10 . Age distribution of influenza B viruses . Density of age distribution of influenza B virus positive samples of Victoria ( black ) and Yamagata ( red ) lineages , collected from Australia and New Zealand during 2002–2013 .", "Patient age was available for 5260 samples .", "The age distributions by lineage were compared by histogram using 2-year bins .", "Also see Table 2 for comparison by age categories and Dryad source data for mean and median age for each year . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 01510 . 7554/eLife . 05055 . 016Figure 10—figure supplement 1 . Year-wise age distribution of influenza B viruses . Mean and median of age distribution of influenza B viruses ( A ) .", "Box-whisker plot with mean ( square ) and age distribution of all influenza B viruses cases ( jitter plot ) are shown for years with greater than 100 samples for either lineage ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 01610 . 7554/eLife . 05055 . 017Table 2 . Age distribution by groupDOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 017VictoriaYamagataAgen%n%p value*<6100728 . 847326 . 86–1713613940222 . 7≥18112432 . 289350 . 5Total34921001768100<0 . 0001*Age categories were compared by lineage using a χ2 test .", "A direct consequence of antigenic drift is the possibility for previously infected individuals to become reinfected .", "Subsequently , higher rates of antigenic drift in the Victoria lineage should lead to a more even age distribution of cases , whereas lower rates of antigenic drift should lead to an age distribution of cases that are skewed towards younger individuals .", "Although viruses of the Victoria lineage were consistently reported at a higher frequency during our surveillance period , the observed skew towards children runs counter to this expectation ( Figure 10 ) .", "One possible explanation is that the higher Re of the Victoria viruses reduces the mean age of infection , as expected in the case of a disease like influenza that imparts some immunity following infection ( Anderson and May , 1992 ) .", "Alternatively , the inability of Victoria viruses to infect an equivalent proportion of other age groups may mean that the relatively older population is better protected against this virus because of a broader immune response .", "The former scenario is supported by an increase in the mean age of infection from 15 years ( median ,", "12 ) in 2008 to 20 . 5 years ( median ,", "14 ) in 2011 for the B/Brisbane/60/2008-like antigenic variant of the Victoria lineage , which coincided with a gradual drop in Re from its peak in 2008 ( Figure 5A ) .", "Finally , we sought to determine whether differences in the evolutionary and epidemiological dynamics between the two influenza B lineages resulted from variation in HA structure and binding preferences .", "First , we compared amino acid substitutions per site within and between influenza virus lineages from 2002 to 2012 and mapped these onto structural models of representative influenza B virus strains ( Figure 11 ) .", "The higher rates of amino acid change observed in the Victoria HA ( Figure 11A ) were consistent with the stronger selective pressures on this viral lineage .", "Importantly , these changes occurred in three major clusters situated around 21 , 29 , and 37 Å to the RBP of the HA domain that also comprises potential antigenic sites .", "Notably , all changes in the closest cluster ( 21 Å ) were comprised exclusively of Victoria lineage amino acid changes , while the few changes observed in Yamagata lineage viruses were distant to the RBP ( Figure 11C ) .", "Overall , however , amino acid changes in both influenza B virus lineages were less frequent than those in influenza A viruses sampled over a similar time period , with the H3N2 viruses showing more extensive structural change ( Figure 11—figure supplement 1 ) . 10 . 7554/eLife . 05055 . 018Figure 11 . Structural view of the HA showing mutational accumulation and lineage differences . Amino acid changes observed within and between influenza B virus lineages ( A ) .", "Arrow colours in ( A ) correspond to inter- ( B ) or intra- ( C ) lineage amino acid changes , based on previously resolved crystal structure ( PDB:4FQM ) .", "Amino acids in red represent differences between the two lineages that were retained over all sampling years; yellow represents differences that are newly observed in 2012 compared to 2002; and magenta represents changes lost in 2012 compared to 2002 .", "Amino acids in blue and green represent changes that occurred in Victoria and Yamagata viruses between 2002 and 2012 , respectively; whereas cyan represents difference between 2002 and 2012 shared between both lineages .", "These amino acid changes occur in regions that cluster around 21 , 29 , and 37 Å distant from the RBP ( C ) .", "Structural differences in RBP among recent Victoria ( B/Brisbane/60/2008 ) and Yamagata ( B/Florida/4/2006 ) strains with a human-like α-2 , 6 host receptor analogue ( magenta ) modeled within the viral RBP ( D ) .", "D was based on crystal structures PDB:4FQM and PDB:4FQJ with side-chains minimized after addition of ligand from PDB:2RFU through superposition .", "Regions differing in backbone conformation are shown in orange for Victoria and cyan for Yamagata , while conserved regions are shown in gray .", "Residues with conserved backbone structure but different amino acid side-chains are shown in red for Victoria and blue for Yamagata .", "Side-chains are shown only for residues within 5 Å of the receptor ligand and differing between the lineages .", "Structural view of receptor binding pocket with α-2 , 6- ( green ) and α-2 , 3-linked ( red ) host receptor and glycans ( blue ) ( E ) .", "E was based on crystal structure PDB:4FQM , with the addition of ligands from PDB:2RFU and PDB:2RFT through superposition and no minimization .", "The presence of a glycan on site 212 allows binding only to 2 , 6-linked receptors , while loss of the glycan allows binding to both α-2 , 3- and α-2 , 6-linked receptors .", "Brown arrows ( B and C ) indicate relative position of receptor binding pocket ( RBP ) , whereas black arrow heads ( C and D ) point to site of known antigenic cluster transition ( Koel et al . , 2013 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 01810 . 7554/eLife . 05055 . 019Figure 11—figure supplement 1 . Structural view of mutational drift in influenza A and B viruses . Amino acid mutations accumulated over 10 years ( red ) using different rotations of the hemagglutinin monomer structure of influenza B Victoria ( 2002–2012 ) ( PDB:4FQM ) ( A ) , Yamagata ( 2002–2012 ) ( PDB:4FQM ) ( B ) in comparison to seasonal influenza A H3N2 ( 1999–2009 ) ( PDB:2YP4 ) ( C ) , and H1N1 ( 1997–2007 ) ( PDB:3UBE ) ( D ) viruses .", "Arrows point to receptor binding pocket . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 019 Notably , we also observed fundamental structural differences between the lineages ( Figure 11B ) .", "Crystal structures showed extensive backbone differences around amino acid sites 165 and 180 that lie near the RBP as well as residue differences in the helix close to where α-2 , 3 and α-2 , 6 ligands differ structurally , thereby potentially influencing receptor binding ( Figure 11D ) .", "Previous experiments suggest that Yamagata viruses bind predominantly to α-2 , 6-linked sialic acid host receptors while Victoria viruses have both α-2 , 3 and α-2 , 6 binding capacities ( Wang et al . , 2012; Velkov , 2013 ) .", "Binding differences may also originate in part from differences in N-glycosylation patterns between the lineages ( Figure 11E , 12 ) .", "While both lineages share a possible glycan at Asn 160 , only Victoria has a functional N-glycosylation site at Asn 248 , although its distance from the receptor may account for only a limited role in binding differences .", "On the other hand , N-glycosylation at Asn 212 occurs in both lineages but has a lower overall frequency in Victoria strains .", "In light of the positive selection acting on codon sites 212 and 214 in the Victoria lineage , it is interesting to note that amino acid changes in either site would abolish the N-glycosylation at 212 , thereby highlighting a possible functional consequence of gain or loss of a glycan at this site .", "Furthermore , position 212 is located at the exit of the RBP which is used by α-2 , 3-linked sialic acid host receptors , and loss of N-glycosylation at 212 consequently adds capacity to bind α-2 , 3 and not just α-2 , 6-linked sialic acid host receptors ( Figure 11E ) .", "Importantly , all our sequenced viruses have been passaged in MDCK cells to avoid egg adaptation artifacts in this context ( Gambaryan et al . , 1999 ) .", "Interestingly , we observed that loss of N-glycosylation at site 212 was associated with an increased proportion in the younger ( 0–5 years ) age group ( Figure 12 ) .", "We therefore hypothesize that subtle differences in the prevalence of α-2 , 3- and α-2 , 6-linked glycans on the cells of the respiratory tract of young children compared to adults ( Nicholls et al . , 2007; Walther et al . , 2013 ) , combined with partial changes in glycosylation patterns , could account for the observed differential age distribution of the two influenza B lineages . 10 . 7554/eLife . 05055 . 020Figure 12 . Glycosylation at Asn 212 and correlation with age groups for Victoria viruses . Yamagata viruses showed five instances of glycosylation loss at 212 , compared to 71 instances in Victoria , hence Victoria lineage strains have been analyzed in detail here .", "Temporal distribution of age groups and glycosylation at 212 for all Victoria strains ( A ) .", "Summary of odds ratio ( OR ) for association of glycosylation loss at 212 with the different age groups ( B ) .", "OR values >1 indicate that it is more likely to find a 212 loss in the respective age group , whereas values <1 indicate that 212 losses are less likely to be found in the respective groups .", "The following guideline helps judging significance of OR: strong positive association >3; moderate positive association 1 . 5–3; moderate negative association 0 . 33–0 . 66; strong negative association <0 . 33 . DOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 020 The genomic and epidemiological data analyzed here provide important insights into the phylodynamics of the two lineages of influenza B virus currently circulating in humans .", "In particular , we find significant differences in the evolutionary and epidemiological dynamics between the Victoria and Yamagata lineages ( Table 3 ) .", "Central to this is the observation that the phylodynamic pattern of the Victoria lineage HA gene is indicative of a virus population under greater selection pressure that escapes host immunity by accruing beneficial amino acid substitutions in the HA gene .", "Indeed , theory predicts that the highest rate of viral adaptation occurs at intermediate levels of immune pressure ( Grenfell et al . , 2004 ) which may characterize the Victoria lineage .", "Such an evolutionary pattern ensures that there is a constant supply of susceptible individuals for Victoria lineage viruses—both naïve and reinfected individuals which in turn increases Re—which then exhibit a pattern of genomic diversity and lineage turnover that is significantly faster and more periodic than Yamagata lineage viruses . 10 . 7554/eLife . 05055 . 021Table 3 . Summary of evolutionary and epidemiological characteristics of influenza B virus lineagesDOI: http://dx . doi . org/10 . 7554/eLife . 05055 . 021CharacteristicsVictoriaYamagataAge distributionyounger ( mean 16 . 8 , median 11 ) older ( mean 26 . 6 , median 18 ) Genetic diversitystrong seasonal changesweak seasonal changesR ( medians ) higher ( 1 . 13–1 . 27 ) lower ( 1 . 08–1 . 14 ) Positive selectionstrongerweakerAntigenic driftrelatively strongrelatively weakReassortmenthigh inter-sublineage reassortment , with lower intra-sublineage reassortmentlow inter-sublineage reassortment , with greater intra-sublineage reassortmentReceptor binding preferenceα-2 , 3- and α-2 , 6-linked sialic acidmainly α-2 , 6 linked sialic acid In contrast , the phylodynamic patterns exhibited by Yamagata viruses are indicative of a virus population that exhibits slower and less periodic dynamics , reflected in a lower and more consistent Re , in turn suggesting that these viruses are under weaker immune selection pressure and accordingly experience weaker antigenic drift .", "Interestingly , clinical trials of influenza B virus vaccination in children ( Skowronski et al . , 2011 ) and experimental infection of mice ( Skowronski et al . , 2012 ) showed that the Yamagata antigens produced a stronger immune response than the Victoria antigens .", "If natural infection with influenza B virus was similar , this would imply that Yamagata viruses are less able to evolve through antigenic drift and therefore escape the immune response ( Grenfell et al . , 2004 ) .", "We propose that these fundamental differences in evolutionary and epidemiological dynamics are driven by differences in hemagglutinin binding preferences .", "Specifically , Victoria viruses appear to have both α-2 , 3- and α-2 , 6-linked sialic acid binding capacities ( Wang et al . , 2012; Velkov , 2013 ) , while Yamagata viruses predominantly bind to α-2 , 6-linked glycans on cells in the human respiratory tract .", "Experimental studies in children ( aged up to 7 ) ( Nicholls et al . , 2007 ) and adults have shown that the respiratory tissue of children mainly have α-2 , 3-linked receptors with a lower level of α-2 , 6-linked receptors than adults , and these differences among the different age groups may in part account for the different age distribution of the two B lineages .", "In turn , the greater propensity to infect children will increase Re , initiating the epidemiological and evolutionary pattern that characterizes the Victoria lineage .", "It remains to be determined whether the broadly equivalent phylodynamic differences between the H3N2 and seasonal H1N1 types of influenza A virus are similarly due to basic differences in the structure of their respective HA proteins .", "Furthermore , to better understand the bimodal age distribution in Yamagata , where a significant reduction of infection was observed among the older children–young adult group ( <25 years ) , additional experimental studies of the receptor distribution in all age groups are necessary .", "These observations have implications for the future control of influenza B virus in the human population .", "While the co-circulation of divergent Yamagata viruses reported here has and can confound the accurate selection of vaccine strains , our analyses also indicate that the Yamagata viruses are under weaker positive selection and antigenic drift , and , on average , infect an older group of people who are more likely to have a higher level of cross-reactive antibodies to the B lineage viruses compared to children .", "As a consequence , there is a greater chance that , given sufficient coverage , Yamagata viruses might experience a major drop in prevalence over time through targeted control methods , such as the extensive use of quadrivalent influenza vaccines containing both B lineages , in contrast to the more adaptable Victoria viruses ." ], [ "Influenza B positive samples collected between 2002 and 2013 from subjects in eastern Australia ( Victoria , New South Wales and Queensland ) and from New Zealand and associated metadata , including date of isolation and age of host , were sent to the WHO Collaborating Centre for Reference and Research on Influenza , Melbourne , from National Influenza Centres and other laboratories as part of the World Health Organization Global Influenza Surveillance and Response System ( WHO GISRS ) .", "Data deposited in Dryad data repository under DOI: 10 . 5061/dryad . n940b ( Vijaykrishna et al . , 2015 ) .", "Influenza B viruses were isolated or re-isolated in MDCK cells ( ATCC-CCL 34 ) from original clinical samples or virus isolates and typed as B/Yamagata or B/Victoria using HI analysis or by molecular assay ( Deng et al . , 2013 ) .", "Viruses were stored at −80°C until sequenced .", "We sequenced the complete genomes of 908 laboratxory confirmed influenza B virus MDCK or MDCK-SIAT cell propagated isolates passaged 1–4 times from eastern Australia and New Zealand using a novel methodology ( Zhou et al . , 2014 ) .", "Influenza B virus genomes were amplified using the universal influenza B genomic amplification strategy that enables amplification of the complete genome of any influenza B virus in a one-step single tube/well reaction .", "Specifically , RNA was isolated from 130 μl of culture supernatant using ZR-96 Viral RNA Kit ( Zymo Research , Irvine , CA ) and eluted in 30 μl of RNase-free water .", "3 μl of the RNA was mixed with FluB Universal Primer Cocktail ( Zhou et al . , 2014 ) and converted to cDNA and amplified with the SuperScript III One-Step RT-PCR System ( Life Technologies , Grand Island , NY ) .", "The amplicons were fragmented , flanked by sequencing adaptors , clonally amplified onto IonSphere particles , and sequenced on the Ion Torrent PGM platform following manufacturer's instruction .", "The sequence reads were sorted by bar code to separate different viruses and used to assemble viral genomes ( sequence accession numbers are available in the Dryad data repository under DOI: 10 . 5061/dryad . n940b ) .", "Sequences were curated , and maximum likelihood ( ML ) phylogenetic trees were inferred for each gene segment independently from the samples described above .", "ML trees were estimated using iqtree v0 . 9 . 5 ( Minh et al . , 2013 ) using the best-fit nucleotide substitution model , chosen by the Bayesian Information Criterion ( BIC ) .", "The data were further divided into separate lineages ( i . e . , Victoria and Yamagata ) and time-scaled phylogenies and rates of nucleotide substitution for each were inferred using a relaxed molecular clock model in a Bayesian Markov Chain Monte Carlo ( MCMC ) framework with the program BEASTv1 . 8 ( Drummond et al . , 2012 ) that incorporates virus sampling dates to concurrently estimate phylogenetic trees , rates of nucleotide substitution , and the dynamics of population genetic diversity using a coalescent based approach .", "The analysis was conducted with a General Time Reversible ( GTR ) model with a gamma ( Γ ) distribution of among-site rate variation and a time-aware linear Bayesian skyride coalescent tree prior ( Minin et al . , 2008 ) .", "We performed at least two independent analyses per data set for 100 million generations sampled every 10 , 000 runs .", "After the appropriate removal of burn-in ( 10–20% of samples in most cases ) , a summary Maximum Clade Credibility ( MCC ) tree was inferred and visualized with Figtree v1 . 4 ( Rambaut , 2014 ) .", "Support for individual nodes is reflected in posterior probability values , and statistical uncertainty is given by 95% Highest Posterior Density ( HPD ) intervals .", "The MCC trees were also used to estimate the genealogical pairwise diversity by averaging the time distance between contemporaneous sample pairs with a 1 month window ( Zinder et al . , 2013 ) .", "The past population dynamics of each linage were compared using a Bayesian skyride analysis in BEAST , which utilizes a Gaussian Markov Random Field ( GMRF ) smoothing prior to estimate the changes in relative genetic diversity in successive coalescent intervals ( Minin et al . , 2008 ) .", "In the absence of natural selection ( i . e . , under a strictly neutral evolutionary process ) , the genetic diversity measure obtained reflects the change in effective number of infections over time ( Net , where t is the average generation time ) .", "However , because natural selection can play a major role in the evolution of the influenza HA , these are interpreted as ‘relative genetic diversity’ , and which is consistent with previous studies of influenza A virus ( Rambaut et al . , 2008 ) .", "Sequence alignments with input parameters are available under Dryad data repository under DOI: 10 . 5061/dryad . n940b .", "We used a continuous-time Markov chain ( CTMC ) phylogeographic process ( Minin and Suchard , 2008; Lemey et al . , 2009 ) to estimate counts of migration to and from Australia and New Zealand , similar to previous studies ( Nunes et al . , 2012; Bahl et al . , 2013 ) .", "Briefly , global influenza B virus HA sequences and their associated spatial locations and isolation dates were downloaded from GenBank for the years for which we estimated an effective reproductive number in the phylodynamic analysis ( see below ) .", "Spatial locations of the isolates were transformed to represent two discrete states: the region of interest ( Australia and New Zealand ) and the rest of the world .", "Phylogeographic events were estimated independently for each of the identified years using an asymmetric CTMC process ( Minin and Suchard , 2008 ) , with the estimated state transition counts ( import and export ) between the two discrete states estimated using a Markov Jump count approach .", "This phylogeographic inference was implemented in BEAST 1 . 8 ( Drummond et al . , 2012 ) similar to the temporal phylogenies described above .", "The resulting log files were used in extracting the net migration counts and mean non-zero transition rates .", "To estimate epidemiological parameters ( specifically the effective reproductive number , Re ) for each epidemic of virus lineages in Australia and New Zealand , we used the birth–death susceptible-infected-removed ( BDSIR ) model ( Kühnert et al . , 2014 ) .", "The BDSIR analysis was also conducted with a GTR + Γ substitution model , with epidemiological dynamics estimated jointly with the phylogenies for each virus lineage .", "The model assumes a closed SIR epidemic in each season for the underlying host population .", "The initial number of susceptible individuals S0 could not be estimated and was therefore initially fixed to 4 , 000 , 000 ( results reported in the main text ) .", "Analysis under different S0 values , ranging from 40 , 000 to 10 million , showed that the estimates of reproductive numbers ( Re ) are robust to the choice of S0 .", "The BDSIR analyses utilized m = 100 intervals for the approximation of the SIR dynamics .", "Incidence and prevalence were computed from the posterior distributions of the SIR trajectories , and the relevant plots show their median values .", "Selection pressures for each gene segment , lineage , and individual codon were estimated as the ratio of the number of nonsynonymous substitutions per nonsynonymous site ( dN ) to the number of synonymous substitutions per synonymous site ( dS ) .", "Estimates were obtained using the Single Likelihood Ancestor Counting ( SLAC ) ( Kosakovsky Pond and Frost , 2005 ) and Fast Unconstrained Bayesian AppRoximation ( FUBAR ) ( Murrell et al . , 2013 ) methods , accessed through the Datamonkey webserver of the HyPhy package ( Delport et al . , 2010 ) .", "In addition , the dN/dS ratio for the internal and external branches of the Victoria and Yamagata HA phylogenies was estimated separately using the CODEML program ( two-ratio model ) available in the PAML suite ( Yang , 2007 ) .", "Representative viruses from each lineage were sub-sampled and tested for antigenic reactivity by a hemagglutination inhibition ( HI ) assay using a panel of reference ferret antisera that were available for each influenza B lineage ( raw HI titers are available in the Dryad data repository under DOI: 10 . 5061/dryad . n940b ) and the subsequent antigenic profile was used to generate antigenic maps ( Cai et al . , 2010 ) for each lineage .", "HI assays were performed as described previously ( WHO Global Influenza Surveillance Network , 2011 ) using panels of post-infection ferret sera raised against representative viruses from both B/Victoria lineage or the B/Yamagata lineage collected from 2000 to 2013 .", "Turkey red blood cells were used to detect unbound virus and the HI titer was determined as the reciprocal of the last dilution that contained non-agglutinated RBC .", "Normalized titers from the HI assay were compiled for antigenic cartography analysis .", "The HI matrix was used in a multi-dimensional scaling ( MDS ) plot algorithm to chart the antigenic distances between isolates tested in a two-dimensional map ( Cai et al . , 2010 ) , through the AntigenMap webserver ( Wan , 2010 ) .", "To identify residues contributing most to HI titer changes , pairwise comparison of sequences with a single amino acid difference were conducted .", "Finally , sequence data of the HA segment from each lineage were used to construct structural models ( Krieger et al . , 2009; Webb and Sali , 2014 ) .", "To identify those residues that contribute most to antigenic drift in Victoria viruses , we compared the HA amino acid sequences of all pairs of HI assay tested strains using the Smith-Waterman algorithm .", "If only a single mutation difference was found , we calculated the respective average HI titer change for occurrences of this mutation .", "These amino acid sites were then mapped on the crystal structure PDB:4FQM ( Dreyfus et al . , 2012 ) and visualized using YASARA ( Krieger et al . , 2009 ) .", "Amino acid substitutions per site between pairs of HA sequences were calculated using MEGA5 ( Tamura et al . , 2011 ) under the Jones-Taylor-Thornton ( JTT ) amino acid substitution model .", "We constructed structural models using MODELLER ( Webb and Sali , 2014 ) ( five models each with and without ligand , best model selected by DOPE quality score ) , structural alignments were conducted using MUSTANG ( Konagurthu et al . , 2006 ) and visualized using YASARA ( Krieger et al . , 2009 ) .", "To identify structural changes occurring on the HA proteins of influenza A subtypes and influenza B virus lineages over a 10-year period , we selected the HA protein sequences of the following virus strains: influenza B Victoria lineage , B/Sydney/1/2002 and B/Sydney/205/2012; Yamagata lineage , B/Victoria/341/2002 and B/Victoria/831/2012; influenza A H1N1 virus , A/Brisbane/59/2007 and A/Malaysia/11641/1997 and influenza A H3N2 virus , A/Perth/16/2009 and A/Moscow/10/1999 .", "Crystal structure templates used for computational modeling include PDB:4FQM ( Dreyfus et al . , 2012 ) ( influenza B virus ) , PDB:3UBE ( Xu et al . , 2012 ) ( H1N1 ) , and PDB:2YP4 ( Lin et al . , 2012 ) ( H3N2 ) .", "Differences in the receptor binding pocket region of the two influenza B lineages were visualized using B/Brisbane/60/2008 ( PDB:4FQM [Dreyfus et al . , 2012] ) and B/Florida/4/2006 ( PDB:4FQJ [Dreyfus et al . , 2012] ) with the addition of an α-2 , 6-linked host receptor analogue ligand from a known complex ( PDB:2RFU [Wang et al . , 2007] ) and targeted side-chain minimization of residues within 8 Å of the ligand through short simulated annealing molecular dynamic simulations in YASARA ( Krieger et al . , 2009 ) as previously benchmarked to ensure realistic results .", "We also used YASARA ( Krieger et al . , 2009 ) to visualize the role of glycosylation on Asn at position 212 for α-2 , 3- vs α-2 , 6-linked host receptor ligands by schematically superimposing both ligands ( PDB:2RFT [Wang et al . , 2007] and PDB:2RFU [Wang et al . , 2007] ) into their respective positions within the receptor binding pocket of a fully glycosylated influenza B HA head ( PDB:4FQM [Dreyfus et al . , 2012] ) ." ] ]

[ "A complex interplay of viral , host , and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses .", "Although considerable attention has been paid to influenza A viruses , a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses , despite their significant disease burden .", "Through the analysis of over 900 full genomes from an epidemiological collection of more than 26 , 000 strains from Australia and New Zealand , we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus ( Victoria and Yamagata ) , showing that their individual dynamics are determined by a complex relationship between virus transmission , age of infection , and receptor binding preference .", "In sum , this work identifies new factors that are important determinants of influenza B evolution and epidemiology ." ]

[ "To develop new therapies against infections caused by a virus , it is important to understand the virus's history—where , when , and why it has caused disease and how it has changed over time .", "For example , new human strains of the influenza type A virus originate from strains that infect animals and rapidly can become common in human populations .", "In contrast , influenza type B virus strains almost exclusively infect humans and are continuously present in human populations .", "Both types have a detrimental impact on global health , but the type B viruses are less well understood , partly because outbreaks have not been as extensively documented .", "Vijaykrishna et al . have now investigated the history of the two strains of the influenza type B virus—called Victoria and Yamagata—that currently circulate in humans .", "To do this , they inspected the genetic sequences of 908 viruses taken from samples of confirmed type B infections collected across Australia and New Zealand over 13 years .", "Individual virus particles of the same strain have genetic sequences that are very similar , but not completely identical .", "Vijaykrishna et al . showed that the diversity of the genetic sequences from the Victoria strain fluctuated between seasons , and particular genetic variants of Victoria only persisted in the population for 1–3 years .", "This indicates that Victoria viruses are under a lot of pressure to evolve , which results in so-called ‘bottlenecks’ whereby only the viruses carrying particular varieties of genetic sequence survive .", "This fluctuating pattern resembles that of the better-understood type A seasonal flu strain H3N2 .", "On the other hand , there was little change in the genetic diversity of the Yamagata strains sampled over the same 13-year period .", "The Yamagata viruses have diversified to a greater extent and several different ‘varieties’ of the virus tend to circulate together for long periods of time .", "For example , the three varieties of Yamagata virus circulating in 2013 evolved from a common parent virus that was circulating around 10 years ago .", "Vijaykrishna et al . found that between disease outbreaks , there was greater variation in the ability of Victoria viruses to be transmitted in humans , but that they were generally more easily transmitted than the Yamagata viruses .", "Victoria viruses tend to infect younger patients than Yamagata viruses , which is thought to be due to differences in the molecules that help the viruses enter the cells of the respiratory tract .", "These findings suggests that it might be possible to eradicate the more slowly evolving influenza B Yamagata virus through mass vaccination programs using existing vaccines .", "This would then allow researchers to focus on developing effective vaccines to target the other strains of influenza virus ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "The cerebellum is critical for coordinating movement and for learning sensorimotor relationships ( Ito , 2006 , 2008 ) .", "Sensory and motor-related afference to the cerebellum is largely conveyed by mossy fiber inputs to granule cells , which notably constitute over half the neurons of the mammalian brain ( De Schutter and Bjaalie , 2001; Herculano-Houzel and Lent , 2005; Watson et al . , 2012 ) .", "Granule cells distribute this sensory and motor information to the rest of cerebellar cortex through crystalline circuitry which has been well-characterized over the past 100 years ( Sotelo , 2008 , 2011 ) .", "However , the nature of the computations which granule cells perform on incoming afferents remains unresolved .", "The input structure to granule cells constrains their potential functions .", "Granule cells are simple neurons , having on average only four dendrites , each of which receives a single , large , excitatory mossy fiber input ( Gray , 1961; Eccles et al . , 1967; Palay and Chan-Palay , 1974; Jakab and Hamori , 1988 ) .", "Mossy fibers project widely in the cerebellar cortex from a disparate set of sensory and motor-related relay structures throughout the brainstem and spinal cord .", "Knowing whether multiple types of mossy fibers synapse onto the same granule cell is key to understanding the types of operations they perform ( Ekerot and Jorntell , 2008; Arenz et al . , 2009 ) .", "For example , a granule cell receiving all synaptic inputs from the same mossy fiber source ( i . e . , ‘unimodal’ ) may serve to filter noise by requiring the summation of inputs in order to fire action potentials ( Jorntell and Ekerot , 2006; Ekerot and Jorntell , 2008; Bengtsson and Jorntell , 2009 ) .", "A multimodal arrangement , in which an individual granule cell mixes inputs from different mossy fiber origins ( e . g . , one sensory and one motor-related precerebellar nucleus ) , enables more complex functions .", "Such mixing of mossy fiber inputs was a critical aspect of the expansion recoding of cerebellar afference performed by granule cells in the influential Marr-Albus theory of cerebellar function ( Marr , 1969; Blomfield and Marr , 1970; Albus , 1971 ) .", "If granule cells can indeed be multimodal , mapping these convergences across the cerebellum will be critical in uncovering their role in particular cerebellar functions .", "Controversy surrounds the multimodal capacity of granule cells ( Ekerot and Jorntell , 2008; Arenz et al . , 2009 ) .", "Electrophysiological evidence was found for multimodal granule cells in the electrosensory lobe of the mormyrid fish ( Sawtell , 2010 ) .", "Whether this granule cell feature extends to the cerebellum and to the mammal remained to be determined .", "Recently , mammalian granule cell input structure has been tested by in vivo receptive field mapping studies ( Jorntell and Ekerot , 2006; Arenz et al . , 2008; Bengtsson and Jorntell , 2009 ) .", "Such studies in the mammal have failed to positively identify more than one input source converging onto individual cerebellar granule cells ( Jorntell and Ekerot , 2006; Arenz et al . , 2008; Bengtsson and Jorntell , 2009 ) .", "This lack of evidence for multimodality has led to a diminution of models of granule cell function from a recoder to a noise filter ( Ekerot and Jorntell , 2008 ) .", "However , these electrophysiological studies did not systematically examine granule cells across cerebellar cortex , nor did they test the most numerous mossy fiber inputs , those originating from the basilar pontine nucleus ( BPN ) ( Brodal and Bjaalie , 1992 ) .", "The majority of BPN input originates from output messages of the cerebral cortex , superior colliculus , red nucleus , and other motor centers ( Burne et al . , 1981; Mihailoff et al . , 1989; Panto et al . , 1995; Schwarz and Thier , 1999; Leergaard et al . , 2000; Leergaard , 2003; Tziridis et al . , 2012 ) .", "Therefore BPN mossy fibers are in a position to carry copies of motor commands—corollary discharges—into the cerebellum ( Sperry , 1950; von Holst and Mittelstaedt , 1950; Poulet and Hedwig , 2007; Glickstein and Doron , 2008; Sommer and Wurtz , 2008 ) .", "In the central nervous system , corollary discharges are found in circuits involving the intersection of motor and sensory pathways ( Wolpert and Miall , 1996; Sommer and Wurtz , 2008 ) .", "While the granular layer receives both of these types of mossy fiber inputs , the controversy about the multimodal nature of individual granule cells raises questions about their capacity to mediate the intersection of pontine and sensory pathways at the cellular level .", "To test both the multimodal nature of granule cells and their specific role in merging pontine and sensory streams , we examined the intersection of the BPN pathway and a primary sensory precerebellar pathway related to motor output—forelimb and upper-trunk proprioceptive information projecting to the cerebellum through the external cuneate nucleus ( ECN ) of the hindbrain ( Campbell et al . , 1974; Akintunde and Eisenman , 1994; Quy et al . , 2011 ) .", "Combining genetics , viral tracing , and large scale confocal microscopy allowed us to take advantage of the unique mossy fiber/granule cell structure to generate synapse-resolution maps of ECN and BPN projections across the entire cerebellar cortex .", "We found that ECN and BPN inputs synapse onto the same granule cells , with a reproducible , region-specific , cerebellar topography .", "For a cerebellar area receiving upper body proprioceptive information , we show that the BPN input receives cortical afferents from an area associated with upper body motor control .", "Therefore , pontine and proprioceptive streams related to somatotopically similar motor output commands may integrate in multimodal granule cells of the cerebellum ." ], [ "To explore the intersection of sensory and pontine pathways , we used a combined genetic/viral strategy to trace the projection patterns of ECN and BPN inputs to the cerebellum .", "The genetic component of the strategy was used to distinguish the ECN and BPN from other nearby precerebellar sources , and the viral component was used to distinguish them from each other .", "First we searched for genes exhibiting regional selectivity for both the ECN and the BPN .", "Literature and Allen Institute Anatomic Gene Expression Atlas database searches produced candidates fitting this expression profile ( Hisano et al . , 2002; Ng et al . , 2009 , 2010 ) .", "The solute carrier family 17 ( sodium-dependent inorganic phosphate cotransporter ) , member 7 ( Slc17a7 ) gene was selected and a knock-in mouse , Slc17a7-IRES-Cre , expressing Cre under the control of this locus was generated .", "The Cre-dependent reporter expression in this mouse recapitulates the selective expression pattern of the Slc17a7 locus as reported in the Allen Institute Anatomic Gene Expression Atlas database ( Figure 1—figure supplement 1 ) .", "Taking advantage of selective Cre expression and the approximate 4-mm separation between the ECN and the BPN , we stereotaxically injected different Cre-dependent reporter viruses into each nucleus of the Slc17a7-IRES-Cre mice ( Figure 1A ) .", "This strategy resulted in selective and distinguishable labeling of the ECN and the BPN ( Figure 1B ) .", "Axons of ECN and BPN were intensely labeled ( Figure 1B , white arrowheads ) and could be identified at their terminations in the cerebellum ( Figure 1C , D ) . 10 . 7554/eLife . 00400 . 003Figure 1 . Termination patterns of ECN and BPN mossy fibers in the cerebellum .", "( A ) Genetic and viral scheme to specifically label ECN and BPN mossy fibers .", "Cre-dependent AAVs expressing tdTomato and EGFP are stereotaxically injected into ECN and BPN respectively in the Slc17a7-IRES-Cre mouse brain .", "CX , cortex; CB , cerebellum; DRG , dorsal root ganglia .", "( B ) Confocal images of viral injection sites ( ECN and BPN ) .", "White arrowheads , ECN and BPN axonal tracts; cst , corticospinal tract .", "D: dorsal; L: lateral; M: medial .", "Scale bars , 100 µm .", "( C ) Projection pattern of ECN ( red ) and BPN ( green ) mossy fibers in the cerebellum .", "Montage confocal images of the cerebellum from rostral to caudal ( Bregma −5 . 8 to −7 . 1 mm ) positions .", "Vermis ( II , III , IV/V , VI VIII , IX , X ) ; Copula of the pyramis ( Cop ) ; lateral vermis ( LV ) ; Paraflocculus ( PFl ) ; Paramedian lobule ( PM ) ; simple lobule ( Sim ) .", "Scale bar , 1 mm . ( D ) Magnified co-termination fields of ECN ( red ) and BPN ( green ) mossy fibers in selected cerebellar lobules .", "Boxed area shows high density of ECN and BPN mossy fiber terminations in the paramedian lobule .", "Scale bars , 100 µm; 10 µm in boxed area . DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 00310 . 7554/eLife . 00400 . 004Figure 1—figure supplement 1 . Expression pattern of Slc17a7-IRES-Cre mouse line .", "( A ) Targeting strategy for generating Slc17a7-IRES-Cre mice .", "Exons: blue boxes .", "HA1 and HA2: homology arms; FRT: flippase recognition target; PGKneo: neomycin resistance cassette; UTR: untranslated region .", "( B ) β-Galactosidase activity in four coronal levels ( low and high magnification ) of a Slc17a7-IRES-Cre; Rosa26-loxP-Stop-loxP-lacZ animal .", "Left scale bars , 1 mm; right scale bars , 250 µm .", "CX: cortex; CB: cerebellum; HC: hippocampus . DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 004 Distinguishable labeling of the ECN and the BPN allowed examination of the spatial relationship of these cerebellar afferent systems .", "As has been previously reported , at a gross level the ECN and BPN target mostly non-overlapping regions of the cerebellum ( Akintunde and Eisenman , 1994; Serapide et al . , 1994 , 2001 ) .", "ECN inputs primarily project to the ipsilateral cerebellar vermis and BPN inputs bilaterally terminate in the cerebellar hemispheres ( Figure 1C , D ) .", "However , strong viral-reporter expression and high-resolution , large-area confocal microscopy revealed many areas of the cerebellum where ECN and BPN axons co-terminate .", "Such areas include the vermis , lateral vermis , simple lobule , paraflocculus , Crus1 , Crus2 , paramedian lobule , and copula of the pyramis ( Figure 1D ) .", "Within these areas , ECN and BPN axons terminate within very close proximity ( <50 µm ) , well within the length of the dendritic arbors of granule cells ( Figure 1D , inset ) .", "Therefore , various cerebellar microcircuits potentially process both proprioceptive and pontine information .", "Co-termination within the spatial range of granule cell dendrites does not necessarily indicate the convergence of ECN and BPN inputs onto the same granule cell .", "A mossy fiber terminal synapses with granule cell dendrites within a glomerulus ( Ekerot and Jorntell , 2008; Arenz et al . , 2009 ) .", "Inside a glomerulus , the claw-footed dendrites of granule cells wrap around and make multiple synaptic contacts with the single , large , mossy fiber termination .", "This synaptic arrangement allows us to determine , with light microscopy , whether ECN and BPN mossy fibers converge onto single granule cells .", "Anatomical assessment of convergence requires simultaneous and separate labeling of granule cell morphology and of the two types of mossy fibers .", "The genetic/viral approach described in Figure 1A accomplishes the labeling of the ECN and BPN mossy fibers .", "However , cell density and the spatial expanse of the cerebellum precluded a similar approach for revealing the morphology of individual granule cells throughout the cerebellum .", "Alternatively , we screened enhancer-trap mouse lines for an expression pattern where granule cells are labeled in a sparse , ‘Golgi stain-like’ fashion .", "This screen identified one mouse line , TCGO , which expresses tetracycline transactivator and mCitrine in a subset of granule cells .", "While the density of labeled cells is consistent across TCGO animals , we lack evidence to distinguish if the labeling is stochastic or marks a particular class of granule cells .", "Strong expression of the mCitrine reporter , a low density of marked cells , and labeling spanning all cerebellar cortical regions allowed manual segmentation of the dendritic morphology of neighboring granule cells over the entire expanse of the cerebellum ( Figure 2A–C ) .", "Simultaneous labeling of the two types of mossy fibers and the granule cells was accomplished by injecting Cre-dependent viruses into both the ipsilateral ECN and the contralateral BPN of Slc17a7-IRES-Cre; TCGO bitransgenic animals ( Figure 2D ) . 10 . 7554/eLife . 00400 . 005Figure 2 . Convergence of ECN and BPN mossy fibers on cerebellar granule cells .", "( A ) TCGO transgene expression in a representative section of cerebellum .", "Scale bar , 1 mm . ( B ) TCGO mCitrine expression in boxed area of ( A ) , simple lobule .", "Scale bar , 100 µm .", "( C ) Maximum projection of labeled granule cells in TCGO mice ( white arrowhead: dendritic arborization ) in boxed areas of ( B ) .", "Scale bar , 5 µm .", "( D ) Co-termination of ECN ( red ) and BPN ( green ) mossy fibers in paramedian lobule of a Slc17a7-IRES-Cre; TCGO mouse .", "Scale bar , 10 µm .", "( E ) Maximum projection of a labeled granule cell that receives mossy fiber inputs from ECN ( red arrowhead ) and BPN ( green arrowhead ) in a Slc17a7-IRES-Cre; TCGO mouse .", "Scale bar , 5 µm .", "( F ) – ( H ) 3D reconstruction of granule cells with associated mossy fiber terminations .", "E granule cell ( GrC ) , granule cell with one ECN input and one other traceable dendrite; EB GrC , granule cell with ECN and BPN input ( s ) ; E+ GrC , granule cell with two or more ECN inputs but no BPN input .", "Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 005 To test the convergence of sensory and pontine pathways , we first identified proprioceptive granule cells , defined as those with at least one input originating from the ECN .", "Once classified , we next tested if these proprioceptive granule cells also synapse with BPN mossy fibers .", "In areas of ECN and BPN co-termination , the dendritic morphology of each proprioceptive granule cell was traced and the identity of any unambiguous synaptic input was cataloged .", "Based on results from one annotator , 40% of proprioceptive granule cells ( 2429/5997 in two animals ) made obvious synapses ( see ‘Materials and methods’ ) with both ECN and BPN inputs ( Figure 2E ) .", "Therefore , subsets of ECN and BPN information streams converge at their first opportunity in the cerebellum—onto granule cells .", "Because of the known functional specialization of cerebellar subregions ( Chambers and Sprague , 1955a , 1955b; Apps and Garwicz , 2005; Cerminara and Apps , 2011 ) , we surveyed where ECN-BPN convergence occurs , and does not occur , throughout the cerebellum .", "We were able to generate such maps due to the cerebellum-wide expression of the TCGO transgene and the ability to image large expanses of cerebellar neuropil .", "For each cerebellar region receiving input from the ECN ( Figures 3–5 ) , two locations along the anterior-posterior axis were analyzed .", "Each proprioceptive granule cell was classified into one of three classes ( Figure 2F–H ) : those receiving one ECN input only ( E ) , those receiving at least one ECN and at least one BPN input ( EB ) , and those receiving more than one ECN input but no BPN inputs ( E+ ) .", "Granule cells without ECN inputs but synapsing with one or multiple BPN axons ( B , B+ , respectively ) were only analyzed for select sections due to the abundance of such granule cell types .", "Both mouse brains were examined by two annotators; the pattern and percentage of granule cell types largely agreed across animals and annotators ( Figures 3–5 ) . 10 . 7554/eLife . 00400 . 006Figure 3 . Cerebellar areas not exhibiting convergence of sensory and pontine inputs . Survey of ECN and BPN convergence in the anterior vermis .", "( A ) Vermis III .", "( B ) Vermis IV/V .", "( i ) Granule cell ( GrC ) classification and distribution .", "Red cross , E GrC; green cross , EB GrC; blue cross , E+ GrC .", "Scale bars , 100 µm .", "( ii ) density contour map of E , EB and E+ granule cells .", "D: dorsal; V: ventral; M: medial; L: lateral .", "Red , green and blue lines in the contour map represent density of E , EB , and E+ granule cells respectively .", "( iii ) upper , percentage of E , EB and E+ granule cells of two Slc17a7-IRES-Cre; TCGO cerebella .", "Lower , comparison between annotators in percentage of E , EB and E+ granule cells in a selected section .", "( C ) Pontocentric view of vermis III .", "( D ) Pontocentric view of vermis IV/V .", "( C and D )", "Same organization as in ( A and B ) but B replaces E and B+ replaces E+ granule cells .", "B GrC: granule cell with one BPN input and one other traceable dendrite; B+ GrC: granule cell with two or more BPN inputs but no ECN input .", "EB GrC is the same as in ( A and B ) DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 00610 . 7554/eLife . 00400 . 007Figure 4 . Cerebellar areas exhibiting mixtures of convergence and separation of sensory and pontine inputs . Survey of ECN and BPN convergence in the lateral vermis , simple lobule , copula of the pyramis and Crus1 .", "( A ) Lateral vermis , anterior section .", "( B ) Lateral vermis , posterior section .", "( C ) Simple lobule , anterior section .", "( D ) Simple lobule , posterior section .", "( E ) Copula of the pyramis , anterior section .", "( F ) Copula of the pyramis , posterior section .", "( G ) Crus1 , anterior section .", "( H ) Crus1 , posterior section .", "( i ) Granule cell ( GrC ) classification and distribution .", "Red cross: E GrC; green cross: EB GrC; blue cross: E+ GrC .", "Scale bars , 100 µm .", "( ii ) Density contour map of E , EB and E+ granule cells .", "D: dorsal; L: lateral .", "Red , green and blue lines in the contour map represent density of E , EB , and E+ granule cells , respectively .", "( iii ) Upper , percentage of E , EB and E+ granule cells of two Slc17a7-IRES-Cre; TCGO cerebella .", "Lower , comparison between annotators in percentage of E , EB and E+ granule cells . DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 00710 . 7554/eLife . 00400 . 008Figure 5 . Cerebellar areas exhibiting abundant convergence of sensory and pontine inputs . Survey of ECN and BPN convergence in the hemispheric regions .", "( A ) Paramedian lobule , anterior section .", "( B ) Paramedian lobule , posterior section .", "( C ) Paraflocculus , anterior section .", "( D ) Paraflocculus , posterior section .", "( E ) Crus2 , anterior section .", "( F ) Crus2 , posterior section .", "( i ) Granule cell ( GrC ) classification and distribution .", "Red cross: E GrC; green cross: EB GrC; blue cross: E+ GrC .", "Scale bars , 100 µm .", "( ii ) Density contour map of E , EB and E+ granule cells .", "D: dorsal; L: lateral .", "Red , green and blue lines in the contour map represent density of E , EB , and E+ granule cells respectively .", "( iii ) Upper , percentage of E , EB and E+ granule cells from two Slc17a7-IRES-Cre; TCGO cerebella .", "Lower , comparison between annotators in percentage of E , EB and E+ granule cells in a selected section .", "( E and F ) do not have comparisons across the two cerebella in", "( iii ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 008 We observed three types of granule cell terrain across cerebellar areas .", "Some cerebellar areas , such as medial vermis , were composed mostly of E and E+ granule cell ( Figure 3A , B ) .", "In some cases the lack of EB cells was due to the scarcity of BPN inputs to the region ( compare Figure 3A , C ) , but in other regions the number of granule cells receiving BPN inputs ( B , B+ ) was roughly equivalent to the number receiving ECN inputs ( compare Figure 3Bcb1 , Figure 3Dcb1 ) .", "Therefore , vermal areas harbor proprioceptive pathways which are segregated from BPN inputs at the cellular level; this segregation would not be evident with traditional neuro-anatomical techniques .", "Other areas intermingle all three granule cell types , this category includes lateral vermis , simple lobule , copula of the pyramis and Crus1 ( Figure 4 ) .", "The densities of granule cell type varied in these areas along the dorsal-ventral , and medial-lateral axes .", "Thus , these areas deliver multiple types of information to the overlying Purkinje cells and the combinations of these types can vary locally .", "Lastly , some cerebellar areas are dominated by EB granule cells .", "These areas include paramedian lobule , the paraflocculus , and Crus2 ( Figure 5 ) .", "These areas are ‘hotspots’ for the intersection of ECN and BPN pathways , where the majority of proprioceptive granule cells also receive BPN inputs .", "Consequently , many parallel fiber inputs to Purkinje cells in these areas carry both ECN and BPN information .", "Differences in the extent and patterns of ECN and BPN convergence suggest that the cerebellum handles proprioceptive information with regional specificity .", "ECN inputs carry forelimb and upper body proprioceptive information to cerebellar granule cells ( Campbell et al . , 1974; Akintunde and Eisenman , 1994; Quy et al . , 2011 ) .", "We tested if the BPN inputs that converge with ECN signals are capable of delivering corollary discharges relating to similar regions of the body .", "To demonstrate the cortical nature of pontine inputs , we combined anterograde labeling from forelimb/upper body primary motor cortex ( M1 ) and retrograde labeling of BPN neurons ( Figure 6A ) .", "We focused on the paramedian lobule as the projection target since nearly every paramedian , proprioceptive granule cell receives BPN inputs .", "For retrograde labeling , we employed a mCherry-expressing ASLV-A envelope glycoprotein ( EnvA ) pseudotyped , glycoprotein-deleted rabies virus ( SADΔG-mCherry ( EnvA ) ) , whose tropism is restricted to avian tumor virus receptor A ( TVA ) -expressing cells ( Wickersham et al . , 2007; Wall et al . , 2010 ) .", "Cre-dependent viral expression of TVA in the BPN in Slc17a7-IRES-Cre mice selectively sensitizes BPN neurons to SADΔG-mCherry ( EnvA ) infection .", "To selectively label paramedian-projecting BPN neurons , we forced SADΔG-mCherry ( EnvA ) rabies infection to originate from BPN distal axons by delivering the rabies virus to the paramedian lobule .", "To anterogradely label M1 , we stereotaxically injected Cre-dependent EGFP-expressing viruses into a region of M1 known to contain neurons which drive forelimb/upper body movement ( Ayling et al . , 2009; Harrison et al . , 2012 ) .", "EGFP-labeled neurons were restricted to lateral agranular cortex , consistent with M1 identity ( Figure 6B ) ( Tennant et al . , 2011 ) .", "We then examined if motor cortical axons synapse with paramedian-projecting BPN neurons .", "The majority of pontine inputs to the paramedian lobule originate from the medial-ventral BPN ( Figure 6C , upper ) .", "Retrogradely-labeled BPN neurons are situated in dense fields of M1 axons ( Figure 6C , lower; Figure 6D ) .", "Cortical axons containing presynaptic vesicle proteins ( Bellocchio et al . , 1998 ) were in close proximity to retrogradely labeled pontine neurons; these M1 terminals were also associated with post-synaptic densities of BPN neurons ( Naisbitt et al . , 1999 ) ( Figure 6E ) .", "Putative M1 synaptic inputs were identified on rabies-labeled BPN neurons ( minimum 10 synaptic partners analyzed per mouse ) in 3/3 animals .", "We did not attempt to quantify the percentage of retrogradely labeled neurons receiving M1 inputs due to the high false negative rate originating from the intentionally incomplete labeling of cortical axons and the inefficiencies of immunostaining synaptic proteins .", "Taken together , these findings suggest synaptic arrangements between forelimb/upper body motor cortex and paramedian-projecting pontine neurons .", "Therefore , forelimb/upper body pathways separately carrying corollary discharges and proprioceptive information are aligned in cerebellar cortex . 10 . 7554/eLife . 00400 . 009Figure 6 . Cortical inputs to paramedian-projecting BPN neurons . Combining M1 anterograde tracing and paramedian lobule retrograde tracing .", "( A ) Scheme to anterogradely label forelimb/upper body M1 cortical axons and retrogradely label paramedian-projecting BPN neurons ( BPNPM ) .", "Cre-dependent AAVs expressing EGFP and mTagBFP2-2A-TVA were stereotaxically injected into M1 and BPN respectively in the Slc17a7-IRES-Cre mouse and SADΔG-mCherry ( EnvA ) rabies virus was injected into the paramedian lobule of the cerebellum .", "CB , cerebellum; M1 , primary motor cortex .", "( B ) Location of EGFP-expressing neurons with relation to cortical cytoarchitecture .", "cc: corpus callosum .", "D: dorsal; M: medial .", "Scale bar , 1 mm . ( C ) Upper , relationship of BPNPM and TVA-expressing BPN neurons ( BPNTVA ) sensitive to rabies infection .", "Lower , colocalization of M1 axons and BPNPM neurons .", "D: dorsal; M: medial .", "Scale bars , 500 µm; 50 µm in magnified areas .", "( D ) High-magnification image of BPNPM neurons and the M1 axon termination field .", "Scale bar , 10 µm .", "( E ) Synaptic arrangement between M1 axons and BPNPM .", "Left , apposition of a M1 axon expressing the presynaptic marker SLC17A7 and a dendrite of a BPNPM in a single confocal slice .", "Right , apposition of a M1 axon and a SHANK1-containing postsynaptic density of a BPNPM in a single confocal slice .", "Scale bars , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00400 . 009" ], [ "Since the number of granule cells far exceeds the number of their mossy fiber inputs , granule cells are in a position to potentially permute combinations of afferent inputs in the cerebellum ( Marr , 1969; Blomfield and Marr , 1970; Albus , 1971 ) .", "David Marr and James Albus proposed that by mixing mossy fiber inputs , granule cells could perform such ‘expansion recoding’ , enabling them to contribute to pattern separation and consequently to be fundamental for motor learning ( Marr , 1969; Blomfield and Marr , 1970; Albus , 1971 ) .", "In this model , the associative capacity of granule cells is maximized if different modalities of mossy fibers are mixed onto individual granule cells .", "To test potential mixing of granule cell inputs , several groups performed receptive field mapping studies .", "In the C3 region of the anterior paravermis of the cerebellum , failure to find evidence for mixing led Jorntell and Ekerot to disfavor the multimodal view and relegate granule cell function from expansion recoding to noise reduction ( Jorntell and Ekerot , 2006; Ekerot and Jorntell , 2008 ) .", "Such reassignment of function implies a marked reduction in the pattern discrimination capability of the cerebellum .", "In the cerebellar flocculus , Arenz et al . ( 2008 , 2009 ) provided indirect evidence for multimodal inputs to three granule cells .", "For these cells , some inputs were modulated by horizontal movement of the animal while others did not show this influence .", "However , whether the non-modulated inputs originated from a different mossy fiber source was not tested in these experiments , stopping short of demonstrating multimodality in granule cells .", "Relevant to our study , both of these electrophysiological mapping experiments failed to test the convergence of pontine and sensory pathways because they were performed in animals where the cortico-ponto-cerebellar pathway was either interrupted or suppressed .", "We avoided difficulties in electrophysiologically assigning input identities by instead investigating granule cell presynaptic partners using a combination of mouse genetics , viral tracing , and anatomy .", "This analysis has yielded the first direct evidence that a large number of mammalian cerebellar granule cells receive inputs from both pontine and primary sensory sources .", "Our results also provide a map of where this variety of multimodal convergence occurs and does not occur over the expanse of the cerebellar cortex .", "Multimodal claw-footed granule cells have also been observed in the electrosensory lobe of the mormyrid fish , suggesting multimodality is a feature of this morphological cell type that is conserved across vertebrate species and across brain regions ( Sawtell , 2010 ) .", "The observation that our labeling strategy rarely accounted for all presynaptic partners of an individual granule cell suggests that multimodality is likely a more prevalent feature of cerebellar granule cells than indicated by the convergence of the ECN and BPN pathways reported here .", "By examining other precerebellar sources , systematic continuation of our mapping strategy will more fully establish the input structure and thus the associative potential of the cerebellum .", "The associative capacity of the cerebellum is thought to largely depend on Purkinje cell plasticity , allowing temporal relationships among inputs to alter the strengths of postsynaptic responses ( Marr , 1969; Albus , 1971; Boyden et al . , 2004; Gao et al . , 2012 ) .", "Unimodal granule cells supply segregated input channels to Purkinje cells , allowing the molecular layer to independently adjust the weights of each modality .", "In addition to this independent modality control , the present finding of multimodal granule cells suggests that some parallel fiber inputs already represent information types .", "Prefabricating simple associations in granule cells may enable Purkinje cells to perform more complex learning operations , analogous to the enhanced learning capacity of a multilayer perceptron over a single layer perceptron ( Minsky and Papert , 1969 ) .", "In order to perform skilled behaviors , motor control systems require knowledge of the environment , the position of the body , and how it moves .", "By signaling muscle stretch and tendon tension , proprioception is particularly well suited to provide input to the brain's sensory model of body position and kinematics ( Bosco and Poppele , 2001; Dietz , 2002 ) .", "Proprioceptive and other sensory afferents deliver accurate post-hoc reports of movements , but many behaviors require predicting the likely consequences of a motor plan prior to , or without , movement ( Wolpert and Miall , 1996; Bastian , 2006; Shadmehr et al . , 2010 ) .", "Forward models have been proposed to use information about intended movements to predict the likely consequences of a voluntary action ( Robinson , 1975; Jordan and Rumelhart , 1992; Wolpert and Miall , 1996 ) .", "Forward models require two inputs: sensory inputs ( peripheral afference ) that update the state of the model and corollary discharge signals related to intended actions .", "To predict sensory consequences , corollary discharges are thought to mimic self-generated ( reafferent ) sensory information .", "To accomplish this mimicry , corollary discharges must be converted from motor to sensory coordinates , but where and how this occurs in the nervous system is largely unknown .", "Theory and perturbation studies suggest somatic forward models reside in the cerebellum ( Miall et al . , 1993 , 2007; Miall , 1998; Wolpert et al . , 1998; Blakemore et al . , 2000; Pasalar et al . , 2006; Ebner and Pasalar , 2008; Ito , 2008; Lesage et al . , 2012 ) .", "We show that proprioceptive and corollary discharge pathways converge on individual granule cells .", "Granule cells can generate action potentials in response to a single mossy fiber input ( Rancz et al . , 2007 ) .", "Therefore , inputs of different modalities can potentially substitute for one another to fire a granule cell .", "This interchangeability suggests a plausible mechanism for converting motor corollary discharges into sensory coordinates , as outlined below .", "A motor command is initiated in forelimb/upper body M1 and delivered to motor output centers and the BPN by corticofugal axons .", "Since these BPN neurons will be driven by cortical motor commands that do not directly participate in movement generation , by definition they can be considered to carry corollary discharges .", "Resulting BPN output is next sent to the cerebellum to synapse with multimodal granule cells that also receive self- and externally-generated forelimb/upper body proprioceptive inputs from the ECN .", "Pontine corollary discharges will stimulate these granule cells , generate parallel fiber output to Purkinje cells , and may be processed similar to proprioceptive signals that synapse on the same granule cells .", "Although speculative , hijacking this pathway could effectively convert the corollary message from motor to sensory coordinates , and thus in the appropriate reference frame to produce proprioceptive predictions .", "Corollary discharge-driven predictions will contain some degree of error , which is thought to be computed by comparing pure sensory models against forward models ( Mazzoni and Krakauer , 2006; Shadmehr et al . , 2010 ) .", "As described above , areas containing granule cells where corollary and sensory information converge might represent components of forward models .", "Other areas where proprioceptive streams are isolated from corollary/pontine information may represent what actually occurred and thus may drive pure sensory models .", "Common downstream targets of these identified forward and sensory models may represent loci where intended and actual actions are compared and thus where error signals are generated .", "The proprioceptive pathway appears to be intersected by corollary discharges at multiple stages .", "Previously , corollary and sensory information has been shown to converge in the first stage of proprioceptive processing—in the precerebellar neurons of the spinal cord ( Hongo and Okada , 1967; Hongo et al . , 1967; Hantman and Jessell , 2010 ) .", "The present findings indicate that one synapse further up the proprioceptive pathway—at cerebellar granule cells—this convergence occurs again .", "Neurons of each of these stages are distinguished by their inputs and intrinsic properties .", "Therefore , motor inputs at each level will produce unique transformations of the sensory streams and thus potentially embody nodes within a hierarchical motor-informed sensory processing system .", "Our findings confirm a key prediction made nearly a half a century ago by David Marr and James Albus about the associative faculty of the most abundant neuron type in the mammalian brain .", "The multimodal capacity of cerebellar granule cells calls for a systematic investigation of the possible mixtures of mossy fiber inputs throughout the cerebellum .", "Finally , by defining the areas of the cerebellum where corollary and sensory information potentially converge , we have uncovered a rich new system to understand the logic of corollary discharges in motor control and perhaps other brain functions ." ], [ "Slc17a7-IRES-Cre mice were generated by the Janelia Farm-Gene Targeting and Transgenics Facility .", "The targeting vector contains a positive selection cassette , an IRES-Cre cassette and two arms homologous to exon 10 to 12 and the 3′-untranslated region of Slc17a7 ( Figure 1—figure supplement 1 ) .", "Embryonic stem cells that correctly recombined with the targeting vector were injected into blastocysts , resulting chimeras were screened for germline transmission , and the positive selection cassette was removed by breeding F1 progeny with a codon-optimized FLP recombinase ( FLPo ) germline deleter strain ( The Jackson Laboratory , Bar Habor , ME ) .", "TCGO transgenic mice were generated at Brandies University ( Shima et al . , under revision ) by random insertion of enhancer-trap lenti-viral vectors through mouse zygote infection ( Kelsch et al . , 2012 ) .", "Both mouse lines were backcrossed to the C57/B6 background .", "Rosa26-loxP-Stop-loxP-lacZ mice were obtained from The Jackson Laboratory .", "Adult mice ( 2–6 months old ) were anesthetized with 2% isoflurane/95% oxygen mixture ( VetEquip , Pleasanton , CA ) and placed in a stereotaxic apparatus ( David Kopf Instruments , Tujunga , CA ) .", "Following a scalp incision , small holes were drilled into the skull and the dura was exposed .", "Coordinates for the BPN were 4 . 0 mm posterior to Bregma , 0 . 4 mm lateral to the midline and 5 . 8/5 . 5/5 . 2/5 . 0 mm deep from dura; coordinates for the ECN were 7 . 2–7 . 4 mm posterior to Bregma , 1 . 3 mm lateral to the midline and 3 . 0/2 . 8 mm deep from dura; coordinates for M1 were 0 . 7 mm anterior to Bregma , 1 . 7 mm lateral to the midline and 0 . 75 mm deep from dura .", "A pulled-glass pipette ( 20 µm tip diameter ) driven by a micromanipulator ( Scientifica , Uckfield , United Kingdom ) was inserted into the target area and one to four injections ( 50 nl per injection site ) were made using a Nanoliter 2000 injector ( World Precision Instruments , Sarasota , FL ) .", "For each penetration , a 2-min waiting period was imposed between sites and the pipette was slowly withdrawn 5 min after the final injection .", "After the surgery , the scalp was sutured , betadine was applied for antiseptic purposes , and ketoprofen ( 5 mg/kg ) analgesic was provided subcutaneously .", "Mice were housed for 21–28 postoperative days in order to achieve optimal viral reporter expression and then were perfused for histology .", "AAV2/1 CAG-FLEX-EGFP-WPRE-bGH ( 1 × 1013 particles per ml ) and AAV2/1 CAG-FLEX-tdTomato-WPRE-bGH ( 2 × 1013 particles per ml ) were produced by The Gene Therapy Program at the University of Pennsylvania ( Philadelphia , PA ) .", "Tag-blue fluorescent protein ( mTagBFP2; Evrogen , Moscow , Russia ) and the avian virus receptor , TVA , were subcloned into a CAG-FLEX-2A viral vector .", "AAV2/1 CAG-FLEX-mTagBFP2-2A-TVA ( 9 × 1012 particles per ml ) was made at the Janelia Farm-Molecular Biology Shared Resource and purified through cesium-chloride density gradients .", "Pseudotyped SADΔG-mCherry ( EnvA ) rabies virus was produced as previously described ( Wickersham et al . , 2007 , 2010 ) and were acquired from the Systems Neurobiology Laboratories ( E . Callaway ) at The Salk Institute for Biological Studies ( San Diego , CA ) .", "Conditional expression of TVA in the BPN was achieved through stereotaxic injection of AAV2/1 CAG-FLEX-mTagBFP2-2A-TVA in Slc17a7-IRES-Cre mice .", "Pseudotyped SADΔG-mCherry ( EnvA ) rabies virus was injected into the paramedian lobule 2 weeks after the initial AAV injection .", "The paramedian lobule ( 7 . 0–7 . 4 mm posterior to Bregma , 2 . 3 mm lateral to the midline and 1 . 8/1 . 5 mm deep from dura ) was chosen based on the convergence pattern observed from our anterograde mapping results .", "Animals were housed in a BSL-2 room for seven postoperative days and then perfused for histology .", "Mice were deeply anesthetized with isoflurane and perfused with phosphate buffered saline ( PBS ) followed by 4% paraformaldehyde ( PFA ) in PBS .", "Brains were dissected and post-fixed in 4% PFA for 4 hr .", "Tissues were transferred to 30% sucrose in PBS for 48 hr and then embedded in Tissue-Tek OCT compound ( Sakura Finetek , Torrance , CA ) .", "Brain sections ( 50 µm ) were made using a cryostat ( Leica ) and mounted on glass slides in glycerol/PBS mix .", "Immunohistochemistry on cryostat sections was performed by sequential exposure to primary antibodies: chick anti-GFP ( Abcam , Cambridge , MA ) , guinea pig anti-SLC17A7 ( gift from the Jessell lab ) , rabbit anti-SHANK1 ( Synaptic Systems , GmbH , Goettingen , Germany ) , and fluorophore-conjugated secondary antibodies ( Jackson Immunoresearch , Laboratories , West Grove , PA and Invitrogen , Carlsbad , CA ) .", "NeuroTrace ( Invitrogen ) and 4′ , 6-diamidino-2-phenylindole ( DAPI ) ( Vector Labs , Burlingame , CA ) were used to achieve Nissl and nuclear staining .", "Standard X-Gal staining protocols were used to assess β-galactosidase activity .", "Cerebellar areas receiving ECN inputs were analyzed in two Slc17a7-IRES-Cre; TCGO bitransgenic animals .", "For each area , two positions were selected along the anterior-posterior axis .", "Crus2 was damaged in the second animal and was not included in this analysis .", "Confocal stacks were acquired by a Zeiss LSM 710 confocal microscope using 10× ( 0 . 45 N . A . ) air , 20× ( 0 . 8 N . A . ) air , 40× ( 1 . 3 N . A . ) oil , 63× ( 1 . 4 N . A . ) oil , and 100× ( 1 . 4 N . A . ) oil objectives .", "Section boundaries were selected and stitched using the MultiTime ( version 25 ) macro plug-in for Zen 2010 ( Carl Zeiss Microimaging , Thornwood , NY ) .", "Images were acquired using a 405-nm diode laser line for mTagBFP2 , DAPI , and NeuroTrace ( filter setting , 391–453 nm ) ; a 488-nm argon laser line for EGFP ( 488–514 nm ) ; a 514-nm argon laser line for mCitrine ( 524–563 nm ) ; a 561-nm diode-pumped solid-state laser for tdTomato ( 584–691 nm ) ; a 594-nm helium-neon laser for mCherry ( 589–696 nm ) ; and a 633-nm helium-neon laser for Alexa647 ( 638–755 nm ) .", "Tiled image stacks were analyzed by three annotators ( two for each animal ) with the Zen program .", "Annotators identified mCitrine-positive , claw-footed structures completely embedded within a tdTomato-positive ECN rosette and manually traced back to the granule cell somata .", "From the soma , each of the other dendrites was manually traced to its termination and associated mossy fiber synapses were identified .", "Granule cells lacking another traceable dendrite were excluded from further analysis .", "Granule cells with at least one tdTomato ECN input and at least one other identifiable dendritic claw foot without a labeled input were marked with red crosses ( E granule cell ) .", "Granule cells with more than two ECN inputs were labeled with blue crosses ( E+ granule cells ) .", "Granule cells with at least one ECN and at least one EGFP-positive rosette ( BPN input ) were annotated with green crosses ( EB granule cells ) .", "For anterior vermal areas , annotators also identified mCitrine-positive dendritic arborizations embedded within an EGFP-positive BPN rosette and traced back to the granule cell soma .", "All other dendrites were traced from the soma and associated mossy fiber synapses were identified .", "In these select vermal areas , granule cells with one EGFP BPN input and at least one other identifiable dendritic claw foot without a labeled input were marked with red crosses ( B granule cells ) .", "Granule cells with more than two BPN inputs were labeled with blue crosses ( B+ granule cells ) .", "Graphs showing percentage of granule cell types between animals and annotators were produced using Excel ( Microsoft , Redmond , WA ) .", "X-Y coordinates of annotation markers were exported from Zen and density contour maps were made using custom Python ( Enthought , Austin , TX ) scripts .", "3D reconstruction of granule cells and associated mossy fibers were made using Fiji-TrakEM2 ." ] ]

[ "Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells .", "The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved .", "Using cell-type-specific projection mapping with synaptic resolution , we observed the convergence of separate sensory ( upper body proprioceptive ) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex .", "These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function .", "We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas .", "Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control ." ]

[ "Learning a new motor skill , from riding a bicycle to eating with chopsticks , involves the cerebellum—a structure located at the base of the brain underneath the cerebral hemispheres .", "Although its name translates as ‘little brain' in Latin , the cerebellum contains more neurons than all other regions of the mammalian brain combined . Most cerebellar neurons are granule cells which , although numerous , are simple neurons with an average of only four excitatory inputs , from axons called mossy fibers . These inputs are diverse in nature , originating from virtually every sensory system and from command centers at multiple levels of the motor hierarchy . However , it is unclear whether individual granule cells receive inputs from only a single sensory source or can instead mix modalities . This distinction has important implications for the functional capabilities of the cerebellum . Now , Huang et al . have addressed this question by mapping , at extremely high resolution , the projections of two pathways onto individual granule cells—one carrying sensory feedback from the upper body ( the proprioceptive stream ) , and another carrying motor-related information ( the pontine stream ) . Using a combination of genetic and viral techniques to label the pathways , Huang and co-workers identified regions where the two types of fiber terminated in close proximity . They then showed that around 40% of proprioceptive granule cells formed junctions , or synapses , with two ( or more ) fibers carrying different types of input . These cells were not uniformly distributed throughout the cerebellum but tended to occur in ‘hotspots’ .", "Lastly , Huang et al . examined the type of information conveyed by the sensory and motor-related input streams whenever they contacted a single granule cell .", "They confirmed that when the sensory input consisted of feedback from the upper body , the motor input consisted of copies of motor commands related to the same body region .", "Because it is thought that the cerebellum converts sensory information into representations of the body's movements , directing motor commands to these same circuits may allow the cerebellum to predict the consequences of a planned movement prior to , or without , the actual movement occurring .", "The work of Huang et al . provides evidence to support the previously controversial idea that granule cells in the mammalian cerebellum receive both sensory and motor-related inputs .", "The labeling technique that they used could also be deployed to study the inputs to the cerebellum in greater detail , which should yield new insights into the functioning of this part of the brain ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cell biology" ]

[ [ "The conserved PCP signaling pathway regulates the orientation of cells and organelles within the plane of an epithelium and is crucially important for developmental patterning as well as organ morphogenesis , homeostasis , and physiology ( Seifert and Mlodzik , 2007; Wang and Nathans , 2007; Peng and Axelrod , 2012; Wallingford , 2012 ) .", "Pioneering studies in Drosophila and Xenopus have revealed that global , non-cell autonomous , and cell intrinsic signaling mechanisms act in concert to establish tissue polarity .", "Core PCP molecules including Van Gogh-like ( Vangl1-2 ) , Cadherin EGF LAG seven-pass G-type receptor ( Celsr1-3 ) , Frizzled ( Fzd3 , 6 ) , Dishevelled ( Dvl1-3 ) , and Prickle ( Pk1-2 ) are localized asymmetrically at the cell cortex to provide polarity information for morphogenesis and oriented cell division .", "Significant progress has been made in understanding the asymmetric core PCP localization in vertebrates but it is less clear how this regulates cytoskeletal rearrangements that drive morphogenesis via tissue specific downstream effector molecules ( Wallingford , 2012 ) .", "Thus , the identification of novel PCP effectors that indicate pathway activity and mediate signaling and/or morphogenesis will be the key to unravel the function of this molecular pathway in development and disease .", "Besides the Rho family of GTPases , which are also implicated in apical–basal ( A–B ) polarity establishment , the best-studied PCP effector molecules are Inturned ( Intu ) and Fuzzy ( Fuz ) ( Collier and Gubb , 1997; Park et al . , 2006 , 2008; Gray et al . , 2009 ) .", "Both directly regulate ciliogenesis by mediating the assembly of the apical actin cytoskeleton but are not required for the polarized accumulation of core PCP components .", "The core PCP molecule Dvl2 localizes near the base of cilia and functions together with Intu and Rho GTPases to dock and polarize BBs for cilia formation and directed ciliary beating ( Park et al . , 2008 ) .", "BBs are amplified deep in the cytoplasm of multiciliated cells ( MCCs ) and apical plasma membrane ( PM ) transport depends on Dvl and the vesicle trafficking protein Sec8 .", "Up-to-date it is not understood how core PCP molecules physically connect to effector molecules , how this leads to asymmetric membrane polarization and cytoskeletal rearrangements , and if these mechanisms are conserved among different cell types in various organs and during evolution .", "First functional evidence for PCP in lung development came from the analysis of Celsr1 , Vangl2 , and Scribble ( Scrib ) mutant mice , which showed defects in branching morphogenesis and narrowed lung airways due to cytoskeletal and junctional defects ( Yates et al . , 2010 ) .", "Multiciliated lung cells first arise at embryonic day ( E ) 14 . 0 in the trachea as well as in the main bronchi ( Jain et al . , 2010 ) .", "Similar to the mucociliary epithelium in frog , differentiation depends on BB amplification , docking , and orientation that allows the formation of hundreds of motile cilia .", "The differentiation of multiciliated lung cells and the dynamics of the underlying cell biological processes can be modeled in air liquid interface ( ALI ) cultures of primary mouse tracheal epithelial cells ( mTECs ) ( You et al . , 2002; Vladar and Stearns , 2007; Vladar et al . , 2012 ) .", "Asymmetric localization of core PCP molecules at apical junctions regulates the orientation of motile cilia along the longitudinal tissue axis for directed beating and mucus clearing .", "This likely interdepends on non-cell autonomous cues and intrinsic polarized microtubule ( MT ) network topology ( Vladar et al . , 2012 ) .", "Currently , PCP effector molecules that link core molecules , BBs , polarized MTs , and the actin cytoskeleton have not been identified .", "A better understanding of these molecular processes could provide further insight into a multitude of ciliary dysfunction syndromes of the lung and other organs .", "The best-established model to study PCP in vertebrates is the organ of Corti in the inner ear ( IE ) .", "Mechanosensory hair cells ( HCs ) are arranged in one inner ( IHC ) and three outer HC ( OHC ) rows .", "The lateral polarization of the V-shaped actin-based stereocilia bundles on HCs strongly depends on ciliogenesis and PCP for proper sound perception ( Montcouquiol et al . , 2003; Wang et al . , 2005 , 2006; Jones and Chen , 2008 ) .", "Core PCP molecules like Celsr1 , Dvl2/3 , Fz3/6 , and Vangl2 are localized to distinct apical membrane compartments of HCs and supporting cells ( Ezan and Montcouquiol , 2013 ) .", "This differential localization seems not sufficient to instruct morphogenesis of actin-rich hair bundles in mammals ( Jones and Chen , 2008 ) .", "Instead , it depends on opposing localization of evolutionarily conserved spindle positioning and apical polarity proteins that serve as a blueprint for kinocilium migration and bundle formation ( Ezan et al . , 2013; Tarchini et al . , 2013 ) .", "It remains unclear , how core PCP molecules couple to spindle positioning complexes and the actin cytoskeleton to orchestrate morphogenesis .", "Spindle positioning proteins as well as the actin and MT cytoskeleton act together with cues from the cell cortex , such as apical junctions and polarity proteins to direct spindle positioning in mammalian epithelial cells ( Kunda and Baum , 2009 ) .", "In addition to Inscuteable ( mInsc in mammals ) , Partner of Inscuteable ( mPins/LGN in mammals ) and the G-protein coupled receptor GαI and other evolutionary conserved proteins , such as the basolateral determinants and septate junction localized proteins Dlg and Scrib , regulate A–B spindle positioning ( Knoblich , 2008; Siller and Doe , 2008 ) .", "In mammals , five Dlgs have been identified and we have recently shown that their function has diverged during evolution ( Van Campenhout et al . , 2011 ) .", "Interestingly , Dlg3 is the only family member that is involved in apical transport and required for tight junction ( TJ ) consolidation .", "Taken together , the function of Drosophila Dlg in spindle positioning ( Bellaiche et al . , 2001; Johnston et al . , 2009; Bergstralh et al . , 2013 ) and mammalian Dlg3 in PCP establishment ( Van Campenhout et al . , 2011 ) , suggested to us that Dlg3 could mediate PCP-dependent BB positioning .", "In this study , we identified Fltp as a gene expressed in regions of active PCP signaling including the node , the MCCs of the lung , and the sensory hair cells of the inner ear .", "Knock-out analysis revealed that Fltp is required for BB docking and cilia formation in the lung as well as BB and kinocilium positioning in the IE .", "Using ALI cultures , we show that Fltp expression is induced while BBs are amplified and docked at the apical PM in differentiating MCCs .", "Fltp localizes next to BBs , and MT plus ends in the apical actin network and is required for efficient BB docking and cilia formation .", "We provide evidence that Dlg3 functions together with Fltp to position BBs and kinocilia in the inner ear .", "Dlg3 and Fltp physically interact with each other , the core PCP protein Dvl2 , and the pericentriolar matrix protein γ-Tubulin , suggesting that we have identified a novel BB positioning complex in the inner ear .", "Together , our data implicate that Fltp is a novel regulator important for BB docking and positioning in mono- and multiciliated cell types that acquire PCP ." ], [ "We discovered Fltp as a functionally non-annotated gene ( 1700009P17RiK ) in a screen to identify Foxa2 target genes involved in polarity establishment ( Burtscher and Lickert , 2009 ) and expressed in monociliated node cells ( Tamplin et al . , 2008; Kinzel et al . , 2010 ) .", "The enrichment of the transcription factor FOXA2 on conserved binding sites in the promoter region of the human FLTP ortholog ( C1Orf192 ) , suggests that FLTP is a direct target of FOXA2 ( Figure 1A ) ( Weedon et al . , 2014 ) .", "The murine Fltp gene consists of six exons and the spliced mRNA codes for an open-reading frame ( ORF ) of 567 nucleotides that translates into a protein of 189 amino acids ( Figure 1B , C ) .", "An alignment of the proteins of different species reveals a high conservation during evolution as well as an N-terminal SH3 binding domain and a C-terminal proline-rich repeat ( PRR ) ( Figure 1C ) .", "Interestingly , the mouse 1700009P17RiK cDNA was also identified in a screen for mRNAs highly abundant in ciliated tissues ( McClintock et al . , 2008 ) . 10 . 7554/eLife . 03842 . 003Figure 1 . FLTP has active FOXA2 binding sites in its promoter and is conserved among species .", "( A ) A high amount of the endodermal transcription factor FOXA2 binds the human FLTP promoter in pancreatic progenitors and in adult islets , indicating that FLTP is a direct target of FOXA2 and expressed in these cells .", "( B ) Fltp shows predicted ( Genomatix ) Foxj1 , Foxa1 , and Foxa2 binding sites in its promoter ( clear red boxes: exons ( E1–E6 ) ; yellow box: promoter; TSS: transcriptional start site; light blue boxes: Foxj1 , Foxa1 , Foxa2 binding sites ) .", "( C ) Fltp protein alignment shows high conservation between different species ( highest conservation in the first 76 amino acids ) .", "The mouse and human proteins are highly homologous ( yellow box: SH3 binding domain; green box: predicted proline rich repeat ( PRR ) ; red filled box: peptide sequence of the Fltp116-1 epitope; red empty box: peptide sequence of the Fltp1 epitope; dark blue indicates conservation over 80%; lighter colors indicate less conservation ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 00310 . 7554/eLife . 03842 . 004Figure 1—source data 1 . Mendelian ratio of Fltp intercrosses on different backgrounds .", "( A–C )", "Fltp animals are born roughly at the expected Mendelian ratio in C57Bl6/6NCrl , 129S6/SvEvTac , or CD1 background .", "Note: FltpZV/ZV animals are slightly underrepresented on the C57Bl6 and 129S6 background . DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 00410 . 7554/eLife . 03842 . 005Figure 1—figure supplement 1 . Fltp antibody specificity .", "( A ) Western blot of Strep Flag ( SF ) Tag , Fltp SF-tagged transiently transfected HEK293T cells and testis lysate of Fltp+/+ , FltpZV/+ , and FltpZV/ZV animals incubated with Fltp116-1 antibody .", "Note , Fltp1 ( Figure 1—figure supplement 2D ) detects one specific and Fltp116-1 two bands at approximately the calculated size of Fltp , likely representing post-translationally modified Fltp protein .", "Anti-alpha-Tubulin antibody was subsequently used on the same blot to confirm equal loading .", "( B–D )", "Laser scanning microscopy ( LSM ) of HEK293T cells transiently transfected with a vector encoding for Fltp-Venus ( B–B″ ) , Venus ( C–C′ ) , and an untransfected control ( D ) stained with Fltp1 and GFP ( for Venus ) .", "Fltp-Venus can be detected in the cytoplasm by both antibodies .", "( E–G )", "LSM of HEK293T cells transiently transfected with a vector encoding for Fltp-Venus ( E–E″ ) , Venus ( F–F′ ) , and an untransfected control ( G ) stained with Fltp116-1 and GFP .", "Fltp-Venus can be detected in the cytoplasm by both antibodies .", "Scale bars; 5 µm ( C–G ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 00510 . 7554/eLife . 03842 . 006Figure 1—figure supplement 2 . Fltp construct and confirmation of the knock-out strategy .", "( A ) The FltpZV targeting strategy deletes the entire open reading frame ( ORF ) from exon 2 to 6 ( exons: black and orange boxes; orange boxes: ORF , primers for genotyping: 418 , 420 , 565 , 566; for primer sequences see Supplemental ‘Materials and methods’ ) .", "The external 5′- as well as the 3′-probe are indicated .", "Restriction enzyme sites for DraIII and EcoRV are shown .", "Homology regions for recombination of the targeting construct are indicated as 5′- and 3′-Retrieval .", "The figure is on scale ( NLS-LacZ: nuclear localization signal-beta-galactosidase; 2A: viral T2A sequence; H2B: histon-2B; Venus: yellow fluorescent reporter gene; SV40-pA: Simian Virus 40 polyadenylation signal; loxP: site of Cre-mediated recombination; bGHpA: bovine Growth Hormone polyadenylation signal; neo: neomycin resistance cassette; PGK: phospho-glycerate kinase; UTR: untranslated region ) .", "( B ) Southern blot of Fltp+/+ embryonic stem ( ES ) cells vs FltpZV/+ ES cells digested with DraIII and hybridized with the external 5′ Southern probe showing the Bl6 ( 16 , 443 bp ) , 129 WT allele , and the Bl6 targeted allele ( 11 , 469 bp ) .", "Notice the shift of the WT band due to restriction length polymorphism .", "( C ) Genotyping PCR to discriminate between Fltp+/+ , FltpZV/+ , and FltpZV/ZV ( primers: 418 , 565 , 566; WT band ( 317 bp ) ; targeted Δneo band ( 387 bp ) ) .", "( D ) Western blot of testis lysate of Fltp+/+ , FltpZV/+ , and FltpZV/ZV animals incubated with Fltp1 antibody .", "Fltp protein band is detectable at around 25 kDa ( calculated weight 20 kDa ) .", "Fltp protein is absent in FltpZV/ZV lysate .", "The asterisk marks an unspecific band . DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 006 To analyze the Fltp protein in more detail , we raised two polyclonal rabbit antibodies against a central and a C-terminal epitope ( Figure 1C ) .", "We confirmed the specificity of these antibodies in western blot analysis and immunocytochemistry in transiently transfected HEK293T cells ( Figures 1—figure supplement 1A–G , 2D ) .", "Endogenous Fltp protein localizes to the apical PM , the BB , and the primary cilium in monociliated mouse node cells , suggestive for a function of Fltp in BB transport , positioning , and/or ciliogenesis ( Figure 2F , G ) . 10 . 7554/eLife . 03842 . 007Figure 2 . Fltp reporter and protein is detectable in mono- and multiciliated tissues .", "( A and B ) mRNA ( Fltp in situ hybridization ) ( A ) and lacZ reporter expression ( B ) are restricted to the node ( n ) at E7 . 5 .", "( C–E ) Whole-mount lacZ stained and benzyl alcohol/benzyl benzoate ( BABB ) cleared FltpZV/+ embryo and organs .", "( C ) E11 . 5 embryo reveals reporter expression in the choroid plexi ( cp ) , prechordal plate ( pcp ) , eye ( e ) , and floor plate ( fp ) .", "( D ) E17 . 5 IE shows reporter expression in posterior crista ampullaris ( pca ) , lateral crista ampullaris ( lca ) , anterior crista ampullaris ( aca ) , utricular macula ( uma ) , and saccular macula ( sma ) of the vestibular part as well as in the organ of Corti ( oc ) .", "( E ) E17 . 5 lung shows lacZ reporter activity in multiciliated lung epithelial cells .", "( F and G )", "Whole-mount antibody stained embryo ( node , E7 . 75 ) analyzed by LSM reveals Fltp protein in vesicular fashion in the cytoplasm and along primary cilia ( white arrow heads ) .", "( H–I′ )", "Immunohistochemistry on cryosections combined with LSM analysis reveals Fltp at the apical plasma membrane and at cilia in multiciliated lung epithelial cells of Fltp+/+adult animals ( H and H′ ) .", "No Fltp immunoreactivity is detected in FltpZV/ZV lungs ( I and I′ ) .", "ZO-1 marks apical TJs , α-Tubulin ( alpha Tub ) , and acetylated-Tubulin ( aT ) , the tubulin network and cilia; nuclei are marked by DAPI and Fltp by Fltp1 ( F and G ) and Fltp116-1 ( H–I′ ) .", "Scale bars; 100 µm ( A and B ) , 500 µm ( C , D , E ) , 10 µm ( F ) , 4 µm ( G ) , and 10 µm ( H–I′ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 00710 . 7554/eLife . 03842 . 008Figure 2—figure supplement 1 . Additional Fltp reporter expression in mono- and multiciliated tissues .", "( A–H )", "Whole-mount lacZ stained and benzyl alcohol/benzyl benzoate ( BABB ) cleared ( D , F , H ) FltpZV/+ embryos and organs .", "β-gal activity is restricted to the node ( n ) ( A ) .", "( B ) Cross-section of the neural tube ( dotted line ) reveals reporter expression in the floor plate ( fp ) .", "( C ) Reporter expression can be detected in the four choroid plexi ( cp ) , the fp , and the inner ear ( ie ) .", "( D ) lacZ expression analysis reveals β-gal activity in the developing cp of the first , second , third , and fourth ventricle , in the prechordal plate ( pcp ) , in the developing eye ( e ) , and in the fp .", "( E and F )", "LacZ reporter is expressed in the 6 sensory regions of the IE .", "Onset of reporter expression is at E12 . 5 .", "( G and H )", "LacZ staining of whole-mount lungs shows onset of reporter expression in the main stem bronchi at E14 . 5 ( G ) .", "Later , expression is restricted to the lung epithelium ( H ) .", "( I ) Histological section through a lacZ stained adult lung showing β-gal activity in the lung epithelium .", "( J ) Laser scanning microscopy of lung cryosections reveals Fltp reporter expression in epithelial lung cells .", "Expression directly stops at the broncho-alveolar-duct junction ( BADJ ) .", "The alveoli are absent of Fltp reporter staining .", "Fltp reporter positive cells are marked by GFP and nuclei by DAPI .", "Scale bars; 200 µm ( A ) , 250 µm ( B ) , 500 µm ( C–H ) , 150 µm ( I ) , 75 µm ( J ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 008 To explore Fltp expression and function in vivo , we generated an FltpZV knock-in/knock-out allele by replacing the entire ORF by a multicistronic lacZ-Venus reporter cassette ( Figure 1—figure supplement 2A and Supplemental ‘Materials and methods’ ) .", "PCR genotyping as well as Southern and western blotting analysis confirmed the targeted homologous recombination and the generation of a null allele ( Figure 1—figure supplement 2B–D ) .", "Fltp homozygous knock-out mice are born at roughly the expected Mendelian ratio and are viable and fertile on CD1 , C57BL/6NCrl and 129S6/SvEvTac background ( Figure 1—source data 1 ) .", "Next , we characterized the temporal and spatial expression pattern of the Fltp reporter during embryonic development .", "LacZ reporter activity in FltpZV/+ animals is first detectable in the embryonic node at E7 . 5 ( Figure 2B ) , which accurately reflects endogenous mRNA expression ( Figure 2A ) and confirmed our previous findings ( Tamplin et al . , 2008; Lange et al . , 2012 ) .", "At E8 . 5–13 . 5 , strong reporter activity is detected in the eye , notochord , floor plate of the neural tube , and all four choroid plexi ( Figure 2C , Figure 2—figure supplement 1A–D ) , tissues that are either mono- or multiciliated and depend on active PCP signaling .", "Consistent with a potential function in the PCP pathway , LacZ reporter activity in the IE coincides with BB docking and kinocilium positioning at E13 . 5 ( Figure 2—figure supplement 1E ) ( McKenzie et al . , 2004; Ezan and Montcouquiol , 2013 ) .", "The Fltp reporter remains expressed during development and early postnatal life in all six sensory regions of the IE ( Figure 2D , Figure 5A , Figure 2—figure supplement 1C , E , F ) .", "Furthermore , Fltp reporter activity strongly correlates with the spatial and temporal onset of ciliogenesis in the lung trachea and the main bronchi at E14 . 5–17 . 5 ( Figure 2E , Figure 2—figure supplement 1G , H ) .", "In adult lungs at postnatal day ( P ) 30 , the dual lacZ and H2B-Venus reporter remains expressed in MCCs of the bronchioles , but not in non-ciliated cells of the alveoli ( Figure 2—figure supplement 1I , J ) .", "To confirm the reporter studies , we analyzed cell type-specific and subcellular localization of the endogenous Fltp protein in the embryonic node and adult lung epithelium .", "Whole-mount antibody staining combined with laser scanning microscopy ( LSM ) on gastrula-stage embryos revealed that the Fltp protein is localized at the apical PM , TJs , primary cilia , and in the cytoplasm of node cells ( Figure 2F , G ) .", "Immunohistochemistry on lung sections further shows that Fltp is highly enriched at the apical cortex and in cilia of the multiciliated bronchial epithelial cells ( Figure 2H , Hʹ ) .", "The absence of Fltp immunoreactivity in FltpZV/ZV lung sections further confirmed antibody specificity ( Figure 2I , Iʹ ) .", "Taken together , Fltp reporter and endogenous expression accurately correlate with onset of PCP establishment in the embryonic node , floor plate , choroid plexi , IE , and lung , which is indicative for a function in or downstream of the PCP pathway .", "The embryonic and adult lung depends on PCP signaling for branching morphogenesis , BB docking , and cilia formation ( Yates et al . , 2010; Vladar et al . , 2012 ) .", "First , we investigated the highly stereotypic branching pattern in cleared lacZ stained lungs at P60 ( Metzger et al . , 2008 ) .", "No difference was detectable in FltpT2AiCre/+; R26R/+ ( Gt[ROSA]26Sor ) controls lungs with normal levels of Fltp protein ( Soriano , 1999; Lange et al . , 2012 ) , FltpZV/+ heterozygous and FltpZV/ZV homozygous mutant lungs , indicating that Fltp has no function during branching morphogenesis ( Figure 2E , Figure 3A–C ) .", "In contrast , measurement and quantification of terminal lung bronchiole diameters revealed a dose-dependent and statistically significant constriction of the distal airways ( Figure 3A–D ) , suggesting that normal Fltp levels are important for lung airway homeostasis .", "To better understand the etiology of the airway constrictions , we analyzed Fltp expression and function on cellular level in Fltp+/+ , FltpZV/+ , and FltpZV/ZV adult lungs at P7 and P30 ( Figure 3E–L ) .", "Fltp-driven H2B-Venus reporter activity was confined to the nuclei of multiciliated epithelial cells in FltpZV/+ and FltpZV/ZV lungs at P7 and P30 .", "LSM analysis further confirmed that cilia formation was significantly affected in a dose-dependent fashion ( Figure 3E–L ) , similar to PCP effector knock-down phenotypes in the mucociliary epithelium of Xenopus laevis ( Park et al . , 2006 , 2008 ) . 10 . 7554/eLife . 03842 . 009Figure 3 . Loss of Fltp leads to constricted distal airways and cilia formation defects in the lung .", "( A–C )", "Whole-mount lacZ stained and BABB cleared distal airways of left lung lobes at P60 .", "Red lines show how diameters were measured .", "( A ) FltpT2AiCre/+; R26R/+is used as control ( Ctrl ) .", "( B and C )", "FltpZV/+ and FltpZV/ZV animals show constricted distal airways .", "( D ) Average distal airway diameter of Ctrl animals is 132 . 25 µm ( n = 4; 125 bronchi ) , of FltpZV/+ animals is 99 . 68 µm ( n = 3; 90 ) , and of FltpZV/ZV animals is 90 . 06 µm ( n = 3; 95 ) .", "( E–G and I–K )", "Immunohistochemistry on cryosections of lung distal airway epithelium combined with LSM analysis .", "( E and I )", "In Fltp+/+animals BBs project cilia at the apical surface .", "( F and J )", "In FltpZV/+ animals less and shorter cilia are detectable .", "( G and K )", "FltpZV/ZV animals often show absence of or shorter cilia at the apical surface .", "Note that only very few BBs are docked at the apical surface .", "( H and L )", "FltpZV/ZV animals show significant shorter cilia than Fltp+/+ animals .", "In total we examined n = 3 ( 81 cells ) for Fltp+/+ , n = 2 ( 22 ) for FltpZV/+ , and n = 2 ( 93 ) for FltpZV/ZV P7 animals .", "For P30 we examined n = 3 ( 121 cells ) for Fltp+/+ , n = 3 ( 134 ) for FltpZV/+ , and n = 2 ( 138 ) for FltpZV/ZV .", "BBs are marked by γ-Tubulin ( γTub ) , cilia by acetylated-Tubulin ( aT ) , cell membrane by β-Catenin ( βCat ) , nuclei by DAPI , and nuclei of Fltp reporter expressing cells by GFP .", "Statistical analysis uses an one way ANOVA ( **p = 0 . 0028; ***p < 0 . 0001 for ( D ) and *p = 0 . 0143; **p = 0 . 0025 for ( H and L ) ) .", "Error bars show the 95% confidence interval of the mean ( D ) and the standard error of the mean ( H and L ) .", "Scale bars; 200 µm ( A–C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 009 To analyze the dynamic cellular and molecular requirement for Fltp during BB docking , cilia formation , and PCP establishment , we employed the mTEC culture system ( You et al . , 2002; Vladar et al . , 2012 ) .", "Switching mTEC culture to ALI conditions induces a differentiation process of lung progenitor cells towards a MCC phenotype .", "During differentiation the progenitor cells pass through different maturation stages ( Figure 4A ) : Monociliated lung progenitor cells ( stage I ) start centrosome/BB amplification followed by BB transport and docking ( stage II–III ) and subsequent cilia formation ( stage IV ) ( Vladar et al . , 2012 ) .", "In these cultures , Fltp-driven H2B-Venus reporter activity is activated at the transition from stage I to stage II when centrosomes/BBs are amplified and docked in heterozygous and homozygous Fltp ALI cultures ( Figure 4B ) .", "Fltp-H2B-Venus expression increases during maturation until cells are terminally differentiated .", "This correlates with active PCP signaling and initial asymmetric core component localization ( Vladar et al . , 2012 ) .", "Subcellular co-localization studies further revealed that Fltp is localized to the apical , but not sub-apical actin cytoskeleton and fills the gaps in the actin network from where MT-based cilia project ( Figure 4C–Cʹʹʹ , E ) .", "Moreover , Fltp co-localizes with newly synthesized MT plus ends that are labeled with anti-EB1 antibodies ( Figure 4D ) .", "Together with the direct adjacent localization of Fltp next to the pericentriolar matrix stained by γ-Tubulin ( Figure 4E ) , these data suggest that Fltp connects BBs and ciliary MT plus ends to the cortical actin cytoskeleton .", "To test this idea , we examined mTECs switched to ALI conditions from Fltp+/+ and FltpZV/ZV mice at day two and four of differentiation qualitatively and quantitatively .", "Fltp+/+ and FltpZV/ZV differentiated with comparable efficiencies and speed as measured by Fltp-driven H2B-GFP expression and centrosome amplification ( Figure 4—figure supplement 1A ) , and a comparable number of centrosomes are formed in differentiating Fltp+/+ and FltpZV/ZV mTECs ( Figure 4F , G ) , whereas BB docking and cilia formation was delayed at ALI+2 and +4 days of differentiation ( Figure 4F–I , Figure 4—figure supplement 1B ) .", "Taken together , these data suggest that Fltp functions in the process of BB docking and cilia formation . 10 . 7554/eLife . 03842 . 010Figure 4 . Fltp is expressed and necessary during BB docking .", "( A ) Scheme of multiciliated cell ( MCC ) maturation .", "A MCC progenitor projects a primary cilium at the apical surface ( stage I ) .", "Centrosome amplification is the first sign of differentiation ( stage II ) followed by apical transport and docking of BBs ( stage III ) .", "Fully differentiated cells project multiple motile cilia at the apical surface ( stage IV ) .", "Staging according to Vladar and Stearns ( 2007 ) .", "The green nucleus indicates Fltp reporter gene expression .", "( B–G )", "LSM of ALI culture of FltpZV and WT mTECs .", "( B ) Onset and level of Fltp reporter expression correlate with the onset of BB amplification and ciliogenesis ( stage II–IV ) in cultured ALI mTECs as shown in the scheme in ( A ) .", "( C–C′′′ )", "Confocal sections of a single cell from the sub-apical actin level ( C ) over the apical actin level ( C′ and C″ ) to the cilia level ( C′′′ ) ( ALI day 4 ) .", "Fltp co-localizes with the MT-plus end binding protein EB-1 ( D ) ( ALI day 4 ) next to the pericentriolar matrix ( E ) ( ALI day 4 ) and to cilia in multiciliated Fltp+/+ cells ( C′′′ ) .", "( F ) Fltp+/+ cell showing all BBs ( red dots ) projecting cilia ( white stripes ) at the apical surface ( ALI day 4 ) .", "( G ) In many FltpZV/ZV cells the majority of BBs are not docked at the level of tight junctions marked by ZO-1 and do not project cilia ( ALI day 4 ) .", "( H and I )", "Side view IMARIS surface rendering shows that all BBs are docked at the apical surface in Fltp+/+ ( H ) ( ALI day 4 ) in contrast to FltpZV/ZV cells where most BBs stay in the cytoplasm ( I ) ( ALI day 4 ) .", "BBs are marked by γ-Tubulin ( γTub ) and pericentrin ( Peric ) , cilia and the tubulin network by tyrosinated-Tubulin ( Tyr Tub ) and acetylated-Tubulin ( aT ) , the actin network by Phalloidin ( Phall ) , MT plus ends by EB-1 , Fltp protein by Fltp116-1 , nuclei by DAPI , and nuclei of Fltp reporter expressing cells by GFP .", "Scale bars; 2 µm ( C–C′′′ , D , E ) , 5 µm ( F and G ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 01010 . 7554/eLife . 03842 . 011Figure 4—figure supplement 1 . ALI differentiation efficiency and quantification of BB docking defect .", "( A ) Graph showing the percentage of cells in stage II–IV for WT and FltpZV/ZV ( HOM ) ALIs at day zero ( before the switch to ALI condition ) , one , two , and four measured by GFP and pericentrin ( HOM ) and only pericentrin ( WT ) staining compared to the total amount of cells in the ALI culture stained by DAPI ( representative images were analyzed ) .", "For WT analysis we counted n = 1 ( 1493 cells ) for day 0 , n = 1 ( 1345 ) for day 1 , n = 2 ( 1635 ) for day 2 , n = 2 ( 1262 ) for day 4 and for HOM n = 1 ( 1367 cells ) for day 0 , n = 1 ( 1542 ) for day 1 , n = 3 ( 942 ) for day 2 , n = 4 ( 1123 ) for day 4 .", "( B ) Shows the percentage of ciliation ( measured by acetylated-Tubulin staining ) of cells with amplified centrosomes ( measured by pericentrin staining ) for WT and HOM ALIs at day two and four categorized into stage II–III and stage IV .", "For WT analysis we counted n = 4 ( 609 cells ) for day 2 , n = 4 ( 231 ) for day 4 and for HOM n = 10 ( 611 ) for day 2 , n = 7 ( 286 ) for day 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 011 To analyze the function of Fltp in PCP , BB positioning , and cilia formation in vivo , we employed the best-characterized mammalian PCP model system , the organ of Corti in the IE .", "Morphologically the IE looked normal and the cochlear duct is not significantly shortened or widened in FltpZV/+ and FltpZV/ZV mice , indicating that PCP-mediated convergent extension movements are not affected at P0 ( data not shown ) .", "However , significant deviations of the polarized arrangement of stereocilia bundles can be seen in IHC and OHC rows with increasing severity closer to the apex along the apico-basal axis ( Figure 5B–C , J–N ) .", "In addition , scanning electron microscopy ( SEM ) analysis uncovered severe stereocilia bundle morphogenesis defects and mispositioning of the MT-based kinocilium , very similar to phenotypes observed in mutants of spindle positioning proteins , such as Gαi3 , mPins , mInsc , and LGN ( Figure 5D–I ) ( Ezan et al . , 2013; Tarchini et al . , 2013 ) . 10 . 7554/eLife . 03842 . 012Figure 5 . Fltp is required for kinocilium positioning and stereocilia bundle formation .", "( A ) Whole-mount LSM of an FltpZV/+ organ of Corti ( OC ) showing its different regions .", "Fltp reporter expression is restricted to all sensory HCs ( IHC and OHC ) of the OC ( red dashed line ) .", "( B–M )", "Whole-mount SEM pictures of an E18 . 5 OC .", "( B and C )", "Fltp+/+ ( WT ) HCs at 50% of the OC length are perfectly polarized ( B ) whereas FltpZV/ZV ( HOM ) HCs show orientational defects ( C ) .", "( D–I )", "Enlargements of HCs reveal perfect polarized kinocilia as well as highly ordered stereocilia in WT ( D ) .", "HOM cells are misaligned and show split stereocilia bundles ( E , G–I ) , general bundle morphology defects ( E–I ) , as well as detached kinocilia ( E ) .", "Polarity and stereocilia bundles disruption increases in HOM hair cells from 75% ( K ) to >75% ( M ) of OC length compared to WT cells ( J and L ) .", "( N ) Deviation of HCs from the polarity axis for WT and HOM animals significantly differs in all OC regions ( <50% , 50% , 75% , >75% ) .", "Quantification of HC rotation was performed by measuring the angle from the normal tissue polarity ( measured by the medial to distal alignment of the HC rows ) to the middle of the stereocilia bundle .", "In total we examined n = 4 ( 219 HCs ) of WT and n = 7 ( 426 ) of HOM animals .", "Statistical analysis uses circular statistics ( ****p < 0 . 0001 ) .", "Error bars show the standard deviation .", "Kinocilia are marked by the red dashed lines , nuclei by DAPI , and nuclei of Fltp reporter expressing cells by GFP .", "Abbreviations: inner hair cell ( IHC ) , outer hair cell ( OHC1-3 ) .", "Scale bars; 10 µm ( B and C ) , 1 µm ( D–I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 012 To seek first evidence if Fltp functions in the PCP pathway , we investigated a potential genetic interaction of Fltp with the core PCP component Celsr1 ( Curtin et al . , 2003 ) .", "As Fltp and Celsr1 are co-expressed in the cochlea of the IE ( Shima et al . , 2002 ) , we compared stereocilia bundle morphogenesis in Celsr1crsh/crsh single mutant and FltpZV/ZV; Celsr1crsh/crsh double mutant cochleae at E18 . 5 .", "On the mixed genetic background analyzed , Celsr1crsh/crsh animals showed rather normal bundle alignment in IHCs and OHCs from the base to the middle part of the cochlea ( <50% ) , but the alignment was significantly disturbed closer to the apex ( >50% ) ( Figure 6A , C , D ) .", "In contrast , FltpZV/ZV; Celsr1crsh/crsh double mutant cochleae revealed an increased degree of misaligned stereocilia bundles , most pronounced in the <50% region , and showed an additional fourth OHC row ( Figure 6B–D ) .", "These data suggest that Fltp and the core PCP gene Celsr1 either act together in the PCP pathway or in two parallel pathways important for the morphogenesis of the cochlea .", "To clarify if Fltp is necessary to establish PCP in the IE according to the current model ( Figure 6E ) ( Ezan and Montcouquiol , 2013 ) , we further investigated the localization of core PCP components .", "No differences in the asymmetric distribution of Vangl1 and Dvl2 at the cell cortex along the medio-lateral axis were detected in Fltp+/+and FltpZV/ZV cochleae at E18 . 5 ( Figure 6F–I ) , consistent with a function of Fltp downstream of core PCP molecules . 10 . 7554/eLife . 03842 . 013Figure 6 . Fltp is a potential downstream mediator of PCP signaling .", "( A–B )", "LSM of an E18 . 5 Fltp+/+; Celsr1crsh/crsh ( WT ) organ of Corti ( OC ) ( A ) revealed rotated outer ( OHC ) and inner hair cells ( IHC ) .", "FltpZV/ZV; Celsr1crsh/crsh ( HOM ) OC ( B ) shows more severely rotated IHCs and OHCs as well as an additional OHC row in comparison to ( A ) .", "( C and D )", "Hair cells of HOM ( red ) animals show a more pronounced PCP phenotype compared to WT ( blue ) in the region <50% and >50% of the OC ( for cochlea region nomenclature and quantification method see Figure 5 ) .", "In total we analyzed n = 1 ( 130 cells ) at <50% , n = 1 ( 90 ) at >50% for WT , n = 5 ( 298 ) at <50% , n = 5 ( 743 ) at >50% for HOM .", "Statistical analysis uses a Kruskal–Wallis test ( *p = 0 . 0375; ***p = 0 . 0003 ) .", "Error bars show the 95% confidence interval of the mean .", "( E ) Model illustrating PCP molecule localization in IE hair cells .", "( F–I )", "Whole-mount IE ( E18 . 5 ) LSM of 3 OHC rows revealed Vangl1 localization at the lateral side of supporting cells in Fltp+/+ ( F ) and FltpZV/ZV ( G ) animals and Dvl2 localization at the lateral side of IE hair cells in Fltp+/+ ( H ) and FltpZV/ZV ( I ) animals indicating that core PCP protein localization is not disrupted .", "The actin network and stereocilia are marked by Phalloidin ( Phall ) and core PCP proteins by Vangl1 and Dvl2 .", "Abbreviations: inner phalangeal cells ( PhC ) , inner pillar cells ( IPC ) , outer pillar cells ( OPC ) , Deiters' cells ( DC1-3 ) .", "Scale bars; 10 µm ( A and B ) , 5 µm ( F–I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 013 BB positioning and stereocilia bundle morphogenesis depend on spindle positioning proteins ( Ezan et al . , 2013; Tarchini et al . , 2013 ) , but how these connect to core PCP molecules and actin-rich stereocilia bundles is unknown .", "Due to PCP defects in the IE ( Van Campenhout et al . , 2011 ) , we hypothesized that the mammalian scaffold protein Dlg3 might be a cortical adaptor for PCP-mediated BB positioning .", "Due to the lack of good antibodies and tools , we generated a transgenic mouse line that constitutively expresses a Venus fluorescent protein fused to the N-terminus of Dlg3 ( Dlg3-Venus ) ( see Supplemental ‘Materials and methods’ ) .", "Next , we examined the subcellular localization of the Dlg3-Venus fusion protein in relation to BB , kinocilium , stereocilia bundle , core PCP molecules , and TJs in cochlea HCs using LSM and three-dimensional reconstruction ( Figure 7A–Bʹ , F–H ) .", "Dlg3 specifically localizes in a crescent shape to the bare zone around the BB and co-localizes with the core PCP component Dvl2 at lateral TJs ( Figure 7A–Bʹ , G ) , the area where mPins/LGN , mInsc , and Gαi are localized ( Ezan et al . , 2013; Tarchini et al . , 2013 ) .", "This suggests that Dlg3 might have a conserved function in spindle/BB positioning ( Johnston et al . , 2009 ) .", "Interestingly , Fltp localizes at the interface between Dlg3 and the actin-based stereocilia bundles ( Figure 7C–Eʹ ) , further suggesting that Fltp might be an adaptor protein linking spindle/BB positioning proteins to the apical actin cortex . 10 . 7554/eLife . 03842 . 014Figure 7 . Fltp is located at the interface of apical actin and Dlg3 in IE hair cells .", "( A , B , C–D′ , F–H )", "Single section LSM of outer HCs of an Fltp+/+; Dlg3-Venus animal at E18 . 5 reveals that Dlg3-Venus is located at the lateral membrane and at the medial membrane ( or the lateral membrane of the supporting cell ) of IE HCs ( A and B ) .", "Fltp is localized lateral to the cuticular plate ( CP ) and the stereocilia bundles ( SC ) ( C and D ) .", "Dlg3-Venus is located in a lateral crescent overlapping with Fltp localization ( C′ and D′ ) .", "The unstained area marks the region of the BB ( C′ and D′ ) .", "Dlg3-Venus is co-localized with Dvl2 ( G ) at the most lateral membrane directly opposite of Vangl1 ( F ) and with ZO-1 at the apical membrane ( H ) .", "( A′ , B′ , E , E′ )", "IMARIS wireframe animation of a Dlg3-Venus IE HC showing Dlg3-Venus co-localization with the BB , the kinocilium , and actin ( A′ and B′ ) and a Fltp+/+; Dlg3-Venus IE HC showing Fltp , Dlg3-Venus , and Phalloidin co-localization ( E and E′ ) .", "The actin network , the CP , and the SC are marked by Phalloidin ( Phall ) , Fltp protein by Fltp116-1 ( Fltp ) , the kinocilium by acetylated-Tubulin ( aT ) , the BB by pericentrin ( Peric ) , the apical cell membrane by ZO-1 , core PCP proteins by Vangl1 and Dvl2 , and Dlg3-Venus fusion protein by GFP .", "Scale bars; 2 µm ( A–E′ ) , 3 µm ( F–H ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 014 We tested this idea by analyzing Dlg3 and BB localization in Fltp mutants .", "When compared to WT ( Figure 8A–Dʹ , J , Figure 8—figure supplement 1A ) , the lateral crescent of Dlg3 in Fltp mutants is disturbed and BBs are not correctly positioned in the middle of the apical Dlg3 crescent ( Figure 8E–Hʹ , J , Figure 8—figure supplement 1A ) , implicating that Fltp binds to Dlg3 to position BBs , kinocilia , and stereocilia bundles .", "If Dlg3 and Fltp cooperate to position BBs , one would predict that these proteins physically interact .", "Dlg3 contains a central SH3 domain which could bind to the SH3 binding or PRR domain of Fltp .", "We tested this by co-immunoprecipitation experiments of Streptavidin-Flag ( SF ) -tagged Dlg3 variants and Fltp-myc variants from serum starved and ciliated HEK293T cells ( Figure 9A , B ) .", "These experiments suggest a physical interaction of Fltp with Dlg3 , which requires the highly conserved N-terminal SH3 binding domain of Fltp , but not the SH3 domain of Dlg3 ( Figure 9B ) .", "As Dvl2 localizes directly adjacent to Dlg3 and Fltp in the inner ear , we next examined the association of endogenous Dvl2 and γ-Tubulin with overexpressed SF full-length Fltp and SF-Dlg3 ( Figure 9C ) .", "The interaction of Dvl2 with Dlg3 and Fltp and γ-Tubulin with Dvl2 and Dlg3 suggests that these proteins form a complex during BB transport and positioning .", "Finally , we tested the predicted requirement of Dlg3 for BB positioning by knock-out analysis ( Figure 8K , L , Figure 8—figure supplement 1B , C ) .", "Taken together , these results suggest that Dlg3 is a BB positioning protein that is anchored at apical TJs ( Van Campenhout et al . , 2011 ) and cooperates with Fltp to position BB at the apical cortex of HCs in the cochlea at E18 . 5 . 10 . 7554/eLife . 03842 . 015Figure 8 . Loss of Fltp or Dlg3 leads to BB mispositioning in IE hair cells .", "( A–D )", "LSM of Fltp+/+; Dlg3-Venus ( WT ) IE HCs at E18 . 5 reveals BBs in the middle of the lateral Dlg3-Venus crescent .", "( B′–D′ )", "IMARIS surface rendering pictures of WT HCs .", "( E–H )", "LSM of FltpZV/ZV; Dlg3-Venus ( HOM ) IE HCs at E18 . 5 .", "BBs are located at the edge of the lateral Dlg3-Venus crescent .", "The crescent itself often shows defective localization .", "The red asterisk marks some affected cells .", "( F′–H′ )", "IMARIS surface rendering pictures of HOM HCs .", "For quantification see Figure 8—figure supplement 1A .", "( I ) For BB mispositioning analyses the angle between the middle of the Dlg3-Venus crescent and the BB location was measured .", "( J ) BB position in affected HCs of HOM animals significantly differs from the position in the WT .", "In total we analyzed n = 2 ( 73 cells ) for WT and n = 2 ( 119 ) for HOM .", "( K ) LSM of Dlg3−/− ( Dlg3tm1Grnt ) IE HCs reveals mislocalized BBs and rotated hair cells .", "For quantification Figure 8—figure supplement 1B , C .", "( L ) Analysis of BB mispositioning was performed as described in ( I ) .", "BB position in affected HCs of Dlg3−/− ( HOM ) animals significantly differs from the Dlg3+/+ ( WT ) position .", "In total we analyzed n = 7 ( 470 cells ) for WT and n = 13 ( 804 ) for HOM .", "Statistical analysis uses an one-way ANOVA ( J ) or a Kruskal–Wallis test ( L ) ( ****p < 0 . 0001 ) .", "Error bars show the 95% confidence interval of the mean .", "The actin network and stereocilia are marked by Phalloidin ( Phall ) , BBs by pericentrin ( Peric ) , cilia by acetylated-Tubulin ( aT ) , and Dlg3-Venus fusion protein by GFP .", "Abbreviation: inner hair cell ( IHC ) , outer hair cell ( OHC 1-3 ) .", "Scale bars; 10 µm ( A and E ) , 1 µm ( B–D′ , F–H′ , K ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 01510 . 7554/eLife . 03842 . 016Figure 8—figure supplement 1 . BB mispositioning in FltpZV; Dlg3-Venus and Dlg3 animals .", "( A ) In FltpZV/ZV; Dlg3-Venus ( HOM ) IE significantly more hair cells across all rows show BB mispositioning over 8° ( red ) compared to controls Fltp+/+; Dlg3-Venus ( WT ) .", "( B ) The same analysis as in ( A ) only for Dlg3−/− ( HOM ) .", "HOM animals show higher number of cells with mispositioned BBs compared to Dlg3+/+ ( WT ) animals .", "( C ) The deviation of hair cells from the polarity axis shows a clear PCP defect in Dlg3−/− ( HOM ) animals ( WT = blue , HOM = red ) .", "In total we analyzed n = 4 ( 1769 cells ) for WT and n = 8 ( 583 ) for HOM .", "Statistical analysis uses a Kruskal–Wallis test ( ****p < 0 . 0001 ) .", "Abbreviation: inner hair cell ( IHC ) , outer hair cell ( OHC 1-3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 01610 . 7554/eLife . 03842 . 017Figure 9 . Fltp interacts with proteins associated with the TJ complex ( Dlg3 ) , the BB ( γ-Tubulin ) and with the core PCP protein Dvl2 . ( A ) Dlg3 and Fltp constructs used for interaction domain mapping .", "( B ) N-terminus of Fltp is essential for interaction with Dlg3 .", "HEK293T cells were transfected with SF Dlg3 variants and with Fltp-myc variants .", "SF-tagged Dlg3 was immunoprecipitated using Streptavidin beads ( Strep-IP ) .", "Full-length Fltp-myc was detected in the Strep-IP in the presence of full-length SF-Dlg3 , SF-Dlg3ΔSH3 , and SF-Dlg3ΔPDZ .", "FΔNT cannot be co-immunoprecipitated with SF-Dlg3 .", "( C ) Fltp and Dlg3 are found in a complex together with Dvl2 and γ-Tubulin .", "HEK293T cells were transfected with SF Dlg3 and SF Fltp .", "In a Strep-IP for Fltp and Dlg3 , endogenous Dvl2 and γ-Tubulin were co-immunoprecipitated .", "Abbreviations: D: Dlg3; F: Fltp; SBD: SH3 binding domain; PRR: proline rich repeat . DOI: http://dx . doi . org/10 . 7554/eLife . 03842 . 017" ], [ "Several lines of evidence suggest that Fltp is a potential PCP effector regulating the cytoskeleton .", "First , the Fltp gene and reporter are expressed in several tissues known to depend on active PCP signalling , such as the node , ependymal cells , floor plate , lung , eye , and IE .", "Second , Fltp expression kinetic correlates with dynamic PCP acquisition , terminal differentiation , and cellular morphogenesis in the lung and the IE .", "Third , the double knock-out of Fltp and the core PCP gene Celsr1 show a more severe PCP phenotype than both single mutants , raising the possibility that both genes act in the same pathway , which needs further confirmation .", "If this is the case , then Fltp seems to be a downstream effector molecule in the PCP pathway , as it is not required for PCP establishment , but rather for cell intrinsic PCP transduction leading to cytoskeletal rearrangements .", "This is supported by the fact that phenotypes observed for the PCP effectors Intu and Fuz in the mucociliary epithelium of the frog and Fltp in the murine lung are strikingly similar ( Park et al . , 2006 , 2008 ) .", "Additionally , spindle positioning rather than core PCP mutant phenotypes mirrors the Fltp mutant phenotype in the IE ( Ezan et al . , 2013; Tarchini et al . , 2013 ) .", "Finally , Fltp is localized at the interface of spindle positioning complexes that capture MT plus ends and connect to the PM and the apical actin cytoskeleton .", "Loss of Fltp leads to failure in BB docking and positioning most probably through disconnection of the BB , MT , and actin cytoskeleton in the mammalian lung and IE .", "Although the mechanisms of Fltp action in these two tissues might differ , a common theme seems that Fltp acts at the interface of the MT and actin cytoskeleton for BB docking and positioning , which we discuss in the following sections .", "PCP is best studied in the mammalian IE , but is also apparent in MCCs of the lung and mucociliary epithelium of the frog skin ( Wallingford , 2012 ) .", "In both terminally differentiated cell types , core PCP molecules are asymmetrically localized at the apical PM and translate long-range PCP signals into cell intrinsic cytoskeletal changes that coordinate BB docking and positioning as well as cilia formation in mono- and multiciliated cells .", "In MCCs of the frog skin the PCP effector proteins Intu and Fuz have been shown to control ciliogenesis and together with Dvl control apical docking and planar polarization of BBs by coordinating apical actin assembly ( Park et al . , 2006 , 2008 ) .", "It was further suggested that the PCP effector Fuz and Dvl2 control membrane trafficking and vesicle fusion at the BB essential for ciliogenesis ( Park et al . , 2008; Gray et al . , 2009 ) .", "Studying Fltp function in the ALI primary cell culture system allowed us to link the onset of Fltp expression to the time of BB amplification , docking , and cilia formation .", "In MCCs , we have shown a close association of Fltp with the apical actin–MT cytoskeleton and BBs .", "Fltp localization at the apical surface is reminiscent of Dvl , Sec8 , and Intu localization in frog multiciliated mucociliary epithelium ( Park et al . , 2006 ) .", "Lack of Fltp function leads to BB docking defects and reduced number of cilia formed at the apical PM in ALI cultures and in vivo .", "Fltp physically interacts with Dlg3 and the core PCP molecule Dvl2 , which regulates BB transport together with proteins of the secretory pathway ( Park et al . , 2008 ) .", "We have previously shown that Dlg3 is the only Dlg family member that is monoubiquitinated by Nedd4 and Nedd4-like E3 ligases .", "This is required for apical PM transport mediated by the motor protein Dynein IC and Sec8 , a component of the exocyst complex ( Van Campenhout et al . , 2011 ) .", "Thus , taken together , these findings suggest that Fltp cooperates with Dlg and Dvl family members to regulate apical BB transport and docking of BBs in multiciliated tissues .", "However , there is likely not a strong parallel between BB docking in MCCs and BB positioning in the IE , a topic which needs to be addressed in the future .", "To better understand the function of Fltp , we further focused our analysis on the IE as a well-described model for PCP .", "It was previously shown that core PCP molecules localize to distinct apical membrane compartments , but differential localization seems not sufficient to instruct morphogenesis of actin-rich hair bundles ( Jones and Chen , 2008 ) .", "Instead , BB as well as kinocilium formation and positioning is essential for hair bundle morphogenesis , which is regulated by opposing localization of spindle positioning proteins and apical polarity proteins ( Ezan et al . , 2013; Tarchini et al . , 2013 ) .", "mInsc , mPins/LGN , and Gαi localize in a microvilli-free zone ( bare zone ) at the lateral side , whereas the apical polarity complex consisting of Partitioning defective 3 and 6 ( Par3 , 6 ) as well as atypical protein kinase C ( aPKC ) localize at the medial side .", "Surprisingly , localization of spindle proteins was not affected in the classical PCP mutants ( Ezan et al . , 2013 ) , which either suggests redundancy in the PCP pathway or alternative mechanisms that position spindle proteins and BBs in the IE .", "We provide evidence that Fltp and Dlg3 position BBs and stereocilia bundles in the IE in addition to Gαi , mPins/LGN , and mInsc , suggesting that we have identified important new molecules for BB/spindle positioning in mammals .", "Dlg3 and Fltp together surround the BB and anchor this organelle asymmetrically to the apical cortex likely via Dvl2 and TJ-associated proteins on the one side and attachment to the apical actin cytoskeleton on the other side .", "We have recently shown that the scaffold protein Dlg3 is important for apical polarity and TJ formation by interaction with a number of TJ-associated proteins ( Van Campenhout et al . , 2011 ) .", "In the fruit fly , Dlg , Mushroom body defective ( Mud ) , and Pins are absolutely necessary for spindle positioning in various cell types in vitro and in vivo ( Bellaiche et al . , 2001; Johnston et al . , 2009; Bergstralh et al . , 2013 ) .", "The co-localization of Gαi , mPins/LGN and mInsc , and Dlg3-Venus in the bare zone further suggests that also in mammals these proteins act in concert to position BBs and spindles .", "Furthermore , the basolateral polarity complex consisting of Dlg , Lgl , and Scrib is known to establish membrane domains by reciprocal inhibitory interactions with the apical aPKC-Par3/6 polarity complex and to regulate A–B spindle positioning ( Knoblich , 2008; Siller and Doe , 2008 ) .", "These reciprocal inhibitory interactions of the apical and basolateral polarity complex at the apical surface further stabilized the positioning of BBs at the apical PM .", "But how spindle positioning complexes are linked to the actin cytoskeleton is still a mystery in mammals .", "In yeast , MT plus ends are captured by Kar9a , which binds directly to a type V myosin motor bound to actin filaments ( Korinek et al . , 2000; Lee et al . , 2000 ) .", "As described above , Fltp functions very likely as a molecular adaptor protein localized between the interface of spindle positioning complexes and the apical actin cytoskeleton .", "Loss of Fltp function leads to disconnection of MT-based kinocilium and actin-based stereocilia bundles .", "Together , this suggests that Fltp couples BB/spindle positioning proteins to core PCP molecules and to the actin cytoskeleton in the IE .", "Further studies have to be conducted to precisely clarify how this complex is anchored to the cytoskeleton and core PCP components .", "Knock-out analysis of Fltp revealed an important function in the terminal differentiation of multiciliated lung cells and the morphogenesis of stereocilia bundles on IE HCs .", "This emphasizes that PCP acquisition and remodeling of the actin- and MT-based cytoskeleton is crucially important for the maturation and function of terminally differentiated cell-types in tissues and organs .", "Consequently , mutations of both core PCP and effector molecules lead to a wide spectrum of ciliary dysfunction syndromes ( Gerdes et al . , 2009; Wallingford , 2012 ) .", "For instance , in primary cilia dyskinesia , lack of directed mucociliary clearance and recurrent respiratory tract infections can eventually progress to permanent lung damage ( Storm van's Gravesande and Omran , 2005 ) .", "The Fltp knock-out mouse shows BB docking and cilia formation defects in MCCs of the lung as well as terminal airway constrictions .", "This suggests that human FLTP might be mutated in lung disease , a hypothesis that we are currently testing with our clinical partners .", "Moreover , mutation of Fltp leads to a stereocilia morphogenesis defect in the cochlea of the IE , which suggests that mutation of FLTP can cause hearing loss in human .", "Mutations in human DLG3 are associated with X-linked mental retardation ( Lickert and Van Campenhout , 2012 ) and we have previously identified that the scaffolding protein Dlg3 regulates apical polarity and TJ formation as well as PCP in the IE ( Van Campenhout et al . , 2011 ) .", "In this study , we have shown that Fltp and Dlg3 cooperate in BB positioning in the IE .", "As Drosophila Dlg acts as a tumor suppressor regulating proliferation and asymmetric cell division ( Johnston et al . , 2009 ) , it is well possible that the Fltp–Dlg3 complex is involved in similar cellular processes in mammals .", "Indeed , we have evidence that Fltp regulates cell division of intestinal stem cells ( Böttcher and Lickert , in preparation ) , suggesting that we identified a novel molecule with brought implication for ciliary disease and stem cell-mediated tissue homeostasis ." ], [ "Mouse keeping was done at the central facilities at HMGU in accordance with the German animal welfare legislation and acknowledged guidelines of the Society of Laboratory Animals ( GV-SOLAS ) and of the Federation of Laboratory Animal Science Associations ( FELASA ) .", "Post-mortem examination of organs was not subject to regulatory authorization .", "Fltp antibodies were generated as described previously ( Lange et al . , 2012 ) .", "Two affinity purified polyclonal antibodies ( Fltp1 , Fltp116-1 ) against mouse Fltp using the peptide sequence: DNPDEPQSSHPSAGHT for Fltp1 and KPFDPDSQTKQKKSVTKTVQ for Fltp116-1 were raised in rabbit ( Pineda , Berlin , Germany ) .", "The Fltp1 epitope locates to the less well conserved C-terminal PRR ( Figure 1C , red empty box ) .", "The Fltp116-1 epitope ( Figure 1C , red filled box ) resides N-terminal to the Fltp1 epitope and is less conserved in human .", "Primary antibodies used were rabbit anti-Fltp1 ( Pineda , Berlin ) , rabbit anti-Fltp116-1 ( Pineda , Berlin ) , mouse anti-ZO-1 ( 33–9100: Invitrogen , Carlsbad , CA ) , mouse anti-α-Tubulin ( T6199; Sigma ) , mouse anti-acetylated Tubulin ( T7451; Sigma ) , mouse anti-γ-Tubulin ( ab11316; Abcam ) , rabbit anti-β-Catenin ( C2206; Sigma ) , chicken anti-GFP ( GFP-1020; Aves Labs ) , rat anti-tyrosinated Tubulin ( MAB1864; Millipore ) , rabbit anti-pericentrin ( PRB-432C; Covance ) , rabbit anti-Vangl1 ( HPA025235; Sigma ) , rabbit anti-Dvl2 ( 3216; Cell Signaling ) , and Alexa Fluor 546 Phalloidin ( A22283; Invitrogen ) .", "Immunostainings were performed as described in the Supplemental ‘Materials and methods’ .", "Western blot analysis was performed by standard procedures .", "Following antibodies were used; mouse anti-Flag ( A8592; Sigma ) , rabbit anti-Fltp1 ( Pineda , Berlin ) , rabbit anti-Fltp116-1 ( Pineda , Berlin ) , rabbit anti-Dvl2 ( 3216; Cell Signaling ) , mouse anti-γ-Tubulin ( ab11316; Abcam ) , mouse anti-GAPDH ( CB1001; Merck Bioscience ) .", "β-gal staining of whole-mount embryos and organs were performed as previously described ( Liao et al . , 2009 ) .", "Some tissues were further processed .", "Not BABB treated whole-mount embryos/organs were fixed , washed in PBS , and photographed .", "BABB treated embryos/organs were left in BABB for photographing .", "For SEM , inner ears were fixed in 2 . 5% glutaraldehyde in cacodylate buffer and then treated using standard procedures .", "The knock-in/knock-out construct was designed as shown in Figure 3A .", "5ʹ and 3ʹ HR for the Fltp gene were amplified by PCR ( 449 , 450 , 451 , 452 ) using a C57BL/6J BAC clone ( RP23-333P11 ) as template .", "These two PCR products were subcloned into the pL254 vector ( Liao et al . , 2009 ) .", "The resulting vector was digested with HindIII , SpeI and electroporated into electrocompetent EL350 bacteria containing the Fltp BAC clone to retrieve the WT sequence between PCR homology arms resulting in the Fltp retrieval vector .", "For cloning of the knock-in/knock-out cassette in pBKS− 5ʹ and 3ʹ HR for the knock-in into the ATG of exon , two of Fltp were generated by PCR ( 453 , 454 , 455 , 456 ) using the previously mentioned BAC as a template and subcloned into pBKS− using the introduced restriction sites , resulting in pBKS−-Fltp-HomArms .", "The targeting vector was generated by ligating the loxP flanked neomycin ( neo ) resistance cassette ( PL-452 ) ( Liu et al . , 2003 ) into the pBKS−-H2B-Venus-intron-SV40pA plasmid resulting in pBKS−-H2B-Venus-intron-SV40pA-loxP-bGHpA-neo-EM7-PGK-loxP ( pBKS−-H2B-Venus-neo ) .", "The T2A sequence from Thosea asigna virus was introduced into the NotI site of pBKS−-H2B-Venus-neo by annealing the following oligos 2A_fwd; 2A_rev , which created a NotI compatible overhang resulting in pBKS−-2A-H2B-Venus-neo .", "NLS-lacZ ( nuclear localization signal-β-galactosidase fusion protein ) was ligated into the pBKS−-2A-H2B-Venus-neo vector resulting in pBKS−-NLS-lacZ-2A-H2B-Venus-neo .", "To finish the minitargeting construct , we cloned pBKS−-NLS-lacZ-2A-H2B-Venus-neo into pBKS−-Fltp-HomArms ( both cut with NotI and SalI ) .", "The minitargeting construct was cut out by SacII and KpnI , electroporated in EL350 bacteria , and introduced into PL254 via bacterial homologes recombination resulting in the final targeting construct ( PL254-Fltp-NLS-lacZ-2A-H2B-Venus-intron-SV40pA-loxP-bGHpA-neo-EM7-PGK-loxP ) which was confirmed by sequencing and is ready for electroporating into embryonic stem ( ES ) cells ( after linearization by AscI ) .", "To generate targeted ES cells , we electroporated IDG3 . 2-F1 ES cells ( C57BL/6J x 129S6/SvEvTac ) ( Hitz et al . , 2007 ) with the AscI-linearized FltpZV targeting vector and neomycin resistant clones were selected using 300 µg/ml G418 ( Invitrogen ) .", "Homologous recombination at the Fltp locus was confirmed by Southern blot analysis of DraIII-digested genomic DNA using the Fltp 5ʹ-probe ( 620 bp ) ( 5ʹ S Fltp FWD XhoI; 5ʹ S Fltp REV XbaI ) .", "8 out of 100 clones ( in total: 22 positive clones out of 289 ) showed homologous recombination .", "Due to restriction fragment length polymorphisms ( RFLP ) of the Bl/6J and 129S6 alleles and an isogenic Bl6 targeting vector , homologous recombination occurred preferentially on the Bl/6J allele and reduced the size of the restriction fragment from 16 . 443 bp to 11 . 469 bp .", "The 129S6 WT band is smaller in size than the Bl/6J WT band and is not targeted .", "Both ES cell clones gave birth to Δneo animals , but for the analysis we concentrated on one clone and founded the animal colony on this .", "After deletion of the neo cassette mice were backcrossed to C57Bl/6J to eliminate the Cre allele .", "Only those mice negative for the Cre allele were used for backcrossings to C57BL/6NCrl , 129S6/SvEvTac , or CD1 and further analyses were carried out with FltpZVΔneo/+ mice backcrossed several generations in C57BL/6NCrl , 129S6/SvEvTac , or CD1 .", "The Dlg3-Venus mouse line was generated by electroporating a pCAG-Venus-Dlg3 construct into IDG3 . 2-F1 ES cells ( C57BL/6J x 129S6/SvEvTac ) .", "Positive ES cells were aggregated with CD1 morulae and the resulting chimeras gave germ-line transmission of the Dlg3-Venus allele .", "Isolation of mouse tracheal epithelial cells ( mTECs ) and air liquid interface ( ALI ) culture was performed as described in Vladar and Brody ( Vladar and Brody , 2013 ) .", "Mice were sacrificed and the trachea dissected out .", "The trachea was put into ice cold HamF12 medium supplemented with penicillin/streptomycin ( P/S ) .", "Tissue surrounding the trachea was removed under the dissecting microscope in HamF12 medium .", "The trachea was digested in HamF12 medium supplemented with 1 . 5 mg/ml pronase ( protease 14 ) at 4°C ON .", "On the next day the tube was put on ice , FBS ( fetal bovine serum ) was added to a concentration of 10% and the tube was inverted at least 12× .", "The tracheas were transferred into a new tube with HamF12 P/S 10% FBS medium and again inverted 12× .", "Tracheas of the same genotype can be pooled and centrifuged for 10 min at 4°C at 400×g .", "200 µl HamF12 P/S per trachea were supplemented with 0 . 5 mg/ml DNaseI and 10 mg/ml BSA ( bovine serum albumin ) , the trachea were resuspended and incubated for 5 min on ice .", "Subsequently , the tubes were centrifuged for 5 min at 4°C at 400×g .", "The cells were resuspended in MTEC basic medium ( DMEM F12 [#21331-020; Invitro] , 15 mM HEPES [1M stock] , 3 . 6 mM sodium bicarbonate , 4 mM L-Glut [200 mM stock] , P/S [100Glu× stock] , Fungizone [500× stock] ) with 10% FBS and incubated for 3–4 hr at 37°C .", "The supernatant was collected , centrifuged for 5 min at 400×g , and resuspended in 100–200 µl MTEC plus medium ( MTEC basic , 10 μg/ml insulin ( 5 mg/ml stock ) , 5 μg/ml transferrin ( 5 mg/ml stock ) , 0 . 1 μg/ml cholera toxin ( 100 μg/ml stock ) , 25 ng/ml EGF ( 25 μg/ml stock ) , 30 μg/ml BPE , 5% FBS , 0 . 01 μM RA ) for counting .", "75 . 000 cells per cm2 were seeded on a transwell filter in MTEC plus medium .", "The medium was changed every 2 days .", "When the cells were confluent the MTEC culture was changed to an ALI culture by removing the medium on top of the filter and providing only medium supply from the bottom .", "For the interaction analysis , we used HEK293T cells transfected with Fltp-TAP-TAG and as a control HEK293T cells only transfected with TAP-TAG .", "The cells were rinsed with warm PBS and lysed with 1 ml ice cold lysis buffer ( 50 mM Tris/HCl , pH 7 . 4 , 150 mM sodium chloride , 2 mM EDTA , pH 8 , 1% Nonidet P-40 , filtrate sterile ) per 10 cm dish on ice .", "Cells were spinned down for 10 min at 10000×g at 4°C .", "Lysates were cleaned by filtration through syringe filters ( MILLEX GP , 0 . 22 µm , Millipore ) .", "1 mg of the filtered lysate was incubated with 50 µl Strep-Tactin superflow resin ( IBA ) in 1 ml of lysis buffer for 1 hr at 4°C in an overhead tumbler .", "The protein–Strep-Tactin solution was spinned down for 30 s at 7000×g and transferred to microspin columns ( GE-Healthcare ) .", "The supernatant was spinned down for 5 s at 100×g , washed three times with 500 µl TBST ( 100 mM Tris/HCl , pH 7 . 4 , 1 . 5 M sodium chloride , 1 . 0% Tween20 ) , and centrifuged for 5 s , 100×g .", "The protein was eluted by 500 µl desthiobiotin elution buffer ( IBA ) and incubated for 10 min while mixing the resin for several times .", "The elute can now be used for western blot analysis .", "Dissected tissues were fixed in 4% paraformaldehyde ( PFA ) and cryoprotected by incubation in a sucrose gradient for at least 1 hr each ( 5% , 15% , 30% ) .", "Tissues were frozen in OCT ( optimal cutting temperature ) after which immunohistochemical staining was carried out on 8- to 12-µmthick sections , mounted on glass slides .", "Briefly , sections were rehydrated in PBS , permeabilised for 10 min in 0 . 1 M glycine/0 . 1% Triton X-100 in PBS , and blocked for 1 hr in 5% donkey serum/PBS-Tween 0 . 1% ( PBS-T ) .", "Finally , the sections were incubated with the primary antibody in blocking solution ON at 4°C .", "The slides were washed with PBS-T , incubated with the secondary antibody in PBS for 2 hr at RT , washed with PBS , incubated with DAPI , and finally mounted with ProLong Gold antifade reagent ( P36930; Invitrogen ) .", "Whole-mount immunohistochemistry was performed as previously described ( Nakaya et al . , 2005 ) .", "Briefly , embryos were isolated , fixed for 20 min in 2% PFA in PBS , and then permeabilized in 0 . 1% Triton X-100 in 0 . 1 M glycine pH 8 . 0 .", "After blocking in 10% FCS , 3% goat serum , 0 . 1% BSA , 0 . 1% Tween 20 for 2 hr , embryos were incubated with the primary antibody ON at 4°C in blocking solution .", "After several washes in PBS-T , embryos were incubated with secondary antibodies in blocking solution for 3 hr .", "During the final washes with PBS-T , embryos were stained with DAPI , transferred into 40% glycerol , and embedded between two coverslips using 120 µm Secure-Seal spacers ( S24737; Invitrogen ) and ProLong Gold antifade reagent .", "Whole-mount inner ears were isolated , fixed for 20 min in 4% PFA in PBS , then the cochlea was dissected out , permeabilized in 0 . 1% Triton X-100 in 0 . 1 M glycine pH 8 . 0 .", "After blocking in 10% FCS , 3% goat serum , 0 . 1% BSA , 0 . 1% Tween 20 for 25 min , ears were incubated with the primary antibody ON at 4°C in blocking solution .", "After several washes in PBS-T , embryos were incubated with secondary antibodies in blocking solution for at least 3 hr .", "During the final washes with PBS-T , embryos were stained with DAPI .", "Alternatively ears were fixed/permeabilized for 10 min in 0 . 25% glutaraldehyde , 3 . 7% PFA , 3 . 7% sucrose , and 0 . 1% Triton X-100 in PHEM buffer ( 60 mM Pipes , 25 mM Hepes , 5 mM EGTA , 1 mM MgCl ) , the cochlea was dissected out and again fixed/permeabilized for 10 min in the same solution as above .", "After PBS washes blocking as described above was carried out .", "Finally , ears were embedded in a spacer between two cover slips .", "Antibody staining of ALI cultures was performed by cutting out , washing ( PBS ) , and fixing ( ice cold MeOH , 4% PFA , or fixed/permeabilised in 0 . 25% glutaraldehyde , 3 . 7% PFA , 3 . 7% sucrose , and 0 . 1% Triton X-100 in PHEM buffer depending on the primary antibody used ) the Transwell membrane .", "Subsequently , membranes were treated as described above .", "Finally , membranes were embedded in a spacer between two cover slips .", "Genotyping of the FltpZV mouse line ( after Cre-mediated excision of the neomycin selection cassette ) was performed using the forward primer 566 and 418 as well as the reverse primer 565 .", "With an annealing temperature of 57°C and 35 cycles , this PCR amplified a WT product of 317 bp and a targeted of 387 bp .", "For genotyping of the Dlg3:Venus mouse line , the forward primer 180 and the reverse primer 181 were used .", "A PCR performed with an annealing of 60°C and 32 cycles amplifies a product of 312 bp .", "Genotyping of Dlg3 mouse line was performed using the forward primer 534 and the reverse primer 535 as well as the reverse primer 536 .", "Amplification of 33 cycles with an annealing of 58°C yielded a WT product of 535 bp and a 215 bp product for the targeted allele .", "The gene trap clone P038A02 ( R1 on a pure 129Sv6 genetic background ) was obtained from the German Gene Trap Consortium .", "Dlg3tm1Grnt/Y male and Dlg3tm1Grnt/+ female mice on a C57Bl/6 background were genotyped as previously described ( Cuthbert et al . , 2007 ) .", "For genotyping of the FltpZV; Celsr1Crsh mouse line , we first performed a PCR with the forward primer 779 and the reverse primer 780 resulting in a 321 bp band .", "Next , we purified the PCR product via the PCR purification kit and sequenced the product in both directions .", "The adenine of the WT sequence was replaced by a guanine in the mutated sequence .", "For the FltpZV genotyping , we used the protocol described above .", "Dissections of embryos and organs were carried out according to Nagy and Behringer ( ‘Manipulating the mouse embryo: a laboratory manual’ ) .", "Embryos were staged according to Downs and Davies ( Downs and Davies , 1993 ) .", "Tissues were dehydrated through a methanol/H2O series: 2 hr in 25% methanol/H2O , 2 hr in 50% methanol/H2O , 2 hr in 75% methanol/H2O , and ON in 100% methanol .", "Finally the tissue was transferred to BABB for clearing .", "Whole-mount in situ hybridization was performed as previously described and Fltp mRNA was transcribed from a sequence-verified cDNA clone ( Tamplin et al . , 2008 ) .", "After in situ hybridization or lacZ staining , the embryos were dehydrated via a methanol series ( 25% , 50% , 75% , 2 × 100% ) for 10 min per step .", "To clear the embryos , we incubated 2× in xylol for 5–10 min ( depending on the thickness of the specimen ) .", "For tissue penetration of the paraffin , the specimen was left in paraffin at 65°C ON .", "On the next day , they were transferred into fresh paraffin and incubated at 65°C ON .", "The embryos were orientated and embedded into a mold .", "The cooled down paraffin blocks were mounted onto a grid and sectioned on a microtome .", "The sections were mounted on glass slides , dried ON at 37°C , dewaxed in xylol ( 2 × 15 min ) , fixed with mounting medium , and covered with a cover slip .", "After one night at 4°C , the sections were ready for microscopical analyses .", "First , paraffin sections on glass slides were dewaxed twice for 15 min in xylene .", "An alcohol row ( 100% , 90% , 80% , 70% , 1 min each ) for rehydration followed and ended in H2O .", "Afterward , the slides were dipped into NFR for 1 min and thoroughly washed with dist .", "H2O .", "Then , a dehydration step followed within an afferent alcohol row ( 70–100% , see above ) .", "Finally , the slides were incubated twice in xylene for 15–30 min and once in Roti-Histol for 15–30 min .", "After incubation in Roti-Histol , slides were put on a paper towel , sprinkled with a few drops mounting medium , and covered with a cover slip .", "Statistical significance was calculated using Prism ( GraphPad Software ) .", "Circular statistics were done using MATLAB and R . Image acquisition was performed on a Leica DMI 6000 confocal microscope or on a Zeiss Lumar . V12 Stereo using an AxioCam MRc5 camera .", "Image analysis was performed with Leica LAS AF software , AxioVision ( Zeiss ) , and Imaris 7 . 6 . 4 ( Bitplane , Zürich , Switzerland ) .", "5ʹ S Fltp FWD: 5ʹ-NNNCTCGAGGAGCCCTTACGCACACTTAAG-3ʹ5ʹ S Fltp REV: 5ʹ-NNNTCTAGACGGGACATTAACTGCATCTTATCTGAGGTTG-3ʹ011: 5ʹ-NNNACTAGTAGGTAAGTGTACCCAATTCGCCCTATAG-3ʹ012: 5ʹ-NNNGGATCCACGCGTTAAGATACATTGATGAGTTTGGAC-3ʹ013: 5ʹ-NNNTCTAGAATGGTGAGCAAGGGCGAGGAGCTGTTC-3ʹ014: 5ʹ-NNNACTAGTTTACTTGTACAGCTCGTCCATGCCGAGAG-3ʹ025: 5ʹ-NNNGCGGCCGCGCCACCATGCCAGAGCCAGCG-3ʹ026: 5ʹ-NNNTCTAGACTTAGCGCTGGTGTACTTGGTGATGG-3ʹ340: 5ʹ-NNNGCGGCCGCGCCACCATGAACCTTGAAGCTCGAAAAACAAAG-3ʹ341: 5ʹ-NNNGGCGCGCCTTTTTGACACCAGACCAACTGGTAATGGTAGC-3ʹ449: 5ʹ-NNNGGCGCGCCAGTCAGGAAGTGGAAGAGAAGAACACAG-3ʹ450: 5ʹ-NNNAAGCTTACTAGTGTGGTGGAGTGCCTGTCTACATGTG-3ʹ451: 5ʹ-NNNAAGCTTCACGACAGTCAAAGCTGCAATAGAAC-3ʹ452: 5ʹ-NNNGGATCCGGTAATTTGGCAATTATAGAACTCAGGC-3ʹ453: 5ʹ-NNNCCGCGGAGCAGACTTAACTATGTTGGGGAAACAGC-3ʹ454: 5ʹ-NNNGTCGACGCGGCCGCTGTTTACACTTGTTGCCTGGCAACTG-3ʹ455: 5ʹ-NNNGTCGACGGTCCTAGTCTAGCTGAGGTCCAGATC-3ʹ456: 5ʹ-NNNGGTACCATGCTGTGGGAGTCACTGACATTCTTG-3ʹ 180: 5ʹ-GTGAACCGCATCGAGCTGAAGG-3ʹ181: 5ʹ-GAACTCCAGCAGGACCATGTG-3ʹ418: 5ʹ-AGCCATACCACATTTGTAGAGG-3ʹ534: 5ʹ-GGTCTCTGATGAAGCAGTGATTTTT-3ʹ535: 5ʹ-TGATGACCCATAGACAGTAGGATCA-3ʹ536: 5ʹ-CTAAAGCGCATGCTCCAGAC-3ʹ565: 5ʹ-CAGCATGGCATAGATCTGGAC-3ʹ566: 5ʹ-GAGGCTGACTGGGAACAATC-3ʹ779: 5ʹ-ACAACCTTTGGGCTCTCG-3ʹ780: 5ʹ-TATAGTCCCTCCGGACCTC-3ʹ" ] ]

[ "Planar cell polarity ( PCP ) regulates basal body ( BB ) docking and positioning during cilia formation , but the underlying mechanisms remain elusive .", "In this study , we investigate the uncharacterized gene Flattop ( Fltp ) that is transcriptionally activated during PCP acquisition in ciliated tissues .", "Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells .", "Furthermore , Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells .", "In these cells , the core PCP molecule Dishevelled 2 , the BB/spindle positioning protein Dlg3 , and Fltp localize directly adjacent to the apical plasma membrane , physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton .", "Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear .", "Taken together , the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types , ciliary disease , and asymmetric cell division ." ]

[ "Epithelial tissues are sheets of cells that line the surface of many parts of the body , including the airways and the inner ear .", "Small hair-like structures called cilia can be found on the top surface of many epithelial cells and are arranged in a precise , ordered pattern .", "Such patterning ensures that cilia can work in a co-ordinated manner , for example by beating together to help clearing mucus from airways .", "Cilia grow out from ‘basal bodies’ and , like many other important structures in a cell , these basal bodies must be oriented along the correct side of an epithelial tissue .", "This is achieved by ‘planar cell polarity signaling’ , which makes sure that the structures inside a cell are correctly aligned , and ensures that polarized cells themselves are correctly oriented across the epithelial tissue .", "Disruption of this signaling can result in developmental defects .", "Some proteins help to establish polarity in a cell by altering the cell's cytoskeleton—the structural support and transport network of the cell .", "A ‘core’ complex of proteins then coordinates how the cells are arranged throughout the epithelial tissue .", "Although many of the proteins involved in each of these roles are known , how they interact with each other to establish planar cell polarity remains poorly understood .", "Now , Gegg et al . report that , in mice , a protein called Flattop functions to position basal bodies—and thus cilia—by working together with another protein called Dlg3 .", "In mice that cannot produce Flattop , cilia formation is defective in the lung , and the cilia in the inner ear are positioned incorrectly .", "Gegg et al . found that in the inner ear , Flattop and Dlg3 physically interact with each other and two other proteins—including one of the core proteins involved in planar cell polarity .", "This protein complex then surrounds the basal bodies at the point where they connect to the cell's cytoskeleton .", "Future challenges will be to clarify how the protein complex anchors to the cytoskeleton and how it interacts with other core planar cell polarity proteins in the cells of the inner ear .", "It will also be important to see whether this protein complex fulfills a similar role in other ciliated epithelial tissues ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "cancer biology" ]

[ [ "Lung adenocarcinoma ( LUAD ) , the most common cause of cancer death worldwide , exhibits significant heterogeneity in tumor cell identity and overall differentiation state ( Travis et al . , 2011 ) .", "The state of LUAD differentiation correlates closely with prognosis , intrinsic therapeutic sensitivity , and drug resistance ( Campos-Parra et al . , 2014; Rotow and Bivona , 2017; Russell et al . , 2011 ) .", "Recent work by our lab and others has shown that the pulmonary lineage specifier NKX2-1/TTF1 is a central regulator of LUAD growth and identity ( Maeda et al . , 2012; Snyder et al . , 2013 ) .", "NKX2-1 is expressed in ~75% of human LUAD , and NKX2-1 negative tumors confer a worse prognosis than NKX2-1-positive tumors ( Barletta et al . , 2009 ) .", "The specific role of NKX2-1 in LUAD depends , in part , on the driver oncogene ( Maeda et al . , 2012; Skoulidis and Heymach , 2019; Snyder et al . , 2013 ) .", "The majority of LUADs harbor mutually exclusive mutations in driver oncogenes that signal through the mitogen-activated protein kinase ( MAPK ) pathway , including EGFR ( 14% of cases ) , KRAS ( 29% ) , and BRAF ( 7% ) ( early stage cases , reviewed in Skoulidis and Heymach , 2019 ) .", "In this disease , active forms of the RAS family of small GTPases stimulate the RAF/MEK/ERK kinase cascade to drive proliferation , survival , and invasion .", "Treatment-naive EGFR-mutant LUADs almost always express NKX2-1 , and genetically engineered mouse models ( GEMMs ) suggest that NKX2-1 is required for optimal growth of EGFR-mutant LUAD ( Maeda et al . , 2012 ) .", "Intriguingly , case reports suggest NKX2-1 expression can be lost when EGFR-mutant LUADs acquire drug resistance during a lineage switch to squamous cell carcinoma ( Levin et al . , 2015 ) .", "In contrast to patient tumors that harbor EGFR mutations , loss of NKX2-1 expression is seen in a subset of KRAS and BRAF mutant tumors ( Skoulidis et al . , 2015; Zhang et al . , 2015 ) .", "In a KrasG12D-mutant LUAD GEMM , Nkx2-1 deletion enhances tumor growth and causes pulmonary to gastric transdifferentiation ( Snyder et al . , 2013 ) , which is driven by the transcription factors FoxA1 and FoxA2 ( Camolotto et al . , 2018 ) .", "Stochastic NKX2-1 loss has a similar effect on a distinct Kras-mutant LUAD GEMM ( Maeda et al . , 2012 ) .", "Nkx2-1-deficient murine tumors closely resemble invasive mucinous adenocarcinoma ( IMA ) , a subtype of NKX2-1-negative human LUAD that also expresses gastric markers .", "Human IMAs often harbor mutations in KRAS ( 62% of cases ) , occasionally BRAF ( 3% , including point mutations and fusions ) , but rarely EGFR ( 0 . 6% ) ( Cha and Shim , 2017 ) .", "These studies suggest that there can be selective pressure for LUAD to either retain or downregulate NKX2-1 expression depending on the specific signaling networks activated by a given driver oncogene .", "MAPK signaling is generally considered to drive proliferation and survival in LUAD , and has become a therapeutic target in some contexts ( Ferrara et al . , 2020 ) .", "For example , small molecule inhibitors of BRAF and its downstream kinase MEK have recently shown clinical efficacy in BRAF-mutant LUAD ( Khunger et al . , 2018 ) .", "However , mechanisms underlying heterogeneous responses remain to be identified .", "MAPK signaling also regulates differentiation state in a variety of tissues , including the stomach , depending on the magnitude and context of its activity ( Mendoza et al . , 2011; Osaki and Gama , 2013 ) .", "We have previously shown that Nkx2-1 deletion in KRASG12D-driven LUAD leads to IMA with high levels of MAPK activity , as assessed by levels of active ( phosphorylated ) ERK1/2 ( pERK ) .", "In contrast , Nkx2-1 deletion in alveolar type-2 pneumocytes causes hyperplasia with low levels of pERK ( Snyder et al . , 2013 ) .", "IMA and NKX2-1-negative hyperplasia both express some common pan-gastric markers ( e . g . HNF4A ) , demonstrating that loss of NKX2-1 causes pulmonary to gastric transdifferentiation independent of elevated ERK activity .", "However , the cells in each lesion have a discrete morphology and form lesions with distinct architectures , suggesting that oncogenic signaling downstream of KRASG12D dictates the specific identity adopted by an NKX2-1-deficient lung cell .", "We have sought to dissect the role of NKX2-1 in Braf-mutant LUAD using a GEMM of this disease .", "Here , we show that NKX2-1 regulates cellular identity , oncogenic signaling , and response to MAPK pathway inhibition in Braf-mutant adenocarcinoma .", "Our data show that the level of MAPK pathway activity dictates the specific identity adopted by NKX2-1-negative tumor cells within the gastric lineage .", "This identity shift is mediated in part by WNT signaling , which is increased after MAPK inhibition in IMA models ." ], [ "To dissect the role of NKX2-1 in mutant BRAF-induced lung adenocarcinoma , we utilized two established mouse strains bearing recombinase-activatable alleles of BrafV600E .", "In the first mouse model , Cre-sensitive conditional alleles of BrafLSL-V600E ( Dankort et al . , 2007 ) , Trp53f/f ( Jonkers et al . , 2001 ) , Rosa26LSL-tdTomato ( Madisen et al . , 2010 ) , and Nkx2-1f/f ( Kusakabe et al . , 2006 ) were recombined in the mouse lung epithelium upon intratracheal delivery of virus expressing Cre recombinase .", "Recombination of these alleles simultaneously activates BRAFV600E and tdTomato expression while eliminating p53 and NKX2-1 expression .", "Mice of the genotype BrafLSL-V600E/+;Trp53f/f;Nkx2-1f/+;Rosa26LSL-tdTomato/LSL-tdTomato are hereafter referred to as BP mice and mice with the genotype BrafLSL-V600E/+;Trp53f/f;Nkx2-1f/f;Rosa26LSL-tdTomato/LSL-tdTomato are hereafter referred to as BPN mice .", "Initial evaluation of histological ( H and E ) and molecular features revealed mucin production and gastrointestinal differentiation state in Nkx2-1-deficient tumors , including expression of HNF4A , PDX1 , Gastrokine 1 , Cathepsin E , and Galectin-4 ( Figure 1A , Figure 1—figure supplement 1A ) .", "In lung tumors , NKX2-1 is required for sustained expression of pulmonary state marker genes such as those encoding the surfactant proteins ( Sftpb and Sftpc Snyder et al . , 2013 ) and the cell surface protein CD36 ( Camolotto et al . , 2018 ) .", "Accordingly , immunohistochemical analysis showed that mucinous NKX2-1-negative tumors in BPN mice lack expression of pro-surfactant proteins B and C as well as CD36 ( Figure 1—figure supplement 1B ) .", "Overall , NKX2-1-negative tumors in the BrafLSL-V600E/+ model bore a close resemblance to those previously seen in the KRASG12D GEMM and human IMA ( Snyder et al . , 2013 ) .", "Despite similar morphologic phenotypes in the BRAFV600E and KRASG12D models , Nkx2-1 deletion at tumor initiation had a profoundly distinct effect when combined with either of these oncogenes .", "Whereas Nkx2-1 deletion augmented tumorigenesis initiated by KRASG12D ( Snyder et al . , 2013 ) , it significantly impaired the early stages of BRAFV600E-driven tumorigenesis .", "Nkx2-1 deletion led to a lower tumor burden 8 weeks after initiation ( Figure 1B , Figure 1—figure supplement 1C ) and significantly prolonged overall survival ( Figure 1C ) .", "We also quantitated tumor grade to better characterize tumor progression in this study and determine the predominant tumor type at the time of euthanasia .", "For BP tumors , we utilized the grading criteria described in Winslow et al . , 2011 .", "Almost all tumors were grade 1–2 ( average ~98% of overall tumor burden ) , and grade three tumors were rare ( Figure 1D ) .", "As BPN tumors are morphologically distinct from BP tumors , we needed to develop a grading scheme specifically for the cellular morphologies observed in this genotype .", "Accordingly , we characterize endpoint BPN tumors as ‘low grade’ if they are essentially identical to BPN tumors at early timepoints: tumor cells contain abundant intracellular mucin , low nuclear to cytoplasmic ratio , and small nuclei with minimal pleomorphism ( Figure 1A ) .", "In contrast , we consider BPN tumors to be ‘high grade’ if they have diminished or absent mucin production , increased nucleus to cytoplasm ratio , and increased nuclear size and pleomorphism ( Figure 1—figure supplement 1D ) .", "Based on these grading criteria , we found that high-grade BPN tumors comprised a much greater proportion of the NKX2-1-negative tumor burden than low-grade BPN tumors ( ~74% vs . 26% ) ( Figure 1D ) .", "Of note , a small minority of tumor burden ( ~4% on average ) was NKX2-1-positive , indicative of a low rate of incomplete recombination ( Figure 1—figure supplement 2 ) .", "Thus , in this model , loss of NKX2-1 is compatible with tumor progression to a high-grade state , despite its impairment of tumor initiation .", "These data suggest that BP mice succumb primarily to an abundance of low-grade tumors .", "In contrast , the lower initial tumor burden in BPN mice provides more time for tumors to grow and progress to a high-grade state before compromising pulmonary function .", "In the KRASG12D model , we previously showed that inducing loss of lineage specifiers in established neoplasms can have distinct consequences when compared to lineage specifier loss at the time of tumor initiation ( Camolotto et al . , 2018 ) .", "Models that enable gene manipulation in established tumors may be more physiologically relevant to tumor progression than models in which all genetic perturbations are present during tumor initiation .", "We therefore used a dual recombinase system in the present study to assess the role of NKX2-1 in the established BRAFV600E lung adenocarcinomas .", "Mice harboring FlpO-recombinase-sensitive alleles of BrafFSF-V600E ( Shai et al . , 2015 ) , Trp53frt/frt ( Lee et al . , 2012 ) , and Rosa26FSF-CreERT2 ( Schönhuber et al . , 2014 ) were transduced with FlpO expressing adenovirus to recombine the above Flp-sensitive alleles and establish BRAFV600E-induced lung lesions .", "After 3 or 6 weeks of tumor growth , Nkx2-1f/f alleles underwent Cre-based recombination upon injection of mice with tamoxifen ( Figure 2A ) .", "To generate NKX2-1-positive controls , a cohort of mice with the same genotype were injected with vehicle ( corn oil ) .", "In contrast to vehicle controls , Nkx2-1 deletion drove transition of established tumors to invasive mucinous adenocarcinoma ( IMA ) ( Figure 2B , Figure 2—figure supplement 2A ) .", "Collectively , these results and previous work from other labs indicate that Nkx2-1 deletion promotes a cell lineage switch in LUAD driven by either KRASG12D or BRAFV600E ( Maeda et al . , 2012; Snyder et al . , 2013 ) .", "We next evaluated the effect of NKX2-1 loss on the growth of established BRAFV600E -driven tumors .", "At early timepoints ( 3 weeks after tamoxifen treatment ) , Nkx2-1 deletion had no significant effect on tumor burden ( Figure 2C ) .", "In contrast , Nkx2-1 deletion in established KRASG12D-expressing cells at the same time point greatly enhanced tumor burden , as described previously ( Figure 2D; Snyder et al . , 2013; Young et al . , 2011 ) .", "At this same timepoint , we also compared proliferation rates using MCM2 positivity in tumors and found that depletion of NKX2-1 did not impact cell proliferation ( Figure 2—figure supplement 1B , C ) .", "Long-term survival analysis revealed no significant difference between tumor-bearing mice treated with tamoxifen and controls ( Figure 2E ) .", "Histopathologic analysis of the survival study revealed that control mice harbored predominantly non-mucinous NKX2-1-positive tumors , whereas tamoxifen-treated mice harbored predominantly NKX2-1-deficient tumors , many of which were high grade , further supporting the notion that NKX2-1 loss is permissive for malignant progression in BRAF-driven lung neoplasia ( images and quantitation in Figure 2—figure supplement 1D , E ) .", "( A minority of tumor burden ~18% on average ) was NKX2-1-positive in the survival study ( Figure 2—figure supplement 2 ) .", "These data show that loss of NKX2-1 in established BRAFV600E lung adenocarcinoma is tolerated but does not augment tumor growth , in contrast to NKX2-1 loss KRASG12D-driven tumors .", "This likely explains why KRAS mutations are enriched in IMA ( relative to LUAD overall ) , whereas BRAF mutations are diminished in frequency , but not excluded altogether like EGFR mutations .", "We have previously shown that Nkx2-1 deletion augments ERK activity in KRASG12D-driven lung adenocarcinoma ( Snyder et al . , 2013 ) , and feedback inhibition of the MAPK pathway is known to be rate-limiting for the growth of KRASG12D-driven tumors in vivo ( Shaw et al . , 2007 ) .", "We therefore asked whether the Nkx2-1 deletion also alters MAPK activity in BRAFV600E-driven lung tumors .", "We initially hypothesized that pERK levels would be similar in BP and BPN tumors , thus explaining the fact that Nkx2-1 deletion does not augment BRAFV600E-driven lung tumorigenesis .", "Surprisingly , Nkx2-1 deletion in both the concomitant and sequential tumor models led to increased MAPK signaling downstream of oncogenic BRAF as assessed by IHC .", "Whereas NKX2-1-positive ( BP ) tumors generally exhibited weak , patchy staining for phosphorylated ERK1 and ERK2 ( pERK ) , we observed strong pERK staining in NKX2-1-negative ( BPN ) tumors ( Figure 3A , Figure 3—figure supplement 1A ) .", "In contrast , phosphorylated levels of MEK1/2 were similar in BP and BPN tumors , suggesting that activation of ERK1/2 in BPN tumors is largely due to regulation downstream of MEK1/2 activity ( Figure 3A ) .", "Moreover , in BPN tumors , we found increased phosphorylation of p90RSK ( a direct target of active ERK ) and 4E-BP1 and S6 proteins - downstream targets of mTORC1 , through which mTORC1 stimulates protein synthesis and cell growth ( Carriere et al . , 2011; Mendoza et al . , 2011; Roux et al . , 2007; Figure 3A , B; Figure 3—figure supplement 1B ) .", "We confirmed differences in degree of pathway activation between BP and BPN tumors by IHC quantitation ( Figure 3C , Figure 3—figure supplement 1B ) .", "Therefore , BPN tumors exhibit hyperactivation of both MAPK and mTORC1 oncogenic pathways .", "Importantly , several of these distinguishing features observed in vivo , such as the association between low NKX2-1 and high pERK levels are conserved in vitro in primary tumor spheroid cultures from autochthonous BP and BPN tumors ( Figure 3—figure supplement 1C ) .", "Finally , we performed IHC for pERK on a panel of mucinous ( n = 17 , see Materials and methods for inclusion criteria ) and NKX2-1-positive primary human lung adenocarcinomas ( n = 51 ) .", "Strong pERK staining was detectable in the majority of mucinous tumors ( 14/17 ) .", "Among positive cases , seven tumors exhibited diffuse staining ( >90% of tumor cells positive ) , and another seven exhibited partial positivity ( 20–90% of tumor cells positive ) .", "In contrast , NKX2-1-positive LUAD showed a trend toward lower pERK staining than the mucinous adenocarcinomas .", "Only 20% of NKX2-1-positive tumors were diffusely positive ( vs . 41% of mucinous tumors ) , and 35% of NKX2-1-positive tumors were negative for pERK ( vs . 18% of mucinous tumors ) .", "Quantitation and representative pictures are shown in Figure 3D , Figure 3—figure supplement 1D .", "The ERK phosphorylation site is more labile than epitopes recognized by antibodies that detect total protein levels .", "Therefore , staining patterns likely reflect both biologic heterogeneity and variable processing of clinical specimens .", "Nevertheless , these data show that the high levels of pERK in IMA mouse models can be observed in many cases of human IMA .", "The increase in MAPK activity we observed in both in vivo and in vitro models led us to ask whether NKX2-1 status influenced response to MAPK pathway inhibition .", "To investigate this , we directly compared the effect of RAF/MEK inhibitors on the growth and proliferation of autochthonous BP and BPN lung tumors .", "We chose dual inhibition of MEK and BRAF because this combination is the standard of care for BRAFV600E-mutant human lung adenocarcinoma ( Planchard et al . , 2016 ) and because in other genetically engineered mouse models driven by BRAFV600E example for thyroid cancer ( McFadden et al . , 2014 ) , the combination of the two inhibitors is more effective than either one alone at inhibiting the MAPK pathway in vivo .", "We found that administration of BRAFV600E inhibitor ( PLX4720 ) in combination with MEK inhibitor ( PD0325901 ) effectively suppressed ERK phosphorylation in lung tumors of both genotypes ( Figure 4A ) .", "Combined BRAF/MEK inhibition led to a dramatically lower tumor burden in both BP and BPN mice when assessed after 2 weeks ( Figure 4B ) and 4 weeks ( Figure 4C ) of MAPKi treatment .", "The decrease in tumor burden in BPN mice was greater by two-fold compared to that in BP mice , suggesting that BPN tumors were overall more sensitive to MAPK inhibition .", "In fact , we noted that residual MAPK-inhibited BPN tumors resembled hyperplasia induced by Nkx2-1 deletion alone with respect to both morphology and low pERK levels ( Figure 4A ) .", "NKX2-1-positive , BRAFV600E-driven lung tumors have been shown to regress when treated with MEK inhibitor , but regrow rapidly after drug cessation ( Trejo et al . , 2012 ) .", "We therefore asked whether drug-treated residual BP and BPN cells retain tumorigenic potential .", "Histopathologic analysis of BP and BPN tumors treated for 4 weeks with BRAF/MEK inhibitor followed by drug removal showed that the residual cells readily grew back over the course of 1–4 weeks , adopting a morphology similar to untreated tumors ( Figure 4—figure supplement 1A ) .", "To gain insights into the mechanisms of drug response in each model , we performed transcriptome profiling by sequencing RNA from whole tumors and single-cell RNA sequencing .", "Using the Cre-activated tdTomato reporter ( Madisen et al . , 2010 ) in the BrafLSL-V600E/+ model , we enriched tdTomato-positive tumor cells via fluorescence activated cell sorting ( FACS ) .", "Tumor cells were isolated from mice of both genotypes ( BP and BPN ) that had been placed on BRAF/MEK inhibitor-diet or control chow for one week .", "We first performed Principal Component Analysis ( PCA ) on RNA sequencing data from whole tumors using the top 500 most variable genes .", "This analysis revealed that replicate samples cluster together but that the four groups ( control and MAPKi-treated BP and BPN tumors ) could be distinguished from each other at the transcriptomic level .", "As represented in Figure 4D , the greatest source of transcriptome diversity was related to deletion of Nkx2-1 and is reflected by PC1 .", "PC2 was related to MAPK inhibition-imposed transcriptomic changes , which appeared to more strongly distinguish BPN tumors than BP tumors .", "Regardless of treatment , BP tumors expressed higher levels of pulmonary genes ( e . g . Sftpb and Sftpc , Figure 4—figure supplement 1B , Supplementary file 1 ) and BPN tumors expressed higher levels of gastric genes ( e . g . Pdx1 as well as a significant subset of HNF4A-target genes as revealed by the Illumina Correlation Engine [Figure 4—figure supplement 1C , Supplementary file 1] ) .", "Furthermore , Dusp6 , a transcriptional readout of active ERK , was significantly downregulated in MAPK-inhibitor-treated BP and BPN tumors compared to their respective controls ( Figure 4—figure supplement 1D , Supplementary file 2 ) .", "Despite these differences , NKX2-1 targets ( e . g . Sftpc ) were detected at higher levels than expected in sorted BPN tumor cells .", "We had previously observed that a minority of tumors in BPN mice were non-mucinous and retain NKX2-1 expression by IHC .", "We therefore inferred that a subset of these tumor cells may retain NKX2-1 due to incomplete recombination ( Figure 1 and data not shown ) .", "Since the presence of incomplete recombinants could compromise our ability to accurately analyze the transcriptional consequences of NKX2-1 deficiency , we performed additional single cell RNA-seq analysis of FACS-sorted BPN tumors ( following 1 week of BRAF/MEK inhibitor or vehicle treatment ) using the 10X Genomics platform .", "We predicted that this would enable us to circumvent the technical difficulty of resolving complete from incomplete recombinants and also investigate therapy response at the single-cell level .", "We characterized the transcriptome of 5065 control and 5563 MAPKi drug-treated single BPN cells ( Figure 4—figure supplement 1E ) .", "In our initial analysis , rare non-tumor cells clustered separately from tumor cells , and their identity was further validated by the expression of well-known stromal markers including Vim , Ptprc , Trpm5 , Pecam1 , Mgp , Cd79a , Itgam , Adgre1 , Cd3g , and Marco .", "Filtering against contaminating stromal , endothelial , hematopoietic , and low-quality cells reduced the dataset to a total of 6807 high-quality tumor cells for further analysis ( Figure 4—figure supplement 1E ) .", "We visualized these cells in reduced dimensionality using UMAPs and identified eight major tumor clusters ( Figure 4E , Supplementary file 3 ) .", "The majority of these cells ( 70% ) fell into three NKX2-1-low clusters ( 1-3 ) and expressed multiple gastric markers ( Figure 4F , Supplementary file 3 , 4 ) .", "Examination of bam files confirmed that the rare Nkx2-1 transcript counts in Clusters 1–3 correspond to residual flanking sequences from complete recombinants at the Nkx2-1 locus and did not contain reads mapping to splice junctions of Cre-deleted exons in the Nkx2-1 gene ( Figure 4—figure supplement 1F ) .", "Cluster three exhibited high expression of Aurkb , Plk1 , and other genes that suggest that these cells are in G2/M phase ( Figure 4F and see Figure 5 for more detail ) .", "Cluster 1 and cluster 2 overwhelmingly contain cells from control and drug-treated mice , respectively .", "In contrast , the remaining cells ( 30% ) exhibited frequent and abundant Nkx2-1 expression , including splicing from Cre-target exons indicating a failure to recombine ( Figure 4F , Figure 4—figure supplement 1F ) .", "These cells also frequently expressed the NKX2-1 target Sftpc ( Figure 4F ) .", "We interpret these cells , which fall into clusters 4–8 , to be the incomplete recombinants that we have directly observed microscopically and inferred from RNA-seq data from whole tumors .", "The variable levels of Nkx2-1 and Sftpc in clusters 4–8 ( Figure 4F ) suggests that some of these cells represent higher grade tumor cells that stochastically lose NKX2-1 activity and expression , as has been documented in KRASG12D-driven GEMMs ( Snyder et al . , 2013; Winslow et al . , 2011 ) .", "For example , we noted that cluster 5 cells express high levels of the embryonic marker Hmga2 , a previously characterized marker of high-grade tumor cells ( Figure 4—figure supplement 1G ) .", "Similar to cluster 3 , cluster 8 exhibited high expression of Aurkb , Plk1 , and other genes that suggest that these NKX2-1-positive tumor cells are in G2/M phase .", "In contrast to cluster 3 , we note that cluster 8 is comprised almost exclusively of untreated cells , consistent with cell cycle exit of MAPKi-treated cells that retain NKX2-1 ( Figure 4E , F and see Figure 5 for further analysis ) .", "We quantified differences in MAPK pathway activation between BP and BPN tumors in our single-cell sequencing data by applying the MEK activity transcriptional signature published in Dry et al . , 2010 .", "This analysis substantiates our conclusions that BPN tumors display higher MAPK signaling than BP tumors ( Figure 4—figure supplement 1H ) .", "Furthermore , using the Dry et al . signature , we find that RAF/MEK inhibition reduces pathway output in both genotypes ( Figure 4—figure supplement 1H ) .", "The MAPK pathway regulates proliferation by multiple mechanisms , including activating expression of the D-type cyclins , which can drive cells out of quiescence and into the cell division cycle ( Lavoie et al . , 1996; Tuveson et al . , 2004 ) .", "We therefore evaluated the impact of MAPKi drug treatment on cell cycle status of BP and BPN lung tumors using IHC for cell cycle markers as well as analysis of RNA-seq datasets .", "We first evaluated drug response by IHC for MCM2 , a helicase detectable throughout the cell cycle but not in quiescence ( G0 ) .", "In BP tumors , 2 weeks of MAPKi treatment led to a significant decline in the percentage of MCM2-positive cells ( Figure 5A , B ) .", "Additional IHC analysis showed a decline in the percentage of BP cells positive for BrdU incorporation and phospho-histone H3 ( pHH3 , M phase marker ) ( Figure 5—figure supplement 1A–C ) .", "Taken together , these data show that MAPKi treatment of BP tumors blocks proliferation and induces cell cycle exit , thereby increasing the proportion of cells in quiescence .", "In contrast to BP tumors , MAPKi treatment of BPN tumors led to a paradoxical increase in the percentage of MCM2-positive cells ( Figure 5A , B ) despite decreased tumor burden ( Figure 4B ) .", "In contrast , the percentage of BPN cells positive for BrdU and pHH3 declined to the same extent as BP cells after MAPKi treatment ( Figure 5—figure supplement 1A–C ) .", "These data suggest that even though BPN cell proliferation is impaired by BRAF/MEK treatment , most residual BPN cells fail to exit the cell cycle and enter quiescence in the absence of MAPK activity .", "Analysis of RNA-seq data from whole tumors further demonstrated that BP and BPN tumor cells exhibit a differential cell cycle response to MAPK inhibition .", "Drug-induced transcriptomic changes were highly distinct between genotypes .", "Pathway analysis ( via Illumina Correlation Engine ) demonstrated key differences between BP and BPN MAPK-inhibitor treated lung tumors in the most significant Gene ontology ( GO ) terms .", "Specifically , multiple cell cycle-related pathways decline in treated BP tumors , but not in treated BPN tumors , relative to their respective untreated controls ( Figure 5C; Supplementary file 5 , 6 ) .", "Instead , the top GO pathway terms in MAPK-inhibited BPN tumors relative to vehicle controls included pathways related extracellular reorganization and cell motility ( Figure 5D; Supplementary file 6 ) .", "We calculated cell cycle scores of control and MAPKi-treated tumor cells in scRNA-seq data using the Seurat package and phased cell cycle gene signatures previously defined in mouse cells ( Mizuno et al . , 2009 ) .", "The results of this analysis are consistent with the general conclusion that BP and BPN cells have distinct cell cycle responses to RAF/MEK inhibition ( Figure 5E ) .", "However , the Seurat cell cycle score approach is not well adapted to distinguish G0 from G1 phase cells .", "We therefore developed a novel methodology for analyzing quiescence in scRNA-seq data using diffusion mapping ( Haghverdi et al . , 2015 ) and the complete set of fine-scale cell cycle phase signatures from Mizuno et al . , 2009 , which includes both quiescent and cycling cells ( Figure 5—figure supplement 1D ) .", "Using this novel approach , cells with high expression of S-phase or G2/M-phase signatures mapped to successive extremes at the right of the cyclic graph ( Figure 5F ) , while the majority of cells ( >80% ) mapped to the left-hand portion corresponding to G0 and G1 signature enrichments ( Figure 5F ) .", "Remarkably , these scRNA-seq analyses also revealed NKX2-1-dependent effects of drug treatment on cell cycle status .", "Treatment of BP cells caused a marked redistribution toward the left hand extreme of the graph , which is most distal from cycling cells and is enriched for the G0 signature , while the distribution of BPN cells was minimally affected by MAPK-inhibition ( Figure 5G ) .", "Given the ability of the MAPK pathway to drive cell cycle entry by activation of D-type cyclin expression , we evaluated the expression patterns of Ccnd1-3 in whole tumor and single-cell RNA sequencing data .", "Suppression of MAPK activity led to decreased Ccnd1 transcript and protein levels in both BP and BPN tumors ( Figure 5H , Figure 5—figure supplement 1E , F ) suggesting that the MAPK pathway is a major activator of Ccnd1 expression in tumors of both genotypes .", "In contrast , Ccnd2 mRNA levels were significantly higher in BPN tumors than BP tumors , and BRAF/MEK inhibition in BPN tumors led to a further increase in Ccnd2 levels ( Figure 5H , Figure 5—figure supplement 1E ) .", "At the protein level , we have only been able to detect Cyclin D2 by IHC in MAPKi-treated BPN tumors ( Figure 5—figure supplement 1G ) .", "Ccnd3 levels were low and relatively stable across all four conditions ( Supplementary file 1 and 4 ) .", "These data raise the possibility that increased Cyclin D2 levels in BPN tumors upon MAPK inhibition might maintain CDK4/6 activity , thereby preventing cells from entering quiescence despite their MAPK-low state .", "To test this idea further , we used IHC to evaluate RB phosphorylation at S807/S811 , two sites that can be phosphorylated by CDK4/6 ( Figure 5I ) .", "Two weeks of combined BRAF/MEK inhibition significantly decreased the percent of phospho-RB-positive cells in BP tumors ( Figure 5I ) .", "In contrast , MAPK-inhibition caused no change in phospho-RB S807/S811 levels in BPN tumors .", "To evaluate specifically the potential role of Cyclin D2 in the cell cycle response to MAPK inhibitors , we knocked down Cyclin D2 in BPN organoids using two different shRNAs ( Figure 5—figure supplement 1H ) .", "We then transplanted these lines into NSG mice subcutaneously , allowed tumors to form and grow for 8 weeks , and treated mice with RAF/MEK inhibitor for 4 weeks .", "Histopathologic analysis revealed that organoid-derived tumors exhibited a triphasic morphology in vivo , including large cystic structures , glandular adenocarcinoma , and small foci of poorly differentiated adenocarcinoma .", "Cystic structures appeared to accumulate fluid at a variable rate , precluding the use of tumor size as a metric of drug response .", "We quantitated MCM2 levels in the glandular adenocarcinomas , the component with the greatest similarity to the autochthonous model .", "We found that MAPK inhibition failed to reduce the MCM2 rate in control adenocarcinomas , whereas the MCM2 rate declined in tumors with Cyclin D2 knockdown upon RAF/MEK inhibitor treatment ( Figure 5J ) .", "Although organoid-derived subcutaneous tumors do not perfectly mimic the autochthonous BPN model , these data provide additional orthogonal evidence that Cyclin D2 plays a role in the response of NKX2-1-negative lung adenocarcinoma to MAPK inhibition .", "Taken together , these data show that NKX2-1 status has a profound effect on the cell cycle response of BRAFV600E-driven lung tumors to targeted therapy .", "We propose that Cyclin D2 helps maintain CDK4/6 activity and RB phosphorylation in MAPKi-treated BPN tumors , thus preventing cell cycle exit .", "The inability of residual BPN cells to exit the cell cycle suggests that distinct therapeutic strategies may be needed to eliminate BP versus BPN tumor cells after MAPK inhibition .", "Further analysis of gene expression changes in BPN tumors revealed surprising switch-like changes in the expression of lineage markers associated with specific gastric cell types .", "BPN tumor cells normally express high levels of transcripts that mark surface mucous cells in the stomach , such as Muc5ac and Gkn1 .", "We find that these surface mucous cell markers decline significantly upon MAPK pathway inhibition ( Figure 6A ) .", "In parallel , two of the most highly upregulated genes in RAF/MEK inhibitor-treated BPN tumor cells were Pgc and Cym ( Figure 6A , Figure 4—figure supplement 1D ) , which encode digestive enzymes normally expressed in chief cells found at the base of the stomach gland ( Han et al . , 2019; Leushacke et al . , 2017; McCracken et al . , 2017 ) .", "Additional markers of murine gastric chief cells including Pga5 and Clps were also induced by MAPK inhibition in these tumors ( Figure 6A ) .", "Cym is expressed predominantly during the neonatal phase of mouse stomach development ( Chen et al . , 2001; Fernandez Vallone et al . , 2016 ) , suggesting that these MAPKi-treated tumor cells may adopt an immature chief cell-like phenotype .", "MAPK inhibition in NKX2-1-negative tumor cells was also associated with induction of several markers of chemosensory tuft cells , including the lineage specifier Pou2f3 ( Kaji and Kaunitz , 2017 ) as well as Gfi1b and Gnat3 ( Figure 6A ) .", "We next utilized gene set enrichment analysis ( GSEA ) to compare the transcriptome of drug treated BPN tumors to published/curated gene expression datasets for gastric epithelial cell types and tuft cells ( Haber et al . , 2017; Leushacke et al . , 2017; Montoro et al . , 2018; Ting and von Moltke , 2019; Zhang et al . , 2019; Supplementary file 7 , 8 ) .", "GSEA showed that signatures of both Lgr5-expressing gastric chief cells as well as tuft cells were significantly enriched in MAPK-inhibited BPN cells ( Figure 6B ) .", "A shift in expression of gastric lineage markers was also evident at the single-cell level .", "Our single-cell transcriptomics confirmed that gastric surface mucous markers like Gkn1 and Tff1 are downregulated in NKX2-1-negative cells by BRAF/MEK inhibitors ( Figure 6C ) , whereas chief markers were induced .", "Many BRAF/MEK inhibitor-treated BPN cells have higher levels of chief markers ( Cym , Pgc , Pga5 ) than the control BPN cells .", "This suggests true induction of transcription rather than simple selection for a pre-existing population of chief-high cells ( Figure 6C ) .", "In contrast , MAPK inhibition appears to increase the fraction of cells positive for the tuft cell marker Pou2f3 , rather than the absolute levels of transcript per cell ( though expression of this marker is low and somewhat sporadically detected in our single cell RNA-seq data ) ( Figure 6C ) .", "Tff2 transcripts , which mark progenitors located in the isthmus of the normal stomach ( Quante et al . , 2010 ) , were also increased in BPN cells after MAPKi treatment ( Figure 6C ) .", "Given this apparent lineage switch , we used IHC to evaluate a subset of lineage markers at the protein level in vivo .", "GKN1 protein ( surface mucous marker ) was abundant in control BPN tumors , but was depleted in MAPK inhibitor-treated BPN tumors ( Figure 6—figure supplement 1A ) .", "Alcian Blue staining for mucin production showed the same pattern as GKN1 protein .", "Control BPN tumors were entirely negative for Pepsinogen C , whereas a subset of drug-treated BPN cells were Pepsinogen C-positive ( Figure 6D , upper row ) .", "In contrast , a small subset of control BPN tumor cells were POU2F3 positive , and the fraction of positive cells was higher upon MAPK inhibition ( Figure 6D , lower row ) .", "These data are concordant with our single-cell analysis of these two markers in terms of changes in proportion of positive cells vs . induction of absolute levels on a per-cell basis .", "Both Pepsinogen C and POU2F3-positive cells were found in alveolar hyperplasia induced by Nkx2-1 deletion , consistent with the microscopic similarity between these hyperplasia and residual MAPKi-treated BPN tumor cells .", "As expected , all three gastric lineage markers evaluated ( GKN1 , Pepsinogen C and POU2F3 ) were undetectable in BP tumors .", "We also analyzed the durability of cell identity changes in BPN induced by BRAF/MEK inhibitors .", "IHC analysis demonstrated that GKN1 and mucin production ( Alcian Blue ) return to the same levels as controls within 2 weeks of drug-withdrawal after a month of treatment with BRAF/MEK inhibitors ( Figure 6—figure supplement 2 ) .", "At this timepoint , PGC becomes undetectable and the percentage of POU2F3-positive cells is similar to controls .", "Thus , based on these markers of major cell state , we conclude that BPN cells readily transition back to their original identity after drug withdrawal .", "We also examined POU2F3 by IHC in a panel of primary human LUAD tissues ( Figure 6—figure supplement 1B ) .", "We found that almost all IMA tumors ( 15/16 ) harbor a minority population of POU2F3-positive cells ( Figure 6—figure supplement 1B ) .", "Like in BPN tumors , these POU2F3-positive cells were relatively rare ( ~5% or less of tumor cells overall ) .", "In contrast , most NKX2-1-positive human lung adenocarcinomas were entirely POU2F3-negative ( 44/51 ) ( Figure 6—figure supplement 1B , C ) .", "Interestingly , we detected a minor population of POU2F3-positive tumor cells in seven NKX2-1-positive cases ( Figure 6—figure supplement 1B ) .", "This expands upon other recent observations that POU2F3 can be upregulated in specific subtypes of lung cancer ( Huang et al . , 2018 ) .", "Thus , a rare population of POU2F3-positive tumors cells is readily detectable in both human IMA and murine models .", "We next asked whether regulation of gastric lineage by the MAPK pathway is a general feature of IMA , or limited to BRAFV600E-driven tumors .", "To address this question , we generated organoids from autochthonous IMA models driven by KRASG12D ( KPN ) or BRAFV600E ( BPN ) .", "IHC for the tdTomato reporter on sections of primary organoid cultures ( Figure 6—figure supplement 1D ) showed that all cultures examined are ~90–100% positive for tdTomato .", "Rare tdTomato-negative organoids have an exclusively epithelial morphology , and thus are unlikely to represent stromal cell contamination in these cultures .", "We treated BPN and KPN organoids with a MEK inhibitor ( Cobimetinib ) for 3 days and found that this was sufficient to stimulate the expression of chief ( Cym , Gif , Pgc ) and tuft ( Pou2f3 and Gfi1b ) cell markers in both KPN and BPN organoid cultures ( Figure 6—figure supplement 1E , F ) .", "Furthermore , induction of chief cell markers in BPN organoids occurred rapidly following MEK inhibition ( 2 hr for Cym and 6 hr for Pgc ) , before any change in cell number occurred ( Figure 6E ) .", "Thus , the kinetics of Cym induction in vitro are more consistent with a true lineage switch rather than selection for a pre-existing Cym-high cells , and are also consistent with our interpretation of single-cell data from tumors in vivo .", "Finally , we treated BPN organoids with additional drugs that target MAPK signaling including the ERK-inhibitor GDC-0994 , MEK inhibitor PD0325901 , and the BRAF inhibitor PLX4720 , all of which induce Cym ( Figure 6—figure supplement 1G ) .", "In aggregate , our data show that drug-induced tumor phenotypic changes are not dependent on the non-specific effects of any one inhibitor .", "Thus , the MAPK pathway modulates gastric lineage in IMA cells driven by distinct oncogenes ( KRAS and BRAF ) and in distinct settings ( i . e . in vivo and in vitro ) .", "LGR5 marks a subset of normal murine chief cells that can function as facultative stem cells ( Leushacke et al . , 2017 ) .", "Lgr5 is also a canonical WNT target gene , and WNT signaling is thought to promote the regenerative capacity of LGR5-positive chief cells .", "Given that MAPK inhibitors led to an enrichment for the signature of LGR5-positive chief cells in BPN mice , we evaluated our gene expression data to determine whether this was indicative of a general increase in WNT signaling .", "We examined a set of WNT pathway genes derived from the ‘WNT signaling’ ontology category on AmiGO ( Carbon et al . , 2009 ) in our whole-tumor RNA sequencing of BP and BPN tumors .", "Several of these genes have previously been identified as direct transcriptional targets of canonical WNT signaling , including Axin2 , Lgr5 , Wnt11 , Fzd , Fgf9 , Notum , Sox9 , Lrp1 , Sox4 , Znrf3 , Wnt3a ( ‘positive regulation of canonical WNT signaling’ category on AmiGO , Carbon et al . , 2009 ) and were significantly activated in MAPK-inhibited BPN tumors ( Figure 7A ) .", "Indeed , applying Ingenuity Pathway Analysis ( IPA ) Upstream Regulator detection to RNA-seq data from whole tumors identified β-catenin as the second top upstream regulator underlying the gene expression changes between control and drug-treated BPN tumors ( Figure 7—figure supplement 1A ) .", "The activation z-score suggests an activated state of β-catenin due to MAPK-inhibition and a more inhibited state in control BPN tumors .", "Consistent with this , Regulatory Motif analysis in Illumina Correlation Engine uncovered significant differences in TCF1 binding site genesets between control and MAPKi-treated BPN tumors ( Figure 7—figure supplement 1B ) .", "WNT targets such as Axin2 ( Jho et al . , 2002 ) and Lgr5 ( Barker et al . , 2007 ) were also induced in our single-cell analysis ( Figure 7—figure supplement 1C ) .", "Although their raw values are at the detection limit in our scRNA-seq data , imputation analysis to recover false negatives further supports a robust increase in Axin2 and Lgr5 levels after BRAF/MEK inhibition ( Figure 7—figure supplement 1D ) .", "Intriguingly , the pattern of induction was distinct for these two WNT target genes ( Figure 7—figure supplement 1C , D ) .", "Cells with the highest levels of Lgr5 are found only in treated BPN tumors , a pattern consistent with our observations of chief cell markers ( Figure 6C ) .", "In contrast , we identified cells with high levels of Axin2 in both control and treated BPN groups , despite the fact that overall Axin2 levels are higher in treated BPN .", "This raises the question of whether WNT-high cells pre-exist in control BPN tumors , or whether MAPK inhibition leads to induction of WNT target genes in WNT-low cells .", "We therefore developed a WNT13 transcriptional signature ( see Materials and methods for derivation ) that integrates the WNT target genes induced by MAPK inhibitors in BPN tumors .", "Scoring of cells in scRNA-seq using this WNT13 signature showed a pattern more similar to Axin2 than Lgr5 ( Figure 7B ) .", "Taken together , these data suggest that WNT-high cells can be detected in control BPN tumors , at least as defined by a WNT13 signature or Axin2 levels .", "However , Lgr5-high cells are not detectable pre-treatment , showing that even WNT-high cells lack high level expression of certain target genes .", "This further supports our observations from analysis of gastric lineage markers that MAPK inhibition modulates the transcriptional cell state of BPN tumor cells ( including WNT target genes ) , and does not purely select for pre-existing populations .", "To further investigate this question , we performed in vitro time course experiments , which showed that MEK inhibition induces Lgr5 and Axin2 within 4 hr of treatment before changes in cell number or viability take place ( Figures 7C and 6E ) .", "In a different approach to study the emergence of WNT-high cells , we stably transduced a BPN organoid line with a WNT-reporter construct , 7TGP , that contains seven TCF binding sites driving eGFP expression ( Figure 7D , E ) .", "When cultured in standard WNT-pathway activating 50% L-WRN media , we found that these cells consisted of a mixture of high and low reporting cells ( WNTrep-high and WNTrep-low cells respectively ) .", "In contrast , most cells were WNTrep-low when cultured in 5% L-WRN media .", "We then sorted the WNTrep-low subpopulation from standard culture conditions ( 50% L-WRN media ) and found that these cells re-equilibrated to a mix of WNTrep-high and WNTrep-low cells when cultured in 50% L-WRN media ( Figure 7E ) .", "Furthermore , culture of the sorted cells in 5% L-WRN media reduced the WNTrep-high percentage , whereas MEK inhibitor increased the WNTrep-high percentage .", "Taken together , these experiments indicate that WNT signaling can be induced in WNTrep-low cells with rapid kinetics ( i . e . prior to a change in cell viability or selection ) .", "Although we cannot exclude the possibility that WNTrep-high cells tolerate MEK inhibition to a greater extent than WNTrep-low cells , these data argue that pure selection for a WNTrep-high subpopulation ( in the absence of changes in gene expression on a per cell basis ) is unlikely to account for our observations on the overall state change of tumors .", "Previous studies in other cancer types have documented negative feedback loops between the MAPK and WNT signaling pathways .", "Mechanistically , these have been reported to be mediated by changes in AXIN1 ( Zhan et al . , 2019 ) , TCF4 isoform levels ( Heuberger et al . , 2014 ) or phosphorylation of FAK ( Chen et al . , 2018 ) after MAPK inhibition .", "However , we have not been able to detect consistent changes in any of these parameters after treatment of our organoids with MEK inhibitors ( Figure 7—figure supplement 1E ) .", "To identify potential sources of WNT production , we analyzed the levels of Wnt transcripts in all cell clusters identified in scRNA-seq , including stromal cells ( see Figure 4—figure supplement 1E ) .", "As presented in Figure 7—figure supplement 2 , several Wnt genes were induced by MAPKi treatment in BPN tumor cells , including Wnt2b , Wnt4 , Wnt8b , and Wnt10b .", "Additional Wnt ligands such as Wnt2 , Wnt5b and Wnt6 were more highly expressed in stromal cells that were co-isolated during lung tumor preparation .", "Thus , there are multiple potential in vivo sources of WNT ligand , including both treated BPN tumor cells and the tumor stroma .", "WNT/β-catenin signaling plays an essential role in gastric epithelial patterning during embryogenesis and promotes chief cell differentiation ( McCracken et al . , 2017 ) .", "We therefore asked whether the WNT pathway played a role in the lineage switch induced by MAPK inhibition in IMA .", "We used two different approaches to modulate WNT signaling in organoid cultures .", "First , we cultured organoids in 5% conditioned media ( L-WRN ) , corresponding to a 10 fold reduction in the amount of exogenous WNT3A and R-spondin three in the culture media relative to standard , 50% L-WRN , conditions .", "Second , we used the small molecule LGK-974 , which blocks Porcupine-mediated posttranslational modification of WNT ligands that is required for their secretion and paracrine signaling .", "Levels of Axin2 and Lgr5 were lower in organoids cultured in 5% L-WRN than 50% L-WRN , and treatment with LGK-974 led to a further reduction in these transcripts suggesting endogenous WNT ligand production and signaling complements L-WRN media in these cultures ( Figure 8—figure supplement 1A ) .", "Consistent with prior analyses ( Figure 7C ) , MEK inhibitor stimulated canonical WNT signaling in BPN and KPN organoids as indicated by increased expression of the Axin2 and Lgr5 transcripts within the first 24 hr ( Figure 8—figure supplement 1A ) .", "The relative effect of this stimulation ( i . e . fold induction ) was similar whether the organoid media composition contained high or low levels of exogenous WNT3A .", "Induction of chief markers ( Cym , Pgc , Gif ) by Cobimetinib was partially impaired when WNT signaling was reduced by using 5% L-WRN media alone or in combination with LGK-974 ( Figure 8A ) .", "Levels of some chief markers ( e . g . Cym ) were responsive to WNT signaling levels even in the absence of MEK inhibitor .", "In contrast , tuft cell markers ( Pou2f3 and Gfi1b ) were largely unaffected by modulation of WNT signaling .", "These data show that one component of lineage switching ( induction of chief cell markers ) is partially WNT-dependent , whereas another component ( tuft cell marker induction ) is largely WNT-independent .", "Previously , our lab reported that FoxA1 and FoxA2 are required for gastric differentiation in mouse models of IMA , including surface mucous cell marker expression ( Camolotto et al . , 2018 ) .", "Here , using a novel NKX2-1-negative organoid line that harbors conditional alleles of Foxa1 and Foxa2 , we show that Foxa1/2 deletion abrogates the induction of chief cell markers ( but not tuft cell markers ) upon MEK inhibition ( Figure 8B , Figure 8—figure supplement 1B ) .", "Thus , the surface to chief gastric lineage switch is driven by both WNT signaling and FoxA1/2 activity .", "Finally , we asked whether WNT signaling might play a role in failure of BPN tumors to exit the cell cycle after RAF/MEK inhibition .", "WNT signaling contributes to oncogenesis in a variety of settings ( Zhan et al . , 2017 ) , including in NKX2-1-positive LUAD GEMMs driven by BRAFV600E ( Juan et al . , 2014 ) or KRASG12D ( Tammela et al . , 2017 ) .", "Further , β-catenin activation upon Apc deletion in the murine intestinal epithelium specifically upregulates cyclin D2 to augment proliferation and tumorigenesis ( Cole et al . , 2010 ) .", "This raises the possibility that activation of the WNT pathway might be sufficient to induce Ccnd2 in MAPKi-treated BPN tumors and thereby prevent cell cycle exit , despite the lack of MAPK activity .", "To investigate this possibility , we first correlated known signatures of MEK ( Dry et al . , 2010 ) and WNT activity ( ‘positive regulation of canonical WNT signaling’ category on AmiGO , Carbon et al . , 2009 ) with levels of Ccnd1 and Ccnd2 in our single-cell data .", "MEK activity was positively correlated to Ccnd1 levels across the single-cell data set .", "Conversely , Ccnd2 levels were inversely correlated to the MEK signature score and positively correlated to the signature for WNT activity ( Figure 8—figure supplement 1C ) .", "This suggests that Ccnd1 levels may be primarily dependent on MAPK activity , whereas Ccnd2 levels may be more directly dependent on WNT activity in this specific context .", "Given the relationship between WNT activity and Ccnd2 levels , we asked whether pharmacological WNT inhibition would be sufficient to drive RAF/MEK-inhibited BPN cells into quiescence .", "To address this question , we initiated lung tumors in a cohort of BPN mice and assigned mice to four groups at 6 weeks after initiation ( vehicle; MAPKi; LGK-974; or MAPKi + LGK-974 ) .", "MAPK inhibitor was administered for 4 weeks ( i . e . 6–10 weeks after tumor initiation ) and LGK-974 was administered for the last 2 weeks of MAPKi treatment ( i . e . 8–10 weeks after tumor initiation ) .", "Four weeks of treatment with RAF/MEK inhibitor led to a significant increase in the percentage of MCM2-positive cells ( Figure 8C ) , consistent with the effects we observed after 1–2 weeks of treatment .", "In contrast , treatment with LGK-974 alone had no effect on MCM2 in BPN tumors .", "Strikingly , additional treatment with LGK-974 was sufficient to block the induction of MCM2 by RAF/MEK inhibition in BPN tumors , resulting in an MCM2-positive rate similar to controls ( Figure 8C ) .", "This demonstrates that induction of WNT signaling after RAF/MEK inhibition acts to prevent residual BPN tumor cells from entering quiescence .", "Although the addition of short-term LGK-974 to RAF/MEK inhibition did not reduce tumor burden beyond RAF/MEK inhibition alone ( Figure 8—figure supplement 1D ) , our data show that the biology of residual tumor cells can be manipulated by modulating WNT signaling .", "We also assessed the status of β-catenin in drug-treated BPN tumors in vivo .", "Using an antibody that recognizes non-phosphorylated ( active ) β-catenin or a second antibody for total β-catenin ( Figure 8—figure supplement 2A , B ) , we found that vehicle and LGK-974-treated tumors exhibit predominantly membranous staining by IHC , with no evidence of nuclear β-catenin .", "MAPK inhibition alone elicited accumulation of β-catenin throughout the cell , including the nucleus .", "Levels of nuclear/cytoplasmic β-catenin in tumors diminished in the presence of combined MAPK and LGK-974 .", "These findings are consistent with the possibility that β-catenin mediates MAPK-inhibition-induced WNT pathway activation and lineage switching in BPN tumors .", "In contrast , control BP tumors had lower levels of active β-catenin than BPN tumors , and there was no appreciable increase in staining with MAPK inhibition .", "Finally , we performed a larger experiment in which we combined RAF/MEK inhibition with either WNT inhibition ( LGK-974 ) or CDK4/6 inhibition ( Palbociclib ) for 4 weeks .", "We analyzed a subset of mice at the end of 4 weeks of drug treatment and performed a survival study on the remaining mice .", "In mice analyzed immediately after the final dose of drug , we found that adding either LGK-974 or Palbociclib to RAF/MEK inhibitors increased the number of cells in quiescence compared to RAF/MEK inhibitor alone ( Figure 8D ) .", "Addition of Palbociclib to the RAF/MEK inhibitor also reduced the amount of phospho-RB ( S807/811 ) in the tumors ( Figure 8E ) .", "These data suggest a model in which elevated WNT signaling in MAPKi-treated BPN tumors prevents cell cycle exit by maintaining higher levels of CDK4/6 activity than in BP tumors treated with the same drug .", "Our gene expression data suggest that Cyclin D2 is the CDK4/6 partner that is most likely to maintain its activity in MAPKi-treated BPN tumors .", "Despite the short term effects of adding a third drug , we did not see a significant increase in survival when either LGK-974 or Palbociclib was added to RAF/MEK inhibitor ( Figure 8—figure supplement 1E ) .", "This indicates that driving residual BPN cells out of the cell cycle is not sufficient to prevent tumor rebound after drug cessation ." ], [ "LUAD progression is driven not only by activation of MAPK signaling but also by changes in cellular identity .", "However , the direct impact of dysregulated MAPK activity on LUAD identity itself remains an unexplored problem in lung cancer .", "This problem is important not only because of the correlation between cellular identity and intrinsic malignant potential , but also because lineage switching has become an increasingly recognized , common mechanism of resistance to therapies targeting the MAPK pathway .", "A better understanding of this phenomenon would provide new insights into the natural history of LUAD progression as well as the changes in cellular identity that occur as a result of targeted therapy .", "Analyses of the histologic spectrum of LUAD in patients and mouse models have identified two cellular differentiation programs that impose barriers to lung tumor progression ( Camolotto et al . , 2018; Snyder et al . , 2013; Winslow et al . , 2011 ) .", "Initially , NKX2-1/FOXA1/2 transcriptional networks maintain a well-differentiated pulmonary identity .", "We have previously shown that downregulation of NKX2-1 and subsequent relocalization of FOXA1 and FOXA2 to the regulatory elements of gastrointestinal lineage genes allows for loss of surfactant proteins , aberrant upregulation of HNF4A and conversion to gastric cell identity .", "Loss of both differentiation programs can lead to poorly differentiated tumors containing high levels of HMGA2 .", "Human IMAs are associated with adverse clinical outcomes and harbor a distinct spectrum of driver mutations .", "Compared to LUAD overall , KRAS mutations are much more common in IMA , whereas BRAF mutations are somewhat less frequent ( Cha and Shim , 2017 ) .", "There is emerging evidence that tumor suppressor gene mutations may also be distinct in IMA .", "For example , CDKN2A mutations appear to be more common than TP53 mutations in human IMA ( Shim et al . , 2015; Skoulidis et al . , 2015 ) .", "Intriguingly , a case report demonstrated that TP53 mutation correlated with progression of a human IMA to a higher grade adenocarcinoma with decreased mucin production ( Kawai et al . , 2019 ) .", "This suggests that TP53 mutations can occur in human IMA but can facilitate progression to a non-mucinous differentiation state .", "This mirrors what we have observed in BPN and KPN models , and further supports their clinical relevance .", "GEMMs have been valuable in explaining the distinct role of NKX2-1 in LUAD driven by KRAS and EGFR mutations ( Maeda et al . , 2012; Snyder et al . , 2013 ) .", "In the present study , we show that NKX2-1 is required for optimal LUAD initiation by BRAFV600E , but it is dispensable for growth and progression to high grade states in established tumors .", "We speculate that concomitant BRAFV600E activation and Nkx2-1 deletion leads to a high level of ERK activity that is not well tolerated by normal cells .", "BRAFV600E-driven neoplastic cells , which have already equilibrated to moderately increased ERK activity , might more readily tolerate a further increase in ERK activity caused by Nkx2-1 deletion .", "In contrast to our results in the BRAFV600E models , loss of NKX2-1 consistently promotes tumorigenesis in KRASG12D-driven GEMMs .", "Taken together , these data explain why KRAS mutations are enriched in IMA , whereas BRAF mutations are not .", "It remains to be determined whether co-mutations can render NKX2-1 loss advantageous ( rather than neutral ) in BRAF-mutant LUADs .", "For example , activating codon 201 mutations in GNAS , which are predicted to drive cAMP-mediated Protein Kinase A ( PKA ) activity , occur more frequently in IMA than other LUAD subtypes ( Ritterhouse et al . , 2017 ) .", "Although functional studies of GNAS mutations in IMA have not been reported , codon 201 GNAS mutants function as oncogenes in KRAS-driven GEMMs of pancreatic cancer , which expresses some of the same foregut markers as IMA ( Patra et al . , 2018; Taki et al . , 2016 ) .", "In our RNA sequencing , several mRNAs encoding negative regulators of cAMP-mediated signaling to PKA , including Prkar2b , Rgs7 , and Adra2a ( Garg et al . , 2016; Ghavami et al . , 2004; Taylor et al . , 2008 ) , were significantly upregulated in BPN cells compared to BP .", "Interestingly , these transcripts were not elevated in Nkx2-1 deleted KRAS-driven tumor cells ( Camolotto et al . , 2018 ) and decreased cAMP-dependent PKA activity may be one reason why tumor initiation is impaired in BPN tumors .", "We have previously shown that the gastric differentiation state of IMA is driven by transcription factors such as FoxA1/2 and HNF4α .", "Here , we show that MAPK and WNT signaling provide an additional layer of regulation , modulating the specific cell type that IMA cells most closely resemble within the gastric lineage .", "Our data reveal that high MAPK activity drives a gene expression program characteristic of the surface mucous cells of the stomach .", "In contrast , low ERK levels , in concert with high WNT activity , activate gene expression signatures associated with other gastric cell types , including LGR5-positive chief cells and tuft cells .", "POU2F3-positive tuft-like cells have also been identified in pancreatic neoplasia , where they restrain tumorigenesis ( DelGiorno et al . , 2020 ) .", "Interestingly , hyperactivation of MAPK signaling ( via TGF-α mediated activation of EGFR ) is observed in Menetrier’s disease , a hypertrophic gastropathy characterized by hyperproliferation of isthmus progenitors and the preferential differentiation of these progenitors into surface mucous cells at the expense of parietal and chief cells ( Fiske et al . , 2009 ) .", "Blockade of EGFR results in regression of overgrown surface epithelium and restoration of lineage fidelity in progenitors .", "Importantly , our results identify crosstalk between the MAPK and WNT pathways in IMA tumors , as suppression of MAPK pathway rapidly activates WNT target gene expression .", "In the mammalian intestine , reciprocal gradients of WNT and phosphorylated ERK1/2 control the balance between proliferation and differentiation of intestinal stem cells ( Kabiri et al . , 2018 ) .", "Some molecular mechanisms driving the crosstalk between WNT and ERK signaling have been reported .", "In one study , reduction of MEK activity via gut-specific ablation of Ptpn11 decreased abundance of TCF4M/S isoforms while favoring the binding of β-catenin to TCF4E variants , which are more efficient drivers of WNT target gene activation ( Heuberger et al . , 2014 ) .", "Alternatively , treatment of BRAF mutant colorectal cancer models with MAPK inhibitors stimulated WNT pathway through the activation of cytoplasmic focal adhesion kinase ( FAK ) ( Chen et al . , 2018 ) or depletion of AXIN1 protein ( Zhan et al . , 2019 ) , resulting in the stabilization of β-catenin .", "Although we find no evidence for the above three mechanisms as mediators of ERK/WNT cross-regulation in our lung tumor model , we did identify three candidate genes induced by MAPK inhibition ( whole-tumor and single cell RNA-seq ) that might account for the increased WNT signaling: Dixdc1 , Sox4 and Sox9 ( Figure 7A ) .", "DIXDC1 ( aka CCD1 ) is a scaffolding protein that promotes assembly of Dishevelled into its biologically active , trimeric , form capable of interacting with AXIN and recruiting the destruction complex to the plasma membrane ( Liu et al . , 2011 ) .", "SOX4 is a transcription factor that can interact with AXIN-APC destruction complex and interfere with the phosphorylation of β-catenin , thereby increasing its stability ( Bhattaram et al . , 2014 ) .", "On the other hand , SOX9 can directly transactivate WNT target genes in specific contexts ( Ma et al . , 2016 ) .", "Our preliminary in vitro data indicates that these transcripts are induced by at least 1 . 5 fold within one hour of Cobimetinib treatment , preceding the upregulation of Axin2/Lgr5 , and continue to accumulate over the course of 24 hr .", "However , establishing whether any of these factors are required for the interplay between ERK/WNT signaling axes in drug-treated IMAs requires further experimental investigation .", "Our results also have translational implications for LUAD treatment .", "BRAF-mutant LUADs exhibit a heterogenous response to BRAF/MEK inhibition in clinical trials ( Planchard et al . , 2016; Planchard et al . , 2017 ) .", "Here , we identify NKX2-1 as a determinant of treatment response in a GEMM of this disease .", "In both NKX2-1-positive and NKX2-1-negative tumors , combined BRAF/MEK inhibition leads to substantial tumor regression , but drug-tolerant cells persist with the potential to relapse .", "In the absence of NKX2-1 , residual tumor cells fail to exit the cell cycle .", "The different cell cycle positions of residual BP and BPN tumor cells suggest that different therapeutic approaches will be needed to eradicate these cells .", "Indeed , we were able to identify factors mediating these differences , including elevated Cyclin D2-CDK4/6 activity and sustained phosphorylation of RB .", "We verified that the cell cycle position of MAPK-inhibited tumor cells can be manipulated by downregulating Cyclin D2 or blocking CDK4/6 activity via Palbociclib .", "However , these tripartite drug combination strategies were not efficacious in achieving durable tumor regression or survival benefit .", "It is interesting to consider these results in the context of two recent studies where the combinatorial effect of Palbociclib and MEK inhibitor ( Trametinib ) was tested in models of KRAS-driven NKX2-1-positive LUAD ( Ruscetti et al . , 2018 ) and in KRAS-driven pancreatic ductal adenocarcinoma ( PDAC ) ( Ruscetti et al . , 2020 ) .", "Consistent with our findings , Trametinib/Palbociclib co-administration in both types of mice elicited a potent inhibition of RB phosphorylation .", "In addition , combination therapy in KRAS-driven lung tumors resulted in prominent induction of cellular senescence and infiltration of tumors with activated NK cells , which drove tumor cell clearance , and markedly increased survival in dual-treatment mice compared to single agent treated mice ( Ruscetti et al . , 2018 ) .", "In PDAC models , however , dual treatment with Trametinib/Palbociclib elicited tumor senescence but not NK cell recruitment and failed to elicit robust tumor regression ( Ruscetti et al . , 2020 ) .", "Importantly , BPN tumors have a gastric/foregut-like differentiation state that is much closer to PDAC than to KRAS-driven NKX2-1-positive LUAD .", "Thus , our results expand upon these published observations that lineage state can influence the response to combined inhibition of MAPK and CDK4/6 activity .", "It appears that immune cell composition of NKX2-1-positive and NKX2-1-negative LUAD GEMMs can be distinct ( Mollaoglu et al . , 2018 ) .", "Thus , investigations into the tumor microenvironment of IMAs and how it is impacted by combination therapies may also provide important clues as to the mechanisms that maintain drug tolerant states .", "Additional work is clearly needed to identify specific vulnerabilities of residual IMA cells after MAPK inhibition .", "The lineage switch we observe in both KRAS and BRAF-driven IMA models is likely to be relevant to the identification of such vulnerabilities .", "MAPK inhibition causes IMA cells to undergo an identity shift from a surface mucous cell-like phenotype toward a more gastric stem cell-like state or tuft cell identity .", "These observations support the broader notion that tumor cell plasticity includes the capacity to explore myriad cell states from the developmental repertoire .", "Lineage switching may be particularly beneficial for tumor cells under the selective pressure of targeted therapy , as diverse lineage programs have distinct dependencies on specific growth and survival mechanisms .", "We speculate that cell identity changes within the gastric lineage may render residual IMA cells susceptible to specific therapeutic interventions .", "This would be reminiscent of prior studies of basal cell carcinoma of the skin showing that an identity switch confers resistance to inhibition of the Hedgehog pathway while rendering the cells susceptible to blockade of WNT signaling ( Biehs et al . , 2018; Sánchez-Danés et al . , 2018 ) .", "In summary , this study characterizes the context-dependent role of NKX2-1 in LUAD and identifies novel mechanisms of cell identity regulation and therapeutic response in this disease ( Figure 8F ) .", "The data provide new insights into the complex interplay between lineage specifiers , oncogenic signaling pathways and the susceptibility of lung cancer cells to targeted therapy .", "Thus , in the interest of developing fully effective therapies , these studies call for deep cataloguing of cell states sampled by tumor cells as well as their state-specific vulnerabilities ." ], [ "All animal work was done in accordance with a protocol approved by the University of Utah Institutional Animal Care and Use Committee .", "The following mouse strains were used .", "BrafLSL-V600E/+ ( Dankort et al . , 2007 ) , BrafFSF-V600E/+ ( Shai et al . , 2015 ) , Trp53f/f ( Jonkers et al . , 2001 ) , Trp53frt/frt ( Lee et al . , 2012 ) , KrasLSL-G12D/+ ( Jackson et al . , 2001 ) , KrasFSF-G12D/+ ( Young et al . , 2011 ) , Nkx2-1f/f ( Kusakabe et al . , 2006 ) , Rosa26LSL-tdTomato ( Madisen et al . , 2010 ) , Rosa26FSF-CreERT2 ( Schönhuber et al . , 2014 ) , Foxa1f/f ( Gao et al . , 2008 ) , Foxa2f/f ( Sund et al . , 2000 ) .", "All animals were maintained on a mixed C57BL/6J × 129SvJ background .", "Tumors were generated by administering mice with PGK-Cre lentivirus at 5 × 103 plaque forming units ( pfu ) /mouse , Ad5CMVFlpO at 2 × 107 pfu/mouse; Ad5CMVCre 2 . 5 × 107 pfu/mouse , Ad5SpcCre 5–8 × 108 pfu/mouse .", "Adenovirus was obtained from University of Iowa Viral Vector Core .", "Rodent Lab Diet ( AIN-76A ) was formulated with the vemurafenib-related compound PLX4720 at 200 mg/kg ( Tsai et al . , 2008 ) and PD0325901 7 mg/kg ( Trejo et al . , 2012 ) .", "Drug formulation was by Plexxikon and chow manufacture was by Research Diets .", "AIN-76A was used as control chow .", "Mice were maintained on the indicated drug pellets for the indicated time periods .", "BrdU incorporation was performed by injecting mice at 50 mg/kg ( Sigma ) intraperitoneally 1 hr prior to necropsy .", "Mice in survival studies were monitored for lethargy and respiratory distress , at which time animals were euthanized .", "LGK-974 and Palbociclib ( Selleck Chemicals ) were delivered to mice via oral gavage .", "LGK-974 was formulated in 0 . 5% ( w/v ) methylcellulose/0 . 5% ( v/v ) Tween-80 solution and given at 7 . 5 mg/kg dose with 5 days ON/2 days OFF schedule for 2 weeks ( Figure 8C , S8D ) or 4 weeks ( Figure 8D and E , S8E ) .", "Weights were monitored once or twice weekly and no toxicity was observed in mice .", "Palbociclib was formulated in 50 mM Sodium Lactate and initially given at 120 mg/kg dose with 5 days ON/2 days OFF schedule .", "Weights were monitored once or twice weekly .", "Due to weight loss in female mice receiving Palbociclib + MAPK-inhibitor chow combination , the following modifications were made for all mice receiving Palbociclib as single or dual agent .", "On week 2: 120 mg/kg dose with 4 days ON/3 days OFF schedule; week 3: Palbociclib was skipped altogether; week 4: 100 mg/kg dose with 4 days ON/3 days OFF schedule .", "Tumor-specific activation of CreERT2 nuclear activity was achieved by intraperitoneal injection of tamoxifen ( Sigma ) dissolved in corn oil at a dose of 120 mg/kg .", "Mice received a total of six injections over the course of 9 days .", "For survival experiments , mice were additionally given pellets supplemented with 500 mg/kg tamoxifen ( Envigo ) .", "HEK-293T cells were cultured in DMEM/High Glucose medium ( Gibco ) .", "L-WRN cells ( ATCC ) were cultured in DMEM/High Glucose medium containing G418 ( Sigma ) and Hygromycin ( InvivoGen ) media .", "All media contained 10% FBS ( Sigma ) .", "To eliminate mycoplasma contamination , cell lines were treated with 25 µg/mL Plasmocin ( InvivoGen ) for 2–3 weeks .", "To maintain cultures mycoplasma free , media were supplemented with 2 . 5 µg/mL Plasmocin .", "Cell line identity was authenticated using STR analysis at the University of Utah DNA Sequencing Core .", "Eight to 10 weeks after tumor initiation with PGK-Cre , tumor bearing mice were euthanized and lungs were isolated .", "Lungs were then minced under sterile conditions and digested at 37°C for 30 mins with continuous agitation in a solution containing the enzymes , Collagen Type I ( Thermo Fisher Scientific , at 450 U/ml final ) ; Dispase ( Corning , at 5 U/ml ) ; DNaseI ( Sigma , at 0 . 25 mg/ml ) and Antibiotic-Antimycotic solution ( Gibco ) .", "The digested tissue was passed through an 18 or 20-gauge syringe needle .", "Enzyme reactions were then stopped by addition of cold DMEM/F-12 with 10% FBS and the cell suspension was dispersed through 100 µm , 70 µm , and 40 µm series of cell strainers to obtain single-cell suspension .", "Subsequently , cell pellets were treated with erythrocyte lysis buffer ( eBioscience ) .", "Finally , cell pellets were reconstituted in Advanced DMEM/F-12 ( Gibco ) supplemented with L-glutamine , 10 mM HEPES , and Antibiotic-Antimycotic .", "Thereafter , 100 , 000 tumor cells were mixed with 50 µl of Matrigel ( Corning ) at 1 to 10 ratio and plated in 24-well plates .", "For the first week of organoid initiation , Matrigel droplets were overlaid with recombinant organoid medium ( Advanced DMEM/F-12 supplemented with 1X B27 ( Gibco ) , 1X N2 ( Gibco ) , 1 . 25 mM nAcetylcysteine ( Sigma ) , 10 mM Nicotinamide ( Sigma ) , 10 nM Gastrin ( Sigma ) and containing growth factors ( 100 ng/ml EGF [Peprotech] , 100 ng/ml R-spondin1 [Peprotech] , 100 ng/ml Noggin [Peprotech] , 100 ng/ml FGF10 [Peprotech] , as well as the ROCK inhibitor Y27632 [R and D Systems] and the TGF-β type I receptor-inhibitor SB431542 [R and D Systems] ) as described in Barker et al . , 2010; Sato et al . , 2009 ) .", "After the initial spheroid culture propagation , organoids were switched to 50% L-WRN conditioned media generated as detailed in Miyoshi and Stappenbeck , 2013 .", "Briefly , L-WRN cells ( ATCC ) seeded and maintained in 10% FBS , DMEM high glucose containing 500 µg/ml G418 ( Sigma ) and 500 µg/ml Hygromycin ( InvivoGen ) media until confluency .", "Once confluent , L-WRN cells were switched to Advanced DMEM/F-12 containing 20% FBS , 2 mM L-glutamine , 100 U/ml penicillin , 0 . 1 mg/ml streptomycin .", "Daily , conditioned supernatant medium was collected from L-WRN cultures , filtered through 0 . 2 µm vacuum filters and saved at −20°C as stock ( 100% ) L-WRN medium .", "To use for culturing spheroids , stock L-WRN was diluted with equal volume Advanced DMEM/F-12 ( final concentration 50% ) .", "Where specified , stock L-WRN media was diluted with Advanced DMEM/F-12 to a final concentration of 5% while still keeping FBS concentration at 10% as described in VanDussen et al . , 2015 .", "For drug studies , organoid media was supplemented with Cobimetinib ( GDC-0973 ) , GDC-0994 ( Genentech ) , LGK-974 , PD0325901 , PLX-4720 ( Selleck Chemicals ) at indicated concentrations .", "Drug containing media was refreshed every 48–72 hr .", "HEK293T cells were transfected with CRE-encoding lentiviral vector , d8 . 9 packaging vector and VSV-G envelope vector mixed with TransIT-293 ( Mirus Bio ) .", "Virus containing supernatant was collected at 36 , 48 , 60 , and 72 hr after transfection .", "Ultracentrifugation at 25 , 000 r . p . m . for 2 hr was necessary to concentrate virus for in vivo infection ( DuPage et al . , 2009 ) .", "For stable transduction of organoids , organoid cultures were first prepared into single cell suspensions by subjecting them to successive incubations with Cell Recovery Solution ( Corning ) and TrypLE ( Gibco ) .", "Cells were then resuspended in a 1:1 mixture , by volume , of 50% L-WRN and lentivirus containing supernatant .", "After supplementation with 8 µg/ml polybrene , cells were incubated for 2 hr .", "Cells were then pelleted , mixed back with Matrigel , and seeded .", "72 hr later , Puromycin selection for 1 week was performed to achieve stable lines .", "For subcutaneous allograft experiments , 3 × 105 single-cell suspension of 988 BPN organoid cells stably expressing either the Mission pLKO . 1-puro Non Target shRNA control ( shscr ) or one of the validated Ccnd2 Mission shRNAs , TRCN0000054764 ( #64 ) or TRCN0000054766 ( #66 ) , were mixed with in 1:1 vol with 50 μl Matrigel .", "Cells were injected subcutaneously into the right or left flanks of NOD/SCID-gamma chain deficient ( NSG ) mice .", "Tumor dimensions were measured with calipers .", "Tumor volume was calculated using the ( L x W2 ) /2 formula .", "When the majority of the tumors reached ~200 mm3 , mice were randomized into control or drug groups , the latter of which received RAF/MEK inhibitor compounded chow for 4 weeks .", "Tumor volume measurements were not reliable as the tumors formed from organoid cultures became fluid-filled cysts rather than solid .", "At end of treatment period , tumors were removed and processed for histological analyses .", "Total RNA was isolated using TRIzol followed by PureLink RNA Mini Kit ( Thermo Fisher Scientific ) .", "RNA was treated with RNase-Free DNAse I ( Invitrogen ) on-column .", "For low cell numbers ( e . g . after FACS ) , Animal Tissue RNA Purification Kit ( Norgen Biotek ) was used instead .", "RNA was converted to cDNA using iScript Reverse Transcription Supermix ( BioRad ) .", "cDNA was analyzed either by SYBR green real-time PCR with SsoAdvanced Universal SYBR Green Supermix ( BioRad ) or by Taqman real-time PCR with SsoAdvanced Universal Probes Supermix ( BioRad ) using a CFX384 Touch Real-Time PCR Detection System ( BioRad ) .", "Gene expression was calculated relative to Ppia ( loading control ) using the 2- ( dCt . x-average ( dCt . control ) ) method and normalized to the control group for graphing .", "Primer sequences and TaqMan Assays used in this study are listed below .", "For organoids , cells were seeded at a density of 2000 cells per 15 µl Matrigel dome , four domes per line per time point , each dome in a single well of a 96-well plate .", "After overnight culture , organoids were treated with different concentrations of the indicated inhibitors and incubated at 37°C for indicated times .", "Subsequently , the relative number of viable cells was measured by by CellTiter-Glo 3D Cell Viability Assay ( Promega ) , according to the manufacturer’s instructions .", "Luminescence was then measured by a microplate reader ( EnVision 2105 Multimode Plate Reader , PerkinElmer ) .", "Replicate values for each experimental group were averaged and all values were normalized to control treatment group for each line .", "Cells were lysed on ice using RIPA buffer ( 50 mM HEPES , 140 mM NaCl , 1 mM EDTA , 1% triton X-100 , 0 . 1% sodium deoxycholate and 0 . 1% SDS ) supplemented with protease and phosphatase inhibitor cocktails ( Roche ) .", "Protein extracts were clarified and concentrations were measured with Pierce Coomassie Plus Protein Assay reagent ( Thermo Fisher Scientific ) .", "Lysates were then resolved on SDS-PAGE gels ( BioRad ) , and transferred to Nitrocellulose blots .", "Membranes were probed with primary antibodies against AXIN1 ( 2087 , CST ) , TCF4 ( 2569 , CST ) pFAK Y397 ( SAB4504403 , Sigma ) , DUSP6 ( EPR129Y , Abcam ) , SPRY2 ( EPR4318 ( 2 ) ( B ) , Abcam ) , pERK ( 4370 , CST ) , tERK ( 4696 , CST ) , Vinculin ( EPR8185 , Abcam ) , and β-tubulin ( E7 , DSHB ) .", "Blots were subsequently incubated with HRP conjugated secondary antibodies .", "For signal development , Supersignal West Femto Substrate kit ( Thermo Fisher Scientific ) was used , followed by image acquisition using darkroom development .", "ImageJ was used for band intensity quantitation .", "Lung tissues were fixed overnight in 10% neutral buffered formalin , processed through 70% ethanol , embedded in paraffin , and sectioned at 4 µm thickness .", "Staining of hematoxylin and eosin , as well as detection of mucin by Alcian Blue were carried out at the HCI Research Histology Shared Resource .", "Immunohistochemistry was performed manually as detailed in Camolotto et al . , 2018 .", "The following primary antibodies were used .", "BrdU ( BU1/75 , Abcam ) , Cathepsin E ( LS-B523 , Lifespan Biosciences ) , Galectin 4 ( AF2128 , R and D Systems ) , Gastrokine 1 ( 2E5 , Abnova ) , Histone H3 pSer10 ( ab5176 , Abcam ) , HNF4A ( C11F12 , CST ) , MCM2 ( ab31159 , Abcam ) , Muc5AC ( SPM488 , Abnova ) , NKX2-1 ( EP1584Y , Abcam ) , PDX1 ( F109-D12 , DSHB ) , RFP ( 600-401-379 , Rockland Immunochemicals ) , pERK1/2 ( D13 . 14 . 4E , CST ) , pRSK S380 ( D3H11 , CST ) , p4EBP1 T37/46 ( 2855 , CST ) , pS6 S235/236 ( D57 . 2 . 2E , CST ) , pS6 S240/244 ( D68F8 , CST ) , pMEK1/2 S221 ( 166F8 , CST ) , Pepsinogen C ( HPA031718 , Sigma ) , POU2F3 ( HPA019652 , Sigma ) , pRB S807/811 ( 8516 , CST ) , Cyclin D1 ( SP4 , Abcam ) , Cyclin D2 ( DCS-3 . 1 , NeoMarkers ) , CD36 ( 324205 , R and D Systems ) , pro-SPB ( AB3430 , Millipore ) , pro-SPC ( AB3786 , Millipore ) , β-catenin ( 610153 , BD Biosciences ) , active β-catenin ( D13A1 , CST ) .", "To visualize Cyclin D2 signal , IHC was performed using the CSA II , Biotin-Free Catalyzed Amplification System ( Dako ) following manufacturer’s instructions .", "Pictures were taken on a Nikon Eclipse Ni-U microscope with a DS-Ri2 camera and NIS-Elements software .", "Tumor quantitation was performed on hematoxylin and eosin-stained or IHC-stained slides using NIS-Elements software .", "All histopathologic analyses were performed by a board-certified anatomic pathologist ( E . L . S . ) .", "De-identified formalin fixed , paraffin-embedded ( FFPE ) tumors were obtained from the Intermountain Biorepository , which collects samples in accordance with protocols approved by the Intermountain Healthcare Institutional Review Board .", "Mucinous tumors were included in the study based on the following criteria:", "1 . Diagnosis of invasive mucinous adenocarcinoma; OR", "2 . Diagnosis of mucinous bronchioloalveolar carcinoma ( older diagnostic term for IMA ) ; OR", "3 . Mucinous lung adenocarcinoma with strong morphologic features of IMA , such as abundant intracellular mucin .", "Colloid adenocarcinomas were excluded , as these are considered to be a discrete subtype of lung cancer .", "Tumor bearing lungs were isolated in ice-cold PBS .", "Lungs were enzymatically digested as described above in the procedure for establishing primary organoids .", "Once single-cell suspensions were obtained , cells were reconstituted in phenol red-free DMEM/F-12 with HEPES containing 2% FBS , 2% BSA and DAPI .", "Cells were sorted using BD FACS Aria for tdTomato-positive and DAPI-negative cells into DMEM/F-12% and 30% serum .", "After sorting , cells were pelleted and flash frozen or resuspended TRIzol then frozen at −80°C .", "To study the dynamics of WNT signaling in vitro , BPN organoids were stably transduced with the β-catenin/TCF-dependent GFP reporter lentiviral construct 7TGP ( Addgene plasmid #24305 ) .", "Single cells were isolated from Matrigel using Cell Recovery Solution and TrypLE incubations .", "Cells were analyzed on BD LSRFortessa or BD FACSAria and sorted on BD FACSAria .", "Events first passed through a routine light-scatter and doublet discrimination gates , followed by exclusion of DAPI-positive dead cells .", "Viable tumor organoids were further identified as tdTomato-high cells .", "7TGP organoids grown in 5% L-WRN media for 2–3 days were used as control for setting the GFP-negative gate .", "This gating strategy was applied to 7TGP organoids that were cultured in standard 50% L-WRN media in order to identify and sort for the WNT-reporter-low fraction .", "This fraction was collected in 50% L-WRN and expanded for ~1 week .", "After expansion , sorted 7TGP organoids were seeded and treated with 50% L-WRN ( ± Cobimetinib ) or 5% L-WRN ( ± Cobimetinib ) for 24 hr , 48 hr , and 72 hr .", "Then , the fraction of WNT-reporter-high population under the above four conditions was analyzed .", "Flow cytometric data analyses was performed using FlowJo software .", "Library preparation was performed using the TruSeq Stranded mRNA Library Preparation Kit with poly ( A ) selection ( Illumina ) .", "Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay .", "The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit .", "Individual libraries were normalized to 5 nM and equal volumes were pooled in preparation for Illumina sequence analysis .", "Libraries were sequenced on Illumina HiSeq 2500 ( 50 cycle single-read sequencing v4 ) .", "Adapters in raw FASTQ files containing 50 bp single-end reads were trimmed with cutadapt .", "QC metrics were generated for each sample with FastQC and Picard’s CollectRnaSeqMetrics after aligning reads to genome with STAR ( Dobin et al . , 2013 ) .", "QC metrics were summarized by MultiQC .", "From this QC analysis , one sample ( id 14489 × 13 ) did not group with other samples in the MultiQC report .", "It was later confirmed that very limited starting material was a contributing factor to its difference from the others .", "This sample was removed from downstream analysis because the noise in the sample was likely a larger negative trade-off than the gains from increasing biological replicates for that condition .", "Next , Salmon v0 . 9 . 1 quantified RNA transcript expression with two steps ( Patro et al . , 2017 ) ; first , the mouse transcriptome downloaded from Gencode ( release 26 ) was indexed using k-mers of length 19 with type ‘quasi’; second , Salmon quasi-mapped reads and corrected for sequence-specific bias ( see option –seqBias ) .", "Salmon-based transcript expression estimates were converted to gene expression estimates with R package tximport ( Soneson et al . , 2015 ) .", "Differential gene expression modeling with DESeq2 ( Love et al . , 2014 ) and EdgeR ( Robinson et al . , 2010 ) evaluated four distinct contrasts testing", "( i ) genotype effects within control groups [BP C vs . BPN C];", "( ii ) genotype effects within treatment groups [BP Tx vs . BPN Tx];", "( iii ) treatment effects within BP genotype [BP C vs . BP Tx];", "( iv ) treatment effects within BPN genotype [BPN C vs . BPN Tx] .", "Prior to fitting a single-factor DESeq2 regression ( four factor levels: BP C , BPN C , BP Tx , BPN Tx ) , genes were filtered if there were less than five total counts across all samples .", "PCA was done on the top 500 most variable genes subject to a regularized log transform to confirm within-group variation was similar .", "A Wald test was applied to the fitted model for each of the four contrasts specified above , where the null hypothesis was gene expression differences less than or equal to log2 ( 2 ) fold change in absolute value and the alternative hypothesis was that differences exceed log2 ( 2 ) .", "Significance of gene-wise differences was controlled by a false discovery rate of 10% ( Benjamini and Hochberg , 1995 ) .", "Differential gene expression modeling with EdgeR for volcano plots used package edgeR_3 . 24 . 3 with ggplot2_3 . 1 . 1 .", "Significance of gene-wise differences was calculated by tag-wise exact test .", "WNT signaling genes were delineated in the whole-tumor sequencing matrix using AmiGO pathway annotations ( http://amigo . geneontology . org ) for ‘Wnt Signaling’ ( Carbon et al . , 2009 ) .", "WNT genes were restricted to those annotated for Mus musculus with direct experimental evidence .", "The most differentially expressed genes between treated and untreated sample groups ( Bonferonni corrected Student’s t-test ) were thresholded at >2 fold change between means of treated and untreated samples .", "The 13 genes meeting this criterion and showing upregulation in RAF/MEK inhibitor-treated cells ( BPN Tx ) was designated as the WNT13 signature in this study .", "Gene set enrichment analysis ( GSEA ) was carried out on RNA-seq data from whole tumors ( and single cell data ( below ) ) by comparing gene expression profiles with archived gene sets from Hallmarks [ftp . broadinstitute . org://pub/gsea/gene_sets/h . all . v7 . 0 . symbols . gmt] and Oncogenic signatures [ftp . broadinstitute . org://pub/gsea/gene_sets/c6 . all . v7 . 0 . symbols . gmt] as well as with the cell type specific genes sets described in Haber et al . , 2017; Leushacke et al . , 2017; Montoro et al . , 2018; Zhang et al . , 2019 .", "All protocols to generate scRNA-seq data on 10x Genomics Chromium platform including library prep , instrument and sequencing setting can be found at: https://support . 10xgenomics . com/single-cell-gene-expression .", "The Chromium Single Cell Gene Expression Solution with 3’ chemistry , version 3 ( PN-1000075 ) was used to barcode individual cells with 16 bp 10x Barcode and to tag cell specific transcript molecules with 10 bp Unique Molecular Identifier ( UMI ) according to the manufacturer’s instructions .", "The following protocol was performed at High-Throughput Genomics Shared Resources at Huntsman Cancer Institute , University of Utah .", "First , FACS sorted single-cell suspensions of tdTomato-positive lung tumors were resuspended in phosphate buffered saline with 0 . 04% bovine serum albumin .", "The cell suspension was filtered through 40 micron cell strainer .", "Viability and cell count were assessed on Countess I ( Thermo Scientific ) .", "Equilibrium to targeted cell recovery of 6 , 000 cells , along with 10x Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell A ( PN-120236 ) to form Gel Beads-in emulsions ( GEMs ) , the nano-droplets .", "Within individual GEMs , cDNA generated from captured and barcoded mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min .", "Subsequent A tailing , end repair , adaptor ligation and sample indexing were performed in bulk according to the manufacturer’s instructions .", "he resulting barcoding libraries were qualified on Agilent D1000 ScreenTape on Agilent Technology 2200 TapeStation system and quantified by quantification PCR using KAPA Biosystems Library Quantification Kit for Illumine Platforms ( KK4842 ) .", "Multiple libraries were then normalized and sequenced on NovaSeq 6000 with 2 × 150 PE mode .", "Average expression of an 18 gene signature from Dry et al . , 2010 was determined per cell across the single cell data set to model MEK activation state .", "Enrichment for the WNT13 signature described above was similarly examined across the single-cell dataset ." ] ]

[ "Cancer cells undergo lineage switching during natural progression and in response to therapy .", "NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma ( IMA ) , a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes .", "In murine BRAFV600E-driven lung adenocarcinoma , NKX2-1 is required for early tumorigenesis , but dispensable for established tumor growth .", "NKX2-1-deficient , BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors .", "Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence , NKX2-1-negative cells fail to exit the cell cycle after the same therapy .", "BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage , which is driven in part by WNT signaling and FoxA1/2 .", "These data elucidate a complex , reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies ." ]

[ "When cells become cancerous they grow uncontrollably and spread into surrounding healthy tissue .", "As the cancer progresses different genes are switched on and off which can cause tumor cells to change their identity and transition into other types of cell .", "How closely tumor cells resemble the healthy tissue they came from can influence how well the cancer responds to treatment .", "Many lung cancers have an identity similar to normal lung cells .", "However , some turn off a gene that codes for a protein called NKX2-1 , which leads to a type of cancer called invasive mucinous adenocarcinoma ( or IMA for short ) .", "Cells from this type of cancer develop an identity similar to mucous cells that line the surface of the stomach .", "But it was unclear how IMA tumor cells that developed from a mutation in the BRAF gene are affected by this loss in NKX2-1 , and how transitioning to a different cell type impacts their response to treatment .", "To investigate this , Zewdu et al . studied lung cells from patients with IMA tumors driven by a mutation in BRAF and cells from mice that have been genetically engineered to have a similar form of cancer .", "This revealed that the NKX2-1 protein is needed to initiate the formation of cancer cells but is not required for the growth of already established BRAF-driven tumors .", "Further experiments showed that removing the gene for NKX2-1 made these cancer cells less responsive to drugs known as BRAF/MEK inhibitors that are commonly used to treat cancer .", "These drugs caused the IMA cancer cells to change their identity and become another type of stomach cell .", "This identity change was found to depend on two signaling pathways which cells use to communicate .", "This study provides some explanation of how IMA lung cancers that lack the gene for NKX2-1 resist treatment with BRAF/MEK inhibitors .", "It also shows new relationships between key genes in these cancers and systems for cell communication .", "These findings could lead to better therapies for lung cancer , particularly for patients whose tumor cells are deficient in NKX2-1 and therefore require specialized treatment ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "Developing neuronal dendrites and axons navigate through complex environments before establishing their final morphologies ( Tessier-Lavigne and Goodman , 1996; Dickson , 2002; Jan and Jan , 2010 ) .", "Extracellular cues provide neurites with instructive spatial signals to guide their growth , turning and branching decisions .", "Different neurons can interpret the same cue differently based on the receptors they express .", "Many guidance cues support both attractive and repulsive responses , which are mediated by different classes of receptors ( Tessier-Lavigne and Goodman , 1996 ) .", "Axon guidance is achieved not only through cell-specific expression of receptors but also by dynamic regulation of those receptors during neural development .", "A classical example of spatial and temporal regulation of guidance receptors is midline crossing in the Drosophila melanogaster central nervous system ( CNS ) .", "In the fly nerve cord , many neurons extend their axons across the midline to the contralateral side while others remain on the ipsilateral side ( Harris et al . , 1996; Kolodziej et al . , 1996; Mitchell et al . , 1996 ) .", "This process critically depends on the midline repellent Slit , its receptor Roundabout ( Robo ) and another protein Commissureless ( Comm ) ( Kidd et al . , 1998a; 1998b; 1999; Brose et al . , 1999; Wang et al . , 1999 ) .", "Slit is secreted by midline cells and repels growth cones expressing the Robo receptor .", "Longitudinal axons that do not cross contain high levels of Robo and are kept away from the midline ( Kidd et al . , 1999 ) .", "Comm , on the other hand , functions in the contralaterally projecting commissural neurons to keep the level of Robo low before commissural axons cross the midline , during which the growth cones need to ignore the repellent Slit .", "The expression of Comm diminishes after growth cones cross the midline , allowing the Robo receptors to localize onto the plasma membrane of postcrossing growth cones so that the axons can be repelled away from the midline and prevented from recrossing ( Keleman et al . , 2002 ) .", "It has been shown that Comm sorts the Robo receptors to endosomal pathways before they reach the plasma membrane ( Keleman et al . , 2002; 2005 ) .", "Similarly , in the vertebrate spinal cord , where no Comm homologue is found , differential responses to the midline repellant Slit is achieved by regulated expression and alternative splicing of the Robo3 receptor isoforms ( Sabatier et al . , 2004; Chen et al . , 2008 ) .", "Pre-crossing axons contain high levels of Robo3 . 1 , which prevents the activation of the Robo1 and Robo2 receptors , thereby allowing axons to grow toward the highly repulsive midline .", "Upon crossing , Robo3 . 2 expression is switched on , and it acts in concert with Robo1 and Robo2 to keep axons from recrossing the midline .", "It remains unknown , however , how neurons achieve such spatiotemporal specificity , and the mechanisms by which Comm and Robo3 are regulated remain unclear ( Dickson and Gilestro , 2006; Ypsilanti et al . , 2010 ) .", "We have previously reported that dendrites of the PVD neurons in Caenorhabditis elegans ( C . elegans ) follow precisely localized guidance signals SAX-7 and MNR-1 to form highly organized dendritic structures ( Dong et al . , 2013; Salzberg et al . , 2013 ) .", "SAX-7 is the ortholog of the vertebrate neuronal adhesion molecule L1CAM ( Zallen et al . , 1999; Chen et al . , 2001 ) .", "PVD neurons first grow two longitudinally extending 1° dendrites , from which numerous 2° dendrites emerge roughly perpendicular to the 1° dendrites ( Smith et al . , 2010; Albeg et al . , 2011 ) .", "Upon reaching two sublateral lines where the guidance molecule SAX-7 is highly enriched ( arrows in Figure 1A ) , the PVD dendrites form stereotyped 'T'-shaped 3° branches .", "SAX-7 and MNR-1 are both necessary and sufficient to instruct dendrite morphogenesis through direct interactions with the PVD receptor DMA-1 ( Liu and Shen , 2012; Dong et al . , 2013; Salzberg et al . , 2013 ) .", "SAX-7 , MNR-1 and DMA-1 form a tripartite ligand-receptor signaling complex that enables stereotyped dendritic branch formation and stabilization .", "A close examination of SAX-7 localization revealed that , in addition to the sublateral stripes where 3° branches form , SAX-7 was also enriched in a zone that roughly lined up with the 1° branches ( Figure 1A , arrowhead ) ( Dong et al . , 2013 ) .", "Since SAX-7 is expressed and localized well before PVD dendrite morphogenesis begins ( Dong et al . , 2013 ) , the 2° dendrites encounter the SAX-7 domain as they emerge from the 1° dendrite .", "In wild-type animals , the vast majority of 2° dendrites grow out of the SAX-7 domain to reach the sublateral line and form 3° branches there .", "How emerging 2° dendrites 'escape' one attractive SAX-7 domain but grow along another SAX-7 domain is conceptually similar to how the commissural neurons dynamically adjust their axon guidance response to Slit in Drosophila and mouse during midline crossing . 10 . 7554/eLife . 11008 . 003Figure 1 . The kpc-1 mutants showed severe trapping of PVD dendrites .", "( A ) Fluorescent images showing ( red ) morphology of the PVD neuron , ( green ) localization of SAX-7 in the hypodermal cell , ( blue ) seam cells and overlay between the three in wild-type worms .", "SAX-7 was enriched in two sublateral longitudinal lines and at the lateral midline around the seam cell-hypodermal junctions .", "Arrows: Sublateral stripes of enriched SAX-7 that co-localize with PVD 3° dendrites .", "Arrowhead: SAX-7 enriched near the 1°dendrites , where it was encountered by the 2° branches as they emerge .", "The images in the lower panels are zoomed-in views of the regions indicated by the boxes .", "Dotted lines indicate the 'trap zone' marked by enriched SAX-7 around seam cells .", "( B ) In kpc-1 ( gk8 ) mutants , almost all branches failed to grow out of the trap zone between the dotted lines indicated by enriched SAX-7 .", "Scale bar: 10 μm .", "( C ) Quantification of the percentage of 2° branches trapped around the 1°dendrite .", "*** is p<0 . 001 , n . s . is p>0 . 05 by Student’s T-test .", "N=50 for each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 00310 . 7554/eLife . 11008 . 004Figure 1—figure supplement 1 . Heat shock expression of KPC-1 at distinct time points during development produced different phenotypes .", "( A ) Expressing KPC-1 during L2 failed to rescue the kpc-1 ( gk8 ) mutant phenotype .", "( B ) Expressing KPC-1 during early L3 completely rescued the mutant phenotype .", "( C ) Expressing KPC-1 during L4 or later stages could partially rescue some menorahs , but still many branches were trapped in the trap zone .", "Scale bar: 10 μm .", "( D ) Quantification of the percentage of worms heat shocked at various time points showing no rescue , partial or full rescue . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 004 Two recent studies have identified the worm Furin homologue KPC-1 as another key regulator of PVD dendritic development ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) .", "KPC-1 is a member of the paired basic amino acid cleaving enzyme ( PACE ) family of proprotein convertases and is shown to be required autonomously in the PVD neurons for menorah formation .", "Genetic analyses showed that mutations in the SAX-7/MNR-1/DMA-1 complex were epistatic to kpc-1 mutations ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) .", "However , the functional mechanism of KPC-1 and its relationship with the receptor-ligand complex remain elusive .", "It is also unclear why weak hypomorphic alleles of kpc-1 exhibit defects in self-avoidance of the 3° branches ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) .", "Here , we show that KPC-1 , instead of promoting branch outgrowth as proposed in the previous papers , enables the dendrites to move away from the high level of SAX-7 at intermediate targets .", "Our genetic , biochemical , and cell biological analyses indicate that KPC-1 supports dendrite pathfinding by maintaining a proper level of the DMA-1 receptor on the plasma membrane of dendrites .", "Loss of KPC-1 causes unregulated and excessive DMA-1 , leading to erroneous dendrite guidance choices .", "These findings present a new mechanism that modulates the neuronal dendrites’ response to extracellular adhesion molecules through controlling the trafficking of dendrite guidance receptors ." ], [ "We reported previously that one component of the PVD dendrite guidance complex , SAX-7 , was enriched at specific sub-cellular locations in hypodermal cells ( Dong et al . , 2013 ) .", "As shown in Figure 1A , YFP tagged SAX-7 expressed in the hypodermal cell was highly enriched in two longitudinal sublateral stripes ( arrows ) which precisely co-localized with the PVD 3° branches labeled by mCherry .", "In addition to these lines , SAX-7 also showed strong enrichment at ring-shaped structures that likely represented the junctions between seam cells ( labeled by CFP using a seam cell-specific promoter Pnhr-81 ) and the major hypodermal syncytium ( Figure 1A , arrowhead ) .", "The seam cells are two rows of specialized hypodermal cells that line up along the lateral midlines of the worms and cover a region in which longitudinal PVD 1° dendrites extend ( Sulston and Horvitz , 1977 ) .", "The location of this SAX-7 zone dictated that PVD 2° branches would encounter an area with a high level of the adhesion molecule SAX-7 as they grow away from the 1° dendrite .", "Since PVD 2° dendrites do not elaborate extensive branches in this SAX-7-rich 'trap zone' , developing PVD dendrites appear to ignore SAX-7 here .", "We reasoned that disabling the normal escaping mechanism would lead to trapping of PVD dendrites within the zone .", "Indeed , in our forward genetic screen for mutants with defects in PVD morphology , we isolated three mutants , wy916 , wy920 and wy936 , which showed a dramatic phenotype in which PVD dendrites were completely trapped near the 1° dendrites .", "This phenotype resembled that of a previously reported mutant allele kpc-1 ( gk8 ) ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) .", "We confirmed that wy916 , wy920 and wy936 were alleles of kpc-1 by complementation test .", "In contrast to the wild-type 2° dendrites , which extended away from the 1° dendrite , 2° branches in kpc-1 mutants remained restricted to a narrow region close to the 1° dendrite ( Figure 1B ) .", "Co-labeling of the PVD neuron , hypodermal SAX-7-YFP and the seam cells revealed that the 2° dendrites in kpc-1 mutants were trapped inside the area with enriched SAX-7 ( Figure 1B ) .", "We quantified the percentage of 2° dendrites that failed to extend beyond the seam cell zone near the 1° dendrite and found that the kpc-1 mutants had a significantly larger portion of dendrites that were trapped in this zone than wild-type ( Figure 1C ) .", "Expressing KPC-1 in PVD using the PVD-specific ser2prom3 promoter fully rescued the dendritic defects , while expressing KPC-1 in hypodermal and muscle cells failed to rescue the phenotypes , consistent with previous reports that KPC-1 was expressed and functioned autonomously in the PVD neuron ( Figure 1C ) ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) .", "We next examined the temporal requirement for KPC-1 during dendritic development using the hsp16 . 48 heat shock promoter ( Stringham et al . , 1992 ) .", "Heat shock expression of KPC-1 at different developmental time points caused drastically different effects ( Figure 1—figure supplement 1D ) .", "Expressing KPC-1 during L1 or L2 larval stages before the outgrowth PVD 2° branches did not modify the kpc-1 ( gk8 ) mutant phenotype ( Figure 1—figure supplement 1A , D ) .", "Expression of KPC-1 during the early L3 stage , in contrast , during 2° branch outgrowth , produced robust rescue of all dendritic branches: 2° branches were able to grow out of the high SAX-7 region around the seam cells and formed full menorahs ( Figure 1—figure supplement 1B , D ) .", "Heat shock expression during later stages produced an altered phenotype in which the branches that were trapped and stabilized before KPC-1 expression remained trapped in the region while the newly developed branches could grow out and form 3° and 4° branches ( Figure 1—figure supplement 1C , D ) .", "These results agreed with a previous report using temporal RNAi ( Salzberg et al . , 2014 ) and demonstrated that KPC-1 was required during a stringent time window when the 2° branches synchronously bypassed the high level SAX-7 region to allow proper outgrowth .", "If the 2° dendrites in the kpc-1 mutants were indeed 'trapped' in the region by the locally enriched SAX-7 , we expected that removing the SAX-7 ligand , its cofactor MNR-1 , or their cognate receptor DMA-1 , would release the dendrites from the trap zone .", "Alternatively , if KPC-1 was required for dendritic outgrowth per se , double mutants between kpc-1 and any member of the ligand-receptor complex would show a PVD dendritic phenotype that was similar to that of the kpc-1 mutants .", "Consistent with previous reports ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) , we observed that the kpc-1; sax-7 , kpc-1; mnr-1 and kpc-1; dma-1 double mutants showed PVD morphologies indistinguishable from those of the sax-7 , mnr-1 or dma-1 single mutants ( Figure 2 , Figure 2—figure supplement 2 ) .", "Instead of remaining within a tight zone close to the 1° dendrites like in the kpc-1 single mutants ( Figure 2A ) , 2° dendrites of the double mutants extended further out in both dorsal and ventral directions ( Figure 2B , Figure 2—figure supplement 1 , 2 ) .", "Since the dendritic morphologies of the sax-7 , mnr-1 and dma-1 mutants were highly disorganized with many short ectopic 2° branches , there were more 2° branches in the 'trap zone' compared with the wild-type animals ( Figure 2—figure supplement 2H ) .", "Nevertheless , the percentages of trapped dendrites in the double mutants were comparable to those of single mutants of the tripartite complex but were significantly lower than that in the kpc-1 single mutants ( Figure 2—figure supplement 2H ) .", "This result suggested that defects in the kpc-1 mutants arose as a result of ectopic activation of ligand-receptor signaling near the seam cells . 10 . 7554/eLife . 11008 . 005Figure 2 . The SAX-7/MNR-1/DMA-1 tripartite complex was causal for the trapping phenotype in kpc-1 mutants .", "( A ) Left: Fluorescent image showing PVD morphology of a kpc-1 ( gk8 ) null mutant .", "Middle: Zoomed-in view of the boxed area in the left panel .", "Dotted lines indicate the 'trapping zone' with enriched SAX-7 .", "Almost all 2° dendrites were trapped in this region .", "Right: Schematic illustration of the phenotype .", "( B ) PVD morphologies of sax-7 ( nj48 ) ; kpc-1 ( gk8 ) double mutants were indistinguishable from sax-7 ( nj48 ) single mutants .", "Dendrites could escape from the trap zone .", "( C ) Expressing SAX-7 in the seam cells restored the trapping phenotype .", "( D ) Left: Fluorescent image showing the PVD morphology of a dma-1 ( wy686 ) ; kpc-1 ( gk8 ) double mutant .", "Middle: Schematic illustration showing the initial phase of 2° branch outgrowth during early L3 when the dendrites pass the 'trap zone' .", "Right: Later in development , dendrites of the dma-1; kpc-1 mutants had escaped the trap zone but failed to form menorahs due to lack of DMA-1 .", "( E ) Expressing DMA-1 during early L3 in dma-1 ( wy686 ) ; kpc-1 ( gk8 ) double mutants completely restored the trapping phenotype .", "( F ) Expressing DMA-1 during L4 or later stages generated a striking rescue of menorahs .", "Since the dendrites had already escaped from the trap zone , supplying DMA-1 enabled the dendrites to respond to sublateral SAX-7 and MNR-1 signal and form normal 3° and 4° branches at the right place .", "Scale bar: 10 μm .", "( G-H )", "Quantification of the percentage of 2° branches that were trapped around the 1°dendrite .", "*** is p<0 . 001 , n . s . is p>0 . 05 by Student’s T-test .", "N=50 for each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 00510 . 7554/eLife . 11008 . 006Figure 2—figure supplement 1 . The SAX-7/MNR-1/DMA-1 ligand-receptor complex was causal for the trapping phenotype in kpc-1 mutants .", "( A–D )", "Fluorescent images showing hypodermal SAX-7 localization in green and PVD morphology in red of ( A ) kpc-1 ( gk8 ) single mutant , ( B ) sax-7 ( nj48 ) ; kpc-1 ( gk8 ) , ( C ) mnr-1 ( wy758 ) ; kpc-1 ( gk8 ) and ( D ) dma-1 ( wy686 ) ; kpc-1 ( gk8 ) double mutants .", "In the kpc-1 single mutant , almost all 2° branches failed to grow out of the trap zone indicated by the enriched SAX-7 ( between dotted lines ) , whereas in double mutants , despite strong morphology defects , 2° dendrites grew out of the trap zone and extended toward the sublateral lines .", "Full length SAX-7::YFP was used in panels A , C and D while SAX-7ΔFnIII::YFP , a non-functional form of SAX-7 which still showed correct subcellular localization , was used in panel B to prevent rescuing of the sax-7 mutant phenotype .", "The images in the right 2 columns are zoomed-in views of the regions indicated by the dashed boxes on the left . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 00610 . 7554/eLife . 11008 . 007Figure 2—figure supplement 2 . SAX-7 , MNR-1 and DMA-1 were epistatic to KPC-1 . ( A–G ) Fluorescent images showing PVD morphologies of ( A ) kpc-1 ( gk8 ) , ( B ) dma-1 ( wy686 ) ; kpc-1 ( gk8 ) , ( C ) dma-1 ( wy686 ) , ( D ) sax-7 ( nj48 ) ; kpc-1 ( gk8 ) , ( E ) sax-7 ( nj48 ) , ( F ) mnr-1 ( wy758 ) ; kpc-1 ( gk8 ) and ( G ) mnr-1 ( wy758 ) mutants .", "The PVD dendritic phenotypes of the double mutants were indistinguishable from the sax-7 , mnr-1 and dma-1 single mutants but different from the kpc-1 single mutants .", "Scale bar: 10 μm .", "( H ) Quantification of the percentage of 2° branches that were trapped around the 1°dendrite .", "*** is p<0 . 001 , n . s . is p>0 . 05 by Student’s T-test .", "N=50 for each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 00710 . 7554/eLife . 11008 . 008Figure 2—figure supplement 3 . PLM and ALM neurons expressing SAX-7-YFP and MNR-1 caused the PVD dendrites of sax-7; kpc-1 double mutants to follow these neurons .", "( A–C )", "Fluorescent images of ( A ) overlay , ( B ) PVD neuron and ( C ) PLM and ALM neurons expressing SAX-7::YFP and MNR-1 .", "Right panels show the zoomed-in views of boxed regions in the left panels .", "Arrow points to multiple PVD dendrites fasciculating on the PLM neurite .", "Scale bar: 10 μm ( D ) Schematic illustration of the phenotype . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 008 Our 'trapping' model inferred that the SAX-7 molecule was required locally near the primary dendrite to restrict 2° branches around the 1° dendrite .", "We next directly tested this hypothesis by ectopically expressing SAX-7 in seam cells in kpc-1; sax-7 double mutants .", "In striking contrast to the double mutants themselves ( Figure 2B ) , the PVD dendrites in the kpc-1; sax-7 double mutants expressing SAX-7 in seam cells were trapped in the seam cell area , with very few 2° branches reaching far from the 1° dendrite ( Figure 2C , G ) .", "These results further supported the notion that the dendritic trapping phenotype in the kpc-1 mutants was due to the excessive activity of the SAX-7/MNR-1/DMA-1 complex near the seam cells .", "We also ectopically expressed SAX-7 and MNR-1 in the ALM and PLM neurons in the sax-7; kpc-1 double mutants ( Figure 2—figure supplement 3 ) .", "ALM and PLM neurons have long neurites that extend in the dorsal and ventral sublateral nerve cords and overlap with the locations where PVD 3° branches form and grow .", "This ectopic expression converted the sax-7 mutant-like dendritic pattern ( Figure 2B ) to a striking new pattern: the entire PVD dendritic arbor followed the PLM and ALM neurites ( Figure 2—figure supplement 3A ) .", "Similar to sax-7; kpc-1 , PVD morphologies of the double mutants between dma-1 and kpc-1 also clearly resembled the dma-1 but not kpc-1 single mutants , with disrupted organization and complete loss of menorahs but no trapping phenotype ( Figure 2D , Figure 2—figure supplement 1D , Figure 2—figure supplement 2B , C and H ) .", "The trapping model also predicted that resupplying DMA-1 to dma-1; kpc-1 double mutants during the outgrowth of 2° branches within the trapping zone would restore the trapping phenotype ( Figure 2D , middle panel ) while expression after the 2° branches reached beyond the trapping zone would not ( Figure 2D , right panel ) .", "To test this , we utilized the heat shock promoter to express DMA-1 at different time points in dma-1; kpc-1 mutants .", "Consistent with our hypothesis , providing DMA-1 during early L3 when the 2° branches grew within the trap zone led to robust restoration of the trapping phenotype ( Figure 2E , H ) .", "On the contrary , expressing DMA-1 at later time points , when most of the branches in dma-1 mutants had already extended beyond the trapping zone , led to a striking rescue of 3° and 4° branches ( Figure 2F , H ) , again demonstrating strongly that dendrites in the kpc-1 mutants did not lack outgrowth capability and maintained intact SAX-7/MNR-1/DMA-1 signaling .", "Instead , the kpc-1 mutant PVD dendrites responded excessively to the guidance cues SAX-7 and MNR-1 near the seam cells , which limited their growth to the 'trap zone' .", "In an independent genetic screen for mutants with PVD dendrite self-avoidance defects , we isolated another allele of kpc-1 , xr58 , which resulted in an amino acid substitution P440S ( Figure 3D ) .", "Unlike a previously reported mutation of the same amino acid ( kpc-1 ( my24 ) , P440L ) which gave rise to a null phenotype , the PVD neurons of the kpc-1 ( xr58 ) mutants showed a fully penetrant , complex phenotype ( Figure 3B ) ( Schroeder et al . , 2013 ) .", "Similar to the complete loss-of-function kpc-1 ( gk8 ) mutants ( Figure 1B , 3D ) , some 2° dendrites in kpc-1 ( xr58 ) mutants were trapped near the 1° dendrites ( Figure 3B , asterisks , Figure 1C ) .", "The 2° dendrite trapping phenotype was much weaker than that in the gk8 allele yet significantly different from that in the wild-type animals ( Figure 1C , 3A–B ) .", "Unlike the PVD dendrites of the kpc-1 null mutants , which failed to make any intact menorahs , many 2° dendrites in the kpc-1 ( xr58 ) mutants , especially those in the proximal region near the cell body , could successfully extend away from the 1° dendrite and formed full menorahs with T-shaped 3° branches and many 4° branches on them .", "However , the 3° branches in this mutant had a striking self-avoidance defect ( Figure 3B , arrowheads ) .", "In wild-type animals , 3° branches showed stringent self-avoidance .", "Neighboring 3° dendrites almost never overlapped with each other and instead showed gaps in between ( Figure 3A , arrows ) .", "The self-avoidance phenotype of the xr58 allele was similar to that of two previously reported weak alleles of kpc-1 ( Salzberg et al . , 2014 ) whose 3° dendrites remained in the same 2D plane but showed extensive overlap .", "We quantified the percentage of 'T'-shaped 3° dendrites that made contact with their neighbors and found that around 70% of 3° branches had self-avoidance defects in the kpc-1 ( xr58 ) mutants , compared to only 2% in wild-type worms ( Figure 5C ) .", "In addition to the self-avoidance defects of the 3° branches , kpc-1 ( xr58 ) also had a reduced number of 4° branches .", "Except in the most proximal menorah ( closest to the cell body ) , the 4° dendrites were largely absent from the menorahs ( Figure 3B , stars , Figure 5D ) . 10 . 7554/eLife . 11008 . 009Figure 3 . Partial loss of KPC-1 caused defects in higher order dendritic branches .", "( A–C )", "Fluorescent images showing PVD morphologies of ( A ) wild-type ( B ) kpc-1 ( xr58 ) mutant and ( C ) kpc-1 ( gk8 ) mutant animals expressing a low concentration ( 0 . 5ng/μL ) of full length , functional KPC-1 .", "The xr58 mutants had severe defects in self-avoidance of 3° branches and reduced number of 4° branches .", "The phenotype was mimicked by expressing low-level wild-type KPC-1 in the gk8 null allele of kpc-1 .", "Arrows: Gaps between 3° branches in wild-type neurons .", "Arrowheads: 3° branches that overlapped with their neighbors in mutants .", "Star: Defective menorah with no 4° branches .", "Asterisks: Trapped 2° branches .", "( D ) Schematic of the KPC-1 protein showing locations of mutations in gk8 and xr58 mutants .", "( E–F )", "Fluorescent images of ( E ) SAX-7 localization in the hypodermal cell , and ( F ) overlay with a PVD marker in red .", "SAX-7 was highly enriched in the sublateral lines where 3° branches formed and grew and was also localized to vertical stripes followed by the 4° branches but at much lower concentration .", "Scale bar: 10 μm .", "( G ) Schematic figure of PVD outgrowth .", "2° branches emerging from the 1° dendrites and 4° branches growing away from the 3° branches faced similar challenges to go from regions with higher levels of SAX-7 to places that were less attractive .", "Compromised function of KPC-1 led to defects in escaping . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 00910 . 7554/eLife . 11008 . 010Figure 3—figure supplement 1 . Activation of KPC-1 required self-cleavage .", "( A ) Fluorescent image showing PVD morphology of kpc-1 ( wy1060 ) mutants generated by CRISPR .", "Both self-cleavage sites were mutated ( R136A , R143A ) .", "Schematic figure on the right shows the locations of the mutated self-cleavage sites .", "( B ) KPC-1 lacking the N-terminal Pro domain could fully rescue the null kpc -1 ( gk8 ) mutants .", "Scale bar: 10 μm .", "( C ) KPC-1ΔProP440S lacking its Pro domain and also carrying the P440S point mutation in the protease domain rescued the kpc-1 ( xr58 ) mutant phenotype .", "( D ) Quantification of the percentage of 2° branches that were trapped around the 1°dendrite .", "*** is p<0 . 001 , n . s . is p>0 . 05 by Student’s T-test .", "N=50 for each genotype .", "( E ) Quantification of the total number of 4° branches per animal .", "*** is p<0 . 001 , n . s . is p>0 . 05 by Student’s T-test .", "N=50 for each genotype .", "( F ) Western blot showing HA-tagged KPC-1 expressed in S2 cells .", "Two bands were detected corresponding to full length and Pro domain-cleaved KPC-1 proteins in size .", "The R136A , R143A mutant form with both self-cleavage sites mutated lacked the ΔPro band . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 01010 . 7554/eLife . 11008 . 011Figure 4 . The level of the DMA-1 receptor was increased in kpc-1 mutants .", "( A–B )", "Fluorescent images of DMA-1::GFP in PVD neurons in ( A ) wild-type and ( B ) kpc-1 ( gk8 ) mutant animals .", "DMA-1 showed diffuse staining in the entire dendritic arbor but was more enriched in vesicles in the cell body and 1° dendrites in wild-type PVD .", "Diffuse membrane localization of DMA-1::GFP was significantly increased and vesicle-like puncta were reduced in the kpc-1 ( gk8 ) mutant .", "The images on the right are zoomed-in views of the regions indicated by the dashed boxes .", "Scale bar: 10 μm .", "( C ) Quantification of fluorescent intensity of diffuse DMA-1::GFP on the 2° dendrites .", "*** is p<0 . 001 by Student’s T-test .", "N=50 for each genotype .", "( D ) Upper panels: Western blot against GFP in wild-type worms without transgene , wild-type worms expressing DMA-1-GFP and kpc-1 ( gk8 ) mutant worms expressing DMA-1-GFP .", "Lower panels: Western blot against FLAG in wild-type animals , dma-1 ( 1041 ) mutants with 2xFLAG inserted into the dma-1 cytosolic domain of the endogenous genomic locus using CRISPR/Cas9 , and dma-1 ( 1041 ) ; kpc-1 ( gk8 ) double mutants .", "( E ) Quantification of relative band intensity normalized to α-tubulin .", "** is p<0 . 01 by Student’s T-test .", "N=4 ( F ) Schematic figure of the proposed model in which loss of KPC-1 caused increased membrane DMA-1 , leading to defects in escaping from the high levels of ligands around the 1° and 3° dendrites . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 01110 . 7554/eLife . 11008 . 012Figure 4—figure supplement 1 . KPC-1 caused specific down-regulation of DMA-1 receptor .", "( A–B )", "Fluorescent images of HPO-30::GFP in ( A ) wild-type and ( B ) kpc-1 ( gk8 ) mutant PVD neurons .", "The level of HPO-30 on the membrane was not changed in kpc-1 mutants .", "Scale bar: 10 μm .", "( C ) Quantification of HPO-30-GFP intensity on the branches .", "n . s . is p>0 . 05 by Student’s T-test .", "N=20 for each genotype .", "( D ) Quantification of DMA-1-YFP fluorescent intensity of an endogenously tagged dma-1 ( wy1000 ) allele and its double mutants with kpc-1 ( gk8 ) .", "*** is p<0 . 001 by Student’s T-test .", "N=20 for each genotype .", "( E ) Quantification of dma-1 mRNA level relative to unc-104 , snb-1 , cdc-42 and tba-1 measured by qPCR .", "n . s . is p>0 . 05 by Student’s T-test .", "N=3 ( F ) .", "Full Western blot against GFP in wild-type worms without transgenes , wild-type worms expressing DMA-1-GFP and kpc-1 ( gk8 ) mutants expressing DMA-1-GFP .", "No additional or absent bands were detected in the kpc-1 mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 01210 . 7554/eLife . 11008 . 013Figure 4—figure supplement 2 . ( A ) Overexpressing DMA-1 caused more dendrites to escape from the trap zone .", "( B ) Overexpressing SAX-7 and MNR-1 in the hypodermal cells trapped the dendrites back around the 1° dendrites .", "The images on the right are zoomed-in views of the region indicated by the dashed boxes on the left .", "Dashed lines indicate the 'trap zone' with high level SAX-7 .", "Scale bar: 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 01310 . 7554/eLife . 11008 . 014Figure 5 . Overexpression of DMA-1 generated kpc-1 mutant-like phenotypes .", "( A ) Overexpression of DMA-1 in PVD neurons caused similar defects to those of the kpc-1 ( xr58 ) mutants shown in Figure 3B .", "Arrowheads: 3° branches that overlapped with their neighbors in mutants .", "Star: Defective menorah with no 4° branches .", "Asterisks: Trapped 2° branches .", "( B ) Overexpressing DMA-1 in kpc-1 ( xr58 ) enhanced the 3° self-avoidance and 4° outgrowth phenotypes .", "( C ) Quantification of the percentage of 3° branches that made contact with their neighbors .", "( D ) Quantification of the total number of 4° branches per animal .", "*** is p<0 . 001 by Student’s T-test .", "N=50 for each genotype .", "( E ) Overexpressing truncated DMA-1 without its cytosolic domain produced dramatic trapping phenotype .", "Arrows: Trapped dendrites .", "Dotted lines indicated the trap zone .", "Scale bar: 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 01410 . 7554/eLife . 11008 . 015Figure 5—figure supplement 1 . Regulation of DMA-1 by KPC-1 did not require the cytosolic domain of DMA-1 . ( A ) Fluorescent image showing PVD morphology of the wy908 cytosolic deleted mutant allele of dma-1 .", "2° and 3° dendrites were mostly intact but the number of 4° branches was reduced .", "( B ) Dendrites of dma-1 ( wy908 ) ; kpc-1 ( gk8 ) double mutants were still trapped .", "( C ) Schematic of the DMA-1 protein showing the cytosolic domain deleted in the wy908 allele .", "( D ) Cytosolic domain-truncated DMA-1 expressed in dma-1 ( wy686 ) null mutants as a transgene at low concentration gave rise to the same phenotype as wy908 .", "When expressed at a higher concentration , this construct caused robust trapping phenotype shown in Figure 5E .", "Scale bar: 10μm . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 015 Several lines of evidence suggested that kpc-1 ( xr58 ) was a partial loss-of-function allele of kpc-1 .", "First , KPC-1 encodes a conserved proprotein convertase and is a homolog of mammalian Furin ( Schroeder et al . , 2013; Hung et al . , 2014 ) .", "kpc-1 ( xr58 ) had a missense point mutation in the catalytic domain of KPC-1 , which likely affected its protease function .", "Injecting a mutated version of kpc-1 cDNA containing the P440S mutation into the null kpc-1 ( gk8 ) deletion mutants produced a dendritic phenotype that was milder than gk8 but indistinguishable from that of kpc-1 ( xr58 ) , suggesting that the xr58 allele was a hypomorphic allele ( data not shown ) .", "Second , expressing the wild-type kpc-1 cDNA at a very low concentration in the kpc-1 ( gk8 ) null allele resulted in a PVD morphology that was similar to that of the xr58 mutants ( Figure 3C , Figure 3—figure supplement 1D and E ) .", "Third , RNAi against kpc-1 also gave rise to a similar PVD dendritic phenotype to that of xr58 ( data not shown ) .", "Since RNAi was often inefficient in worm neurons , this result further supported the notion that the kpc-1 ( xr58 ) phenotype represented a partial loss of KPC-1 activity in the PVD neuron .", "Despite the seemingly different phenotypes between the null allele and partial loss-of-function allele of kpc-1 , both phenotypes could be regarded as trapping of the dendritic branches .", "High levels of SAX-7 were present both around the primary dendrite and along the 3° branch sites ( Liang et al . , 2015 ) , where dendritic branches preferentially grew in kpc-1 null and partial loss-of-function alleles , respectively ( Figure 3E ) .", "In other words , in wild-type animals , the growth of both the 2° and 4° dendrites required them to move away from the high SAX-7 regions ( Figure 3F , G ) .", "Insufficient KPC-1 function led to failures of dendrites to leave the high SAX-7 intermediate targets .", "The full activation of mammalian Furin requires proteolytic cleavage of its N-terminal Pro domain by its own catalytic domain ( Thomas , 2002 ) .", "Indeed , when the worm KPC-1 was expressed in cultured Drosophila S2 cells , we found two protein products that corresponded to full-length and Pro domain-cleaved KPC-1 based on size ( Figure 3—figure supplement 1F ) .", "With sequence analysis , we identified two adjacent putative cleavage sites in the N-terminus of KPC-1 ( Figure 3—figure supplement 1A , right panel ) .", "We mutated both sites with R to A mutations and found that the resulting mutant protein completely lacked the self-cleavage product when expressed in S2 cells ( Figure 3—figure supplement 1F ) , suggesting that these two sites were indeed responsible for the self-cleavage of KPC-1 .", "To assess the physiological function of these cleavage sites , we introduced these two mutations into the endogenous kpc-1 locus using the CRISPR/Cas9 system ( Paix et al . , 2014 ) .", "The mutants exhibited a PVD dendritic phenotype indistinguishable from that of the gk8 null mutants ( Figure 3—figure supplement 1A ) , demonstrating the necessity of Pro domain cleavage for the activation of KPC-1 .", "Furthermore , we artificially deleted the Pro domain and expressed this mutant form in the kpc-1 ( gk8 ) null allele to test if this truncated version of KPC-1 still retained its activity .", "Consistent with our hypothesis , KPC-1 lacking the Pro domain could fully rescue the gk8 mutants .", "Both secondary trapping and menorah outgrowth phenotypes were restored to wild-type level ( Figure 3—figure supplement 1B , D and E ) .", "Since the xr58 mutation was in the protease domain , we hypothesized that the partial loss of function might be due to inefficient self-cleavage .", "To test this , we engineered the same P440S mutation into the Pro domain deleted version of KPC-1 and expressed this construct in kpc-1 ( xr58 ) mutants ( Figure 3—figure supplement 1C , right panel ) .", "Since it was no longer necessary to remove the Pro domain and self-activate , we expected this mutant form to bypass the catalytic defect of xr58 and restore normal dendritic morphology .", "KPC-1ΔProP440S fully rescued the 2° branch trapping phenotype ( Figure 3—figure supplement 1C and D ) and partially restored the number of 4° branches ( Figure 3—figure supplement 1C and E ) .", "This was consistent with our model that , similar to mammalian Furin , KPC-1 also needed to cleave its own Pro domain to be fully activated .", "The dendritic morphology of the kpc-1 ( xr58 ) mutant was reminiscent of the phenotype caused by overexpression of DMA-1 in PVD ( Figure 5A ) ( Liu and Shen , 2012 ) : First , neurons overexpressing DMA-1 had severe self-avoidance defects .", "Their 3° dendrites often failed to avoid each other and formed a continuous fascicle that covered the entire sublateral line ( Figure 5A , arrowheads , Figure 5C ) .", "In addition , many 2° dendrites were trapped around the 1° dendrite ( Figure 5A , asterisks , Figure 1C ) , and the total number of 4° branches was decreased ( Figure 5A , star , Figure 5D ) .", "All three aspects of the DMA-1 overexpression phenotype were similar to those of the kpc-1 ( xr58 ) mutants , suggesting that having too much DMA-1 led to similar consequences as insufficient KPC-1 activity .", "Since both KPC-1 and DMA-1 functioned autonomously in the PVD neuron , we hypothesized that KPC-1 might affect the PVD dendrites through regulating DMA-1 .", "We utilized several approaches to directly examine DMA-1 in kpc-1 mutants .", "First , we visualized the DMA-1 protein with an integrated reporter expressing gfp-tagged dma-1 genomic DNA driven by the PVD-specific ser2prom3 promoter .", "This transgenic reporter allowed us to monitor the amount of DMA-1 receptors at precise locations in the entire PVD dendritic arbor .", "In wild-type animals , diffuse DMA-1-GFP fluorescence was localized to all dendritic compartments including the higher order branches ( Figure 4A ) .", "Discrete GFP puncta could also be found in the cell body and 1° dendrites , which were likely secretory or endocytic vesicles that carry DMA-1 ( Figure 4A , right panel ) .", "In wild-type neurons , DMA-1-GFP intensity in the discrete puncta was significantly higher than the diffuse staining in the dendrites ( Figure 4A ) .", "In the kpc-1 ( gk8 ) mutants , we observed a striking increase in the diffuse DMA-1-GFP intensity in the entire dendritic arbor ( Figure 4B ) .", "Quantification of the brightness of diffuse DMA-1-GFP on the dendrite , which was likely membrane-localized DMA-1-GFP , showed a dramatic increase in the kpc-1 ( gk8 ) allele compared with wild-type ( Figure 4C ) .", "A similar but less dramatic increase in DMA-1-GFP intensity was observed for the weak allele kpc-1 ( xr58 ) as well ( Figure 4C ) .", "Such up-regulation could be completely rescued by PVD autonomous expression of KPC-1 ( Figure 4C ) .", "To quantify the total DMA-1-GFP protein level , we performed Western blot analysis using an antibody against GFP and confirmed that there was indeed an overall increase in DMA-1 protein level in the kpc-1 mutants ( Figure 4D ) .", "Furthermore , we monitored endogenous DMA-1 by engineering a YFP into the dma-1 genomic locus using CRISPR ( Paix et al . , 2014 ) .", "Despite dim fluorescence intensity , we were able to visualize the endogenous expression and observed a DMA-1 localization pattern similar to what we saw using high copy transgenes , with both membrane and vesicular distribution of the protein ( data not shown ) .", "When we crossed the dma-1::yfp knockin strain into kpc-1 ( gk8 ) , we again observed dramatic increase in DMA-1-YFP intensity in the double mutants ( Figure 4—figure supplement 1D ) .", "Similarly , a 2xFLAG tag was inserted in frame into the endogenous locus of dma-1 using CRISPR ( Paix et al . , 2014 ) , and we again measured significant up-regulation of DMA-1 protein level in kpc-1 mutants ( Figure 4D , E ) .", "As a control , we examined the fluorescent intensity and localization of HPO-30 , another transmembrane protein important for PVD development ( Smith et al . , 2013 ) , and observed no significant change in its dendritic levels in the kpc-1 ( gk8 ) mutants despite severe morphology defects ( Figure 4—figure supplement 1A–C ) .", "We also performed qPCR and found that there was no difference in dma-1 transcript level between wild-type and kpc-1 ( gk8 ) mutants ( Figure 4—figure supplement 1E ) .", "These results further supported the notion that KPC-1 specifically down-regulated the DMA-1 protein level without affecting transcription .", "We noticed an increase in branching in the proximal region when we overexpressed DMA-1 in the kpc-1 mutants ( Figure 4B , Figure 4—figure supplement 2A ) .", "Although most dendrites were still trapped around the 1° neurite ( Figure 4—figure supplement 2A , right panel ) , some 2° dendrites close to the soma extended away to form 3° branches .", "We suspected that the very high level of DMA-1 on the dendritic membrane caused by both the lack of KPC-1 and the DMA-1 overexpression transgene might saturate the SAX-7/MNR-1 ligands in the region , allowing the dendrites to escape .", "To test this hypothesis , we further overexpressed SAX-7 and MNR-1 in the hypodermal cells in this genetic background , and indeed saw that the dendrites were trapped near the seam cells again ( Figure 4—figure supplement 2B ) .", "Genetic interactions between kpc-1 and dma-1 were also in support of this model .", "First , overexpressing DMA-1 in the kpc-1 ( xr58 ) allele further enhanced the dendritic phenotypes ( Figure 5B ) .", "The 3° branches in these animals showed an even more severe self-avoidance defect than the kpc-1 ( xr58 ) worms , and the number of 4° branches was further reduced ( Figure 5C , D ) .", "Second , we found that overexpression of a truncated version of DMA-1 lacking its entire cytosolic domain generated a dramatic trapping phenotype that mimicked that of the kpc-1 ( gk8 ) null mutant ( Figure 5E ) .", "In these animals , 2° dendrites were short and trapped around the 1° dendrite .", "We suspected that the cytosolic domain of DMA-1 may contain signaling motifs that were important for its endocytosis , so overexpressing a DMA-1 construct which lacked these motifs led to higher level of DMA-1 on the dendritic membrane than overexpression of the full length DMA-1 did , thereby produced a more severe trapping phenotype .", "The same construct , when expressed at a much lower level could partially rescue the dma-1 mutant phenotype by allowing the formation of 3° and a small number of 4° branches ( Figure 5—figure supplement 1D ) .", "We also generated a similar mutant allele of dma-1 lacking its cytosolic domain using the CRISPR/Cas9 system ( Friedland et al . , 2013 ) and observed a similar phenotype .", "2° and 3° branches of these mutants developed normally , suggesting that this truncated protein was partially functional ( Figure 5—figure supplement 1A , C ) .", "Interestingly , removing KPC-1 in this genetic background resulted in a severe trapping phenotype , indicating that KPC-1 did not regulate DMA-1 through its cytosolic domain and that the extracellular domain of DMA-1 was sufficient to respond to SAX-7/MNR-1 and to generate trapping ( Figure 5—figure supplement 1B ) .", "The striking similarity between the kpc-1 null and the strain overexpressing the truncated DMA-1 shown in Figure 5E , together with the observation that the loss of DMA-1 suppressed the trapping phenotype of the kpc-1 ( gk8 ) mutant allele , argued strongly that the overabundance of the DMA-1 receptor was responsible for the phenotypes in the loss-of-function alleles of kpc-1 ( Figure 4F ) .", "To test whether KPC-1 directly affected the interaction between DMA-1 and its ligands SAX-7 and MNR-1 , we co-expressed KPC-1 and DMA-1 in Drosophila S2 cells and asked whether these cells could still form aggregates with cells expressing SAX-7/MNR-1 .", "As we have reported previously , when DMA-1-RFP expressing cells were mixed with cells co-expressing SAX-7-GFP and MNR-1-GFP , the red and green cells formed clusters that indicated direct interaction between the DMA-1 receptor and the SAX-7/MNR-1 complex in trans ( Figure 6A , arrows , Figure 6C ) ( Dong et al . , 2013 ) .", "Co-transfection of KPC-1 with DMA-1 in the same cells completely blocked cell aggregation ( Figure 6B , C ) .", "We then directly examined the amount of DMA-1 in the cells by probing for Myc-tagged DMA-1 with an anti-Myc antibody on Western blots , and found that , consistent with what we have observed in worms , the DMA-1 level was diminished in the cells co-expressing KPC-1 ( Figure 6D , E ) .", "Another type I transmembrane protein mCD8 was co-transfected in the same cell cultures and was not affected , showing that the effect of KPC-1 on DMA-1 was specific .", "This result showed that KPC-1 down-regulated DMA-1 not only in worm neurons but also in Drosophila S2 cells . 10 . 7554/eLife . 11008 . 016Figure 6 . KPC-1 interrupted the interaction between DMA-1 and SAX-7/MNR-1 by down-regulating membrane DMA-1 . ( A ) Drosophila S2 cells co-expressing SAX-7-GFP and MNR-1-GFP formed aggregates with cells expressing DMA-1-RFP alone .", "( B ) Cell aggregation failed when KPC-1-GFP is co-transfected with DMA-1-RFP .", "( C ) Quantification of percentages of fluorescent cells in aggregates after 3 hr .", "*** is p<0 . 001 by Student’s T-test .", "The experiment was repeated three times for quantification .", "( D ) Immunoblot showing that the amount of DMA-1 was significantly reduced when co-transfected with KPC-1 while that of another co-transfected type I transmembrane protein , mCD8 , was not affected .", "( E ) Quantification of band intensity on the Western blots .", "*** is p<0 . 001 and n . s . is p>0 . 05 by Student’s T-test .", "Each experiment was repeated three times for quantification . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 01610 . 7554/eLife . 11008 . 017Figure 6—figure supplement 1 . KPC-1 prevented DMA-1 from localizing to the plasma membrane .", "( A ) Fluorescent images showing S2R+ cells expressing DMA-1-RFP and mCD8-Venus .", "DMA-1 localized to the plasma membrane .", "( B ) Co-transfection with KPC-1-CFP caused depletion of DMA-1 from the membrane while mCD8-Venus was unaffected .", "Scale bar: 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 017 Since DMA-1 functioned as the membrane receptor for dendritic branching , we examined the membrane localization of DMA-1 and how it was modified by KPC-1 in adhesive S2R+ cells .", "DMA-1-RFP alone localized to the plasma membrane , which could be seen particularly clearly on the filopodial and lamellipodial structures ( Figure 6—figure supplement 1A ) .", "However , when KPC-1-CFP was co-transfected with DMA-1-RFP , membrane-localized DMA-1-RFP was diminished ( Figure 6—figure supplement 1B ) .", "In contrast , neither the level nor the localization of mCD8 was affected by KPC-1 .", "Receptor down-regulation is often achieved by targeting the transmembrane proteins to endosomal pathways either from the trans-Golgi network ( TGN ) or via endocytosis from the plasma membrane ( Katzmann et al . , 2002; Maxfield and McGraw , 2004 ) .", "We first asked whether DMA-1 was targeted to late endosomes and lysosomes in wild-type worms .", "An extrachromosomal array expressing mCherry-RAB-7 marker which labeled the late endosomes/lysosomes ( Poteryaev et al . , 2010 ) was co-expressed with the integrated DMA-1-GFP line to visualize DMA-1 and the late endosomes simultaneously .", "Indeed , the intracellular DMA-1-GFP puncta showed a high degree of co-localization with mCherry-RAB-7 in wild-type PVD neurons ( Figure 7A ) .", "Around 80% of RAB-7 vesicles co-localized with DMA-1-GFP ( Figure 7E ) .", "This result indicated that DMA-1 was indeed targeted to late endosomes in wild-type animals .", "In contrast , in kpc-1 ( gk8 ) mutant animals , concomitant with the increased DMA-1-GFP level on the dendritic membrane , there was reduced co-localization between DMA-1-GFP and RAB-7 vesicles ( Figure 7B , E ) .", "We hypothesized that surface DMA-1 could be down-regulated via two distinct mechanisms: One that required the cytosolic domain of DMA-1 but was independent of KPC-1 , and the KPC-1 pathway .", "The fact that overexpression of cytosolic truncated DMA-1 produced a much stronger gain-of-function phenotype than full length DMA-1 ( Figure 5E ) argued that cytosolic region-mediated endocytosis of DMA-1 might help to down-regulate its level on the plasma membrane .", "Indeed , DMA-1Δcyto-GFP exhibited much stronger membrane localization when expressed at comparable levels as the full length protein ( Figure 7C ) .", "We then quantified the co-localization between mCherry-RAB-7 and DMA-1Δcyto-GFP and observed a reduction in the percentage of RAB-7 vesicles containing GFP ( Figure 7C and E ) .", "Furthermore , when we crossed the integrated transgene expressing DMA-1Δcyto-GFP into kpc-1 ( gk8 ) mutants , the GFP signal appeared almost exclusively on the plasma membrane ( Figure 7D ) .", "Very few vesicles were seen throughout the dendritic arbor and only 10% of late endosomes labeled by mCherry-RAB-7 contained DMA-1Δcyto-GFP ( Figure 7D and E ) .", "Since both DMA-1 and KPC-1 were predicted to be type I transmembrane proteins , and since the down-regulation of DMA-1 by KPC-1 did not require the cytosolic domain , we reasoned that KPC-1 might target DMA-1 for degradation through directly interacting with its extracellular domain .", "To test this hypothesis , DMA-1 and KPC-1 ectodomains were expressed using baculoviruses in High Five cells and purified .", "Direct binding between these proteins was detected with an apparent dissociation constant of 33 µM using biolayer interferometry ( Figure 7F , blue circles ) .", "No binding was detected between DMA-1 and the negative control , the ectodomain of human GPR56 ( Figure 7F , open rectangles ) . 10 . 7554/eLife . 11008 . 018Figure 7 . KPC-1 targeted DMA-1 to endocytic vesicles through direct interaction with its ectodomain .", "( A–B )", "Fluorescent images of DMA-1-GFP ( upper panels ) , mCherry-RAB-7 ( middle panels ) and overlay ( lower panels ) in PVD neurons of ( A ) wild-type and ( B ) kpc-1 ( gk8 ) mutant animals .", "Many DMA-1-GFP puncta co-localized with late endosomes/lysosomes labeled by mCherry-RAB-7 in wild-type PVD while the co-localization was reduced in the kpc-1 ( gk8 ) mutants .", "kpc-1 mutants showed enhanced DMA-1-GFP fluorescence on the membrane of dendritic branches but less in vesicles .", "( C–D )", "Fluorescent images of DMA-1Δcyto-GFP ( upper panels ) , mCherry-RAB-7 ( middle panels ) and overlay ( lower panels ) in PVD neurons of ( C ) wild-type and ( D ) kpc-1 ( gk8 ) mutants .", "DMA-1 lacking its entire cytosolic domain showed brighter signal on the plasma membrane but still localized to endocytic vesicles .", "However , in the kpc-1 ( gk8 ) mutants , DMA-1Δcyto-GFP was almost exclusively on the plasma membrane but was absent from RAB-7-positive versicles .", "Scale bar: 10 μm .", "( E ) Quantification of the percentages of mCherry-RAB-7 vesicles that showed DMA-1-GFP or DMA-1Δcyto-GFP fluorescence .", "*** is p<0 . 001 , * is p<0 . 05 by Student’s T-test .", "N=20 for each genotype .", "( F ) DMA-1 binding on KPC-1-immoilized surface using biolayer interferometry .", "Blue circles represent DMA-1 binding responses on KPC-1 , with the black curve as the fit to a Langmuir isotherm model .", "Open rectangles show DMA-1 binding on a negative-control surface with the SA tip decorated with biotynlated ectodomain of human GPR56 .", "( G-I )", "Fluorescent images showing the localization of ( G ) KPC-1-CFP , ( H ) Venus-RAB-7 and ( I ) overlay in S2R+ cells .", "Arrow: KPC-1-CFP puncta that co-localized with Venus-RAB-7 .", "Scale bar: 10 μm .", "( J ) Schematic illustration of the model .", "Plasma membrane DMA-1 is down-regulated via two synergistic mechanisms: KPC-1 binds to the ectodomain of DMA-1 and targets it to endosomes , while other endocytic pathways signal through the cytosolic domain of DMA-1 .", "LRR: Leucine rich repeats domain , cyto: cytosolic domain , LE/LY: Late endosomes/lysosomesDOI: http://dx . doi . org/10 . 7554/eLife . 11008 . 018 We made several attempts to visualize KPC-1 in the PVD neuron .", "KPC-1 tagged on both its N- and C-termini produced very weak fluorescent signal .", "We thus turned to the S2R+ cells , in which a similar down-regulation effect of KPC-1 on DMA-1 had been observed ( Figure 6—figure supplement 1A , B ) .", "We co-expressed KPC-1-CFP and Venus-RAB-7 in these cells and observed strong co-localization between KPC-1 and RAB-7 ( Figure 7G–I , arrows ) .", "Together , these data were consistent with a model in which KPC-1 bound directly to the ectodomain of DMA-1 and targeted the receptor to late endosomes and lysosomes for degradation ( Figure 7J ) .", "In kpc-1 mutants , the level of DMA-1 was increased on the dendritic plasma membrane , leading to excessive responsiveness to the guidance signals SAX-7 and MNR-1 .", "Consequently , PVD dendrites became trapped at intermediate targets with enriched SAX-7 and failed to move away ." ], [ "Receptor-ligand interaction is an important theme in neural development .", "It has been shown by the classic studies on axon guidance that the same ligand can trigger a variety of responses in different neuronal types depending on the membrane receptor contents of their growth cones .", "Netrin functions as a chemoattractant when binding to the UNC-40/DCC receptor but repels axons containing an UNC-5 and UNC-40/DCC receptor complex ( Hedgecock et al . , 1990; Tessier-Lavigne and Goodman , 1996; Hong et al . , 1999; Powell et al . , 2008 ) .", "In regions of the nervous system that are wired in a topographic fashion such as the superior colliculus in the visual system , the relative level of Eph receptors dictates where in the Ephrin gradient an axon stops ( Cheng et al . , 1995; Wilkinson , 2001; Mann et al . , 2002 ) .", "Even the same neuron can respond to a ligand differently at different decision points during development .", "In Drosophila , Comm functions in pre-crossing commissural axons to keep the Robo receptor level low by sorting the receptors into endosomes , whereas Comm is turned off after the axon has crossed the midline and the Robo receptor is restored on the surface of growth cones to gain responsiveness to the midline repellent Slit and prevent axons from recrossing ( Keleman et al . , 2002; Keleman et al . , 2005; Dickson and Gilestro , 2006 ) .", "Similarly , commissural axons in the mammalian spinal cord change their responses to Slit by altering their expression of the Robo3 splicing isoforms .", "( Sabatier et al . , 2004; Chen et al . , 2008 ) .", "Hence , the temporal and spatial regulation of receptors is critical to achieving precise neural development .", "We present a new case in neurite morphogenesis in which dynamic regulation of the guidance receptor is required for proper development .", "Like axons , dendrites face the challenge of ignoring and escaping from intermediate targets with high affinity guidance cues after reaching them .", "We show that KPC-1/Furin is responsible for down-regulation of the DMA-1 receptor to allow dendrites to move away from an area with enriched SAX-7/MNR-1 ligands .", "KPC-1 is a worm proprotein convertase known to process and activate proteins and peptides by proteolytic cleavage at a conserved consensus sequence ( Thomas , 2002 ) .", "For example , KPC-1 was shown to be a major convertase to cleave and activate insulin peptides in neurons for the activation of the dauer formation pathway ( Hung et al . , 2014 ) .", "Our results , like previous studies , indicate that KPC-1 functions in multi-dendritic neurons to assist dendritic morphogenesis and is in the same genetic pathway as the SAX-7/MNR-1/DMA-1 signaling complex ( Schroeder et al . , 2013; Salzberg et al . , 2014 ) .", "However , our results provide strong genetic and cell biological evidence that the morphological defects of the PVD dendrites in the kpc-1 mutants are due to enhanced , rather than decreased , response to the SAX-7/MNR-1 ligands .", "First , we showed a causal relationship between the presence of the SAX-7/MNR-1/DMA-1 tripartite complex and the dendritic trapping phenotype of the kpc-1 mutants .", "Dendrites of the kpc-1 mutants were trapped in regions with high levels of SAX-7 .", "In the null allele of kpc-1 , almost all 2° dendrites were unable to extend away from the hypodermal-seam cell junction region where SAX-7 was highly enriched .", "Removal of SAX-7 or other components of the tripartite complex completely suppressed the trapping phenotype in the kpc-1 null mutants .", "Dendritic morphologies of the kpc-1; sax-7/mnr-1/dma-1 double mutants were indistinguishable from the sax-7 , mnr-1 or dma-1 single mutants but were drastically different from the kpc-1 null mutants .", "Thus , the outgrowth defect of kpc-1 mutants was due to the trapping of dendrites caused by excessive response to SAX-7 and MNR-1 .", "To further confirm this model , we used ectopic expression of SAX-7 to manipulate ligand distribution and tested if that was sufficient to cause a predictable trapping pattern .", "Indeed , we could trap the kpc-1 mutant dendrites ectopically by expressing SAX-7 and MNR-1 in seam cells or PLM and ALM neurons ( Figure 2 , Figure 2—figure supplement 3 ) .", "Also , when we expressed the DMA-1 receptor at a late developmental stage in dma-1; kpc-1 double mutants when dendrites had 'escaped' from the trap zone , menorah structures could be largely rescued , showing that PVD’s ability to extend dendrites and respond to the ligand complex was intact in kpc-1 mutants .", "Second , we showed that the DMA-1 receptor was down-regulated by KPC-1 both endogenously in PVD neurons and in heterologous cell systems ( Figure 4 , Figure 6 ) .", "Overexpression of a cytoplasmic domain deleted version of DMA-1 mimicked the kpc-1 ( gk8 ) phenotype ( Figure 5E ) , showing that the dendritic defects of kpc-1 mutants could be attributed to excessive amounts of DMA-1 on the membrane .", "In the weak kpc-1 ( xr58 ) allele , overexpression of DMA-1 enhanced the 3° self-avoidance and the 4° outgrowth phenotypes , which further supported the notion that the phenotypes arose as a result of elevation in DMA-1 level .", "How does KPC-1 down-regulate DMA-1 ?", "Both proteins are predicted to be type I transmembrane proteins , with KPC-1 having a C-terminal transmembrane domain but no cytosolic domain .", "The topology of these proteins suggested that KPC-1 could cleave DMA-1’s ectodomain at specific sites and inactivate DMA-1 .", "We could not , however , identify any consensus Furin cleavage sites in the DMA-1 protein sequence .", "Likewise , we did not observe any truncated product of DMA-1 in the presence of KPC-1 ( Figure 4—figure supplement 1F ) .", "Instead , we observed a reduction in the total amount of DMA-1 when KPC-1 was co-expressed in systems that we examined .", "These results suggested that KPC-1 did not cleave DMA-1 but rather down-regulated DMA-1 via a different mechanism .", "We also found that , consistent with the proteins’ topologies , the regulation of DMA-1 by KPC-1 did not require its cytosolic domain , since double mutants between a dma-1 allele lacking its cytosolic domain dma-1 ( wy908 ) and kpc-1 ( gk8 ) still showed strong trapping phenotype ( Figure 5—figure supplement 1B ) .", "We therefore hypothesized that KPC-1 functioned similarly to another member of the mammalian convertase family , PCSK9 , which down-regulated the LDL receptor through directly binding to its extracellular EGF domain and targeting the receptor to the endocytic pathway for degradation ( Lagace et al . , 2006; Zhang et al . , 2007; Poirier and Mayer , 2013 ) .", "Consistent with this model , we detected direct interaction between the ectodomains of DMA-1 and KPC-1 and observed that KPC-1 localized to the late endosomes and lysosomes when expressed in S2R+ cells .", "In summary , we show that KPC-1 down-regulates the DMA-1 receptor through direct interaction with its extracellular domain in PVD neurons .", "This function is necessary to allow developing dendrites to move away from the intermediate targets where the guidance cues SAX-7 and MNR-1 are highly enriched before reaching their final destinations .", "These results demonstrate that precise regulation of receptor levels is critical for dendrite branching and patterning ." ], [ "N2 Bristol was used as the wild-type strain .", "Worms were raised on OP50 Escherichia coli ( E . coli ) -seeded nematode growth medium ( NGM ) plates at 20°C or room temperature , following standard protocol ( Brenner , 1974 ) .", "All transgenes and plasmids are listed in Supplementary file 1-Tables S1 and S2 .", "To generate new dma-1 alleles by CRIPSR/Cas9-mediated genome editing , Peft-3::cas9 ( 50 ng/μL ) , pU6::dma-1 sgRNA ( 50 ng/μL ) , Punc-122::rfp ( 50 ng/μL ) and Pmyo-3::mCherry ( 5 ng/μL ) were injected into wyIs592 ( ser2prom3:: myr::gfp ) worms .", "F1 worms with abnormal PVD dendrite morphologies were identified through fluorescence microscopy and rescued .", "8 of 24 F1 worms carried germ line mutations ( point mutations , small insertions/deletions ) in dma-1 , which were revealed by sequencing .", "wy908 contained an 8 base pairs deletion that caused a frame shift leading to premature stop codons in the cytosolic domain of dma-1 .", "A similar approach was used to insert 2xFLAG into the cytosolic domain of dma-1 using CRISPR/Cas-9 combined with homologous repair .", "A single-stranded repair template carrying 2xFLAG and 50 base pairs of homology sequences on each end was co-injected at 50ng/μL and successful insertion was confirmed by PCR genotyping and sequencing .", "The FLAG tag was inserted between the third and fourth amino acids after the transmembrane domain , and we confirmed with fluorescent microscopy that these mutants had normal PVD morphology and thus the edited dma-1 allele was still fully functional .", "The kpc-1 ( wy1060 ) allele was generated using a 113 base pairs single-stranded repair template .", "The 136th and the 143th Arginines were mutated to Alanines .", "The entire coding region of kpc-1 was sequenced to confirm precise homologous recombination and no other mutations were identified .", "Sequences of all sgRNAs and repair oligos are listed in Supplementary file 1-Table S3 .", "Images of DMA-1-GFP and HPO-30-GFP were acquired in live animals using a Zeiss Axio Observer Z1 microscope equipped with a Plan-Apochromat 63X/1 . 4NA objective , Yokagawa spinning disk head , 488 nm and 561 nm diode lasers , and a Hamamatsu ImagEm EMCCD camera driven by MetaMorph ( Molecular Devices , Sunnyvale , CA ) .", "Other fluorescent images were captured using a Plan-Apochromat 40X/1 . 3NA objective on a Zeiss LSM710 confocal microscope ( Carl Zeiss , Germany ) .", "Worms were immobilized on 2% agarose pads using a mixture of 225 mM 2 , 3-butanedione monoxime and 2 . 5 mM levamisole ( Sigma-Aldrich , St . Louis , MO ) .", "Z-stacks were collected and maximum intensity projections were used for additional analysis .", "Drosophila S2 and S2R+ cells were obtained from the Drosophila Genomics Resource Center and cultured in Schneider’s insect medium ( Sigma ) according to the manufacturer’s description and transfected using Effectene ( Qiagen , Valencia , CA ) .", "S2 cell aggregation assays were performed as previously described ( Zorio et al . , 1994 ) .", "All plasmids used for transfection are listed in Supplementary file 1-Table S2 .", "For the aggregation assay , S2 cells were transfected with Pactin::sax-7::gfp +Pactin::mnr-1::gfp , Pactin::dma-1::rfp or Pactin::dma-1::rfp+ Pactin::kpc-1::gfp .", "3 days after transfection , cells were washed with 5 mL phosphate buffered saline ( PBS ) and resuspended in S2 medium at 106 cell/mL .", "500 μL of green cells were mixed with 500 μL red cells and rotated at 30 rpm for 3 hours at room temperature .", "3 μL of each mixture was immediately spotted on glass slides for imaging and quantification .", "Similarly , S2R+ cells were cultured in 4-well chamber slides ( Fisher Scientific , Waltham , MA ) and transfected with Pactin::dma-1::rfp , Pactin::mCD8::Venus , Pactin::kpc-1::cfp and Pactin::Venus::rab-7 .", "The cells were washed with PBS and imaged on a Zeiss LSM710 confocal microscope ( Carl Zeiss ) 2 days after transfection .", "For Figure 3—figure supplement 1E , S2 cells were transfected with Pactin::kpc-1::HA or Pactin::kpc-1R136AR143A::HA and for Figure 6D , with Pactin::dma-1::Myc alone or with Pactin::kpc-1::HA .", "3 days after transfection , cells were collected from T25 tissue culture flasks and lysed in lysis buffer ( 1xPBS , 1% Triton X-100 and 1% protease inhibitor cocktail ( Sigma ) ) for 20 min on ice .", "Cell lysates were spun at 13 , 000 rpm for 10 min and supernatants were collected and detected using Western blot analysis with mouse antibody to HA ( 1:1000 , Roche ) or rabbit antibody to Myc ( 1:2000 , Santa Cruz Biotechnology , Dallas , TX ) and HRP-conjugated goat antibodies to mouse or rabbit ( 1:20 , 000 , Jackson Immuno Research West Grove , PA ) .", "For Figure 4D , twenty 10 cm NGM plates of each worm strain were lysed in RIPA lysis buffer ( Sigma ) with 1% protease inhibitor cocktail ( Sigma ) using Lysing Matrix C and the FastPrep-24 tissue homogenizer ( MPbio , Santa Ana , CA ) .", "Lysates were spun at 13 , 000 rpm for 10 min .", "Supernatants were collected and analyzed using Western blots with mouse antibody to GFP ( 1:1000 , Roche , Indianapolis , IN ) , mouse antibody to FLAG ( 1:5000 , Sigma ) and HRP-conjugated goat antibody to mouse ( 1:20 , 000 , Jackson Immuno Research ) .", "For protein expression , the ectodomain of DMA-1 ( amino acids 20–507 ) was cloned into the baculoviral transfer plasmid pAcGP67-A ( BD Biosciences Pharmingen , San Jose , CA ) with a C-terminal hexahistidine tag , and the ectodomain of KPC-1 ( amino acids 34–673 ) was cloned into pAcGP67-A with a C-terminal biotinylation-acceptor peptide and a hexahistidine tag .", "Proteins were expressed using the baculoviruses in High Five cells , and secreted into expression media ( Insect-XPRESS , Lonza , Walkersville , MD , supplemented with 10 μg/mL gentamicin ) .", "Proteins were purified using affinity against Ni2+-NTA agarose resin ( Qiagen ) , followed by gel filtration chromatography with a Superdex 200 10/300 column in HEPES-buffered saline ( HBS , 10 mM HEPES pH 7 . 2 , 150 mM NaCl ) .", "Both proteins eluted as single , monodisperse peaks at volumes indicative of being monomeric .", "KPC-1 was further treated with the E . coli enzyme BirA ( biotin ligase ) for biotinylation , and re-purified with gel filtration chromatography .", "We measured binding between KPC-1 and DMA-1 using biolayer interferometry ( BLItz System , ForteBio , Pall Life Sciences , Port Washington , NY ) with streptavidin tips ( SA Biosensors; Fortebio , Menlo Park , CA ) to capture biotinylated DMA-1 .", "DMA-1 was immobilized to saturation at ~4 . 6 nm BLI responses .", "KPC-1 in HBS at varying concentrations from 0 . 45 µM to 218 µM was titrated on the biosensor tip .", "Association and dissociation kinetics were observed to be fast; equilibrium binding responses were used to fit the data to the one-site Langmuir absorption isotherm model ( Figure 7F , blue circles ) .", "No corrections were applied for non-specific binding .", "The maximum response calculated from the isotherm ( 2 . 0 nm ) indicates a biosensor surface with ~60% active protein , an experimentally sensible value .", "Dissociation constant ( KD ) was calculated to be 33 . 5 ± 5 . 3 µM and 31 . 6 ± 7 . 2 µM over duplicate runs .", "As a negative control , the SA tip was decorated with biotinylated ectodomain of human GPR56 ( a kind gift from Celia Giulietta Fernandez ) to ~11 . 5 nm BLI response levels .", "DMA-1 was observed not to bind to GPR56 ( Figure 7F , open rectangles ) ." ] ]

[ "Extracellular adhesion molecules and their neuronal receptors guide the growth and branching of axons and dendrites .", "Growth cones are attracted to intermediate targets , but they must switch their response upon arrival so that they can move away and complete the next stage of growth .", "Here , we show that KPC-1 , a C . elegans Furin homolog , regulates the level of the branching receptor DMA-1 on dendrites by targeting it to late endosomes .", "In kpc-1 mutants , the level of DMA-1 is abnormally high on dendrites , resulting in trapping of dendrites at locations where a high level of the cognate ligand , the adhesion molecule SAX-7/L1 , is present .", "The misregulation of DMA-1 also causes dendritic self-avoidance defects .", "Thus , precise regulation of guidance receptors creates flexibility of responses to guidance signals and is critical for neuronal morphogenesis ." ]

[ "Neurons are the principal cells in the nervous system that send and receive information .", "A vast network of neurons helps transmit information throughout the brain and body .", "The end of the neuron that receives messages forms branched structures called dendrites , the shapes of which determine the signals the neuron receives .", "Therefore , establishing the correct shape of the dendrites is critical for the neurons to work correctly .", "As dendrites grow during development , signals from the environment tell them where to branch and where to stop .", "For example , the neurons that transmit information about touch respond to signals from skin cells to guide the growth of their dendrites .", "These signals bind to receptor proteins on the surface of the neuron .", "However , the environment around the neurons also contains many guidance signals that the neurons must ignore .", "Dong et al . now show that touch neurons control how they respond to signals by adjusting the abundance of the receptors on their surface .", "First , genetic mutations were identified that distort the shape of the dendrites of touch-sensing neurons in a simple worm called Caenorhabditis elegans .", "These neurons lacked the equivalent of an enzyme called Furin and had abnormally high amounts of a receptor protein called DMA-1 on their surfaces .", "This suggests that controlling the receptor level on dendrites creates flexibility in the guidance choices of dendrites .", "Furin usually cuts up proteins .", "However , Dong et al . found that Furin prevents DMA-1 from inserting into the membrane of neurons by binding to the receptors and sending them to the lysosomes , cellular compartments where proteins are destroyed .", "Reducing the number of receptors at the surface of the cell in this way prevents the neuron from responding to the guidance signals at wrong locations .", "In the future , more studies are needed to understand how the neuron checks and balances this process and how it eventually is turned off ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology" ]

[ [ "Proteins are not static , but exist as an ensemble of conformations in dynamic equilibrium ( Wei et al . , 2016 ) .", "Characterization of conformational heterogeneity can be an essential step towards interpreting function , understanding pathogenicity , and exploiting pharmacological perturbation of target proteins ( Ferguson and Gray , 2018; Latorraca et al . , 2017; Lu et al . , 2016 ) .", "Biophysical techniques such as X-ray crystallography ( Shi , 2014 ) , nuclear magnetic resonance ( NMR ) ( Huang and Kalodimos , 2017 ) , and cryo-electron microscopy ( Fernandez-Leiro and Scheres , 2016 ) mainly provide static snapshots of highly-populated conformational states .", "While complementary techniques such as relaxation-dispersion NMR can resolve a limited number of low-population states , they are incapable of providing detailed structural information ( van den Bedem and Fraser , 2015 ) .", "By contrast , molecular simulations provide atomistic detail---a prerequisite to structure-guided rational ligand design---and insight into relevant conformational transitions ( Wei et al . , 2016 ) .", "The emergence of Markov state models ( MSMs ) has shown the power of distributed molecular simulations in resolving complex kinetic landscapes of proteins ( Husic and Pande , 2018; Plattner et al . , 2017 ) .", "By integrating simulation datasets with MSMs , functionally relevant conformational dynamics as well as atomistic details can be extracted ( Plattner et al . , 2017 ) .", "Recently , MSMs have been used to identify key intermediates for enzyme activation ( Shukla et al . , 2014; Sultan et al . , 2017 ) and allosteric modulation ( Bowman et al . , 2015 ) .", "However , these approaches are limited by the number of seed structures and timescales accessible by molecular simulations ( generally microseconds for one structure ) relative to the reality of complicated conformational transitions ( up to milliseconds for multiple structures ) ( Klepeis et al . , 2009 ) .", "To overcome the limitations of individual techniques , efforts have been made to combine simulation with experiment to characterize and experimentally validate conformational landscape models of proteins that provide insight into functions ( Hart et al . , 2016; Knoverek et al . , 2019; Latallo et al . , 2017; Zimmerman et al . , 2017 ) .", "Protein lysine methyltransferases ( PKMTs ) comprise a subfamily of posttranslational modifying enzymes that transfer a methyl group from the cofactor S-adenosyl-L-methionine ( SAM ) ( Luo , 2018 ) .", "PKMTs play epigenetic roles in gene transcription , cellular pluripotency , and organ development ( Allis and Jenuwein , 2016; Murn and Shi , 2017 ) .", "Their dysregulation has been implicated in neurological disorders and cancers ( Dawson , 2017; Flavahan et al . , 2017 ) .", "SETD8 ( SET8/Pr-SET7/KMT5A ) is the sole PKMT annotated for monomethylation of histone H4 lysine 20 ( H4K20me ) ( Fang et al . , 2002; Nishioka et al . , 2002 ) and many nonhistone targets such as the tumor suppressor p53 and the p53-stabilizing factor Numb ( Dhami et al . , 2013; Shi et al . , 2007 ) .", "Disruption of endogenous SETD8 leads to cell cycle arrest and chromatin decondensation , consistent with essential roles for SETD8 in transcriptional regulation and DNA damage response ( Beck et al . , 2012; Liu et al . , 2010; Veschi et al . , 2017 ) .", "SETD8 has also been implicated in cancer invasiveness and metastasis ( Yang et al . , 2012 ) .", "High expression of SETD8 is associated with pediatric leukemia and its overall low survival rate ( Hashemi et al . , 2014 ) .", "While there is enormous interest in elucidating functional roles of SETD8 in disease , it has been challenging to develop potent , selective , and cellularly active SETD8 inhibitors ( Blum et al . , 2014; Milite et al . , 2016a; Milite et al . , 2016b ) .", "Given the essential roles of conformational dynamics in enzymatic catalysis ( Schramm , 2011; Wei et al . , 2016 ) and our current limited knowledge of conformational landscapes of PKMTs , we envisioned characterizing the dynamic conformational landscapes of SETD8 and its cancer-associated mutants with atomic resolution .", "To access previously-unseen , less-populated conformational states of SETD8 to seed parallel distributed molecular dynamics ( MD ) simulations , we envisioned trapping these conformations with small-molecule ligands .", "Here we solved four distinct crystal structures of SETD8 in alternative ligand-binding states with covalent SETD8 inhibitors and native ligands .", "With the aid of these new structures , we generated an aggregate of six milliseconds of unbiased explicit solvent MD simulation data for apo- and SAM-bound SETD8 .", "Using a machine learning approach to select features and hyperparameters for MSMs via extensive cross-validation , we clustered apo-SETD8 conformers into 24 kinetically distinct , likely functionally relevant metastable conformational states and annotated how the conformational landscape is remodeled upon SAM binding .", "We then explored these conformational landscape models experimentally with stopped-flow kinetics and isothermal titration calorimetry by examining SAM binding , characterizing rationally-designed SETD8 variants with increased catalytic efficiency , and resolving multiple timescales associated with transitions among these conformers .", "The resulting model furnishes key insights into how these dynamic conformations play a role in catalysis of SETD8 and how cancer-associated SETD8 mutants alter this process allosterically through reshaping the conformational landscape rather than directly affecting the catalytic site .", "These findings suggest the importance of referencing conformational landscapes for elucidating enzymatic catalysis and allosteric regulation of SETD8 and likely other PKMTs ." ], [ "To identify hidden high-energy conformational states of SETD8 , we envisioned a strategy of trapping the associated conformers with small-molecule ligands .", "The development of high-affinity SETD8 inhibitors with canonical target-engagement modes is challenging ( Milite et al . , 2016b ) , and led us to exploit covalent inhibitors ( Blum et al . , 2014; Butler et al . , 2016 ) .", "These compounds can overcome the high energy penalties associated with hidden conformers through the irreversible formation of energetically-favored inhibitor‒SETD8 adducts .", "Our prior efforts led to the development of covalent inhibitors containing 2 , 4-diaminoquinazoline arylamide and multi-substituted quinone scaffolds by targeting Cys311 ( Blum et al . , 2014; Butler et al . , 2016 ) .", "Upon further optimization of these scaffolds , we identified MS4138 ( Inh1 ) and SGSS05NS ( Inh2 ) ( Luo et al . , 2015 ) , two structurally distinct covalent inhibitors with the desired potency against SETD8 ( Figure 1a , Figure 1—figure supplement 1 ) .", "X-ray crystal structures of SETD8 were then solved in complex with Inh1 and Inh2 , respectively ( Figure 1b , c , Figure 1—figure supplements 2 and 3 , Table 1 ) .", "Notably , despite the overall structural similarity of the pre-SET , SET , and SET-I motifs , the Inh1- and Inh2-SETD8 binary complexes ( BC-Inh1 and BC-Inh2 ) differ from the SETD8-SAH-H4 ternary complex ( TC ) ( Couture et al . , 2005; Couture et al . , 2008; Xiao et al . , 2005 ) by the distinct conformations of their post-SET motifs .", "The post-SET motif of TC was characterized by its U-shaped topology with a double-kinked loop-helix-helix architecture , which appears to be optimally oriented for binding both SAM and a peptide substrate ( Figures 1c and", "2 ) ( Couture et al . , 2005; Couture et al . , 2008; Xiao et al . , 2005 ) .", "In comparison , BC-Inh1 and BC-Inh2 rotate their post-SET motifs by 140° and 60° , respectively ( Figure 2 ) .", "Moreover , the post-SET motifs of BC-Inh1 and BC-Inh2 adopt more extended configurations with a less structured loop and a singly-kinked helix , respectively ( Figures 1c and 2 ) .", "Whereas multiple factors may influence the overall conformations , the formation of Cys311 adducts likely made the key contribution to the discovery of these hidden post-SET motif conformers .", "To reveal additional hidden conformers that are structurally distinct from TC , we also solved crystal structures of SETD8 upon depleting native ligands and obtained structures of the SAM-SETD8 binary complex ( BC-SAM ) and apo-SETD8 ( APO ) ( Figure 1c , Figure 1—figure supplements 4 and 5 , Table 1 ) .", "Strikingly , BC-SAM and APO differ from TC by their distinct SET-I motifs in the context of the otherwise similar SET-domain ( Figure 2 ) .", "Furthermore , the post-SET motif of APO structurally resembles an intermediate state between BC-Inh1 and BC-Inh2 but is distinct from those of BC-SAM and TC ( Figure 2 ) .", "In contrast to the structurally diverse SET-I ( I1-3 ) and post-SET motifs ( P1-4 ) in these structures , their pre-SET motifs show only slightly altered configuration ( Figure 2 ) .", "The differences between these structures highlight the conformational plasticity of the SET-I and post-SET motifs .", "Collectively , these observations provide strong structural rationale for the existence of a dynamic conformational landscape of SETD8 .", "The BC-SAM , BC-Inh1 , BC-Inh2 , APO , and TC structures can be readily classified into three distinct SET-I configurations ( I1-3 ) and four distinct post-SET configurations ( P1-4 ) ( Figure 2 ) .", "Given the relative spatial separation between the SET-I and post-SET motifs , we envisioned additional combinations of discrete motifs might be accessible to yet-unobserved conformations of SETD8 .", "We thus constructed putative ‘structural chimeras’ of apo-SETD8 containing orthogonal I1-3 and P1-4 in a combinatorial ( 3 × 4 ) manner ( Figure 3a , Figure 3—figure supplement 1 ) .", "Among the twelve structural chimeras as potential seeds for MD simulations , five were crystallographically-determined conformers ( BC-Inh1 , BC-Inh2 , BC-SAM , TC with ligands removed , and APO ) , four were new structurally-chimeric conformers , and three were excluded because of obvious steric clashes ( Figure 3a , Figure 3—figure supplement 2 ) .", "The four structurally-chimeric conformers were included to seed MD simulation with the intention to uncover the conformational landscape more effectively , although this operation proved to be redundant for the discovery of new conformations in the validation process ( see details below ) .", "With seed conformations prepared as above , we envisioned illuminating the conformational landscape with distributed long-timescale MD simulations and resolving its kinetic features with Markov state models ( MSMs ) ( Figure 3b , Figure 3—figure supplement 1 ) .", "Because there is no prior report of the conformational landscapes of PKMTs that can be used as the reference of SETD8 , we leveraged extensive computational power for MD simulation with the intention to not only uncover the conformational landscape of SETD8 in an unbiased manner but also cross-validate the completeness of the dataset .", "Here we conducted approximately 500 × 1 µs explicit-solvent MD simulations from each seed and accumulated 5 milliseconds of aggregate data in 10 million conformational snapshots for apo-SETD8 ( Appendix 1—figure 1 , Supplementary file 1a ) .", "To identify functionally relevant conformational states and their transitions , we built MSMs using a pipeline that employs machine learning and extensive hyperparameter optimization to identify slow degrees of freedom and structural and kinetic criteria to cluster conformational snapshots into discrete conformational states ( Appendix 1—figures 2–9 , Supplementary file 1b , 1c ) ( Husic et al . , 2016 ) .", "This approach identified 24 kinetically metastable conformations ( macrostates ) from an optimized , cross-validated set of 100 microstates ( Figure 4a , Figure 5—figure supplement 1 , Supplementary file 1d , 1e ) .", "These macrostates are remarkably diverse , spanning up to 10 . 5 Å Cα RMSD from APO .", "To visualize the kinetic relationships between functionally important conformations , dimensionality reduction was used to project the landscape into 2D while preserving log inverse fluxes between states ( Figure 4b ) .", "The relative populations of these macrostates were also calculated , resolving rare conformational states up to 6 kT in free energy ( Figures 4b and 5a ) .", "Given the success in constructing the dynamic conformational landscape of apo-SETD8 , we applied the same strategy to SAM-bound SETD8 .", "With the two crystal structures of SETD8 in complex with SAM ( BC-SAM and TC ) as the seed conformations , we conducted ~500 × 1 µs explicit solvent MD simulations from each structure and accumulated 1 millisecond of aggregate data ( 2M snapshots ) ( Appendix 1—figure 10 ) .", "The MSM of the conformational landscape of SAM-bound SETD8 was constructed using the same degrees of freedom as that of apo-SETD8 to facilitate direct comparison of the models ( Appendix 1—figures 11-13 ) .", "The resulting MSM for SAM-bound SETD8 contained 10 kinetically metastable macrostates arising from 67 microstates ( Figure 5—figure supplement 2 , Supplementary file 1f , 1g ) .", "Similar to those of apo-SETD8 , the relative macrostate populations of SAM-bound SETD8 and their flux kinetics were computed and embedded into 3D/2D scatter plots and a chord diagram ( Figures 4a , b and 5b ) .", "The smaller number of metastable states identified for SAM-bound SETD8 is anticipated given that specific conformations are required for optimal interaction between SAM and SETD8’s post-SET motif ( Couture et al . , 2005; Couture et al . , 2008; Xiao et al . , 2005 ) .", "We also compared the timescale structure of the apo- and SAM-bound SETD8 MSMs , as well as an MSM constructed from the subset of apo-SETD8 trajectories originating from the same conformations as the SAM-bound trajectories ( Appendix 1—figure 14 ) .", "We found a large decrease in the number of slow processes seen in the SAM-bound model compared to the other two ( respectively for the apo , SAM-bound , and subset of apo MSMs there are 14 , 4 , and 9 processes slower than 1 μs ) .", "SAM binding thus restricts overall conformational accessibility of SETD8 .", "Upon uncovering the dynamic conformational landscapes of apo- and SAM-bound SETD8 for the first time of the PKMT family of enzymes , we were able to extract new structural information and designed experiments to further examine this model ( Figure 6 ) .", "Comparison of the conformational ensembles between apo- and SAM-bound SETD8 revealed that SAM binding dramatically alters the environment of Trp390 ( Figure 6a , blue sticks ) , the sole tryptophan residue in the catalytic domain of SETD8 .", "This residue is flexible and mainly solvent-exposed in apo-SETD8 conformational ensembles but restricted in a hydrophobic environment through SAM-mediated pi-pi stacking in SAM-bound SETD8 conformational ensembles ( Figure 6a ) .", "Such environmental changes upon SAM binding are expected to quench fluorescence of Trp390 ( Royer , 2006 ) .", "To verify this prediction , we designed rapid-mixing stopped-flow kinetic experiments with 5 ms dead time and 0 . 1 ms resolution to track the fluorescence change of Trp390 upon SAM binding ( Figure 6b ) .", "We observed SAM-dependent biphasic kinetics of the fluorescence decrease within 1 s with >80% of the change occurring in the fast phase ( 0–0 . 1 s ) ( Figure 7a ) .", "In the context of the conformational landscape of apo-SETD8 , we interpreted the major decrease in fluorescence intensity ( fast-phase kinetics ) as a consequence of the collective changes of Trp390 from the solvent-exposed hydrophilic environment in apo conformations to the hydrophobic environment in SAM-bound conformations ( Figure 6a ) .", "In contrast , the minor decrease in fluorescence intensity ( slow-phase kinetics ) reflects the slow conformational changes of Trp390 in the SAM-bound SETD8 conformational ensembles ( Figure 7a ) .", "With unsupervised global fitting to this two-step model , we obtained forward and reverse rate constants for the fast- and slow-phase kinetics , which are in agreement with conventional fitting to double exponential kinetics ( Johnson , 1992 ) ( Figures 7a , b and 8a , Figure 7—figure supplement 1 , Supplementary file 1h ) .", "The k-1 value was also confirmed independently by rapid-mixing stopped-flow dilution of SAM-bound SETD8 ( Agafonov et al . , 2014 ) ( ‘ES +E'S’ , Appendix 1—figure 15 , Supplementary file 1h ) .", "Here the k-1/k1 ratio ( Figure 7b ) of 309 ± 6 µM corresponds to the average SAM dissociation constant Kd1 of apo-SETD8 conformers , which is consistent with independently determined ITC Kd of 251 ± 16 µM ( Figure 7b and c , Figure 7—figure supplement 2 ) .", "In contrast , the large k-2/k2 ratio ( Figure 7b ) of 30 ± 11 suggests that the second phase corresponds to a slow equilibrium between ES and E'S with minimal contribution of E'S to the overall SAM dissociation constant Kd ( Figure 7c ) .", "The conformational ensembles we identified for apo- and SAM-bound SETD8 demonstrate the statistical nature of its SAM-binding process .", "Therefore , the observed fluorescence changes and herein determined macroscopic kinetic constants represent an ensemble-weighted average of microscopic behaviors of all species that exist in the solution .", "A rigorous mathematical description of microscopic kinetics of SAM binding was thus obtained under the consideration of interconversion of the metastable conformational states of apo- and SAM-bound SETD8 ( Figure 7—figure supplement 3 ) .", "We then proposed to confirm our understanding of functionally-relevant conformations and their thermodynamics by identifying SETD8 variants with increased affinity for SAM .", "We uncovered a collection of characteristic kink motifs around Lys382 in the post-SET motif of SAM-bound SETD8 conformational ensembles ( Figure 6c ) , while this region is less structured in apo-SETD8 conformational ensembles .", "We hypothesized that a proline mutation ( K382P ) could better stabilize the conformational ensembles of SAM-bound SETD8 than apo-SETD8 ( Figures 6c and 8b ) .", "We also considered the characteristic α-helix in the SET-I motif , which adopts flexible and diverse configurations in the apo ensembles but becomes constrained and elongated in SAM-bound ensembles ( Figure 6c ) .", "We proposed that the replacement of I293 or E292 adjacent to the α-helix with a flexible glycine should relax this distortion to better stabilize SAM-bound ensembles ( Figures 6c and 8b ) .", "We therefore characterized the SAM-binding kinetics and affinities of K382P , I293G , and E292G variants of SETD8 with stopped-flow kinetics and ITC ( Figures 6c , 7a , b and c , Figure 7—figure supplements 1 and 2 , Appendix 1—figure 15 ) .", "While exhibiting biphasic kinetics similar to that of wild-type SETD8 , the stopped-flow mixing experiment revealed the three variants showed a significant two-fold decrease of Kd , SAM ( Figures 7a , c and 8a ) .", "The stopped-flow data further revealed that the two-fold change of Kd , SAM mainly arises from increased SAM-binding rates k1 with relatively unchanged k-1 ( Figure 8a ) .", "These results are consistent with independently determined Kd and k-1 from ITC and stopped-flow dilution , respectively ( Figure 7b and c , Figure 7—figure supplement 2 , Appendix 1—figure 15 , Supplementary file 1h ) .", "Collectively , these observations confirm the robustness of our conformational landscape model for apo- and SAM-bound SETD8 .", "We systematically investigated how the choices of seed structures and simulation time---key computational parameters---influence microstate discovery and quality of conformational landscapes of SETD8 ( Figures 9 and 10 ) .", "The simulations of apo-SETD8 initiated from any single X-ray structure ( BC-Inh1 , BC-Inh2 , BC-SAM , APO , or TC in Figure 1c ) only reveal a partial conformational landscape ( 28–61% microstate coverage , Figure 9a , Supplementary file 1i ) .", "To achieve >90% microstate coverage , at least two crystal structures---BC-SAM in combination with either BC-Inh1 or BC-Inh2---must be included ( Figure 9a ) .", "If three crystal structures are included , BC-SAM in combination with TC and APO can provide >90% coverage ( Figure 9a ) .", "In terms of the structural motifs ( I1-3 or P1-4 , Figures 2 and 3a ) , simulations originating from the SET-I motif I1 , I2 , or I3 alone led to the discovery of 69 , 58 , or 39 of the 100 microstates , respectively ( Figure 9b , Supplementary file 1j ) .", "The combination of I1 and I2 is sufficient to cover all 100 microstates , arguing for the redundant character of I3 .", "For the post-SET motif , any combination of two post-SET configurations except P2∪P3 leads to >90 microstate coverage ( Figure 9b , Supplementary file 1j ) .", "These findings are in agreement with the key requirement of structural motif conformations I1 ( equivalent to BC-Inh1 , BC-Inh2 , or TC ) , I2 ( equivalent to BC-SAM ) , and any two of P1−4 except P2∪P3 ( e . g . P1∪P3 is equivalent to the combination of APO with BC-SAM or TC ) to achieve >90% microstate coverage .", "For SAM-bound SETD8 , the seed conformations derived from BC-SAM and TC structures contribute 31 and 38 of 67 microstates ( Figure 9c and d , Supplementary file 1k ) .", "These findings argue for the importance of using multiple structures to construct the landscape within achievable computer time .", "The seed conformations prepared from ligand-trapped SETD8 structures are essential to discovering the complete conformational landscapes of SETD8 .", "For simulation time , we observed that the fewer seed conformations of apo-SETD8 were employed , the more computing power ( the product between the number of simulation trajectories and the time length per trajectory ) was required to reach a comparable level of microstate coverage ( Figure 9e , Supplementary file 1l , 1m ) .", "When computing power is fixed , comparable microstate coverages of apo- and SAM-bound SETD8 can be obtained by running either multiple short trajectories or few long trajectories ( Figure 10 , Figure 10—figure supplement 1 ) .", "While the current aggregate simulation time ( 5 ms for apo-SETD8 and 1 ms for SAM-bound SETD8 ) appears sufficient to discover essentially all relevant conformations in the landscapes of SETD8 and estimate their relative populations and corresponding uncertainties , more data would yet be needed to improve estimates of inter-macrostate kinetics in order to develop a fully kinetically accurate model ( Supplementary file 1h , 1l ) .", "Collective contributions of the number of seed structures and the overall simulation time determined the efficiency of uncovering conformational landscapes of SETD8 .", "The conformational landscape of apo-SETD8 can be revealed upon implementing a minimum of two seed structures ( TC and BC-SAM ) or 10% of the current simulation time .", "With the two seed structures ( TC and BC-SAM ) and sufficient simulation time , apo-SETD8 sampled 22 more microstates than SAM-bound SETD8 ( 89 states with 750 μs simulation versus 67 states with 850 μs simulation , Figure 9d , e ) , consistent with the conformational restriction of SETD8 upon SAM binding .", "We also noted that it is redundant to include the four structurally-chimeric conformers because this operation contributes less than 10% of microstate coverage and the comparable conformational landscape of apo-SETD8 can be generated with the subsets of seeds solely prepared from the X-ray structures ( Supplementary file 1l ) .", "After experimentally corroborating the conformational landscapes of apo- and SAM-bound SETD8 , we explored the dynamic details of these landscapes with the focus on the connectivity and equilibrium fluxes between kinetically metastable macrostates ( henceforth referred to as the ‘network’ ) .", "When projected into two dimensions , the conformational landscape of apo-SETD8 takes the form of a dumbbell-like shape containing two lobes , each composed of about 12 macrostates primarily connected via a single hub-like central macrostate A11 ( Figures 4b and 11 , Supplementary file 1e ) .", "The conformational landscape also consists of other multiply-connected macrostates , including A1−A4 , A9 , and A14 , as characterized by their rapid kinetic interconversion with multiple other macrostates ( Figures 4b and 5a ) .", "Most low-populated macrostates ( A17−A24 ) appear as satellite macrostates in the periphery of the network with few high-flux channels of interconversion to other macrostates ( Figures 4b and 5a ) .", "The remaining states were classified as basin-like macrostates including {A5 , A10} , A7 , A8 , {A12 , A13 , A16} and A15 , because these macrostates are highly populated and either are relatively isolated or appear in tightly interconnected but globally isolated groups .", "The hub-like macrostate A11 consists of two structurally distinct microstates with comparable populations ( Figures 4b and 11a ) .", "One microstate structurally resembles the conformation of APO ( I3P3 ) , while the other microstate represents a conformer with the I1P23 feature for its SET-I and post-SET motifs ( Figure 11b , Supplementary file 1d ) .", "Rapid conformational interconversions within A11 are consistent with its hub-like character , centered between the two lobes of the dumbbell-like network .", "Interestingly , macrostates kinetically adjacent to A11 have structurally similar SET-I motifs within each lobe but distinct SET-I motifs between the two lobes ( {I2 ~3} for the left and {I1 ~2} for the right ) ( Figures 4b and 11b ) .", "Therefore , A11 is a transition-type state essential for the conformational fluxes of the macrostates between the two lobes , involved in a key step of conformational changes of the SET-I motif between {I1 ~2} and {I2 ~3} .", "The intermediate-like macrostates A1−A4 , A9 , and A14 each contains multiple structurally distinct but kinetically associated microstates ( Figures 4b , 11a and b ) .", "The satellite macrostates A17−A24 are less populated and more structurally homogeneous ( Figures 4b , 11a and b ) .", "Conformers in the macrostates A22 , A24 , and A20 are structurally similar to TC and BC-SAM with slightly different but well-defined SAM-binding pockets , suggesting minimal conformational reorganization of A22 , A24 , and A20 is required to accommodate the cofactor ( Figure 11a , b , c ) .", "Interestingly , A22 and A24 , whose overall structures are similar to each other ( TC-like ) , rarely interconvert in the apo landscape ( Figure 4b ) .", "In contrast , the basin-like macrostates {A5 , A10} , A7 , A8 , {A12 , A13 , A16} and A15 do not contain a well-defined SAM-binding pocket ( Figure 11a , b , c ) .", "Here the conformers in macrostate A12 are similar to APO , the conformers in the macrostate A6 are similar to BC-Inh1 , and the conformers in the macrostate A10 are similar to BC-Inh2 ( Figure 11d ) .", "The structural similarity between the simulated conformers and BC-Inh1/2 suggests that the two covalent inhibitors successfully trapped key hidden conformers of apo-SETD8 .", "Similar to that of apo-SETD8 , the interconversion network of the macrostates of SAM-bound SETD8 also displays a dumbbell-like shape with S9 as the hub-like state connecting the two lobes of the network ( Figures 4b and 11a ) .", "The macrostates S1 and S3−S5 are multi-connected states; S6 , S8 , and S10 are satellite-like states; S2 and S7 are basin-like states ( Figure 11a , b ) .", "Notably , the complexity of the overall conformational landscape of SAM-bound SETD8 is significantly reduced in comparison with those of apo-SETD8 ( Figures 4b and 11a ) .", "The conformers in S1 , S2 , and S10 are structurally similar to those of A20 , as well as BC-SAM; the conformers in S4 , S6 , and S8 are structurally similar to those in A22 and A24 , as well as TC ( Figure 11d , e ) .", "The structural similarities between these apo and SAM-bound macrostates suggest possible pathways for connecting the two conformational landscapes upon SAM binding .", "Sequences from tumor samples retrieved from cBioPortal ( Cerami et al . , 2012; Cheng et al . , 2015; Gao et al . , 2013 ) contain two dozen point mutations in the catalytic domain of SETD8 ( Figure 12a and b , Supplementary file 1n ) .", "We expect that some of these mutations perturb SETD8 function .", "Because of conformational heterogeneity , it has historically been challenging for in silico approaches to annotate how mutations---in particular those structurally remote from functional sites---allosterically affect a target protein on the basis of its static structure ( s ) ( Campbell et al . , 2016; Klinman and Kohen , 2014; Stefl et al . , 2013 ) .", "Here , we envisioned addressing this challenge with reference to the conformational ensemble of wild-type SETD8 .", "To characterize mutations remote from catalytic sites ( 20 out of 24 known mutations ) , 40 independent microsecond-long MD simulations for each of the cancer-associated apo-SETD8 mutants were conducted with seed structures prepared from one ternary complex ( TC ) conformer---a structure resembling the enzymatic transition state and thus essential for SETD8-catalyzed methylation reaction ( Linscott et al . , 2016 ) .", "We then constructed a differential residue-contact map for each variant ( Figure 12c , d ) and extracted snapshots representing the largest conformational deviations from the wild-type conformational ensembles ( Figure 12e ) .", "Even with modest simulation time , 8 of the 20 examined cancer-associated mutants displayed neo-conformations that were not observed in the 5 ms wild-type dataset and cannot be predicted from static X-ray crystal structures .", "Interestingly , all of the neo-conformations display distinct reorganizations at the SET-I motif ( Figure 12e ) .", "For instance , a single point mutation A296T , ~16 Å remote from the active site , yields five distinct neo-conformations ( Figure 12e , f ) .", "In addition , relative to wild-type apo-SETD8 , this mutant populates several conformations with a structurally relaxed α-helix at the SET-I motif ( Figure 12e ) .", "C324del , ~20 Å from the SET-I motif , is associated with three neo-conformations and displays the largest changes in the differential contact map ( Figure 12d , panel 13 ) .", "The remote H340D mutation is associated with one neo-conformation as well as more populated conformations containing spatially compressed active sites ( Figure 12d , panel 7; Figure 12e ) .", "Using in vitro radiometric assays , the A296T and H340D mutants were characterized by loss of the methyltransferase activity on H4K20 peptide substrate ( Figure 12g ) .", "The failure to purify recombinant C324del also supports the impact of this deletion on SETD8 function .", "H388Q , which mutates a histidine involved in substrate binding , is also associated with neo-conformations as well as loss of the methyltransferase activity ( Figure 12e , g ) .", "These observations provide potential molecular rationale for how remote mutations can alter the active sites and the SET-I motif---and hence catalysis allosterically---via modulating the overall conformational landscape rather than directly affecting specific residues at the catalytic site .", "Exceptions are T274I , R279W , R279Q , and A368V , which yielded neo-conformations but showed activity comparable to wild-type SETD8 ( Figure 12e , g ) , suggesting that certain neo-conformations must either still be catalytically competent or their population may not significantly alter the ability to populate conformations relevant for catalysis .", "The exceptions suggest that a more complete picture of the conformational ensembles might be necessary to uncover quantitative correlations with the relative methyltransferase activities of these SETD8 mutants .", "The differential residue-contact maps further revealed that 8 out of the 20 remote mutations alter conformational landscapes by changing populations of pre-existing conformations ( Figure 12c , d ) .", "For instance , E257K , G280S , A301V , T309M , E330Q , D352Y mutations populate conformations containing spatially compressed active sites ( Figure 12—figure supplement 1 ) ; E372D populates conformations containing a constrained post-SET motif; R333C populates conformations with reorganized SET motifs adjacent to the peptide binding pocket .", "All of these mutations showed partial loss of methyltransferase activity ( Figure 12g ) .", "Notably , these structural alterations are often remote from the corresponding mutation sites ( Figure 12b ) .", "In contrast , R244S and V356I ( 2 out of 20 ) showed no significant conformational change on the basis of their differential contact maps , consistent with their comparable methyltransferase activity to wild-type SETD8 ( Figure 12g ) .", "Likely due to insufficient simulation time ( 40 × 1 μs/mutant ) , R333L and L334P variants , characterized by partial-to-complete loss of the methyltransferase activity ( Figure 12g ) , showed similar conformational landscapes to that of wild-type apo-SETD8 .", "These exceptions , though only a small portion of all mutants studied , point to the necessity of a more extensive exploration of the conformational ensembles to obtain quantitative correlations of the atomistic structure with activities of this collection of SETD8 mutants .", "Exploring these conformational landscapes is thus an effective strategy to reveal structural alterations associated with majority of remote-site mutations of SETD8 for qualitative functional annotation .", "More importantly , this change provides a mechanistic rationale of the allosteric effect of remote residues of SETD8 with reference to its conformational landscape ." ], [ "Here we have demonstrated that tight integration of structural determination---using covalent probes and multiple ligand-binding states to trap hidden conformations ( Figure 1 ) ---with distributed molecular simulations and the powerful framework of Markov state models ( Figure 3b ) can provide insights into the detailed conformational dynamics of an enzyme .", "The current work demonstrates the merit of an approach that leverages multiple X-ray structures with distinct diverse conformations of a PKMT for MD simulations and machine-learning-based MSM construction to elucidate complex conformational dynamics , and corroborates the resulting model experimentally with testable biophysical predictions ( Figures 6–8 ) .", "Previously , individual components of our integrative strategy have been employed to study the dynamics of transcriptional activators ( Wang et al . , 2013 ) , kinases ( Shukla et al . , 2014; Sultan et al . , 2017 ) , and allosteric regulation ( Bowman et al . , 2015 ) .", "Several efforts have also been made to combine experimental and computational approaches to explore conformational landscapes of proteins and their utilities ( Hart et al . , 2016; Knoverek et al . , 2019; Latallo et al . , 2017; Zimmerman et al . , 2017 ) .", "However , it is the first time that these diverse approaches are consolidated explicitly with the goal of illuminating conformational dynamics of a PKMT in a comprehensive and feasible manner .", "Assessment of key computational parameters concluded that we have utilized sufficient or even redundant seed structures and simulation time for essentially complete microstate discovery ( Figures 9 and 10 ) .", "This implementation is essential for the current work because of the lack of the conformational landscapes of PKMTs as reference or for validation .", "Notably , we relied on a unique computational resource---Folding@home---to collect six-millisecond of aggregate simulation data ( see Materials and methods ) .", "Without access to Folding@home , contemporaneous progress on developing adaptive Markov state model construction algorithms---where iterative model building guides the collection of additional simulation data ( Hruska et al . , 2018; Shamsi et al . , 2017; Zimmerman et al . , 2018 ) ---will still allow research groups to achieve this feat on local GPU clusters or cloud resources in the near future .", "Furthermore , the concept of adaptive model construction can be extended to identify which new structural or biophysical data would be valuable in reducing uncertainty ( Dixit and Dill , 2018; Matsunaga and Sugita , 2018; Olsson et al . , 2017 ) and producing refined MSMs .", "Utilizing the slow collective variables identified here , advanced sampling methods such as metadynamics ( Saladino and Gervasio , 2012 ) or umbrella sampling ( Meng and Roux , 2014 ) can be applied to more efficiently compute the free energy landscape for SETD8 and its mutants .", "With a transfer learning approach ( Sultan et al . , 2017 ) , it is also possible to adapt these collective variables to other members of the PKMT protein family .", "This work represents the first time that conformational dynamics of a protein methyltransferase have been definitively characterized with atomic details .", "SETD8 adopts extremely diverse dynamic conformations in apo and SAM-bound states ( 24 and 10 kinetically metastable macrostates , respectively , Figure 4 ) .", "Interconversions between metastable conformers cover a broad spatio-temporal scale in particular associated with motions of SETD8’s SET-I and post-SET motifs ( Figures 1 , 2 and 11 ) .", "In the apo landscape , the general structural features of the X-ray structures of BC-Inh1 , BC-Inh2 , APO , BC-SAM and TC ( Figure 1 ) are recapitulated by a subset of macrostates ( e . g . A6 for BC-Inh1; A10 for BC-Inh2; A12 for APO; A20 for BC-SAM; A22 , A24 for TC , 6 of 24 macrostates , Figure 11 ) .", "Such observation indicates that these X-ray structures trapped in the different ligand-binding states are not ligand-induced artifacts but indeed relevant snapshots of hidden conformations of apo-SETD8 .", "Similarly , a few macrostates in the SAM-bound landscape also recapitulate major structural features of the two cofactor-bound X-ray structures ( e . g . S1 , S2 , S10 for BC-SAM , S4 , S6 , S8 for TC , 6 of 10 macrostates , Figure 11 ) .", "Meanwhile , our results also demonstrate that X-ray crystallography alone is insufficient to capture all metastable conformations of SETD8 .", "In addition , there is no correlation of overall structural similarity and interconversion rates between metastable conformers .", "As observed previously in other studies of protein dynamics ( Bowman and Pande , 2010 ) , in addition to fast transitions between structurally similar conformers and slow transitions between structurally distinct conformers ( e . g . microstates within individual satellite macrostates A17−A24 of apo‒SETD8; S6 , S8 , and S10 of SAM-bound SETD8 , Figure 11 ) , we frequently observed fast kinetics of transitions between structurally distinct microstates ( e . g . microstates within hub-like macrostates A11 and S8; multi-connected states A1−A4 , A9 , A14 , S1 and S3−S5 ) and vice versa ( e . g . macrostates A22 and A24 ) ( Figures 4 and 11 ) .", "It is thus interesting to examine how other factors such as specific residue contacts and cooperative long-range motions of certain structural motifs play roles in interconversion kinetics .", "Meanwhile , utilizing the power of Markov state models to stitch together multiple short ( microseconds long ) trajectories and generate synthetic trajectories orders of magnitude longer ( milliseconds ) , we visualized the MSMs of apo- and SAM-bound SETD8 via 2 ms long ( enough to visit all macorstates ) movies ( Videos 1 and 2 ) .", "Functional annotation of the landscapes revealed that the SET-I motif adopts diverse conformations ( Figures 4 and 5 ) , and its overall configuration is a key feature that differentiates the lobes of the dumbbell-like conformational landscape of SETD8 .", "The conformational dynamics within the hub-like macrostate A11 primarily involve motions of the SET-I motif , secondarily coupling a shift of the post-SET motif .", "Two gain-of-function I293G and E292G variants of SETD8 were designed for relaxing constrained elongate helix configurations of the SET-I motif upon SAM binding ( Figure 6 ) .", "These findings argue the functional essentiality of the intrinsically dynamic motions of SET-I motif for SETD8 SAM binding and catalysis .", "Importance of dynamic conformational modulation of the SET-I motif has also been shown for other SET-domain PKMTs .", "For instance , the SET domains of MLLs and EZH1/2 alone are catalytically inert but active in the presence of binding partners WDR5-RbBP5-Ash2L-Dpy30 ( referred as MLL-WRAD ) and EED-Suz12 ( referred as PRC2 ) , respectively ( Luo , M . , 2018 ) .", "Recent structural evidence implicated that the formation of these complexes regulates the conformational dynamics of the SET-I motif , which is essential for catalysis ( Justin et al . , 2016; Li et al . , 2016 ) .", "Interestingly , this region has also been exploited by cancer-associated mutants of PKMTs .", "For instance , NSD2’s E1099 is located in its SET-I motif and its E1099K mutant was characterized as a hot-spot cancer mutation with the gain-of-activity of H3K36 methylation ( Oyer et al . , 2014 ) .", "Additionally , many mutations of PKMTs have been mapped in their SET-I motifs , implicating their potential roles in alternation of function ( Figure 12—figure supplement 2 , Supplementary file 1o ) .", "In contrast to static X-ray structures , this analysis greatly facilitated the characterization of cancer-associated SETD8 mutants ( Figure 12 ) .", "Among the 20 examined SETD8 mutations , eight deplete the pre-existing conformations of TC and showed the partial loss of activity in comparison with wild-type SETD8 ( 8 out of 8 ) ; eight have neo-conformations with four characterized with the partial loss of methyltransferase activity ( 4 out of 8 ) ; four do not affect the conformational landscape with two characterized for no loss of methyltransferase activity ( 2 out of 4 ) .", "Collectively , comparing the conformational landscapes between SETD8 mutations and wild-type TC allows us to predict the methyltransferase activity with 70% accuracy ( 14 out of 20 ) .", "However , we could not quantitatively correlate the amounts of the neo- or altered conformations of these SETD8 mutants with their methyltransferase activities .", "We reason that certain nonnative conformations can still be catalytically active .", "A significant portion of cancer-associated , loss-of-function SETD8 mutations , though remote from active sites , were revealed to perturb the SET-I motif and thus catalysis allosterically via altering the conformational landscape , which is relevant to the formation of the ternary complex and likely the transition state of native SETD8 ( Figure 12 ) .", "We also discovered significant changes in the connective networks and a large decrease in conformational heterogeneity of SETD8 upon SAM binding ( Figures 4 and 5 ) .", "This finding highlights how SETD8-SAM interactions reshape conformational landscapes .", "The conformational landscapes of SETD8 thus provide a platform for virtual screening of ligand candidates as inhibitors via exploring different modes of interaction ( SAM-competitive , substrate-competitive , covalent or allosteric ) .", "Uncovering hidden conformations can thus be essential for developing potent and selective SETD8 inhibitors by targeting these conformations .", "Furthermore , it seems feasible that additional simulation effort---if appropriately allocated among poorly-sampled transitions---can produce a statistically precise kinetic model of the conformational dynamics of apo- and SAM-bound SETD8 , and that these landscapes could be used to seed simulations for the construction of atomistic models of the rest of the catalytic cycle .", "Furthermore , the structural information in the resulting models and the kinetic experimental observables could be reconciled using the dynamical fingerprints framework ( Noé et al . , 2011 ) .", "This approach can also be used to design new experiments by proposing locations of site-specific labels for optimal experimental probing of the molecular relaxation processes of interest .", "Future work could therefore furnish a quantitative atomistic explanation of the experimentally observed kinetics .", "Additionally , these metastable states could be paired with alchemical free energy calculations ( Gapsys et al . , 2016 ) to rapidly assess the impact of point mutations on the populations of each metastable state in each stage of the catalytic cycle to aid the annotation of the functional impact of these mutations .", "A prerequisite of our approach was the determination of conformationally diverse structures as seeds for molecular simulations .", "Here , this was achieved with Cys-covalent inhibitors and native ligand depletion because of the lack of conventional structural probes of SETD8 .", "Given the significant interest in exploring PKMT catalysis and developing selective inhibitors to study functions ( Luo , 2018 ) , we envision applying similar strategies to other native or disease-associated PKMTs ( Nacev et al . , 2019 ) ." ], [ "Human SETD8 catalytic domain ( Uniprot Q9NQR1-1 positions 232–393 , SRKSKAELQSEERKRIDELIESGKEEGMKIDLIDGKGRGVIATKQFSRGDFVVEYHGDLIEITDAKKREALYAQDPSTGCYMYYFQYLSKTYCVDATRETNRLGRLINHSKCGNCQTKLHDIDGVPHLILIASRDIAAGEELLDYGDRSKASIEAHPWLKH ) with an N-terminal 6 × His tag in pHIS2 vector was overexpressed in E . coli Rosetta 2 ( DE3 ) in LB medium in the presence of 100 μg/ml of ampicillin .", "Cells were grown at 37°C to an OD600 of 0 . 4 ~ 0 . 6 and the expression of SETD8 was induced by 0 . 4 mM isopropyl-1-thio-D-galactopyranoside ( IPTG ) at 17°C overnight .", "Harvested cells were suspended in a lysis buffer ( 50 mM Tris-HCl , pH = 8 . 0 , 25 mM NaCl , 10% Glycerol , 25 mM imidazole ) supplemented with EASY pack protease inhibitor ( one tablet/10 mL solution ) , a tip amount of lysozyme and DNAase I .", "The mixture was lysed by FrenchPress .", "SETD8 ( aa 232–393 ) was purified by a Ni-NTA column subjected to a washing buffer ( 50 mM Tris-HCl , pH = 8 . 0 , 25 mM NaCl , 10% glycerol , 25 mM imidazole ) and then an eluting buffer ( 50 mM Tris-HCl , pH = 8 . 0 , 25 mM NaCl , 10% glycerol , 400 mM imidazole ) .", "The protein was further purified by a Superdex-75 gel filtration column with a buffer containing 25 mM Tris-HCl ( pH = 8 . 0 ) , 200 mM NaCl , and 10% glycerol .", "The elution fractions were pooled , supplemented with 5 mM of tris ( 2-carboxyethyl ) phosphine ( TCEP ) , and concentrated to about 60 mg/mL for storage at −80°C .", "All purification was conducted at 4°C .", "The N-terminal 6 × His SETD8 ( aa 232–393 ) construct was used to measure IC50 of SETD8 inhibitors .", "Plasmids of SETD8 mutants were generated by QuickChange site-directed mutagesis kit ( Stragaene ) according to manufacturer’s instructions and validated by DNA sequencing .", "Primer sequences for mutagesis were designed by PrimeX and listed in Supplementary file 1p .", "SETD8 mutants were expressed and purified as described above for wild-type SETD8 .", "The IC50 of SETD8 inhibitors were measured by a previously reported filter plate assay ( Blum et al . , 2014; Ibanez et al . , 2012 ) with some modifications .", "DMSO stock solutions of SETD8 inhibitors with different concentrations were prepared through series dilution .", "The final assay mixture ( a total volume of 20 μL ) contains 300 nM SETD8 protein ( N-terminal 6 × His taged , amino acid 232–393 ) , 10 μM H4K20 peptide ( aa 10–30 , prepared by Rockefeller University Proteomics Resource Center , New York , NY ) , 1 . 5 μM [3H-Me]-SAM ( PerkinElmer Life Sciences ) , and various concentrations of inhibitors in a reaction buffer ( 50 mM HEPES , pH = 8 . 0 , 0 . 005% Tween-20 , 5 μg/mL BSA , 1 mM TCEP and 0 . 5% DMSO ) .", "Prior to each reaction , 10 μL of a reaction mixture containing 2 × concentrations of SETD8 and inhibitors was pre-incubated at ambient temperature ( 22°C ) for 2 hr . 10 μL of another reaction mixture containing 2 × concentrations of peptide and [3H-Me]-SAM was then added to initialize the reaction .", "The resulting mixture was allowed to react at ambient temperature ( 22°C ) for 2 hr . 3 × 6 μL ( total 18 μL ) of this mixture were spotted onto 3 wells of MultiScreenHTS PH Filter plate ( Millipore ) to immobilize 3H-labeled peptide .", "After drying in ambient air overnight , each well was washed 6 times with 200 μL of 50 mM Na2CO3/NaHCO3 buffer ( pH = 9 . 2 ) , followed by the addition of 30 μL Ultima Gold scintillation cocktail ( PerkinElmer Life Sciences ) .", "The plate was sealed and the mixture was further equilibrated for 30 min .", "The immobilized radioactivity of 3H-labeled peptide was quantified by 1450 Microbeta liquid scintillation counter .", "The inhibition curve was generated according to the equation: Percentage of inhibition = [ ( \"CPM of no inhibitor control\" – \"CPM of a reaction mixture\" ) / ( \"CPM of no inhibitor control\" – \"CPM of background\" ) ]×100% .", "The IC50 values were obtained by fitting inhibition percentage versus concentrations of inhibitors using GraphPad Prism .", "Data presented are best fitting values ± s . e . SAM-free SETD8 was prepared as described previously ( Linscott et al . , 2016 ) .", "Briefly , the concentrated N-terminal 6 × His tagged SETD8 protein ( aa 232–393 , ~60 mg/mL ) was diluted by about 1:10 ratio ( v/v ) with a stripping buffer ( 25 mM Tris-HCl , pH = 8 . 0 , 35 mM KCl , and 5% glycerol ) .", "Activated charcoal was added into the solution ( 1:1 w/w ratio of protein versus charcoal ) .", "The resulting mixture was incubated for 45 min .", "The charcoal-treated sample was then centrifuged and filtered to afford SAM-free SETD8 .", "All these steps were performed at 4°C .", "SAM-free SETD8 mutants were prepared in a similar manner .", "Dissociation constants of SETD8 with SAM ( Sigma-Aldrich ) were measured using an Auto-iTC200 calorimeter ( MicroCal ) at 20°C .", "Both SAM and SAM-free SETD8 proteins were dissolved into an assay buffer containing 50 mM HEPES ( pH = 8 . 0 ) , 0 . 005% Tween-20 , 5 µg/mL BSA , 0 . 00125% TFA , and 1 mM TCEP .", "2 . 5 mM SAM was titrated into 125 µM SETD8 through 20 injections .", "Experimental data were analyzed by Origin 7 . 0 after correcting the heat generated upon injecting SAM into the assay buffer .", "Best fits were obtained with a fixed stoichiometry ( N = 1 ) .", "Data are shown as mean ± s . e . of at least three biological replicates .", "The binary binding kinetics of SAM to SETD8 ( wild-type and mutants ) were studied using stopped flow spectrometry ( SX20 , Applied Photophysics ) .", "The slit widths of the entrance and exit of the monochromator were set to 2 . 0 mm .", "Equal volume of samples from two 2 . 5 mL syringes were driven into a 20-μL observation cell to mix at ambient temperature ( 22 °C ) , to reach the final concentration of 1 μM SAM-free SETD8 and serial concentrations of SAM ( 16 μM to 2000 μM ) in a mixing buffer containing 50 mM HEPES-HCl ( pH = 8 . 0 ) , 0 . 005% Tween 20 , and 1 mM TCEP .", "6–8 shots ( drives ) were taken for each SAM concentration .", "Trp fluorescence change was recorded for 1 second upon mixing with an excitation wavelength of 295 nm and a wavelength cutoff emission filter ( ≥ 320 nm ) .", "10000 data points were collected with Pro-Data SX20 software for each stopped-flow experiment .", "Data analysis was performed using KinTek Explorer ( Johnson et al . , 2009 ) .", "For the global fitting , the signal traces for all concentrations of SAM were simultaneously fitted to a two-step binding model with an initial binding step followed by the step of further conformational changes: \"E + SAM\" , \"ES\" and \"E'S\" , in which E , ES , and E'S correspond to different states of SETD8 .", "The fluorescence signal was defined as the expression F = a× [E] + b× [ES] + c× [E'S] + bkg , in which F is the detected total fluorescence intensity , a , b , and c are fluorescence coefficients of E , ES , and E'S , respectively , and bkg is the background fluorescence intensity .", "For the calculation of equilibrium constants , the equations of Kd1 = k-1/k1 , Keq = k-2/k2 , and Kd = Kd1 × Keq/ ( 1+ Keq ) were followed .", "For conventional fittings , the fluorescence data were fitted into Equation .", "1 , in which F is the fluorescence intensity , A1 and A2 are the amplitudes of the signal changes for fast and slow phases , respectively , kobsfast and kobsslow are the observed rate constants for two phases , and t is time .", "The plot of kobsfast and kobsslow versus SAM concentrations were fitted with Equation .", "2 and Equation .", "3 , respectively , where [S] is the concentration of SAM , ki and k-i are the association and dissociation rate constants for step i ( i = 1 or 2 ) , respectively .", "For individual rate constants , data are best fitting values ± s . e . from KinTek .", "Uncertainties of Kd1 , Keq , Kd are shown as s . e . calculated by the propagation of s . e . from individual rate constants and dissociation constants , respectively .", "Meanwhile , the data was also globally fitted into a conformational-selection model ( E = E' + SAM = E'SAM ) and failed to generate good fitting results ( Appendix 1—figure 22 ) .", "( 1 ) F=A1×exp ( −kobsfast×t ) +A2×exp ( −kobsslow×t ) +C ( 2 ) kobsfast=k1×[S]+k−1+k2+k−2 ( 3 ) kobsslow≈k1×[S]+ ( k−2+k2 ) +k−1×k−2 ( k1×[S]+k−1+k2+k−2 ) ( plateau≈k−2+k2 ) 25 μM SAM-free SETD8 ( wild-type and mutants ) was pre-mixed with serial concentrations of SAM ( 1000 μM to 2000 μM ) in the mixing buffer and incubated for 10 min at ambient temperature ( 22°C ) .", "The pre-mixed samples were loaded into a 100 μL syringe , and the mixing buffer was loaded into a 2 . 5 mL syringe .", "The two syringes were then driven into the observation cell and mixed to achieve a 1:25 dilution of the pre-mixed samples .", "The time-dependent fluorescence signal changes were recorded up to 3 s under the same setting as described above for the binding assay .", "Total of 11333 points were collected with 10000 points for the first 1 s and 1333 points for 1–3 s .", "Conventional fitting of results was performed using KinTek Explorer following equation: F = A1×exp ( -k-1×t ) +C , in which A1 is the amplitude of the signal change , k-1 is the dissociation constant for the first step in rapid quenching experiment , and t is time .", "Signals from different concentrations of SAM are fitted separately , and the average k-1 is calculated accordingly .", "Data are best fitting values ± s . e . from KinTek .", "The methyltransferase activities of wild-type and cancer-associated SETD8 mutations were characterized by a previously described filter paper assay ( Blum et al . , 2014; Ibanez et al . , 2012 ) with some modifications .", "Briefly , 50 nM SETD8 protein ( N-terminal 6 × His tag , amino acids 232–393 , wild-type or mutants ) , 1 . 5 μM [3H-Me]-SAM , and 30 μM histone H4 peptide ( amino acids 10–30 ) were incubated in a reaction buffer containing 50 mM HEPES ( pH = 8 . 0 ) , 0 . 005% Tween 20 , 5 µg/mL BSA , and 1 mM TCEP at ambient temperature ( 22°C ) for 3 hr .", "Each reaction mixture was split into three aliquots and quenched by spotting on phosphor cellulose ( P-81 ) filter paper , followed by 2 hr air-dry .", "The dried filter paper was then washed 5 times with 50 mM Na2CO3/NaHCO3 solution ( pH = 9 . 2 ) .", "The washed filter paper was then transferred into a scintillation vial , well mixed with 0 . 5 ml ddH2O and 5 ml Ultima Gold , and analyzed by a Liquid Scintillation Analyzer ( Perkin Elmer Tri-Carb 2910 TR ) .", "The methyltransferase activities of SETD8 mutants relative to that of wild-type SETD8 were calculated with the following equation: Percentage of relative activity = [ ( CPM of mutant – CPM of background ) / ( CPM of wild type – CPM of background ) ]×100% .", "Data are presented as mean ± s . d . of 3 biological replicates .", "To quantify how the diversity of starting conformations influences the number of microstates observed out of the total of 100 , the apo-SETD8 discrete trajectories were split into nine sets corresponding to their starting conformations .", "All logical relations between the sets were generated and the numbers of microstates explored in each intersection were counted in order to produce Venn diagrams of microstate coverage .", "Analogically , the SAM-bound SETD8 discrete trajectories were split into two sets and the microstate coverage was evaluated .", "Further , to quantify how the number and the length of trajectories influence the number of microstates observed in addition to the diversity of starting conformations , all combinations of all possible lengths of the five apo-SETD8 sets started from crystal structures were enumerated .", "Appropriate sets out of the four originating from structural chimeras were added to those combinations which contained the appropriate SET-I and post-SET motif configurations for the formation of those chimeras .", "Also , if a combination contained either of the BC-Inh1 or BC-Inh2 sets ( SET-I configuration I1 ) , and the BC-SAM set ( post-SET configuration P1 ) , the TC set ( configurations I1-P1 ) was added as it could be generated as a structural chimera .", "For all combinations that resulted , the microstate coverage was assessed at all trajectory numbers between 0 to all trajectories in the combination at intervals of 50 trajectories , and simultaneously at all maximum trajectory lengths between 0 to the length of the longest trajectory in the combination at intervals of 50 ns .", "The desired number of trajectories was randomly drawn from all trajectories in the combination without replacement and the trajectories were trimmed to the desired maximum length .", "The number of microstates observed was then calculated .", "This was repeated five times with different draws of trajectories and the results of the five draws were averaged .", "Analogically , the microstate coverage at increasing trajectory numbers and trajectory lengths was evaluated for the two SAM-bound SETD8 sets .", "The Anaconda Python ( Millman and Aivazis , 2011; Oliphant , 2007 ) distribution with Python 2 . 7 , 3 . 5 , or 3 . 6 was used for all programming .", "Conda was used for package management .", "The IPython ( Perez and Granger , 2007 ) shell and the Jupyter Notebook ( Kluyver et al . , 2016 ) environment were used for interactive scripting and data analysis .", "Data were managed with the NumPy ( multiple versions ) ( Oliphant , 2006 ) and pandas ( multiple versions ) ( McKinney , 2010 ) libraries .", "Mathematical operations were performed with the NumPy library or Python built-in functions .", "Figures were made with PyMOL 1 . 8 . 4 ( Schrödinger LLC , 2019 ) , matplotlib 2 . 2 . 2 ( Hunter , 2007 ) , seaborn 0 . 8 . 1 ( Waskom et al . , 2017 ) , MSMExplorer 1 . 1 ( Harrigan et al . , 2017 ) , and PyEMMA 2 . 5 . 1 ( Scherer et al . , 2015 ) .", "The molecular dynamics datasets generated and analyzed in this study are available via the Open Science Framework at https://osf . io/2h6p4 .", "The code used for the generation and analysis of the molecular dynamics data is available via a Github repository at https://github . com/choderalab/SETD8-materials ( Wiewiora , 2019; a copy archived at https://github . com/elifesciences-publications/SETD8-materials ) ." ] ]

[ "Elucidating the conformational heterogeneity of proteins is essential for understanding protein function and developing exogenous ligands .", "With the rapid development of experimental and computational methods , it is of great interest to integrate these approaches to illuminate the conformational landscapes of target proteins .", "SETD8 is a protein lysine methyltransferase ( PKMT ) , which functions in vivo via the methylation of histone and nonhistone targets .", "Utilizing covalent inhibitors and depleting native ligands to trap hidden conformational states , we obtained diverse X-ray structures of SETD8 .", "These structures were used to seed distributed atomistic molecular dynamics simulations that generated a total of six milliseconds of trajectory data .", "Markov state models , built via an automated machine learning approach and corroborated experimentally , reveal how slow conformational motions and conformational states are relevant to catalysis .", "These findings provide molecular insight on enzymatic catalysis and allosteric mechanisms of a PKMT via its detailed conformational landscape ." ]

[ "Our cells contain thousands of proteins that perform many different tasks .", "Such tasks often involve significant changes in the shape of a protein that allow it to interact with other proteins or ligands .", "Understanding these shape changes can be an essential step for predicting and manipulating how proteins work or designing new drugs .", "Some changes in protein shape happen quickly , whereas others take longer .", "Existing experimental approaches generally only capture some , but not all , of the different shapes an individual protein adopts .", "A family of proteins known as protein lysine methyltransferases ( PKMTs ) help to regulate the activities of other proteins by adding small tags called methyl groups to specific positions on their target proteins .", "PKMTs play important roles in many life processes including in activating genes , maintaining stem cells and controlling how organs develop .", "It is important for cells to properly control the activity of PKMTs because too much , or too little , activity can promote cancers and neurological diseases .", "For example , genetic mutations that increase the levels of a PKMT known as SETD8 appear to promote the progression of some breast cancers and childhood leukemia .", "There is a pressing need to develop new drugs that can inhibit SETD8 and other PKMTs in human patients .", "However , these efforts are hindered by the lack of understanding of exactly how the shape of PKMT proteins change as they operate in cells .", "Chen , Wiewiora et al . used a technique called X-ray crystallography to generate structural models of the human SETD8 protein in the presence or absence of native or foreign ligands .", "These models were used to develop computer simulations of how the shape of SETD8 changes as it operates .", "Further computational analysis and laboratory experiments revealed how slow changes in the shape of SETD8 contribute to the ability of the protein to attach methyl groups to other proteins .", "This work is a significant stepping-stone to developing a complete model of how the SETD8 protein works , as well as understanding how genetic mutations may affect the protein’s role in the body .", "The next step is to refine the model by integrating data from other approaches including biophysical models and mathematical calculations of the energy associated with the shape changes , with a long-term goal to better understand and then manipulate the function of SETD8 ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "During the past several decades , multiple arboviruses , including dengue ( DENV ) , chikungunya ( CHIKV ) , and Zika ( ZIKV ) viruses , have re-emerged to cause widespread epidemics affecting millions of people ( Weaver , 2018 ) .", "For example , DENVs are estimated to cause ~390 million infections per year resulting in ~500 , 000 cases of dengue hemorrhagic fever/dengue shock syndrome that can result in death ( Bhatt et al . , 2013 ) .", "Since 2004 , CHIKV has infected millions of people and expanded into Europe , Asia , the Pacific region , and the Americas ( Moro et al . , 2010; Volk et al . , 2010; Zeller et al . , 2016 ) .", "Joint pain , swelling , and stiffness , and tenosynovitis , can endure for months to years after CHIKV infection ( Borgherini et al . , 2008; Couturier et al . , 2012; Gérardin et al . , 2011; Rodriguez-Morales et al . , 2016; Schilte et al . , 2013 ) .", "As chronic CHIKV disease is debilitating , large epidemics have severe economic impact ( Cardona-Ospina et al . , 2015; Soumahoro et al . , 2011; Vijayakumar et al . , 2013 ) .", "ZIKV caused outbreaks on Yap Island ( Duffy et al . , 2009 ) and islands of French Polynesia ( Musso et al . , 2018 ) in 2007 and 2013 , respectively , before being introduced to the Americas ( Metsky et al . , 2017 ) .", "Here , ZIKV caused nearly one million confirmed and suspected cases and became recognized as a cause of congenital neurodevelopmental defects ( Pierson and Diamond , 2018 ) .", "The lack of safe and effective vaccines and therapeutics limits prevention and treatment of many arboviral diseases .", "The magnitude and duration of viremia in vertebrate hosts are important determinants of arboviral emergence , transmission efficiency , and geographic spread ( Weaver , 2018 ) .", "A subset of arboviruses , including CHIKV , DENV , and ZIKV , are capable of sustained human-mosquito-human transmission cycles , allowing for rapid spread through urban environments .", "This is in part due to the capacity of these viruses to generate a magnitude and duration of viremia in humans sufficient to infect mosquito vectors and propagate the transmission cycle ( Weaver , 2018 ) .", "Moreover , for many arboviruses , viremia levels correlate with disease severity ( Chow et al . , 2011; de St Maurice et al . , 2018; Pozo-Aguilar et al . , 2014; Vaughn et al . , 2000; Waggoner et al . , 2016 ) .", "Despite the critical role for viremia during arboviral infections , innate host defenses that determine the magnitude and duration of arboviral viremia in vertebrate hosts are poorly defined .", "Phagocytic cells are strategically positioned within organs such as the spleen and liver to detect and capture circulating self and non-self molecules .", "In the murine spleen , blood released from terminal arterioles drains into the marginal zone , which separates the white and red pulp ( Lewis et al . , 2019 ) .", "Within the marginal zone , tissue resident marginal zone ( MZM ) and metallophilic ( MMM ) macrophages surveil the blood for circulating antigens , apoptotic cells , debris , and pathogens ( Borges da Silva et al . , 2015; Lewis et al . , 2019 ) .", "In addition , splenic red pulp macrophages ( RPM ) also function to surveil the circulation by removing aberrant red blood cells as well as some pathogens , such as malaria parasites ( Borges da Silva et al . , 2015 ) .", "Similar to the marginal zone of the spleen , cell populations in liver sinusoids , including tissue resident macrophages ( i . e . , Kupffer cells ( KCs ) ) , effectively survey blood as it percolates through the liver ( Hickey and Kubes , 2009 ) .", "For example , following intravenous inoculation , bacteria are captured within seconds by KCs ( Lee et al . , 2010; Zeng et al . , 2016 ) , whereas the absence of KCs results in persistent bacteremia ( Lee et al . , 2010 ) .", "Although the surveillance function of these tissues and cells is well-established , how they influence viremia following arbovirus infections has not been defined .", "In this study , we demonstrate that phagocytic cells are essential for efficient clearance of multiple alphaviruses from the circulation , thus limiting viremia and impeding viral dissemination .", "Experiments in splenectomized mice showed that the spleen is dispensable for clearance of these circulating alphaviruses .", "Consistent with this , we found that virus accumulates in the liver , and liver resident KCs play a dominant role in removing circulating alphavirus particles .", "Mechanistically , we identified SR-A6 ( MARCO ) as the receptor required for efficient alphavirus clearance .", "We also found that discrete lysine ( K ) to arginine ( R ) mutations in the E2 glycoproteins of CHIKV and o’nyong ‘nyong virus ( ONNV ) ( E2 K200R ) , as well as Ross River virus ( RRV ) ( E2 K251R ) , abrogated clearance of circulating alphavirus particles , and promoted rapid viral dissemination .", "Moreover , substitution of the lysine residues at CHIKV E2 200 or RRV E2 251 with a variety of other amino acids also allowed for clearance evasion , indicating an essential requirement for key lysine residues in E2 for efficient viral clearance from the circulation .", "These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts ." ], [ "To explore whether phagocytic cells participate in the clearance of circulating arboviruses , we treated mice intravenously ( i . v . ) with PBS- ( control ) or clodronate-loaded liposomes ( PLL or CLL , respectively ) to deplete phagocytic cells in the spleen , liver , and circulation that are in direct contact with blood ( Seiler et al . , 1997; Van Rooijen and Sanders , 1994 ) .", "The uptake of CLL by phagocytic cells results in intracellular release of the encapsulated clodronate which is cytotoxic ( Van Rooijen , 1989 ) .", "At 42 hr ( h ) post-treatment , mice were i . v . inoculated with clinical isolates of CHIKV , ONNV , or RRV , all pathogenic alphaviruses ( Morrison , 2014; Suhrbier et al . , 2012 ) , and viral RNA or infectious virus in the serum was analyzed at 45 minutes ( min ) post-inoculation .", "In PLL-treated mice , viral particles ( CHIKV and RRV ) or plaque forming units ( ONNV ) were nearly undetectable in the serum by 45 min post-inoculation ( Figure 1 ) .", "In stark contrast , CLL-treated mice had 3–4 orders of magnitude higher amounts of viral particles ( CHIKV and RRV ) or plaque-forming units ( ONNV ) in the serum at this time point ( Figure 1 ) .", "Viral RNA in PLL-treated mice also was absent from the clotted fraction of the blood ( Figure 1—figure supplement 1 ) , demonstrating that CHIKV particles had been removed from the circulation .", "These findings suggest that blood-exposed phagocytic cells , such as those in the spleen or liver , are required for clearance of blood-borne alphaviruses .", "The spleen and liver contain phagocytic cell populations that are strategically positioned to capture circulating pathogens .", "To determine the relative contribution of the spleen , viral clearance was evaluated in mice that underwent sham or splenectomy surgeries .", "CHIKV was efficiently cleared from the circulation of splenectomized mice ( Figure 2A ) , demonstrating that the spleen is not required for clearance of circulating CHIKV .", "Moreover , CLL-pretreatment blocked CHIKV clearance in splenectomized mice ( Figure 2B ) , suggesting that the remaining phagocytic cells in the liver are sufficient to clear CHIKV from the circulation .", "Consistent with this , CHIKV RNA accumulated in the liver of control PLL-treated mice , and this specific accumulation was lost in CLL-treated mice ( Figure 2C ) .", "In contrast , viral RNA levels increased in the spleen and lung of CLL-treated mice compared with control PLL-treated mice ( Figure 2C ) , likely due to the high amounts of virus present in contaminating blood .", "Moreover , CHIKV RNA is readily detectable by in situ hybridization in the livers of PLL-treated mice at 45 min post-inoculation , but not in CLL-treated mice ( Figure 2D ) .", "While both dendritic cells ( DCs ) and macrophages can sequester pathogens in the liver ( Jenne and Kubes , 2013 ) , treatment of mice with CLL resulted in depletion of F4/80+ liver macrophages ( Figure 2E , Figure 2—figure supplement 1 and Figure 2—figure supplement", "2 ) but had minimal impact on CD11c+ DCs ( Figure 2—figure supplement 2 ) , suggesting macrophages are responsible for alphavirus clearance .", "Collectively , these data suggest that Kupffer cells ( KCs ) , the tissue resident macrophage population in the liver ( Jenne and Kubes , 2013 ) , clear alphavirus particles from the circulation .", "Clearance of circulating bacteria and other particles from the circulation often involves opsonins such as antibodies and complement component 3 ( C3 ) ( Guilliams et al . , 2014; Kubes and Jenne , 2018; van Lookeren Campagne et al . , 2007 ) .", "However , CHIKV and RRV particles were efficiently cleared from the circulation of C3-/- ( Figure 3A ) and B cell deficient μMT mice ( Figure 3B ) , which lack C3 and circulating IgM and IgG , respectively ( Kitamura et al . , 1991; Wessels et al . , 1995 ) .", "These data suggest that clearance of circulating alphavirus particles is independent of receptors for fragments of C3 and of Fc receptors .", "Scavenger receptors ( SRs ) are a large family of transmembrane proteins that capture and eliminate a broad range of endogenous and microbial ligands via non-opsonic mechanisms ( Canton et al . , 2013; Mukhopadhyay and Gordon , 2004 ) .", "To test the hypothesis that one or more SRs mediate clearance of alphavirus particles from the circulation , 5 min prior to inoculation of virus , mice were pre-treated i . v . with the polyanionic SR competitive inhibitor poly ( I ) , or poly ( C ) , a polyanion that is not a competitive inhibitor of SRs ( Pearson et al . , 1993 ) .", "Similar to pretreatment with CLL , poly ( I ) pretreatment potently inhibited the clearance of circulating CHIKV and RRV particles ( Figure 3C ) .", "Similar results were observed following pretreatment of mice with dextran sulfate ( DS ) , another SR ligand ( Platt and Gordon , 1998 ) ( Figure 3D ) .", "To identify SRs that are expressed by KCs , we mined publicly available single-cell transcriptomic data of the mouse liver ( Tabula Muris Consortium et al . , 2018 ) , and found SR-A1 ( MSR1 ) , SR-A6 ( MARCO ) , SR-B1 ( SCARB1 ) , SR-B2 ( CD36 ) , and SR-I1 ( CD163 ) mRNAs to be robustly expressed in KCs ( Figure 3—figure supplement 1 ) .", "Of these , only SR-A1 and MARCO are known to bind poly ( I ) and DS ( Chen et al . , 2006; Platt and Gordon , 1998 ) .", "To investigate the relative contributions of SR-A1 and MARCO , CHIKV clearance was evaluated in SR-A1-/- and MARCO-/- mice .", "CHIKV particles were efficiently cleared from the circulation of SR-A1-/- mice , and this clearance was inhibited by pre-treatment of SR-A1-/- mice with poly ( I ) ( Figure 3E ) , indicating that CHIKV clearance was still occurring through a SR-dependent mechanism .", "In contrast , CHIKV particles remained readily detectable in the serum of MARCO-/- mice at 45 min post-inoculation ( Figure 3F ) .", "Moreover , whereas pretreatment with CLL blocked clearance of circulating CHIKV particles in WT mice , this treatment had no effect on the level of CHIKV in the circulation of MARCO-/- mice ( Figure 3G ) .", "Furthermore , CHIKV RNA was detectable by in situ hybridization in the liver of PLL-treated WT mice , but not in the liver of PLL-treated MARCO-/- mice or CLL-treated WT and MARCO-/- mice ( Figure 3H ) .", "Despite the virological differences in the circulation and liver of PLL-treated WT and MARCO-/- mice , these mice had a similar number of KCs in the liver ( Figure 3I ) .", "Finally , both WT and MARCO-/- KCs were efficiently depleted by CLL ( Figure 3H and I ) , suggesting that MARCO-/- KCs retain phagocytic capacity .", "Collectively , these findings demonstrate that MARCO is essential for clearance of CHIKV particles from the circulation .", "We next investigated which viral features promote clearance of alphaviral particles from the circulation .", "In prior studies , DENV and West Nile virus ( WNV ) clearance from the circulation of mice was found to be influenced by N-linked glycans on viral envelope glycoproteins ( Fuchs et al . , 2010 ) .", "CHIKV E2 is predicted to be glycosylated at N345 , N263 and N273 ( Metz et al . , 2011; Voss et al . , 2010 ) .", "N345 is located in the E2 stem near the transmembrane domain , making it unlikely that glycans at this position mediate interactions with MARCO ( Voss et al . , 2010 ) .", "To investigate if the more surface exposed glycosylation sites influenced clearance , we introduced asparagine to glutamine mutations at N263 and N273 to disrupt glycosylation .", "Mutation of positions N263 or N273 alone , or in combination , had no effect on the clearance of CHIKV particles from the circulation ( Figure 4—figure supplement 1 ) , demonstrating that clearance of CHIKV particles does not rely on glycosylation at these sites .", "Previously , we identified a mutation in the CHIKV E2 glycoprotein ( E2 K200R ) in virus persistently circulating in Rag1-/- mice ( Hawman et al . , 2017 ) .", "This mutation enhanced CHIKV dissemination in mice following subcutaneous inoculation and caused more severe disease outcomes .", "These effects were associated with a strongly elevated viremia .", "Having identified an important role for phagocytic cells and MARCO in the clearance of CHIKV particles from the circulation , we hypothesized that the E2 K200R mutation allows for viral escape from phagocytic cell-mediated clearance .", "Indeed , unlike WT CHIKV , which is efficiently cleared from the circulation , CHIKV E2 K200R particles remained stably detectable in the serum for at least 1 hr post-inoculation ( Figure 4A ) .", "Importantly , the differential clearance was not dependent on the cell type from which the viral particles were derived , as differential clearance of WT and E2 K200R CHIKV particles was maintained when virions were derived from human fibroblasts , a clinically relevant cell type ( Couderc et al . , 2008 ) ( Figure 4B ) .", "The E2 K200R mutation did not impact the stability of viral particles in serum , demonstrating that this effect was not due to differential stability of WT and E2 K200R CHIKV particles ( Figure 4—figure supplement 2A ) .", "Moreover , the levels of E2 K200R CHIKV particles in control PLL-treated mice were indistinguishable from WT and E2 K200R CHIKV particles in the circulation of mice pre-treated with CLL ( Figure 4C ) , demonstrating that CHIKV E2 K200R allows for evasion of phagocytic cell-mediated clearance .", "The ability of E2 K200R to evade this response was not limited to WT C57BL/6J mice ( Figure 4C ) , as evasion of clearance also was observed in 129S1/SvImJ , and BALB/cJ mouse strains ( Figure 4—figure supplement 2B ) .", "To test if the effects of the E2 K200R mutation were unique to the CHIKV strain tested ( AF15561; Asian genotype ) or functioned within broader viral genetic contexts , we introduced an E2 K200R mutation into the genome of CHIKV strains representative of all phylogenetic lineages: Asian-American ( strain 99659; Jones et al . , 2017; Lanciotti and Valadere , 2014 ) , East , Central , South African ( strain SL15649; Morrison et al . , 2011 ) , and West African ( strain 37997; Tsetsarkin et al . , 2006; Vanlandingham et al . , 2005a ) .", "In addition , we introduced an E2 K200R mutation into the genome of the SG650 strain of ONNV ( Lanciotti et al . , 1998 ) .", "Similar to CHIKV AF15561 , all WT strains of CHIKV and ONNV were cleared from the circulation at 45 min post-inoculation , whereas the levels of all E2 K200R mutant particles were substantially higher ( Figure 4D ) .", "These results demonstrate that an E2 K200R mutation broadly facilitates evasion of phagocytic-cell mediated clearance in multiple CHIKV strains , as well as the related ONNV .", "To evaluate if phagocytic-cell mediated clearance of blood-borne virus has implications following a more natural route of infection , we inoculated mice subcutaneously with WT or E2 K200R viruses and evaluated viral burdens in various tissues at 1 d post-inoculation .", "Viral burdens in the serum were significantly higher in mice inoculated with E2 K200R strains ( Figure 5A ) , indicating that phagocytic cell-mediated clearance is an important immune mechanism to control viremia following subcutaneous inoculation .", "Moreover , although viral burdens were similar in tissues proximal to the site of inoculation ( i . e , the L . ankle ) , significantly elevated levels of virus were detected in the contralateral right ankle of mice infected with E2 K200R strains ( Figure 5A ) , demonstrating that this mutation facilitates more rapid viral dissemination .", "To investigate if expression of MARCO also influences viremia and viral dissemination following subcutaneous inoculation of virus , we inoculated MARCO-/- mice subcutaneously with WT CHIKV and evaluated viral burdens at 1 d post-inoculation .", "Similar to infection of WT mice with CHIKV E2 K200R , infection of MARCO-/- mice with WT CHIKV resulted in an elevated viremia and more rapid viral dissemination to distal sites including the right ankle and quadriceps compared with infection of WT mice ( Figure 5B ) .", "Collectively , these findings indicate that MARCO-dependent clearance of circulating CHIKV particles controls viremia and impedes viral dissemination .", "As arboviruses , alphaviruses must maintain efficient replication in both vertebrate hosts and mosquito vectors .", "Given this , we evaluated whether the E2 K200R mutation influenced viral replication and/or dissemination in Ae .", "aegypti mosquitoes .", "Following blood-feeding inoculation of mosquitoes , similar levels of WT CHIKV and E2 K200R were detected in the bodies , legs and saliva of infected mosquitoes at 3 d post-infection , suggesting that in contrast to mice , the E2 K200R mutation had no impact on viral dissemination in mosquitoes ( Figure 6A ) .", "To more rigorously test if this mutation affects viral fitness in mosquitoes , we performed in vitro and in vivo competition experiments with a WT CHIKV strain genetically marked with an ApaI restriction site ( CHIKV-ApaI ) .", "Control 1:1 competitions of CHIKV and CHIKV-ApaI in C6/36 cells ( Figure 6B ) and microinjected Ae .", "Aegypti mosquitoes ( Figure 6C and D ) demonstrated that the genetic marker does not influence viral fitness .", "Similar results were observed following direct 1:1 competition of CHIKV and CHIKV E2 K200R , ( Figure 6B , C and D ) , suggesting that the E2 K200R mutation has no selective disadvantage to WT virus in the mosquito vector .", "Collectively , these findings suggest that while the E2 K200R mutation dramatically influences CHIKV dissemination in the vertebrate host , it is neutral in the mosquito vector .", "The critical lysine at position E2 200 of CHIKV and ONNV is not conserved within RRV strains , which led us to investigate viral features that promote clearance of circulating RRV particles .", "In addition to RRV strain SN11 ( Figure 1C ) , a 2009 clinical isolate ( Liu et al . , 2011 ) , we found that RRV strain DC5692 ( Jupille et al . , 2011; Lindsay , 1996 ) also was efficiently cleared from the circulation by phagocytic cells ( Figure 7A ) .", "In contrast , RRV strain T48 ( Doherty and Whitehead , 1963 ) was resistant to clearance , and clearance was relatively unaffected by prior depletion of phagocytic cells ( Figure 7A ) .", "Through chimeric virus analysis of DC5692 and T48 strains , we mapped the genetic determinant of RRV susceptibility to clearance to a region of the genome encoding a portion of capsid , E3 and E2 ( Figure 7—figure supplement 1A ) .", "Within the E2 glycoprotein , the T48 and DC5692 RRV strains differ by only six amino acids ( Figure 7B ) .", "Remarkably , mutating position E2 251 of T48 from arginine to lysine ( R251K ) was sufficient to make this strain more susceptible to clearance , while the corresponding K251R mutation in DC5692 conferred resistance to clearance ( Figure 7B ) .", "Moreover , sequencing analysis confirmed the presence of a lysine at E2 251 in RRV strain SN11 .", "The R251K mutation had no impact on the stability of RRV T48 in serum ( Figure 7—figure supplement 1B ) .", "In addition , inoculation of purified viral particles generated in serum free media maintained the differential clearance of T48 and T48 E2 R251K , indicating that viral clearance is not mediated by factors in fetal bovine serum ( Figure 7—figure supplement 1C ) .", "Collectively , these findings demonstrate that , in three independent alphaviruses , lysine to arginine mutations at distinct sites in the E2 glycoprotein ( Figure 7C ) allow for viral escape from phagocytic cell-mediated clearance .", "The impact of lysine to arginine mutations was surprising as this is a conservative amino acid substitution .", "To further map biochemical features of amino acid side chains that associate with efficient viral clearance by phagocytic cells , we introduced a variety of amino acid substitutions at CHIKV E2 200 .", "The amino acid panel was designed to evaluate positively charged ( K , R , H ) , negatively charged ( D ) , hydrophobic ( A , L ) , and polar , uncharged ( Q , S ) amino acids ( Figure 8A ) .", "In addition , the amino acids selected varied in side chain length ( Figure 8A ) .", "Remarkably , all substitutions tested prevented efficient clearance of circulating CHIKV particles following i . v . inoculation ( Figure 8B ) .", "Moreover , a subset of these mutations was introduced at position E2 251 of RRV , and again all substitutions away from lysine allowed for RRV escape from phagocytic cell-mediated clearance ( Figure 8C ) .", "These findings suggest that clearance of these alphavirus particles from the circulation is strictly dependent on the presence of a lysine residue at E2 200 ( CHIKV ) or E2 251 ( RRV ) .", "The critical lysine residue that mediates clearance in RRV ( E2 K251 ) is conserved in CHIKV ( E2 K252 ) .", "To investigate if this lysine residue also contributes to CHIKV clearance , we introduced an E2 K252R mutation .", "However , we found that CHIKV E2 K252R remained susceptible to clearance by phagocytic cells ( Figure 8D ) , suggesting that , in contrast to RRV , a lysine at this position in E2 is not required for CHIKV clearance .", "Collectively , these findings demonstrate that specific lysine residues in the E2 glycoproteins of CHIKV , ONNV , and RRV facilitate phagocytic cell-mediated clearance of viral particles from the circulation of a vertebrate host ." ], [ "Our findings reveal a previously uncharacterized innate immune pathway that rapidly clears a number of distinct alphavirus particles from the circulation .", "While liver KCs have been implicated as a critical innate defense against blood-borne bacterial pathogens ( Jenne and Kubes , 2013; Krenkel and Tacke , 2017; Kubes and Jenne , 2018 ) , this work expands their role to additionally serve as sentinels for circulating CHIKV , ONNV , and RRV .", "Mechanistically , this innate clearance pathway occurs independently of opsonization with natural antibody or fragments of C3 , and instead is mediated by the scavenger receptor MARCO .", "While our data suggest that KCs in the liver are primarily responsible for clearance of these alphavirus particles from the circulation , MARCO is known to be expressed by other macrophage populations exposed to the blood , including MZM in the spleen ( Borges da Silva et al . , 2015 ) .", "Although we found that the spleen is not necessary for the clearance of circulating CHIKV particles , it remains possible that MZM contribute to the clearance of this group of alphaviruses in intact mice .", "Further work is needed to evaluate whether KCs are required for the clearance of circulating CHIKV , ONNV , and RRV particles , or whether other macrophage populations can compensate in their absence .", "Beyond the antiviral antibody response , the virus-host interactions that determine the magnitude and duration of vertebrate viremia following an arbovirus infection are poorly understood .", "In some cases , alphavirus particles interact directly with glycosaminoglycans ( GAGs ) , such as heparan sulfate ( Bernard et al . , 2000; Byrnes and Griffin , 1998; Gardner et al . , 2011; Heil et al . , 2001; Klimstra et al . , 1998; Smit et al . , 2002 ) , that are ubiquitously found on the surface of mammalian cells .", "Following serial passage in cell culture , alphaviruses frequently acquire adaptive mutations that enhance interactions between viral particles and GAGs .", "( Bernard et al . , 2000; Byrnes and Griffin , 1998; Heil et al . , 2001; Klimstra et al . , 1998; Silva et al . , 2014 ) .", "Alphaviruses that have acquired enhanced in vitro GAG binding are rapidly cleared from the circulation of mice ( Bernard et al . , 2000; Byrnes and Griffin , 2000 ) , suggesting that virion-GAG interactions are one mechanism that can determine the magnitude and duration of arboviral viremia .", "However , the extent to which specific GAGs contribute to clearance of circulating alphavirus particles has not been defined in vivo .", "Beyond GAGs , previous work demonstrated that opsonization of DENV particles by mannose binding lectin ( MBL ) , which can neutralize particles via a C3-dependent mechanism , accelerated their clearance from the circulation of mice ( Fuchs et al . , 2010 ) .", "However , we found that the innate clearance of circulating CHIKV and RRV was maintained in mice lacking C3 and in mice lacking antibodies , suggesting that the clearance of these circulating alphavirus particles occurs through a non-opsonic mechanism .", "SRs are expressed on a variety of tissue resident macrophage populations , including those in the liver and spleen that surveil the blood ( Kubes and Jenne , 2018; Lewis et al . , 2019 ) .", "SRs mediate non-opsonic clearance of a variety of circulating self and non-self molecules due to their capacity to recognize a diverse range of ligands , including modified endogenous proteins or lipoproteins , as well as bacterial surface proteins , lipopolysaccharide ( LPS ) , and lipotechoic acid ( LTA ) ( Areschoug and Gordon , 2009; Canton et al . , 2013 ) .", "We found that the clearance of circulating CHIKV and RRV particles was inhibited by pre-treatment of mice with the SR competitive inhibitors poly ( I ) and dextran sulfate ( Platt and Gordon , 1998 ) , supporting a role for SRs in clearance of these alphaviruses from the circulation .", "Poly ( I ) and dextran sulfate are known ligands for the class A scavenger receptors SR-A1 and MARCO ( Chen et al . , 2006; Platt and Gordon , 1998 ) , both of which are transcriptionally expressed in liver KCs ( Tabula Muris Consortium et al . , 2018 ) .", "SR-A1 and MARCO play a prominent role in host defense by functioning as phagocytic receptors for multiple bacterial pathogens .", "For example , mice deficient in SR-A1 are more susceptible to infection with L . monocytogenes , S . aureus , S . pneumonia and N . meningitides , and MARCO-/- mice have enhanced sensitivity to infection with S . pneumonia ( Areschoug and Gordon , 2009 ) .", "However , less is known about the role of class A scavenger receptors during viral infections .", "SR-A1 has a protective role during systemic herpes simplex virus type 1 ( HSV-1 ) infection of mice ( Suzuki et al . , 1997 ) , and HSV-1 can utilize MARCO for attachment and entry into epithelial cells ( MacLeod et al . , 2013 ) .", "Similarly , vaccinia virus ( VacV ) binding to MARCO on keratinocytes enhances infection of these cells in vitro ( MacLeod et al . , 2015 ) .", "MARCO also mediates transduction of macrophages by adenovirus in vitro , and appears to promote innate immune responses to the infection ( Maler et al . , 2017 ) , suggesting that MARCO-virus interactions can regulate host cell responses to infection .", "Our studies in SR-A1-/- and MARCO-/- mice identified critical roles for MARCO in the efficient clearance of circulating CHIKV particles and for limiting viral dissemination and the magnitude and duration of viremia , suggesting that MARCO may limit disease severity following CHIKV infection .", "Beyond identification of the cell type and specific receptor involved in the clearance of alphavirus particles from the circulation , we also defined viral features that facilitate host recognition and clearance of viral particles by phagocytic cells .", "N-linked glycosylation of DENV and WNV glycoproteins was previously found to influence clearance of these viral particles from the circulation of mice ( Fuchs et al . , 2010 ) .", "However , we found that mutation of the predicted surface-exposed CHIKV E2 glycosylation sites had no impact on the clearance of CHIKV particles from the circulation , suggesting that clearance occurs independent of E2 glycosylation .", "To define the viral features that mediated clearance of circulating alphavirus particles , we identified CHIKV and RRV strains that evade phagocytic cell-mediated clearance .", "CHIKV E2 K200R was isolated from the serum of a Rag1-/- mouse chronically infected with an Asian genotype CHIKV strain ( Hawman et al . , 2017 ) , suggesting that this mutation arose as a mechanism to evade phagocytic cell-mediated clearance from the circulation .", "Introduction of an E2 K200R mutation into CHIKV strains representative of the Asian-American , ECSA , and West African CHIKV clades also facilitated escape of viral particles from phagocytic cell-mediated clearance and enhanced viral dissemination following subcutaneous inoculation .", "Moreover , E2 K200 is conserved in the closely related alphavirus ONNV , and introduction of an E2 K200R mutation in this independent alphavirus species also allowed for escape from clearance from the circulation .", "Collectively , these data demonstrate that an E2 K200R mutation can function in a variety of distinct genetic backgrounds to facilitate escape of circulating alphavirus particles from MARCO-dependent clearance .", "While we found that circulating RRV particles also are susceptible to clearance by phagocytic cells , RRV has an asparagine at E2 200 , suggesting that a viral feature other than E2 K200 promotes the clearance of circulating RRV particles .", "Analysis of a panel of RRV strains revealed that the RRV T48 strain , in contrast to strains SN11 and DC5692 , was relatively resistant to clearance by phagocytic cells .", "Through chimeric and mutational analysis of susceptible ( DC5692 ) and resistant ( T48 ) RRV strains , we discovered that a lysine residue at E2 251 is essential for efficient clearance of RRV particles from the circulation .", "Thus , discrete lysine residues in the E2 glycoprotein of three independent alphaviruses promote their efficient clearance from the circulation .", "We were surprised that single lysine to arginine mutations at discrete sites in E2 allowed circulating alphavirus particles to escape from clearance by phagocytic cells , as the side chains of lysine and arginine are both positively charged and similar in length .", "To more thoroughly evaluate biochemical features of the residue at CHIKV E2 200 or RRV E2 251 that promote particle clearance or particle escape from clearance , we generated panels of CHIKV and RRV strains encoding amino acids at positions E2 200 or 251 with positively-charged , negatively-charged , hydrophobic , or polar uncharged side chains .", "Moreover , this panel of amino acids also had a wide variety of side chain lengths and structures .", "Remarkably , with the exception of lysine , all amino acid substitutions at CHIKV E2 200 or RRV E2 251 allowed for evasion of clearance from the circulation .", "These findings suggest that a lysine residue at CHIKV E2 200 or RRV E2 251 is essential for MARCO-dependent clearance of circulating viral particles .", "Taken together , these observations suggest that these lysine residues may be post-translationally modified to facilitate capture and clearance of circulating alphavirus particles .", "A number of observations from this study and previously published studies support this idea , including ( 1 ) an E2 K200R mutation functions in a variety of distinct genetic backgrounds to facilitate escape of circulating CHIKV and ONNV particles from MARCO-dependent clearance; ( 2 ) a lysine residue at a distinct site in RRV E2 is essential for phagocytic-cell mediated clearance of circulating RRV particles; ( 3 ) while the lysine that mediates RRV clearance is conserved in CHIKV , mutating this lysine has no impact on clearance of circulating CHIKV particles; ( 4 ) lysine is frequently post-translationally modified , and can be modified with a diverse range of post-translational modifications ( PTMs ) ( Azevedo and Saiardi , 2016 ) ; and ( 5 ) SRs were initially identified based on their capacity to capture lysine acetylated low density lipoprotein ( Platt and Gordon , 1998 ) .", "However , whether specific features of unmodified lysine residues within particular structural contexts of E2 , or post-translational modifications of these lysine residues , allow for alphavirus recognition by phagocytic cells requires additional biochemical and structural analyses , and thus remains to be determined .", "Despite the apparent benefit for the virus to encode an arginine or other amino acid at E2 200 or 251 , lysine residues at CHIKV E2 200 , ONNV E2 200 , and RRV E2 251 are commonly found in virus isolates .", "Analysis of E2 protein sequences in the ViPR database ( Pickett et al . , 2012 ) revealed that 1 , 333/1 , 335 CHIKV sequences encode a lysine at position E2 200 ( 2/1 , 335 encode an arginine ) , 8/8 ONNV sequences encode a lysine , and 187/204 available RRV sequences encode a lysine at position E2 251 , while the remaining 17 encode an arginine .", "One possible explanation for this conservation could be that a lysine residue at this position provides a fitness advantage in the mosquito vector .", "However , we found that CHIKV E2 K200R replication and dissemination was indistinguishable from that of WT CHIKV in Ae .", "aegypti mosquitoes , and no selective disadvantage of an arginine residue at this position was observed in direct competition experiments , suggesting that the conservation of lysine at CHIKV E2 200 is not due to constraints in the mosquito vector .", "Alternatively , the susceptibility or resistance of circulating alphavirus particles to clearance may be influenced by polymorphisms in SRs .", "Genes encoding SRs , including MARCO , exhibit substantial genetic variation in both humans and mice ( Bowdish and Gordon , 2009 ) and this variation can have functional consequences .", "For example , position 252 of MARCO is known to be polymorphic in humans , and the less common variant has a decreased capacity to phagocytize bacteria in vitro ( Novakowski et al . , 2018 ) .", "Consistent with this , SNPs within human SRs associate with differential susceptibility to various pathogens ( Bowdish et al . , 2013; High et al . , 2016; Lao et al . , 2017; Ma et al . , 2011; Westhaus et al . , 2017 ) .", "Following alphavirus infections , human patients exhibit a high degree of variability in peak viremia and disease severity ( Chow et al . , 2011; de St Maurice et al . , 2018; Musso et al . , 2017; Thiberville et al . , 2013; Vaughn et al . , 2000; Waggoner et al . , 2016 ) .", "Given this , inter- or intra-host variability within SRs may influence the susceptibility of alphaviruses to SR-dependent clearance from the circulation , contributing to variability in viremia and disease outcomes .", "Similar species-specific effects have been observed with multiple pathogen recognition receptors ( Daugherty and Malik , 2012 ) .", "Furthermore , MARCO is evolving under positive selection ( Yap et al . , 2015 ) , a signature often associated with host-viral conflicts .", "Thus , circulating alphaviruses may have adapted to their cognate hosts and can readily evade this defense mechanism in certain vertebrates , but the MARCO allele in the mouse strains evaluated in this study allows these alphaviruses to be efficiently cleared from the circulation .", "More work is needed to determine whether polymorphisms within or between vertebrate species influence SR-dependent clearance of circulating alphaviruses .", "The efficient clearance of alphaviruses from the circulation by phagocytic cells has important implications for the course of infection .", "The CHIKV E2 K200R strain that escapes MARCO-dependent clearance generates a higher viremia , disseminates to distal sites more rapidly , and causes more severe disease outcomes in mice ( Hawman et al . , 2017 ) .", "Analogously , infection of MARCO-/- mice with WT CHIKV also resulted in enhanced viremia and more rapid viral dissemination to distal sites , suggesting that the E2 K200R mutation allows the virus to escape from MARCO-dependent clearance .", "Consistent with our data , prior studies found that some strains of Venezuelan equine encephalitis virus accumulated in the liver following i . v . inoculation of hamsters ( Jahrling and Gorelkin , 1975 ) and that depletion of phagocytic cells with clodronate liposomes resulted in a higher magnitude and duration of viremia following subcutaneous CHIKV infection of mice ( Gardner et al . , 2010 ) .", "Similarly , clodronate liposome depletion of phagocytic cells enhanced WNV viremia and disease severity in mice ( Bryan et al . , 2018 ) , suggesting that phagocytic cell-mediated clearance of circulating arboviruses has implications that span virus genera .", "Beyond disease outcomes , the magnitude and duration of viremia also greatly influences transmission cycles .", "While some arboviruses , such as WNV and Japanese Encephalitis virus , are unable to generate a viremia of sufficient magnitude in humans to infect a mosquito vector and propagate the transmission cycle ( Weaver , 2018 ) , others like CHIKV , DENV , and Zika virus are readily amplified through human-mosquito-human cycles ( Weaver , 2018 ) .", "Susceptibility or resistance to clearance by liver macrophages , other scavenging cell types such as liver sinusoidal endothelial cells , and SRs may contribute to whether humans are dead-end hosts for an arbovirus or can fuel widespread epidemics ." ], [ "This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health .", "All of the animals were handled according to approved institutional animal care and use committee ( IACUC ) protocols ( #00026 ) of the University of Colorado School of Medicine ( Assurance Number A3269-01 ) .", "Experimental animals were humanely euthanized at defined endpoints by exposure to isoflurane vapors followed by thoracotomy .", "Cell lines were obtained directly from ATCC .", "Vero cells ( ATCC CCL81 ) were cultured in Dulbecco’s Modified Eagle medium ( DMEM ) -F-12 ( Life Technologies ) supplemented with 10% fetal bovine serum ( FBS , Lonza ) , 1x nonessential amino acids ( Life Technologies ) , sodium bicarbonate , 2 mM L-glutamine , and penicillin-streptomycin .", "BHK-21 cells ( ATCC CCL10 ) were cultured in α-minimum essential medium ( Life Technologies ) supplemented with 10% FBS ( Lonza ) , 10% tryptone phosphate broth , and penicillin-streptomycin .", "Human dermal fibroblasts ( ATCC PCS-201–012 ) were cultured in high-glucose DMEM supplemented with 10% FBS and penicillin-streptomycin .", "C6/36 cells ( ATCC CRL-1660 ) were cultured in minimum essential medium with Earle’s salts ( Life Technologies ) supplemented with 5% FBS ( Lonza ) and penicillin-streptomycin .", "All mammalian cells were cultured at 37°C , and C6/36 mosquito cells were incubated at 30°C .", "Cells tested negative for mycoplasma .", "CHIKV strains used include Asian strain AF15561 ( Ashbrook et al . , 2014 ) , Asian-American strain 99659 ( Jones et al . , 2017 ) , ECSA stain SL15649 ( Morrison et al . , 2011 ) , and WA strain 37997 ( Vanlandingham et al . , 2005b ) .", "All CHIKV stocks were derived from infectious cDNA clones by electroporating RNA into BHK-21 cells , as previously described ( Morrison et al . , 2011 ) , with the exception of virus used in Figure 4B , which was a propagated P1 stock of AF15561 generated in human dermal fibroblasts .", "Unless otherwise noted , AF15561 was used as the representative CHIKV strain .", "RRV strains used include SN11 ( Liu et al . , 2011 ) , T48 ( Doherty and Whitehead , 1963; Kuhn et al . , 1991 ) , and DC5692 ( Jupille et al . , 2011; Lindsay , 1996 ) .", "SN11 is a clinical isolate that was passed 1x on C6/36 cells before we propagated a stock in BHK-21 cells ( Liu et al . , 2011 ) , while T48 and DC5692 were derived from cDNA clones by electroporation of viral RNA into BHK-21 cells as previously described ( Morrison et al . , 2006 ) .", "For some experiments ( Figure 7—figure supplement 1C ) , viral particles were propagated in serum free media and purified by centrifugation through a 20% sucrose cushion at 24 , 000 x g for 6 hr .", "Unless otherwise noted , SN11 was used as the representative RRV strain .", "ONNV SG650 was derived from a cDNA clone through electroporation into BHK-21 cells , and propagated on BHK-21 cells for one passage to increase titer .", "Mutant variants used were described previously , ( AF15561 E2 K200R [Hawman et al . , 2017]; T48-DC5692 E1/6K [RR73] [Jupille et al . , 2011] , T48-DC5692 E2/E3 [RR100] [Jupille et al . , 2011] , and T48 E2 R251K [Jupille et al . , 2013] ) or were generated through site-directed mutagenesis of cDNA clones .", "Mutant virus stocks were sequenced to confirm the presence of introduced mutations .", "All viruses were titered by plaque assay on BHK-21 cells or by RT-qPCR .", "The cDNA clone of CHIKV 99659 was a gift from Mark Heise ( University of North Carolina ) , the cDNA clones of CHIKV 37997 and ONNV SG650 were gifts from Stephen Higgs ( Kansas State University ) , and RRV SN11 was a gift from John Aaskov ( Queensland University of Technology ) .", "Viral point mutants were constructed through site-directed mutagenesis ( Agilent #200517 ) of cDNA clones , using the primers provided in Supplementary file 1 .", "A sequence verified fragment containing the desired mutation was then subcloned into the unmutagenized parental plasmid .", "For AF15561 E2 K200 mutants , SL15649 E2 K200R , 99659 E2 K200R , and AF15561 glycosylation mutants , subcloning was performed using restriction sites XhoI and XmaI .", "37997 E2 K200R was not subcloned , but the E2 gene was sequence verified to only contain the K200R mutation .", "For ONNV E2 K200R , BstBI and BamHI were used for subcloning .", "RRV DC6592 E2 K251R was subcloned using site PspOMI and XmaI .", "RRV T48 mutants E2 R251A , E2 R251D , E2 R251Q , and E2 R251S were not subcloned , but the E2 gene was sequence verified to only contain the specified E2 251 mutation .", "BALB/cJ , 129S1/SvlmJ , C57BL/6J , sham or splenectomized C57BL/6J , and congenic μMT ( Kitamura et al . , 1991 ) , C3-/- ( Wessels et al . , 1995 ) , and SR-A1-/- ( Suzuki et al . , 1997 ) mice were obtained from The Jackson Laboratory .", "MARCO-/- mice ( Arredouani et al . , 2004 ) were provided by Dawn Bowdish ( McMaster University ) .", "All experiments were performed in 3–4 week-old mice , except those involving sham or splenectomized mice , which were 6 weeks-old at the time of virus inoculation .", "All WT mice used were male , with the exception of Figure 2C , where five mice were female .", "WT mice were purchased commercially and were age-matched , and distributed randomly across groups .", "For mouse strains bred in house ( μMT , C3-/- , SR-A1-/- , MARCO-/- ) , mice were distributed randomly into groups containing approximately equal division of the sexes .", "All experiments were performed under Biosafety level 2 or 3 conditions , as appropriate .", "Masking was not used .", "Phagocytic cell depletions were achieved by inoculating mice i . v . with 100 μl/10 g body weight of PBS- or clodronate-loaded liposomes ( Clodronateliposomes . org ) 42 hr prior to inoculation with virus .", "For clearance blockade experiments , mice were treated i . v . with 200 μg of poly ( I ) or poly ( C ) ( Sigma 26936-41-4 and 26936-40-3 ) or 200 μg of dextran or dextran sulfate ( Sigma 31392 and D6001 ) 5 min prior to inoculation with virus .", "For serum clearance experiments , mice were inoculated i . v . with a defined number of particles ( CHIKV and RRV: 107 or 108 viral genomes depending on experiment; ONNV: 105 PFU ) diluted in a total volume of 100–200 μl of diluent ( PBS with 1% FBS ) .", "Viral inoculum was reserved to quantify input genomes .", "At the time of termination , mice were euthanized through inhalation of isoflurane vapors , followed by thoracotomy , and serum was collected .", "In some experiments , mice were then perfused by intracardiac injection of PBS prior to collecting the indicated tissues in RPMI for flow cytometry or in TRIzol Reagent ( Life Technologies ) for RNA analysis .", "Tissues for RNA analysis were first homogenized with a MagNA Lyser ( Roche ) .", "For viral dissemination experiments , mice were inoculated in the left rear footpad with 10 μl containing 103 PFU of virus diluted in PBS with 1% FBS .", "At 24 hpi , mice were euthanized and blood was collected .", "Mice were then perfused by intracardiac injection of PBS , and the indicated tissues were collected in in vitro diluent ( PBS with 1% FBS , 1× Ca2+ , and 1× Mg2+ ) and homogenized with a MagNA Lyser ( Roche ) for analysis by focus formation assay or plaque assay .", "To quantify viral genomes , RNA was isolated from 20 μl of serum or homogenized tissues in TRIzol using the PureLink RNA mini kit ( Life Technologies ) .", "CHIKV cDNA was generated using random primers ( Invitrogen ) with SuperScript IV reverse transcriptase ( Life Technologies ) .", "CHIKV genome copies were quantified by RT-qPCR using a CHIKV specific forward primer ( 5′-TTTGCGTGCCACTCTGG-3′ ) and reverse primer ( 5′-CGGGTCACCACAAAGTACAA-3′ ) with an internal TaqMan probe ( 5′-ACTTGCTTTGATCGCCTTGGTGAGA-3′ ) , as previously described ( Hawman et al . , 2013 ) .", "To generate RRV cDNA , a sequence-tagged ( indicated with lower case letters ) RRV-specific RT primer ( 5′-ggcagtatcgtgaattcgatgcAACACTCCCGTCGACAACAGA-3′ ) was used with SuperScript IV reverse transcriptase ( Life Technologies ) .", "To quantify RRV genomes by RT-qPCR , a tag sequence-specific reverse primer ( 5′-GGCAGTATCGTGAATTCGATGC-3′ ) was used with a RRV sequence-specific forward primer ( 5′-CCGTGGCGGGTATTATCAAT-3′ ) and an internal TaqMan probe ( 5′-ATTAAGAGTGTAGCCATCC-3′ ) , as previously described ( Stoermer et al . , 2012 ) .", "In some experiments , infectious virus was quantified by plaque assay or focus formation assay , as described previously ( Hawman et al . , 2017 ) .", "In brief , for plaque assays , BHK-21 cells were seeded in a 6-well plate and inoculated with 10-fold serial dilutions of serum or tissue homogenate in in vitro diluent .", "Following a 1 hr adsorption , cells were overlayed with 1% immunodiffusion agarose ( MP Biomedical ) and incubated at 37°C for 40–44 hr before staining with neutral red stain to enumerate plaques .", "For focus formation assays , Vero cells were seeded in a 96-well plate and serial dilutions of serum or tissue homogenate were adsorbed to the cells for 2 hr , after which the inoculum was removed and an overlay of 0 . 5% methylcellulose in MEM-alpha with 10% FBS was added to the cells .", "Following an 18 hr incubation at 37°C , the cells were fixed with 1% paraformaldehyde and probed with CHK-11 monoclonal antibody at 500 ng/ml in Perm Wash ( 1x PBS , 0 . 1% saponin , 0 . 1% BSA ) , followed by a secondary goat anti-mouse IgG conjugated to horseradish peroxidase at 1:2000 in Perm Wash .", "Foci were visualized with TrueBlue substrate ( Fisher ) and counted with a CTL Biospot analyzer using Biospot software ( Cellular Technology ) .", "WT or MARCO-/- C57BL/6 mice were treated i . v . with 100 μl/10 g body weight of PLL or CLL 42 hr prior to i . v . inoculation with 2 . 5 × 109 genomes of CHIKV AF15561 diluted in 100 μl of diluent .", "At 45 min post-inoculation , mice were sacrificed , perfused with 4% paraformaldehyde ( PFA ) , and liver lobes were collected and fixed in 4% PFA for 24 hr .", "Tissues were paraffin-embedded and sectioned .", "RNA in situ hybridization was performed using the RNAscope 2 . 5 HD Assay-Brown ( Advanced Cell Diagnostics ( ACD ) Cat .", "No . 322300 ) according to the manufacturer’s protocol .", "In brief , paraffin-embedded tissue sections were incubated at 60°C for 1 hr followed by deparaffinization .", "Endogenous peroxidase activity was quenched by pretreatment with hydrogen peroxide for 10 min at room temperature ( RT ) , and slides were boiled in RNAscope Target Retrieval Solution for 15 min .", "Slides were then treated with Protease III at 40°C for 30 min in the ACD Ez II Hybridization Oven ( ACD Cat . No . 321710 ) , followed by hybridization of V-CHIKV-sp-01 probe ( ACD Cat . No . 481891 ) for 2 hr at 40°C in the EZ II Oven .", "Following hybridization of AMP1-6 , signal was detected using DAB substrate and tissues were counterstained with Gill’s hematoxylin and visualized using standard bright-field microscopy .", "On sections from the same liver lobes , immunohistochemistry was used to visualize F4/80 positive cells .", "Tissues were deparaffinized and target retrieval was performed by incubating slides in Citrate pH 6 . 1 Target Retrieval Solution ( Dako Cat . No . S1699 ) in a Biocare Medical decloaking chamber at 110°C for 20 min .", "Tissues were then incubated with Dual Endogenous Enzyme Block ( Dako Cat . No . S2000389 ) for 10 min at RT , followed by incubation with Protein Block ( Dako Cat . No . X090930 ) for 10 min at RT .", "F4/80 antibody clone Cl:A3-1 ( BioRad Cat . No . MCA497 ) was diluted 1:100 and added to tissues and incubated for 1 hr at RT , followed by biotinylated Anti-Rat IgG ( Vector Laboratories BA-9401 ) for 30 min at RT .", "Tissues were then incubated with VECTASTAIN Elite ABS reagent ( Vector Laboratories , PK-6100 ) at RT for 30 min , signal was detected using DAB substrate , and tissues were counterstained with Gill’s hematoxylin and imaged .", "To quantify F4/80 positive cells in WT and MARCO-/- livers , positively-stained cells in 10 randomly selected high power fields per section were counted in a blinded manner and used to calculate the average number of F4/80 positive cells per field .", "106 PFU of virus ( CHIKV WT or E2 K200R , AF15561 strain; RRV T48 or T48 E2 R251K ) were incubated in triplicate in normal mouse serum ( Thermo Fisher Scientific #10410 ) at either 4°C or 37°C .", "Aliquots from 0 , 1 , 8 , 24 and 48 hr time points were titered by plaque assay ( RRV ) or Focus Formation Assay ( CHIKV ) , as described above .", "Livers were minced and incubated in digestion buffer ( RPMI 1640 with 10% FBS , 15 mM HEPES , 2 . 5 mg/ml Collagenase Type 1 ( Worthington Biochemical ) , 1 . 7 mg/ml DNase I ( Roche ) , 1% penicillin/streptomycin ) for 30 min at 37°C with shaking ( 130 rpm ) .", "Digested livers were passed through a 100 μm cell strainer ( BD Falcon ) .", "Liver cells were pelleted and re-suspended in a 35% Percoll solution and centrifuged for 15 min at 450 x g at 4°C with medium acceleration and no break to isolate lymphocytes , and washed 1X with RPMI .", "Isolated cells from livers were blocked with anti-mouse FcγRII/III ( 2 . 4G2; BD Pharmingen ) for 20 min on ice and then stained in FACS buffer ( 1x PBS , 2% FBS ) for 1 hr on ice with the following antibodies from Biolegend ( most ) or eBioscience ( NK1 . 1 ) : anti-MHC-II ( M5/114 . 14 . 2 ) , anti-Ly6C ( HK1 . 4 ) , anti-F4/80 ( BM8 ) , anti-CD11b ( M1/70 ) , anti-NK1 . 1 ( PK136 ) , anti-TCRB ( H57-597 ) , anti-CD19 ( 6D5 ) , anti-CD11c ( N418 ) , anti-CD45 ( 30-F11 ) , anti-Ly6G ( 1A8 ) .", "Liver cells were fixed in 1% paraformaldehyde ( PFA ) for 15 min .", "Samples were analyzed on an LSRFortessa using FACSDiva software ( Becton Dickinson ) .", "Further analysis was done using FlowJo Software ( Tree Star ) .", "Aedes aegypti mosquitoes from Mexico ( Poza Rica [Vera-Maloof et al . , 2015] , F18-20 ) were used for vector competence studies .", "Mosquitoes were fed citrated sheep blood for colony maintenance and provided with sugar and water ad libitum .", "Larvae and adults were reared and maintained under controlled conditions of 28°C temperature , 70% relative humidity and a 12:12 ( L:D ) diurnal light cycle .", "Mosquitoes were provided an infectious blood meal containing either CHIKV AF15561 WT or CHIKV AF15561 E2 K200R .", "Defibrinated calf blood was mixed 1:1 with DMEM containing 2 . 2 × 106 PFU/mL virus , resulting in a final blood meal titer of 1 . 1 × 106 PFU/mL .", "On day 3 post-infection , mosquitoes were cold-anesthetized and legs and wings were removed and placed into a 2 mL tube containing 250 µL mosquito diluent ( 1x PBS containing 20% FBS , antibiotics and antifungals ) and a sterile stainless-steel bead .", "Mosquito saliva was then collected by placing the mosquito proboscis into a capillary tube filled with immersion oil to salivate for 30 min .", "Each capillary tube end containing saliva was broken off into a microcentrifuge tube containing 100 µL mosquito diluent , centrifuged for 3 min at 15 , 000 x g and stored at −80°C .", "After salivation , mosquito bodies were also placed in a 2 mL tube containing 250 µL mosquito diluent and a sterile stainless-steel bead .", "Tissues ( legs/wings and bodies ) were homogenized using a Retsch Mixer Mill MM400 ( Germany ) at 24 cycles/second for 1 min , centrifuged at 15 , 000 x g for 3 min and stored at −80°C .", "To determine whether samples contained virus or not , Vero cells grown in 24-well plates were inoculated with 50 µL mosquito sample for 1 hr before a semi-solid overlay ( 0 . 6% Tragacanth gum in EMEM ) was added .", "Cells were fixed and stained with crystal violet three dpi to visualize plaques .", "Samples found to be positive for virus were further evaluated by focus formation assay , as described above , to quantify the amount of infectious virus present in the sample .", "To perform viral competition assays , we generated a genetically marked clone of CHIKV AF15561 using sight- directed mutagenesis ( Forward Primer: 5′-ctaaactaaaggggcccaaagcagcagcgctgt-3′; Reverse Primer: 5′-acagcgctgctgctttgggcccctttagtttag-3′ ) to introduce a silent mutation that generates an ApaI restriction site in nsP4 , as described previously ( Chen et al . , 2013 ) .", "For in vitro competition experiments , C6/36 cells were seeded in 24-well dishes and inoculated in triplicate at an MOI of 1 PFU/cell with a 1:1 ratio of CHIKV AF15561-ApaI and CHIKV AF15561 or CHIKV AF15561-ApaI and CHIKV AF15561 E2 K200R .", "Following a 1 hr adsorption , virus inoculum was removed , cells were washed , and fresh media was added .", "At 24 hpi , 1/10th of the supernatant was passaged onto a new well of cells , and this was repeated for a total of five passages .", "RNA was extracted from the supernatant , and cDNA was generated as described above for CHIKV quantification .", "For in vivo competition experiments , Aedes aegypti mosquitoes were microinjected intrathoracically with 138 PFU of a 1:1 mixture of CHIKV AF15561-ApaI and CHIKV AF15561 or CHIKV AF15561-ApaI and CHIKV AF15561 E2 K200R .", "At seven dpi , mosquito bodies and saliva were collected and processed as described above and frozen at −80°C .", "RNA was extracted from 50 µL of each sample using the Mag-Bind Viral DNA/RNA 96 kit ( Omega Bio-Tek ) on the KingFisher Flex Magnetic Particle Processor ( Thermo Fisher Scientific ) following the manufacturer’s instructions .", "cDNA was generated as described above for CHIKV quantification .", "Relative amounts of each virus in samples were evaluated as described previously ( Chen et al . , 2013 ) .", "In brief , cDNA was subjected to PCR using forward primer ( 5′-atatctagacatggtgga-3′ ) and reverse primer ( 5′- tatcaaaggaggctatgtc-3′ ) to amplify the region of nsP4 ( 6106–6794 ) containing the ApaI site .", "PCR products were digested with equal amounts of ApaI and PspOMI ( isoschizomers ) at room temperature for 30 min , and at 37°C for 4 hr .", "Complete digestion was confirmed using control PCR products amplified from cDNA plasmid clones of CHIKV AF15561 WT or CHIKV AF15561-ApaI .", "Digested products were run on a 1% TAE gel , stained with ethidium bromide ( Sigma ) , and imaged using Syngene G:Box .", "The percent band intensity was quantified using Syngene GeneTools .", "To determine the sample size to be used , population variance from pre-existing sample sets were used in a power calculation ( 80% power , 0 . 05 type I error ) to detect a 4–5-fold effect .", "The defined sample size ( N ) listed in each figure legend refers to biological replicates of individual mice , mosquitoes , or cell wells .", "Data are represented as mean ± SD .", "The statistical tests used for each data set are indicated in the figure legends .", "Statistical analysis was conducted using GraphPad Prism 7 . 0 .", "Two-sided t-tests ( parametric ) or Mann Whitney tests ( nonparametric ) were used to compare two groups .", "One-way ANOVA with Bonferroni’s multiple comparison test ( parametric ) or Kruskal-Wallis with Dunn’s multiple comparisons test ( nonparametric ) were used to compare three or more groups , and two-way ANOVA with Bonferroni’s multiple comparison test was used to compare two groups at multiple time points ." ] ]

[ "The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission , geographic spread , and disease severity .", "We find that multiple alphaviruses , including chikungunya ( CHIKV ) , Ross River ( RRV ) , and o’nyong ‘nyong ( ONNV ) viruses , are cleared from the circulation of mice by liver Kupffer cells , impeding viral dissemination .", "Clearance from the circulation was independent of natural antibodies or complement factor C3 , and instead relied on scavenger receptor SR-A6 ( MARCO ) .", "Remarkably , lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV ( E2 K200R ) as well as RRV ( E2 K251R ) allowed for escape from clearance and enhanced viremia and dissemination .", "Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions .", "These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts , ultimately influencing disease severity and likely transmission efficiency ." ]

[ "Viruses transmitted by blood-feeding insects , such as mosquitoes and ticks , cause serious human diseases .", "In recent years these viruses ( also known as arboviruses ) have re-emerged at an unprecedented scale , leading to global outbreaks of diseases such as Zika or chikungunya fever .", "The severity of these diseases and how easily they can be transmitted depends , in part , on the level of virus in the host’s bloodstream following infection .", "The more viral particles present in the blood , the easier it is for other insects that bite the host to become infected and help spread the disease .", "Yet , the mechanisms that hosts use to control the amount of virus present in the blood and how long it persists are poorly understood .", "To investigate this further , Carpentier et al . used a combination of molecular and genetic techniques to study how mice clear particles of arbovirus from their bloodstream .", "Surgically removing the spleen from infected mice revealed that this organ , which filters out unwanted or damaged materials from blood , is not required to clear some arbovirus particles .", "Carpentier et al . found that removing these arboviruses from the blood instead required Kupffer cells , a type of immune cell found in the liver .", "Genetically manipulating mice so they no longer produced a protein on the surface of Kupffer cells known as MARCO revealed that this receptor is needed to clear chikungunya viral particles .", "When MARCO was genetically deleted this led to an increase in the number of viral particles in the mice’s bloodstream , and allowed the virus to spread more rapidly throughout the bodies of the mice .", "Further experiments on three different types of arboviruses showed that in order to be cleared by MARCO , each of these viruses needed a lysine residue – one of the building blocks that makes up proteins – at defined positions within their protein sequence .", "These findings reveal a previously unknown mechanism for how hosts remove arbovirus particles from their bloodstream .", "Future studies could use this information to identify new ways to control the transmission and reduce the severity of these viral diseases ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "During development bone morphogenetic proteins ( BMPs ) , comprising at least twenty structurally-related members of the transforming growth factor β ( TGF-β ) superfamily , are involved in many growth and differentiation events essential for determining body structure ( Wu and Hill , 2009 ) .", "Establishing temporospatial gradients or restricted distributions of BMP signaling is important for many of these processes , which are regulated by a number of mechanisms: ligand binding by extracellular BMP antagonists ( Brazil et al . , 2015 ) , intracellular feedback inhibition downstream of the BMP receptor ( Massagué et al . , 2005 ) , spatially restricted proteolytic processing ( Thomas et al . , 2006 ) , and promotion or restriction of diffusion by interactions with extracellular matrix proteins such as collagen type IV ( Umulis et al . , 2009 ) and heparan sulfate proteoglycans ( HSPGs ) ( Matsumoto et al . , 2010; Belenkaya et al . , 2004 ) .", "In addition , BMP signaling is also influenced via communication with other signaling pathways , particularly those that act through mitogen-activated protein kinases ( MAPKs ) .", "MAPK-directed phosphorylation of BMP receptor-regulated Smad 1/5/8 in the linker region inhibits BMP signaling by blocking Smad translocation to the nucleus ( Alarcón et al . , 2009; Sapkota et al . , 2007 ) .", "We showed previously that SMOC , a matricellular protein associated with basement membranes ( Vannahme et al . , 2002 ) and expressed in developing brain , branchial arches , eye , pronephros , limb bud cartilage condensations , and joint interzones ( Okada et al . , 2011; Rainger et al . , 2011; Thomas et al . , 2009 ) , inhibits BMP signaling ( Thomas et al . , 2009 ) ; following the addition of BMP2 to NIH3T3 fibroblasts transfected with SMOC , downstream phosphorylation of Smad 1/5/8 is blocked ( Thomas et al . , 2009 ) .", "In Xenopus ectodermal explants ( animal caps ) , SMOC was shown to activate MAPK signaling and inhibit Smad 1/5/8-mediated BMP signaling downstream of the constitutively active BMP receptor , BMPR1B ( Thomas et al . , 2009 ) .", "Although the exact mechanism is not known , the activity was lost in the presence of non-phosphorylatable linker-mutant Smad , suggesting that BMP inhibition results from activation of MAPK signaling and subsequent ubiquitination and degradation of Smad following linker Smad phosphorylation ( Sapkota et al . , 2007 ) .", "In Drosophila , the SMOC orthologue pentagone ( pent ) is expressed in developing wing imaginal discs and has also been shown to inhibit BMP signaling ( Vuilleumier et al . , 2010 ) .", "However , Pent did not appear to inhibit BMP signaling in the presence of the constitutively active zebrafish BMPR1 receptor , Alk8 ( Vuilleumier et al . , 2010 ) .", "Structurally , SMOC and Pent are similar , containing an N-terminal follistatin-like domain ( FS ) , followed by two thyroglobulin-like domains separated by a non-homologous domain , and a C-terminal extracellular calcium binding ( EC ) domain ( Vannahme et al . , 2002; Vuilleumier et al . , 2010 ) .", "Although Pent contains an additional EC domain located between the two thyroglobulin-like domains , their phylogenetic conservation would predict similar functions .", "In Drosophila , Pent binds to the cell surface HSPG , Dally , which is required for Pent to extend the range of BMP signaling during Drosophila wing patterning ( Vuilleumier et al . , 2010 ) .", "While SMOC has also been shown to bind to HSPGs ( Klemenčič et al . , 2013 ) , there have been no reports of SMOC promoting BMP signaling at a distance from its source .", "Indeed , the dual function of SMOC/Pent as a BMP inhibitor and an expander of BMP signaling have not been reconciled .", "Here , we present the first evidence showing how different domains within SMOC function either to inhibit BMP signaling locally or expand its range of effect ." ], [ "To support the applicability of functional information from Pent to the vertebrate SMOC ( Figure 1A ) , we first confirmed that a pent cDNA construct was biologically active in Drosophila .", "As reported previously ( Vuilleumier et al . , 2010 ) , compared to controls , flies homozygous for pent mutations display a characteristic truncation of the L5 longitudinal vein of the adult wing ( Figure 1—figure supplement 1 ) .", "When pent was expressed in its normal location during wing development , using the Gal4/UAS system , the mutant phenotype was rescued completely , demonstrating that the pent construct had full biological activity ( Figure 1—figure supplement 1 ) .", "Initial injections of pent mRNA into Xenopus embryos produced no apparent effects ( not shown ) ; however overexpression of a synthetic pent mRNA ( co-pent ) optimized for codon usage and translation efficiency in Xenopus ( Villegas and Kropinski , 2008 ) ( Figure 1—figure supplement 2 ) produced a dorsalized phenotype indistinguishable from that observed following overexpression of Xenopus SMOC-1 ( XSMOC-1 ) ( Figure 1B ) .", "The ability of Pent to inhibit BMP signaling downstream of the BMP receptor was analyzed in Xenopus ectodermal explants ( animal caps ) following co-injection of mRNAs for co-pent and constitutively active BMP receptor1B ( caBMPR1B ) ; the caBMPR1B , containing an intracellular activating mutation ( Q203D ) , promotes phosphorylation of Smad 1/5/8 and subsequent BMP signaling events independent of ligand binding ( Zou et al . , 1997 ) .", "As expected , XVent , a direct downstream target of BMP signaling ( Gawantka et al . , 1995 ) , was strongly expressed in animal caps from embryos injected with caBMPR1B alone ( Figure 1C ) .", "In contrast , XVent was markedly reduced in caps from embryos co-injected with caBMPR1B and either co-pent or XSMOC-1 ( Figure 1C ) ; these results suggested that , as we had shown previously for SMOC ( Thomas et al . , 2009 ) , Pent can inhibit BMP signaling downstream of the BMPR1B receptor .", "It is unclear why , in a previous report ( Vuilleumier et al . , 2010 ) , Pent was able to rescue ventralization caused by overexpression of Bmp2b in Zebrafish , but not following overexpression of the Zebrafish caBMPR1 , Alk8 .", "As both BMPR1B ( Alk6 ) and Alk8 are type I BMP receptors that activate Smad 1/5/8 , a possible reason could be the amounts of mRNA injected and/or the amounts of protein produced .", "In our previous study ( Thomas et al . , 2009 ) , SMOC transfected NIH3T3 cells were able to inhibit BMP signaling in the presence of excess BMP2 ( 100 ng/ml ) ; where the limiting factor would be the number of BMP receptors on the cells .", "Conversely , in order for SMOC/Pent to inhibit BMP signaling in the presence of caBMPR1B , SMOC/pent needed to be overexpressed at a 2:1 ratio ( Figure 1C ) ; SMOC was not able to inhibit BMP signaling in the presence of an excess of caBMPR1B ( not shown ) . 10 . 7554/eLife . 17935 . 003Figure 1 . Xenopus SMOC-1 and Drosophila pent are orthologues that inhibit BMP signaling downstream of the BMP receptor .", "( A ) Schematic representation of vertebrate SMOC and Drosophila Pent: SP- signal peptide , FS – Follistatin-like domain , Tg1 – Thyroglobulin type I-like domain , EC- Extracellular calcium binding domain ( B ) Dorsalized phenotypes of stage 35 Xenopus embryos following overexpression of mRNAs for XSMOC-1 or codon-optimized pent ( co-pent ) : Xenopus embryos were injected bilaterally at the two-cell stage with 200 pg of mRNA for either GFP ( control ) , XSMOC-1 , or co-pent .", "The exaggerated dorsal/anterior structures and diminished posterior structures observed following XSMOC-1 or co-pent overexpression were observed in 95% of embryos in four independent experiments ( n = 130 ) .", "( C ) RT-PCR analysis of animal cap ( AC ) explants removed from stage 8/9 embryos injected bilaterally at the two-cell stage with either 450 pg of GFP ( Control ) , 150 pg of constitutively active BMP receptor IB ( caBMPR-IB ) plus GFP ( 300 pg ) , or 150 pg of caBMPR-1B plus XSMOC-1 or co-pent ( 300 pg ) mRNAs .", "The AC explants were incubated until stage 20 before RNA extraction .", "Induction of the BMP signaling target gene , XVent , by caBMPR-1B was blocked by co-expression of either XSMOC-1 or co-pent .", "–RT control , without reverse transcriptase . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 00310 . 7554/eLife . 17935 . 004Figure 1—figure supplement 1 . Pent constructs are biologically active in Drosophila , but XSMOC-1 protein is not detected in Drosophila following mRNA overexpression .", "( A–C )", "Adult Drosophila longitudinal wing vein ( L1 to L5 ) patterns in ( A ) wild type; arrow indicates the position of the anterior crossvein between L4 and L5 .", "( B ) pent2-5/pentA17 mutant lacking pent where L5 is missing ( C ) Rescue of pent2-5/pentA17 phenotype by expression of Pent in a pent2-5/pentA17 background using the UAS/Gal4 system with brinker ( brk ) /Gal4 as the driver .", "( D ) Immunoblot of Drosophila embryo extracts ( 10 μg/lane ) from control yellow white ( YW ) Drosophila embryos spiked with XSMOC-1 ( Lanes 1–5 ) as indicated , XSMOC-1 only ( Lane 6 ) , or Drosophila lines 4 ( Lane", "7 ) and 5 ( Lane", "8 ) overexpressing XSMOC-1 mRNA .", "( E ) Coomasie-stained SDS-PAGE gel following protein transfer shown in ( D ) , showing residual protein .", "The amount of protein in lane seven is notably more than that in other lanes , but the XSMOC-1 signal is nevertheless undetectable . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 00410 . 7554/eLife . 17935 . 005Figure 1—figure supplement 2 . Nucleotide alignment of pentagone and codon optimized pentagone ( co-pentagone ) .", "Amino acid identity is 100%; nucleotide identity is 73% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 005 Having established that Pent can function as a BMP antagonist in Xenopus assays , we wanted to determine whether SMOC could function as an expander of BMP signaling in Drosophila .", "However , attempts to express XSMOC-1 in Drosophila using a synthetic construct optimized for codon usage in Drosophila were unsuccessful; immunoblot analysis of two Drosophila lines generated to overexpress XSMOC-1 demonstrated that XSMOC-1 was below the level of detection ( 5 ng ) for the assay ( Figure 1—figure supplement 1 ) , suggesting that despite codon optimization , XSMOC-1 was not translated in amounts sufficient to be effective .", "As SMOC and Pent are structurally similar , SMOC may function as an expander of BMP signaling in vertebrates .", "To address this possibility we developed assays using SMOC expressed in bacteria and refolded together with two SMOC deletion mutant constructs; XSMOC-1∆EC lacking the EC domain , and XSMOC-1EC containing the EC domain only .", "The EC domain was of interest as hSMOC-1 binds to heparan sulphate proteoglycans ( HSPGs ) via the EC domain ( Klemenčič et al . , 2013 ) and the expander function of Pent is associated with binding to the cell surface-associated HSPG , Dally ( Vuilleumier et al . , 2010 ) .", "For expression of XSMOC-1 in bacteria , the predicted signal peptide ( 2-24 ) was omitted and a C-terminal hexahistidine-tag added .", "When first expressed , two predominant induced proteins were observed on SDS-PAGE ( Figure 2A ) ; one migrating at 49 kDa , the other at approximately 24 kDa .", "Protein sequencing revealed the 49 kDa protein to be XSMOC-1 , whereas the 24 kDa protein was a partial XSMOC-1 sequence beginning at V235 .", "The base sequence ( GTG ) was consistent with an alternative start codon ( Villegas and Kropinski , 2008 ) ; when changed to GTA by site-directed mutagenesis only the expected 49 kDa product was produced ( Figure 2A ) .", "All subsequent XSMOC-1 and XSMOC-1∆EC constructs contained GTA at V235 . 10 . 7554/eLife . 17935 . 006Figure 2 . Expression and refolding of recombinant mature XSMOC-1 , XSMOC-1∆EC , and XSMOC-1EC .", "( A ) Coomassie stained SDS-PAGE of wild type ( V235-GTG ) and silent mutant ( V235-GTA ) recombinant mature XSMOC-1 following size exclusion chromatography ( SEC ) .", "The product migrating at 24 kDa is a partial XSMOC-1 sequence beginning at the cryptic start site encoded by GTG at V235 .", "( B–E )", "Solid lines: SEC profiles obtained following refolding of XSMOC-1 ( B , C ) , XSMOC-1∆EC ( D ) and XSMOC-1 EC ( E ) either in the absence ( B ) or presence of 2 mM Calcium Chloride ( C–E ) .", "Dashed line: SEC profile ( C ) obtained for human SMOC-1 refolded in the presence of calcium .", "Asterisk symbols indicate the peaks corresponding to each schematic diagram . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 00610 . 7554/eLife . 17935 . 007Figure 2—figure supplement 1 . Dimeric XSMOC-1 is not dissociated by chelation or reduction .", "( A ) SEC profile showing that XSMOC-1 , refolded in the presence of 2 mM CaCl2 , continues to migrate as a dimer ( ↓ ) upon the subsequent chelation of calcium ions by dialysis in the presence of 10 . 5 mM EDTA .", "( B ) SDS-PAGE analysis of XSMOC-1 , XSMOC-1 ∆EC , and XSMOC-1 EC in the presence ( + ) and absence ( − ) of β-mercaptoethanol shows that the XSMOC-1 , XSMOC-1 ∆EC dimers are not linked by disulfide bonds . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 007 Initial XSMOC-1 refolding studies were conducted using the protocol described previously , where calcium is absent , and produces hSMOC-1 that is monomeric ( Novinec et al . , 2008 ) .", "Analysis by S-200 size-exclusion chromatography ( SEC ) showed a mixture of poorly separated peaks , one of which had a calculated molecular weight of 45 . 5 kDa , approximate to the predicted 49 . 6 kDa of monomeric XSMOC-1 ( Figure 2B ) .", "However , when tested in the Xenopus animal cap assay the protein was inactive ( not shown ) .", "As occupancy of the calcium binding sites may be necessary for biological activity , we modified the refolding buffer to include 2 mM CaCl2 .", "With this change , in addition to poorly-separated higher molecular weight material , both XSMOC-1 ( Figure 2C ) and XSMOC-1∆EC ( Figure 2D ) migrated as single , symmetrical peaks .", "Their calculated molecular weights of 95 . 4 kDa and 53 kDa , respectively , were approximately twice the predicted monomeric sizes ( 49 . 6 kDa and 32 . 3 kDa ) .", "This suggested that XSMOC-1 and XSMOC-∆EC refolded in the presence of calcium form dimers .", "Furthermore , hSMOC-1 , shown previously to elute as a monomer ( Novinec et al . , 2008 ) , also migrated as an apparent dimer under these conditions with a calculated molecular weight of 90 . 3 kDa ( Figure 2C ) ; no peak was observed at the expected monomeric size of 46 . 6 kDa .", "In contrast , XSMOC-1EC eluted at the calculated molecular weight of 23 . 7 kDa ( Figure 2E ) , consistent with that predicted for a monomer ( 18 . 4 kDa ) .", "Dimeric XSMOC-1 could not be dissociated by chelation of Ca++ ions and continued to elute as a dimer following dialysis in the presence of 10 . 5 mM EDTA ( Figure 2—figure supplement 1 ) .", "Indeed , a dimer was still observed in the presence of 50 mM EDTA and 1 mM nitriloacetic acid ( not shown ) .", "Analysis by SDS-PAGE under non-reducing and reducing conditions demonstrated that dimeric XSMOC-1 and XSMOC-∆EC were not formed through disulfide linkages ( Figure 2—figure supplement 1 ) .", "The biological activity of the SMOC proteins was assessed using the Xenopus animal cap explant assay in which overexpression of XSMOC-1 mRNA was shown previously to induce anterior neural markers ( Thomas et al . , 2009 ) .", "XSMOC-1 , XSMOC-1∆EC , or XSMOC-1EC proteins were incubated with stage 9 ( Nieuwkoop and Faber , 1994 ) late blastula animal caps , at equimolar concentrations , until wild type embryos reached the late neurula stage ( stage 20–21 ) .", "RT-PCR demonstrated the induction of anterior neural markers in the presence of XSMOC-1 and XSMOC-1∆EC , but not XSMOC-1EC ( Figure 3A ) ; hSMOC-1 was also effective in this assay ( not shown ) .", "For these studies , the optimal concentration of SMOC was found to be 100 μg/ml , which appears relatively high .", "However , SMOC is a matricellular protein and though it is difficult to estimate the effective concentration in vivo , its affinity for HSPGs suggests that its diffusion will be restricted unless HSPG sites are saturated .", "Consequently , it may remain concentrated near the site of secretion and thus achieve high levels locally .", "Temporal analyses showed that a two-hour pulse of XSMOC-1 or XSMOC-1∆EC protein was sufficient to commit the naïve ectoderm of the Xenopus animal cap to an anterior neural fate sixteen hours later ( Figure 3B ) ; a one-hour pulse was not .", "This suggests that , following a two hour exposure to SMOC , a duration of exposure ( Rogers and Schier , 2011 ) is reached whereby sufficient changes in gene transcription occur in SMOC-responsive cells to convert their fate from epidermal to neural .", "While it is well established that inhibition of endogenous BMP activity in Xenopus ectodermal explants promotes a neural fate ( Vonica and Brivanlou , 2006 ) and a number of genes have been implicated in neural fate specification ( Kishi et al . , 2000; Ueno et al . , 2008; Milet et al . , 2013; Green and Vetter , 2011; Gammill and Sive , 2001 ) , the sequence of events resulting in commitment to the neural lineage is not known .", "It should now be feasible to design an unbiased genome-wide screen to identify the early transcriptional changes , following a two hour exposure to SMOC , that initiate neural differentiation . 10 . 7554/eLife . 17935 . 008Figure 3 . XSMOC-1 and XSMOC-1∆EC , but not XSMOC-1EC convert the fate of naïve Xenopus ectoderm explants ( animal caps ) to anterior neural tissue within two hours .", "( A ) RT-PCR analysis of animal caps removed at stage 8/9 and incubated in 0 . 7X MMR/0 . 1% BSA ( control ) containing equimolar amounts of XSMOC-1 ( 100 μg/ml ) , XSMOC-1∆EC ( 75 μg/ml ) or XSMOC-1EC ( 50 μg/ml ) until sibling embryos reached the late neurula stage ( 20 ) ; anterior neural markers ( N-CAM , Nrp-1 , Otx2 , Xag-1 ) were induced by both XSMOC-1 and XSMOC-1∆EC , but not by XSMOC-1EC .", "Expression of the ectodermal marker Keratin was suppressed by both XSMOC-1 and XSMOC-1∆EC , but not by XSMOC-1EC .", "( B ) Animal caps removed at stage 8/9 were incubated in 0 . 7X MMR/0 . 1% BSA ( control ) in the presence of XSMOC-1 ( 100 μg/ml ) for six minutes , one hour , or two hours before replacing with 0 . 7X MMR/0 . 1% BSA and incubating until sibling embryos reached stage 20 .", "RT-PCR analysis shows that a two hour exposure to XSMOC-1was sufficient to induce the naïve ectoderm to express anterior neural markers 16 hr post-pulse; a one hour exposure was not .", "The continual presence of XSMOC-1 ( 16 hr ) was used as a positive control . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 008 The ability of the SMOC proteins to inhibit BMP signaling was assessed in mammalian cell lines .", "Serum-starved NIH-3T3 and HEK-293 cells were incubated with either XSMOC-1 ( 100 μg/ml ) or equimolar amounts of XSMOC-1∆EC or XSMOC-1EC for thirty minutes , followed by the addition of BMP2 ( 50 ng/ml ) for an additional thirty minutes .", "As expected , BMP2 treatment alone caused phosphorylation of Smad 1/5/8 ( Figure 4A , B ) .", "This was blocked in the presence of XSMOC-1 and XSMOC-1∆EC , whereas XSMOC-1EC significantly enhanced BMP2-mediated Smad phosphorylation ( Figure 4A , B ) .", "Potentiation of Smad phosphorylation by the EC domain was not due to an additive effect , as the addition of XSMOC-1EC alone to NIH-3T3 cells did not result in Smad1/5/8 phosphorylation ( Figure 4C ) . 10 . 7554/eLife . 17935 . 009Figure 4 . The two thyroglobulin-like domains are necessary for BMP inhibition , whereas the EC domain promotes BMP signaling .", "( A ) Immunoblot showing phosphorylation of Smad 1/5/8 ( pSmad ) by BMP2 ( 50 ng/ml ) in HEK-293 cells is inhibited by the addition of XSMOC-1 ( 100 μg/ml ) or XSMOC-1∆EC ( 75 μg/ml ) , but is enhanced by the addition of XSMOC-1EC ( 50 μg/ml ) .", "Total Smad is shown as a loading control .", "( B ) Graph showing the relative fluorescence of pSmad 1/5/8 obtained on immunoblots from four separate experiments using both HEK293 and NIH3T3 cells; each treatment is displayed as the percent difference from control .", "The inhibition of Smad 1/5/8 phosphorylation by XSMOC-1 or XSMOC-1 ∆EC and the potentiation of BMP signaling by XSMOC-1EC are both significant ( p=≤0 . 01 ) .", "( C ) Immunoblot of HEK293 cell extracts showing that XSMOC-1 EC alone does not promote Smad phosphorylation .", "( D ) RT-PCR analysis of Xenopus animal cap ( AC ) explants removed at stage nine from embryos injected bilaterally at the two-cell stage with mRNAs for GFP ( Control ) , caBMPR1B ( 120 pg ) , or caBMPR1B and equimolar amounts of XSMOC-1 ( 600 pg ) , XSMOC-1 ∆FS ( 540 pg ) , XSMOC-1 ∆EC ( 420 pg ) , XSMOC-1 Tg1 ( 330 pg ) , XSMOC-1 ∆Tg1 ( 360 pg ) orXSMOC-1 EC ( 240 pg ) .", "The AC explants were incubated until stage 20 ( late neurula ) before RNA extraction and analysis .", "The induction of the direct BMP signaling target gene , XVent , by caBMPR1B was blocked by co-expression with XSMOC-1 , XSMOC-1 ∆FS , or XSMOC-1 ∆EC , but not by XSMOC-1 Tg1 , XSMOC-1 ∆Tg1 , or XSMOC-1 EC .", "H4: Histone loading control , –RT: Negative control . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 00910 . 7554/eLife . 17935 . 010Figure 4—source data 1 . Source data file for generating Figure 4B . Absorbance values obtained from pSmad immunoblots in four separate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 010 Having established that the SMOC EC domain is not required for the inhibition of BMP signaling we designed deletion constructs for use in mRNA overexpression studies to determine which domain ( s ) of SMOC are required for BMP inhibition; XSMOC-1∆FS contained a 49 amino acid deletion of the N-terminal FS-like domain ( ∆Q43 to A91 ) ; XSMOC-1∆Tg1 contained only the follistatin-like domain and EC domain ( ∆K95 to S304 ) ; XSMOC-1∆FS∆EC ( referred to as XSMOC-1Tg1 ) contained deletions of the FS-like and EC domains ( ∆Q43 to A91 and ∆N310 to end ) , leaving only the two Tg1-like domains .", "The effect on BMP inhibition was analyzed in Xenopus embryos following overexpression of mRNAs for each deletion construct and caBMPRIB .", "Analysis of animal caps following co-injection of Xenopus embryos with caBMPRIB and XSMOC-1 , XSMOC-1∆FS , or XSMOC-1∆EC showed inhibition of BMP signaling , indicated by the suppression of XVent expression ( Figure 4D ) .", "Whereas these data suggest that the FS and EC domains are not required for BMP inhibition overexpression of XSMOC-1Tg1 , containing the Tg1-like domains only , did not inhibit BMP signaling in this assay ( Figure 4D ) ; deletion of the Tg1-like domains ( XSMOC-1∆Tg1 ) produced a similar result .", "While we believe the Tg1-like domains to be important for BMP inhibition , by process of elimination , the effects of large deletions on protein folding are difficult to predict; in experiments of this type , where protein function is lost , improper folding is common ( Valastyan and Lindquist , 2014 ) .", "Consequently , we consider misfolding of the protein produced by XSMOC-1∆FS∆EC as the most likely reason for the inability of this construct to block BMP signaling .", "Additional evidence for the importance of the Tg1-like domains in SMOC comes from studies of human Ophthalmo-Acromelic ( Waardenburg Anophthalmia ) Syndrome , an autosomal disorder caused by mutations in SMOC-1 ( Rainger et al . , 2011 ) .", "The phenotype of anopthalmia , oligodactyly , and joint abnormalities was found to be the same in patients with nonsense or frameshift mutations and those with missense mutations in the second Tg1-like domain ( Rainger et al . , 2011 ) .", "As the nonsense and frameshift mutations were predicted to result in a complete loss of SMOC-1 function , the two pedigrees harboring two different single amino acid missense mutations in the Tg1-like domain suggests this domain is indeed essential for SMOC-1 function .", "Alternative approaches will be required to elucidate the exact role of the Tg1-like domains in BMP inhibition .", "Many proteins contain Tg1-like domains , including thyroglobulin , insulin-like growth factor binding proteins ( IGFBPs ) 1–6 , the proteoglycan testican , and the basement membrane associated protein nidogen/entactin ( Novinec et al . , 2006 ) .", "However , there have been no reports of any of these proteins inhibiting BMP signaling .", "The potentiation of BMP signaling by the EC domain was examined further by investigating the relative affinities of XSMOC-1EC and BMP2 for each other and for HSPGs .", "The binding of SMOC/Pent and BMP2/4 to HSPGs is known , as evidenced by the co-purification of SMOC and BMPs following heparin affinity chromatography of bovine cartilage extracts ( Chang et al . , 1994 ) , and the binding of BMP2/4 and the EC domain of hSMOC/Pent to heparin/HSPGs ( Vuilleumier et al . , 2010; Klemenčič et al . , 2013; Ruppert et al . , 1996 ) .", "In addition , the basic amino acid-rich putative heparin-binding region identified within the EC domain of SMOC ( Klemenčič et al . , 2013 ) is highly conserved in Pent ( Figure 5—figure supplement 1 ) .", "Using the Protein Homology/analogY Recognition Engine ( PHYRE ) , an unsupervised homology model for XSMOC-1EC was constructed based on the structure of the EC domain of the related family member BM-40 ( Hohenester et al . , 1996 ) .", "XSMOC-1EC aligned well with the BM-40-EC model ( Figure 5—figure supplement 1 ) and the electrostatic surface potential map predicted an area of positive charge similar to that reported in the EC domain of hSMOC1 ( Klemenčič et al . , 2013 ) ( Figure 5—figure supplement 1 ) .", "As monomeric hSMOC-1 , refolded in the absence of calcium , was used in previous heparin-binding studies ( Klemenčič et al . , 2013 ) , we first determined whether dimeric XSMOC-1 can bind heparin .", "XSMOC-1 and XSMOC-1EC bound to heparin Sepharose in the presence of 0 . 5M NaCl , whereas XSMOC-1∆EC did not ( Figure 5A ) , confirming the EC domain to be the site of HSPG binding .", "Comparison of the heparin-binding affinities of XSMOC-1EC and BMP2 showed a striking similarity , with both eluting between 0 . 65M and 0 . 7M NaCl ( Figure 5B ) .", "The possibility that XSMOC-1 and BMP2 bind to each other was discounted ( Figure 5C ) ; when BMP2 was incubated with XSMOC-1 or XSMOC-1 EC , pre-bound to heparin-Sepharose , BMP2 was only present in the unbound fraction ( Figure 5C; lanes 2 and 5 ) and did not co-elute with XSMOC-1 or XSMOC-1 EC ( Figure 5C; lanes 3 and 6 ) .", "The lack of interaction of SMOC with BMP2 agrees with an analogous finding observed for Pent and the Drosophila BMP , decapentaplegic ( Dpp ) ( Vuilleumier et al . , 2010 ) .", "This , combined with SMOC and BMP2 having similar heparin-binding affinities , suggests that XSMOC-1EC could compete with BMPs for HSPG binding on HEK293 cells and thereby increase BMP bioavailability . 10 . 7554/eLife . 17935 . 011Figure 5 . XSMOC-1EC and BMP2 have similar binding affinities for heparin sepharose ( HS ) , but do not bind to each other . SDS-PAGE analysis ( Coomassie staining ) of HS elution profiles showing ( A ) binding of XSMOC-1 , XSMOC-1∆EC , and XSMOC-1EC in PBS or PBS/0 . 5M NaCl; binding of XSMOC-1 to HS requires the EC domain .", "( B ) XSMOC-1EC and mature BMP2 ( A284-R396 ) in a NaCl gradient ( 400–700 mM ) have equivalent HS binding affinities .", "( C ) BMP2 does not bind directly to XSMOC-1 ( lanes 1–3 ) or XSMOC-1EC ( lanes 4–6 ) at physiological ionic strength ( PBS ) ; BMP2 ( 4 μg ) incubated with HS ( 0 . 3 μl ) saturated with XSMOC-1 or XSMOC-1EC ( 6 μg ) , did not co-elute with XSMOC-1 or XSMOC-1EC ( lanes 3 and 6 ) and was only present in the unbound fraction ( lanes 2 and 5 ) .", "Saturation of HS by XSMOC-1 and XSMOC-1EC was confirmed by their presence in the unbound fractions ( lanes 1 and 4 ) prior to incubation with BMP2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 01110 . 7554/eLife . 17935 . 012Figure 5—figure supplement 1 . The EC domains of SMOC and Pent are conserved and share structural homology to BM-40 . ( A ) Predicted heparin binding sequence of hSMOC-1 aligned with the same region of XSMOC-1 and Pent .", "Conserved amino acids are shown in bold and basic amino acids are shown in red .", "( B ) Unsupervised homology model for the XSMOC-1 EC domain , based on the structure of the EC domain of BM-40 ( 1SRA . pdb ) , constructed using the Protein Homology/analogy Recognition Engine ( PHYRE , RRID:SCR_010270 ) .", "The XSMOC-1 EC domain aligns in the exact orientation with the BM-40 model; alpha helices E and F are noted .", "( C ) Electrostatic surface potential maps ( based on solvent accessibility ) of the EC domains of XSMOC-1 and BM-40 showing an area of positive charge ( shown in blue ) in XSMOC-1 that is absent in the equivalent region of BM-40 . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 012 We designed an in vitro assay to test the hypothesis that SMOC can expand the range of BMP signaling by competing with BMP2/4 for HSPG-binding .", "BMP4-soaked beads represented a cellular source of BMPs and agarose gels ( 0 . 7% ) containing heparan sulfate ( HS ) ( 10 μg/ml ) represented an extracellular matrix ( ECM ) capable of binding SMOC and BMPs .", "Chamber slides containing BMP4-soaked beads embedded in the agarose/HS/XSMOC-1EC matrices were seeded with the stable reporter cell line C33A-2D2-09 , harboring luciferase under the control of a BMP response element ( BRE ) .", "After 24 hr , immunohistochemical analysis of cells in fields of view adjacent to the matrices showed many luciferase positive cells ( 59% ) adjacent to the agarose-only gel ( Figure 6A , D ) , whereas when HS was present only a few ( 8% ) were detected ( Figure 6B , E ) .", "In contrast , when the matrix contained both HS and XSMOC-1EC , the number of BMP-responsive cells ( 64% ) was similar to that observed in the absence of HS ( Figure 6C , F ) .", "The result showed that only a relatively small amount of BMP4 diffused through the HS-containing agarose gel , activating luciferase expression in only a few BMP-responsive cells .", "When XSMOC-1EC was present , sufficient BMP4 diffused through the HS-containing matrix to activate a greater number of cells .", "The assay demonstrated that binding of BMPs to HSPGs restricts their range of effect and that BMP diffusion can be enhanced by the binding of SMOC to HSPGs , effectively expanding the range of effect . 10 . 7554/eLife . 17935 . 013Figure 6 . Immunofluorescence assay demonstrating that XSMOC-1 can promote BMP signaling at a distance from its source by competitive binding to HSPGs . BMP4-soaked beads ( 100 μm ) were implanted into 0 . 5 μl drops of 0 . 7% low melting point ( LMP ) agarose ( A , D ) , LMP agarose containing 10 μg/ml heparan sulfate ( B , E ) , or LMP agarose containing heparan sulfate ( 10 μg/ml ) and 100 μg/ml XSMOC-1EC ( C , F ) on 8-well chamber slides .", "C33A-2D2-09 cells , harboring luciferase under the control of a BMP response element ( BRE ) were seeded at 2 × 104 cells/well and incubated for 48 hr in serum-free medium .", "Luciferase immunofluorescence ( green ) indicates cells positive for BMP signaling .", "Cell nuclei ( blue ) were stained with DAPI .", "Dashed lines in A-C indicate the boundaries between C33A-2D2-09 cells and the agarose drops .", "Analysis of four fields of view in three separate experiments demonstrated the number of luciferase-positive cells to be lower ( 16% ± 8% ) adjacent to matrices containing HS alone ( B , E ) compared to those containing both HS and XSMOC-1EC ( 69% ± 26% ) ( C , F ) .", "Representative fields are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 01310 . 7554/eLife . 17935 . 014Figure 6—source data 1 . Percentage of luciferase positive cells per field of view . The number of Luciferase positve cells in five fields of view were calculated as a percentage of the total number of cells in each field . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 014 We designed an assay to assess the ability of SMOC to affect the range of BMP signaling in vivo using Xenopus ectodermal conjugates , from stage 9 to 9 . 5 Xenopus embryos , in which endogenous BMP signaling is absent ( Faure et al . , 2000 ) .", "Animal caps , removed at stage nine from embryos co-injected with mCherry and/or SMOC mRNAs , were grafted onto the animal poles of BMP2 mRNA-injected embryos from which the caps had been removed ( Figure 7A ) .", "The conjugates were allowed to heal for two hours before the ectoderm from the entire animal half of the chimeric embryos was removed for immunostaining for phospho-Smad 1/5/8 ( Figure 7B–F ) .", "In non-injected controls , no pSmad signal was detected either in the host tissue or the mCherry mRNA-injected donor tissue ( Figure 7B ) .", "When host embryos were injected with a high concentration of BMP2 mRNA ( 300 pg ) , pSmad signaling was observed throughout the host and donor graft tissue ( Figure 7C ) .", "Following a series of titration experiments we found that injection of 30 pg BMP2 mRNA at the two cell stage ( 15 pg into each blastomere ) was sufficient to induce BMP signaling in the host tissue , while pSmad was observed only at a few cell diameters into the donor graft ( Figure 7D ) .", "This amount of BMP2 mRNA was used in subsequent experiments to test whether SMOC can extend the range of BMP signaling .", "Next , donor animal caps from embryos injected with different amounts of SMOC mRNA were grafted onto the animal poles of embryos injected with 30 pg BMP2 mRNA .", "Donor caps from embryos injected with a low amount of SMOC mRNA ( 10 pg ) had an increased number of pSmad positive nuclei compared to non-SMOC injected caps ( Figure 7E ) .", "Conversely , donor caps from embryos injected with a high amount of SMOC mRNA ( 300 pg ) were devoid of pSmad ( Figure 7F ) .", "The results demonstrate that , in an intact tissue , low concentrations of SMOC can promote diffusion of BMP from its source of synthesis , thereby extending its range of effect , whereas high SMOC concentrations inhibit BMP signaling .", "These data would be consistent with low levels of SMOC binding to HSPGs , but not inhibiting BMP signaling , and high amounts of SMOC both binding HSPGs and inhibiting BMP signaling .", "Closer examination of the host tissue surrounding the donor grafts expressing the highest level of SMOC showed BMP signaling was both absent in the graft and decreased in the BMP2-expressing ectoderm immediately adjacent to the SMOC expressing graft ( Figure 7F ) .", "This would be consistent with SMOC inhibiting BMP signaling within the graft and also diffusing into the immediate adjacent tissue at levels sufficient to inhibit BMP signaling . 10 . 7554/eLife . 17935 . 015Figure 7 . In vivo assay demonstrating that XSMOC-1 can expand the range of BMP2 signaling .", "( A ) Schematic diagram of the host/donor animal cap ( AC ) transfer assay .", "( B–F )", "Donor AC grafts expressing mCherry ( red ) and host/donor immunofluorescent nuclear staining of pSmad 1/5/8 ( green ) .", "( B ) Control host + mCherry mRNA ( 200 pg ) -injected donor AC ( mCherry AC ) ; endogenous pSmad is not detectable , ( C ) BMP2 mRNA ( 300 pg ) -injected host + mCherry AC; pSmad is detected throughout the host tissue and donor AC , ( D ) BMP2 mRNA ( 30 pg ) -injected host ( BMP2-30 pg host ) + mCherry AC; pSmad is detected in the host tissue and at the host tissue/AC boundary , ( E ) BMP2-30 pg host + mCherry/XSMOC-1 mRNA ( 10 pg ) -injected AC; pSmad is detected in the host tissue and 4–5 cell diameters into the AC ( F ) BMP2-30pg host + mCherry/XSMOC-1 mRNA ( 300 pg ) -injected AC; pSmad is not detected in the AC and is also absent at the host tissue/AC boundary . DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 015 In addition to the data we present here , there are substantial in vivo data from previous work that are consistent with SMOC/Pent acting both as a BMP antagonist and by expanding the range of BMP signaling .", "During Drosophila wing development , while dpp expression is restricted to a stripe of medial cells in the wing disc , the morphogenetic gradient expands across the anterior/posterior ( A/P ) axis ( Nellen et al . , 1996 ) .", "Long-range Dpp signaling in the wing has been shown to involve both pent and the cell membrane-anchored HSPG , dally ( Vuilleumier et al . , 2010; Fujise et al . , 2003 ) ; the absence of either causes severe contraction of the range of Dpp signaling , and wing patterning defects ( Vuilleumier et al . , 2010 ) .", "However , the mechanism by which Pent and Dally cooperate to expand the range of Dpp signaling is not clear .", "It has been speculated that the Pent/Dally interaction may reduce the affinity of Dpp for its receptor , Thickveins , leading to an increase in Dpp diffusion ( Ben-Zvi et al . , 2011 ) .", "However , the results we obtained from cell culture studies suggest that this is not the case .", "When XSMOC-1EC is added to HEK293 cells at 50 μg/ml , in serum-free media it will bind to the many cell surface HSPG binding sites .", "Consequently , following the subsequent addition of BMP2 , any impairment of BMP2/receptor affinity caused by SMOC/HSPG binding would result in a reduction of BMP signaling .", "Instead , a potentiation of BMP signaling was observed ( Figure 4A , B ) .", "Based on the expression patterns of dpp , pent , and dally , their known biological activities , and the new information presented here , we propose the following model ( Figure 8A–C ) . 10 . 7554/eLife . 17935 . 016Figure 8 . Schematic diagrams showing the proposed mode of action of Pent in the Drosophila wing disc and SMOC in vertebrate joint interzones .", "( A ) Representations of the expression patterns of pent ( Vuilleumier et al . , 2010 ) dally ( Fujise et al . , 2003 ) and Dpp signaling ( pMad ) ( Vuilleumier et al . , 2010 ) in wild type Drosophila wing discs , compared with ( B ) pent−/− or ( C ) dally−/− mutant discs .", "The expression patterns observed in the absence of pent or dally ( Vuilleumier et al . , 2010 ) are consistent with Pent/Dally binding being required to expand the range of Dpp signaling .", "( D ) Expression patterns of SMOC ( Okada et al . , 2011; Rainger et al . , 2011 ) , Syndecan-3 ( Koyama et al . , 1995 ) , and BMP signaling ( pSmad ) [4949] in a developing vertebrate autopod joint , compared with that predicted for ( E ) the absence of HSPGs ( HS−/− ) , or ( F ) the absence of SMOC ( SMOC−/− ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17935 . 016 Dally , expressed both medially and laterally in the wing disc ( Fujise et al . , 2003 ) , encodes a cell autonomous membrane-associated protein; pent , expressed laterally , and dpp , expressed medially , encode secreted proteins able to diffuse away from their source ( Thomas et al . , 2009; Vuilleumier et al . , 2010 ) .", "Indeed , we showed previously that SMOC can diffuse across many cell diameters in Xenopus animal caps ( Thomas et al . , 2009 ) .", "At the lateral border , Dpp signaling will be inhibited by a combination of the expression of pent ( Figure 7A ) and the BMP repressor , brinker ( Minami et al . , 1999 ) .", "As Pent diffuses across the wing disc it will create a lateral-medial gradient and compete with Dpp for Dally binding .", "Dpp , diffusing in an opposing medial-lateral gradient , will encounter gradually increasing levels of Pent .", "At low levels , Pent will occupy some Dally binding sites , promoting further Dpp diffusion; at high levels , Pent will occupy many Dally binding sites and also inhibit Dpp signaling downstream of its receptor ( Figure 8A ) .", "This model is supported by the observations made either in the absence of pent ( Vuilleumier et al . , 2010 ) , or the absence of dally ( Fujise et al . , 2003 ) , where there is a contraction of the Dpp signaling gradient .", "In the absence of pent , medial-lateral diffusion of Dpp will be restricted by Dally because there is no Pent to compete for Dally binding sites ( Figure 8B ) .", "The resulting high level of medially localized Dpp signaling , indicated by Mad phosphorylation ( pMad ) ( Vuilleumier et al . , 2010 ) , has been shown to induce expression of the inhibitory Smad , daughters against dpp ( dad ) , promoting a negative feedback loop to limit the range of Dpp signaling ( Tsuneizumi et al . , 1997; Ogiso et al . , 2011 ) .", "In the absence of dally , Dpp diffusion will also be restricted as Dally-binding is required to establish both Dpp and Pent gradients ( Vuilleumier et al . , 2010 ) .", "Medially , dally mutant discs show abnormally high levels of pMad ( Fujise et al . , 2003 ) , which would promote a negative feedback loop in Dpp signaling similar to that observed in the absence of pent ( Figure 8C ) .", "Our proposed mode of action of Pent in the Drosophila wing disc is consistent with the role of glypicans in the ‘restricted extracellular diffusion’ model for establishing a Dpp signaling gradient ( Schwank et al . , 2011 ) ; the inclusion of the Pent/glypican interaction provides for further diffusion of Dpp to expand the morphogenetic field .", "During the review of this manuscript a study was published ( Norman et al . , 2016 ) showing that binding of Pent to Dally in Drosophila induces endocytosis of the complex , effectively reducing the amount of Dally on cell surfaces .", "Although it remains to be determined whether binding of SMOC to HSPGs also induces endocytosis , this finding is consistent with the model we propose: both competitive binding of SMOC/Pent to HSPGs and removal of HSPG binding sites for BMP/Dpp by endocytosis would promote BMP/Dpp diffusion .", "This idea is further supported by the observation that removal of the heparin binding site within BMP4 has also been shown to promote the BMP diffusion in Xenopus embryos ( Ohkawara et al . , 2002 ) .", "The binding of Pent to Dally also modulates Wingless ( Wg ) signaling in Drosophila , where a reduction in Wg signaling is observed in the presence of high levels of Pent ( Norman et al . , 2016 ) .", "The interaction of Wg with Dally is known to potentiate Wg binding to its receptor Frizzled ( Tsuda et al . , 1999; Lin and Perrimon , 1999 ) ; therefore , the sequestration of Dally by Pent would be consistent with a reduction in Wg signaling .", "These data could also provide an explanation for our previous observation that SMOC activates MAPK signaling and inhibits BMP signaling via a mechanism requiring phosphorylation of Smad in the linker region ( Thomas et al . , 2009 ) .", "Linker Smad phosphorylation at MAPK sites has been shown to be followed by phosphorylation at Glycogen Synthase Kinase 3 ( GSK3 ) sites ( Fuentealba et al . , 2007 ) , resulting in Smad ubiquitination and degradation ( Sapkota et al . , 2007 ) .", "As the activity of GSK3 is inhibited by Wnt signaling , a reduction in Wnt signaling by Pent/SMOC could potentiate GSK3-mediated phosphorylation of linker Smad .", "The spatial distributions and interactions of pent , dally , and dpp in the Drosophila wing disc can also be applied to explain the role of SMOC , HSPGs , and BMPs during development of vertebrate skeletal joints ( Figure 8D–F ) .", "SMOC-1 ( Okada et al . , 2011 ) , the cartilage-specific BMP called growth and differentiation factor 5 ( GDF5 ) ( Storm and Kingsley , 1996 ) or cartilage-derived morphogenetic protein-1 ( CDMP-1 ) ( Chang et al . , 1994 ) , BMP2/4 ( Francis-West et al . , 1999 ) , and HSPGs ( Koyama et al . , 1995 ) are all expressed at the sites of future joint interzones; the absence of any one of these results in dysmorphogenesis of autopod joints ( Okada et al . , 2011; Rainger et al . , 2011; Chang et al . , 1994; Storm and Kingsley , 1996; Mundy et al . , 2011; Thomas et al . , 1996 , 1997 ) .", "Joint development in the digits occurs by segmentation of existing cartilage anlagen in regions where BMP signaling is inhibited by the BMP antagonist Chordin ( Francis-West et al . , 1999 ) and , based on our studies , by SMOC .", "Once formed , the joint interzone acts as a signaling center to promote appositional growth of the opposing cartilage epipheses ( Archer et al . , 2003; Ray et al . , 2015 ) .", "Based on information from existing in vivo mutational analyses and the data presented here , we propose the following model for the role of SMOC in promoting joint formation and subsequent appositional epiphyseal growth ( Figure 8D , E ) .", "Within the interzone , Chordin and SMOC will both bind to HSPGs ( Klemenčič et al . , 2013; Jasuja et al . , 2004 ) such as syndecan-3 ( Koyama et al . , 1995 ) , allowing diffusion of GDF5 and BMP2/4 into the epiphyses , where BMP signaling promotes chondrocyte proliferation and appositional epiphyseal growth ( Francis-West et al . , 1999; Ray et al . , 2015 ) via the BMP receptor BMPR1B ( Baur et al . , 2000 ) .", "This hypothesis is consistent with the skeletal and joint abnormalities observed in the absence of HS following conditional knockdown of the glycosyltransferase , Ext1 , in joint interzones ( Mundy et al . , 2011 ) .", "Ext1 is essential for HS synthesis and binding of Chordin to HSPGs and is necessary for its BMP inhibitory activity ( Jasuja et al . , 2004 ) .", "Consequently , in the absence of HS within the interzone , the BMP antagonist activity of Chordin will be reduced .", "While SMOC will still contribute to the inhibition of BMP signaling , the imbalance caused by the reduced Chordin activity is consistent with the increase in BMP signaling ( pSmad ) observed within the interzone in the absence of HS ( Mundy et al . , 2011 ) ; as a result , in response to BMP signaling , the interzone cells undergo chondrogenesis and the joint does not form ( synostosis ) .", "In the absence of SMOC , we predict that the increased availability of HSPG binding sites will restrict diffusion of GDF/BMPs out of the interzone .", "In addition , the imbalance in BMP inhibition within the interzone will lead to an increase in BMP signaling resulting in chondrogenesis and failure of joint formation .", "This hypothesis is supported by oligodactyly and joint synostosis observed in patients with Ophthalmo-Acromelic ( Waardenburg Anophthalmia ) Syndrome ( Okada et al . , 2011; Rainger et al . , 2011; Abouzeid et al . , 2011 ) .", "In conclusion , based on the data we present here together with existing literature , we propose a model to explain how vertebrate SMOC ( Thomas et al . , 2009 ) and Drosophila Pent ( Vuilleumier et al . , 2010 ) regulate BMP signaling .", "SMOC/Pent can inhibit BMP signaling locally , downstream of the BMP receptor , probably via the region containing the two Tg1-like domains .", "In addition , binding of SMOC/Pent to cell surface HSPGs via the C-terminal EC domain prevents BMP binding , promoting BMP diffusion , thereby expanding its morphogenetic field ." ], [ "UAS-pentagone ( UAS pent ) was constructed by PCR amplification ( forward primer: ATCTCGAGCCGAAGCACAGTAACAGTT; reverse primer: TATCTAGACGACGACATCTAATGAGTTG ) from a cDNA clone corresponding to CG2264 ( Drosophila Genomics Resource Center ) , and subcloned into pUAST using XhoI and XbaI .", "Sequence verification of the UAS-pent subclone followed by sequencing of the original cDNA indicated a base pair change was present in both , which resulted in a W to R amino acid change in a highly conserved region of the protein .", "Site directed mutatgenesis was performed with the QuikChange Lightning Site-Directed Mutagensis Kit ( Stratagene ) to correct pent cDNA to the database sequence .", "Drosophila transgenic lines were prepared by standard P-element transformation ( Spradling , 1986 ) and mapped to a specific chromosome .", "Rescue was carried out by creating flies homozygous for the pentagone mutants: pent2-5/pentA17 , and carrying the Gal four line brinker -Gal 4 ( brk-Gal4 ) and the UAS-pent construct .", "Adult wings were dissected , fixed in 70% ethanol , mounted in Euperal ( Asco Laboratories , Manchester , U . K . ) and photographed on a Nikon E-800 microscope .", "XSMOC-1∆FS ( ∆Q43-A91 ) was created from two separate PCR fragments obtained using pCS2-XSMOC-1 ( Thomas et al . , 2009 ) as template .", "The 5’ fragment was obtained using the pCS2 forward primer and the reverse primer 5’-GAGAGGATTGCACCCGGGGTCTCTGTCC-3’; the 3’ fragment was obtained using the forward primer 5’-AGGTGCAAAGATCCCGGGCAGAGCAAGTGT-3’ and the pCS2 reverse primer .", "A SmaI site ( underlined ) was incorporated to facilitate ligation of the two fragments .", "XSMOC-1∆EC ( ∆N310 to end ) was amplified using pCS2-XSMOC-1 as template and the primer set 5’-GGCAACATGACCCCAAGA-3’ , 5’-CTTTAAATAGGCCTTCTCAGTCCGTATTTTTCCA-3’ ( incorporating a StuI restriction site , underlined , and stop codon , italics ) .", "XSMOC-1∆FS and XSMOC-1∆EC PCR products were cloned into PCR-TOPO ( Invitrogen ) , sequenced , and then subcloned into pCS2 .", "XSMOC-1EC ( ∆Q43 to W308 ) , XSMOC-1Tg1 ( ∆Q43-A91 and ∆N310 to end ) and XSMOC-1∆Tg1 ( ∆K95 to S304 ) in pCS2 were obtained using a primer design method to generate large deletions ( Liu and Naismith , 2008 ) .", "For XSMOC-1EC and XSMOC-1∆Tg1 , the template was pCS2-XSMOC-1 and the respective primer sets 5’-CTCAGTGTTTCGGCCAAAGAGCGACTG-3’ , 5’-CAGTCGCTCTTTGGCCGAAACACTGAG-3’ and 5’- GCAAAGATGCTGGTCAGAGCGATGCCAGATGGAAAAATACG-3’ , 5’-CGTATTTTTCCATCTGGCATCGCTCTGACCAGCATCTTTGC-3’ .", "For XSMOC-1Tg1 , the template was pCS2-XSMOC1∆FS and the primer set 5’-GATGCCAGATGGAAATAGGAGAGCCGGCCAGAAG-3’ , CTTCTGGCCGGCTCTCCTATTTCCATCTGGCATC-3’ .", "All pCS2 constructs were linearized with Not1 prior to mRNA transcription using the mMESSAGE mMACHINE SP6 kit ( Life Technologies ) .", "Full length XSMOC-1 without the predicted signal peptide ( 2-24 ) was amplified by PCR using the forward primer 5’-TGCCATGGCCAAAGAGCGACTGGC-3’ ( containing a NcoI restriction site , underlined ) , the reverse primer 5’-CTCTCGAGCGCAAGGCGACTGAAGGGG T-3’ ( containing a XhoI restriction site , underlined ) , and full length XSMOC-1 in pCS2 ( Thomas et al . , 2009 ) as the template .", "The PCR product was cloned into PCR-TOPO ( Invitrogen ) , sequenced , and subcloned into the pET-28b ( + ) expression vector ( Novagen ) in frame with a C-terminal hexahistidine tag .", "An alternative start site located within XSMOC-1 at V235 ( GTG ) was removed by changing the codon to GTA by site directed mutagenesis ( QuikChange II kit , Agilent Technologies ) using the forward primer 5’-CCAAGAGAGGGAATTGTAATTCCAGAATGTGC-3’ , reverse primer 5’-GCA CATTCTGGAATTACAATTCCCTCTCTTGG-3’ , and XSMOC-1 in pET-28b ( + ) as template .", "Unlike XSMOC-1 , the conserved Valine in hSMOC-1 is encoded by GTA in the human sequence , making the site-directed mutagenesis unnecessary .", "Using a primer design method to generate large deletions ( Liu and Naismith , 2008 ) , XSMOC-1∆EC ( ∆N310 to end ) lacking the EC domain was obtained using the forward primer 5’- GATGCCAGATGG AAACACCACCACCACCACCACTG-3’ , the reverse primer 5’-CAGTGGTGGTGGTGGTGGTGT TTCCATCTGGCATC , and XSMOC-1 in pET-28b ( + ) as template .", "Similarly , XSMOC-1EC containing the EC domain only ( ∆T2 to W308 ) was obtained using the same starting template , the forward primer 5’-GTGATC GGGACAGAGACAAAAATACGGACGCTGAAGACCC-3’ , and the reverse primer 5’-GGGTCT TCAGCGTCCGTATTTTTGTCTCTGTCCCGATCAC-3’ .", "Several E . coli strains were evaluated for XSMOC-1 expression and the ShuffleT7 Express strain C3029 ( New England Biolabs ) , which allows the formation of disulfide bonds , was found to produce the highest yield of bioactive protein .", "For hSMOC-1 expression the host strain was BL21DE3 , as described previously ( Novinec et al . , 2008 ) .", "hSMOC-1 ( pET28-SMOC-1-HT ( Novinec et al . , 2008 ) ) , kindly provided by B . Lenarcic , University of Ljubljana , Slovenia , was expressed in E . coli strain BL21DE3 .", "Shaker cultures ( 3 L ) were grown at 30°C in LB broth supplemented with 30 μg/ml kanamycin .", "When cell densities reached OD600 >0 . 5 , recombinant protein expression was induced by the addition of IPTG ( 0 . 1 mM ) for 5 hr .", "Cells were collected by centrifugation at 9000 g for 5 min .", "Bacterial expression and refolding was based on that described previously ( Novinec et al . , 2008 ) with some modifications .", "Bacterial pellets were washed by suspension in 40 ml of 20 mM Tris-HCl/20% ( w/v ) sucrose/5 mM EDTA pH 7 . 5 , centrifuging and resuspending in 40 ml of ice-cold deionized water .", "Following centrifugation the pellets were resuspended in phosphate-buffered saline ( PBS ) /5 mM EDTA and disrupted under high pressure ( 15 , 000 psi ) using the EmulsiFlex-C3 high pressure homogenizer ( Avestin , Inc , Ottawa , Canada ) .", "Lysates were centrifuged at 14 , 000g and inclusion bodies solubilized in 40 ml of solubilization buffer ( 50 mM sodium phosphate buffer , pH 8 . 0 , 8M urea , 500 mM NaCl , 10 mM imidazole , 20 mM 2-mercaptoethanol ) before applying to a pre-equilibriated 20 ml gravity-flow Ni-NTA agarose column ( Qiagen ) .", "After washing with solubilization buffer bound protein was eluted with solubilization buffer/300 mM imidazole , concentrated to 5 ml ( Vivaspin 20 , GE Healthcare ) and refolded by rapid dilution into 500 ml of refolding buffer ( 100 mM Tris/HCl , pH 9 . 0 , 600 mM L-Arginine , 6 mM reduced L-Glutathione , 0 . 6 mM oxidized L-Glutathione , and 2 mM CaCl2 ) .", "Following slow stirring overnight at 4°C the refolded protein solutions were concentrated to 20 ml and dialyzed against 20 mM Tris-HCl pH 7 . 5 , 300 mM NaCl , 2 mM CaCl2 .", "Non-soluble precipitate was removed and the dialysates concentrated to 2 ml before injecting onto a 1 . 6 × 60 cm HiLoad Superdex-200 PG gel filtration column ( GE Healthcare ) pre-equilibrated in S-200 buffer ( 20 mM Tris/HCl pH 7 . 5 , 300 mM NaCl , 2 mM CaCl2 , 10% glycerol ) .", "Fractions containing major peaks , collected using the ÄKTA purifier 10 system and UNICORN control software ( GS Healthcare ) , were pooled , concentrated , and analyzed by SDS-PAGE , and , when necessary , sequenced .", "From three separate 3L cultures the total yields of dimeric XSMOC-1 were 1 . 3 mg , 2 . 5 mg , and 5 . 1 mg and for dimeric XSMOC-∆EC 3 . 4 mg , 4 . 1 mg , and 12 . 6 mg .", "The yields of monomeric XSMOC-1EC were 8 . 2 mg , 4 . 1 mg , and 3 . 25 mg .", "NIH-3T3 Fibroblasts ( ATCC CRL-1658 ) and HEK 293 ( ATCC CRL-1573 ) cells were cultured in DMEM medium supplemented with 10% fetal bovine serum ( FBS ) .", "Prior to the addition of recombinant protein cells were serum-starved in their respective media for one hour .", "The stable human cervical carcinoma clonal cell line C33A-2D2 , containing a multimerized BMP-responsive element ( BRE ) for the BMP response gene inhibitor of differentiation-1 ( Id1 ) ( Korchynskyi and ten Dijke , 2002 ) linked to luciferase was a kind gift from Martine Roussel ( Vrijens et al . , 2013 ) .", "C33A-2D2 cells were maintained in Eagle’s minimum essential medium ( EMEM ) supplemented with 10% FBS .", "The parental C33A cell line was purchased from ATCC ( ATCC HTB-31 ) .", "HEK-293 and parental C33A cells were authenticated by ATCC via STR profiling .", "While each cell line was confirmed to be free of mycoplasma contamination by ATCC , cells were not tested for mycoplasma in the experiments we describe .", "For BMP response assays , a sub-clone containing over 80% responsive cells ( C33A-2D2-09 ) was prepared by single cell dilution .", "In these assays C33A-2D2-09 cells were maintained in serum-free Prime XV MSC expansion medium ( Irvine Scientific ) that does not contain BMPs .", "Wherever protein was added , XSMOC-1 , XSMOC-1∆EC , and XSMOC-1EC were used at molar equivalent amounts based on their predicted molecular weights .", "BMP2 ( Cell Signaling #4697 ) was added at 50 ng/ml .", "Frogs ( Xenopus laevis ) , purchased from NASCO ( Fort Atkinson , WI ) , were housed and maintained in aquaria approved by the FDA White Oak Campus Animal Care and Use Committee ( ACUC ) .", "Prior to testes collection , male frogs were euthanized by anesthesia in a 2% solution of tricaine methane-sulphonate , a protocol approved by the ACUC .", "Frog embryos were manipulated using standard methods ( Gurdon , 1967; Sive et al . , 2000 ) and euthanized by anesthesia when the required developmental stage was reached ( the study was approved by the ACUC ) .", "Injection of mRNAs was performed by standard procedures as described previously ( Moos et al . , 1995 ) .", "Perturbations of axial patterning were quantified by Dorso-Anterior Index ( DAI , ( Kao and Elinson , 1988 ) ) and dark field images of embryos were photographed with low angle oblique illumination and a Zeiss Stemi-6 dissecting microscope .", "For animal cap assays , animal caps were removed from stage nine embryos and cultured in 0 . 7 x Marc’s Modified Ringer’s ( MMR ) solution ( Sive et al . , 2000 ) , 1 mg/ml BSA/50 µg/mL gentamicin until non-injected siblings reached stage 20 .", "Where indicated , caps were incubated in 0 . 7 X MMR containing XSMOC-1 , XSMOC-1∆EC , or XSMOC-1EC , at molar equivalent amounts , for different time periods .", "Pools of animal cap explants from at least two different fertilizations were prepared and analyzed for each condition reported .", "Total RNA isolation , reverse transcription ( RT ) , and PCR amplification were performed as described previously ( Thomas et al . , 2009 ) .", "PCR products were analyzed on 1 . 5% agarose gels in TAE buffer , stained with SYBR Green 1 ( Molecular Probes ) , and scanned using a Fluorimager ( Molecular Dynamics ) .", "Cell lysates prepared by extraction in 6 M Urea , 25 mM Tris base , 2% SDS , 2% β-mercaptoethanol , and 5% glycerol were analyzed by SDS-PAGE ( 10 µg/lane ) using Novex 10% Nu-PAGE gels ( Invitrogen ) and the MES buffer system .", "Immunoblot analyses were performed using the Novex XCell SureLock Mini-Cell system ( Life Technologies ) and nitrocellulose membranes ( Invitrogen ) .", "Transferred proteins were detected using IRDye-labeled secondary antibodies and the Odyssey infrared imaging system ( Li-COR Biosciences ) .", "The primary antibodies used were , phospho-Smad 1/5/8 ( Cell Signaling Technology Cat# 9511 , RRID:AB_331671 ) and Smad1 ( Cell Signaling Technology Cat #9517 , RRID:AB_10699149 ) .", "Peptide antibodies specific to XSMOC-1∆EC ( SDRDRDPQCNPHCTRPQHK ) or XSMOC-1EC ( GSFPPGKRPGSNPFSR ) were produced in rabbits ( Biomatik Corp . ) .", "For heparin-binding studies , 5 µg of XSMOC , XSMOC∆EC , XSMOC-EC or human BMP2 ( R and D Systems ) in 50 μL of 1X PBS/0 . 5M NaCl was added to 20 μL of pre-equilibrated heparin Sepharose high performance beads ( GE Life Sciences ) and mixed with rotation for 15 min at room temperature .", "The beads were centrifuged ( 350 g for 2 min ) and the supernatant removed .", "The protein-heparin bead mixture was then washed twice with 500 µL of 1X PBS or 1X PBS/NaCl ( 0 . 4 to 0 . 7M ) before elution with 20 µL of 1 X Lithium Dodecyl Sulphate ( LDS ) sample buffer ( Invitrogen ) /0 . 1 M DTT for 5 min at 95°C .", "The supernatants were analyzed on a 10% NuPAGE gel and visualized by Coomassie staining .", "Affi-Gel Blue beads ( BioRad ) approximately 100 µm in diameter were soaked in 100 µg/ml BMP4 ( R and D Systems # 314 BP-010/CF ) for 3 hr in a humidified chamber .", "Drops ( 0 . 5 µl ) of 0 . 7% low melting point agarose ( Life Technologies ) with or without heparan sulfate ( 10 µg/ml; Sigma Aldrich #H7640 ) and either XSMOC-1EC ( 100 µg/ml ) or S-200 buffer were placed on Millicell EZ chamber slides ( Millipore ) .", "The slides were placed in a humidification chamber and incubated at room temperature for 5 min .", "Individual BMP4-soaked beads were placed into the partially gelled matrices and incubation was continued until gelling was complete .", "C33A-2D2-09 cells ( 2 × 104 ) in Prime XV MSC expansion medium ( 400 µl ) were seeded into each well .", "The slides were incubated at 37°C in 5% CO2 for 48 hr prior to detection of luciferase .", "Briefly , following fixation ( 15 min ) in 4% paraformaldehyde/PBS , cells were washed and permeabilized ( 0 . 5% Triton-X-100/PBS ) for 10 min .", "Slides were washed ( TBS/0 . 05% Tween ) and incubated in blocking solution ( Duolink , Olink Biosciences ) for 1 hr in a humidified chamber prior to incubation with goat anti-luciferase ( 20 µg/ml ) primary antibody ( Promega #G745A , RRID:AB_2335880 ) for 1 hr .", "After washing ( Duolink Wash Buffer A ) , slides were incubated in donkey anti-goat Alexa-488 ( Thermo Fisher Scientific Cat# A-11055 , RRID:AB_2534102 ) secondary antibody ( 5 µg/ml ) for 45 min .", "Slides were then washed three times each in Duolink Wash Buffers A and B , mounted in Duolink mounting medium with DAPI , imaged by confocal microscopy ( Zeiss LSM710 ) , and analyzed with ImageJ ( NIH ) software .", "Each blastomere of Xenopus embryos at the two cell stage was injected with mRNAs for BMP2 ( 15 pg or 150 pg ) , or SMOC ( 5 , 15 , or 150 pg ) and/or mCherry ( 50 pg ) .", "pCS2 +8 NmCherry ( Gökirmak et al . , 2012 ) was a gift from Amro Hamdoun ( Addgene plasmid # 34936 ) .", "Animal caps were removed at stage nine and grafted into the animal poles of control or BMP2-injected embryos from which the animal poles had been removed .", "Following a two hour incubation the animal halves of the embryos were removed and fixed for 25 min in PBS/4% paraformaldehyde .", "The explants were permeabilized in 0 . 5% TritonX-100 for 10 min , washed in PBS , then transferred to blocking solution ( PBS/5% goat serum ) for 1 hr before incubating overnight at 4°C in a 1/1500 dilution of anti-pSMAD 1/5/8 antibody ( Cell Signaling Technology Cat# 9511 , RRID:AB_331671 ) .", "The tissue was washed in PBS and prepared for immunofluorescence by incubating in goat anti-rabbit Alexa 488 ( Thermo Fisher Cat#A11008 , RRID:AB_143165 ) secondary antibody ( 5 µg/ml ) for 2 hr .", "The tissue was then washed three times in PBS , transferred to a glass slide , mounted in Duolink mounting medium with DAPI , and imaged by confocal microscopy ( Zeiss LSM710 ) ." ] ]

[ "The matricellular protein SMOC ( Secreted Modular Calcium binding protein ) is conserved phylogenetically from vertebrates to arthropods .", "We showed previously that SMOC inhibits bone morphogenetic protein ( BMP ) signaling downstream of its receptor via activation of mitogen-activated protein kinase ( MAPK ) signaling .", "In contrast , the most prominent effect of the Drosophila orthologue , pentagone ( pent ) , is expanding the range of BMP signaling during wing patterning .", "Using SMOC deletion constructs we found that SMOC-∆EC , lacking the extracellular calcium binding ( EC ) domain , inhibited BMP2 signaling , whereas SMOC-EC ( EC domain only ) enhanced BMP2 signaling .", "The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding .", "Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation ." ]

[ "During the development of an embryo , a group of proteins known as growth factors stimulate cells to divide and direct how organs and limbs form .", "One family of growth factors called bone morphogenetic proteins ( BMPs ) regulate the formation of bone and many other tissues in the embryo .", "BMPs are released from cells , diffuse away and are then detected by other cells .", "When BMPs attach to docking station-like structures on the cell surface , called receptors , they stimulate a signaling process inside the cell .", "In 2009 , researchers found that a protein called SMOC blocks BMP activity in animals with backbones by triggering an interfering signal inside the cell .", "In flies , however , the equivalent protein can make BMP diffuse further from the cell that releases it .", "To find out how SMOC can do both of these things , Thomas et al . – including some of the researchers involved in the 2009 study – conducted experiments to see which parts of SMOC are required to either block BMP signaling or encourage the diffusion of BMP .", "These experiments revealed that one end of SMOC can stick to molecules on the cell surface that are not receptors but are molecules where BMP can also bind .", "When this end of SMOC attaches to these sites , BMPs cannot bind and so diffuse further away .", "Thomas et al . then produced complete or shortened versions of SMOC proteins to see how this affected BMP activity in frogs .", "These experiments indicated that the opposite end of SMOC is required for short-circuiting the BMP signal .", "The results also showed that , at lower concentrations , SMOC stimulates BMPs to diffuse , and that higher concentrations are required to block BMP signaling .", "These findings suggest that similar to flies , SMOC can also stimulate BMPs to diffuse from the cell in animals with backbones .", "The next step will be to identify the cell surface receptor for SMOC to better understand the molecular mechanisms that inhibit BMP .", "The SMOC pathway could be targeted for therapeutic strategies to combat diseases associated with errors in BMP signaling like osteoarthritis , or in cell-based therapies where BMP signaling must be inhibited to produce cells needed to repair damaged tissues ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology" ]

[ [ "In order to serve as a bridge from prebiotic chemistry to the RNA world ( Patel et al . , 2015; Powner et al . , 2009 ) , the nonenzymatic copying of RNA templates by suitably activated nucleotides ( Figure 1a ) must occur quickly enough to replicate functional RNA sequences faster than they degrade .", "However , the nonenzymatic copying of sequences containing all four nucleotides has not yet been possible for a number of reasons ( Szostak , 2012 ) , most notably the slow rate of primer extension with adenosine and uridine monomers ( Joyce et al . , 1987; Wu and Orgel , 1992b; Deck et al . , 2011 ) .", "Indeed , two adjacent A or U residues in the template will stop polymerization entirely ( Wu and Orgel , 1992b ) under normal conditions; even partial copying requires extreme conditions , such as the eutectic phase of frozen samples ( Vogel and Richert , 2007 ) , which are not compatible with replication within protocells .", "Extensive optimization of reaction conditions with the aim of improving the rate of primer extension with all four monomers , including varying the choice of divalent cation and leaving group , improved the rate and regiospecificity of polymerization for G and C but not A and U ( Wu and Orgel , 1992b , c; Deck et al . , 2011; Inoue and Orgel , 1981; Hagenbuch et al . , 2005; Lohrmann et al . , 1980 ) .", "Recent biophysical studies suggest that low A and U monomer affinity for the template is not the only reason for this problem ( Szostak , 2012; Izgu et al . , 2015 ) since A binds only three-fold more weakly than G , but primer extension with A is at least 100-fold slower ( Joyce et al . , 1987; Wu and Orgel , 1992b; Deck et al . , 2011; Heuberger et al . , 2015 ) than with G . Moreover , Deck et al . have shown that downstream helper oligonucleotides ( Deck et al . , 2011 ) that provide incoming monomers with an additional binding surface improve the binding of A and U monomers , but the underlying difference in rates remains .", "The use of helper oligonucleotides allowed primer extension to proceed on a template containing all four bases over the course of several weeks , where previously such templates could not be copied at all . 10 . 7554/eLife . 17756 . 003Figure 1 . Catalysis of nonenzymatic primer extension by activated downstream nucleotides .", "( a ) Structure of 2-methylimidazole-activated guanosine-5′-monophosphate , and its schematic representation .", "( b ) Schematic of the RNA primer extension reaction .", "N1 represents an individual ribonucleotide monomer in position to react with the primer , N2 represents either a monomer or an oligomer downstream , with a leaving group capable of interacting with N1 .", "Dashed lines: Potential interactions between leaving groups or between the downstream leaving group and the upstream nucleotide N1 .", "( c ) Schematics of the RNA primers , templates , monomers and oligomers used in d–f .", "Templates are complementary to the displayed monomers and oligomers .", "Template 2 has a U following the C to which the G monomer is bound to prevent downstream binding of G .", "( d ) Primer extension by polymerization or ligation on templates 1 or 3 , respectively .", "Fits describe ln ( fraction primer remaining ) vs time , giving an apparent first-order rate constant ( Figure 1—figure supplement 1 ) .", "( e ) Primer extension assay for the experiments described in c , showing reaction progress after 10 min .", "In lane 6 , a primer + 4 band can be observed , representing the slow addition of the activated trimer after the monomer .", "( f ) Pseudo-first order rates of the reactions described in c .", "Error bars indicate S . E . M; all experiments were performed in triplicate or greater .", "Reaction conditions: 10 μM primer , 11 μM template , 200 mM CHES pH 9 . 0 , 200 mM MgCl2 , 50 mM monomer , 1 mM trimer . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 00310 . 7554/eLife . 17756 . 004Figure 1—figure supplement 1 . Determination of the rates of primer extension reactions .", "( a ) Schematic of a primer extension reaction .", "( b ) Gel showing primer ( starting material ) and extended primer .", "( c ) Plot showing the decay of primer as a percentage of total fluorescence intensity on the gel .", "( d ) Following the assumption of a pseudo-first order rate curve , the ln ( fraction of primer remaining ) plotted over time gives a plot where the slope is the rate for the reaction . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 00410 . 7554/eLife . 17756 . 005Figure 1—figure supplement 2 . Rates of additional trimer ligations .", "( a ) Schematic of trimer ligation reaction .", "( b ) Calculated rates of ligation of various trimer sequences . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 005 Here we show that the presence of a leaving group at the 5′ end of a helper oligonucleotide can be a source of significant catalysis .", "Activated helper oligonucleotides facilitate the addition of all four RNA monomers individually and in sequence in a single reaction .", "Finally , when a template is immobilized by attachment to a bead , sequential addition of activated monomers and helper oligonucleotides successfully promoted the synthesis of a significant part of an active hammerhead ribozyme ." ], [ "Given the historical and continuing problems in demonstrating efficient nonenzymatic template directed primer extension , one might wonder whether this biologically inspired model is appropriate for replication within primordial cells .", "An alternative scenario that might seem more reasonable in the context of prebiotic chemistry involves template copying by the initial formation of short oligonucleotides , followed by ligation events that generate longer oligonucleotide intermediates and eventually a full length copy ( Szostak , 2011; James and Ellington , 1999 ) .", "To test the viability of this hierarchical assembly model , we compared the rate of primer extension with activated monomers to the rate of ligation of two template bound oligonucleotides ( Figure 1b , c reactions 1 and 3 ) .", "Nucleotides activated with 2-methylimidzole on the 5′-phosphate have been used extensively to model nonenzymatic template copying ( Deck et al . , 2011; Wu and Orgel , 1992c ) , irrespective of their prebiotic plausibility , and were used in all experiments reported here .", "In both the nucleotide polymerization and the oligonucleotide ligation reactions , the chemical reaction is the same , i . e . attack of the 3′-hydroxyl of an oligonucleotide on the 5′-phosphate of a downstream nucleotide , with displacement of the 2-methylimidazole leaving group ( Figure 1b ) .", "Moreover , the reacting nucleotides are the same in both cases: a G at the 3′-end of the primer and a G monomer or a G at the 5′-end of an oligonucleotide .", "We expected the ligation reaction to be faster , because the oligonucleotides to be ligated are both stably bound to the template , and because the fully base-paired nicked duplex should be largely in the optimal A-type conformation ( Kozlov and Orgel , 2000 ) , whereas the primer/template complex with template bound monomers would be significantly more disordered .", "To our surprise , the primer extension reaction proceeded almost 100 times more rapidly than the ligation reaction ( Figure 1d ) .", "This was true for a variety of trimer sequences ( Figure 1—figure supplement 2 ) .", "In our search for an explanation for this unexpected observation , we reasoned that one of the major differences between the two scenarios was the presence of multiple adjacent 5′-activated nucleotides downstream of the primer in the case of monomer addition , but not ligation , where there is only a single activated nucleotide .", "Indeed , the case for a role of a second downstream activated nucleotide is consistent with the well known difficulty of extending a primer to the last nucleotide of a template ( Wu and Orgel , 1992c ) .", "Furthermore , Orgel et al . showed over 20 years ago that efficient primer extension requires the presence of two activated monomers adjacent to the primer .", "Remarkably , the reaction is fastest when both monomers are activated with 2-methylimidazole instead of either or both being activated with imidazole ( Wu and Orgel , 1992a ) , suggesting a possible catalytic role for a physical interaction of the leaving groups of adjacent monomers .", "In order to directly test the possibility that a 2-methylimidazole leaving group on the 5′-phosphate of a downstream monomer or oligonucleotide ( Figure 1b ) would increase the rate of reaction between a primer and an adjacent monomer , we designed a series of templates for nonenzymatic RNA polymerization and ligation ( Figure 1c ) .", "We then measured the rate of addition of a monomer to a primer in the presence or absence of downstream nucleotides , with or without 5′-activation of the downstream nucleotides ( Figure 1c–f ) .", "Rates were calculated assuming a pseudo-first order rate equation ( Figure 1—figure supplement 1 ) , because the concentrations of primer and template were far below that of the monomer , which would not change significantly during the reaction .", "Primer extension by addition of a single monomer , in the absence of any additional downstream mono- or oligo-nucleotides , was very slow and comparable in rate to the ligation of the primer to a 5′-activated GAG trinucleotide ( Figure 1c–f , reactions 2 and 3 ) .", "The binding of an unactivated AGC trimer , with either a 5′ hydroxyl or a 5′ phosphate , downstream of an activated G monomer conferred only a modest increase in the rate of addition of the G monomer to the primer ( Figure 1c–f , reactions 4 and 5 ) , consistent with previous reports of unactivated downstream ‘helper’ oligonucleotides ( Deck et al . , 2011; Jauker et al . , 2015; Kervio et al . , 2010 ) .", "Such helper oligonucleotides are thought to act largely by increasing monomer affinity to the primer/template complex by providing an additional base-stacking surface .", "In contrast , when the same AGC trimer was activated as a 5′-phosphoro-2-methylimidazolide and used as a downstream helper oligonucleotide , the rate of primer extension ( by addition of a G monomer ) was increased by over two orders of magnitude ( Figure 1c–f , reaction 6 ) compared to reactions with unactivated trimers .", "The rate of primer extension with a single G in the presence of an activated downstream trinucleotide was about twice as fast as the rate when the primer was followed by up to four sequential activated G monomers ( Figure 1c–f , reaction 1 and 6 ) .", "To systematically study the rate of nonenzymatic primer extension as a function of the length of the downstream activated ‘helper’ oligonucleotide , we synthesized a series of activated oligonucleotides of different lengths ( Figure 2a ) .", "At saturating concentrations ( Figure 2—figure supplement 1 ) of each of the oligonucleotides , the rate of primer extension improved as the length of the helper increased from mononucleotide to dinucleotide to trinucleotide , and then stayed approximately constant with the tetranucleotide ( Figure 2a ) .", "At subsaturating concentrations of helper oligonucleotide , the rate of the primer extension reaction increased with increasing concentrations of di- or tri-nucleotide helper ( Figure 2—figure supplement 1 ) .", "Because the maximum rate of the trimer-assisted reaction was significantly faster than that of the dimer-assisted reaction , we proceeded with trimers as the downstream helper oligonucleotides for the remainder of this study . 10 . 7554/eLife . 17756 . 006Figure 2 . Nonenzymatic primer extension using all four monomers .", "( a ) RNA primer extension assay using alternative 'helper' oligomers and corresponding , complementary templates .", "The respective rates are shown at bottom .", "Concentration curves are provided for AG and AGC in Figure 2—figure supplement 1 .", "( b ) Primer extension assay for each of the four monomers , as well as 2-thiouridine , in the absence and presence of activated trimer .", "Shown at bottom of both a and b is a bar graph with the respective rates .", "Reaction conditions: 10 μM primer , 11 μM template , 200 mM Tris pH 8 . 0 , 100 mM MgCl2 , 50 mM monomer , 10 mM 2-MeImpAG , 1 mM 2-MeImpAGC , 1 mM 2-MeImpAGGC .", "The gels in a and b show reaction progress after 10 min and 1 hr respectively .", "Error bars indicate S . E . M; all experiments were performed in triplicate or greater . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 00610 . 7554/eLife . 17756 . 007Figure 2—figure supplement 1 . Rate of primer extension vs . concentration of downstream activated di- and tri-nucleotides .", "( a ) Schematic of the primer extension reaction .", "( b ) Saturation curves of primer extension reactions with either AG or AGC as the helper oligomers . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 007 We then tested the ability of a downstream 2-methylimidazole-activated AGC trimer to catalyze template-directed RNA primer extension with all four individual 2-methylimidazole activated monomers – A , G , C and U . We also tested the 2-thiouridine monomer , which we have previously shown to be superior to uridine in nonenzymatic primer extension in terms of both increased rate and improved fidelity ( Heuberger et al . , 2015 ) .", "In each case , the rate of primer extension increased by at least two orders of magnitude compared to the corresponding reactions without any helper oligonucleotides ( Figure 2b ) .", "Remarkably , whereas the rate of primer extension with A and U monomers was previously too slow to measure ( Wu and Orgel , 1992b; Heuberger et al . , 2015 ) – conservatively estimated to be at least three orders of magnitude slower than primer extension with G or C monomers – the difference in polymerization rates was reduced to roughly one order of magnitude when assisted by downstream activated trimers ( Figure 2b ) .", "In order to assess the fidelity of nonenzymatic trimer-assisted primer extension , we measured the rates of monomer addition for all monomer-template combinations , including both matches and mismatches ( Figure 3a ) .", "We calculated the fidelity for each template as the rate of matched monomer addition divided by the sum of matched and mismatched monomer addition rates .", "By averaging the fidelities of the four templates we found the overall fidelity of trimer-assisted polymerization to be 98% .", "Assuming an error threshold of one mutation per genome ( Eigen , 1971 ) , this value of fidelity allows for effective genome sizes of 50 nucleotides , long enough to produce functional ribozymes ( Ferré-D'Amaré and Scott , 2010 ) .", "The fastest mismatch reaction was primer extension by G during copying of a template U , which was approximately 5% as fast as primer extension with A on the same template ( Figure 3a , Figure 3—figure supplement 1 ) .", "This is likely due to the formation of a G:U wobble base pair ( Heuberger et al . , 2015 ) , and is consistent with previous studies of nonenzymatic primer extension reactions ( Rajamani et al . , 2010 ) .", "All other mismatches were at least two orders of magnitude slower than their matched counterparts .", "We also tested the effect of replacing the canonical U monomer with 2-thiouridine and 2-thioribothymine on the polymerization rate and found , in line with previous reports ( Heuberger et al . , 2015 ) , that this sulfur substitution improved the rates by approximately one order of magnitude ( Figure 3b ) , bringing the rate of primer extension closer to the rates of G and C addition .", "More rigorous tests of fidelity , in which monomers can compete for binding on a template in all possible sequence contexts , are ongoing . 10 . 7554/eLife . 17756 . 008Figure 3 . Fidelity of trimer-assisted primer extension .", "( a ) Schematic of the fidelity assay .", "Using the same RNA primer and trimer , four different templates , one with each base , were paired with each of the four 2-methylimidazole activated monomers to test the relative rates of each matched and mismatched pair .", "( b ) Heat map showing the relative rates of primer extension for each template:monomer pairing .", "Black indicates a rate of 0 . 001 h−1 , white indicates a rate of 100 h−1 .", "A bar graph and a table of the same data can be found in Figure 3—figure supplement 1 .", "( c ) Relative rates of primer extension with the modified U monomers , 2-thiouridine and 2-thioribothymidine compared to that of a canonical , activated U monomer on a template containing an A . Reaction conditions as in Figure 2 . Error bars indicate S . E . M . All experiments were performed in triplicate or greater . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 00810 . 7554/eLife . 17756 . 009Figure 3—figure supplement 1 . Rates of trimer-assisted primer extension for all monomer/template combinations .", "( a ) Schematic of the primer extension reaction .", "( b ) Rates of monomer additions on different templates for all monomer-template pairs .", "( c ) The rates from part b on the grid from Figure 3b . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 009 Encouraged by the relatively fast and accurate addition of all four monomers to a primer , we then attempted to copy short RNA templates containing all four nucleotides .", "In order to iterate the process of trimer assisted monomer addition , after a downstream activated trimer catalyzes the addition of the first monomer to the primer , the helper trimer must dissociate to allow for the binding of the next monomer-trimer pair .", "To test the feasibility of this mode of primer extension , we designed a template that contained binding sites for all four monomers and synthesized the appropriate activated trimers ( Figure 4a ) .", "We used 2-thiouridine monomer in place of uridine ( see above ) , and all four monomers ( A , G , C and 2-thiouridine ) were present in every reaction .", "In the absence of activated trimers , we observed almost no primer extension ( lane 1 Figure 4b ) ; even though all four monomers were present , their combined rate of addition was not measurable .", "Adding just the first helper trimer resulted in rapid addition of the first monomer ( C ) to the primer , followed by slow addition of the first trimer ( AGC ) to the growing primer .", "Similarly , adding two , three or four trimers together resulted in the generation of primers extended by two , three or four monomers respectively ( Figure 4b ) .", "These reactions converted primer into a product with four sequentially added monomers with an ~80% yield after 16 hr ( Figure 4c , d ) ; single monomer addition reactions reached >95% yield after only 10 min ( Figure 2b ) .", "The difference between the fast rates observed in the trimer-assisted addition of a single base ( Figure 2b ) and the slower rate of trimer-assisted polymerization of all four bases simultaneously ( Figure 4b ) may be due to the competition of trimers for overlapping binding sites on the template .", "This hypothesis is supported by the decreased rate of trimer assisted primer extension with a single monomer in the presence of additional overlapping downstream trimers ( Figure 4—figure supplement 1 ) .", "The copying of templates containing two consecutive A or U monomers is also made possible by the use of activated helper oligonucleotides ( Figure 5 ) . 10 . 7554/eLife . 17756 . 010Figure 4 . Primer extension with all four monomers in a one-pot reaction .", "( a ) Schematic of a primer extension reaction incorporating all four RNA monomers , with four respective downstream helper trimers .", "( b ) All four monomers and the activated trimers indicated below the gel were added to each reaction .", "For example , in lane 3 all four monomers and the activated AGC and GCG trimers were mixed with primer , template , buffer and Mg2+ .", "Higher bands represent either the ligation of activated trimers or the polymerization of G monomers without trimer assistance .", "The gel shows reaction progress after 16 hr .", "( c ) Timecourse of the reaction with all four trimers .", "A 48 hr timepoint of the reaction without any trimers ( left lane of", "b ) is contrasted with a timecourse ( timepoints at 5 and 30 min , 2 , 24 and 48 hr ) .", "The percentage of polymerization products that have been extended by at least four nucleotides is plotted over time .", "( d ) A gel displaying the timepoints plotted in part c .", "A timecourse showing the effect of overlapping trimers on reaction rate can be found in Figure 4—figure supplement 1 . Reaction conditions: 2 . 5 μM primer , 2 . 5 μM template , 100 mM HEPES pH 8 . 0 , 100 mM MgCl2 , 20 mM monomer , 100 μM trimer . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 01010 . 7554/eLife . 17756 . 011Figure 4—figure supplement 1 . Inhibition of primer extension by overlapping helper-trimers .", "( a ) Schematic of the primer extension reaction .", "( b ) Rate of primer extension as a function of increasing GCG trimer concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 01110 . 7554/eLife . 17756 . 012Figure 5 . Extension of primers by multiple consecutive A or U nucleotides .", "( a ) Schematic of a primer extension reaction wherein two consecutive A nucleotides can polymerize .", "The gel shows extension without trimers and with one or two trimers after 16 hr .", "( b ) Same as in", "( a ) but with two U additions .", "In the right half 2-MeImpU is replaced with 2-MeImp-2-thioU . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 012 If nonenzymatic RNA polymerization did indeed precede the RNA world , then it would have been necessary to synthesize RNA sequences long enough to function as ribozymes ( Deck et al . , 2011 ) .", "While our method can only produce sequences >7 nucleotides in one reaction in low yields ( Figure 6—figure supplement 1 ) due to the cross-inhibitory effect of trimers with overlapping binding sites ( Figure 4—figure supplement 1 ) , we reasoned that a ribozyme could be synthesized by this approach if monomers were added , along with their attendant trimers , one at a time ( Figure 6 ) .", "To that end , we immobilized a template that codes for the sequence of one half of the hammerhead ribozyme ( HH2 ) – on a bead and added matched pairs of monomers and trimers sequentially ( Figure 6a ) .", "Primer extension was monitored by gel electrophoresis .", "Despite the fact that no 2-thiouridine monomers were used ( in order to protect the catalytic capacity of the ribozyme ) a high conversion rate was observed at each step ( 75–95% ) ( Figure 6b ) .", "The reaction durations required to achieve these conversion rates varied widely , for example , the seventh monomer addition required 21 hr to reach 95% while the eighth addition reached the same level of conversion after just 30 min .", "Generally , G and C additions were much faster than A or U additions; the reason for the discrepancy between the rates of these reactions on beads and those in solution is unknown .", "The use of s2U or s2T might offset some of the discrepancy .", "The final product was released from the bead by hybridization of an excess of a DNA oligonucleotide of identical sequence to the immobilized template .", "After DNase treatment , the hammerhead substrate , HH1 , was added to the crude synthesized pool of oligonucleotides in the presence of 200 mM MgCl2 for 16 hr ( Figure 6c ) .", "Despite the fact that this was a crude reaction product , with full-length HH2 comprising only 6% of the reaction products , the product mixture was able to function as a catalyst , with 50% ribozyme cleavage yield , thus demonstrating that a significant portion of an active ribozyme was synthesized nonenzymatically . 10 . 7554/eLife . 17756 . 013Figure 6 . Nonenzymatic RNA synthesis of an active hammerhead ribozyme by template dependent primer extension .", "( a ) Schematic of the bead-assisted primer extension reaction .", "Synthesis of the hammerhead ribozyme strand HH2 by the sequential addition of matched pairs of activated monomers and trimers .", "( b ) Each lane shows primer extension by one nucleotide , catalyzed by the corresponding downstream activated trimer .", "Final yield of the +12 product was 6% .", "Reaction conditions: 100 mM MgCl2 , 200 mM Tris-HCl pH 8 , 25 mM monomer and 2 mM trimer .", "The beads were washed between steps in 2 M NaCl , 1 mM EDTA and 10 mM Tris-HCl pH 7 .", "For steps 2 , 5 and 7 , 50 mM monomer and 4 mM trimer were added .", "Each step was monitored by gel electrophoresis until it reached >75% yield .", "Steps varied in length from 30 min to 22 hr; the total time required was 150 hr .", "( c ) The product of the last bead-assisted reaction was removed from the bead by the addition of the an excess of the DNA sequence of the extended primer at 95°C over 2 min .", "After DNase treatment , the target hammerhead RNA , HH1 , was added .", "The third lane shows the extent of the nuclease reaction , 50% , after 16 hr at room temperature .", "For comparison , pure HH2 made from DNA and RNA served as negative and positive controls respectively .", "Reaction conditions: Extended primer from the beads was added to 200 mM MgCl2 , 200 mM Tris-HCl pH 8 and 250 nM Cy5 labeled HH1 RNA . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 01310 . 7554/eLife . 17756 . 014Figure 6—figure supplement 1 . One pot synthesis of hammerhead ribozyme .", "( a ) Schematic of the primer extension reaction .", "( b ) Gel showing primer extension after 18 hr .", "Reaction conditions: 5 μM primer , 5 . 5 μM template , 100 mM Tris pH 8 , 100 mM MgCl2 , 12 . 5 mM monomers , 500 μM trimers . DOI: http://dx . doi . org/10 . 7554/eLife . 17756 . 014" ], [ "The experiments described above demonstrate for the first time a simple and robust means of nonenzymatically copying mixed sequence RNA templates .", "Short , activated oligonucleotides – themselves plausibly generated by either templated or untemplated monomer polymerization – are efficient catalysts of high fidelity primer extension with all four RNA monomers .", "Thus short activated oligonucleotides can play dual roles , acting either as catalysts of chain growth by monomer addition , or directly as replication intermediates , since they can also be incorporated into a growing chain by ligation .", "Template copying in a complex but realistic milieu containing both activated monomers and oligomers could therefore occur via a hybrid process combining primer extension with monomers and oligonucleotide ligation .", "The disparity in rates between the reactions catalyzed by activated vs . unactivated trimers indicates a key role for the leaving group downstream of the polymerizing monomer .", "The slow rate of activated oligonucleotide ligation is also explained by the lack of a leaving group one base downstream of the site of the primer extension reaction: in this case the downstream position corresponds to the second monomer in the oligonucleotide which necessarily has no leaving group as it is linked to the upstream monomer by a phosphodiester linkage .", "The nature of the interaction between the downstream leaving group and the reacting monomer remains unclear .", "It is possible that the downstream leaving group interacts attractively with the reacting leaving group , positioning it optimally for attack by the 3′ hydroxyl of the primer .", "As observed by Wu et al . ( Wu and Orgel , 1992a ) this interaction could be very sensitive to steric effects , such that 2-methylimidazole at the downstream position would facilitate polymerization much more effectively than imidazole , though the authors ultimately concluded that such subtle effects are not likely to adequately explain the strength of the interaction .", "Wu et al . ( Wu and Orgel , 1992a ) also suggested that the catalytic effect could be due to acid-base catalysis .", "Another possibility is that the upstream leaving group forms a covalent bond with the phosphate of the downstream monomer , a reaction that is only possible if there is a leaving group on the downstream monomer ( Kervio et al . , 2016 and T . Walton and J . W . Szostak , unpublished data ) .", "This covalent bond could form before the attack of the 3′ hydroxyl of the primer , creating an imidazolium intermediate , or in a concerted reaction where the leaving group of the upstream monomer replaces the leaving group of the downstream monomer .", "Further understanding of the reaction mechanism could lead to concomitant improvements in the rate and extent of template copying .", "Further advances are required to enable the copying of longer and potentially functional RNA sequences in one pot , protocell-compatible conditions .", "However , the copying of long mixed template sequences under our conditions compares favorably , both in terms of rate and efficiency with reactions in which arbitrary RNA templates are copied by highly evolved ribozyme polymerases ( Wochner et al . , 2011 ) ( Figure 6—figure supplement 1 ) .", "In fact , if the rates and fidelities of nonenzymatic RNA polymerization were sufficiently high , the need for a ribozyme polymerase in the RNA world may be circumvented: RNA could be copied by nonenzymatic chemistry alone until the advent of protein polymerases .", "We suggest that the identification of a plausible source of chemical energy that could drive the re-activation of monomers and oligomers following hydrolytic loss of the leaving group is a key missing component in efforts to reconstitute nonenzymatic RNA replication .", "A chemical environment that could maintain a fully activated pool of substrates , and avoid accumulation of the inhibitory by-products resulting from hydrolysis , might be sufficient to drive high yielding and complete copying of longer RNA templates in one pot , thus setting the stage for the emergence of Darwinian evolution ." ], [ "Guanosine 5′-monophosphate was purchased as the free acid from Santa Cruz Biotechnology ( Dallas , TX ) .", "2-thiouridine-5′-phosphoro-2methylimidazolide and 2′ , 3′-diacetyl-nucleosides were purchased from ChemGenes ( Wilmington , MA ) .", "Phosphoramidite nucleotides and all other oligonucleotide synthesis reagents were purchased from either ChemGenes or Bioautomation ( Plano , TX ) .", "Tris ( hydroxymethyl ) aminomethane ( Tris ) -HCl , ThermoScript reverse transcriptase as well as reaction buffer and NTPs were purchased from Thermo Fisher Scientific ( Waltham , MA ) .", "DNA and RNA primers and were purchased from Integrated DNA Technologies ( Coralville , IA ) .", "All oligonucleotide sequences are listed below .", "All other chemicals were purchased from Sigma Aldrich Corporation ( St . Louis , MO ) .", "Gels were prepared using the SequaGel – UreaGel system from National Diagnostics ( Atlanta , GA ) .", "Gels were prepared to 20% acrylamide and scanned using a Typhoon Scanner 9410 ( GE Healthcare , Little Chalfont , Buckinghamshire , UK ) .", "RNA oligonucleotides were prepared by standard phosphoramidite oligonucleotide synthesis using a MerMade 6 DNA/RNA synthesizer ( Bioautomation , Plano , TX ) or starting with 2′ , 3′-diacetyl nucleosides using standard manual coupling procedures ( Beaucage and Caruthers , 1981 ) .", "5′-phosphates were installed using bis-cyanoethyl-N , N-diisopropyl phosphoramidite from ChemGenes by standard phosphoramidite coupling chemistry .", "Mononucleotide monophosphates and oligonucleotide monophosphates were activated using a modified published protocol ( Joyce et al . , 1984 ) .", "As an example , 2-MeImpAGC ( the 2-methylimidazolide of the trimer 5′-phosphoro-AGC ) was synthesized by first dissolving 5 mg ( 5 μmole ) of 5′-phosphoro-AGC in 1 mL of dimethyl sulfoxide ( DMSO ) .", "To that mixture 160 mg ( 2 mmoles ) of 2-methylimidazole , 56 mg triphenylphosphine ( 260 μmole ) , 64 mg 2 , 2′-dipyridyldisulfide ( 290 μmole ) and 40 μL triethylamine ( 550 μmole ) were added .", "After stirring overnight at room temperature , the mixture was precipitated in 10 mL of a 400:250:30:1 mixture of acetone:diethylether:triethylamine:saturated solution of NaClO4 in acetone .", "The precipitate was pelleted by centrifugation ( 3000 rpm , 5 min ) and washed twice with a 1:1 mixture of acetone:diethylether and once with pure diethylether .", "After decanting the solvent , the pellet was dried under vacuum , resuspended in deionized water and purified by high-performance liquid chromatography ( HPLC ) over a C18 column ( Alltima C18 5 μm , Thermo Fisher , 250 × 10 mm ) with 25 mM triethylammonium bicarbonate ( TEAB ) pH adjusted to 7 . 5 in 2% ( v/v ) acetonitrile for mobile phase A and acetonitrile for mobile phase B over a gradient beginning at 100% A and falling to 80% A over 20 min with a flow rate of 3 mL/minute .", "The fraction containing the product was verified by electrospray ionization mass spectrometry ( ESI/MS ) in negative mode ( m/z = 996 ) , frozen in liquid nitrogen and lyophilized overnight to yield a white powder .", "The powder was dissolved in water and the concentration of the activated trimer was determined using a NanoDrop 2000c spectrophotometer ( Thermo Fisher Scientific , Waltham , MA ) and calculated assuming ε260 = 34 , 170 M−1 cm−1 29 .", "Monomer stocks were adjusted to pH 7 before adding into the reaction mixture .", "Nonenzymatic RNA primer extension reactions exploring the effect of downstream nucleotides and oligonucleotides on 2-MeImpG addition were conducted under the following conditions: 200 mM MgCl2 , 200 mM sodium N-cyclohexyl-2-aminoethanesulfonic acid ( CHES ) pH 9 , 50 mM monomer , 1 mM trimer , 10 μM primer and 11 μM template .", "The primer sequence was the same for all reactions ( 6-carboxyfluorescein ( FAM ) -5′-GAC UGG-3′ for kinetics , cyanine 3 ( Cy3 ) -5′-GCG UAG ACU GAC UGG-3′ for gels ) .", "The templates used in Figure 1 had the following sequences:", "1 ) 5′-AAC CCC CCA GUC -3′", "2 ) 5′-G CUC CCA GUC -3′ 3 ) 5′-AA CUC CCA GUC -3′ The templates in Figure 1—figure supplement 2 had the following sequences: For GGG: 5′-AA CCC CCA GUC -3′ For GCG: 5′-AA CGC CCA GUC -3′ For GAG: 5′-AA CUC CCA GUC -3′ For AGC: 5′-GCU GCU GCU GCU CCA GUC AGU CUA CGC-3′ The templates in Figure 2 had the following sequences: Part a lanes 1–4: 5′-G CUC CCA GUC -3′ Part a lane 5: 5′-GCC UCC CAG UC -3′ Part b: 5′-GCU N CCA GUC -3′ The templates in Figure 3 had the same sequences as Figure 2b .", "The template in Figure 4 had the following sequence: 5′-CCC GCU ACC AGU C -3′ The template in Figure 4—figure supplement 1 had the following sequence: 5'- GCU C GCU C GCU C GCU C CCAGUCAGUCUACGC -3' The templates in Figure 5 had the following sequences: Part a: 5′-CCC GCU UCC AGU C -3′ Part b: 5′-AUC AAC CAG UC -3′ The template in Figure 6 had the following sequence: 5′-biotin – AAC CCC GCG CCU CAU CAG CCA GUC -3′ The template in Figure 6—figure supplement 1 had the following sequence: 5′ - GCG CCU CAU CAG CCA GUC -3′ Reaction progress was assessed by gel electrophoresis ( 20% acrylamide denaturing urea gel ) after 10 min .", "Reaction rates were calculated by quantifying primer conversion to products using a Typhoon Scanner 9410 ( GE Healthcare , Little Chalfont , Buckinghamshire , UK ) .", "Band intensities were quantified using ImageQuant TL software ( GE Healthcare , Little Chalfont , Buckinghamshire , UK ) .", "The negative log of the fraction of unreacted primer was plotted against time , in hours .", "A linear regression was performed and the slope of the fit as plotted was reported as the pseudo-first order rate kobs .", "Primer extension reactions exploring the effect of different monomers and different oligomers on the rate of polymerization were conducted under the following conditions: 100 mM MgCl2 , 200 mM Tris-HCl pH 8 , 25 mM monomer , 1 mM trimer , 10 μM primer and 11 μM template .", "The downstream helper oligomers were added in the following concentrations: 25 mM 2-MeImpA and 1 mM activated oligonuleotide ( AG , AGC or AGGC ) .", "The reaction progress was assessed by gel electrophoresis ( 20% acrylamide denaturing urea gel ) after 10 min .", "Reaction rates were calculated as above .", "Primer extension reactions exploring the addition of multiple monomers and trimers in one reaction had the following conditions: 100 mM MgCl2 , 200 mM Tris-HCl pH 8 , 25 mM monomers , 500 μM 2-methylimidazole-activated AGC , CGG and GGG , 100 μM activated GCG , 10 μM primer and 11 μM template .", "The reaction progress was assessed by gel electrophoresis ( 20% acrylamide denaturing urea gel ) after 18 hr . 30 μL of Dynabeads MyOne Streptavidin T1 ( Invitrogen Dynal AS , Oslo , Norway ) beads were prepared as specified by the manufacturer .", "Primer ( Cy3 - 5′- GCG UAG ACU GAC UGG - 3′ ) and template ( biotin - 5′- AAC CCC GCG CCU CAU CAG CCA GUC AGC UCU ACG C - 3′ ) were bound to the beads at 250 μM and 275 μM , respectively , in 2 M NaCl , 1 mM EDTA and 10 mM Tris-HCl pH 7 .", "Monomer additions were conducted under the following conditions: 100 mM MgCl2 , 200 mM Tris-HCl pH 8 , 25 mM monomer and 2 mM trimer .", "The beads were washed between steps in 2 M NaCl , 1 mM EDTA and 10 mM Tris-HCl pH 7 .", "For steps 2 , 5 and 7 , 50 mM monomer and 4 mM trimer were added .", "Each step was monitored by gel electrophoresis until it reached >75% yield .", "Steps varied in length from 30 min to 22 hr; the total time required was 150 hr . 10% of the product of the bead-immobilized reactions was washed twice in 100 μL water containing 0 . 01% ( v/v ) Tween 20 .", "174 μM DNA complement was added in 0 . 01% ( v/v ) Tween 20 and the beads were incubated at 95°C for 1 min .", "The solution was removed from the beads and 2 units of DNaseI along with DNaseI buffer ( NEB ) were added .", "Finally , the reaction was incubated at room temperature with 200 mM MgCl2 , 200 mM Tris-HCl pH 8 and 250 nM Cy5 labeled HH1 RNA ( Cy5 - 5′ - CGC GCC GAA ACA CCG UGU CCC AGU C - 3′ ) for 20 hr .", "The products of both the bead immobilized reactions and the hammerhead reaction were analyzed by gel electrophoresis ( 20% acrylamide denaturing urea gel ) ." ] ]

[ "The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes ( ribozymes ) catalyze replication of the RNA genomes of primordial cells .", "However , the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides .", "We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension , and allow for the one-pot copying of mixed sequence RNA templates .", "The fidelity of the primer extension products resulting from the sequential addition of activated monomers , when catalyzed by activated oligomers , is sufficient to sustain a genome long enough to encode active ribozymes .", "Finally , by immobilizing the primer and template on a bead and adding individual monomers in sequence , we synthesize a significant part of an active hammerhead ribozyme , forging a link between nonenzymatic polymerization and the RNA world ." ]

[ "Though defining what makes something “alive” has proved challenging , one crucial feature of living things is the ability to copy genetic information and pass it on to the next generation .", "Nowadays , enzymes called polymerases copy genetic information encoded within the DNA of living cells .", "However , when life on Earth began approximately four billion years ago , polymerases had not evolved yet .", "This means that the genetic information of the first cells had to be copied some other way .", "The earliest life on Earth is unlikely to have used DNA to store its genetic information , and probably used a closely related molecule called RNA instead .", "Like DNA , RNA is made up of four smaller building blocks joined together to form long chains .", "The building blocks of RNA are commonly referred to using single letters: A , C , G and U . Previous studies have shown that it is possible to copy RNA without enzymes , but for only two of the four RNA letters , namely C and G . Prywes et al . wanted to know if it was possible to create a chemical system , without polymerases , in which all four RNA letters could be copied .", "The experiments showed that strings of RNA that were three letters long could catalyze RNA copying , just as long as they were chemically activated .", "That is to say , these short RNA strings allowed RNA to be copied without enzymes if they had a chemical group at one end that made them more reactive . Each short catalyst helped copy one of the four RNA letters , and adding several into one reaction meant that longer sequences containing all four RNA letters could be copied .", "Prywes et al . then used these short catalysts to copy an RNA molecule that itself acts a bit like an enzyme , and confirmed that a significant portion of this molecule could be copied without any polymerases .", "Further work is now needed to see if it is possible to copy other RNA sequences , and especially longer ones , without enzymes .", "Another challenge for the future would be to attempt to copy an RNA sequence multiple times without enzymes; a challenge that the earliest ancestors of cells on Earth must have overcome to pass their genetic information down through the generations ." ]

[ "Introduction", "Materials and methods", "Results", "Discussion" ]

[ "epidemiology and global health", "genetics and genomics" ]

[ [ "At the current time , the coronavirus disease 2019 ( COVID-19 ) pandemic is implicated in the deaths of more than 4 million people worldwide ( Dong et al . , 2020 ) .", "Although effective vaccines have been developed to substantially reduce mortality and morbidity due to severe COVID-19 , the emergence of mutated strains of the SARS-CoV-2 virus has challenged the effectiveness of existing vaccines and raised the urgency of identifying alternate therapeutic pathways to target the virus ( Tegally , 2020; Erik et al . , 2020 ; Collier et al . , 2021 ) .", "Nevertheless , it is likely that the mutated strains of SARS-CoV-2 will continue to exploit the same vulnerable host biology to bind onto and infect cells and , in susceptible individuals , evade immune defences and promote the excessive host inflammatory response that is characteristic of severe COVID-19 ( Gordon et al . , 2020a ) .", "Therefore , the identification of host proteins that play roles in COVID-19 susceptibility and severity remains crucial to the development of therapeutics as host protein mechanisms are independent of genomic mutations in the virus .", "An improved understanding of these therapeutically relevant virus-host pathways may also be important in combating viruses beyond SARS-CoV-2 ( Perrin-Cocon et al . , 2020 ) .", "Several large-scale systematic experimental efforts have identified key host proteins that interact with viral proteins in the pathogenesis of severe COVID-19 ( Gordon et al . , 2020a; Gordon et al . , 2020b; Bouhaddou et al . , 2020 ) .", "These notably include efforts to identify direct interactions with the spike protein of SARS-CoV-2 , which mediates virus attachment onto receptors to infect host cells and is also the basis of most vaccines ( Shang et al . , 2020; Harvey et al . , 2021 ) .", "To complement in vitro host protein characterisation efforts , several groups have leveraged genetic datasets of human proteins and COVID-19 disease to identify therapeutically actionable candidate host proteins that are likely to play roles in enhancing COVID-19 susceptibility or to be involved in the pathogenesis of severe COVID-19 ( Pairo-Castineira et al . , 2021; Zhou et al . , 2021 ) .", "One of the approaches used was Mendelian randomisation ( MR ) .", "MR simulates the design of randomised trials , with the underlying principle that randomisation of alleles at conception offers the opportunity to examine approximate differences in average risk of disease between comparable groups in a population that differ only in the distribution of the risk factor of interest ( Davies et al . , 2018 ) , for example , protein abundance ( Zheng et al . , 2020 ) .", "This allows the use of alleles as genetic instruments representing genetically predicted protein levels to proxy effects of pharmacological modulation of the protein .", "Some of the clinically actionable proteins identified by the MR approach are part of type I interferon signalling ( encoded by genes: IFNAR2 , TYK2 , OAS1 ) and interleukin-6 ( IL-6 ) signalling pathways ( IL6R ) .", "Only one of these proteins ( encoded by OAS1 ) had any evidence of genetic colocalisation , that is , evidence that genetic associations of the protein and COVID outcomes shared the same causal genetic signal ( Zhou et al . , 2021 ) .", "An additional protein that was supported by both MR and genetic colocalisation tests was ABO ( Zhou et al . , 2021 ) , reported in several published genome-wide association studies ( GWAS ) of COVID-19 ( Pairo-Castineira et al . , 2021; Ellinghaus et al . , 2020 ) .", "In response to the first published GWAS of COVID-19 , we reported findings that link the ABO signal with a number of clinically actionable targets including coagulation factors ( von Willebrand factor [vWF] , and Factor VIII [F8] ) , IL-6 , and CD209/DC-SIGN ( Karim et al . , 2020 ) .", "However , in most of the previous MR studies ( Pairo-Castineira et al . , 2021; Zhou et al . , 2021 ) , investigators only used curated cis-acting variants ( genetic variants near or in the gene encoding the relevant protein ) as genetic instruments to represent effects of genetically predicted protein concentrations , rather than genome-wide instruments .", "While the use of cis-acting variants can minimise the risk of horizontal pleiotropic effects ( i . e . associations driven by other proteins not on the causal pathway for the disease ) , it can suffer from lower power than a genome-wide analysis due to fewer available instruments ( Zheng et al . , 2020 ) .", "Furthermore , in previous protein-COVID-19 MR studies , genetic colocalisation tests were carried out only for protein-phenotype associations that were significant in the MR analysis , potentially excluding many protein-phenotype associations that may share the same causal genetic signal but are underpowered in a proteome-wide MR approach .", "In the present study , we expanded on these previous reports by undertaking a proteome-wide two-sample pan- and cis-MR analysis using the Sun et al . GWAS ( Sun et al . , 2018 ) of plasma protein concentrations and several COVID-19 GWAS phenotypes from the ICDA COVID-19 Host Genetics Initiative ( October 2020 release ) ( Huang et al . , 2020 ) .", "First , we showed that genetically predicted circulating ABO protein was associated with COVID-19 susceptibility and severity and the lead ABO signal was associated strongly with plasma concentrations of soluble CD209 .", "Second , we collected evidence for a direct mechanism of interaction between the SARS-CoV-2 spike protein and human CD209 protein .", "Third , we performed proteome-wide genetic colocalisation tests , followed by single-instrument cis-MR analysis , and we report additional novel targets of therapeutic relevance .", "Finally , we examined associated phenotypes using the colocalising signals from the Open Targets Genetics portal ( http://genetics . opentargets . org ) to shed light on the biological basis of association of the proteins with the COVID-19 phenotypes ." ], [ "We primarily used Sun et al . protein GWAS data ( Sun et al . , 2018; Emilsson et al . , 2018 ) for the pan-/cis-MR analyses and for performing genetic colocalisation tests ( described below ) .", "The pan-/cis-MR effects were expressed per standard deviation ( SD ) higher genetically predicted plasma protein concentrations .", "Two additional proteomic datasets ( Emilsson et al . , 2018; Suhre et al . , 2017 ) were used to identify proteins associated with the ABO locus .", "The genotyping protocols and QC of these proteomic studies have been described previously ( Sun et al . , 2018; Emilsson et al . , 2018; Suhre et al . , 2017 ) .", "All three of the proteomic studies have used the SOMAscan assay platform ( an aptamer-based protein detection platform ) to detect and quantify protein abundance ( Gold et al . , 2012 ) .", "We used seven meta-analysed COVID-19 datasets from the October 2020 release of the ICDA COVID-HGI group ( https://www . covid19hg . org/results/r4/ ) .", "These seven COVID-19 outcomes are A1 ( very severe respiratory confirmed COVID vs . not hospitalised COVID ) , A2 ( very severe respiratory confirmed COVID vs . population ) , B1 ( hospitalised COVID vs . not hospitalised COVID ) , B2 ( hospitalised COVID vs . population ) , C1 ( COVID vs . lab/self-reported negative ) , C2 ( COVID vs . population ) , and D1 ( predicted COVID from self-reported symptoms vs . predicted or self-reported non-COVID ) .", "Definitions of these outcomes are provided in Supplementary file 1 .", "Prior to analyses , we performed a liftover of datasets that reported genomic coordinates using the GRCh37 assembly to GRCh38 .", "We also checked and ensured that the effect allele in a GWAS locus is the alternative allele in the forward strand of the reference genome .", "To infer strand for palindromic variants ( variants with A/T or G/C alleles , i . e . variants with the same pair of letters on the forward strand as on the reverse strand ) , we first checked the orientation of all non-palindromic variants with respect to the reference genome to assess whether there was a strand consensus of 99% or more .", "For example , for a given GWAS , if ≥99% of the non-palindromic variants were on the forward strand , we assumed that the palindromic variant would also be on the forward strand; otherwise , they were excluded from analyses .", "Details of the harmonisation workflow are provided in our GitHub pages ( EBISPOT , 2020; Opentargets Inc , 2021 ) .", "To construct genetic instruments for MR analysis , we selected near-independent ( r² = 0 . 05 ) genetic variants from across the genome ( ‘pan’-instruments ) or from within ±1 Mbp from the transcription start site ( TSS ) of the gene encoding the protein ( ‘cis’-instruments ) associated with the encoded protein abundance at p≤5 × 10–8 for pan-MR analyses and at a less stringent p ≤ 1 × 10⁻⁵ for cis-MR analyses ( this p-value corrects for the number of proteins in the druggable genome Schmidt , 2020 ) .", "We used the generalised summary data-based Mendelian randomisation ( GSMR ) approach with the heterogeneity-independent instrument ( HEIDI ) -outlier flag turned on to carry out the pan- and cis-MR analyses ( Zhu et al . , 2018 ) .", "The GSMR software , using the HEIDI-outlier method , removes potentially pleiotropic instruments and accounts for the residual correlation between instruments ( important as we are using near-independent genetic instruments ) .", "To select near-independent genetic instruments and account for linkage disequilibrium ( LD ) in the MR analyses , we used genotype data from 10 , 000 randomly sampled UK Biobank participants to create a reference LD matrix , which is ancestry-matched to the pQTL data we used .", "For each COVID-19 outcome , we used the Benjamini–Hochberg FDR ( False Discovery Rate ) threshold of 5% for significance , adjusting for 2042 tests in cis-MR analyses and 1286 tests in pan-MR analyses .", "For trans-acting instruments in pan-MR associations , variants were mapped to their respective cis-gene that had the highest overall V2G score in the Open Targets Genetics portal ( Ghoussaini , 2021; Mountjoy , 2020; Open Targets Genetics , 2019a ) .", "To identify shared causal genetic signals between protein and COVID outcomes , we used the Bayesian method of genetic colocalisation implemented in the coloc R package ( Giambartolomei , 2014 ) using the marginal association statistics for each trait ( i . e . assuming one independent signal in each region ) .", "We used beta and standard errors of cis-pQTLs of phenotype pairs as inputs .", "The default priors in coloc were used , that is , the prior of an SNP ( single nucleotide polymorphism ) -trait association is 1 × 10–4 , and the prior of an SNP associating with both traits is 1 × 10–5 .", "For each COVID-19 outcome , a posterior probability for shared causal genetic signal ( PP . H4 ) threshold of more than 0 . 8 was used to identify shared causal genetic variants .", "For colocalising signals , we carried out a phenome-wide association study ( PheWAS ) using GWAS summary statistics ( n = ~ 3000 GWAS ) from the Open Targets Genetics portal ( Ghoussaini , 2021; Mountjoy , 2020 ) .", "For variants associated with proteins due to aptamer or epitope binding artefacts ( which tend to be missense variants ) ( Joshi and Mayr , 2018 ) , we first assessed whether genetic instruments for MR or coloc-based single-SNP MR analysis were associated with corresponding gene expression ( i . e . whether they were also cis-eQTLs ) .", "This used gene expression data from the Open Targets Genetics portal ( Ghoussaini , 2021 ) .", "SNPs that were not cis-eQTLs were investigated further by identifying whether they were ( or were in LD at r2 = 0 . 8 with ) missense variants .", "To query if variants were missense or in LD with missense variants , we used the functional consequence data from Open Targets Genetics ( Ghoussaini , 2021 ) ( which used gnomAD v2 for variant effect prediction annotation , Lek , 2016 ) .", "The reasoning was , if missense variants also had effects on corresponding gene expression , the causal inference using the missense variants as genetic instruments was unlikely to be biased even if the effect estimates were invalid .", "Where cis-pQTLs were not cis-eQTLs and were missense variants ( or in LD with missense variants at r2 = 0 . 8 ) affecting the respective genes , these proteins were flagged and excluded from any further downstream analyses on the basis that the missense variant ( s ) might influence aptamer binding and produce biased effect estimates .", "Where cis-pQTLs were also cis-eQTLs and were missense variants ( or in LD with missense variants ) for the respective genes , although the effect estimates would not be valid , the causal inference using the instruments is unlikely to be biased; hence , these variants were retained in supplementary files and estimates of probes represented by these variants were flagged ( using an asterisk ) in the main figures .", "The rest , where cis-pQTLs had an effect on gene expression but were not missense variants or in LD with missense variants , were included in all analyses and presented without restrictions .", "Recombinant human receptors and SARS-CoV-2 spike protein extracellular domains were expressed and purified as previously described ( Shilts et al . , 2021 ) .", "Briefly , the full extracellular domain sequences of each were expressed as soluble secreted proteins in HEK293 cells .", "All proteins were affinity-purified using their hexahistidine tags .", "For biotinylated proteins , co-transfection of secreted BirA ligase in the presence of 100 µM D-biotin resulted in the covalent addition of a biotin group to an acceptor peptide tag , also as described previously ( Kerr and Wright , 2012 ) .", "The extracellular domain of CD209 ( Q9NNX6 ) was defined as beginning at Pro114 , while the full cDNA sequence was acquired from OriGene ( #SC304915 ) .", "The binding of biotinylated human receptor extracellular domains to pentameric SARS-CoV-2 spike protein was measured using the avidity-based extracellular interaction assay ( AVEXIS ) as previously described ( Bushell et al . , 2008 ) .", "Briefly , the wells of a streptavidin-coated 96-well plate were saturated with biotinylated bait of either CD209 , ACE2 , or a previously described negative-control construct consisting only of the C-terminal protein tags shared by all other recombinant proteins ( rat Cd4 ( d3 +4 ) -linker-Bio-6xHis ) ( Voulgaraki , 2005; Galaway and Wright , 2020 ) .", "Across these baits , we applied a dilution series of the full SARS-CoV-2 spike protein extracellular domain pentamerised by a peptide sequence from the cartilage oligomeric matrix protein with a beta lactamase reporter .", "After washing , binding was measured by hydrolysis of a colorimetric nitrocefin substrate whose product was quantified by light absorbance at 450 nm .", "HEK293 cells were transiently transfected as described previously ( Bartholdson , 2012 ) with expression plasmids encoding full-length cDNA of CD209 ( Origene SC304915 ) , or a mock transfection lacking the expression plasmid .", "Separately , recombinant biotinylated spike protein was tetramerised around streptavidin conjugated to phycoerythrin as previously described ( Sharma et al . , 2018 ) .", "Cells were incubated with tetramers of spike or a control construct of protein tags before being analysed on a flow cytometer as previously described ( Shilts et al . , 2021 ) .", "HEK293 cell lines were provided by Yves Durcohcer ( National Research Council , Canada ) .", "Cell lines were authenticated upon first receipt by DNA sequencing .", "All cell lines were regularly tested for mycoplasma by PCR ( Surrey Diagnostics ) and found to be negative all throughout experiments .", "These cell lines are not listed by ICLAC as 'commonly misidentified’ .", "Codes used to harmonise summary statistics are provided in https://github . com/EBISPOT/gwas-sumstats-harmoniser ( EBISPOT , 2020 ) .", "Codes for pan- and cis-MR analyses are provided on the GSMR website ( https://cnsgenomics . com/software/gcta/#GSMR ) .", "Codes for genetic colocalisation analyses are provided on the coloc GitHub page ( https://github . com/chr1swallace/coloc; Wallace , 2021 ) .", "All codes used in the paper to reproduce results are provided in https://github . com/mohdkarim/covid_paper ( copy archived at swh:1:rev:4ab9f9b17ffde57f7831ea555394290ba240a2b9; Anisul , 2021 ) ." ], [ "Our multi-instrument MR analysis used both genetic variants from across the genome ( pan-MR ) and genetic variants near or in the gene encoding the relevant protein ( cis-MR ) to investigate associations of genetically predicted plasma protein concentrations with the risk of COVID-19 outcomes .", "The COVID-19 outcome definitions are provided in Supplementary file 1 .", "Although the pan-MR analysis leveraged genetic data from both cis- and trans-acting pQTLs ( with a selection of pQTLs from across the genome automated by GSMR’s built-in HEIDI-outlier exclusion method ) , for some protein-COVID-19 pairs that were associated at 5% FDR , the associations with COVID-19 outcomes were exclusively driven by trans-acting pQTLs or cis-acting genetic instruments .", "For example , although six proteins were represented by both cis- and trans-acting genetic instruments , two ( ABO and IL6R ) were represented only by cis-acting variants and one ( SELE ) was driven entirely by trans-acting instruments ( mainly ABO trans-pQTLs ) ( Supplementary file 2 ) .", "Overall , the pan-MR analysis revealed nine distinct protein probes associated with four COVID outcomes at an FDR of 5% ( Figure 1A ) .", "The pQTLs selected by GSMR to represent these nine probes were also cis-eQTLs ( as curated for the Open Targets Genetics portal Open Targets Genetics , 2019 ) and , except the ABO signal via rs8176719-insertionC ( will be referred to as rs8176719-insC – a frameshift mutation that inserts a guanine nucleotide in the 258th position of exon 6 ) , were not missense variants or in LD with missense variants ( Supplementary file 3 ) , minimising the possibility that SNPs with artefactual associations with proteins were used as genetic instruments for the majority of significant pan-MR association .", "While the pan-MR analysis used genetic data from across the genome , the cis-MR analysis restricted genetic instrument selection to those near ( within 1 Mb of TSS ) or in the gene encoding the protein .", "Three proteins with pan-MR associations were supported by corresponding cis-MR associations ( Figure 1A and B , Supplementary file 4 ) : ABO , ICAM-1 , and IL-6R .", "Among these three , only ABO and IL-6R proteins had some evidence of genetic colocalisation with posterior probabilities ( PP . H4 ) more than 0 . 9 and 0 . 4 , respectively , of a shared genetic signal between protein and COVID-19 phenotype ( Figure 2 ) .", "Although the PP . H4 of IL-6R was very weak ( 0 . 4 ) , it had a positive ( H4/H3 = 3 . 6 ) , indicating a common signal of the IL-6R protein with the COVID-19 outcome is a more likely scenario than the association driven by two independent signals .", "Genetically predicted ABO concentration was associated with risk in four out of seven COVID-19 outcomes ( Figure 2 ) .", "These four outcomes represented both susceptibility ( e . g . COVID-19 vs . population , cis-MR odds ratio [OR] [95% CI] per SD genetically predicted ABO concentrations: 1 . 08 [1 . 05 , 1 . 10] , p=7 × 10–10 ) and severity ( e . g . hospitalised COVID-19 vs . population , cis-MR OR [95% CI]: 1 . 12 [1 . 08 , 1 . 17] , p=1 × 10–8 ) of COVID-19 .", "Genetically predicted soluble IL-6R was only associated with higher risk of hospitalised COVID-19 compared to population-based controls ( cis-MR OR [95% CI] per SD genetically predicted IL-6R: 0 . 94 [0 . 91 , 0 . 97] , p=8 × 10–4 ) ( Figure 2 ) .", "When examining the SNPs involved in the pan-MR associations of the nine probes , all probes except IL-6R and ABO had at least one trans-acting SNP , and in all these cases , at least one of the trans-acting SNPs were assigned to the ABO gene by the Open Targets Genetics V2G pipeline ( Table 1 ) , re-confirming the pervasive pleiotropy of the ABO genetic signal .", "Furthermore , when examining the consistency of pan-MR associations of these nine probes across all seven COVID-19 outcomes , the protein probes that have trans-acting ABO SNPs exhibited a similar association profile as the ABO protein probe , associated with only COVID-19 outcomes that have population-based controls ( Supplementary file 5 ) .", "The ABO signal ( rs8176719-insC ) contributes to the determination of non-O blood groups and regulates circulating levels of both ABO and several non-ABO proteins; Yamagata University Genomic Cohort Consortium ( YUGCC ) , 2014; Arguinano et al . , 2018 .", "We explored proteome-wide associations of rs8176719-insC in three separate proteomic datasets; Emilsson , 2020 .", "Aside from the ABO protein , the ABO signal rs8176719-insC showed the strongest association ( Sun et al: p=6 . 03 × 10–258 , Emilsson et al: p=1 . 00 × 10–307 , Suhre et al: p=1 . 27 × 10–75 ) with higher plasma concentrations of soluble CD209 in all three datasets ( associations from two datasets illustrated in Figure 3 , and associations from all three datasets tabulated in Supplementary file 6 ) .", "To validate this as a relevant target for COVID-19 , we experimentally tested whether CD209 directly interacts with SARS-CoV-2 , as had been recently proposed based on similarities to SARS-CoV-1 , which was reported to bind CD209 ( Yang , 2020 ) .", "We used human cells to generate recombinant SARS-CoV-2 spike protein , spanning the full-length extracellular domain according to a design previously established to retain functionality ( Shilts et al . , 2021 ) .", "We found that purified spike protein indeed could directly attach onto human cells expressing CD209 but not control cells , suggesting that CD209 could act as a receptor for viral attachment onto host cells ( Figure 4A ) .", "Furthermore in a direct binding assay testing purified soluble CD209 and the viral spike protein , we could detect binding that was specific and comparable to the primary known receptor for SARS-CoV-2 , ACE2 ( Figure 4B ) .", "To identify additional proteins associated with the risk of COVID-19 , we conducted proteome-wide genetic colocalisation tests followed by single-SNP MR analysis ( Supplementary file 7 ) .", "This ‘coloc-first’ approach identified four proteins ( ABO , FAS , OAS1 , THBS3 ) with evidence of genetic colocalisation ( PP . H4 >0 . 8 ) with four out of seven COVID-19 phenotypes ( Figure 5 ) .", "Two of these ( FAS and THBS3 ) are , to the best of our knowledge , not reported in proteomic MR studies of COVID-19 to date which have only examined for colocalisation evidence after MR . Consistent with pan- and cis-MR findings , there was evidence of genetic colocalisation between the ABO protein and six out of seven COVID-19 phenotypes ( Figure 6 ) , with similar MR estimates when the colocalising SNP was used to perform single-SNP cis-MR .", "The coloc-first approach revealed a common genetic signal between OAS1 and COVID-19 in two out of seven COVID-19 phenotypes ( PP . H4 = 0 . 88 in COVID-19 vs . population , and 0 . 82 in hospitalised COVID-19 vs . population ) .", "However , the SNPs representing the common genetic signal between OAS1 and COVID-19 phenotypes ( 12:112919637:G:A and 12:112919388:G:A ) were missense variants in OAS1 gene or in LD with missense variants at r2 >0 . 8 , rendering their effect estimates potentially biased due to aptamer binding effects ( see Materials and methods ) .", "Despite this , the same variants have effects on gene expression ( as assessed in any of several tissues curated by Open Targets Genetics Open Targets Genetics , 2019 ) , which is independent of aptamer binding , suggesting that causal inference regarding OAS1 protein and COVID-19 risk may still be valid .", "We have , therefore , presented OAS1 estimates in Figure 5 but flagged with an asterisk denoting the effect estimates as potentially biased .", "Unlike ABO , OAS1 could not be tested using the multi-instrument MR approach due to insufficient number of valid instruments , highlighting the complementary value of the genetic colocalisation approach alongside multi-SNP MR methods .", "In single-SNP MR analyses , genetically predicted higher OAS1 was associated with lower risk of severe COVID-19 vs . population ( OR [95% CI] per SD genetically predicted OAS1 concentrations: 0 . 52 [0 . 42 , 0 . 65] , p=5 × 10–9 ) , hospitalised COVID-19 vs . population ( 0 . 63 [0 . 53 , 0 . 75] , p=1 × 10–7 ) and susceptibility to COVID-19 vs . population ( 0 . 79 [0 . 71 , 0 . 87] , p=2 × 10–6 ) .", "Proteins that exhibited association only in one of the COVID-19 phenotypes included circulating FAS and THBS3 .", "Genetically predicted elevated FAS ( indicated by two FAS probes: FAS . 9459 . 7 . 3 and FAS . 5392 . 73 . 2 ) and THBS3 were associated with a higher risk of severe COVID-19 ( OR [95% CI] per SD genetically predicted FAS concentrations indicated by FAS . 9459 . 7 . 3: 1 . 38 [1 . 17 , 1 . 62] , p=1 × 10–4 ) and COVID-19 vs . lab/self-reported negative COVID-19 ( OR [95% CI] per SD genetically predicted THBS3 concentrations: 1 . 70 [1 . 30 , 2 . 22] , p=9 × 10–5 ) , respectively .", "For the proteins with evidence of genetic colocalisation between protein and COVID-19 phenotype , we used their lead variants ( or variants they tag at r2 >0 . 6 if a lead variant was not reported in a GWAS ) to identify additional associated phenotypes .", "At p<1 × 10–5 ( Bonferroni corrected for the ~3000 phenotypes in the Open Targets Genetics portal ) , most of the variants exhibited associations with haematological indices , with some , like the ABO signal , also associated with other COVID-19-relevant phenotypes ( Supplementary file 8 ) .", "For example , the ABO signal was associated with monocyte count , deep vein thrombosis ( DVT ) , and pulmonary embolism ( PE ) .", "OAS1 and THBS3 variants were associated with platelet counts .", "For FAS , there were no additional phenotypic associations at p<1 × 10–5 shown by its colocalising variant ." ], [ "Our systematic proteome-wide MR and genetic colocalisation analysis supported several previously proposed proteins and suggested additional clinically actionable targets for COVID-19 ( Table 1 ) .", "Of particular note , we provided pan- and cis-MR evidence with strong genetic colocalisation support for the ABO signal for most COVID-19 phenotypes .", "Although the ABO protein itself is not clinically actionable , the ABO signal was linked to plasma concentrations of several clinically tractable targets .", "We demonstrated that the CD209 protein we had found to have the strongest association with this ABO signal has a direct interaction with the SARS-CoV-2 spike protein , providing further evidence for a plausible mechanism .", "Our analyses also supported the role of soluble IL-6R in hospitalised COVID-19 , with evidence from pan- and cis-MR analyses but limited evidence of genetic colocalisation with hospitalised COVID-19 but supported by the recent COVID-19 clinical trials of tocilizumab ( which is partially mimicked by the IL-6R instrument used in the present study ) .", "Using a proteome-wide ‘colocalisation-first’ approach , we recapitulated previously reported targets ( e . g . OAS1 ) and uncovered additional novel proteins that may play causal roles in COVID-19 susceptibility ( THBS3 ) , or severity ( FAS ) .", "Our proteome-wide genetic colocalisation analysis prioritised soluble Fas ( sFas , also known as soluble CD95 ) receptor protein in the very severe COVID-19 phenotype .", "This finding was not reported in previous proteomic MR studies of COVID-19 most likely because they only assessed evidence of shared signals for targets prioritised by an MR-first approach .", "The soluble Fas receptor is reported to act as a decoy receptor competing with the trans-membrane Fas receptor for Fas ligand ( FasL ) ( Cheng , 1994 ) .", "Genetically predicted higher circulating sFas is , therefore , likely to represent effects of lower Fas-FasL signalling and , in our study , was associated with a higher risk of very severe COVID-19 .", "Fas-FasL signalling typically initiates a cascade of intracellular programmes that result in cell death or apoptosis .", "Fas-mediated apoptosis plays a central role in T- and B-cell homeostasis ( Hao , 2008 ) , preventing the emergence of autoreactive or overactive immune cells ( Hao , 2008; Butt , 2015 ) .", "Excessive inflammation by hyperactive T-cells and autoantibodies was reported to underlie several cases of severe COVID-19 ( Khamsi , 2021 ) .", "To what extent sFas contributes to the excessive pro-inflammatory response in severe COVID-19 remains to be determined .", "Furthermore , Fas-mediated apoptosis of virus-infected cells is a major mechanism of resolution of viral infections ( Thomson , 2001 ) .", "Delayed apoptosis is reported to be one of the strategies exploited by SARS-CoV-2 in the early stages of infection to facilitate viral replication ( Thomson , 2001; Ivanisenko et al . , 2020 ) .", "Additional insights for the role of sFas in COVID-19 can be gleaned from the results of recent drug trials .", "For example , Fas is one of the major targets of lopinavir-ritonavir – a combination HIV protease inhibitor ( Sorbera et al . , 2020 ) ; clinical trials of lopinavir-ritonavir failed to provide any therapeutic benefits beyond standard care in hospitalised COVID-19 patients ( Cao et al . , 2020 ) .", "On the other hand , clinical trials testing dexamethasone demonstrated beneficial effects on survival for COVID-19 patients who were on respiratory support ( RECOVERY Collaborative Group et al . , 2021 ) .", "In addition to its anti-inflammatory effects , dexamethasone downregulates molecules associated with decelerating apoptosis ( Achuthan , 2018 ) , including sFas ( Joashi et al . , 2002 ) .", "An in-depth assessment of the specific role of soluble Fas in COVID-19 , including whether or not it contributes to the beneficial effects of dexamethasone , is warranted in future studies .", "Observational studies were the first to report differences in risk of severe COVID-19 based on ABO blood groups , although with some conflicting reports ( Zhao , 2020; Zietz et al . , 2020; Bhattacharjee et al . , 2020 ) .", "GWAS of COVID-19 susceptibility have , however , consistently reported a signal in the ABO locus ( Bhattacharjee et al . , 2020; Shelton , 2020 ) , despite prior observations that controls used in the first published GWAS of COVID-19 ( Ellinghaus et al . , 2020 ) may be over-represented for blood group O ( the most common blood group ) and can result in associations due to selection bias .", "However , in a meta-analysis of GWAS of COVID-19 , the ABO signal remained even when Ellinghaus et al . was excluded .", "Furthermore , using these GWAS data , we and Katz et al . ( in a preprint ) ( Katz et al . , 2020; Karim et al . , 2020 ) had previously linked the ABO signal with CD209/DC-SIGN protein , clotting factors , coagulation disorders , and concentrations of IL-6 , all potential risk factors for COVID-19 .", "In the present study , we build on previous work and show consistent cis- and pan-MR associations of genetically predicted circulating ABO protein with an expanded list of COVID phenotypes which colocalise with the ABO signal , supporting a shared genetic signal of ABO protein and the COVID-19 phenotypes .", "We show that , next to the ABO protein , the ABO signal had the strongest association with the CD209 protein relative to other proteins and present experimental evidence of binding of CD209 with the full-length spike protein of SARS-CoV-2 , independently but consistent with a concurrent preprint ( Amraie , 2020 ) .", "CD209 is a receptor on monocyte-derived dendritic cells ( moDCs ) that was shown , before this binding interaction was known , to facilitate entry of replication-competent SARS-CoV-2 and demonstrated to switch off the type I interferon signalling pathways necessary for transcription of several antiviral genes ( Yang , 2020 ) .", "The soluble isoforms of CD209 measured in our proteome datasets are known to correlate in expression levels to the membrane isoforms ( Mummidi et al . , 2001; Plazolles , 2011 ) , making it plausible that the signal we observed is associated with greater abundance of CD209 as a cell-surface viral receptor .", "Alternatively , in the context of other viruses known to directly bind CD209 as we show here for SARS-CoV-2 , soluble CD209 has been demonstrated to modulate infection , such as by promoting endocytosis if the soluble CD209 coating the virus acts as opsonins ( Plazolles , 2011 ) .", "Further research would be beneficial to reveal which of these mechanisms explain the association we observed .", "These findings may also help interpret the clinical significance of the higher CD209 gene expression in immune cells ( extracted from bronchoalveolar lavage fluid ) in severe COVID patients than healthy controls ( Gao , 2020 ) .", "It should be noted that the present study developed a therapeutic hypothesis for CD209 based solely on the strong evidence of association of the ABO signal with plasma concentrations of CD209 and evidence from the pan-MR association of CD209 with COVID-19 phenotypes ( the pan-MR associations being driven mainly by trans-acting ABO SNPs ) with no corresponding support of cis-MR or colocalisation .", "This suggests that while cis-MR and colocalisation analyses can support pan-MR associations of a target with disease , the lack of cis-MR or colocalisation for a target is not necessarily evidence against its therapeutic relevance .", "In the present study , we also found that genetically predicted higher OAS1 – an interferon-induced broad-spectrum antiviral enzyme – was associated with lower risk of both susceptibility and severity of COVID-19 , consistent with findings of a recent published report ( Zhou et al . , 2021 ) .", "A large clinical trial of systemically administered interferons failed to show any substantial therapeutic benefits for severe COVID-19 ( WHO Solidarity Trial Consortium et al . , 2021 ) .", "However , the strong evidence from human genetics supports reconsidering the role of interferon-based therapies in a new light , especially with respect to timing of administration ( which current genetic studies are unable to provide any insights on ) and route ( systemic vs . nebulised ) ( Monk , 2020 ) .", "Non-O blood group individuals generally have higher risk of DVT and other coagulation disorders than O blood group individuals ( Groot , 2020 ) .", "The ABO signal , which largely determines the non-O blood groups , was also associated with DVT , PE , and higher levels of vWF and F8; vWF binds to and protects F8 from biological degradation ( Federici , 2003 ) .", "F8 is a key protein in the intrinsic coagulation pathway that activates Factor X and induces the formation of fibrin – the central component of blood clots ( Bhopale and Nanda , 2003 ) .", "Both DVT and PE are reported to affect almost a third of ICU-admitted COVID-19 patients ( Malas , 2020 ) .", "While several clinical trials evaluating the efficacy of anticoagulants for severe COVID-19 are underway , the National Institute of Clinical Excellence in the UK has suggested screening all hospitalised COVID-19 patients for any contraindications to anticoagulant use and offering prophylactic anticoagulation to eligible patients ( National Institute for Health and Care Excellence , 2020 ) .", "We found moderate evidence for the role of IL-6 signalling in COVID-19 in agreement with a previous report ( Bovijn et al . , 2021 ) .", "However , there was ambiguous evidence of genetic colocalisation ( PP . H4: 0 . 46 ) .", "Nevertheless , there was more support for a shared genetic signal between sIL-6R and hospitalised COVID-19 than for them to be driven by independent signals ( H4/ H3 = 3 . 6 ) .", "As noted by Bovign and colleagues ( Bovijn et al . , 2021 ) , with some caveats , the phenotypic consistency of associations between the IL-6R genetic instrument and pharmacological effect of tocilizumab enable potential use of the IL-6R instrument to investigate therapeutic or adverse effects of tocilizumab .", "Although a previous report showed largely neutral effects of tocilizumab compared to placebo in hospitalised COVID-19 patients ( Stone et al . , 2020 ) , two recent trials ( REMAP-CAP Anthony C and Paul R , 2021 and RECOVERY RECOVERY Collaborative Group , 2021 ) with a longer follow-up period showed beneficial effects on survival at 90 days , consistent with the prediction of a protective effect using the tocilizumab-mimicking IL-6R genetic instrument in the present study and the previous report .", "The major strengths of our study include the use of both genome-wide and local genetic instruments for MR analysis , the proteome-wide genetic colocalisation tests to nominate additional proteins of therapeutic relevance , and the expanded list of COVID-19 phenotypes analysed .", "We showed consistency of the association of ABO with the different COVID-19 phenotypes for both instrument selection strategies .", "Proteome-wide colocalisation tests implicated additional proteins that likely lacked sufficient genetic instruments to be detected by the multi-instrument GSMR method .", "For our top-ranked association with the CD209 protein , we provide experimental evidence for a mechanism that implicates CD209 as having a potentially causal role in disease pathology .", "Our experiments provide both direct evidence of biochemical binding between the purified spike protein of SARS-CoV-2 and CD209 , and verification that this interaction occurs in live human cells .", "Host-directed therapies involving pathogen binding receptors have previously been developed against other infectious diseases where pathogen mutations or variants stymied more traditional approaches ( Zenonos et al . , 2015 ) .", "Our study also has several limitations .", "The reliability of the MR approach depends on the selection of the appropriate genetic instruments for the exposure ( Schmidt , 2020 ) .", "Where proteins are the exposure , the use of genetic instruments from across the genome can result in more instruments and potentially higher power to detect associations .", "However , the inclusion of a broader set of genetic instruments for protein-MR analysis can lead to associations not mediated by the protein under investigation ( i . e . horizontal pleiotropy ) .", "In these cases , the use of genetic variants near or in the locus encoding the protein ( cis-acting SNPs ) can provide more specific estimates of risk , albeit at a potential power cost , associated with genetically predicted concentrations of the protein under investigation ( Schmidt , 2020 ) .", "A key problem of the latter approach is the selection of correlated genetic instruments that can lead to numerical approximation errors ( Gkatzionis et al . , 2021 ) .", "In the present study , we leveraged both pan- and cis-MR approaches and used an MR method ( GSMR ) that automates the selection of near-independent genetic instruments and performs MR adjusting for any residual correlation ( Zhu et al . , 2018 ) .", "Nevertheless , horizontal pleiotropy can also affect cis-MR analyses when different variants from the same gene region represent different biological pathways , indicated by heterogeneous effect estimates , or driven by a single variant with a large effect ( e . g . missense variants ) ( Gkatzionis et al . , 2021 ) .", "To prevent the selection of heterogeneous instruments and minimise the selection of variants with large effects , the multi-instrument GSMR method used in the present study implements the HEIDI test which excludes genetic variants with strong or heterogeneous effects .", "The exclusion of missense variants with potential aptamer binding effects is evidenced in our study , where SNPs in 96% of nominally significant protein probes associated with COVID-19 also had effects on corresponding gene expression in different tissues across gene expression datasets as curated by our portal ( Open Targets Genetics , 2019 ) .", "Even while using cis-acting genetic instruments , the MR associations can be confounded due to LD between cis-pQTLs and disease-associated SNPs , and this is at least partially mitigated by genetic colocalisation tests .", "However , the genetic colocalisation tests used in our study assumed a single causal variant in each locus and will , therefore , result in higher false-negative tests if there is more than one trait-associated causal variant .", "An additional issue is related to the selection of COVID-19 GWAS datasets used for analyses .", "Most protein-MR studies have used COVID-19 phenotypes with population-based controls , given their larger number of controls providing additional power to detect signals but at a cost of not being able to distinguish signals relevant to disease progression .", "While study designs with milder/asymptomatic cases as controls are useful to study disease progression , they are frequently underpowered and , because the selection of study participants are conditioned on the outcome , are susceptible to collider-stratification bias ( Griffith , 2020 ) .", "To enable a comprehensive assessment , we used all published COVID-19 phenotypes ( October 2020 freeze ) , irrespective of controls used and , as expected , found most signals in COVID-19 phenotypes with population-based controls .", "For one of the targets ( CD209 ) , although we experimentally demonstrate binding of CD209 with spike protein of SARS-CoV-2 , understanding the functional significance CD209 has on viral entry and any immunological relevance during infection requires further research .", "Finally , although we nominate several targets that may be therapeutically relevant for COVID-19 , clinical trials are required for definitive assessments and to guide therapy .", "For example , the findings related to the ABO signal strongly implicated the adverse role of dysregulated coagulation in COVID-19 specifically in non-O blood group individuals; whether pre-emptive use of anticoagulants guided by blood groups can prevent severe COVID-19 is subject to findings of trials such as the ongoing ACTIV-4 trial ( NCT04505774 ) ( U . S . National Library of Medicine , 2020 ) .", "In conclusion , we integrated genetic investigation with functional assessments of CD209 , a receptor in moDCs , and postulated that this target may convey the COVID-19 risk of the ABO signal .", "Based on proteome-wide genetic colocalisation and MR , we also prioritised sFas for more detailed investigations of its therapeutic relevance to severe COVID-19 risk ." ] ]

[ "The virus SARS-CoV-2 can exploit biological vulnerabilities ( e . g . host proteins ) in susceptible hosts that predispose to the development of severe COVID-19 .", "To identify host proteins that may contribute to the risk of severe COVID-19 , we undertook proteome-wide genetic colocalisation tests , and polygenic ( pan ) and cis-Mendelian randomisation analyses leveraging publicly available protein and COVID-19 datasets .", "Our analytic approach identified several known targets ( e . g . ABO , OAS1 ) , but also nominated new proteins such as soluble Fas ( colocalisation probability >0 . 9 , p=1 × 10-4 ) , implicating Fas-mediated apoptosis as a potential target for COVID-19 risk .", "The polygenic ( pan ) and cis-Mendelian randomisation analyses showed consistent associations of genetically predicted ABO protein with several COVID-19 phenotypes .", "The ABO signal is highly pleiotropic , and a look-up of proteins associated with the ABO signal revealed that the strongest association was with soluble CD209 .", "We demonstrated experimentally that CD209 directly interacts with the spike protein of SARS-CoV-2 , suggesting a mechanism that could explain the ABO association with COVID-19 .", "Our work provides a prioritised list of host targets potentially exploited by SARS-CoV-2 and is a precursor for further research on CD209 and FAS as therapeutically tractable targets for COVID-19 .", "MAK , JSc , JH , AB , DO , MC , EMM , MG , ID were funded by Open Targets .", "J . Z . and T . R . G were funded by the UK Medical Research Council Integrative Epidemiology Unit ( MC_UU_00011/4 ) .", "JSh and GJW were funded by the Wellcome Trust Grant 206194 .", "This research was funded in part by the Wellcome Trust [Grant 206194] .", "For the purpose of open access , the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission ." ]

[ "Individuals who become infected with the virus that causes COVID-19 can experience a wide variety of symptoms .", "These can range from no symptoms or minor symptoms to severe illness and death .", "Key demographic factors , such as age , gender and race , are known to affect how susceptible an individual is to infection .", "However , molecular factors , such as unique gene mutations and gene expression levels can also have a major impact on patient responses by affecting the levels of proteins in the body .", "Proteins that are too abundant or too scarce may mean the difference between dying from or surviving COVID-19 .", "Identifying the molecular factors in a host that affect how viruses can infect individuals , evade immune defences or trigger severe illness , could provide new ways to treat patients with COVID-19 .", "Such factors are likely to remain constant , even when the virus mutates into new strains .", "Hence , insights would likely apply across all virus strains , including current strains , such as alpha and delta , and any new strains that may emerge in the future .", "Using such a ‘natural experiment’ approach , Karim et al . compared the genetic profiles of over 30 , 000 COVID-19 patients and a million healthy individuals .", "Nine proteins were found to have an impact on COVID-19 infection and disease severity .", "Four proteins were ranked as top priorities for potential treatment targets .", "One protein , called CD209 ( also known as DC-SIGN ) , is involved in how the virus enters the host cells , and had one of the strongest associations with COVID-19 .", "Two proteins , called IL-6R and FAS , were involved in the immune response and could be responsible for the immune over-activation often seen in severe COVID-19 .", "Finally , one protein , called OAS1 , formed part of the body’s innate antiviral defence system and appeared to reduce susceptibility to COVID-19 .", "Knowing more about the proteins that influence the severity of COVID-19 opens up new ways to predict , protect and treat patients who may have severe or fatal reactions to infection .", "Indeed , one of the identified proteins ( IL-6R ) had already been targeted in recent clinical trials with some encouraging results .", "Considering CD209 as a potential receptor for the virus could provide another avenue for therapeutics , similar to previously successful approaches to block the virus’ known interaction with a receptor protein .", "Ultimately , this research could supply an entirely new set of treatment options to help combat the COVID-19 pandemic ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "The Niemann-Pick type C ( NPC ) proteins NPC1 and NPC2 bind with cholesterol derived from endocytosed lipoproteins and transport it to the lysosomal membrane ( Kwon et al . , 2009; Wang et al . , 2010 ) .", "Genetic defects in NPC1 and NPC2 cause a neurodegenerative disorder characterized by the accumulation of cholesterol and other lipids in lysosomes , demonstrating the physiological importance of these proteins ( Rosenbaum and Maxfield , 2011 ) .", "The molecular mechanism by which NPC proteins transport cholesterol has been studied extensively: NPC2 , a soluble protein , binds cholesterol and transfers it to NPC1 , a multipass transmembrane protein , which then inserts cholesterol into the lysosomal membrane ( Kwon et al . , 2009; Wang et al . , 2010 ) .", "In parallel with this ‘hand-off’ mechanism , NPC2 is also thought to transport cholesterol directly to the membrane , bypassing NPC1 ( Kennedy et al . , 2012; Xu et al . , 2008 ) .", "Cholesterol that is inserted into the lysosomal membrane is then delivered to other organelles , including the endoplasmic reticulum and the plasma membrane , by mechanisms that are yet to be defined in detail ( Ikonen , 2008 ) .", "At one point during this transport process , cholesterol must be incorporated into the lysosomal membrane .", "Considering the low cholesterol content of the lysosomal membrane under ordinary circumstances ( Kolter and Sandhoff , 2005 ) and the known effect of cholesterol on various membrane properties ( Bloom et al . , 1991; Lingwood and Simons , 2010 ) , the insertion of cholesterol by NPC proteins is likely to influence the lysosomal membrane significantly , but to the best of our knowledge , relatively little attention has been paid to this aspect .", "In the present study , we aimed to investigate this matter through experiments with budding yeast Saccharomyces cerevisiae .", "The use of yeast is legitimate because the functional orthologs of NPC proteins in budding yeast , namely , Ncr1p and Npc2p , can substitute for NPC1 and NPC2 , respectively , in mammalian cells ( Berger et al . , 2005; Malathi et al . , 2004 ) .", "We examined yeast in two different conditions: one is the stationary phase , in which the cell cycle is arrested because of the gradual exhaustion of nutrients , and in which the formation of raft-like domains and lipophagy occur in the vacuole ( Toulmay and Prinz , 2013; Wang et al . , 2014 ) ; the other is acute nitrogen starvation , which causes macroautophagy as well as microautophagy of lipid droplets ( LDs ) and the nucleus ( Roberts et al . , 2003; van Zutphen et al . , 2014 ) .", "We discovered that NPC proteins , especially Npc2p , play a critical role in the formation and expansion of the raft-like domain in the vacuole , and that the domain is engaged in the binding and engulfment of LDs , both in stationary phase and in acute nitrogen starvation .", "This result indicates that a major function of NPC proteins is to expand the sterol-rich raft-like membrane domain in the vacuole to execute microautophagy ." ], [ "In stationary phase yeast , raft-like domain formation and lipophagy have been hypothesized to proceed in a feed-forward manner ( Wang et al . , 2014 ) , but the exact mechanism has not been clear .", "To determine how the raft-like domain is involved in lipophagy , we observed the entire process by means of freeze-fracture electron microscopy ( EM ) .", "The vacuolar membrane in stationary phase showed geometrical patterns ( Moeller and Thomson , 1979 ) ( Figure 1A ) .", "We confirmed that domains that are deficient in the intramembrane particles ( IMPs ) , which represent transmembrane proteins , correspond to the raft-like domain by labeling the non-raft domain marker Vph1p–mRFP in freeze-fracture replicas ( Toulmay and Prinz , 2013 ) ( Figure 1B and Figure 1—figure supplement 1A ) . 10 . 7554/eLife . 25960 . 003Figure 1 . Lipophagy in stationary phase occurs through the expansion of raft-like vacuolar membrane domains .", "( A ) Freeze-fracture EM of the vacuole .", "The vacuolar membrane in stationary phase yeast shows geometrical patterns in both the protoplasmic face ( P face; cytoplasmic leaflet ) and the exoplasmic face ( E face: non-cytoplasmic leaflet ) .", "Intramembrane particles ( IMPs ) , which represent transmembrane proteins , are largely confined to the edges of polygonal areas .", "Bars: 0 . 5 µm ( low magnification ) ; 0 . 2 µm ( high magnification ) .", "( B ) Freeze-fracture labeling of Vph1p–mRFP in the stationary phase vacuole .", "Labels for Vph1p–mRFP ( arrows ) were excluded from the polygonal IMP-deficient area , but were present in the IMP-rich region , indicating that the IMP-deficient area corresponds to the raft-like domain .", "The IMP-rich area , or the nonraft-like domain , is marked by double arrowheads .", "Bar: 0 . 2 µm .", "( C–F )", "Freeze-fracture/etching EM of stationary phase vacuole .", "Bars: 0 . 2 µm .", "The black-and-white contrast is reversed to enhance the three-dimensional features .", "( C ) LDs adhered tightly to IMP-deficient raft-like domain , which bulges toward the lumen .", "The vacuolar membrane is marked by double arrowheads .", "See Figure 1—figure supplement 2 for a picture of the whole cell and Video 1 for a three-dimensional view of the LD-vacuole apposition .", "( D ) Only a limited number of raft-like domains showed bulging .", "Irrespective of the variable degree of bulging in raft-like domains , nonraft-like domains were confined to the edge .", "See Figure 1—figure supplement 3 for additional photos .", "( E ) The raft-like domain formed a balloon-like structure that protrudes into the vacuolar lumen .", "IMPs were observed as a ring-like structure in the constricted portion ( arrows ) .", "The vacuolar membrane is marked by double arrowheads .", "( F ) Vesicles in the vacuolar lumen harbored IMP clusters ( arrow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 00310 . 7554/eLife . 25960 . 004Figure 1—figure supplement 1 . The procedure for quick-freezing and freeze-fracture EM .", "( A ) Quick-freezing and freeze-fracture replica-labeling EM .", "( a ) Quick freezing of live cells without chemical fixation;", "( b ) preparation of freeze-fracture replicas – physical fixation of a phospholipid monolayer with vacuum evaporation of carbon and platinum;", "( c ) SDS treatment to remove extra-membrane materials; and", "( d ) labeling of freeze-fracture replicas with specific probes .", "PtdIns ( 3 ) P and Vph1p–mRFP were labeled by this method in the present study .", "( B ) Freeze-fracture and etching EM .", "After freeze-fracture , quick-frozen samples are kept at –102°C for 2 min before vacuum evaporation .", "During this ‘etching’ procedure , water in the cytosol and the organellar lumen sublimes , while the lipid esters in the LD core do not , thus making LDs stand out among etched structures .", "The photographs show the difference in appearance of LDs in simple freeze-fracture and freeze-fracture/etching EM .", "Bars: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 00410 . 7554/eLife . 25960 . 005Figure 1—figure supplement 2 . Freeze-fracture/etching EM of an entire yeast cell in stationary phase . The two rectangular areas are magnified in Figure 1C .", "Bar , 1 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 00510 . 7554/eLife . 25960 . 006Figure 1—figure supplement 3 . Additional examples of raft-like domain bulging in the vacuole . Adjacent raft-like domains show variable degrees of bulging .", "Bars: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 006 To examine how the vacuolar domains are related to lipophagy , we performed freeze-etching after freeze-fracture to make LDs clearly identifiable in EM ( Figure 1—figure supplement 1B ) .", "This technique revealed that the invaginating IMP-deficient raft-like domain , which is tightly bound to LDs ( Figure 1C , Figure 1—figure supplement 2 , and Video 1 ) , expanded and bulged toward the lumen and formed balloon-like structures , whereas the IMP-rich , nonraft-like domain was confined to the edges of bulges and made a ring-like structure at the neck of the raft-like domain balloon ( Figure 1D , E , and Figure 1—figure supplement 3 ) .", "IMP clusters were also observed in vesicles in the vacuolar lumen ( Figure 1F ) , indicating that the balloon-like structures were pinched off at the nonraft-like domain to complete the microautophagic process ( hereafter the intravacuolar vesicles formed by microautophagy will be called microautophagic vesicles ) . 10 . 7554/eLife . 25960 . 007Video 1 . Tilted images of a freeze-fracture/etching replica of stationary phase yeast . An LD revealing the non-etchable content is adhered to the IMP-deficient domain of the vacuolar membrane . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 007 The distribution of phosphatidylinositol 3-phosphate [PtdIns ( 3 ) P] , as revealed by freeze-fracture replica labeling , was consistent with the microautophagic mechanism .", "That is , PtdIns ( 3 ) P was confined to the cytoplasmic leaflet both in the vacuolar membrane ( Figure 2A and Figure 2—figure supplement 1A ) and in the microautophagic vesicles ( Figure 2B , C , and Figure 2—figure supplement 1B ) .", "This PtdIns ( 3 ) P asymmetry was opposite to that found in autophagic bodies in macroautophagy ( hereafter called macroautophagic bodies ) , in which PtdIns ( 3 ) P is far more abundant in the non-cytoplasmic leaflet than in the cytoplasmic leaflet ( Cheng et al . , 2014 ) ( Figure 2—figure supplement 1C ) , and was utilized to distinguish microautophagic vesicles from other structures in subsequent experiments .", "These results demonstrated that stationary phase lipophagy is a microautophagic process made possible by expansion of the raft-like domain ( Figure 2D ) . 10 . 7554/eLife . 25960 . 008Figure 2 . Microautophagic vesicles show the same PtdIns ( 3 ) P asymmetry as the vacuolar membrane .", "( A , B )", "Freeze-fracture replica labeling of PtdIns ( 3 ) P in stationary phase vacuoles .", "Colloidal gold particles indicate PtdIns ( 3 ) P labeled by recombinant GST-p40phox PX domain .", "Bars: 0 . 2 µm .", "See also Figure 2—figure supplements 1A and B for additional photos .", "( A ) The vacuolar membrane was labeled for PtdIns ( 3 ) P in the cytoplasmic leaflet .", "Note that the domain engulfing an LD is also labeled ( arrows ) .", "( B ) PtdIns ( 3 ) P was labeled densely in the convex protoplasmic face ( P; the cytoplasmic leaflet ) , but scarcely in the concave exoplasmic face ( E; the luminal leaflet ) , of microautophagic vesicles .", "The vacuolar membrane is marked by double arrowheads .", "( C ) PtdIns ( 3 ) P labeling density in the intravacuolar vesicles .", "The density was significantly higher in the P face than in the E face .", "The result of one representative experiment out of three independent experiments .", "**p<0 . 01 .", "( D ) Diagram showing how the vacuolar membrane forms the microautophagic vesicle in stationary phase . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 00810 . 7554/eLife . 25960 . 009Figure 2—figure supplement 1 . PtdIns ( 3 ) P in a stationary phase vacuole .", "( A ) Freeze-fracture replica labeling EM of PtdIns ( 3 ) P in a stationary phase vacuole .", "The labeling in the vacuolar membrane was dense in the cytoplasmic leaflet , whereas it was scarce in the non-cytoplasmic leaflet .", "Bar: 0 . 2 μm .", "( B ) PtdIns ( 3 ) P labeling in the intravacuolar vesicles .", "The dense labeling was observed only in the convex P face , representing the cytoplasmic leaflet .", "Note the presence of IMP clusters ( arrows ) .", "The vacuolar membrane is marked by double arrowheads .", "Bars: 0 . 2 μm .", "( C ) Schematic diagram showing the origin of contrasting PtdIns ( 3 ) P asymmetry in microautophagic vesicles and macroautophagic bodies .", "PtdIns ( 3 ) P in autophagosomes and autophagic bodies is much more abundant in the non-cytoplasmic leaflet than in the cytoplasmic leaflet ( Cheng et al . , 2014 ) , whereas PtdIns ( 3 ) P in microautophagic vesicles , which are derived from the vacuolar membrane , is confined to the cytoplasmic leaflet . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 009 Consistent with the formation of raft-like domains , sterol stained by filipin was observed along the vacuolar membrane in stationary phase , giving a double-ring staining pattern , whereas filipin staining in log-phase yeast was found only in the cell surface ( Figure 3—figure supplement 1A ) .", "Freeze-fracture EM confirmed the presence of filipin-sterol complexes in the vacuolar raft-like domain ( Figure 3—figure supplement 1B ) ( Elias et al . , 1979 ) , indicating that the internal filipin staining in stationary phase yeast is derived from the vacuolar membrane at least partially .", "Although filipin staining shows the relative distribution , but not the absolute amount , of sterols in membranes , the result suggested an increase of sterol in the vacuolar membrane in stationary phase .", "We hypothesized that this probable increase in sterol in the stationary phase vacuolar membrane may not occur through spontaneous incorporation but may involve NPC proteins; accordingly , we examined ncr1△ , npc2△ , and ncr1△npc2△ to test this idea .", "First , domain formation in the vacuolar membrane was compared by freeze-fracture EM ( Figure 3A ) .", "In wild-type cells ( WT ) , a majority of vacuoles showed domains with an inward curvature ( type II ) .", "In ncr1△ , npc2△ , and ncr1△npc2△ , the proportion of vacuoles with no domain was greater , whereas the proportion with the type II vacuole was significantly lower ( Figure 3A ) . 10 . 7554/eLife . 25960 . 010Figure 3 . Sterol transport by NPC proteins is essential for stationary phase lipophagy .", "( A ) Vacuolar domains were classified in three categories: no domain , domains without inward curvature ( type I ) , and domains with inward curvature ( type II ) .", "Bars: 0 . 2 µm .", "In comparison to WT , the type II vacuoles were significantly decreased in NPC-deficient cells .", "Mean ± SD of three independent experiments ( >50 vacuoles were counted for each group in each experiment ) .", "*p<0 . 05; **p<0 . 01 ( B ) Lipophagy in stationary phase .", "LDs were stained with BODIPY493/503 .", "Bar: 5 µm .", "The vacuoles were classified by the number of LDs in the lumen: that is , no LD , 1–2 LDs , and more than 3 LDs .", "There were significantly fewer vacuoles containing LDs in NPC-deficient cells than in WT .", "Mean ± SD of a single representative experiment ( n > 150 for each group ) out of three repeated experiments .", "**p<0 . 01 .", "( C ) Intravacuolar vesicles .", "Microautophagic vesicles ( defined by PtdIns[3]P labeling of more than 100 vesicles/µm2 in the cytoplasmic leaflet; colored in red ) were small in ncr1△ npc2△ ( see Figure 2B for comparison with WT ) .", "Many small vesicles with low levels of PtdIns ( 3 ) P labeling were also observed .", "See Figure 3—figure supplement 2 for ncr1△ and npc2△ .", "Bar: 0 . 2 µm .", "( D ) Sizes of microautophagic vesicles .", "Mean ± SD of three independent experiments .", "See Figure 3—figure supplement 2B for a box plot .", "**p<0 . 01 .", "( E ) Filipin staining .", "The proportion of cells showing the double-ring filipin-staining pattern was significantly lower in NPC-deficient cells than in WT [mean ± SD of three independent samples ( >150 vacuoles each were counted for each group ) ] .", "The vacuole of NPC-deficient cells showed prominent sterol deposition in the vacuolar lumen ( arrows ) .", "Bar: 5 µm .", "**p<0 . 01 .", "( F ) Yeast expressing Npc2p ( P143S ) showed all the defects observed in npc2△ .", "The domain formation , lipophagy , and the size of the microautophagic vesicles in Npc2p ( P143S ) were compared with those in WT as shown in Figure 3A , B and D , respectively .", "*p<0 . 05; **p<0 . 01DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01010 . 7554/eLife . 25960 . 011Figure 3—figure supplement 1 . Filipin staining .", "( A ) Wild-type yeast in log phase was labeled only in the cell surface , but in stationary phase , it showed intense labeling in the vacuolar membrane as well as in the cell surface ( arrows ) , giving rise to a double-ring staining pattern .", "Bar: 5 μm .", "( B ) The vacuolar raft-like domain in the stationary phase WT yeast showed membrane deformation caused by filipin–sterol complexes ( arrows ) .", "Bar: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01110 . 7554/eLife . 25960 . 012Figure 3—figure supplement 2 . The number and sizes of LDs in stationary phase . The number and sizes of LDs were measured by fluorescence microscopy after BODIPY493/503 staining and freeze-fracture EM , respectively .", "Neither of these measurements differed drastically between WT and NPC-deficient cells .", "Mean ± SD .", "Five independent samples and more than 30 cells were counted to assess numbers , whereas three independent samples and more than 30 LDs were counted to assess size .", "*p<0 . 05; **p<0 . 01 .", "DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01210 . 7554/eLife . 25960 . 013Figure 3—figure supplement 3 . Microautophagic vesicles in the lumen of a stationary phase vacuole .", "( A ) Microautophagic vesicles were identified by intense PtdIns ( 3 ) P labeling ( more than 100 gold labels/μm2 ) in the cytoplasmic leaflet ( colored in red ) .", "These were significantly smaller in ncr1Δ and npc2Δ than in WT .", "The vacuolar membrane is marked by double arrowheads .", "Bars: 0 . 2 μm .", "( B ) Box plot of the diameter of microautophagic vesicles .", "Microautophagic vesicles were significantly smaller in NPC-deficient and Npc2 ( P143S ) cells than in WT .", "The result of one representative experiment out of three independent experiments .", "**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 013 Second , lipophagy in stationary phase was examined by fluorescence microscopy as described previously ( Wang et al . , 2014 ) .", "The proportion of cells showing LDs in the vacuole was significantly smaller in NPC-deficient cells than in WT ( Figure 3B ) .", "Here it is worth noting that the number and sizes of LDs were not drastically different between WT and NPC-deficient cells ( Figure 3—figure supplement 2 ) .", "Third , the vesicles in the vacuole were examined by freeze-fracture EM using PtdIns ( 3 ) P to define microautophagic vesicles ( Figure 3C and Figure 3—figure supplement 3A ) .", "The size of microautophagic vesicles was significantly smaller in ncr1△ , npc2△ , and ncr1△npc2△ than in WT ( Figure 3D and Figure 3—figure supplement 3B ) .", "Those small microautophagic vesicles were not likely to accommodate LDs , which have diameters ranging from 0 . 3 µm to 0 . 5 µm both in WT and in NPC-deficient cells .", "The other prominent feature of NPC-deficient vacuoles was the presence of a large number of small PtdIns ( 3 ) P-deficient vesicles ( Figure 3C and Figure 3—figure supplement 3A ) .", "Fourth , the proportion of cells showing the double-ring filipin-staining pattern was significantly lower in NPC-deficient cells than in WT cells , indicating that sterol transport to the vacuolar membrane may be suppressed in NPC-deficient cells ( Figure 3E ) .", "Moreover , NPC-deficient cells showed deposition of sterol-rich materials in the vacuole lumen ( Figure 3E ) , which most probably corresponds to the small PtdIns ( 3 ) P-deficient vesicles observed by freeze-fracture EM ( Figure 3C and Figure 3—figure supplement 3A ) .", "The paucity of PtdIns ( 3 ) P labeling in those vesicles suggested that they were neither microautophagic vesicles nor macroautophagic bodies per se .", "In all of the experiments described above , npc2△ showed a stronger phenotype than ncr1△ .", "Moreover , a single amino acid substitution in Npc2p , that is , replacement of the 143rd proline with serine ( P143S ) , the mutation that abrogates cholesterol binding in the corresponding residue of human NPC2 ( P120S ) ( Wang et al . , 2010 ) , recapitulated all the defects of NPC-deficient cells ( Figure 3F and Figure 3—figure supplement 3B ) .", "These results indicated that NPC proteins , Npc2p in particular , promote the formation and expansion of the vacuolar raft-like domain , causing stationary phase lipophagy .", "Vacuolar domain formation and/or lipophagy in stationary phase were reported to be abrogated in several gene-deletion mutants , but the mechanism underlying the defect was not necessarily clear ( Toulmay and Prinz , 2013; Wang et al . , 2014 ) .", "We suspected that some of the gene deletions may cause abnormalities by altering the distribution of NPC proteins .", "In WT , Ncr1p–GFP and Npc2p–GFP were observed in the membrane and the lumen of log-phase vacuoles , respectively ( Berger et al . , 2005; Zhang et al . , 2004 ) , and a similar distribution was preserved in stationary phase vacuoles ( Figure 4A ) .", "In cells lacking a core atg gene , i . e . , atg1△ , atg2△ , atg3△ , atg5△ , atg7△ , atg8△ , and atg18△ , Ncr1p–GFP and Npc2p–GFP were seen in the vacuole in log phase , but they were occasionally observed in puncta adjacent to the vacuole in post-diauxic phase , and finally , in stationary phase , they were found only in the puncta in a majority of cells ( Figure 4A and Figure 4—figure supplement 1 ) .", "The identity of the NPC puncta is not clear at present , but their aberrant distribution was not likely to be caused by total disorder of protein transport , because Vph1p–mRFP showed normal vacuolar distribution ( Figure 4—figure supplement 2 ) .", "Consistent with the decrease in NPC proteins in the vacuole , the proportion of cells showing the double-ring filipin-staining pattern was lower in atg7△ than in WT ( Figure 4—figure supplement 3 ) . 10 . 7554/eLife . 25960 . 014Figure 4 . Trafficking defect of NPC proteins impairs stationary phase lipophagy .", "( A ) Ncr1p–GFP and Npc2p–GFP in stationary phase were targeted to the vacuole in WT , but showed punctate distribution in a majority of Atg-deficient cells .", "Bar: 5 µm .", "The quantification data show the mean ± SD of a single representative experiment ( n > 150 for each group ) out of two to three replicates .", "( B ) Expression of Atg18 ( FTTG ) −2xFYVE did not correct the defects in atg18△ in stationary phase .", "Aberrant NPC distribution , decreased vacuolar domain formation , decreased lipophagy , and smaller microautophagic vesicles were observed in atg18△ expressing Atg18 ( FTTG ) −2xFYVE .", "Respective data were obtained as in Figures 4A , 3A , B and D . ( C ) fab1△ showed aberrant NPC distribution , decreased vacuolar domain formation , and virtually no microautophagic vesicles .", "The data on NPC proteins and domain formation were obtained as in Figures 4A and 3A .", "The number of microautophagic vesicles was counted in three independent experiments .", "Mean ± SD ( n = 3; >15 vacuoles were counted in each experiment ) .", "*p<0 . 05; **p<0 . 01 .", "DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01410 . 7554/eLife . 25960 . 015Figure 4—figure supplement 1 . Time course of Ncr1p–GFP distributional change in WT and mutants . Ncr1p–GFP in WT distributed to the vacuolar membrane in log phase ( day 0 ) through post-diauxic phase ( day 1 and day 2 ) and stationary phase ( day 3 ) .", "In atg7Δ , Ncr1p–GFP was in the vacuolar membrane in log phase , but it started to appear in puncta in day 2 , and the punctate distribution became prevalent in day 3 .", "In pep4Δ , Ncr1p–GFP showed a vacuolar distribution like that in WT even in stationary phase , whereas in fab1Δ , the punctate distribution of Ncr1p–GFP was already predominant in day", "1 . Bar: 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01510 . 7554/eLife . 25960 . 016Figure 4—figure supplement 2 . Distribution of Vph1p–mRFP . Vph1p–mRFP in stationary phase showed similar distribution in the vacuole in WT and atg18Δ .", "Bar: 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01610 . 7554/eLife . 25960 . 017Figure 4—figure supplement 3 . Filipin staining of atg7Δ in stationary phase . Filipin staining was performed by the method described for Figure 3E , and the proportion of cells showing the double-ring filipin-staining pattern was measured .", "The ratio was significantly lower in atg7Δ than in WT , indicating that the sterol concentration in the vacuolar membrane of atg7Δ was less than that of WT .", "Bar: 5 μm .", "**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01710 . 7554/eLife . 25960 . 018Figure 4—figure supplement 4 . Microautophagic vesicles in atg18Δ with or without expressing Atg18 ( FTTG ) −2xFYVE in stationary phase .", "( A ) The microautophagic vesicles ( colored in red ) defined by the PtdIns ( 3 ) P labeling in the cytoplasmic leaflet appeared to be of similar sizes in atg18Δ with or without expressing Atg18 ( FTTG ) −2xFYVE .", "Bars: 0 . 2 μm .", "( B ) The diameter of microautophagic vesicles was significantly smaller in atg18Δ than in WT irrespective of the expression of Atg18 ( FTTG ) −2xFYVE .", "The result of one representative experiment out of three independent experiments is presented .", "**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01810 . 7554/eLife . 25960 . 019Figure 4—figure supplement 5 . Freeze-fracture replica EM of fab1Δ in stationary phase . The vacuolar lumen was vacant .", "Bar: 0 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 01910 . 7554/eLife . 25960 . 020Figure 4—figure supplement 6 . Distribution of Ncr1p–GFP and Npc2p–GFP in stationary phase . The proportion of cells showing vacuolar distribution of Ncr1p–GFP and Npc2p–GFP was significantly smaller in vps4Δ and nem1Δ than in WT .", "Mean ± SD of one representative experiment ( n > 150 for each group ) out of two repeats .", "Statistical difference was examined in comparison to the WT shown in Figure 4A .", "**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 020 To examine whether the aberrant distribution of NPC proteins in atg mutants occurred as a result of macroautophagy deficiency , we focused on atg18△ .", "Atg18p has two different functionalities , one related to autophagosome biogenesis ( Barth et al . , 2001 ) and the other related to the regulation of vacuolar morphology ( Efe et al . , 2007 ) .", "These two functions require binding to PtdIns ( 3 ) P and PtdIns ( 3 , 5 ) P2 , respectively , and the expression of Atg18 ( FTTG ) −2xFYVE , which binds to PtdIns ( 3 ) P but not to PtdIns ( 3 , 5 ) P2 , restores macroautophagy in atg18△ ( Obara et al . , 2008 ) .", "Even in atg18△ expressing Atg18 ( FTTG ) −2xFYVE , however , NPC proteins in stationary phase showed abnormal distribution , and domain formation and lipophagy , and the size of the microautophagic vesicles remained significantly different from those in WT ( Figure 4B and Figure 4—figure supplement 4 ) .", "Additionally , in pep4△ , which is defective in the recycling of autophagocytosed materials , NPC proteins were present in the vacuole even in stationary phase ( Figure 4—figure supplement 1 ) .", "These results indicated that the restoration of macroautophagy is not sufficient to normalize stationary phase lipophagy in atg18△ and that PtdIns ( 3 , 5 ) P2-dependent Atg18p functionality may be required .", "Yet this does not rule out the possibility that the macroautophagy mechanism is involved in stationary phase lipophagy .", "The importance of PtdIns ( 3 , 5 ) P2 was further confirmed by the observation of PtdIns ( 3 , 5 ) P2-deficient fab1△ , in which the punctate distribution of NPC proteins was already prevalent in post-diauxic phase and the membrane domain formation was reduced ( Figure 4C and Figure 4—figure supplement 1 ) .", "The complete absence of vesicles in the vacuolar lumen of fab1△ suggested that defects besides abnormal NPC trafficking may also exist in this mutant ( Figure 4C and Figure 4—figure supplement 5 ) .", "Among the other mutants that were defective in domain formation ( Toulmay and Prinz , 2013 ) , vps4△ and nem1△ showed the aberrant dot distribution of NPC proteins ( Figure 4—figure supplement 6 ) ( other defects in vps4△ will be shown later ) .", "In these as well as in atg mutants , the mechanism causing the abnormal distribution of NPC proteins in stationary phase is not clear and demands further study .", "In light of the findings in stationary phase , we hypothesized that sterol transport by NPC proteins may also be involved in microautophagy in acute nitrogen starvation ( Roberts et al . , 2003; van Zutphen et al . , 2014 ) .", "In fact , IMP-deficient domains were found in the vacuole of yeast cultured in nitrogen-deficient medium ( Figure 5A ) , and exclusion of Vph1p labeling ( Figure 5—figure supplement 1 ) from those domains indicated that they also have raft-like properties .", "Vacuoles often harbored several raft-like domains , but vacuoles that were entirely covered with them , as in stationary phase , were scarce . 10 . 7554/eLife . 25960 . 021Figure 5 . Microautophagy in acute nitrogen starvation also occurs at NPC-dependent raft-like domains .", "( A ) The IMP-deficient raft-like domains in the vacuolar membrane of WT after nitrogen starvation for 5 hr .", "These domains were observed both in the cytoplasmic leaflet ( P face ) and the non-cytoplasmic leaflet ( E face ) .", "The P face is densely labeled for PtdIns ( 3 ) P both in the IMP-rich and IMP-deficient domains .", "Bars: 0 . 2 µm .", "( B ) IMP-deficient raft-like domains were significantly less frequent in NPC-deficient cells than in WT .", "Cells showing more than one raft-like domain were counted .", "Mean ± SD ( n > 3; >50 vacuoles were counted for each group in each experiment ) .", "*p<0 . 05; **p<0 . 01 .", "( C ) Filipin staining in nitrogen-starved cells .", "Sterol ( magenta ) was stained in the area overlapping with the vacuolar membrane visualized by Vph1p–GFP ( green ) in WT , whereas in ncr1△npc2△ , sterol-rich deposits ( arrows ) were observed in the vacuolar lumen encircled by Vph1p–GFP .", "Bar: 5 µm .", "( D ) The IMP-deficient domain showed close apposition to an LD ( green ) and the nucleus ( blue ) .", "Bar: 0 . 2 µm .", "See also Figure 5—figure supplement", "2 . ( E ) Degradation of organelle markers by nitrogen starvation for 5 hr: Erg6p–GFP ( LD ) , Nvj1p–GFP ( nucleus ) , and Om45p–GFP ( mitochondria ) .", "The intensity of free GFP bands ( black arrowheads ) and the relative ratio of free GFP to the full-length protein ( white arrowheads ) were measured .", "Degradation of Erg6p–GFP and Nvj1p–GFP was significantly reduced in ncr1△npc2△ ( NPC△ ) compared to WT , whereas degradation of Om45p–GFP was not .", "Mean ± SEM ( n = 3 ) .", "**p<0 . 01 .", "( F ) Vacuoles ( Vph1p–mRFP ) showed dumbbell-like ( arrowheads ) and multi-lobular shapes ( arrows ) after nitrogen starvation in WT , but shape change was much less frequent in ncr1△npc2△ .", "Bar: 5 µm .", "Mean ± SD of a single representative experiment ( n > 150 for each group ) out of three repeats .", "*p<0 . 05; **p<0 . 01 .", "( G ) Nvj1p–GFP ( green ) in WT showed marked elongation along the deformed vacuole after nitrogen starvation ( magenta; FM4-64 ) , but this change was minimal in ncr1△npc2△ .", "Bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 02110 . 7554/eLife . 25960 . 022Figure 5—figure supplement 1 . Distribution of Vph1p–mRFP in the vacuolar membrane after nitrogen starvation . Vph1p-mRFP was labeled only in the IMP-rich area ( arrows ) and not in the IMP-deficient domain .", "The result verified that the IMP-deficient domain induced by acute nitrogen starvation has raft-like properties similar to those seen in stationary phase .", "Bar: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 02210 . 7554/eLife . 25960 . 023Figure 5—figure supplement 2 . Close association of the raft-like domain and organelles .", "( A ) Freeze-fracture EM .", "LDs and the nucleus were associated with the IMP-deficient domain of the vacuole in nitrogen starvation .", "LDs were often enclosed within the vacuolar membrane invagination .", "Bar: 0 . 2 μm .", "( B ) Freeze-fracture/etching EM showed LDs engulfed by the vacuolar invagination .", "A close association between the nucleus and the vacuole was also observed .", "Bar: 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 02310 . 7554/eLife . 25960 . 024Figure 5—figure supplement 3 . Effects of NPC deficiency in nitrogen starvation .", "( A ) The number and sizes of LDs .", "Quantification after nitrogen starvation for 5 hr was done by the method described for Figure 3—figure supplement", "2 . Neither number nor sizes of LDs were drastically different between WT and NPC-deficient cells .", "Mean ± SD are shown .", "Five independent samples and more than 30 cells were counted to assess numbers , whereas three independent samples and more than 30 LDs were counted to assess sizes .", "**p<0 . 01 .", "( B ) Pho8Δ60 assay .", "Cells in log phase and after nitrogen starvation for 5 hr were compared .", "The Pho8Δ60 activity was normalized to the activity of WT after starvation .", "Deletion of NPC proteins did not affect bulk autophagic activity .", "Mean ± SD of triplicate samples are shown .", "( C ) Distribution of LDs .", "After nitrogen starvation , LDs ( green ) were aligned along the indented surfaces of dumbbell-like vacuoles ( magenta; Vph1p–mRFP ) in WT .", "Bar: 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 024 In comparison to WT , NPC-deficient cells in nitrogen starvation had far fewer raft-like domains ( Figure 5B ) , and this reduction in domain formation was more prominent in npc2△ than in ncr1△ .", "Filipin staining suggested a relative increase in sterol in the vacuolar membrane of WT , whereas sterol deposition in the vacuolar lumen was prominent in ncr1△npc2△ ( Figure 5C ) , indicating that NPC proteins are also engaged in sterol transport in nitrogen starvation .", "Furthermore , in WT after nitrogen starvation , LDs and the nucleus were frequently associated with the vacuoles , and their association always occurred in the raft-like domain ( Figure 5D and Figure 5—figure supplement 2 ) .", "LDs were often found within deep vacuolar invaginations , suggesting that they were processed by a microautophagic mechanism similar to that in stationary phase .", "The LD-containing invagination was morphologically different from the autophagic tube , which is a tubular extension with an IMP-deficient vesicle at the tip ( Müller et al . , 2000 ) .", "To determine whether NPC proteins are necessary for microautophagy in nitrogen starvation , we examined the degradation of two marker proteins , Erg6p–GFP for LDs and Nvj1p–GFP for the nucleus , by Western blotting .", "After nitrogen starvation , the degradation of Erg6p–GFP was significantly less advanced in ncr1△npc2△ than in WT , as shown both by the amount of free GFP and by the relative proportion of free GFP in the total protein amount ( Figure 5E ) .", "We confirmed that the number and size of LDs are not drastically different between WT and NPC-deficient cells ( Figure 5—figure supplement 3A ) .", "Nvj1p–GFP also showed significantly less generation of free GFP in ncr1△npc2△ than in WT ( Figure 5E ) , but the difference in the proportion of free GFP in the total protein was not significant between WT and ncr1△npc2△ .", "The latter result was apparently produced because the quantity of full-length Nvj1p–GFP in WT increased after nitrogen starvation , reflecting the expansion of the nuclear-vacuolar junction ( NVJ ) ( see Figure 5G ) .", "By contrast , the degradation of the mitochondrial marker Om45p–GFP ( Figure 5E ) and the bulk autophagy activity measured by the Pho8Δ60 assay ( Figure 5—figure supplement 3B ) were at comparable levels in WT and NPC-deficient cells .", "In conjunction with the EM data , this finding confirmed that NPC proteins play a critical role specifically in microautophagy of LDs and the nucleus .", "Vacuoles were largely spherical in log phase both in WT and in NPC-deficient cells , but under nitrogen starvation a majority of the vacuoles showed gross deformation , taking multi-lobular or dumbbell shapes , only in WT ( Figure 5F ) .", "This vacuolar deformation made it difficult to capture microautophagy using light microscopy , but LDs were often observed along the indented surface of the dumbbell-shaped vacuoles , indicative of their close association ( Figure 5—figure supplement 3C ) .", "Notably , NVJs that were marked by Nvj1p–GFP in WT was changed from a short line in log phase to a longer line along a deformed vacuole after nitrogen starvation , whereas such a change was negligible in ncr1△npc2△ ( Figure 5G ) .", "We next asked about the source of sterol that is transported by NPC proteins within the vacuole .", "To investigate this matter , we examined the identity of the sterol-rich material observed in the vacuolar lumen of ncr1△npc2△ ( Figure 5C ) .", "Freeze-fracture EM revealed that smooth vesicles and small rugged structures were constantly present in the vacuolar lumen in ncr1△npc2△ but only scarcely in WT ( Figure 6A , B ) .", "The smooth vesicles were often labeled for PtdIns ( 3 ) P in the cytoplasmic leaflet , indicating that they are microautophagic vesicles .", "On the other hand , the rugged structure was morphologically identified as the intraluminal vesicle ( ILV ) of the multi-vesicular body ( MVB ) ( Figure 6—figure supplement 1A ) .", "Previously , mammalian ILVs have been shown to be enriched with sterol ( Möbius et al . , 2002 ) , and thus we hypothesized that ILVs in yeast may be the major source of sterol to be transported by NPC proteins in nitrogen starvation . 10 . 7554/eLife . 25960 . 025Figure 6 . The MVB pathway supplies sterol for induction of microautophagy .", "( A ) The vacuolar content of ncr1△npc2△ after nitrogen starvation for 5 hr .", "ILVs , seen as small rugged structures ( arrows ) , and microautophagic vesicles labeled for PtdIns ( 3 ) P in the cytoplasmic leaflet ( * ) were observed .", "Bars: 0 . 2 µm .", "( B ) The density of ILVs in the vacuolar lumen after nitrogen starvation was significantly higher in ncr1△npc2△ than in WT .", "Mean ± SD are represented ( n = 3; >30 vacuoles were counted in each experiment ) .", "**p<0 . 01 .", "( C ) Degradation of Erg6p–GFP and Nvj1p–GFP in nitrogen starvation was suppressed in vps4△ .", "The bands of the full-length proteins ( white arrowheads ) and free GFP ( black arrowheads ) are marked .", "( D ) Raft-like domains in the vacuolar membrane were significantly less frequent in vps4△ and pep4△prb1△ than in WT ( see Figure 5B for WT ) .", "*p<0 . 05 .", "( E ) The vacuolar content of pep4△prb1△ after nitrogen starvation .", "The vacuolar lumen was filled with autophagic bodies ( left figure; bar , 0 . 5 µm ) and ILVs ( arrows in the right panel; bar , 0 . 2 µm ) .", "( F ) Filipin staining showed that the vacuolar lumen of atg7△pep4△prb1△ after nitrogen starvation contains abundant sterol-rich structures , whereas that of atg7△ does not ( arrows ) .", "Combined with the result of freeze-fracture EM ( see Figure 6—figure supplement 1D ) , the filipin fluorescence in the vacuolar lumen of atg7△pep4△prb1△ is thought to be derived from ILVs .", "Bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 02510 . 7554/eLife . 25960 . 026Figure 6—figure supplement 1 . Analysis of MVB and vps4Δ .", "( A ) Freeze-fracture EM of MVB in ypt7Δ .", "ILVs in the lumen are observed as rugged structures ( arrows ) .", "Bar: 0 . 2 μm .", "( B ) Filipin staining in vps4Δ after nitrogen starvation .", "Vph1p–GFP ( green ) was observed in the vacuolar membrane ( arrowheads ) and in the class E compartment ( arrows ) .", "Filipin staining was intense in the class E compartment , but it was negligible in the vacuolar membrane area .", "Bar: 5 μm .", "( C ) Distribution of NPC proteins in vps4Δ after nitrogen starvation .", "Ncr1p–GFP and Npc2p–GFP were observed in the vacuolar membrane and in the vacuolar lumen , respectively .", "Bar: 5 μm .", "( D ) The vacuolar content of atg7Δpep4Δprb1Δ and atg7Δ after nitrogen starvation .", "The vacuolar lumen of atg7Δpep4Δprb1Δ after nitrogen starvation contained a large number of ILVs , whereas that of atg7Δ was virtually vacant .", "Bar: 0 . 2 μm .", "( E ) vps4Δ in stationary phase .", "( a ) Domain formation was completely absent in vps4Δ .", "See Figure 3A for comparison .", "Mean ± SD of three independent experiments ( >50 vacuoles were counted for each group in each experiment ) are represented .", "( b ) Lipophagy was also absent in vps4Δ .", "See Figure 3B for comparison .", "Mean ± SD of one representative experiment ( n > 150 for each group ) out of two repeats are shown .", "**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 02610 . 7554/eLife . 25960 . 027Figure 6—figure supplement 2 . are1Δare2Δ in stationary phase .", "( A ) Vacuolar domain formation was observed in are1Δare2Δ , although its frequency was lower than that in WT .", "See Figure 3A for comparison .", "Mean ± SD of three independent experiments ( >50 vacuoles were counted for each group in each experiment ) are represented .", "( B ) Lipophagy in are1Δare2Δ was observed at a level comparable to that in WT .", "See Figure 3B for comparison .", "Mean ± SD of one representative experiment ( n > 150 for each group ) out of two repeats are illustrated .", "*p<0 . 05; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 027 Three lines of evidence support this idea .", "First , in vps4△ , in which the MVB pathway is blocked , hardly any degradation of Erg6p–GFP and Nvj1–GFP was observed ( Figure 6C ) and the number of raft-like domains in the vacuolar membrane was significantly decreased ( Figure 6D ) .", "Consistently , filipin failed to stain the vacuolar membrane in vps4△ after nitrogen starvation ( Figure 6—figure supplement 1B ) .", "Here it is of note that , in vps4△ , macroautophagy is not suppressed ( Babst et al . , 1997 ) and NPC proteins show normal vacuolar distribution ( Figure 6—figure supplement 1C ) .", "Second , in cells in which two major vacuolar proteases ( pep4△prb1△ ) are depleted , the vacuolar lumen harbored a large number of ILVs as well as macroautophagic bodies ( Figure 6E ) , and the raft-like domain in the vacuolar membrane was reduced to a minimum level ( Figure 6D ) .", "This result , together with the persistence of ILVs and microautophagic vesicles in ncr1△npc2△ ( Figure 6A , B ) , suggested that both proteolysis and NPC-mediated sterol extraction are necessary for efficient degradation of sterol-rich membranes such as ILVs .", "Finally , to confirm that yeast ILVs are enriched with sterol , atg7△pep4△prb1△ was examined after nitrogen starvation .", "As expected from the results from pep4△prb1△ , the vacuole of atg7△pep4△prb1△ contained abundant ILVs with few other structures , whereas that of atg7△ was largely vacant ( Figure 6—figure supplement 1D ) .", "Filipin staining yielded a diffuse fluorescence signal in the vacuolar lumen of atg7△pep4△prb1△ , but not of atg7△ ( Figure 6F ) .", "The combined result indicated that ILVs are enriched with sterol .", "On the basis of these results , we concluded that sterol brought into the vacuole by ILVs is crucial for the formation and expansion of the raft-like domain and microautophagy in nitrogen starvation .", "The above result prompted us to ask whether ILVs also play a role in the domain formation in the stationary phase vacuole .", "In stationary phase , sterol esters in LDs were proposed to be a sterol source ( Wang et al . , 2014 ) .", "Consistently , raft-like domain formation and lipophagy were decreased to low levels in are1△are2△ that cannot synthesize sterol esters ( Figure 6—figure supplement 2 ) .", "On the other hand , domain formation and lipophagy in the stationary phase vacuoles were absent in vps4△ ( Figure 6—figure supplement 1E ) .", "Abnormal trafficking of NPC proteins may be a cause of the defect , but the complete absence of the raft-like domain in vps4△ suggests that sterol in ILVs may be important as an initial trigger to induce the stationary phase vacuolar domain .", "Microautophagy of LDs and the nucleus in nitrogen starvation is suppressed in atg mutants ( Roberts et al . , 2003; van Zutphen et al . , 2014 ) .", "We confirmed significant reduction in the degradation of Erg6p–GFP and Nvj1p–GFP in atg mutants , and wanted to determine the point in the process at which microautophagy is aborted in these cells .", "The vacuolar distribution of Ncr1p–GFP and Npc2p–GFP in atg mutants was preserved in acute nitrogen starvation ( Figure 7—figure supplement 1 ) , but the numbers of raft-like domains were significantly reduced ( Figure 7A ) .", "The activation of the MVB pathway in nitrogen starvation has been reported to occur similarly even in the absence of macroautophagy , suggesting that ILVs should reach the vacuolar lumen ( Müller et al . , 2015 ) .", "Nevertheless , few structures were observed in the vacuolar lumen of atg mutants ( Figure 6—figure supplement 1D ) , suggesting that ILV degradation and sterol transport to the vacuolar membrane may be occurring normally .", "This result inferred that raft-like domain formation in nitrogen starvation was suppressed in atg mutants even though sterol is transported to the vacuolar membrane as in WT ( see Discussion for the possible cause of this defect ) . 10 . 7554/eLife . 25960 . 028Figure 7 . Involvement of Atg proteins and actin filaments in microautophagy during acute nitrogen starvation .", "( A ) Raft-like domain formation after nitrogen starvation was significantly less frequent in atg7△ and atg8△ than in WT ( see Figure 5B for WT ) .", "( B ) Degradation of Erg6p–GFP and Nvj1p–GFP in nitrogen starvation was suppressed by latrunculin A ( 0 . 2 mM; latA ) , but not by nocodazole ( 10 µg/ml; noc ) .", "Mean ± SEM ( n = 3 ) are shown .", "The intensity of free GFP bands ( black arrowheads ) and the relative density ratio of free GFP to the full-length protein ( white arrowheads ) were quantified .", "*p<0 . 05; **p<0 . 01 .", "( C ) Raft-like domain formation after nitrogen starvation in WT was reduced by latrunculin A ( latA ) .", "Raft-like domain in ncr1△npc2△ , which was already low , was not further decreased by latrunculin A . The result was compared to that of WT and ncr1△npc2△ shown in Figure 5B .", "( D ) The rate of vacuolar shape change in nitrogen starvation was reduced by latrunculin A ( latA ) .", "Vacuoles were classified as in Figure 5F .", "Latrunculin A further decreased the rate of shape change in ncr1△npc2△ .", "The results ( mean ± SD ) of one representative experiment ( n > 150 for each group ) out of three repeats are shown .", "*p<0 . 05; **p<0 . 01 .", "( E ) The putative mechanism of microautophagy in nitrogen starvation .", "( 1 ) The activated MVB pathway delivers sterol-rich ILVs to the vacuole; ( 2 ) autophagosomes fuse with the vacuolar membrane to supply sphingolipids; ( 3 ) NPC proteins transport sterol from decomposing ILVs to the vacuolar membrane; ( 4 ) the generated raft-like domain adheres to cargo organelles via raft-philic receptors; and ( 5 ) the raft-like domain expands to engulf cargos leading to microautophagy . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 02810 . 7554/eLife . 25960 . 029Figure 7—figure supplement 1 . Distribution of Ncr1p–GFP and Npc2p–GFP after nitrogen starvation . In both WT and atg7Δ , Ncr1p–GFP and Npc2p–GFP were observed in the membrane and in the lumen of the vacuole , respectively .", "Bar: 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 029 To further characterize microautophagy in nitrogen starvation , actin filaments and microtubules were depolymerized by latrunculin A and nocodazole , respectively , and the effect was examined .", "Latrunculin A suppressed degradation of both Erg6p–GFP and Nvj1p–GFP , whereas nocodazole did not ( Figure 7B ) .", "The larunculin A treatment also decreased the number of raft-like domains in the vacuolar membrane in WT , but it did not affect the already reduced number of raft-like domains in ncr1△npc2△ ( Figure 7C ) .", "On the other hand , the gross shape change in vacuoles was reduced in WT treated with latrunculin A and further decreased in ncr1△npc2△ ( Figure 7D ) , indicating that the effects of actin depolymerization and NPC deletion were additive in this respect .", "This result suggested that actin depolymerization may have an additional effect besides suppressing raft-like domain formation .", "On the basis of all of these results , we propose the mechanism for microautophagy in nitrogen starvation , as depicted in Figure 7E ." ], [ "The absence of Ncr1p or Npc2p does not cause overt abnormalities in yeast cells cultured under normal growth conditions ( Berger et al . , 2005; Malathi et al . , 2004; Zhang et al . , 2004 ) .", "This may be because the yeast vacuole does not need to process endocytosed lipoproteins as mammalian cells do , but this possible explanation raises the question of which functions NPC proteins have in yeast .", "The present study showed that NPC proteins play a crucial role in the expansion of raft-like vacuolar membrane domains in microautophagy , probably by transporting sterol to the vacuolar membrane .", "Obviously , NPC proteins are not the only mechanism capable of transporting sterol to the vacuolar membrane .", "This is indicated by the presence of raft-like domains in NPC-deficient cells , albeit at a lower level than in WT .", "Nevertheless , stationary phase lipophagy is prominently suppressed in NPC-deficient cells .", "This result suggests that sterol transport by NPC proteins is important not just for raft-like domain formationbut more critically so for the expansion of those domains , and this latter process appears indispensable for the engulfment of cargo in microautophagy .", "How can NPC proteins expand the raft-like domain and promote microautophagy ?", "In this context , it is notable that depletion of Npc2p causes more significant defects in microautophagy than does depletion of Ncr1 .", "The Npc2p dominance indicates that direct sterol transport by Npc2p ( bypassing Ncr1p ) , as shown for mammalian NPC proteins ( Kennedy et al . , 2012; Xu et al . , 2008 ) , may play a major role in the expansion of raft-like domains .", "Npc2p was shown to bind preferentially with negative-charged membranes ( McCauliff et al . , 2015 ) , and thus the sphingolipid-enriched raft-like domain is a suitable platform for the interaction with Npc2p .", "Hence , it is plausible that recruitment of Npc2p to the raft-like domain facilitates the domain’s expansion through direct sterol transfer .", "Exclusion of Ncr1p from raft-like domains is also consistent with this deduction ( Toulmay and Prinz , 2013 ) .", "The other important finding that contributes to our understanding of the mechanism of NPC proteins’ functioning is the manner in which raft-like domains invaginate .", "Inward budding of raft-like domains has been observed in giant liposomes ( Hamada et al . , 2007 ) , suggesting that it may be an endogenous propensity of those domains .", "However , the vacuolar invagination differs from the liposomal phenomenon in that it occurs only in limited raft-like domains .", "This was particularly obvious in stationary phase vacuoles , where polygonal raft-like domains exist over the entire vacuole surface but invagination occurs only in the domains adhering to cargo .", "This result suggests that tight adhesion to cargo is conducive to invagination .", "We speculate that the inward curvature imposed on raft-like domains by the adhesion may create packing defects in the luminal leaflet of the vacuolar membrane ( Cui et al . , 2011 ) , thereby enhancing the Npc2p-mediated sterol transfer .", "Finally , it is noteworthy that Vac8p , the vacuolar protein interacting with Nvj1p , has an affinity to rafts ( Peng et al . , 2006 ) .", "We hypothesize that this raft-philicity of vacuolar interacting molecules is important for the microautophagic process , because enlargement of the raft-like domain and recruitment of additional interacting proteins may proceed in a feed-forward manner .", "The marked elongation of NVJ in nitrogen starvation is consistent with this idea .", "The MVB pathway and endocytic degradation of plasmalemmal proteins are activated immediately after amino acid starvation; and the resultant proteome remodeling , including upregulation of vacuolar hydrolases , is thought to boost macroautophagic degradation ( Jones et al . , 2012; Müller et al . , 2015 ) .", "The present study showed that this activation of the MVB pathway is also important for microautophagy and that ILVs are a major source of sterol that is transported by NPC proteins .", "Hence , in acute nitrogen starvation , both macroautophagy and microautophagy appear to be coordinated with the MVB pathway to support cell survival .", "The importance of the MVB pathway in microautophagy in the stationary phase vacuole is less clear than that in acute nitrogen starvation because of the longer time course .", "Nevertheless , we think that as cells enter the post-diauxic phase , the MVB pathway is likely to be upregulated because the recycling of endocytosed plasmalemmal proteins from the endosome to the plasma membrane is reduced in glucose starvation ( Lang et al . , 2014 ) .", "Complete lack of raft-like domains in vps4△ , in which ILVs do not form , is consistent with this assumption .", "We thus propose that the MVB pathway may be involved in an early stage of raft-like domain formation in the stationary phase vacuole , whereas LDs may become an important sterol source at later time points after the MVB pathway subsides .", "Microautophagy in acute nitrogen starvation is suppressed in atg mutants ( Roberts et al . , 2003; van Zutphen et al . , 2014 ) , but how this defect is correlated with macroautophagy deficiency has not been clear .", "We found that the raft-like domain formation in nitrogen starvation is significantly down-regulated in atg mutants .", "This is peculiar because the activation of the MVB pathway ( Müller et al . , 2015 ) and the vacuolar distribution of NPC proteins are observed in atg mutants , suggesting that sterol transport to the vacuolar membrane occurs in these mutants on a level comparable to that in WT .", "We speculate that the defect of microautophagy in atg mutants may be caused by deficiency of sphingolipids , the other indispensable component of raft-like domains ( Lingwood and Simons , 2010 ) .", "Considering that the vacuolar membrane is relatively poor in sphingolipids under normal growing conditions ( Hechtberger et al . , 1994; Schneiter et al . , 1999 ) , sphingolipids are thought to be imported to the vacuole to form the raft-like domains in nitrogen starvation .", "Fusion of autophagosomes is the most likely route for such transport ( Yamagata et al . , 2011 ) , and this explains why atg mutants that are deficient in autophagy show few raft-like domains .", "Nevertheless , the possibility that other sphingolipid transport pathways that are downregulated in atg mutants may be involved cannot be excluded .", "Additional defects may also occur in atg mutants after nitrogen starvation .", "In this context , it is notable that microautophagy in nitrogen starvation was suppressed by depolymerization of actin .", "In light of the necessity of actin for selective macroautophagy , but not for bulk autophagy ( Reggiori et al . , 2005 ) , one role of actin may be to facilitate the engagement of cargo organelles with the vacuolar membrane .", "Yet we found that actin depolymerization also decreases raft-like domain formation , which implies that actin has some direct effect on the vacuolar membrane .", "Considering that actin integrity is important in organizing plasma membrane domains in mammalian cells ( Goswami et al . , 2008; Suzuki et al . , 2007 ) , actin that is associated with the vacuolar membrane ( Eitzen et al . , 2002 ) may be involved in generating the raft-like domains .", "This vacuole-associated actin might not be properly organized in atg mutants in nitrogen starvation .", "These speculations need to be tested in further experiments .", "In mammalian cells , microautophagy of cytosolic proteins has been reported to occur in the late endosome by an ESCRT-dependent mechanism ( Sahu et al . , 2011 ) , but it is not known whether microautophagy and the MVB pathway are correlated in the same way that we observed in yeast .", "It is noteworthy , however , that autophagosomes in mammalian cells may often fuse with MVBs to form amphisomes before fusing with lysosomes to form autophagolysosomes , whereas autophagosomes in yeast fuse directly with vacuoles ( Fader and Colombo , 2009 ) .", "The amphisome is unique in that it delivers autophagocytosed cytosolic materials and sterol-rich ILVs to the lysosome simultaneously .", "Moreover , the relative size of an autophagosome ( and an amphisome ) to a lysosome ( or a vacuole ) is much larger in mammals than in yeast , indicating that a single autophagosomal fusion would have a greater impact on the mammalian lysosome than on the yeast vacuole .", "It is thus probable that the mammalian autophago-lysosomal membrane acquires an amount of both sterol and sphingolipids that is sufficient to generate raft-like domains .", "On the basis of this inference , we would predict that microautophagy in mammalian cells occurs preferentially in lysosomes after fusion with amphisomes ." ], [ "The yeast strains used in this study are based on the parent strain SEY6210 ( Table 1 ) .", "Yeast manipulations were performed according to standard protocols ( Kaiser et al . , 1994 ) . 10 . 7554/eLife . 25960 . 030Table 1 . Yeast strains used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 25960 . 030NameGenotypeReference/originSEY6210MATα leu2-3 , 112 ura3-52 his3-∆200 trp1-∆901 lys2-801 suc2-∆9Robinson et al . ( 1988 ) KVY4SEY6210; ypt7Δ::LEU2Kihara et al . ( 2001 ) KVY5SEY6210; atg8Δ::HIS3Kirisako et al . ( 1999 ) KVY135SEY6210; atg7Δ::HIS3Obara et al . ( 2008 ) TKY1001SEY6210; atg18Δ::KanMXCheng et al . ( 2014 ) YT350SEY6210; pep4Δ::hphNT1 , prb1Δ::natNT2This studyYT956SEY6210; fab1Δ::cgTRP1This studyYT1167SEY6210; NVJ1–yeGFP::klTRP1This studyYT1242SEY6210; npc2Δ::cgHIS3This studyYT1276SEY6210; ncr1Δ::cgTRP1 , npc2Δ::cgHIS3This studyYT1278SEY6210; ncr1Δ::cgTRP1 , pho8Δ60::natNT2This studyYT1287SEY6210; VPH1–mRFP::natNT2 , ncr1Δ::cgTRP1 , npc2Δ::cgHIS3This studyYT1288SEY6210; ncr1Δ::natNT2This studyYT1296SEY6210; VPH1–yeGFP::hphNT1This studyYT1325SEY6210; NCR1–yeGFP::klTRP1This studyYT1330SEY6210; npc2Δ::cgHIS3 , pho8Δ60::natNT2This studyYT1331SEY6210; ncr1Δ::cgTRP1 , npc2Δ::cgHIS3 , pho8Δ60::natNT2This studyYT1349SEY6210; npc2 ( P143S ) ::cgTRP1This studyYT1371SEY6210; NPC2–yeGFP::cgTRP1This studyYT1373SEY6210; fab1Δ::cgTRP1 , NPC2–yeGFP::hphNT1This studyYT1385SEY6210; atg18Δ::KanMX , NCR1–yeGFP::klTRP1This studyYT1386SEY6210; atg18Δ::KanMX , NPC2–yeGFP::klTRP1This studyYT1388SEY6210; fab1Δ::cgTRP1 , NCR1–yeGFP::hphNT1This studyYT1467SEY6210; VPH1–mRFP::natNT2This studyYT1474SEY6210; nem1Δ::cgHIS3 , NCR1–yeGFP::klTRP1This studyYT1476SEY6210; nem1Δ::cgHIS3 , NPC2–yeGFP::klTRP1This studyYT1479SEY6210; VPH1–yeGFP::hphNT1 , ncr1Δ::TRP1 , npc2Δ::HIS3This studyYT1481SEY6210; vps4Δ::cgHIS3 , NCR1–yeGFP::klTRP1This studyYT1482SEY6210; vps4Δ::cgHIS3 , NPC2–yeGFP::klTRP1This studyYT1503SEY6210; atg18Δ::KanMX , ATG18p-atg18 ( FTTG ) −3HA-2FYVE , NCR1–yeGFP::klTRP1This studyYT1509SEY6210; ncr1Δ::cgTRP1 , npc2Δ::cgHIS3 , ERG6–yeGFP::klTRP1This studyYT1517SEY6210; atg18Δ::KanMX , ATG18p-atg18 ( FTTG ) −3HA-2FYVE , NPC2–yeGFP::klTRP1This studyYT1523SEY6210; ncr1Δ::cgTRP1 , npc2Δ::cgHIS3 , NVJ1–yeGFP::hphNT1This studyYT1536SEY6210; ncr1Δ::cgTRP1 , npc2Δ::cgHIS3 , Om45–yeGFP::hphNT1This studyYT1542SEY6210; ERG6–yeGFP::natNT2This studyYT1549SEY6210; vps4Δ::natNT2 , NVJ1–yeGFP::hphNT1This studyYT1552SEY6210; atg7Δ::HIS3 , NCR1–yeGFP::klTRP1This studyYT1553SEY6210; atg3Δ::KanMX , NCR1–yeGFP::klTRP1This studyYT1554SEY6210; atg5Δ::LEU2 , NCR1–yeGFP::klTRP1This studyYT1556SEY6210; atg2Δ::LEU2 , NCR1–yeGFP::klTRP1This studyYT1557SEY6210; atg1Δ::KanMX , NCR1–yeGFP::klTRP1This studyYT1558SEY6210; atg7Δ::HIS3 , NPC2–yeGFP::klTRP1This studyYT1559SEY6210; atg3Δ::KanMX , NPC2–yeGFP::klTRP1This studyYT1560SEY6210; atg5Δ::LEU2 , NPC2–yeGFP::klTRP1This studyYT1561SEY6210; atg8Δ::HIS3 , NPC2–yeGFP::klTRP1This studyYT1562SEY6210; atg2Δ::LEU2 , NPC2–yeGFP::klTRP1This studyYT1569SEY6210; atg1Δ::KanMX , NPC2–yeGFP::klTRP1This studyYT1579SEY6210; atg8Δ::HIS3 , NCR1–yeGFP::klTRP1This studyYT1583SEY6210; vps4Δ::cgHIS3 , ERG6–yeGFP::klTRP1This studyYT1584SEY6210; atg8Δ::HIS3 , NVJ1–yeGFP::klTRP1This studyYT1586SEY6210; atg7Δ::HIS3 , ERG6–yeGFP::klTRP1This studyYT1591SEY6210; atg14Δ::LEU2 , ERG6–yeGFP::natNT2This studyYT1592SEY6210; atg14Δ::LEU2 , NVJ1–yeGFP::natNT2This studyYT1597SEY6210; atg8Δ::HIS3 , ERG6–yeGFP::klTRP1This studyYT1598SEY6210; atg7Δ::HIS3 , NVJ1–yeGFP::klTRP1This studyYT1726SEY6210; pep4Δ::hphNT1 , prb1Δ::natNT2 , atg7Δ::HIS3This study For stationary phase lipophagy , cells were cultured in synthetic complete ( SC ) medium ( 0 . 17% yeast nitrogen base without amino acids and ammonium sulfate [Becton Dickinson , Franklin Lakes , NJ , USA] , 0 . 5% ammonium sulfate , 2% dextrose , and 0 . 13% dropout mix ) for three days starting at OD600 nm ≈ 0 . 15 .", "To induce macroautophagy , cells cultured to OD600 nm ≈ 1 in SC medium or YPD medium ( 1% yeast extract , 2% polypeptone , and 2% dextrose ) were washed twice with distilled water and incubated for 5 hr in nitrogen-depleted medium SD ( −N ) ( 0 . 17% yeast nitrogen base without amino acids and ammonium sulfate and 2% dextrose ) .", "In some experiments , the SD ( −N ) medium was added with 0 . 2 mM latrunculin A ( Nacalai Tesque , Kyoto , Japan ) or 10 µg/ml nocodazole ( Sigma-Aldrich , St . Louis , MO , USA ) .", "All cultures were kept at 30°C .", "Yeast cell extracts for Western blotting were prepared using lithium acetate and sodium hydroxide ( Zhang et al . , 2011 ) .", "The precipitated sample was dissolved in SDS sample buffer ( 2% SDS , 10% glycerol , 60 mM Tris-HCl , pH 6 . 8 ) , and after centrifugation , the supernatant was loaded for SDS-PAGE , electrotransferred to PVDF membrane , and subjected to Western blotting using SuperSignal West Dura Extended Duration Substrate ( Thermo Fisher , Waltham , MA , USA ) .", "Images were captured with a Fusion Solo S instrument ( Vilber Lourmat , Eberhardzell , Germany ) and analyzed by the accompanying software .", "For labeling of LDs , yeast cells were incubated with 0 . 5 μg/ml BODIPY493/503 ( Thermo Fisher ) for 10 min , and rinsed with 50 mM Tris-HCl ( pH 7 . 5 ) immediately before microscopy ( Wang et al . , 2014 ) .", "The vacuolar membrane was either visualized with Vph1p–mRFP or stained with FM4-64 ( Biotium , Fremont , CA , USA ) as described previously ( Vida and Emr , 1995; Wang et al . , 2014 ) .", "For sterol staining , yeast cells were fixed for 30 min with 1% glutaraldehyde in 40 mM potassium phosphate buffer ( pH 7 ) and 0 . 5 mM magnesium choloride , and treated for 10 min with 0 . 7% 2-mercaptoethanol in 0 . 2 M Tris and 20 mM EDTA .", "After rinses with 0 . 17 M potassium dihydrogen phosphate and 30 mM sodium citrate ( pH 5 . 8 ) , cells were treated for 5 min with 0 . 025% Nonidet P-40 in PBS , then with 1 mg/ml sodium borohydride in Tris-buffered saline ( pH 8 . 2 ) , and were labeled for 10 min in 10 μg/ml filipin ( Polysciences , Warminster , PA , USA ) in PBS .", "In experiments that looked at filipin staining and Vph1–GFP simultaneously , cells were fixed for 1 hr with 0 . 25% glutaraldehyde and 5% formaldehyde in 100 mM sodium phosphate buffer ( pH 7 . 4 ) .", "They were then treated with 0 . 2 mg/ml Zymolyase 100T ( Nacalai Tesque ) in PBS and 0 . 1% 2-mercaptoethanol for 15 min at 30°C before proceeding to the Nonidet P-40 permeabilization , sodium borohydride treatment , and filipin staining .", "The stained yeast cells , as well as those expressing GFP- or mRFP-tagged proteins , were mounted on a glass slide and observed under an AxioImager or Axiovert microscope using a 63× NA1 . 4 Apochromat objective lens ( Zeiss , Oberkochen , Germany ) .", "Yeast cells were quick-frozen either by metal-contact freezing using a Slammer ( Valiant Instruments , Ellisville , MO , USA ) or by high-pressure freezing using an HPM 010 high-pressure freezing machine ( Leica Microsystems , Wetzlar , Germany ) .", "For metal-contact freezing , cell pellets were placed on a glass coverslip and slammed to a copper block pre-cooled with liquid nitrogen .", "For high-pressure freezing , an EM grid ( 200 mesh ) impregnated with yeast cells was sandwiched between a 20-μm–thick copper foil and a flat aluminum disc ( Engineering Office M . Wohlwend , Sennwald , Switzerland ) and frozen according to the manufacture’s instructions ( Cheng et al . , 2014 ) .", "For freeze-fracture , the specimens were transferred to the cold stage of a Balzers BAF 400 apparatus and fractured at −120 to −100°C under a vacuum of ~1 × 10−6 mbar .", "Replicas were made by electron-beam evaporation in a process involving three steps: carbon ( C ) ( 2–5 nm in thickness ) at an angle of 90° to the specimen surface , platinum-carbon ( Pt/C ) ( 1–2 nm ) at an angle of 45° , and C ( 10–20 nm ) at an angle of 90° ( Fujita et al . , 2010 ) .", "The thickness of the deposition was adjusted by referring to a crystal thickness monitor .", "Thawed replicas were treated with 2 . 5% SDS in 0 . 1 M Tris-HCl ( pH 7 . 4 ) at 60°C overnight .", "To remove the cell wall , yeast replicas were treated for 2 hr at 37°C with one of the following solutions: ( 1 ) 20 μg/ml Zymolyase 100T ( Nacalai Tesque ) in PBS containing 0 . 1% Triton X-100 , 1% BSA , and a protease inhibitor cocktail ( Nacalai Tesque ) or ( 2 ) 0 . 1% Westase ( Takara Bio , Kusatsu , Japan ) in McIlvain buffer ( 37 mM citrate , 126 mM disodium hydrogen phosphate , pH 6 . 0 ) containing 10 mM EDTA , 30% fetal calf serum , and a protease inhibitor cocktail .", "Replicas for labeling PtdIns ( 3 ) P were generally treated with Zymolyase , whereas those for protein labeling were treated with Westase .", "After the enzyme treatment , the replicas were further treated in 2 . 5% SDS and stored in buffered 50% glycerol at −20°C until use .", "For freeze-fracture/etching , the specimens were fractured at −102°C under a vacuum of ~1 × 10−6 mbar and kept at the same temperature for 2 min to induce sublimation of water from the fractured surface .", "The samples were then exposed to rotary evaporation of Pt/C ( 4 nm ) at an angle of 20° , followed by C ( 20 nm ) at an angle of 80° .", "The replicas were treated with household bleach to digest biological materials before mounting on EM grids for observation .", "The method of freeze-fracture/etching is described in detail at Bio-protocol ( Tsuji and Fujimoto , 2017 ) .", "PtdIns ( 3 ) P was labeled as described previously ( Cheng et al . , 2014 ) .", "Briefly , SDS-treated freeze-fracture replicas were incubated at 4°C overnight with GST-tagged p40phox PX domain ( 10 ng/ml ) in PBS containing 1% BSA , followed by rabbit anti-GST antibody ( 10 µg/ml ) and then by colloidal gold ( 10 nm ) -conjugated protein A ( PAG10; University of Utrecht , Utrecht , The Netherlands ) , each for 30 min at 37°C in 1% BSA in PBST .", "For labeling of Vph1p–mRFP , replicas were incubated with rabbit anti-mCherry antibody ( provided by Dr Shuh-ichi Nishikawa of Niigata University and Dr Masahiko Watanabe of Hokkaido University ) followed by PAG10 .", "The labeled replicas were picked up on Formvar-coated EM grids and observed with a JEOL JEM-1011 EM ( Tokyo , Japan ) operated at 100 kV .", "Digital images were captured using a CCD camera ( Gatan , Pleasanton , CA , USA ) and subjected to further analysis .", "Bulk autophagic activity of yeast cells was quantified by measuring phosphatase activity as previously described ( Noda et al . , 1995 ) .", "EM images obtained from more than three independent experiments were used for the analysis .", "The number of colloidal gold particles was counted manually , and areas were measured using ImageJ ( NIH ) .", "The labeling density in the selected structure was calculated by dividing the number of colloidal gold particles by the area .", "Statistical differences between samples were examined by Student’s t-test except for the results concerning PtdIns ( 3 ) P labeling density , the diameter of microautophagic vesicles , and Western blotting .", "Differences in PtdIns ( 3 ) P labeling density ( Figure 2C ) and in the diameter of microautophagic vesicles ( Figure 3—figure supplement 3B and Figure 4—figure supplement 4B ) were examined by Mann-Whitney’s U-test .", "The Western blotting results ( Figures 5E and 7B ) were normalized to the controls in their respective blots and subjected to Bootstrapping analysis ( Clèries et al . , 2012 ) .", "Statistical difference is indicated by *p<0 . 05 and **p<0 . 01 .", "The number of technical replicates , that is , the number of multiples of the same sample that were analyzed , is described in respective figure legends .", "Box plots were prepared using BoxPlotR ( http://boxplot . tyerslab . com/ ) .", "The center lines show the medians , box boundaries indicate the 25th and 75th percentiles , whiskers delineate maximum and minimum data points that are no more than 1 . 5 times the interquartile range , and dots represent individual data points .", "The notches are defined as ±1 . 58 times the interquartile range/square root of the sample number , and represent the 95% confidence interval for each median .", "The average is shown by + ." ] ]

[ "Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins , which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment .", "Using freeze fracture electron microscopy , we show here that the yeast NPC orthologs , Ncr1p and Npc2p , are essential for formation and expansion of raft-like domains in the vacuolar ( lysosome ) membrane , both in stationary phase and in acute nitrogen starvation .", "Moreover , the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism .", "We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole .", "These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions , likely by increasing sterol in the limiting membrane of the vacuole ." ]

[ "Niemann-Pick disease type C is a human disease that is characterized by severe neurological symptoms .", "The brains of children with this disease develop more slowly and adult patients have difficulty walking , coordinating movements , speaking clearly and they develop dementia .", "The disease is caused by mutations in two proteins called NPC1 and NPC2 , which are normally needed to move lipid molecules , especially cholesterol , between different compartments within a cell .", "Lysosomes are compartments within human cells that act as recycling points for many nutrients , including lipid molecules .", "The membranes surrounding lysosomes can bend to form pouches that can engulf the materials to be recycled .", "However , it was not clear exactly how this process , also known as microautophagy , happens and whether the NPC1 and NPC2 are involved .", "Here Tsuji et al . used a method called freeze fracture to study microautophagy in yeast under an electron microscope .", "For the experiments , the yeast cells were exposed to conditions that prevented them from dividing to mimic human nerve cells in the brain .", "The experiments show that NPC1 and NPC2 play important roles in creating and enlarging areas in the lysosome’s membranes that are rich in a lipid molecule called ergosterol , which yeast uses in the same way as animals use cholesterol .", "These areas , also known as rafts , promoted microautophagy of lipid storage compartments in the yeast cells , ensuring the healthy recycling of nutrients .", "Furthermore , when the normal version of NPC2 was replaced with a mutated form of NPC2 that could not bind to ergosterol , the yeast cells were less able to form ergosterol-rich rafts .", "These findings indicate that the symptoms of Niemann-Pick type C may be due , at least in part , to defects in the formation of cholesterol-rich lipid rafts in the membranes of lysosomes .", "Future experiments may investigate whether this microautophagy process , which depends on the NPC proteins in yeast , is exactly the same in human cells ." ]

[ "Introduction", "Materials and methods", "Results", "Discussion" ]

[ "epidemiology and global health", "tools and resources" ]

[ [ "Testing and isolating SARS-CoV-2-positive cases has been one of the main strategies for controlling the rise and spread of the coronavirus pandemic that has affected about 157 million people and 3 million deaths all over the world ( World Health Organization , WHO ) .", "Like all other viruses , the SARS-CoV-2 genome has undergone several mutations during its passage through the human host , and the positive or negative impact of most of them on virus transmission and immune escape are still under investigation ( Rabi et al . , 2020; Pachetti et al . , 2020; Korber et al . , 2020; Zhou et al . , 2021 ) .", "In December 2020 , health authorities in the United Kingdom had reported the emergence of a new SARS-CoV-2 variant ( lineage B . 1 . 1 . 7 ) that is phylogenetically distinct from other circulating strains in the region .", "With 23 mutations , this variant had rapidly replaced former SARS-CoV-2 lineages occurring in the region and has been established to have greater transmissibility through modeling and clinical correlation studies ( Chand , 2020; Horby et al . , 2021; Davies et al . , 2021; Volz et al . , 2021 ) .", "Subsequently , the South African and Brazilian authorities have also reported variants ( B . 1 . 351 and P . 1 , respectively ) that have been associated with a higher viral load in preliminary studies ( Tegally et al . , 2020; Faria et al . , 2021 ) .", "Till May 2021 , at least 13 emerging SARS-CoV-2 lineages with multiple mutations have been reported from sequencing efforts across the globe ( CDC , 2021 ) .", "Variants in lineages of the SARS-CoV-2 genome have been classified either as Variants of Concern ( VOC ) or Variants of Interest ( VOI ) .", "Among these , the VOCs have shown an indication of higher severity , transmissibility , and impact on pandemic spread from ongoing studies .", "VOIs have limited evidence for the same currently but are being monitored for potential impact in the future .", "Till May 2021 , at least five lineages ( B . 1 . 1 . 7 , P . 1 , B . 1 . 351 , B . 1 . 427 , and B . 1 . 429 ) have been classified as VOCs and are being investigated for their susceptibility to diagnosis , response to vaccines , and correlation with disease severity and transmission ( CDC , 2021 ) .", "As air travel has resumed in most countries post lockdown , infected individuals harboring these mutations have been detected in countries far away from their origin , generating concerns about greater disease incidence unless these cases are recognized and isolated .", "That is particularly important as vaccine roll-out has been initiated in several countries , and the studies about vaccine efficacy against the new variants are only in their infancy .", "Among the diagnostic methods that have been employed for identifying coronavirus infected cases , qRT-PCR has been the most widely adopted nucleic-acid-based test due to its ability to identify even low copy numbers of viral RNA in patient samples and has been considered as a gold standard in diagnosis ( Mackay , 2002; Carter et al . , 2020 ) .", "However , probe-based qRT PCR assays rely on amplification of a target gene with real-time analysis of DNA copy numbers and are generally not suited for genotyping point mutations associated with novel CoV2 variants ( Afzal , 2020; Yin , 2020; Vandenberg et al . , 2021 ) .", "Although strategies such as Amplification Refractory Mutation System-quantitative PCR ( ARMS-qPCR ) have been described in the literature for genotyping mutations in nucleic acids , these require substantial design and validation for reproducible readouts and hence are not routinely used for clinical diagnosis ( Little , 2001 ) .", "Similarly , antigen-based SARS-CoV-2 assays which have lower sensitivity than qPCR have not yet been reported to specifically identify mutant variants .", "In the absence of any true diagnostic test for the variants , most countries have resorted to next-generation sequencing of patient samples both for understanding the evolution of mutations in the virus as well as detecting the variants in suspected individuals ( Vandenberg et al . , 2021 ) .", "Using sequencing for routine diagnosis greatly increases both the time and cost of identifying positive individuals ( Jayamohan et al . , 2021 ) .", "We and several others have documented the applicability of CRISPR diagnostics ( CRISPRDx ) for identifying CoV2 signatures in patient samples ( Broughton et al . , 2020; Patchsung et al . , 2020; Ding et al . , 2020; Joung et al . , 2020; Fozouni et al . , 2021; Guo et al . , 2020; Lucia et al . , 2020; Wang et al . , 2021; Azhar et al . , 2021 ) .", "Using the Cas9 from Francisella novicida ( FnCas9 ) , we have recently reported the development of the FELUDA ( FnCas9 Editor Linked Uniform Detection Assay ) platform for SARS-CoV-2 diagnosis with similar accuracy as the gold standard qPCR test ( Azhar et al . , 2021 ) .", "The assay which is performed on a Lateral Flow strip generates a visual readout within an hour that is quantifiable using a smartphone-based application .", "Since FnCas9 has a very high intrinsic specificity to point mismatches in the target ( Acharya et al . , 2019 ) , we speculated that the enzyme can also be used for detecting SARS-CoV-2 variants on a paper strip with high accuracy .", "In this report , we introduce RAY ( Rapid variant AssaY ) , a paper-strip based platform to identify mutational signatures of the coronavirus variants in a sample eliminating the need for sequencing-based diagnostics .", "RAY can successfully detect both SARS-CoV-2 infection as well as the presence of the common N501Y mutation present across the majority of VOCs described so far and distinguish it from the parent CoV2 lineage .", "Thus , it can be employed as a primary surveillance method for isolating cases belonging to these groups and can enable the initiation of appropriate management protocols ." ], [ "The study was designed to evaluate the efficacy of RAY on left-over patient samples .", "The intent of the study was to develop a robust CRISPR diagnostic that can perform with high accuracy for variant detection at significantly low cost and time taken for detection as compared to sequencing .", "For the N501Y detection using RAY , SARS-CoV-2 and control RNA samples were received from the diagnostic laboratory at CSIR-Institute of Genomics and Integrative Biology .", "A list of all oligos ( Merck ) used in the study can be found in Supplementary file 4 .", "Plasmids containing FnCas9-WT and dFnCas9 ( catalytically-inactive , dead ) ( Acharya et al . , 2019 ) sequences were transformed and expressed in Escherichia coli Rosetta 2 ( DE3 ) ( Novagen ) .", "Rosetta 2 ( DE3 ) cells were cultured at 37°C in LB medium ( with 50 mg/ml Kanamycin ) and induced when OD600 reached 0 . 6 , using 0 . 5 mM isopropyl β-D-thiogalactopyranoside ( IPTG ) .", "After an overnight culture at 18°C , E . coli cells were harvested by centrifugation and resuspended in a lysis buffer ( 20 mM HEPES , pH 7 . 5 , 500 mM NaCl , 5% ) glycerol supplemented with 1X PIC ( Roche ) containing 100 µg/ml lysozyme .", "Subsequent cell lysis by sonication , the lysate was put through to Ni-NTA beads ( Roche ) .", "The eluted protein was further purified by size-exclusion chromatography on HiLoad Superdex 200 16/60 column ( GE Healthcare ) in a buffer solution with 20 mM HEPES pH 7 . 5 , 150 mM KCl , 10% glycerol , 1 mM DTT .", "The purified proteins were quantified by Pierce BCA protein assay kit ( Thermo Fisher Scientific ) and stored at −80°C until further use .", "All 48 VOCs/VOIs within 12 emerging SARS-CoV-2 lineages till May 2021 were taken from the Centers for Disease Control and Prevention report ( CDC , 2021; Rambaut et al . , 2020; Hadfield et al . , 2018 ) .", "Further , by having these mutations at 2nd/6th/16th/19th bp upstream to PAM ( NGG ) in the SARS-CoV2- genome , a total of 19 among 48 variants could be targeted with the RAY strategy .", "Next , within the variant nucleotide containing crRNA sequence , a second synthetic mismatch nucleotide at the corresponding 6th/2nd/19th/16th position was added .", "Primers required for IVC assays flanking these crRNAs were designed using Primer3plus python library ( Untergasser et al . , 2012 ) .", "Finally , the crRNAs/primers were checked for off-targets on a representative bacterial genome database ( NCBI ) , virus genome database ( NCBI ) ( Brister et al . , 2015 ) , and human genome/transcriptome ( GENCODE GRCh38 ) ( Frankish et al . , 2019 ) .", "For the benefit of end users with quick design and implementation of the RAY for any targetable SNV/VOC/VOI , we now have also optimized our previously developed web tool JATAYU ( Junction for Analysis and Target Design for Your FELUDA assay ) ( Azhar et al . , 2021 ) , which functions by incorporating mismatches in the sequence provided by the user .", "The server generates sgRNA and flanking primer sequences ready for synthesis ( http://jatayu . igib . res . in ) .", "Purified PCR 228 bp amplicon from HBB gene was used as substrate in in vitro cleavage assays , as optimized in our previous studies ( Acharya et al . , 2019 ) .", "Substrate amplicons were treated with reconstituted dFnCas9 RNP complex ( 100 nM ) in a reaction buffer ( 20 mM HEPES , pH7 . 5 , 150 mM KCl , 1 mM DTT , 10% glycerol , 10 mM MgCl2 ) at 37°C for 10 min , the cleaved products were visualized on a 2% agarose gel and densitometric quantification of images were done ( Figure 1A ) .", "Sequences containing WT and Mutant were reverse transcribed , and amplified using primers with/without 5’ biotinylation from SARS-CoV-2 Viral RNA-enriched samples .", "Purified PCR amplicons containing WT and Mutant sequences were used as substrates in in vitro cleavage assays , as optimized in our previous studies ( Azhar et al . , 2021 ) .", "Substrate amplicons were treated with reconstituted RNP complex ( 100 nM ) in a reaction buffer ( 20 mM HEPES , pH7 . 5 , 150 mM KCl , 1 mM DTT , 10% glycerol , 10 mM MgCl2 ) at 37°C for 10 min , and cleaved products were visualized on a 2% agarose gel .", "A region from the SARS-CoV-2 S gene containing N501Y/T716I/E484K mutations was reverse transcribed and amplified using a single end 5’ biotin-labeled primer .", "In vitro transcription of sgRNAs/crRNAs was done using MegaScript T7 Transcription kit ( ThermoFisher Scientific ) following manufacturer’s protocol and purified by NucAway spin column ( ThermoFisher Scientific ) .", "Chimeric gRNA ( crRNA:TracrRNA ) was prepared by equally ( crRNA:TracrRNA molar ratio , 1:1 ) combining respective crRNAs and synthetic 3'-FAM-labeled TracrRNA in an annealing buffer ( 100 mM NaCl , 50 mM Tris-HCl pH8 and 1 mM MgCl2 ) by heating at 95°C for 2–5 min and then allowed to cool at room temperature for 15–20 min .", "RNP complex was prepared by equally mixing ( Protein:sgRNA molar ratio , 1:1 ) Chimeric gRNA and dead FnCas9 in a buffer ( 20 mM HEPES , pH7 . 5 , 150 mM KCl , 1 mM DTT , 10% glycerol , 10 mM MgCl2 ) and rested for 10 min at RT .", "Target biotinylated amplicons were then treated with the RNP complexes for 10 min at 37°C .", "Finally , 80 µl of Dipstick buffer was added to the mix along with Milenia HybriDetect one lateral flow strip .", "The strips were allowed to stand in the solution for 2–5 min at room temperature and the result was observed .", "The strip images can be processed using the TOPSE application that generates background corrected values from the smart phone acquired images of the strip .", "This application has been previously trained on a large number of CoV-2 samples ( Azhar et al . , 2021 ) .", "In vitro synthesized crRNAs used for double amplicon RAY were a gift from TATA Medical and Diagnostics .", "Detailed description of the protocol can be found in Appendix 1 .", "qRT-PCR was performed using STANDARD M nCoV Real-Time Detection kit ( SD Biosensor ) as per manufacturer’s protocol .", "Briefly , per reaction 3 µl of RTase mix and 0 . 25 µl of Internal Control A was added to 7 µl of the reaction solution .", "Five µl of each of the negative control , positive control , and patient sample nucleic acid extract was added to the PCR mixture dispensed in each reaction tube .", "The cycling conditions on the instrument were as follows: Reverse transcription 50°C for 15 min , Initial denaturation 95°C for 1 min , 5 Pre-amplification cycles of 95°C for 5 s; 60°C for 40 s followed by 40 amplification cycles of 95°C for 5 s; 60°C for 40 s .", "Signal was captured in the FAM channel for the qualitative detection of the new coronavirus ( SARS-CoV-2 ) ORF1ab ( RdRp ) gene , JOE ( VIC or HEX ) channel for E gene , and CY5 channel for internal reference .", "Individuals who were found to be RT-PCR positive for SARS-CoV-2 , were sequenced to determine whether it was a UK variant ( 20I/501Y . V1 ) or non-UK variant .", "With a low number of samples arriving each day in the lab and with quick turnaround time for reporting , Nanopore sequencing was undertaken for most of the samples .", "In brief , 100 ng total RNA was used for double-stranded cDNA synthesis by using Superscript IV ( ThermoFisher Scientific , Cat . No . 18091050 ) for first strand cDNA synthesis followed by RNase H digestion of ssRNA and second strand synthesis by DNA polymerase-I large ( Klenow ) fragment ( New England Biolabs , Cat . No . M0210S ) .", "Double stranded cDNA thus obtained was purified using AMPure XP beads ( Beckman Coulter , Cat . No . A63881 ) .", "The SARS-CoV-2 genome was then amplified from 100 ng of the purified cDNA following the ARTIC V3 primer protocol .", "Sequencing library preparation consisting of End Repair/dA tailing , Native Barcode Ligation , and Adapter Ligation was performed with 200 ng of the multiplexed PCR amplicons according to Oxford Nanopore Technology ( ONT ) library preparation protocol-PCR tiling of COVID-19 virus ( Version: PTC_9096_v109revE_06Feb2020 ) .", "Sequencing in sets of 24 barcoded samples was performed on MinION Mk1B platform by ONT .", "The ARTIC end-to-end pipeline was used for the analysis of MinION raw fast5 files up to the variant calling .", "Raw fast5 files of samples were basecalled and demultiplexed using Guppy basecaller that uses the base calling algorithms of Oxford Nanopore Technologies ( https://community . nanoporetech . com ) with phred quality cut-off score >7 on GPU-linux accelerated computing machine .", "Reads having phred quality scores less than seven were discarded to filter the low-quality reads .", "The resulting demultiplexed fastq were normalized by read length of 300–500 ( approximate size of amplicons ) for further downstream analysis and aligned to the SARS-CoV-2 reference ( MN908947 . 3 ) using the aligner Minimap2 v2 . 17 ( Li , 2018 ) .", "Nanopolish ( Loman et al . , 2015 ) was used to index raw fast5 files for variant calling from the minimap output files .", "To create consensus fasta , bcftools v1 . 8 was used over normalized minimap2 output ." ], [ "In an earlier study , we had successfully established that FnCas9 is unable to bind or cleave targets having two mismatches at the 2nd and 6th position ( PAM proximal ) of the sgRNA with respect to the target ( Azhar et al . , 2021; Figure 1A ) .", "Through in vitro cleavage studies of a double-stranded DNA substrate , we identified that the same outcome is also observed when mismatches are present at the 16th and 19th position ( PAM distal ) ( Figure 1A , Materials and methods ) .", "This implies that if a mismatch exists in any of these position combinations , placing an additional synthetic mutation in the sgRNA at the other positions makes FnCas9 unable to bind or cleave the target ( Figure 1A ) .", "Thus , mutations at any of these positions relative to a NGG PAM site in the SARS CoV-2 variants could be potentially distinguished from the parent strain .", "To develop RAY for identifying SARS-CoV-2 variants , we first analyzed the mutations arising in all SARS-CoV-2 lineages reported so far ( May 2021 ) and looked for the possibility of FnCas9-mediated targeting based on the presence of an NGG PAM site in the vicinity .", "We found that out of the 48 unique SNVs reported across all the VOCs and VOIs reported so far ( CDC , 2021 ) , 19 SNVs ( 12 VOC-associated SNVs and 7 VOI-associated SNVs ) can be targeted by RAY ( Supplementary file 1 ) .", "Interestingly , each VOC/VOI reported so far consists of at least one of the 19 SNVs and can therefore be targeted by RAY design ( Figure 1A ) .", "Among these SNVs , N501Y is present across 3/5 of the VOCs ( B . 1 . 1 . 7 , P . 1 , and B . 1 . 351 ) .", "This mutation affects the receptor-binding domain of the virus and is the subject of numerous studies analyzing the efficacy of vaccines against the mutated form of the virus ( Cheng et al . , 2021; Dejnirattisai et al . , 2021; Ku et al . , 2021; Luan et al . , 2021; Naveca et al . , 2021; Tian et al . , 2021; Xie et al . , 2021; Collier et al . , 2021 ) .", "Owing to its prevalence as a shared marker for the majority of mutated lineages , we took this SNV further for developing a diagnostic assay .", "We designed primer pairs surrounding the N501Y mutation after analyzing the mutational spectrum in SARS-CoV-2 strains obtained from the publicly available sequencing database , GISAID ( Shu and McCauley , 2017 ) .", "Additionally , we ensured that no regions from human or non-human genome as well as transcriptome shared significant homology to the sites to prevent any non-specific amplification during the reverse transcription PCR ( RT-PCR ) reaction ( Materials and methods ) .", "In our earlier study , we have validated two FnCas9 sgRNAs in the N and S gene of SARS-CoV-2 which could detect positive cases with high sensitivity and specificity ( Azhar et al . , 2021 ) .", "We reasoned that including the S-gene sgRNA which lies in the vicinity of the N501Y variant would serve as an internal positive control both for the presence of the SARS-CoV-2 virus in the sample as well as quality control for the amplicons generated in the RT PCR step of the assay .", "We tested the N501Y sgRNA containing the variant mismatch at PAM proximal 2nd position to selectively bind and cleave the mutant substrate , while not affecting the WT substrate due to mismatch at PAM proximal 2nd and 6th positions .", "We found that catalytically active FnCas9 was able to successfully cleave the dsDNA substrate containing the N501Y mutation while leaving the WT sequence intact ( Figure 2A ) .", "Importantly , this leads to a distinct pattern on agarose gel that can distinguish the two variants .", "We established that this approach for variant identification can be performed using a stand-alone or portable electrophoresis apparatus leading to a turnaround time of about 1 . 5 hr from RNA to read-out ( Figure 2A ) .", "The electrophoresis based identification of the N501Y ( A23063T ) mutation can be adapted for COVID-19 detection under laboratory conditions .", "However , dye-based systems have inherent low sensitivity and cannot be used for routine diagnosis .", "We reasoned that for RAY to detect and diagnose variants , we need to adapt the detection to a more sensitive readout .", "In an earlier study , we had reported that using a lateral flow assay , a catalytically inactive ( dead ) FnCas9 generated by mutating the RuvC and HNH domains can be used to accurately identify the presence of SARS-CoV-2 RNA in patient samples ( Azhar et al . , 2021 ) .", "To enable such a readout , the RNA sample first undergoes reverse transcription PCR using biotin-labeled primers , generating labeled amplicons at the end of the PCR reaction .", "The products from the reaction are then incubated with dFnCas9:sgRNA ribonucleoprotein complex where the sgRNA component is labeled with Fluorescein amidite ( FAM ) at the 3’ end .", "Upon identifying its target , the RNP forms a tight complex with the dsDNA substrate .", "This complex is then applied on a paper strip that has gold nanoparticles tagged with antibodies recognizing the FAM label .", "In an aqueous environment , the nanoparticle:RNP:substrate complex moves up the strip by lateral flow and upon reaching a streptavidin line on the strip these get deposited if the RNP is attached to biotin-labeled substrates ( test band ) .", "Residual or unbound AuNP complexes get deposited at a control line where secondary antibodies recognizing the anti-FAM antibody attached to the AuNPs are immobilized on the strip ( Figure 2B , C ) .", "We reasoned that in order to enable RAY to distinguish two samples different by a single mismatch on a paper strip , the intensity of the one mismatched sgRNA ( as the other mismatch corresponds to N501Y SNV ) should be several folds higher than wild-type samples ( having two mismatches with the sgRNA , one at the SNV position and another synthetic mismatch ) .", "To generate a visually distinctive signal between WT and mutant sample , we performed a single-step reverse transcription PCR to generate a biotin-labeled amplified product that can be detected by a single sgRNA ( called SWT ) if the sample is WT and by both sgRNAs ( SWT and SN501Y ) if the sample contains the N501Y variant ( Figure 2B , C ) .", "The presence of the SWT band also serves as a validation for the sample to be SARS-CoV-2 positive .", "We first generated a single amplicon labeled with biotin at one end and performed RAY with both sgRNAs ( SWT and SN501Y ) ( Figure 3A ) .", "We reasoned that labeling biotin in the reverse primer would reduce possible background signal due to non-specific interactions of the unused biotin primers with the streptavidin line on the strip and increase the signal resolution between the mutant and WT samples .", "Among the four amplicon sizes that we investigated , an amplicon of 580 bp gave a clear discernible signal distinguishing the N501Y mutation over the WT sample ( Figure 3A , Materials and methods ) .", "To validate the reproducibility of this method , we took mutant and WT substrates , and performed RAY 10 times , and were able to successfully distinguish them on every occasion ( Figure 3B ) .", "Next , we tested RAY on RNA extracted from samples of eight qRT-PCR-positive SARS-CoV-2-infected individuals who harbored the N501Y mutation ( along with other mutations ) .", "The samples were sequenced in parallel to detect the presence of N501Y mutation .", "RAY was able to correctly identify the variant signature in all eight samples that harbored the mutation ( Figure 3C ) .", "However , we observed variability of signal intensities across the different samples .", "In particular , samples with low viral load ( qRT-PCR Ct >25 ) showed a comparatively faint band ( Figure 3C ) .", "This suggested that the RAY protocol required further optimization to increase the signal intensity , especially for low viral titres .", "Importantly , RAY correctly classified all eight WT samples where neither the N501Y mutation nor any other lineage variants were present as seen by either an absence of a distinct band or an extremely faint band in the test line ( Figure 3C ) .", "In addition , RAY identified all confirmed COVID-19-negative samples ( 12 ) tested simultaneously ( Figure 3C ) .", "Taken together , the single amplicon RAY assay showed good specificity in identifying WT and SARS-CoV-2-negative samples but required improvements to increase the sensitivity for samples with low viral load .", "To improve the performance of the assay for patient samples across a wide range of viral titres , we modified several aspects of the RAY assay .", "Firstly , we used TOPSE ( True Outcome Predicted via Strip Evaluation ) , a smartphone-based application to generate a band intensity score from a lateral flow strip to eliminate bias that can result from visual estimation ( Appendix 1 ) .", "Secondly , we labeled both forward and reverse primers in the assay with 5' Biotin to increase the signal intensity and reduced the amplicon length of the substrate to get a consistent amplification at the end of each PCR run ( Figure 4A , B ) .", "We observed that upon double biotinylation the test band intensity for the 580 bp amplicon was 1 . 5 times higher than single biotin-labeled amplicon ( Figure 4A ) .", "To increase the band intensity further , we reduced the length of the PCR amplicon containing the N501Y mutation and found that within the same sample , amplicons with shorter lengths had higher band intensities ( Figure 4B ) .", "In particular , with an amplicon size of 149 bp , a clearly discernible background-corrected signal intensity could be achieved in the mutant sample over the WT sample ( Figure 4B ) .", "Since the 149 bp amplicon did not contain the SWT sgRNA-binding site , we modified the assay to perform two simultaneous PCR reactions from the same sample , one generating the N501Y specific amplicon ( 149 bp ) , and another corresponding to the SWT sgRNA from the original FELUDA assay ( 287 bp ) ( Figure 4C ) .", "The double amplicon RAY assay results in shorter products and lower overall time for completion , starting from RNA samples while compared to a single amplicon RAY ( reduced to 55 min from 75 min ) .", "Next , we investigated the sensitivity of the assay on serial dilutions of a WT and a N501Y mutant patient sample with moderately high viral load ( Ct <25 ) .", "We observed that RAY was able to give a clearly discernible SN501Y sgRNA signal that was at least 5 . 5 times higher than that of the WT sample up to a Ct value of 34 .", "Upon further dilution , no signal was observed both in RAY or qRT PCR for these samples ( Figure 4E ) .", "Taken together , RAY is sufficiently sensitive in detecting the N501Y VOC in a sample with only a few copies of the virus and is comparable to other sequencing platforms ( Klempt et al . , 2020 ) .", "We then proceeded to test RAY on a set of patient samples containing either the WT ( n = 37 ) or the N501Y variants ( n = 22 ) identified by sequencing .", "For each of these samples , a qRT-PCR and the SWT RAY were first performed to confirm SARS-CoV-2 positivity ( Appendix 1 , Supplementary file 1 ) .", "With a single run of the assay , SN501Y was able to detect 36/37 WT samples and 19/22 N501Y samples corresponding to a sensitivity of 86% and a specificity of 97% across all ranges of Ct values ( Figure 4F , Supplementary file 2 ) .", "Thus , RAY can identify the N501Y SNV with high accuracy in patient samples underscoring its impact as a rapid screening methodology for SARS-CoV-2 VOCs .", "Notably , we were able to successfully validate RAY for two more mutations E484K and T716I in patient samples using corresponding sgRNAs suggesting that the assay can be optimized and adapted for other VOCs in addition to N501Y ( Figure 4G ) .", "Among these , the E484K mutation has been associated with high transmissibility and the possibility of reinfection ( Weisblum et al . , 2020; Xie et al . , 2021; Collier et al . , 2021; Wibmer et al . , 2021; Annavajhala et al . , 2021; Resende et al . , 2021; Nonaka et al . , 2021; Naveca et al . , 2021 ) .", "Importantly , amplicons containing the E484K mutation do not show cross-reactivity with the N501Y sgRNA , highlighting the specificity of the assay between variants ( Figure 4H ) .", "Finally , as seen in our earlier study , synthetic sgRNA containing a phosphorothioate backbone modification leads to higher stability and greater band intensity when compared to in vitro synthesized sgRNAs ( Figure 4I ) ." ], [ "Currently , deep-sequencing of patient samples is being routinely done by health authorities in various countries to track and isolate cases containing the SARS-CoV-2 variants ( Meredith et al . , 2020; Oude Munnink et al . , 2020; McNamara et al . , 2020; Rockett et al . , 2020; Bhoyar et al . , 2021 ) .", "The accuracy of variant calling in any sequencing platform is dependent on the very high quality and coverage of reads to the reference genome .", "Since the process from sample to sequence consists of multiple steps , low viral titres can be missed out during sequencing assays for variant calling .", "The RAY pipeline uses an amplification step followed by enzymatic binding and is reproducible across a wide range of viral titres tested .", "The main advantage of sequencing COVID-19 samples is the generation of cumulative information about all mutations co-existing in a given variant ( Chiu and Miller , 2019 and Vandenberg et al . , 2021 ) .", "This information is valuable to track and trace how lineages evolve and has led to the identification of mutations in the first place .", "RAY can identify only known VOCs and is therefore useful as a diagnostic tool for the early detection of variant signatures in a sample .", "For example , determining whether a sample belongs to the South African lineage through E484K specific RAY can allow early quarantine of the patient and prevent the spreading of a highly transmissible variant .", "Similarly , identifying a WT sample rapidly through RAY negates the necessity of sequencing such a sample thereby reducing the cost of the associated sequencing exercise .", "Together , this can ease the burden of surveillance and ensure both rapid disease control measures as well as the identification of newer variants efficiently .", "In the current format ( lateral flow assay ) , RAY has limitations in multiplexing several VOC candidates in a single assay .", "Although longer amplicons can be used to identify multiple mutations that appear close to each other , we observed a drop in sensitivity probably due to reduced PCR efficiency for longer amplicons .", "Similarly , to increase the throughput of the assay , converting the readout into a fluorescence-based mode might be beneficial .", "Perhaps , the most imperative area of improvement for RAY is increasing the number of possible mutations that can be targeted .", "The necessity of an NGG PAM at a fixed position away from the position of the target mutation limits the total number of targets that RAY can identify .", "This can be significantly expanded by systematically exploring more sites in the sgRNA where a mutation weakens the enzyme:substrate interaction .", "Alternatively , engineering the enzyme to generate PAM-flexible versions can be considered .", "Routine sequencing of patient samples for disease surveillance is fraught with issues such as cost , logistics , and turnaround times .", "A typical deep sequencing cost per sample is several orders of magnitude higher than RAY ( which costs ~7 USD per sample , Supplementary file 3 ) and the minimum turnaround time for sequencing is 36 hr .", "Importantly , sequencing runs are generally done on pooled samples to save on cost , workforce , and machine time , all of which necessitate the advent of alternate solutions that can identify variants through a simple pipeline .", "RAY -on the contrary- is scalable to any number of samples at a given time .", "Sequencing also generates a large amount of data and requires physical storage and high-performance computing methodologies to record , analyze and interpret the data ( Chiu and Miller , 2019 and Vandenberg et al . , 2021 ) .", "In addition , dedicated personnel with a substantial understanding of sequence analysis and programming skills are necessary for identifying variants within reads generated from a sample .", "In contrast , RAY uses a visual readout on a paper strip leading to a binary decision about the sample .", "The automated image acquisition and recording through a smartphone app allows operators with a minimum skill set to process samples .", "This has the advantage of rapid screening , particularly at the point of care settings .", "In this study , we report the development of a CRISPR diagnostic platform for detecting the major VOCs in the SARS-CoV-2 genome .", "The outcomes of this platform can be extended to other pathogenic SNVs and can provide a robust solution for rapid variant calling in patient samples .", "Considering the rise and spread of mutated lineages of the virus in multiple countries , it is imperative to develop technologies that can quickly and accurately detect these variants .", "Since the first-generation vaccines against SARS-CoV-2 have been developed against the parent strain , careful assessment of the variant lineages and their response to the vaccine will become imperative during the course of the pandemic ( Fontanet et al . , 2021; Walensky et al . , 2021 ) .", "Novel variants , particularly those correlated with adverse disease manifestation , need to be controlled so that these do not spread rampantly across populations .", "Testing and isolating them will thus continue to be at the forefront of disease control measures ." ] ]

[ "The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health , causing increased mortality among elderly patients and individuals with comorbid conditions .", "During the passage of the virus through affected populations , it has undergone mutations , some of which have recently been linked with increased viral load and prognostic complexities .", "Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR ( qRT-PCR ) method and necessitates widespread sequencing which is expensive , has long turn-around times , and requires high viral load for calling mutations accurately .", "Here , we repurpose the high specificity of Francisella novicida Cas9 ( FnCas9 ) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples .", "We report the detection of the S gene mutation N501Y ( present across multiple variant lineages of SARS-CoV-2 ) within an hour using lateral flow paper strip chemistry .", "The results were corroborated using deep sequencing on multiple wild-type ( n = 37 ) and mutant ( n = 22 ) virus infected patient samples with a sensitivity of 87% and specificity of 97% .", "The design principle can be rapidly adapted for other mutations ( as shown also for E484K and T716I ) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics ( CRISPRDx ) for disease surveillance even beyond COVID-19 .", "This study was funded by Council for Scientific and Industrial Research , India ." ]

[ "SARS-CoV-2 , the virus responsible for COVID-19 , has a genome made of RNA ( a nucleic acid similar to DNA ) that can mutate , potentially making the disease more transmissible , and more lethal .", "Most countries have monitored the rise of mutated strains using a technique called next generation sequencing ( NGS ) , which is time-consuming , expensive and requires skilled personnel .", "Sometimes the mutations to the virus are so small that they can only be detected using NGS .", "Finding cheaper , simpler and faster SARS-CoV-2 tests that can reliably detect mutated forms of the virus is crucial for public health authorities to monitor and manage the spread of the virus .", "Lateral flow tests ( the same technology used in many pregnancy tests ) are typically cheap , fast and simple to use .", "Typically , lateral flow assay strips have a band of immobilised antibodies that bind to a specific protein ( or antigen ) .", "If a sample contains antigen molecules , these will bind to the immobilised antibodies , causing a chemical reaction that changes the colour of the strip and giving a positive result .", "However , lateral flow tests that use antibodies cannot easily detect nucleic acids , such as DNA or RNA , let alone mutations in them .", "To overcome this limitation , lateral flow assays can be used to detect a protein called Cas9 , which , in turn , is able to bind to nucleic acids with specific sequences .", "Small changes in the target sequence change how well Cas9 binds to it , meaning that , in theory , this approach could be used to detect small mutations in the SARS-CoV-2 virus .", "Kumar et al . made a lateral flow test that could detect a Cas9 protein that binds to a nucleic acid sequence found in a specific mutant strain of SARS-CoV-2 .", "This Cas9 was highly sensitive to changes in its target sequence , so a small mutation in the target nucleic acid led to the protein binding less strongly , and the signal from the lateral flow test being lost .", "This meant that the lateral flow test designed by Kumar et al . could detect mutations in the SARS-CoV-2 virus at a fraction of the price of NGS approaches if used only for diagnosis .", "The lateral flow test was capable of detecting mutant viruses in patient samples too , generating a colour signal within an hour of a positive sample being run through the assay .", "The test developed by Kumar et al . could offer public health authorities a quick and cheap method to monitor the spread of mutant SARS-CoV-2 strains; as well as a way to determine vaccine efficacy against new strains ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "short report", "neuroscience" ]

[ [ "Historically , the sensation of fullness has been documented as far back as Homer's Odyssey .", "Pioneering work by Cannon and Washburn revealed a correlation between stomach expansion and satiety in humans ( Cannon and Washburn , 1911 ) , which was later confirmed in rodents ( Hargrave and Kinzig , 2012 ) .", "Recently , several groups have shown that feeding-related neurons are sensitive to satiety state but not nutrients in Drosophila ( Marella et al . , 2012; Dus et al . , 2013; Pool et al . , 2014 ) .", "These studies argue that non-metabolic inputs such as mechanic tension could regulate feeding ." ], [ "Recent studies in Drosophila have identified neuronal regulation of food intake and nutrient sensing in the central nervous system ( Marella et al . , 2012; Dus et al . , 2013 ) .", "However , the potential contribution of the enteric neurons in the gastrointestinal tract has not been explored .", "To investigate this , we utilized four previously characterized Gal4 lines that are expressed in enteric neurons in the different parts of the Drosophila digestive system ( Figure 1A , B ) .", "While the GMR48A05-Gal4 neurons project to the proventriculus and the anterior midgut , the GMR51F12-Gal4 neurons project to the anterior midgut and the crop ( Figure 1A , B ) ( Jenett et al . , 2012 ) .", "In contrast , both HGN1-Gal4 and Ilp7-Gal4 neurons project to two posterior regions in the gut: ( 1 ) the hindgut pylorus and the connecting posterior midgut and anterior hindgut , and ( 2 ) the rectum pylorus and the rectum ( referred to as the posterior enteric neurons or the PENs , Figure 1A , B ) ( Cognigni et al . , 2011 ) . 10 . 7554/eLife . 04402 . 003Figure 1 . Modulating activities of Drosophila PENs causes metabolic defects .", "( A ) Enteric neural projections of Gal4 lines tested ( red , phalloidin; green , UAS-mCD8::GFP ) and their diagram ( B ) .", "GMR51F12-Gal4 neurons project to the foregut , anterior midgut and crop .", "GMR48A05-Gal4 neurons project to the proventriculus and anterior midgut .", "Both HGN1-Gal4 and Ilp7-Gal4 drive expression in the neurons projecting to the posterior midgut , hindgut pylorus , anterior hindgut , rectal pylorus and the rectum .", "Pr , Proventriculus; C , Crop; Py , Pylorus; RP , Rectal Pylorus; R , Rectum; VNC , Ventral Nerve Cord .", "The effects of activating ( C ) or inactivating ( D ) enteric neurons on hemolymph glucose ( GMR51F12-Gal4 , GMR48A05-Gal4 , Ilp7-Gal4 , or HGN1-Gal4; UAS-TRPA1 or UAS-shiTS1 ) ( n = 6–10 replicates of 10 flies ) .", "( E ) The effect of silencing the PENs in starvation conditions ( Ilp7-Gal4 or HGN1-Gal4; UAS-shiTS1 ) ( n = 6–9 replicates of 10 flies ) .", "* = p < 0 . 05 , compared to corresponding UAS and Gal4 control .", "Significances indicated are based on ANOVA and Tukey post-hoc test .", "Data represent the average ± s . e . m . of the results obtained . DOI: http://dx . doi . org/10 . 7554/eLife . 04402 . 003 We first used these Gal4 lines to activate specific enteric neurons by expressing the temperature-sensitive ion channel , TRPA1 , and measured the effects on hemolymph glucose levels ( UAS-TRPA1 [Hamada et al . , 2008] ) .", "Activation of the Ilp7-Gal4 neurons significantly decreases glucose levels in comparison to the Ilp7-Gal4 and UAS-TRPA1 controls ( Figure 1C ) .", "Since Ilp7-Gal4 is also expressed in the CNS and in neurons projecting to the reproductive organs ( Yang et al . , 2008 ) , we did similar experiment using HGN1-Gal4 , which expresses in the PENSs , but not in the other Ilp7-Gal4 neurons ( Cognigni et al . , 2011 ) , and obtained similar results on glucose levels ( Figure 1C ) .", "We next examined enteric neurons projecting to other regions of the digestive system using GMR48A05-Gal4 and GMR51F12-Gal4 and did not observe any obvious effect ( Figure 1C ) .", "We then assayed the effects of silencing these neurons by expressing the temperature-sensitive Dynamin , ShiTS1 ( UAS-ShiTS1 [Kitamoto , 2013] ) .", "Silencing of the PENs using both Ilp7-Gal4 and HGN1-Gal4 increased glucose levels in comparison to the controls , while no effect was observed by silencing the GMR48A05-Gal4 and GMR51F12-Gal4 neurons ( Figure 1D ) .", "Together , the data indicate that activities of the PENs are integral in the regulation of hemolymph glucose levels .", "The change of glucose levels could result from differences in food intake .", "Previous work by Miguel-Aliaga and colleagues revealed that silencing Ilp7-Gal4 neurons increases defecation ( Cognigni et al . , 2011 ) , which could be the result of increased feeding .", "We therefore examined the hypothesis that the activities of the PENs regulate food intake .", "Consistent with the hypothesis , we silenced the PENs in the absence of food and found that this abolished the gains in glucose levels ( Figure 1E ) .", "We next investigated this directly using the capillary feeding assay ( Ja et al . , 2007 ) .", "Silencing the PENs dramatically increased food intake ( Figure 2A ) , which is consistent with the gains in glucose levels seen earlier .", "Conversely , activating these neurons caused dramatic decreases in feeding ( Figure 2B ) .", "Together , manipulation of the activities of the PENs results in an overall six-fold change in feeding in comparison to the controls .", "This change is significantly larger than alterations by modulation of neuropeptide F signaling ( Hong et al . , 2012 ) .", "These data indicate that the activities of the PENs play a prominent role in feeding . 10 . 7554/eLife . 04402 . 004Figure 2 . PPK1 functions in Drosophila PENs to regulate feeding .", "( A–B )", "Results of capillary feeding assays by either inactivating ( A , Ilp7-Gal4 or HGN1-Gal4; UAS-shiTS1 ) or activating ( B , Ilp7-Gal4 or HGN1-Gal4; UAS-TRPA1 ) the PENs ( n = 4–8 replicates ) .", "( C ) Outside and inside views of the hindgut ( red , phalloidin , muscle ) with posterior enteric neuron projections ( green , 22C10 ) .", "( D ) PPK1 expresses in the PENs projecting to the hindgut pylorus ( left ) and rectum ( right ) ( PPK1-Gal4;UAS-mCD8::GFP ) .", "( E ) The effect of PPK1 knock-down on food intake ( Ilp7-Gal4 or HGN1-Gal4 , UAS-PPK1-RNAi#1 or UAS-PPK1-RNAi#2 ) ( n = 3–8 replicates ) .", "( F ) Food intake results for PPK1 deficiency ( dfb88h49/dfA400 ) and rescued animals ( dfb88h49/dfA400; Ilp7-Gal4 , UAS-PPK1 ) ( n = 4–7 replicates ) .", "( G ) Food intake results when PPK1 is inhibited using benzamil in wild-type or Ilp7 > PPK1 RNAi #1 flies ( n = 8–10 replicates ) .", "* = p < 0 . 05 , compared to corresponding UAS and Gal4 control or indicated controls .", "Significances indicated are based on ANOVA and Tukey post-hoc test .", "Data represent the average ± s . e . m . of the results obtained . DOI: http://dx . doi . org/10 . 7554/eLife . 04402 . 004 As described above , experiments in mammals indicated that mechanic tension in the gastrointestinal tract could be a satiety signal ( Cannon and Washburn , 1911; Hargrave and Kinzig , 2012 ) .", "To determine whether the role of the PENs in feeding is related to mechanosensing activity , we first examined the projections of these neurons and found that they tightly wrap around the muscle layer rather than projecting into the lumen of the gut ( Figure 2C ) .", "This anatomy favors an involvement of mechanosensory activity rather than in detecting nutritional signals in gastrointestinal tract .", "Previous studies in Caenorhabditis elegans , Drosophila , and mice have shown that members of the Degenerin/Epithelial Sodium Channels ( DEG/ENaCs ) function as a conserved family of mechanosensory ion channels ( O'Hagan et al . , 2005; Hwang et al . , 2007; Zhong et al . , 2010 ) .", "Mutants of the C . elegans DEG/ENaC Mec-4 and its Drosophila homolog , PPK1 , are touch-insensitive and the affected neurons fail to generate action potentials in response to mechanic tension ( O'Hagan et al . , 2005; Hwang et al . , 2007; Zhong et al . , 2010 ) .", "This raised the possibility that PPK1 could be involved in regulating feeding in the PENs .", "We thus examined PPK1 expression using PPK1-Gal4 driving mCD8::GFP and confirmed its presence in the PENs ( Figure 2D ) .", "To test whether the function of PPK1 in the PENs is involved in the regulation of feeding , we first assayed the effect of RNAi knockdown of PPK1 expression ( Ilp7-Gal4 or HGN1-Gal4//UAS-PPK1-RNAi ) .", "Knockdown of PPK1 in the PENs , but not knockdown of PPK29 , a related family member ( Thistle et al . , 2012 ) , dramatically increased feeding ( Figure 2E ) , phenocopying the effects of silencing these neurons .", "Next , we examined the effect of PPK1 deficiency on feeding and found that PPK1 deficient flies have increased food intake ( Figure 2F ) .", "This feeding defect is rescued by expressing a PPK1 transgene in the PENs ( Ilp7-Gal4 , UAS- PPK1 ( Ainsley et al . , 2014 ) , Figure 2F ) .", "Finally , pharmacological inhibitor of DEG/ENaCs , benzamil , has been used to antagonize PPK1 in Drosophila and homologs in mice ( Liu et al . , 2003; Page et al . , 2007 ) .", "We therefore investigated the effect of benzamil on feeding and found it increased food consumption when supplemented in fly food , but not in flies where PPK1 is knocked down ( Figure 2G ) .", "Together , these data indicate that the mechanosensory ion channel , PPK1 , plays a critical role in the PENs for regulating feeding .", "The identification of the involvement of the DEG/ENaC mechanosensory ion channels in enteric neurons for regulating feeding lays the groundwork for investigating mechanisms underlying the phenomenon of fullness sensation .", "Pharmacological interventions on appetite stimulation and suppression are important for many diseases including obesity , cancer , and AIDS .", "Currently , all three FDA-approved weight-loss drugs have significant side-effects that target either the hypothalamus or fat absorption ( Manning et al . , 2014 ) .", "Enteric DEG/ENaCs provide an attractive alternative for drug development due to their druggability , pharmacological accessibility , and fewer side-effect complications than the central nervous system .", "Overall , our findings indicate an important role of the DEG/ENaC mechanosensory ion channels in the enteric nervous system in food intake and suggest an exciting therapeutic alternative for fighting obesity ." ], [ "Flies were reared at 25°C on standard cornmeal-molasses medium , unless indicated otherwise .", "The following stocks were used in this study: GMR51F12-Gal4 ( Jenett et al . , 2012 ) ( Bloomington Stock Center ) , GMR48A05-Gal4 ( Jenett et al . , 2012 ) ( Bloomington Stock Center ) , Ilp7-Gal4 and HGN1-Gal4 were gifts from Dr Irene Miguel-Aliaga ( Cognigni et al . , 2011 ) , UAS-ShiTS1 was a gift from Toshihiro Kitamoto ( Kitamoto , 2013 ) , UAS-TRPA1 ( Bloomington Stock Center ) , 1XUAS-cd8::GFP ( [Pfeiffer et al . , 2010] , Bloomington Stock Center ) , PPK1-Gal4 ( [Ainsley et al . , 2014] , Bloomington Stock Center ) , UAS-PPK1 was a gift from Wayne Johnson ( Ainsley et al . , 2008 ) , UAS-PPK1 RNAi #1 ( Bloomington Stock Center ) , UAS-PPK1 RNAi #2 ( Vienna Drosophila RNAi Center , 108683 ) , UAS-PPK29 RNAi ( Bloomington Stock Center ) , b88h49df ( Bloomington Stock Center ) , A400df ( Bloomington Stock Center ) and yw flies .", "Crosses were performed at 18°C .", "2-day old male flies of the indicated genotypes were incubated at 29°C in groups of ten for 24 hr and starved for 5 hr .", "Hemolymph was then collected as described ( Haselton et al . , 2010 ) and subjected to a glucose assay ( Glucose Hexokinase Liquid Stable Reagent , Thermo Scientific , Waltham , Massachusetts , USA ) .", "Flies were raised at 18°C .", "Capillary feeding assays were performed as described ( Ja et al . , 2007 ) on 2-day old males in groups of four at 29°C for 24 hr .", "The diet was a 5% yeast extract and 5% sucrose solution .", "For the benzamil experiment , male yw flies were provided food with 100 mM sucrose supplemented with either 10 mM benzamil or DMSO .", "The concentration was chosen based upon previous work ( Liu et al . , 2003 ) .", "Green food coloring ( 1:100 , McCormick , Sparks , Maryland , USA ) was added to the food to track consumption .", "Drosophila guts were dissected and fixed as previously described ( Cognigni et al . , 2011 ) .", "The following antibodies and fluorescent markers were used: rabbit Anti-GFP antibody ( ab290; 1:1000; Abcam , Cambridge , UK ) .", "Alexa Fluor 488 Goat Anti-Rabbit IgG ( H + L ) ( A11034; 1:800; Life Technologies , Gaithersburg , MD , USA ) , mAb22C10 ( Developmental Studies Hybridoma Bank , University of Iowa ) , and Alexa Fluor 633 phalloidin ( A22284; 1:250; Life Technologies ) .", "For the three-dimensional model of the posterior enteric neuron region , a z-stack series of confocal images were taken from a gut sample immunostained with mAb22C10 and Alexa Fluor 633 phalloidin and then converted into a model using Imaris .", "All images were acquired using a Zeiss LSM510 and analyzed using Imaris ( Bitplane , Zurich , Switzerland ) .", "All data , presented as average ± s . e . m . or average ± s . d . , were analyzed with GraphPad Prism 6 .", "Unless indicated otherwise , unpaired Student's t test was used to determine differences between groups in each panel .", "For the rescue experiment , the results were compared by ANOVA followed by Tukey post hoc test .", "Data for each experiment met the assumption of the statistical tests .", "The sample size , as indicated in the figure legends , was chosen based on similar experiments reported previously , and was large enough to eliminate the variance between the groups before testing .", "No samples or animals were excluded from statistical analysis .", "All studies had been repeated for more than three times .", "The experimental groups were allocated randomly , and no blinding was done during allocation ." ] ]

[ "Regulation of food intake is fundamental to energy homeostasis in animals .", "The contribution of non-nutritive and metabolic signals in regulating feeding is unclear .", "Here we show that enteric neurons play a major role in regulating feeding through specialized mechanosensory ion channels in Drosophila .", "Modulating activities of a specific subset of enteric neurons , the posterior enteric neurons ( PENs ) , results in sixfold changes in food intake .", "Deficiency of the mechanosensory ion channel PPK1 gene or RNAi knockdown of its expression in the PENS result in a similar increase in food intake , which can be rescued by expression of wild-type PPK1 in the same neurons .", "Finally , pharmacological inhibition of the mechanosensory ion channel phenocopies the result of genetic interrogation .", "Together , our study provides the first molecular genetic evidence that mechanosensory ion channels in the enteric neurons are involved in regulating feeding , offering an enticing alternative to current therapeutic strategy for weight control ." ]

[ "Around one third of children and two thirds of adults in the US are thought to be overweight or obese .", "By increasing the risk of disorders such as heart disease , stroke , diabetes and some types of cancer , obesity has become one of the leading causes of preventable death worldwide and accounts for an increasing proportion of all spending on healthcare .", "Given the high costs of obesity for individuals and society , there is widespread interest in the development of drugs to aid weight loss .", "Three compounds are currently approved for this purpose , and they work either by reducing the body's ability to absorb fat or by acting on the brain to suppress appetite .", "However , all three have significant side effects .", "Now , on the basis of experiments in fruit flies , Olds and Xu suggest an alternative strategy , namely targeting the ‘stretch-sensitive’ ion channels in the neurons in the digestive system that signal to the brain that the body has ingested enough food .", "By artificially activating these ion channels , it might be possible to induce feelings of fullness after smaller quantities of food have been consumed .", "These ion channels—known as PPK1 ion channels—are present on posterior enteric neurons , which wrap around the muscles of the gut .", "Silencing these neurons caused fruit flies to eat too much , whereas activating them caused the flies to eat less .", "Deleting the gene that encodes the PPK1 ion channel had the same effect as silencing neurons , suggesting that drugs that act directly on PPK1 could help to regulate food intake .", "Consistent with this , insects ate more when their food was supplemented with a chemical that blocked the PPK1 ion channels .", "By showing that PPK1 ion channels can be targeted pharmacologically , Olds and Xu have opened up a new avenue of anti-obesity research .", "A drug that can activate the equivalent ion channel in mammals would have the potential to aid weight loss , while avoiding the side effects associated with compounds that act directly on the brain ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "immunology and inflammation" ]

[ [ "Antibodies are immunogenic proteins expressed by B cells that play a major role in the adaptive immune response against non-self .", "Upon recognition of target epitopes , these antibodies undergo multiple rounds of somatic hypermutation and affinity maturation inside a germinal center , whereby the amino acid sequence of the epitope-binding surface is selected for optimal binding to the target ( Victora and Nussenzweig , 2012; Eisen and Siskind , 1964; McKean et al . , 1984 ) .", "The longer this affinity maturation process extends , the higher the affinity and specificity of the antibodies toward their target antigen , primarily through mutagenesis of the six complementarity determining region ( CDR ) loops of the antibody ( Victora and Nussenzweig , 2012 ) .", "Using a combination of affinity matured CDR loops , these antibodies bind strongly to the target and aid in invader neutralization .", "While the process of affinity maturation and somatic hypermutation of antibodies results in high-affinity and incredibly specific binders to a particular epitope , some antibodies have been shown to display signs of reactivity toward diverse off-target epitopes .", "This broad but low-affinity binding has been termed ‘polyreactivity’ .", "Antibody polyreactivity has been hypothesized to be beneficial in the early stages of antibody maturation , acting as a pool of diverse binders ready to recognize novel antigens and initiate the more stringent selection process ( Dimitrov et al . , 2013 ) .", "To this end , a majority of B cell receptors and antibodies which have not undergone somatic hypermutation , including those on immature B cells and early ‘natural’ antibodies , have been found to be polyreactive to some extent and are suggested to have an innate-like response to pathogens ( Ochsenbein et al . , 1999; Wardemann et al . , 2003 ) .", "While these mostly unmutated polyreactive antibodies remain at low frequency in antigen-experienced individuals , a distinct population of polyreactive antibodies that have undergone selection are still expressed by mature B cells that circulate in blood ( Tiller et al . , 2007 ) .", "In fact , some studies have found that the polyreactivity status of an antibody is mostly independent of the number of somatic hypermutations in the antibody sequence ( Mouquet et al . , 2010; Prigent et al . , 2016 ) .", "In line with this finding , only 5–10% of the repertoire of naive B cells circulating in the periphery are polyreactive , but this increases to 20–30% in the memory B cell compartment , showing a distinct capability of polyreactivity to survive selection ( Tiller et al . , 2007; Koelsch et al . , 2007 ) .", "These results suggest that polyreactivity can persist , or perhaps even be selected for during the selection process within the germinal center .", "In a few notable cases , polyreactivity may in fact augment the efficacy of a given immune response .", "Polyreactive IgA antibodies have been shown to have an inherent reactivity to microbiota in the mouse gut , with a predicted role in host homeostasis ( Bunker et al . , 2017 ) .", "These previously identified antibodies so far have no known primary ligands , yet play a key role in facilitating the gut immune response to the plethora of exogenous antigens encountered in the dynamic dietary and microbial environment of the gut .", "This implies the existence of antibodies whose primary function is to act as polyreactive sentries in the gut , yet the downstream effects of polyreactive antibodies coating commensal bacteria is so far unclear .", "Similar polyreactive IgA and IgG mucosal antibodies were found in the gut of human immunodeficiency virus ( HIV ) -infected patients , but these antibodies either had low affinity to the virus or lacked neutralization capabilities ( Planchais et al . , 2019 ) .", "The benefit of singular antibody sequences with the ability to sample large portions of the commensal population may represent an improvement in efficiency of the homeostatic machinery of the gut .", "While the precise role of these primarily polyreactive gut antibodies is still a topic of debate , polyreactivity has been suggested to augment the immune response in other immunological niches .", "Broadly neutralizing antibodies ( bnAbs ) , which bind robustly to conserved epitopes on the surface glycoproteins of influenza viruses or HIV are more likely to be polyreactive ( Haynes et al . , 2005; Mouquet et al . , 2011; Andrews et al . , 2015 ) .", "In one study of HIV-binding antibodies , over half of all tested bnAbs were found to be polyreactive ( Prigent et al . , 2018 ) .", "These bnAbs have been the subject of intense study for their potential as the central components of an HIV treatment or as the byproduct of an immune response to a universal Influenza vaccine ( Andrews et al . , 2015; Haynes et al . , 2019; Crowell et al . , 2019; Li et al . , 2012 ) .", "One hypothesized mechanism for the capability of polyreactive antibodies to confer this broad neutralization in the face of a changing viral epitope is heteroligation , the ability of a single antibody to bind the primary target with one binding domain and use the other binding domain to bind in a polyreactive manner ( Mouquet et al . , 2010 ) .", "This heteroligation allows the antibody to take advantage of the significant avidity increase afforded by bivalent binding , despite the low envelope protein density of HIV or a geometry which does not readily lend itself to bivalent binding on the surface of influenza viruses ( Klein and Bjorkman , 2010 ) .", "Although polyreactivity may play a positive role in natural immune responses , oftentimes this same property is considered undesirable from the point of view of generating therapeutic antibodies with high specificity .", "Antibody-based treatments , which generally take the form of an intravenous transfusion , are sensitive to the accelerated systemic clearance of polyreactive antibodies ( Hötzel et al . , 2012; Kelly et al . , 2015; Kelly et al . , 2018; Datta-Mannan et al . , 2015 ) .", "In general , much work has focused on attempting to answer the question of optimizing ‘developability’ of a given antibody .", "These efforts have been dedicated to determining the most critical components of developability through a large array of experimental assays , in silico structural prediction-based methods , sequence-based analysis and their correlations with clearance , sequence-based SASA predictions , and sequence-based aggregation propensity predictors ( Jain et al . , 2017b; Raybould et al . , 2019; Sharma et al . , 2014; Jain et al . , 2017a; Obrezanova et al . , 2015 ) .", "In many of these studies , polyreactivity or non-specificity in general was seen to be a negative indicator of the developability of a drug , suggesting that therapeutic antibodies should strive toward a drug-like specificity ( Starr and Tessier , 2019 ) .", "In line with this goal of understanding the predominant factors involved in the specificity of therapeutic antibodies , many researchers have worked to identify the biophysical underpinnings of polyreactivity in natural immune responses .", "The most popular hypotheses for the primary biophysical predictors of polyreactivity have included CDR3 length ( Prigent et al . , 2016 ) , CDR3 flexibility ( Prigent et al . , 2018 ) , net hydrophobicity ( Lecerf et al . , 2019 ) , and net charge ( Rabia et al . , 2018 ) .", "More observational studies have found an increased prevalence of arginine and tyrosine in polyreactive antibodies ( Kelly et al . , 2018; Birtalan et al . , 2008 ) .", "While these previous studies represent substantial advances in the study of polyreactivity , they have often been limited in scope , focusing on a singular antibody source and primarily focused on CDR3H .", "Comparing across these individual antibody sources highlights discrepancies between the proposed predictors of polyreactivity .", "The aforementioned properties determined to be key to polyreactivity in previous studies were found to be statistically insignificant in studies of HIV-binding and mouse gut polyreactive antibodies ( Bunker et al . , 2017; Mouquet et al . , 2010 ) .", "Clearly , a computational framework that would enable us to predict the polyreactivity of a given antibody a priori , whether evaluating the efficacy of a natural immune response or the potential fate of a therapeutic antibody , would be tremendously useful .", "Such a framework , for example , could be used to assist in the isolation of broadly neutralizing antiviral antibodies , or speed up the process of therapeutic antibody screening .", "To achieve this goal , a thorough understanding of the molecular features behind polyreactive binding interactions is critical .", "Experimental approaches utilizing next-generation sequencing and ELISA allow for the identification of hundreds of polyreactive antibody sequences .", "However , the systematic characterization of these antibodies is difficult .", "Issues immediately arise when defining the conditions by which we determine an antibody to be polyreactive .", "While polyreactivity may exist on some continuous spectrum , we are inclined to frame the problem as binary .", "This binary discretization is useful for the identification of meaningful differences , yet must be recognized as an imperfect assumption .", "In addition to this more philosophical challenge , experimental efforts must also overcome significant hurdles .", "Detailed biochemical studies of polyreactive antibodies via protein crystallography , quantitative binding experiments , and mutagenesis provide exceptional insight but are inherently low throughput .", "Structural modeling of these polyreactive antibodies represent a high-throughput approach , but models of flexible loops are relatively unreliable , and are unlikely to capture nuances in side-chain placement ( Karami et al . , 2019 ) .", "A bioinformatics-based approach , centered around high-throughput analysis that minimizes structural assumptions while maintaining positional context of amino acid sequences would provide a thorough , unbiased analysis of existing data and create a powerful pipeline for future studies .", "In this study , we show that using just the amino acid sequences of antibodies from a database of over 1000 sequences tested for polyreactivity , unifying biophysical properties that distinguish polyreactive antibodies from non-polyreactive antibodies can be identified .", "We find that , while charge and hydrophobicity are in fact important determinants of polyreactivity , the characteristic feature of polyreactive antibodies appears to be a shift toward neutrality of the binding interface .", "In addition , loop crosstalk is more prevalent in the heavy chain of polyreactive antibodies than non-polyreactive antibodies .", "From these properties , a machine learning-based classification software was developed with the capability to determine the polyreactivity status of a given sequence .", "This software is generalizable and can be retrained on any binary classification problem and identify the key differences between two distinct populations of antibodies , T cell receptors , or MHC-like molecules at the amino acid level ." ], [ "Our aggregate database of over 1000 antibody sequences is compiled from our own previously published and new data , in addition to data from published studies by the Mouquet and Nussenzweig labs ( Table 1; Mouquet et al . , 2010; Bunker et al . , 2017; Planchais et al . , 2019; Mouquet et al . , 2011; Prigent et al . , 2018 ) .", "Using an ELISA-based assay , the reactivity of each antibody is tested against a panel of 4–7 biochemically diverse target antigens: DNA , insulin , lipopolysaccharide ( LPS ) , flagellin , albumin , cardiolipin , and keyhole limpet hemocyanin ( KLH ) .", "This panel has become increasingly prevalent in the literature for experimental measures of polyreactivity in antibodies ( Mouquet et al . , 2010; Prigent et al . , 2016; Bunker et al . , 2017; Planchais et al . , 2019; Mouquet et al . , 2011; Andrews et al . , 2015; Prigent et al . , 2018; Jain et al . , 2017b; Neu et al . , 2019; Wrammert et al . , 2011 ) .", "The ligands represent a diverse sampling of biophysical and biochemical properties: for example , enrichment in negative charge ( DNA , insulin , LPS , albumin ) , amphipathic in nature ( LPS , cardiolipin ) , exceptionally polar ( KLH ) , or large in size ( KLH , flagellin ) .", "From this panel , a general rating of ‘polyreactive’ or ‘non-polyreactive’ is given to 529 and 524 antibodies , respectively .", "For the purposes of this study , antibodies are determined to be polyreactive if the authors of the original studies determined a particular clone binds to two or more ligands in the panel .", "Those that bind to one or none of the ligands in the panel are deemed non-polyreactive .", "A limitation of this full polyreactivity dataset is that there exists an intermediate between the two classes .", "As discussed in the introduction , it is not immediately obvious where the line for polyreactivity should be drawn .", "An antibody that binds to 2–3 ligands may not necessarily achieve broad reactivity through the same mechanism as an antibody that binds four or more ligands from a panel of 6 or 7 .", "To remove these ambiguities , a so-called 'parsed' dataset is generated whereby antibodies that bind 4–7 ligands are labeled polyreactive , antibodies that bind 0 panel ligands are labeled non-polyreactive , and those that bind 1–3 are removed from the analysis .", "The results presented below utilize the full dataset of 1053 antibody sequences , unless otherwise noted .", "Analysis of the parsed dataset can be found in the supplementary figures .", "As a first pass at the given dataset , we focus on the most simplistic of the possible explanations for differences between polyreactive and non-polyreactive antibodies , specifically the J- and V-gene usage of each group .", "Figure 1A and B , rendered with code adapted from the Dash et al . derived program TCRdist ( Dash et al . , 2017 ) , represents each antibody V-gene as a line connecting a single heavy and light chain gene for the full human-derived antibody dataset ( 685 sequences ) .", "We repeat this analysis in Figure 1—figure supplement 1 using the parsed human-derived antibody dataset ( 472 sequences ) .", "Direct comparisons between mouse and human derived antibodies is difficult at the gene usage level .", "A similar analysis highlighting differences between mouse polyreactive and non-polyreactive antibodies can be found in the supplement ( Figure 1—figure supplement 2 ) .", "Genes are identified from nucleotide sequences using NCBI’s IgBLAST command line tool ( Ye et al . , 2013 ) .", "Heavy and light chain genes that are shared between polyreactive and non-polyreactive sequences are colored for the top labeled instances .", "Genes that are labeled but not found above a 2% threshold in the opposite population are colored gray , whereas those that do not have a visible name are colored randomly to highlight variation in gene usage .", "From this comparison , it is clear that the variable gene usage is skewed between polyreactive and non-polyreactive sequences , with an enrichment of VH⁢1⁢-⁢69 , VH⁢1⁢-⁢46 , and VH⁢4⁢-⁢59 in the polyreactive population , a trend that persists in the parsed dataset ( Figure 1—figure supplement 1 ) .", "In contrast , no qualitative differences in the J-gene usage are readily discernible between these two groups ( Figure 1—figure supplement 3 ) .", "While the full alignment of these most used heavy chain variable genes shows a high degree of sequence similarity ( Figure 1—figure supplement 4 ) , Figure 1C highlights the regions of highest dissimilarity between the biophysical properties of amino acids in prevalent genes within each population .", "VH⁢3⁢-⁢23 , the most prevalent gene in the non-polyreactive human dataset and the second most prevalent gene in the polyreactive human dataset , can be used as a reference for comparisons between genes enriched in each individual population .", "This reference gene shares a high degree of sequence similarity with the second and third most frequently occurring genes in the non-polyreactive dataset , VH⁢3⁢-⁢7 and VH⁢3⁢-⁢9 , save for a lysine and aspartic acid pair in framework 2 of VH⁢3⁢-⁢7 .", "The genes enriched in the polyreactive dataset , however , are quite different from this reference .", "All three of the polyreactive enriched genes have charged residues where the non-polyreactive enriched genes have hydrophilic residues ( or vice versa ) at IMGT positions 1 , 13 , and 90 .", "These initial results hint at some systematic differences between the polyreactive and non-polyreactive antibody populations .", "Figure 1D quantifies the extent of the difference in gene usage in each population by comparing these most prominent genes from our accumulated dataset of HIV- and influenza virus-reactive antibodies .", "While the two most common genes in the polyreactive dataset account for 27% of the human polyreactive antibodies in this study , the top three most common genes in the non-polyreactive dataset account for just over 17% of the total population .", "In addition to being the most prevalent gene in the polyreactive dataset , VH1−69∗01 has also been found historically to be more prevalent in broadly neutralizing antibodies against influenza viruses , in line with the previously mentioned overlap between bnAbs and polyreactivity ( Andrews et al . , 2015; Wrammert et al . , 2011 ) .", "Overall , there is a noticeable difference between the gene usage frequency of polyreactive and non-polyreactive antibodies , but the overlap in the usage of the two populations suggests that gene usage alone is not sufficient to distinguish the two groups .", "While there exist qualitative differences between framework sequences enriched in the polyreactive dataset compared to the non-polyreactive population , a look at the amino acid usage of the CDR loops of each group shows no significant differences ( Figure 1—figure supplement 5 ) .", "This implies that the positional context of a given amino acid is critical to tease out differences in antibody binding properties .", "To identify deeper trends in the biophysical properties of polyreactive antibodies , we utilize a new methodology to analyze and represent a range of different properties inherent to these sequences .", "Although the framework regions of antibodies are highly conserved , the CDR loops vary significantly in length and show very low conservation between populations .", "This makes alignment of CDR loops difficult without creating subgroups for loops of identical length .", "To overcome this , the sequence data is reorganized into a matrix representation ( Figure 2A ) .", "Each sequence is aligned by the center of each CDR loop , with spaces between the loops set to zero and each amino acid encoded as a number from 1 to 21 .", "While this alignment method excludes the framework regions of the antibodies and slightly averages out some of the properties at the edge of the CDR loops , we reason that most of these differences are evident in the gene usage analysis of the previous section .", "From this simple alignment , no obvious patterns emerge separating polyreactive and non-polyreactive antibodies; however , we can clearly see that mouse gut-derived IgA antibodies have generally shorter CDR3H loops , and more conserved CDR3L sequences when compared to the human-derived antibody sequences .", "All subsequent analysis is derived from this matrix representation of the sequences .", "With this new positionally sensitive and quantitative alignment method , we are able to further dissect the differences in amino acid sequences presented in Figure 1 .", "Figure 2B uses this positional sequence encoding to determine the amino acid frequency difference between polyreactive and non-polyreactive sequences .", "For example , phenylalanine is found at position 93 in roughly 40% of polyreactive sequences and nearly 60% of non-polyreactive sequences .", "Therefore position 93 , amino acid F has an intensity of −0 . 2 in Figure 2B .", "From this panel it is evident that most of the major differences are in the germline encoded regions CDR1H and CDR2H , in line with the observations from Figure 1 that suggest polyreactive antibodies have a distinct gene usage when compared to non-polyreactive antibodies .", "Figure 2C further expands on these differences , showing the largest changes in amino acid frequencies between the two populations .", "We can see that there is a slight decrease of phenylalanine frequency in CDR1H of polyreactive antibodies , in favor of isoleucine .", "Additionally , there is a general shift toward hydrophobicity in CDR2H , as the hydrophilic residue serine at matrix positions 78 and 82 is less prevalent in polyreactive antibodies , instead replaced by the more hydrophobic residues isoleucine and glycine .", "In the parsed dataset , these differences become larger in magnitude , particularly in CDR1L , where phenylalanine is again found less frequently in polyreactive sequences ( Figure 2—figure supplement 1 ) .", "This increased prevalence in loop hydrophobicity of polyreactive antibodies has been suggested before in the literature ( Prigent et al . , 2018 ) along with a net increase in positive charge ( Rabia et al . , 2018 ) , so we next aimed to analyze this matrix systematically using biophysical properties inherent to the loops .", "A simple analysis of the full human and mouse-derived dataset investigating classical parameters explored previously by other groups ( CDR loop length , net charge , net hydrophobicity , and gene usage ) and some new properties ( side chain flexibility , side chain bulk , and the Kidera Factors from Kidera et al . , 1985 ) show some significant differences between polyreactive and non-polyreactive antibodies ( Figure 3A , B ) .", "The versatility of the positionally sensitive amino acid matrix allows for the application of multiple 'property masks' to tease out the specific regions of each CDR loop that contributes most to these significant differences .", "Given a property , amino acid charge for example , we can replace each simple 1–21 representation with a distinct representation based upon amino acid properties .", "In the matrix of Figure 2A leucine , histidine , and arginine are represented by the integers 3 , 16 , and 17 .", "As an example , when the charge property mask is applied , the matrix representations of these three amino acids in all sequences is changed to 0 . 00 , 0 . 091 , and 1 . 00 , respectively .", "We apply 62 such masks to this matrix , including simple metrics like charge , hydrophobicity , side chain flexibility , and side chain bulkiness to go along with more carefully curated metrics from the works of Kidera et al . , 1985; Liu et al . , 2018 .", "A complete description of these properties can be found in the 'Key resources table' and in Appendix 1—table 1 .", "The application of these masks gives an entirely new matrix describing the localization of amino acids with a given property .", "By averaging across all sequences in the polyreactive or non-polyreactive dataset when these masks are applied , we can readily see differences in charge patterning and hydrophobicity when comparing polyreactive and non-polyreactive sequences ( Figure 3C , D ) .", "Including errors obtained via bootstrapping , we see that these differences are most pronounced in the center of CDR3H , with some differences also apparent in the remaining five loops .", "This analysis shows an overall bias toward neutrality in these regions; that is neither positively nor negatively charged , neither strongly hydrophilic nor hydrophobic .", "These results also contextualize the findings of Figure 2C .", "The trend toward hydrophobic residues in CDR2H of polyreactive antibodies importantly does not make these regions net hydrophobic , but instead make these regions slightly less hydrophilic on average .", "This effect is yet again more pronounced in the parsed dataset ( Figure 3—figure supplement 1 ) , with a strong trend toward interface neutrality .", "Conversely , when comparing bootstrap samples drawn from the null distribution , that is the 'polyreactive' or 'non-polyreactive' labels are given to antibody sequences at random , we see no difference between the biophysical properties of the two populations ( Figure 3—figure supplement 2 ) .", "Along with simple property averaging , these masks also give a high dimensional space from which we can determine , in an unbiased way , the primary factors that discriminate polyreactive and non-polyreactive antibodies .", "As a first pass , we apply a principal component analysis ( PCA ) to the matrix of all antibody sequences in an attempt to separate the polyreactive or non-polyreactive populations along the axes of highest variation in the dataset .", "Unfortunately , the principal components of these data do not effectively distinguish between the two populations ( Figure 4—figure supplement 1 ) .", "To further investigate the physical and sequence-based properties of polyreactivity in antibodies in a more targeted manner , we employ linear discriminant analysis ( LDA ) , a common algorithm often applied in classification problems ( Barker and Rayens , 2003; Cordeiro et al . , 2009; Ma et al . , 2013 ) .", "LDA works in a manner conceptually similar to PCA , reducing the dimensionality of a given dataset via a linear combination of the original dimensions .", "However , LDA takes one additional input , the label or class of each sequence .", "Whereas the objective of PCA is to identify the axes which maximize the variance in the dataset , LDA has the dual objective of maximizing the projected distance between two classes while minimizing the variance within a given class .", "While LDA is well adapted for classifying two distinct populations , it is susceptible to overfitting , unlike PCA ( Qiao et al . , 2009 ) .", "Here , we have labeled our two classes in the matrix with either a ‘1’ for polyreactive , or ‘0’ for non-polyreactive .", "In our application of LDA , we parse down the large number of input vectors using either PCA or an algorithm which selects the vectors with the largest average differences between the two populations .", "This reduction in dimensionality ensures the data are not being overfit , and the tunable number of input vectors allows us to control for overfitting in each individual application .", "Figure 4A shows the results of LDA when applied to the parsed dataset comprised of 311 polyreactive antibodies and 362 non-polyreactive antibodies .", "As discussed in the introduction , the framing of polyreactivity as a binary problem is not a perfect assumption .", "The inclusion of intermediate levels of polyreactivity further confounds this issue .", "Indeed , the application of LDA to the full dataset shows a reduced ability to split polyreactive and non-polyreactive antibodies ( Figure 4—figure supplement 2 ) , likely due to this spectrum of polyreactivity .", "By considering only the parsed dataset for these classification analyses , we can improve confidence that the differences identified are those that separate strongly polyreactive and strongly non-polyreactive antibodies .", "LDA analysis is versatile in its applications , and in this work , we utilize the method in two distinct modes .", "In the first mode , all available data is used as input with the output vector representing the features that best distinguish between the two complete populations .", "Plots of the data projected onto this vector ( as in Figure 4A ) represent the maximum achievable separation between the two populations for a defined number of input components from the given biophysical property matrix .", "In the second mode , we utilize LDA as a more canonical classification algorithm separating the data randomly into training and test groups .", "In this classification mode of operation , a combination of correlation analysis coupled with maximal average differences is used to parse input features , and a support vector machine ( SVM ) is used to generate the final classifier from these features .", "Accuracy of the resultant classifiers is assessed via leave one out cross validation , these accuracies are shown in Figure 4B .", "In the first mode , we find that the data can be split more effectively when the parsed dataset is broken up into the distinct ‘reactivity’ groups , that is those antibodies specific for influenza viruses , HIV , or found in the mouse gut ( Figure 4A ) .", "This suggests there may be some bias due to antigen specificity , or lack thereof , whereby influenza virus-specific antibodies take a slightly different path toward polyreactivity compared to HIV reactive or mouse gut IgA antibodies .", "However , when using the classification mode , the classification accuracy is roughly equivalent across all tested datasets ( Figure 4B ) .", "Testing this classifier with a scrambled dataset , where the labels are randomly assigned , shows the expected decrease in classification accuracy for each individual dataset for all ranges of input features .", "When applying LDA in the first mode ( Figure 4A ) , we can directly pull the linear weights of each component comprising linear discriminant one and reveal which biophysical properties at each CDR position best distinguish between the two populations .", "The differences in the linear weights from the heavy chain CDR loops comprising each discriminant show clear differences when comparing the complete parsed dataset ( Figure 4C ) to the HIV only dataset ( Figure 4D ) .", "In the parsed dataset , the discriminating weights are heavily concentrated in CDR2H .", "Whereas in the HIV dataset , these weights are centered around the CDR3H loop .", "Only the top 10 linear weights are shown in Figure 4C , D .", "The full matrix of linear weights can be found in Figure 4—figure supplement 3 .", "The predominant discriminating factors between datasets might be due to the significant difference in CDR3H length between the mouse ( IgA ) and the human datasets , which confounds the analysis in this region .", "However , when examining each individual subset of the complete dataset we do find that there are common properties that seem to be the primary discriminators ( i . e . largest linear weights ) .", "These are hydrophobicity 1 , hydrophobicity 2 , and hotspot variable 6 ( a structural parameter related to α-helix propensity ) .", "While analysis of the biophysical property differences between polyreactive and non-polyreactive sequences provides some insight into the molecular basis for the polyreactivity phenomenon , a broad unifying pattern which could discern the biophysical mechanism behind polyreactivity was not readily evident across all types of antibodies .", "To probe these polyreactive sequences in a quantitative yet more coarse manner , we applied the formalism of information theory to our dataset of antibody sequences .", "Information theory , a theory classically applied to communication across noisy channels , is incredibly versatile in its applications , with high potential for further applications in immunology ( Shannon , 1948; Román-Roldán et al . , 1996; Cheong et al . , 2011; Vinga , 2014; Mora et al . , 2010; Murugan et al . , 2012 ) .", "In this work , we utilize two powerful concepts from information theory , namely Shannon entropy and mutual information .", "Shannon entropy , in its simplest form , can be used as a proxy for the diversity in a given input population .", "This entropy , denoted as H , has the general form shown in Equation 1: ( 1 ) H⁢ ( X ) =-∑Xp⁢", "( x ) ⁢log2⁡p⁢", "( x ) where p⁢", "( x ) is the occurrence probability of a given event , and X is the set of all events .", "We can then calculate this entropy at every position along the CDR loops , where X is the set of all amino acids , and p⁢", "( x ) is the probability of seeing a specific amino acid at the given position .", "In other words , we want to determine , for a given site in a CDR loop , how much diversity ( or entropy ) is present .", "Figure 5A shows this Shannon entropy distribution for the full dataset of polyreactive and non-polyreactive antibodies .", "Given there are only 20 amino acids used in naturally derived antibodies , we can calculate a theoretical maximum entropy of 4 . 2 bits , which assumes that every amino acid occurs at a given position with equal probability .", "Although the observed entropy of the CDR3H loop approaches this theoretical maximum , it hovers below it ( 3 . 5 Bits ) due to the relative absence of the amino acids cysteine and proline in the center of this loop .", "The difference in the entropy distributions in CDR1H are consistent with the bias in amino acid usage in this region , shown previously in Figure 2 .", "Importantly , from this entropy we can calculate an equally interesting property of the dataset , namely the mutual information .", "Mutual information is similar , but not identical to , correlation .", "Whereas correlations are required to be linear , if two amino acids vary in any linked way , this will be reflected as an increase in mutual information .", "In addition , due to some of the highly conserved residues in the non-CDR3H loops , high covariance can be achieved for residues that have not been specifically selected for in the germinal center .", "Using this information theory framework , these conserved residues have a mutual information of 0 .", "Overall , the mutual information can be used to identify patterns in antibody sequences that were not readily evident through the previous analysis in this or other studies .", "If there is some coevolution or crosstalk between residues undergoing some selection pressure in the antibody maturation process , it will be reflected as an increase in the mutual information .", "In this work , mutual information I⁢ ( X;Y ) is calculated by subtracting the Shannon entropy described above by the conditional Shannon entropy H⁢ ( X|Y ) at each given position as seen in Equations 2 and 3: ( 2 ) H⁢ ( X|Y ) =-∑y∈Yp⁢", "( y ) ⁢∑x∈Xp⁢ ( x|y ) ⁢log2⁡p⁢ ( x|y ) ( 3 ) I⁢ ( X;Y ) =H⁢ ( X ) -H⁢ ( X|Y ) To orient ourselves in physical space , Figure 5B gives an example crystal structure ( PDB: 5UGY ) ( Whittle et al . , 2011; Ziegler et al . , 2014 ) highlighting the lateral arrangements of the CDR loops .", "The matrix in Figure 5C shows that the mutual information between CDR loops on this binding surface is increased in the heavy chains of polyreactive antibodies over non-polyreactive ones , suggesting an increase in loop crosstalk in antibodies that exhibit polyreactivity .", "Interestingly , it appears that there is a corresponding decrease of loop crosstalk in the light chains of polyreactive antibodies .", "Importantly , this crosstalk is increased across and within all loops when analyzing the parsed dataset ( Figure 5—figure supplement 1 ) .", "This observed crosstalk persists across all polyreactive antibodies within all subsets of our tested dataset and is evident both in intra-loop and inter-loop interactions .", "Figure 5D highlights some examples of the interesting significant differences of this crosstalk at distinct given positions within CDR1L , CDR1H , and CDR3H .", "A complete plot of the statistically significant differences ( p≤0 . 05 ) of Figure 5C shows that a large portion of these differences are in fact significant ( Figure 5—figure supplement 2 ) .", "The ordering of these entropy and information plots was chosen to reflect the spatial arrangement of the loops on the antibody surface; as such they show also that mutual information between loops drops off with physical distance between these loops .", "In other words , loops ( and residues ) that are located close to each other will have more of an effect on their direct neighbors as opposed to those that are more physically distant .", "This increased mutual information suggests that in the heavy chains of polyreactive antibodies , there is enhanced cooperativity or co-evolution of the amino acids of intra- and inter-CDR loop pairs .", "Given the ability of this analysis pipeline to find nuanced differences between polyreactive and non-polyreactive antibodies , we next sought to expand the range of applications of our approach .", "Extension of the pipeline to the analysis of TCR sequences is trivial , due to the similar arrangement of CDR loops on the binding surface and the capability of IgBLAST to annotate TCR sequences ( Ye et al . , 2013 ) .", "Instead , we sought to significantly expand the scope of this software by applying a similar approach to the analysis of MHC and MHC-like molecules .", "MHC molecules are encoded by a large superfamily of genes that are spread throughout the genome ( Adams and Parham , 2001; Piertney and Oliver , 2006 ) .", "MHC and MHC-like genes are found across a wide range of divergent species , and these genes have diversified extensively over time , making the distinction between orthologs and instances of convergent evolution difficult .", "In some cases , the divergence is extreme enough that phylogenetics cannot provide predictions of function .", "Given that these MHC molecules have evolved to present different antigen subtypes , such as lipid molecules in the case of CD1 proteins ( Borg et al . , 2007; Luoma et al . , 2013; Adams , 2014 ) , we explored the use of our pipeline as a classifier based on biophysical properties rather than phylogeny .", "In achieving this new functionality , the critical step lies in the transformation of the MHC sequences into a numeric form as in Figure 2A .", "To accomplish this , we split the sequences by their most prominent structural features .", "For MHC and MHC-like molecules , these features are the two β-strands and α-helices of the so-called platform domain .", "As a test case , we use two example training classes; a representative list of human MHC Class I molecules , and the output from a BLAST query on CD1d ( Sayers et al . , 2020 ) .", "We can then assess our ability to separate these two training classes , while also introducing a test class derived from the data of Almeida et al . ( Almeida et al . , 2020 ) .", "Each sequence within a given class is globally aligned , and one representative sequence from each class is sent through the Phyre2 structural prediction server ( Kelley et al . , 2015 ) .", "We then use these structural predictions to identify the start and end points of each major structural feature in the alignment space .", "These start and end points then define the boundaries that are numerically encoded into our position-sensitive matrix , as seen in Figure 6A .", "Once the data are in this form , all downstream analysis outlined previously can be applied in a similar manner .", "In this example case , we find that average biophysical properties across these sequences reveal expected differences in hydrophobicity , with the lipid binding CD1 molecules displaying increased hydrophobicity when compared to the peptide binding MHC class I molecules ( Figure 6B ) .", "Interestingly , unlike in the case of the antibody analysis , a simple PCA can effectively discriminate between the two training classes in this example case .", "Figure 6C shows the projection of the biophysical property data of each class onto the first two principal components .", "Here , we see that the peptide binding molecules ( HLA-E , HLA-A2 , H2-D ) and the lipid binding molecules ( Human CD1b , Chicken CD1 ) of the test dataset cluster with the respective peptide and lipid binding training data .", "The majority of the data of Almeida et al . , comprised of non-classical MHC class I molecules from cartilaginous fish , clusters as its own distinct group , likely due to evolutionary distance between these molecules and those derived from mammalian and avian immune systems ." ], [ "Previous research has highlighted the importance of hydrophobicity , charge , and CDR loop flexibility on antibody specificity .", "In this work , we expand upon these previous results with a new bioinformatic and biophysical characterization of polyreactive antibodies .", "The software generated for this study provides a powerful computational tool which can be utilized by researchers interested in discerning differences between populations of adaptive immune molecules in broad contexts .", "Building off of the efforts of our own work and that of experimental collaborators , we were able to aggregate to date one of the largest publicly available datasets of antibodies tested for polyreactivity .", "Differences in the germline gene frequency and amino acid frequencies show there exists some underlying differences between polyreactive and non-polyreactive antibodies .", "A surface level analysis of this dataset is able to discriminate certain features of polyreactive and non-polyreactive antibodies , namely that on average , polyreactive antibodies are less strongly negatively charged , less hydrophilic , and have a higher prevalence of antibodies with longer CDR loops of the heavy chain .", "Importantly , however , these binding surfaces do not have a net positive charge nor are they net hydrophobic .", "Our results highlight an increase in VH⁢1⁢-⁢69 gene usage in polyreactive antibodies , an interesting finding given the substantial literature outlining its importance in diverse immune environments .", "In addition to the aforementioned role of VH⁢1⁢-⁢69 in broadly neutralizing anti-influenza and anti-HIV antibodies ( Haynes et al . , 2005; Mouquet et al . , 2011; Andrews et al . , 2015; Prigent et al . , 2018 ) , autoreactive chronic lymphocytic leukemic B cells commonly express receptors bearing VH⁢1⁢-⁢69 ( Sasso et al . , 1993; Forconi et al . , 2010 ) , and anti-HIV antibodies which target the membrane-proximal external region of HIV-1 envelope glycoproteins frequently utilize VH⁢1⁢-⁢69 ( IAVI Protocol G Investigators et al . , 2019 ) .", "While previous reports suggest that the key feature permitting these auto-reactive or polyreactive interactions of VH⁢1⁢-⁢69 is an exceptionally hydrophobic CDR2H loop ( Chen et al . , 2019 ) our results suggest this does not explain the over-representation of this antibody in the polyreactive dataset , as on average the CDR2H of polyreactive antibodies is strongly hydrophilic .", "Instead , certain structural or dynamic features of the antibody may contribute to its out-sized role in critical biological contexts .", "To dig deeper into the biophysical differences between polyreactive and non-polyreactive antibodies , we created an adaptable software for the automated analysis of large antibody datasets and the application of a new analysis pipeline for the study of polyreactive antibodies .", "Overall , the improvements of this software to the current state of antibody sequence analysis are sufficient to highlight key differences in the two populations with improved spatial resolution .", "The position sensitive sequence alignment is able to further parse through the genetic differences and show that in general , polyreactive antibodies have a tendency to have more hydrophobic residues in CDR2H , and a decreased preference for phenylalanine in CDR1H .", "While these observational differences provided some initial insight , a more rigorous biophysical treatment was necessary .", "With the addition of 62 biophysical properties analyzed using the position sensitive alignment , significant differences between the CDR3H loops in polyreactive and non-polyreactive antibodies become immediately evident , providing a more detailed depiction of the antigen binding surface of polyreactive antibodies .", "These data suggest a movement toward neutrality or ‘inoffensive’ residues in the CDR loops of polyreactive antibodies: amino acids that are neither exceptionally hydrophobic nor hydrophilic and with a net charge close to 0 .", "Previous studies have suggested that polyreactive antibodies tend to have more hydrophobic CDR loops , such that low-affinity Van der Waals interactions might be the primary means of polyreactive interactions ( Prigent et al . , 2018; Starr and Tessier , 2019 ) .", "However , these studies counted the number of hydrophobic residues per sequence or averaged the hydrophobicity of all six CDR loops .", "While our results partially agree with these previous findings , our analysis extends much further into defining the biophysical basis of this phenomenon .", "For example , while our position sensitive representation of the sequences shows that CDR3H does become more hydrophobic in polyreactive sequences , it is still net hydrophilic on average .", "A highly hydrophobic binding surface would provide an avenue for non-specific interactions with other hydrophobic proteins , but it would occlude binding to highly hydrophilic ligands like DNA .", "A slightly hydrophilic , neutral-charged binding surface would permit weak interactions with a wide range of ligands .", "Using these and other biophysical properties as input feature vectors , we were able to generate a generalizable protocol for binary comparisons between two distinct populations of Ig-domain sequences .", "This framework is able to successfully split all tested polyreactive and non-polyreactive antibody datasets .", "Care was taken to not overfit these data and a preliminary classifier built from this algorithm was able to identify the proper number of input vectors for each LDA application .", "While there are general features which best split the polyreactive and non-polyreactive antibodies in these datasets , including charge , hydrophobicity , and α-helix propensity , these features alone are not sufficient to discriminate between the two populations .", "Instead , 75 vectors taken from the position-sensitive biophysical property matrix are necessary to properly split the groups , including both simple properties like charge , hydrophobicity , flexibility , and bulkiness and more carefully curated properties like the often used Kidera factors and the hotspot detecting variables of Liu et al . ( Kidera et al . , 1985; Liu et al . , 2018; Vihinen et al . , 1994 ) .", "The inability to arrive at a core few biophysical properties that could effectively distinguish polyreactive and non-polyreactive antibodies necessitated the application of further approaches , namely information theory .", "The tools provided by information theory proved to be effective in the present study .", "The classic approach to information theory considers some input , communication of this input across a noisy channel , and then reception of a meaningful message from the resultant output .", "We can think of the analogous case for these antibodies , whereby the sequence and structure of the antibodies can be seen as our input , the thermal noise inherent to biological systems can complicate biochemical interactions , and the necessary output is antigen recognition , i . e . binding between the antibody and the ligand .", "Focusing just on the antibody side of this communication channel , we determined the underlying loop diversity through the Shannon entropy of the polyreactive and non-polyreactive datasets .", "This diversity was found to be nearly equivalent while the mutual information , a metric of ‘crosstalk’ across populations , between and within CDR loops was found to be increased in the heavy chain and decreased in the light chain of polyreactive antibodies .", "What this loop crosstalk entails physically is not immediately clear from these measurements .", "The mutual information increase could come from gene usage being somehow coupled , amino acid usage coupling with the cognate ligand , or the amino acids directly interacting physically with each other .", "In some way , this crosstalk appears to be selected for in the polyreactive population .", "If this increase in mutual information manifests as an increase of charge-charge interactions , this could explain why there is a minimal change in net charge of antibodies between the two groups , yet a significant move toward neutrality in the CDR loops of polyreactive antibodies .", "The pairing of two charged groups would help move the binding surface of polyreactive antibodies toward a more ‘inoffensive’ binding surface .", "A binding surface that is neither exceptionally hydrophobic nor hydrophilic , and lacks a significant positive or negative charge , would represent a relatively appealing binding interface for a low-affinity interaction with a large array of diverse ligands .", "A patchwork of hydrophobic and hydrophilic non-charged residues exposed to potential ligands would provide an ideal candidate polyreactive surface .", "The corresponding decrease in the mutual information between the light chain CDR loops of polyreactive antibodies could be caused by a de-emphasis in the involvement of these loops due to differential binding configurations of polyreactive ligands , as has been previously hypothesized ( Dimitrov et al . , 2013; Sethi et al . , 2006 ) .", "While further crystallographic , biochemical , and dynamic studies are necessary to identify the true source of this increase in mutual information across polyreactive antibodies , we can speculate what these results may mean in the context of the results obtained using linear discriminant analysis .", "In addition to standard side chain properties , many of the most important features for splitting polyreactive and non-polyreactive antibodies were structural in nature .", "Specifically , hotspot variables 6 , 24 , 25 , and 41 all correspond to the structural tendencies of a given amino acid .", "Coupled with the increase in side chain interactions that may be implied by the increased mutual information across the loops of polyreactive antibodies , this potential for increased loop structure may suggest more rigid CDR loops in polyreactive antibodies .", "In addition to the insights into polyreactivity , the computational tools developed for this study are broadly applicable to future studies of large antibody or T cell receptor repertoires .", "One of the strengths of this approach is a decreased emphasis on structural information when crystal structures are unavailable .", "Computational prediction of loop conformation is difficult , and drawing inferences from incorrect models regarding side-chain interactions and positioning could be misleading .", "Reliable structural information on these polyreactive antibodies will be critical to a further understanding of the mechanisms of polyreactivity , including complex structures of antibodies bound to various ligands .", "In the high-throughput analysis of antibody sequences , our approach strikes a careful balance of the structural assumptions that should apply consistently across antibody populations .", "Further experimental assays will be necessary to more comprehensively identify the underlying mechanisms of polyreactivity , including further sequencing and biochemical analysis of polyreactive and non-polyreactive antibodies .", "Antibodies specific to other pathogens or those from other organisms tested for polyreactivity will help form a more complete picture and improve the generality of the results .", "As with any machine learning based approach , the classification algorithm is only as good as the data it is trained on .", "Adding further data in the training set , including more mutations and germline reversions that turn a polyreactive antibody non-polyreactive or vice-versa , will be critical for a comprehensive analysis of polyreactivity .", "Additionally , a more robust assay for determining polyreactivity such as a chip based screen to test for binding to many diverse targets , would greatly broaden our perspective and help understand just how broadly reactive these polyreactive antibodies are .", "Lastly , a more complete understanding of the germinal center and the selection processes inherent to the affinity maturation process will assist in the determination of whether polyreactivity is a byproduct or a purposeful feature of the affinity maturation process .", "The software generated for this study is publicly available as a python application ( see Materials and methods ) .", "The unique aspect of this software is its hybrid approach to position-sensitive amino acid sequence analysis .", "Structural information is implicitly encoded by the alignment strategy employed , yet these assumptions are weaker than those imposed by explicit structural prediction .", "Downstream analysis from this positional encoder is streamlined and can be generalized to analyze any binary or higher order classification problems .", "This streamlined analysis allows for the generation of each figure in this study to be applied to thousands of sequences in a matter of minutes .", "The classification capabilities of the software could prove particularly useful when comparing binary classes , such as T cell receptors or antibody sequences derived from healthy and diseased tissue samples .", "Acceptable inputs are not restricted to CDR loops of immunoglobulins , and we have shown that the software can be adapted for the analysis of MHC-like molecules .", "Moving forward , this MHC analysis has the potential to classify the antigen binding properties of highly-divergent MHC sequences from a broad range of species , providing insights where phylogenetic approaches prove difficult .", "This software represents a strong addition to the existing toolkit for repertoire analysis of diverse molecular species ." ], [ "All analyses were performed in python , with code tested and finalized using Jupyter Notebooks ( Kluyver et al . , 2016 ) .", "Figures were generated with MatPlotLib ( Hunter , 2007 ) or seaborn ( Ziegler et al . , 2014 ) , while the majority of data analysis was carried out using Pandas ( McKinney , 2015 ) , SciPy ( Virtanen et al . , 2020 ) , and SciKit-learn ( Pedregosa et al . , 2011 ) .", "All code and data is available at https://github . com/ctboughter/AIMS , including the original Jupyter Notebooks used to generate the data in this manuscript as well as generalized Notebooks and a python-based GUI application for analysis of novel datasets ( Boughter , 2020; copy archived at swh:1:rev:f6c855ef4a7ce63f72dba6b34e9d0e9edd9200ce ) .", "Error bars in all plots are provided by the standard deviation of 1000 bootstrap iterations .", "Statistical significance is calculated using either a two-sided nonparametric Studentized bootstrap or a two-sided nonparametric permutation test as outlined in ‘Bootstrap Methods and Their Application’ ( Davison and Hinkley , 2011 ) .", "For the Studentized bootstrap , the bootstrapped data are drawn from a resampling of the null distribution of the data , with replacement .", "Practically , this entails combining the polyreactive and non-polyreactive antibodies into a single matrix , without labels , and using the Scikit-learn resample module to randomly separate this matrix into two classes , preserving the number of sequences in each population .", "To calculate bootstrapped averages , we draw from the empirical rather than null distribution .", "Statistical significance is estimated by calculating the p-value using the relation: ( 4 ) p=1+♯ ( z2≥z02 ) R+1 Here , we calculate the p-value by counting the number of bootstrap iterations where z2 is greater than or equal to z02 . z2 and z02 are Studentized test statistics taken from the null and empirical and distributions , respectively .", "R is the number of times this bootstrapping process is repeated .", "The general form of z is given by: ( 5 ) z=Y¯2−Y¯1 ( σ22n2−σ11n1 ) 1/2where Y¯ represents the bootstrapped sample mean of each population , σ is the bootstrapped sample standard deviation , and n is the number of samples .", "Populations 1 and 2 in this case correspond to polyreactive and non-polyreactive antibodies .", "To calculate z for the empirical distribution ( z0 ) , all values correspond to the empirical rather than bootstrapped values .", "To calculate p-values for differences in mutual information , the permutation test was used rather than the Studentized bootstrap .", "Here , the test statistic t is set to a simple difference of means , and rather than sampling with replacement from the empirical or null distributions with replacement , we randomly permute the data into ‘polyreactive’ or ‘non-polyreactive’ bins .", "We then count the number of permutations where the randomly permuted test statistic is greater than or equal to the empirical test statistic .", "This count then replaces the count ( # ) in the above equation for p ." ] ]

[ "Antibodies are critical components of adaptive immunity , binding with high affinity to pathogenic epitopes .", "Antibodies undergo rigorous selection to achieve this high affinity , yet some maintain an additional basal level of low affinity , broad reactivity to diverse epitopes , a phenomenon termed ‘polyreactivity’ .", "While polyreactivity has been observed in antibodies isolated from various immunological niches , the biophysical properties that allow for promiscuity in a protein selected for high-affinity binding to a single target remain unclear .", "Using a database of over 1000 polyreactive and non-polyreactive antibody sequences , we created a bioinformatic pipeline to isolate key determinants of polyreactivity .", "These determinants , which include an increase in inter-loop crosstalk and a propensity for a neutral binding surface , are sufficient to generate a classifier able to identify polyreactive antibodies with over 75% accuracy .", "The framework from which this classifier was built is generalizable , and represents a powerful , automated pipeline for future immune repertoire analysis ." ]

[ "To defend itself against bacteria and viruses , the body depends on a group of proteins known as antibodies .", "Each subset of antibodies undergoes a rigorous training regimen to ensure it recognizes a single epitope well – that is , one specific region on the surface of foreign , harmful organisms .", "Most antibodies stick extremely tightly to their one unique epitope , but some can also weakly bind to molecules that are vastly different from their main trained targets .", "This feature – known as polyreactivity – can in some cases help the immune system fight against multiple strains of viruses .", "On the other hand , when antibodies are designed in the laboratory to treat diseases , this characteristic can sometimes lead to the failure of pre-clinical trials .", "Yet it is currently unclear why some antibodies are polyreactive when others are not .", "To investigate this question , Boughter et al . compared over 1 , 000 polyreactive and non-polyreactive antibody sequences from a large database , revealing differences in the physical properties of the region of the antibodies that attaches to epitopes .", "Using these defining features , Boughter et al . went on to design a new piece of freely available , automated software that could predict which antibodies would be polyreactive more than 75% of the time .", "Such software could ultimately help to guide the design of antibody-based treatments , while bypassing the need for costly laboratory tests ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods", "Accession numbers" ]

[ "chromosomes and gene expression" ]

[ [ "The eukaryotic genome is packaged into chromatin , with the basic unit being a nucleosome , consisting of 147 base pairs ( bp ) of DNA wrapped around an octamer of histone proteins .", "Chromatin limits the accessibility of DNA-binding factors to the underlying DNA sequence and presents a strong physical barrier that must be overcome by RNA polymerase II ( PolII ) during transcription .", "In vitro experiments have shown that transcription initiation is the most affected and that elongation is also highly inefficient unless nucleosomes are destabilized ( Lorch et al . , 1987; Hodges et al . , 2009; Jin et al . , 2010; Bintu et al . , 2012 ) .", "Despite this barrier , in vivo elongation rates through chromatin are comparable to rates on naked DNA , indicating that the cell has evolved mechanisms to overcome this chromatin barrier ( Li et al . , 2007; Ardehali and Lis , 2009; Singh and Padgett , 2009; Petesch and Lis , 2012 ) .", "Through the combined action of PolII , elongation factors , nucleosome modifications and chromatin remodelers , the chromatin barrier to transcription is highly dynamic , with nucleosomal turnover correlating with gene expression ( Deal et al . , 2010 ) .", "Nucleosomes are disrupted to allow efficient elongation and are reassembled in the wake of PolII to prevent cryptic initiation from intragenic sequences ( Cheung et al . , 2008; Petesch and Lis , 2008; Quan and Hartzog , 2010 ) .", "In metazoans , PolII is often enriched immediately downstream of the transcription start site ( TSS ) , raising the possibility that this is a significant barrier to promoter escape in the progression from transcriptional initiation to elongation , with an unknown mechanism by which this barrier is overcome ( Guenther et al . , 2007; Mavrich et al . , 2008; Schones et al . , 2008 ) .", "Although the intrinsic structural preferences of DNA contribute substantially to nucleosome occupancy and positioning in vivo , this chromatin landscape is further manipulated by chromatin remodelers ( Segal and Widom , 2009; Kaplan et al . , 2010 ) .", "Chromatin remodelers use the energy from ATP hydrolysis to evict , assemble , and slide nucleosomes in order to guide the proper nucleosome positioning at key sites , such as eukaryotic promoters , where they maintain the nucleosome-depleted region ( NDR ) and phased flanking nucleosomes ( Hartley and Madhani , 2009; Gkikopoulos et al . , 2011; Tolkunov et al . , 2011 ) .", "Chromatin remodelers share a common Snf2 helicase-like ATPase domain that is required for the disruption of histone–DNA contacts ( Hota and Bartholomew , 2011; Petty and Pillus , 2013 ) .", "Furthermore , they can be separated into two groups based upon their requirement for extra-nucleosomal DNA .", "First , ISWI , INO80/SWR1 and CHD require extra-nucleosomal DNA to remodel nucleosomes , whereby they are primarily involved in nucleosome assembly and generating equally spaced arrays by binding to stretches of exposed DNA and shifting nucleosomes into the gap ( Whitehouse et al . , 2003; McKnight et al . , 2011; Udugama et al . , 2011 ) .", "Second , SWI/SNF and RSC remodelers slide or evict nucleosomes irrespective of extra-nucleosomal DNA ( Whitehouse et al . , 1999; Lorch et al . , 2011 ) .", "The potential role of remodelers in overcoming the nucleosomal barrier has not been fully investigated .", "Studies in yeast have suggested the main role for the chromatin remodeler Chd1 to be within the gene body , where Chd1 reassembles and positions nucleosomes in order to prevent cryptic initiation ( Cheung et al . , 2008; Gkikopoulos et al . , 2011; Radman-Livaja et al . , 2012; Smolle et al . , 2012; Zentner et al . , 2013 ) .", "However , the promoter chromatin architecture is different in metazoans , with the TSS embedded in the nucleosome-depleted region , raising the possibility that this differing chromatin landscape contributes to metazoan PolII accumulating to high levels ahead of the first nucleosome ( Mavrich et al . , 2008; Schones et al . , 2008; Rahl et al . , 2010 ) .", "Here , we show that mammalian Chd1 controls nucleosome dynamics at actively transcribed genes .", "We find that Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover at the promoter and is required to allow efficient PolII promoter escape , with PolII becoming stalled in the absence of Chd1 activity .", "Stalling with the loss of Chd1 activity implies that Chd1 is required to overcome the nucleosomal barrier to allow transcription within a chromatin context .", "In contrast , in the gene body , Chd1 reassembles nucleosomes and suppresses histone turnover .", "Interestingly , mammalian Chd1 is required for pluripotency of embryonic stem ( ES ) cells by maintaining euchromatin and is also required for efficient reprogramming , suggesting that Chd1 plays a key role in mammalian cellular identity ( Gaspar-Maia et al . , 2009 ) .", "Through both positively and negatively impacting histone dynamics , Chd1 plays roles in transcription and in regulating pluripotency and reprogramming ." ], [ "To study the role of mammalian Chd1 , mouse embryonic fibroblasts ( MEFs ) were transfected with a transgene expressing FLAG-tagged wild-type mouse Chd1 or a mutant variant harboring the replacement of a conserved lysine to an arginine residue ( K510R ) in the ATP-binding site of the remodeler ( Figure 1A ) .", "The K510R Chd1 mutation eliminates the catalytic activity without disrupting its ability to interact with other proteins , thereby generating a dominant negative ( Corona et al . , 2004 ) .", "Mutation of this conserved lysine residue has been used to functionally investigate various chromatin remodelers including Brahma and ISWI in Drosophila ( Figure 1A; Elfring et al . , 1998; Deuring et al . , 2000 ) .", "In comparison to knocking down Chd1 expression , a dominant negative approach is less likely to enable redundant mechanisms to mask protein functions .", "Western analysis indicated that the FLAG-tagged proteins migrated at the expected size and were only moderately over-expressed relative to endogenous Chd1 in the MEFs ( Figure 1B ) . 10 . 7554/eLife . 02042 . 003Figure 1 . Chd1 is recruited to promoters with high PolII occupancy and requires ATPase activity to extend into the gene body .", "( A ) Schematic of the domain structure of the full-length mouse Chd1 ( 1-1711 ) used to generate the N-terminal FLAG-tagged construct .", "A region corresponding to the Chd1 ATP-binding pocket is shown below and aligned to various homologues from Saccharomyces cerevisiae and Drosophila melanogaster .", "The arrow indicates the conserved lysine residue mutated to form the dominant negative .", "( B ) MEFs stably expressing either FLAG-tagged wild-type Chd1 or the dominant negative K510R were subjected to western analysis with a two-fold dilution series .", "Untransfected MEFs were used as a reference .", "( C ) A representative genome browser snapshot of ChIP-seq data ( all fragment lengths ) indicating the high occupancy of PolII , wildtype-Chd1 and K510R-Chd1 at gene promoters .", "PolII distribution was determined in cells expressing wildtype-Chd1 using the N20 antibody .", "Normalized counts are indicated on the y-axis .", "( D ) Chd1 binding to the 5′ end of genes was determined by ChIP-seq .", "Here , all recovered DNA fragments , irrespective of length , were analyzed .", "Each row of the heatmap represents the binding pattern across the −0 . 5 kb to +2 kb region flanking the TSS .", "Genes were ranked by the level of PolII occupancy in the −100 to +300 bp region , as measured by ChIP-seq in the MEFs expressing wildtype Chd1 , using the N20 antibody that binds to the N-terminus of the largest subunit of PolII .", "( E ) Analyzing only the short DNA fragments recovered ( 35–75 bp ) allows precise mapping of Chd1 , indicating that wildtype Chd1 binding tracks into the gene body , whereas K510R-Chd1 accumulates just downstream of the TSS .", "The genome-wide average is shown across the 4 kb encompassing the TSS . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 00310 . 7554/eLife . 02042 . 004Figure 1—figure supplement 1 . A novel crosslinking ChIP strategy allows near-complete extraction of chromatin-associated proteins and high-resolution mapping of binding sites .", "( A ) Extraction efficiency of mammalian chromatin associated proteins is low using native conditions .", "Nuclei from cells expressing FLAG-tagged wildtype Chd1 were prepared by hypotonic lysis .", "A nuclear extract was prepared by MNase digestion to mononucleosomes , followed by needle extraction in a buffer containing either physiological or high salt concentration .", "The resulting soluble extract was tested for extraction efficiency compared to the total by fluorescent western blotting ( Licor Odyssey ) .", "Extraction efficiency in percentage is indicated below .", "( B ) Micrococcal nuclease digestion of cross-linked chromatin yields protected sub-nucleosomal particles .", "MNase digestion of cross-linked chromatin yields mono-nucleosomes , as expected from digestion of non-cross-linked chromatin , but also shorter fragments as indicated by agarose gel electrophoresis of the extracted DNA .", "A brief sonication pulse was used in order to efficiently solubilize and extract all FLAG-tagged Chd1 and histones .", "The sonication did not significantly affect the size distribution of DNA fragments , as measured by agarose gel electrophoresis .", "A typical input sample resolved by agarose gel electrophoresis is shown .", "( C ) Limited sonication of cross-linked chromatin allows complete extraction of chromatin-associated proteins .", "Cells expressing FLAG-tagged wildtype Chd1 were cross-linked and digested with MNase as detailed .", "Sonication using a Branson Digital Sonifier was used to prepare a soluble extract , which was tested for extraction efficiency compared to the total by fluorescent western blotting ( Licor Odyssey ) .", "Extraction efficiency in percentage is indicated below . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 00410 . 7554/eLife . 02042 . 005Figure 1—figure supplement 2 . Analysis of short fragments provides high-resolution mapping of Chd1 and PolII binding sites at the promoter .", "( A ) Size class comparison to show crosslinking of Chd1 to the promoter proximal nucleosomes .", "Mapped paired-end reads recovered from the ChIP-seq experiment were separated based on size and occupancy plotted from −1 kb to +2 kb surrounding the TSS as a percentage of the dynamic range .", "PolII ChIP-seq was performed in MEFs expressing wildtype Chd1 .", "( B ) K510R Chd1 is enriched at the promoter and depleted in the gene body relative to wildtype Chd1 .", "Average normalized counts were calculated for the promoter region and the gene body and plotted as a percentage of wildtype Chd1 .", "Statistical significance was determined using the two sample Kolmogorov–Smirnov ( KS ) test .", "( C ) To confirm that the high resolution mapping of PolII and Chd1 shows a similar trend irrespective of fragment size ( Figure 1C ) , genes were ranked and split into quintiles based on PolII promoter occupancy ( all fragment sizes; −100 to +300 bp ) .", "Using only the short reads that were recovered ( 35–75 bp ) , averages for each quintile were plotted from −1 kb to +2 kb surrounding the TSS . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 005 First , we determined the genomic localization of Chd1 using chromatin immunoprecipitation in combination with high throughput sequencing ( ChIP-seq ) .", "We found that under native conditions only 14% of Chd1 could be extracted ( Figure 1—figure supplement 1A ) .", "This led us to pursue a formaldehyde cross-linked ChIP strategy , which allows for harsher extraction conditions .", "Sonication is often used in ChIP to fragment the formaldehyde-cross-linked chromatin .", "This , however , only provides low resolution , as the fragment size is typically 100–300 bp in comparison to the footprint of either PolII or Chd1 , which is 35 bp and 20–45 bp respectively ( Samkurashvili and Luse , 1996; Ryan et al . , 2011 ) .", "We therefore used Micrococcal nuclease ( MNase ) digestion of cross-linked chromatin to increase the resolution of ChIP-seq .", "MNase digests unprotected DNA , leaving DNA fragments corresponding to mono-nucleosomes and sub-nucleosomal fragments , presumably protected by proteins cross-linked to the extra-nucleosomal DNA ( Figure 1—figure supplement 1B ) .", "By using a modified Solexa library preparation protocol in combination with paired-end sequencing , we were able to map DNA fragments as small as ∼35 bp ( Henikoff et al . , 2011 ) .", "By using MNase to fragment the chromatin and minimal sonication we were able to achieve essentially complete extraction of chromatin and associated proteins , thereby overcoming a significant limitation of native ChIP ( Figure 1—figure supplement 1C ) .", "This technique is highly transferable , as it requires no special tool development and uses current sequencing technology .", "Initially analysis of all the recovered fragments was performed .", "As expected , wild-type Chd1 was recruited to the 5′ end of genes , similar to what has been shown for endogenous Chd1 in mouse ES cells ( Figure 1C , D; Gaspar-Maia et al . , 2009 ) .", "Furthermore , the dominant negative K510R-Chd1 showed a broadly similar chromatin recruitment pattern , consistent with the mutation not affecting interactions with recruiting factors .", "In addition , we found that the enrichment of Chd1 was primarily just downstream of the TSS , with recruitment correlating with PolII occupancy of the promoter , as measured in the MEFs expressing wild-type Chd1 ( Figure 1D ) .", "PolII has previously been shown to crosslink to nucleosomes ( Koerber et al . , 2009 ) .", "Therefore , mapping the DNA recovered by immunoprecipitation of these cross-linked PolII:nucleosome complexes will not identify the exact binding site of PolII on DNA .", "To precisely map the position of PolII and Chd1 , we mapped 35–75 bp fragments , which more closely corresponds to the footprint of PolII and Chd1 ( Figure 1—figure supplement 2A ) .", "For PolII , there was a shift of the maximal occupancy towards the TSS , from +150 bp for all fragment sizes to a peak centered over the TSS , likely indicating that the larger fragments correspond to PolII crosslinked to the +1 nucleosome .", "Similarly , for both wild-type and K510R Chd1 , larger size classes indicated Chd1 crosslinked to the well-positioned +1 nucleosome .", "Analysis of short fragments , however , indicated wild-type Chd1 was enriched over the beginning of the gene body with a steady decline from +500 bp .", "However , in the absence of ATPase activity , K510R-Chd1 density peaked downstream of the TSS at +60 bp , where it accumulated to higher levels than wild-type Chd1 and was depleted in the gene body ( Figure 1E , Figure 1—figure supplement 2B ) .", "To confirm that mapping of short fragments was similar to that of all fragments , genes were ranked by PolII promoter occupancy as determined from all fragment lengths and separated into quintiles ( Figure 1—figure supplement 2C ) .", "We find a correlation between Chd1 recruitment and PolII occupancy , as seen for all fragment lengths .", "Overall , this novel ChIP-seq strategy based on the MNase digestion of cross-linked chromatin and the mapping of short fragments allows precise mapping of the footprint of PolII and Chd1 .", "We find that Chd1 binds just downstream of the TSS , correlates with PolII occupancy at the promoter and requires ATPase activity for the binding to extend into the gene body .", "Chromatin remodelers are known to have a major role in the correct positioning and occupancy of nucleosomes ( Gkikopoulos et al . , 2011 ) .", "Therefore we determined the nucleosome profile in cells expressing either wild-type or K510R Chd1 by mapping mono-nucleosome-sized fragments recovered from the sequencing of MNase digested , cross-linked chromatin .", "Cells expressing wild-type Chd1 display the classical metazoan nucleosome organization pattern , with a pronounced nucleosome depleted region ( NDR ) containing the TSS , flanked by well positioned nucleosomes , with the +1 nucleosome entry site ∼50 bp downstream of the TSS ( Figure 2A; Mavrich et al . , 2008; Schones et al . , 2008 ) .", "In addition , the position of the +1 nucleosome corresponded to the level of PolII promoter occupancy , with the 5′ edge progressively moving towards the TSS , to approximately +15 bp for genes with the lowest PolII occupancy , as previously indicated by ranking human nucleosome positions by steady-state RNA levels ( Schones et al . , 2008 ) .", "The positioning of the nucleosomes was not affected by the loss of Chd1 activity , but the nucleosome occupancy was reduced both upstream and downstream of the TSS .", "In contrast to Saccharomyces cerevisiae , where deletion of Chd1 had no effect on the +1 nucleosome occupancy but only the surrounding nucleosomes , blocking Chd1 activity in MEFs affected promoter proximal nucleosomes including the +1 nucleosome ( Gkikopoulos et al . , 2011 ) .", "In S . cerevisiae , the TSS is found just within the +1 nucleosome , thereby suggesting that pre-initiation complex formation requires loss of the +1 nucleosome ( Rhee and Pugh , 2012 ) .", "However , metazoans have a different promoter nuclear architecture with the TSS embedded in the nucleosome depleted region and the +1 nucleosome entry site ∼50 bp downstream ( Mavrich et al . , 2008; Schones et al . , 2008 ) .", "Therefore , loss of the mammalian +1 nucleosome occupancy observed here likely reflects a unique role of mammalian Chd1 because of differences to S . cerevisiae in promoter architecture and perhaps the mechanism of transcription through the promoter proximal chromatin .", "Decreased nucleosome occupancy was also apparent throughout the gene body including upstream of the transcriptional end site ( TES ) . 10 . 7554/eLife . 02042 . 006Figure 2 . Chd1 activity is required to maintain nucleosome occupancy in the promoter region and the gene body . Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing .", "Mono-nucleosomal fragments ( 111–140 bp ) were aligned relative to the TSS or TES .", "( A ) Average nucleosome profile for all genes .", "Genes were also ranked and split into quintiles based on PolII promoter occupancy ( all fragment sizes; density within −100 to +300 bp ) .", "The nucleosome map for the highest and lowest quintile is shown .", "( B ) Nucleosome profiles for genes with either the highest or lowest quintile of wildtype-Chd1 binding ( 35–75 bp fragment sizes; density within 0 to +1 kb ) .", "Statistical significance was determined using the two sample Kolmogorov–Smirnov ( KS ) test on the average number of normalized counts within 5 kb downstream of the TSS or upstream of TES .", "All groups were highly statistically significant ( p<1 × 10−9; TES of genes with the highest Chd1 at the promoter was significant p<3 × 10−4 ) with the exception of the TSS and TES of genes with the lowest PolII density and the TSS of genes with the lowest Chd1 density which showed no significant change . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 00610 . 7554/eLife . 02042 . 007Figure 2—figure supplement 1 . Chd1 activity is required to maintain nucleosome occupancy in the promoter region and the gene body .", "( A ) Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing .", "Mono-nucleosomal fragments ( 111–140 bp ) were aligned relative to the TES .", "Nucleosome profiles for genes with either the highest ( p<2 × 10−16 ) or lowest quintile ( no significant difference ) of wildtype-Chd1 binding within the last 3 kb of the gene body ( 35–75 bp fragment sizes ) .", "Statistical significance was determined using the two sample KS test on the average number of normalized counts within 5 kb upstream of the TES .", "( B ) MEFs were transduced with either a lentivirus harboring a short hairpin RNA to Chd1 or an empty vector in order to knockdown expression of Chd1 .", "Western analysis to confirm knockdown of endogenous Chd1 .", "γ-Tubulin was used as a loading control .", "Quantification revealed that 33% of the Chd1 protein remained after knockdown .", "( C ) Upon knockdown of Chd1 , nucleosome occupancy is reduced at both promoters and gene bodies .", "Nucleosome profiles were determined by MNase digestion and paired end sequencing .", "Mapped mono-nucleosomal fragments ( 111–140 bp ) were aligned relative to the TSS and TES .", "The average profile for all genes is shown , as well as for genes with the highest or lowest quintile of wildtype-Chd1 binding ( 35–75 bp fragment sizes; density within 0 to +1 kb ) .", "Statistical significance was determined using the two sample KS test on the average number of normalized counts within 5 kb downstream of the TSS or upstream of the TES .", "All pairwise comparisons were highly statistically significant ( p<1 × 10−8 ) with the exception of those with the lowest Chd1 occupancy . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 007 The decrease in nucleosome occupancy was most apparent at genes with the highest PolII levels at the promoter , as expected given that Chd1 occupancy correlates with PolII promoter occupancy , with no statistically significant change at genes with low PolII levels at the promoter ( Figure 2A ) .", "Genes were ranked by wildtype-Chd1 binding within the beginning of the gene to more directly test the role of Chd1 in maintaining nucleosome occupancy ( Figure 2B ) .", "Genes with the highest recruitment of Chd1 exhibited the most dramatic loss of nucleosomes at the promoter upon expression of K510R-Chd1 , consistent with a direct role of Chd1 .", "Additionally , genes with the lowest recruitment of wild-type Chd1 displayed a markedly different nucleosome landscape , with unphased nucleosomes and the absence of a pronounced NDR ( Figure 2B ) .", "Despite this relatively flat profile at the TSS being insensitive to the expression of K510R-Chd1 , it suggests that the ‘open’ and ‘closed’ promoter architecture , as defined by the existence of an NDR at the TSS , can be categorized based on recruitment of remodelers such as Chd1 ( Cairns , 2009 ) .", "The loss of nucleosome occupancy within the last 1 kb of the gene body was independent of the level of Chd1 recruitment to the promoter ( Figure 2B ) .", "In contrast , the loss of nucleosome occupancy in the gene body correlated with level of Chd1 binding in the gene body ( Figure 2—figure supplement 1A ) .", "This indicates that Chd1 action within the distal part of the gene body is independent of its recruitment to the promoter .", "To establish whether the effects of the K510R mutant can be mimicked by reduced levels of Chd1 , endogenous levels of Chd1 were reduced using a short-hairpin RNA ( Figure 2—figure supplement 1B ) .", "Nucleosome occupancy was reduced at both the promoter and the gene body upon knockdown of Chd1 , as seen with the expression of K510R-Chd1 ( Figure 2—figure supplement 1C ) .", "To determine the concordance between knockdown and expression of the dominant negative , genes were ranked by transgenic wild-type Chd1 occupancy .", "Genes with the highest Chd1 occupancy were most affected by knockdown , suggesting that knockdown of Chd1 phenocopied the expression of the dominant negative .", "Overall , loss of Chd1 activity resulted in reduced nucleosome occupancy at both the promoter and the gene body confirming that Chd1 is involved in defining the chromatin landscape .", "The mapping of MNase-digested mono-nucleosomes provides information regarding steady-state nucleosome occupancy and positioning , but does not yield insight regarding nucleosome dynamics .", "We used CATCH-IT ( covalent attachment of tags to capture histones and identify turnover ) to measure nucleosome turnover ( Deal et al . , 2010 ) .", "CATCH-IT involves the pulse labeling of newly synthesized proteins and the subsequent purification of newly assembled nucleosomes .", "Our results showed that histone turnover was most rapid at the nucleosomes flanking either side of the promoter and progressively decreased towards the gene body ( Figure 3A ) .", "We observed marked changes in nucleosome turnover upon the expression of K510R-Chd1 .", "First , nucleosome turnover was significantly reduced on either side of the TSS ( Figure 3A ) .", "Second , nucleosome turnover was significantly increased within the gene body , evident from ∼500 bp downstream of the TSS and also in the gene body upstream of the TES ( Figure 3A , B , Figure 3—figure supplement 1A ) .", "These results suggest opposing roles for Chd1 in regulating nucleosome turnover at the promoter and the gene body .", "We found that the role of Chd1 in regulating nucleosome turnover at the promoter and the gene body is independent of gene length ( Figure 3—figure supplement 1B ) .", "This is in contrast to yeast , where a previous study indicated Chd1 protects long genes from nucleosome replacement in the gene body ( Radman-Livaja et al . , 2012 ) .", "In yeast , the magnitude of the effect on nucleosome turnover was approximately equivalent at the promoter and the gene body ( Radman-Livaja et al . , 2012; Smolle et al . , 2012 ) .", "However , we find that the primary action of mammalian Chd1 on driving nucleosome turnover is at the promoter , which might suggest a different function of Chd1 in mammals . 10 . 7554/eLife . 02042 . 008Figure 3 . Chd1 activity has opposing effects on nucleosome turnover at the promoter and the gene body . Nucleosome turnover is decreased over the promoter but increased over the gene body .", "The genome-wide average nucleosome turnover was discerned using CATCH-IT at both the ( A ) TSS and ( B ) TES .", "( C ) Nucleosome turnover correlates with elongating PolII density and is increased in cells expressing K510R-Chd1 .", "Genes were ranked by the density of serine-2-phosphorylated PolII within the last 3 kb of the gene body .", "The average density of elongating PolII was calculated with a sliding window of 500 genes plotted against the average CATCH-IT signal within −3 to −0 . 5 kb relative to the TES ( Pearson correlation coefficient >0 . 85 ) .", "The difference for each window was calculated and is shown graphically below .", "( D ) Chd1 is recruited to genes with actively elongating PolII .", "Genes were ranked as per ( C ) and plotted against the average ChIP-seq signal for FLAG-tagged wildtype Chd1 ( 35–75 bp fragment sizes ) within the same region .", "( Pearson correlation coefficient >0 . 9 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 00810 . 7554/eLife . 02042 . 009Figure 3—figure supplement 1 . Chd1 activity suppresses nucleosome turnover in the gene body , and the role of Chd1 is independent of gene length .", "( A ) Chd1 activity suppresses nucleosome turnover in the gene body .", "Nucleosome turnover is increased in the gene body in cells lacking Chd1 activity .", "Average CATCH-IT signal is plotted for the regions +1 to +3 kb relative to the TSS and −3 to −0 . 5 kb relative to the TES .", "Statistical significance was determined using a t-test .", "( B ) The role of Chd1 on nucleosome turnover at the TSS and the gene body is independent of gene length .", "CATCH-IT signal was averaged at the promoter ( left; −150 bp to +150 bp relative to the TSS ) or the gene body ( right; −3 kb to −0 . 5 kb relative to the TES ) and plotted as a function of gene length with a sliding window of 500 genes . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 009 We found that nucleosome turnover in the gene body correlated with increasing levels of elongating PolII ( Figure 3C ) .", "This is consistent with several studies suggesting that there is lower retention of the complete octamer at high transcriptional rates than at lower ones ( Kireeva et al . , 2002; Thiriet and Hayes , 2006; Bintu et al . , 2011 ) .", "Nevertheless , nucleosome turnover was highly variable at low levels of elongating PolII density , indicating that there is significant gene-to-gene variation in the mechanism of PolII transit through nucleosomes at low rates of transcription .", "Nucleosome turnover in the gene body was increased in cells expressing K510R-Chd1 , with the increase approximately constant at all densities of elongating PolII , suggesting an equivalent contribution by Chd1 in the suppression of nucleosome turnover irrespective of transcription rate ( Figure 3C; lower panel ) .", "We sought to further understand the nature of Chd1 action in the gene body by studying potential recruitment mechanisms .", "As previously discussed , the loss of nucleosome occupancy within the distal regions of the gene body did not scale with Chd1 recruitment to the promoter .", "This suggests separable Chd1 recruitment to the promoter and the gene body .", "We found that the level of both wildtype and K510R-Chd1 recruitment to the gene body positively correlated with elongating PolII density within the gene body ( Figure 3D ) .", "Taken together , these observations suggest that occupancy of Chd1 at the promoter and the gene body correlates with the amount of PolII in the respective genomic compartments .", "The surprising lack of correlation between Chd1 occupancy and suppression of nucleosome turnover in the mutant suggests that Chd1 is not absolutely required for turnover at high transcription rates .", "Overall , these results suggest that Chd1 is recruited to actively transcribing chromatin where it is involved in the suppression of nucleosome turnover within the gene body .", "In contrast to nucleosome turnover at the gene body , nucleosome turnover at the promoter was decreased upon the expression of K510R-Chd1 .", "We sought to determine if this reduction correlated with PolII promoter density given that both Chd1 occupancy and loss in nucleosome occupancy was most pronounced at genes with high PolII promoter density .", "Genes with higher levels of PolII displayed increased nucleosome turnover surrounding the TSS ( Figure 4A ) , as might have been expected from the positive correlation between steady-state RNA levels and nucleosome turnover ( Deal et al . , 2010; Yang et al . , 2013 ) .", "Turnover outside of the promoter region remained high in the absence of Chd1 activity , but was markedly reduced within the promoter proximal region ( −350 to +350 bp ) encompassing the −1 nucleosome , the TSS and the +1 and +2 nucleosomes ( Figure 4A , B ) .", "We determined the average CATCH-IT signal within the promoter proximal region as a function of PolII occupancy ( Figure 4C ) .", "At low to intermediate PolII densities , we found that cells expressing wildtype-Chd1 displayed a positive correlation between PolII density and nucleosome turnover , with turnover reaching a ceiling value at high levels of PolII .", "In contrast , expression of the dominant negative Chd1 resulted in the near-complete abrogation of turnover to basal levels for all levels of PolII occupancy .", "Knockdown of endogenous Chd1 resulted in a partial reduction in nucleosome turnover at the promoter ( Figure 4—figure supplement 1 ) , consistent with either incomplete knockdown or redundant factors .", "The striking loss of nucleosome turnover in the K510R-Chd1 mutant suggests that Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover within the promoter proximal region .", "In summary , by analyzing nucleosome dynamics in combination with steady-state nucleosome occupancy , our results suggest that Chd1 is responsible for PolII-directed nucleosome turnover at promoters but suppresses nucleosome turnover within gene bodies .", "Despite these opposite actions of Chd1 in nucleosome turnover kinetics , we find that Chd1 is required to maintain net nucleosome occupancy both around the promoter and within the gene body . 10 . 7554/eLife . 02042 . 010Figure 4 . Chd1 activity is responsible for PolII-directed nucleosome turnover at the promoter .", "( A ) CATCH-IT data represented as a heatmap for the ±1 . 5 kb surrounding the TSS .", "Genes were ranked by the level of PolII occupancy at the promoter ( all fragment sizes; density within −100 to +300 bp ) .", "( B ) Nucleosome turnover is reduced over the promoter proximal region in cells expressing K510R-Chd1 .", "The difference in CATCH-IT signal between cells expressing wildtype and K510R-Chd1 at the TSS ±500 bp is plotted .", "The genome-wide average nucleosome occupancy is shown for reference for cells expressing wildtype-Chd1 .", "( C ) Chd1 is required for PolII-directed turnover at the promoter proximal region .", "Genes were ranked by PolII promoter density in cells expressing wildtype-Chd1 and plotted against the average CATCH-IT signal in the promoter proximal region with a sliding window of 600 genes . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 01010 . 7554/eLife . 02042 . 011Figure 4—figure supplement 1 . Knockdown of endogenous Chd1 shows a partial reduction in nucleosome turnover at the promoter . MEFs were transduced with either a lentivirus harboring a short hairpin RNA to Chd1 or an empty vector in order to knockdown expression of Chd1 .", "CATCH-IT was performed to measure nucleosome dynamics .", "Genes were ranked by PolII promoter density and plotted against the average CATCH-IT signal in the promoter proximal region with a sliding window of 600 genes . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 011 Previous work in yeast has not identified a clear link between the loss of Chd1 and the consequence on transcription .", "In fission yeast , approximately half of Chd1 ortholog bound promoters showed a change in nucleosomal occupancy , with the vast majority increasing histone H3 density upon loss of Chd1 and no significant change in gene expression ( Walfridsson et al . , 2007 ) .", "In vitro analysis of the S . cerevisiae PHO5 gene promoter indicated that Chd1 was required to remove the +1 nucleosome , which occludes the TSS , but no robust change in gene expression was observed in vivo without the synergistic loss of the chromatin remodeler ISWI ( Ehrensberger and Kornberg , 2011 ) .", "To better interpret the transcriptional consequences of the alterations in mammalian chromatin architecture at the promoter , we established the changes in PolII distribution upon K510R-Chd1 expression by performing ChIP-seq with an antibody against the N-terminus of the largest subunit of PolII , which recognizes total PolII independent of phosphorylation ( Figure 5A ) .", "The majority of PolII was bound to the promoter region , as expected given the prevalence of promoter proximal pausing in metazoans and consistent with the idea that promoter escape is a major barrier to productive elongation ( Krumm et al . , 1995; Rahl et al . , 2010 ) .", "This is in contrast to S . cerevisiae , where the majority of transcriptional control is at the level of PolII recruitment to the promoter , with a relatively even distribution of PolII across the gene ( Stargell and Struhl , 1996; Robert et al . , 2004; Rhee and Pugh , 2012 ) .", "Unexpectedly , the expression of K510R-Chd1 resulted in increased PolII occupancy within the promoter region and was most apparent at genes with the highest PolII occupancy , suggesting a role for Chd1 in the distribution of PolII . 10 . 7554/eLife . 02042 . 012Figure 5 . Chd1 activity is required to enable PolII to efficiently escape the promoter proximal region . Genome-wide distribution of PolII was determined using ChIP-seq with antibodies against ( A ) the N-terminus of Rpb1 to map total PolII and ( B ) PolII Ser2phos to map elongating PolII .", "The recovered short reads ( 35–75 bp ) were mapped and the genome-wide average was plotted .", "Genes were also ranked and split into quintiles based on total PolII promoter occupancy in cells expressing wildtype-Chd1 ( all fragment sizes; density within −100 to +300 bp ) .", "The PolII distribution is shown for the highest and lowest quintiles .", "Statistical significance was determined using the KS test on the average counts within ( A ) −100 to +300 bp and ( B ) +1 kb to +5 kb relative to the TSS .", "Differences between wt-Chd1 and K510R-Chd1 were significant ( p<1 × 10−9 ) with the exception of elongating PolII density in genes the lowest promoter PolII occupancy .", "( C ) The elongation rate within the gene body is unaffected by the expression of K510R-Chd1 .", "The fold enrichment in PolII Ser2phos density within 1 kb genic windows was calculated relative to the average density within the first 5 kb of the gene .", "Error bars represent standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 012 The increased PolII occupancy at the promoter could be due to either increased recruitment of PolII to the promoter or alternatively decreased efficiency of promoter escape into the gene body .", "To distinguish these alternatives , we established the distribution of actively elongating PolII using an antibody that recognizes the serine-2-phosphorylated elongating form ( PolII Ser2phos ) ( Figure 5B ) .", "The signal for PolII Ser2phos increased after the promoter region and continued into the gene body , as expected for elongating PolII .", "In cells expressing K510R-Chd1 , the level of PolII Ser2phos was reduced throughout the gene body , with the effect most pronounced at the genes with highest level of PolII occupancy at the promoter , indicating a role of Chd1 in enabling promoter escape .", "Alternatively , Chd1 might be affecting the elongation rate within the gene body .", "To test this possibility , the distribution of PolII Ser2phos was normalized to the average density across the first 5 kb of the gene body ( Figure 5C ) .", "We found no significant change in the distribution of elongating PolII , suggesting that Chd1 does not affect elongation rate in the gene body .", "Altogether , our results showed that Chd1 activity is required for efficient promoter escape by PolII and in its absence , PolII accumulates at the promoter with a concomitant decrease in the gene body .", "As Chd1 modulates PolII release from promoters , we wondered whether this ATP-remodeling complex alleviates PolII stalling .", "Therefore , we calculated the stalling index in the presence of wild-type and mutant Chd1 .", "We defined the stalling index as the ratio of total PolII density at the promoter ( −100 to +300 bp ) relative to the density of PolII Ser2phos within the gene body ( +1 to +5 kb ) ( Figure 6A , Figure 6—figure supplement 1A ) .", "We found that , on average , cells expressing K510R-Chd1 had a two-fold increase in the stalling index in comparison to wildtype ( Figure 6B ) .", "To determine if this increase in stalling predominantly occurred at stalled or processive genes , genes were ranked by increasing stalling index ( Figure 6C ) .", "Cells expressing wild-type Chd1 displayed stalling indices spanning several orders of magnitude .", "Cells expressing K510R-Chd1 had a greatly reduced range of stalling indices , with an overall increase in stalling and the processive genes being most affected .", "This suggests that Chd1 enables PolII to escape from the promoter region to allow processive transcription . 10 . 7554/eLife . 02042 . 013Figure 6 . Chd1 functions to alleviate the nucleosomal barrier to PolII transit at highly processive genes .", "( A ) Schematic describing the calculation of the stalling index .", "The dark gray shading indicates the total PolII density at the promoter ( −100 to +300 bp ) as calculated from the ChIP-seq data using the N20 antibody against the N-terminus of Rpb1 ( black line ) .", "The light grey shading indicates the density of actively elongating PolII in the gene body ( +1 to +5 kb or the end of the gene ) , calculated from the PolII Ser2phos ChIP-seq data ( gray line ) .", "The stalling index is the ratio of these two values .", "( B ) The stalling index is increased ∼twofold in cells expressing K510R-Chd1 .", "Box plot comparing the distribution of stalling indices for all genes .", "Statistical significance was determined using the Kolmogorov–Smirnov test .", "( C ) Lack of Chd1 leads to increased PolII stalling , preferentially affecting genes with processive transcription .", "Genes were ranked by stalling index and plotted against the stalling index in cells expressing wildtype or K510R Chd1 with a sliding window of 200 genes .", "( D ) Chd1 is preferentially recruited to the promoter region of genes with processive transcription compared to stalled genes .", "Wildtype-Chd1 binding profile across highly stalled and processive genes from the highest quintile of PolII promoter occupancy .", "( E ) K510R-Chd1 accumulates near the dyad axis of the promoter proximal nucleosomes .", "The wildtype and K510R Chd1 binding profiles at these highly processive genes are shown .", "Nucleosome occupancy in cells expressing wildtype-Chd1 is shown for reference with gray ovals indicating the 147 bp of DNA protected by the nucleosomes .", "( F ) PolII stalls in front of promoter proximal nucleosomes in the absence of Chd1 activity .", "PolII distribution at highly processive genes , as measured by ChIP-seq using the N20 antibody is shown .", "Nucleosome occupancy in cells expressing wildtype-Chd1 is shown for reference with gray ovals indicating the 147 bp of DNA protected by the nucleosomes . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 01310 . 7554/eLife . 02042 . 014Figure 6—figure supplement 1 . Loss of Chd1 results in an increase in the stalling index , predominantly at processive genes .", "( A ) Stalling index and traveling ratio are closely correlated .", "Genes were ranked by increasing stalling index in cells expressing wildtype-Chd1 and then the average stalling index was calculated with a 500-gene sliding window and plotted against the traveling ratio .", "The traveling ratio has previously been used to measure accumulation of PolII within the promoter region and is defined as the ratio of total PolII density at the promoter ( −30 to +300 bp ) relative to the total PolII density in the gene body .", "There is a potential concern with this metric: alternative promoters could cause a large peak of total PolII density within the gene body .", "The stalling index reduces this problem by using PolII Ser2phos density within the gene body ( +1 to +5 kb ) .", "This graph shows that over a large numbers of genes the stalling index and traveling ratio are however closely correlated ( Pearson correlation coefficient >0 . 85 ) .", "( B ) PolII density at the promoter ( −100 to +300 bp ) in the MEFs expressing wildtype-Chd1 .", "Boxplots were calculated for the top 500 stalled and processive genes as described in ( C ) .", "( C ) Boxplots comparing stalling indices for the different groups analyzed .", "From left to right: ( 1 ) all genes; ( 2 ) genes from the highest quintile of PolII promoter occupancy ( all fragment sizes; density within −100 to +300 bp ) ; ( 3 ) top 500 stalled genes from this quintile; ( 4 ) top 500 processive genes from this quintile .", "Statistical significance was determined using the KS test . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 01410 . 7554/eLife . 02042 . 015Figure 6—figure supplement 2 . The promoters of processive genes have a highly dynamic chromatin barrier to transcription .", "( A ) Distinct nucleosome organization at processive and stalled genes .", "Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing .", "Mono-nucleosomal fragments ( 111–140 bp ) were aligned relative to the TSS .", "MNase-seq for the highly stalled and processive genes from the highest quintile of PolII promoter occupancy .", "Statistical significance was determined using the two sample KS test on the average number of normalized counts within 5 kb downstream of the TSS: p<2 × 10−12 .", "( B ) Nucleosome turnover at the +1 is higher at processive genes than stalled genes .", "Average CATCH-IT signal is plotted for the regions 0 to +150 bp relative to TSS .", "Statistical significance was determined using a t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 015 To confirm that the main function of Chd1 is at processive genes , Chd1 binding to stalled or processive genes was addressed .", "As Chd1 binding scales with PolII density ( Figure 1 ) , only the genes in the highest quintile of PolII promoter occupancy were investigated to minimize the variation due to differences in PolII promoter occupancy .", "We found that processive genes showed a much higher Chd1 occupancy than stalled genes ( Figure 6D ) , despite having a lower level of PolII density ( Figure 6—figure supplement 1B ) .", "In addition , the median stalling index was increased ∼twofold at these processive genes by the expression of K510R-Chd1 , whereas these stalled genes were unaffected ( Figure 6—figure supplement 1C ) .", "This suggests that although Chd1 recruitment largely follows PolII occupancy at the promoter , there must be additional factors that are linked to processive elongation that influence Chd1 recruitment .", "The Chd1-binding profile was plotted for processive genes over the promoter region to understand how Chd1 might be functioning to enable promoter clearance at processive genes ( Figure 6E ) .", "Wildtype-Chd1 displayed a relatively even profile across the promoter , whereas K510R-Chd1 displayed peaks of binding near the dyad axis for each of the promoter proximal nucleosomes , with the peak of Chd1 ∼50 bp inside of the leading edge of the +1 nucleosome .", "Binding at this position is consistent with in vitro studies , which suggest that chromatin remodelers initiate translocation on nucleosomal DNA at an internal position ∼20 bp from the dyad at superhelical location 2 ( Saha et al . , 2005; Zofall et al . , 2006 ) .", "These peaks likely represent the sites of action of wildtype-Chd1 on its nucleosomal substrate , but through the expression of catalytically dead Chd1 we have been able to capture these normally transient interactions .", "Recent work in Drosophila indicated that at the more processive M1BP bound genes , PolII accumulated in front of the well-positioned +1 nucleosome ( Li and Gilmour , 2013 ) .", "In agreement with this , we find that processive genes have higher promoter proximal nucleosome occupancy and furthermore that +1 nucleosome turnover is higher at processive genes than stalled genes ( Figure 6—figure supplement 2A , B ) .", "This raised the possibility that the promoter proximal nucleosomes at processive genes are acting as dynamic barriers to transcription .", "We therefore sought to understand how PolII transits through the promoter proximal nucleosomes in the absence of functioning Chd1 .", "The total PolII ChIP-seq profile was plotted over the promoter region of these highly processive genes , which become stalled following the expression of K510R-Chd1 .", "In contrast to the relatively even profile for PolII in the cells expressing wildtype-Chd1 , the absence of Chd1 activity resulted in clear peaks in the PolII distribution ( Figure 6F ) .", "These peaks precede each of the promoter proximal nucleosomes and are shifted upstream relative to the sites of action of Chd1 at the nucleosomes , with PolII accumulating ∼20 bp inside the entry site of the +1 nucleosome .", "This suggests that Chd1 functions at processive genes to remove the nucleosomal barrier ahead of PolII and in its absence PolII stalls in front of the now static promoter proximal nucleosomes leading to a reduced efficiency of promoter escape ." ], [ "Using a novel high resolution chromatin immunoprecipitation strategy , we report the key role of Chd1 in defining nucleosome dynamics during the transcription cycle ( Figure 7A ) .", "We find that mammalian Chd1 is primarily recruited just downstream of the start site of PolII-bound genes .", "Through the expression of a dominant negative form of the chromatin remodeler Chd1 , we have shown that Chd1 is responsible for the vast majority of nucleosome turnover at the promoter and is required for PolII promoter escape .", "Our analysis identifies promoter proximal nucleosomes as a barrier to PolII promoter escape and suggests how Chd1 overcomes this barrier .", "In addition , we find that Chd1 also functions in later stages of the transcription cycle , with Chd1 binding in the gene body correlating with elongating PolII density , where it suppresses nucleosome turnover and in its absence leads to decreased nucleosome occupancy . 10 . 7554/eLife . 02042 . 016Figure 7 . Model depicting the role of Chd1 in defining nucleosome dynamics during transcription . At the promoter of processive genes , Chd1 is recruited directly to the promoter proximal nucleosomes , where it is required for both eviction of existing histone octamers ( green ) and deposition of new octamers ( red ) .", "In the absence of Chd1 activity , PolII stalls close to the leading edge of the nucleosome .", "In the gene body , Chd1 occupancy correlates with elongating PolII , where it reassembles histone octamers in cis behind the transiting PolII .", "The lack of Chd1 activity leads to increased deposition of new histone octamers through an unknown factor . DOI: http://dx . doi . org/10 . 7554/eLife . 02042 . 016 The role for Chd1 in gene bodies is consistent with in vitro studies indicating that Chd1 actively assembles chromatin and slides nucleosomes into ordered arrays ( Lusser et al . , 2005; Torigoe et al . , 2013 ) .", "In vivo , Chd1 is likely to function in reassembly of nucleosomes displaced during elongation .", "In the absence of Chd1 activity , reassembly is compromised , leading to the deposition of de novo synthesized nucleosomes , consistent with observations in yeast ( Gkikopoulos et al . , 2011; Hennig et al . , 2012; Radman-Livaja et al . , 2012; Smolle et al . , 2012 ) .", "The close correlation between Chd1 occupancy and elongating PolII density indicates a possible mechanism for the recruitment of Chd1 , consistent with studies indicating that Chd1 associates with elongation factors including polymerase-associated factor ( PAF1 ) , DSIF and FACT ( Simic et al . , 2003 ) .", "In vitro remodeling by Chd1 is dependent upon its C-terminal DNA binding domain interacting with extranucleosomal DNA and may also be required for efficient Chd1 recruitment ( Kagalwala et al . , 2004; Stockdale et al . , 2006; McKnight et al . , 2011 ) .", "More specifically , the DNA immediately upstream of the nucleosome has been shown to be important for the initial stage of nucleosome movement in ISWI-type remodelers ( Zofall et al . , 2004 ) .", "Single-molecule optical trap experiments suggest that PolII transcribes through a nucleosome by the in cis transfer of histones , particularly the H3:H4 tetramer , to DNA upstream of PolII via the formation of a transient DNA loop ( Hodges et al . , 2009; Kulaeva et al . , 2009; Bintu et al . , 2011 ) .", "This might suggest that Chd1 is recruited to the gene body via the elongation complex and is then targeted to the nucleosome substrate via binding to the exposed DNA loop immediately adjacent to the histone octamer to facilitate the in cis transfer of histone octamers .", "Chd1 is primarily found at the 5′ end of PolII bound genes and is particularly enriched at genes undergoing processive transcription .", "By increasing the resolution of standard cross-linked ChIP-seq , we find that Chd1 binds within the leading edge of promoter proximal nucleosomes , where it is required for virtually all of PolII-directed nucleosome turnover at the promoter .", "Expression of K510R-Chd1 results in reduced steady-state nucleosome occupancy .", "Despite this , PolII was observed to accumulate just within the entry site of promoter proximal nucleosomes and a concomitant increase in the stalling index , indicative of an increase in the barrier to PolII promoter escape .", "This increased PolII stalling is consistent with the reduced nucleosome turnover at the promoter , indicating a more static chromatin structure forming a barrier to promoter escape .", "The increase in stalling occurs despite a decrease in steady-state nucleosome occupancy , highlighting the importance of kinetic measurements for chromatin structure .", "The apparent discrepancy of an increase in the barrier to PolII accompanying a decrease in steady-state nucleosomal occupancy can be most easily reconciled if Chd1 acts to evict the nucleosomes ahead of PolII transit and then reassemble nucleosomes in the wake of PolII .", "In the absence of Chd1 , nucleosome eviction is likely to still occur as a result of transcription albeit at a lower rate , and the CATCH-IT analysis suggests de novo nucleosome deposition is greatly reduced , overall leading to the reduced steady-state nucleosome occupancy .", "These opposing activities at the gene body and the promoter may perhaps be explained through differing mechanisms by which Chd1 engages with the nucleosomal substrate after the point of recruitment .", "As discussed , the assembly activity in the gene body is likely to be directed via the non-specific DNA binding domain interacting with extra-nucleosomal DNA .", "A recent in vitro study directly tethered yeast Chd1 to the nucleosomal substrate independent of the DNA binding domain and observed nucleosome sliding and eviction more typical of SWI/SNF-type remodelers ( Patel et al . , 2013 ) .", "This suggests that the observed nucleosome eviction by Chd1 at the promoter is likely to reflect activity independent of the DNA binding domain , raising the question of how Chd1 engages with the nucleosomal substrate .", "The tandem chromodomains in human Chd1 have been shown to specifically interact with H3K4me3 , whereas in S . cerevisiae , the chromodomains lack key tryptophan residues required for H3K4me3 binding ( Flanagan et al . , 2005; Sims et al . , 2005 ) .", "Therefore activity dependent on the chromodomains provides a tantalizing explanation for the role of mammalian Chd1 in nucleosome eviction at the promoter in contrast to yeast Chd1 , where Chd1 does not affect occupancy of the +1 nucleosome ( Gkikopoulos et al . , 2011 ) .", "This unique requirement for mammalian Chd1 to overcome the nucleosomal barrier to PolII at the promoter may reflect differences from yeast in both chromatin architecture and transcriptional regulation , with yeast having the TSS buried within the +1 nucleosome and primarily regulating transcription through recruitment ( Robert et al . , 2004; Rhee and Pugh , 2012 ) .", "These functions of mammalian Chd1 in defining nucleosome dynamics during transcription may explain phenomena associated with its loss .", "Given that the majority of post-translational modifications ( PTMs ) are found on either H3 or H4 , the retention of the H3:H4 tetramer during transcription has been proposed to have a role in the maintenance of chromatin states throughout the genome ( Kulaeva et al . , 2009 ) .", "This suggests that the increased histone turnover observed in the gene body in the absence of Chd1 activity could compromise epigenetic regulation , and might explain the observation that Chd1 is required for maintaining euchromatin in ES cells by preventing heterochromatin formation ( Gaspar-Maia et al . , 2009 ) .", "In contrast , the Chd1 dependent turnover observed at the promoter would have the effect of removing histone ‘memory’ and might explain the requirement for Chd1 in reprogramming through the deposition of de novo histones at the promoter to allow the reactivation of pluripotency genes ( Gaspar-Maia et al . , 2009; Jullien et al . , 2012; Skene and Henikoff , 2012 ) .", "Given that doxorubicin , a commonly used anti-cancer drug , enhances nucleosome turnover , the identification of Chd1 in directing nucleosome turnover provides a mechanistic basis for its tumor suppressor role and may indicate the molecular basis for carcinogenesis in the absence of Chd1 ( Huang et al . , 2012; Liu et al . , 2012; Yang et al . , 2013 ) .", "Overall , these observations point to a key role for Chd1 in regulating the mammalian transcriptional landscape ." ], [ "Immortalized MEFs were transfected with a Piggybac expression vector with the CAG promoter driving N-terminal FLAG-tagged expression of Chd1 ( 40086932; Open Biosystems , Pittsburgh PA ) and puromycin resistance .", "The K510R mutation was generated by site directed mutagenesis and verified by sequencing .", "MEFs were cultured in Dulbecco’s modified Eagle’s medium ( DMEM ) ( #11965-092; Invitrogen , Grand Island NY ) supplemented with 10% FBS , 1% penicillin-streptomycin at 37°C and puromycin ( 1 . 5 µg/ml ) .", "Knockdown of endogenous Chd1 was achieved using vectors as previously described ( Gaspar-Maia et al . , 2009 ) .", "Western blots were performed under standard conditions using whole cell extracts and probed with the following antibodies: FLAG ( M2 F1804; Sigma , St . Louis MO ) ; Chd1 ( 39729; Active Motif , Carlsbad CA ) ; γ-Tubulin ( ab11316; Abcam , Cambridge MA ) .", "ChIP was performed as previously described ( Schmiedeberg et al . , 2009 ) with the following modifications .", "Chromatin fragmentation was achieved by adding MNase after cell lysis to produce predominantly mono-nucleosomes .", "Sonication ( 40 s; 30% amplitude; Branson digital sonifier ) was used to ensure complete extraction of Chd1 and histone H3 , as measured by western analysis .", "The following antibodies were used: FLAG ( A2220; Sigma ) ; N20 ( sc-899; Santa Cruz , Dallas Tx ) ; PolII Ser2phos ( ab5095; Abcam ) .", "Libraries were prepared from the isolated DNA and sequenced on the Illumina HiSeq 2000 platform ( Henikoff et al . , 2011 ) .", "Paired-end sequencing data were processed and aligned to the mm9 genome build using Bowtie and the distribution plotted around the 5′ and 3′ ends of genes as previously described ( Henikoff et al . , 2011 ) .", "CATCH-IT was performed as described previously with the following modifications ( Yang et al . , 2013 ) .", "Nuclei were isolated by hypotonic lysis of cells in ice-cold NE1 buffer ( 20 mM HEPES-KOH ( pH 7 . 9 ) , 10 mM KCl , 1 mM MgCl2 , 0 . 1% Triton X-100 , 1 mM DTT , 20% Glycerol ) and then processed as previously described .", "Input and CATCH-IT DNAs were amplified using a whole-genome amplification kit ( Sigma-Aldrich ) , followed by labeling using Cy3 and Cy5 heptamers according to the Roche Nimblegen labeling protocol .", "Labeled samples were hybridized together to mouse 2 . 1-million probe promoter arrays ( Roche Nimblegen , Madison WI ) ." ], [ "Data have been submitted to the Gene Expression Omnibus under accession number GSE52349 ( Skene et al . , 2014 ) ." ] ]

[ "RNA polymerase II ( PolII ) transcribes RNA within a chromatin context , with nucleosomes acting as barriers to transcription .", "Despite these barriers , transcription through chromatin in vivo is highly efficient , suggesting the existence of factors that overcome this obstacle .", "To increase the resolution obtained by standard chromatin immunoprecipitation , we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin .", "We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription , where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover .", "The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes .", "We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape , thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency ." ]

[ "DNA is tightly packaged in a material called chromatin inside the cell nucleus .", "To produce proteins this DNA must first be transcribed to produce a molecule of messenger RNA , which is then translated to make a protein .", "To assist with this process cells ‘unpack’ certain regions of the DNA so that enzymes that catalyze the different steps in this process can have access to the DNA .", "A protein called Chd1 is involved in the unpacking process in yeast , but its role in more complex animals is not clear .", "Now , Skene et al . have shown that this protein is needed to allow the enzyme that catalyzes the transcription of DNA—an enzyme called RNA polymerase II—to do its job .", "Chd1 acts to unpack the tightly packaged DNA from chromatin , thus allowing the transcription of the DNA to proceed .", "In the absence of Chd1 activity , RNA polymerase II stalls at the gene promoter—the region of DNA that starts the transcription of a particular gene .", "This work highlights how the packaging of DNA in the cell is highly dynamic and controls fundamental biological processes .", "Skene et al . modified a well-known genetic technique called ChIP-seq .", "Previous ChIP-seq protocols typically provided a blurry , low-resolution map of where proteins bound to chromatin .", "Skene et al . used an enzyme to ‘chew back’ the DNA to reveal the exact ‘footprints’ of the Chd1 protein and the RNA polymerase II enzyme on the chromatin in mice .", "It will be possible to adapt this new protocol to map the positions of other proteins , which will help to improve our understanding of the ways in which chromatin regulates access to DNA ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "short report" ]

[ [ "The temporal and spatial heterogeneity of any given environment poses a major challenge to all life on Earth .", "Accordingly , a common characteristic of living organisms is the capacity to respond to environmental cues .", "Many of these responses involve nuanced decisions integrating multiple cues or evaluating cue intensity , such as chemotaxis in bacteria and other microbes ( Wadhams and Armitage , 2004 ) .", "Even ‘simple’ organisms make decisions based on the integration of multiple cues or the intensity of particular cues .", "For example , unicellular Dictyostelium discoideum amoebae aggregate into multicellular slugs in the absence of food , and travel at precise , genetically determined angles in the direction of a light source ( Annesley and Fisher , 2009 ) .", "Many complex environmental responses have recently been discovered in plants , including luring in animal predators to attack herbivores in response to herbivory ( De Moraes et al . , 1998 ) , production of toxins in response to distress signals from kin ( Karban et al . , 2013 ) , and dose-dependent responses to insufficient water ( Pandey et al . , 2016 ) or excess salt ( Julkowska and Testerink , 2015 ) in the soil .", "These responses demonstrate an impressive capacity to detect a variety of environmental signals and respond accordingly .", "These instinctive behaviors have been sculpted over evolutionary time , and can be contrasted with learned behaviors that are developed within the lifetime of an individual as a result of their particular interactions with the environment .", "Individuals and groups of organisms leverage learning to exploit patterns in their environment over short timescales , and the capacity to do so frequently confers a selective advantage ( Morand-Ferron , 2017 ) .", "Learning is useful in any environment that contains exploitable regularities and patterns and for which the instincts of the organism have not evolved to perfectly exploit those patterns .", "Learning is ubiquitous among animals , from zebrafish ( Sison and Gerlai , 2010 ) and insects ( Prokopy et al . , 1982 ) to nematodes ( Wen et al . , 1997 ) , and recent work in Caenorhabditis elegans is beginning to unravel its molecular and genetic mechanisms ( Gyurkó et al . , 2015 ) .", "Over the last decade , there has been substantial debate in neurobiology and philosophy of mind regarding what kinds of organisms possess the capacity to learn ( Gagliano et al . , 2016; Gagliano et al . , 2018; Thellier , 2017; Gagliano , 2017 ) , along with thornier issues like cognition ( Garzón , 2007; Gross , 2016; Adams , 2018; Segundo-Ortin and Calvo , 2019 ) and intelligence ( Marder , 2013; Trewavas , 2017 ) .", "Some of these disputes center around particular terminology and language , but some basic empirical questions remain contentious , including the simple question: can plants learn ?", "A series of experiments on associative conditioning have been carried out in plants since the 1960s , notably the efforts of Holmes and Gruenberg , 1965 , Haney , 1969 , Levy et al . , 1970 , and Armus , 1970 .", "These studies generally failed to observe conditioning in plants , and those that concluded in favor of conditioning lacked sufficient controls to rule out other explanations , as reviewed by Adelman , 2018 .", "Gagliano et al . , 2016 provide the most convincing report of conditioning in plants to date , but there are no published reports of replication , from the original lab or others .", "Unlike many previous experiments designed to test for learning in plants , Gagliano et al . lengthened the timelines of traditional animal learning experiments to be more appropriate for plant physiology .", "Training takes place over 3 days with stimuli lasting 60–90 min , with intertrial intervals of 60 min .", "Associative learning is the phenomenon whereby an individual organism associates two stimuli , and thereafter uses one as an indicator for the other .", "In Ivan Pavlov’s classic work , dogs were trained to associate the ringing of a metronome with the imminent arrival of food , and demonstrated the appropriate physiological response of salivation ( Pavlov , 1927 ) .", "Associative learning experiments attempt to pair an unconditioned stimulus for which there is an observable response to a conditioned stimulus which is originally neutral .", "Learning is demonstrated when presentation of the conditioned stimulus can elicit responses normally associated with the unconditioned stimulus .", "In December 2016 , Gagliano et al . published a report claiming plants have the capacity to learn adaptive behaviors during individual lifetimes using an experimental setup analogous to Pavlov’s famous experiment ( Gagliano et al . , 2016 ) ." ], [ "Gagliano et al . adapted Pavlov’s paradigm for pea plants ( Pisum sativum ) using light as the unconditioned stimulus and wind as the neutral stimulus , which through training became the conditioned stimulus ( Gagliano et al . , 2016 ) .", "Plants were grown inside a dark controlled growth chamber with individual PVC Y-mazes with blue LEDs and fans attached on each arm , and plant learning was inferred through the maze arm selection of the plants .", "This experimental setup is particularly suited to pea plants , which are topped during their early stages by a single tendril , which makes maze arm selection quite clear .", "See Materials and methods for growth conditions , germination protocol , and maze dimensions , which are unchanged in this study .", "Figure 1—figure supplement 1 details the construction of the Y maze and the associated lights and fans required for the experiment .", "Gagliano et al . performed two pilots: Stationary Light and Double Light Stationary Fan .", "In stationary light , a LED was fixed on one arm and turned on for three 1-hr periods per day for 3 training days , then turned off for the testing day .", "All plants grew into the arm with the LED , confirming positive phototropism .", "In Double Light Stationary Fan , lights were affixed to both arms and a blowing fan was fixed on one arm .", "Plants grew at random , unaffected by the fan .", "Taken together , these pilots showed light is a suitable unconditioned stimulus and wind is a suitable neutral candidate conditioned stimulus , at least when both stimuli are stationary .", "However , the main experiments involve moving stimuli .", "Therefore , we chose a different set of additional controls to replace the pilot studies , designated Moving Light , Moving Light Fan Opposite , and Moving Light Fan Adjacent .", "The training period for all three of these consisted of a light moving between maze arms in the same pattern as the lights in the four main conditions .", "On testing day , Moving Light was given no stimulus , Moving Light Fan Opposite had a fan placed opposite the last light exposure , and Moving Light Fan Adjacent had a fan placed on the same maze arm as the last light exposure .", "Moving Light is intended to confirm that the training regimen consistently results in growth towards the last presentation of light in the absence of wind , the other two are to check for systematic growth toward or away from the fan as well as any ‘fan-induced amnesia , ’ wherein the fan interferes with the plant’s growth towards the last presentation of light .", "If fans interfere with phototropic growth , this phenomenon would explain the Gagliano et al . results: phototropic growth without the fan on the testing day , random growth with the fan on testing day .", "For the main experiment in both studies , seedlings were randomly assigned to two groups: F vs L presented light and fan on opposite maze arms , F + L presented light and fan on the same arm .", "The plants were trained for 3 days with 3 exposures per day with wind proceeding light by 60 min , overlapping for 30 min , then light only for an additional 30 min .", "These exposures were moved from one arm to the other according to the pattern: day 1 , left ( L ) /right ( R ) /L; day 2 , L/R/R; day 3 R/L/L , tested on R , or the inverse ( plants were randomly assigned to opposite patterns and grouped for analysis ) .", "These 3 days of training were timed with the plants’ growth such that on the fourth day the plants would grow into one arm or the other of the maze .", "Lights were disabled for this day , and the plants were subdivided into control and experimental groups .", "Control plants were left undisturbed and grew towards the arm where light was last presented on day 3 , whereas experimental plants had fans placed so as to suggest light would be presented from the opposite arm .", "Gagliano et al . report that approximately two thirds of experimental plants showed behavior influenced by the fan , thus demonstrating associative learning .", "The setup and results of the two pilots and four main experimental conditions are shown in Figure", "1 . The Gagliano et al . experiment was clever and innovative , and reports a finding of outstanding interest .", "Findings this unexpected have the potential to open up a new field of study , the establishment of which requires independent verification and experimental rigor ( Kuhn , 2012 ) .", "To that end , this study uses a larger sample size and fully blinded scoring .", "Sample size in the Gagliano et al . study was sufficiently large to generate strong evidence , with several p values under 0 . 005 .", "The present study increased sample size significantly to allow detection of a weaker effect and estimate the effect size more precisely .", "The blinding protocol is also more complete .", "In the original study , the two experimenters also performed the scoring: the experimenter who set the experiment up recorded while the other scored the plants .", "In this replication attempt , scoring began with the experimenter removing all plants from the growth chamber , removing the fans and lights , and placing plants still within Y-mazes in a random order along the bench .", "Thereafter , an independent observer who had no other involvement in the experiment scored the plants and recorded scores while the experimenter was in a separate room .", "The scoring protocol was precisely defined as follows: if any part of the plants has grown more than 5 mm into a maze arm the plant was scored as choosing that arm , unless there was any growth into the other arm .", "If any growth was present in both arms or if <5 mm growth was present in either arm , plants were scored as choosing neither and not counted .", "The additional controls Moving Light Fan Opposite and Moving Light Fan Adjacent were used to test for an inherent attraction or repulsion to the fan stimulus , and found no significant effect on growth direction ( p=0 . 1713 , Fisher’s exact test [Fisher , 1935] , two-tailed ) , as reported by Gagliano et al . This supports fans as an acceptable neutral stimulus .", "However , while Gagliano et al . demonstrated plants always grow toward a stationary light , we chose to ask whether plants always grow toward the last presentation of a light moving in the same regimen as the experiment , the Moving Light condition .", "Their growth did not differ significantly from a random 1:1 expectation at this sample size ( p=0 . 5885 , Fisher’s exact test ) .", "The results of the additional control studies in this study and the Gagliano et al . pilots are compared in Figure", "2 . In the four main conditions , the difference in results is fundamentally the same .", "F + L Control and F vs L Control plants did not always grow towards the last presentation of light , although interestingly both , like the Moving Light control , had a slight tendency in that direction .", "This tendency was statistically significant only in F + L control ( also the condition with the largest sample size , n = 61 , p=0 . 0243 ) .", "In other words , there is a tendency to grow toward the last presentation of light , even if the presentation of light was very brief ( 1 hr ) and proceeded by presentations of light from the opposite direction .", "However , we found this effect to be much weaker than the perfectly phototropic response Gagliano et al . report , despite using very similar lighting .", "In the F + L Control and F vs L Control , we also found a slight tendency to grow toward the last presentation of light but far weaker than the perfect directionality reported by Gagliano et al . The noise in the control conditions makes any associative learning more difficult to detect .", "While Gagliano et al . report a significant difference between F + L and F + L Control ( p=0 . 0027 , n = 13 + 10 , Fisher’s exact test , two-tailed ) , we found no significant difference ( p=0 . 335 , n = 61 + 60 ) .", "Similarly , Gagliano et al . report a significant difference between F vs L and F vs L Control ( p=0 . 0017 , n = 13 + 9 ) , whereas we found no significant difference ( p=0 . 387 , n = 42 + 40 ) .", "The results of the main four conditions are shown in Figure 3 ." ], [ "Gagliano et al . reported that in the absence of fan stimuli , pea plants always grow towards the last 1-hr presentation of light , whereas we find their growth to be only slightly biased toward the last presentation of light .", "It is possible that this difference is due to the use of different cultivars of Pisum sativum - Gagliano et al . used cv Massey gem , whereas this study used cv Green Arrow because Massey Gem was not available .", "These cultivars are closely related , are both full sun varieties , and have a similar growth habit .", "There were several other minor differences in materials used , but importantly growth conditions , maze construction , training regimen , and fan and light intensity were identical to the conditions reported by Gagliano et al . Because the capacity for associative learning is a complex trait , it is expected to not have evolved in the few decades since these cultivars shared a common ancestor , and seems equally unlikely to have been lost from one cultivar but not another in the absence of specific selection .", "However , a stronger phototropic response in Massey Gem cannot be excluded , calling for further replication efforts .", "We hope to see these performed using larger sample sizes and fully blinded scoring , and ideally with input and assistance from the original authors .", "Input from the original authors would allow for a more accurate replication , such as using the same pea plant supplier , LEDs , and fans , which may be critical to reproduce the phenomenon .", "If these efforts reproduce the phenomenon , we predict this experimental system will become widely used in experiments on plant learning .", "One shortcoming in the experimental system is that variation in plant growth rates results in substantial attrition .", "Plants were examined the morning of testing day to determine if they had already grown into one arm of the maze , and if they had they were excluded from the analysis .", "Furthermore , after testing day some plants had still not grown into either arm , and were therefore also excluded from the analysis .", "While care was taken to grow plants as uniformly as possible , approximately 40% of the plants were disqualified for growing too fast or too slow , as shown in Table 1 .", "The December 2016 report of associative learning in plants has garnered substantial attention in the press ( WNYC Studios , 2018; Morris , 2018; Berman , 2018 ) , and the reported phenomenon is extremely interesting .", "Gagliano et al . conclude this phenomenon will force us to reconsider the nature of learning ( Gagliano , 2017 ) and we agree , contingent on the phenomenon being reproducible .", "Associative learning in the absence of traditional neurons must have a fascinating molecular mechanism and would open a new field of research .", "Unfortunately , no reports of successful replication have been published since the initial paper .", "Furthermore , this attempt at replication casts some doubt on the underlying premise used for scoring plant response , namely that plants will always grow towards the most recent one-hour light exposure after repeated exposures on different arms of a Y maze .", "At the least , it suggests that the conditions required for the experimental setup to function properly may be more precise than the stated parameters in the original paper .", "Additional work from the original authors and replications in independent labs are needed if this fascinating phenomenon is to move into the scientific mainstream ." ], [ "Peas ( Pisum sativum cv Green Arrow , Botanical Interests , USA ) were germinated hydroponically in round containers kept in the dark .", "Seeds were first soaked in water for 24 hr , then wrapped by wet paper towel surrounded by aluminum foil in vertical rolls .", "These rolls were placed in water and incubated in the dark at 20°C , changing the water daily .", "Roughly three times as many seeds were germinated as plants desired , and after 5 days of water incubation seeds were inspected for germination using a dim red LED headlamp ( ~0 . 8 lux measured at light source , <0 . 2 lux measured at seedlings ) .", "Seeds with radicle >5 mm were considered to be germinated , ungerminated seeds were discarded .", "Among the germinated seeds , those with particularly long or short radicles were discarded to minimize variance in growth stage .", "Roughly twice times as many germinated seedlings were kept as plants desired .", "Each seedling was then planted in the center of round pots ( 5 cm diameter at top , 6 cm deep , 4 cm diameter at bottom , with a single drain hole of ~2 mm diameter in the center ) in Hoffman seed starter potting and planting mix ( Good Earth Inc , NY ) , at a depth of 15 mm .", "Soil was first watered to saturation , seeds were planted , and plants watered again to saturation .", "Soil was allowed to drain for ~30 min , then germinated seedlings were moved into a controlled growth chamber ( PGR15 Growth Chamber , Conviron ) until emergence from soil .", "Chamber was maintained at 20°C , 85% humidity , with blue and red LEDs balanced for an approximation of white light .", "Light was delivered at 50 µmol m−2 s−1 at soil surface with 8:16 hr light:dark cycle with light phase beginning at 09:00 ( identical to Gagliano et al . ) .", "After 3–4 days , most seedlings had emerged from soil .", "Seedlings at similar growth stages were selected , watered to saturation and allowed to drain , and the Y maze attached .", "To correct for some plants being minorly off-center , all plants were rotated to maximize left-right symmetry before the attachment of the maze ( importantly , the relevant growth directions had not been assigned at this stage , so there is no possible bias in the attachment of mazes , which were as centered as possible ) .", "Once all plants had mazes attached to their pots , they were assigned to groups using a random number generator , then grouped in rows and placed into the experimental growth chamber , where the fan and LEDs were attached on top of the maze arms .", "The experimental apparatus consisted of blue LEDs with light intensity ~14 μmol m−2 s−1 ( measured at soil surface ) in the 430–505 nm wavelength range ( CO RODE part#CR150514E156 , Dr . Gagliano did not specify a particular LED model and did not respond to requests for model/part numbers ) .", "Fans were 35 mm 10 , 000 RPM computer cooling fans ( Gdstime XH2 . 54-2Pin 3510S 35 × 10 mm brushless fan ) .", "The fans generated a semi-turbulent airflow of 0 . 6–0 . 8 m s−1 at all points in the Y maze , including soil surface , the branching point of the maze , and on the fan arm and the opposite arm .", "Flow was approximately downward in the fan arm ( parallel to the arm of the maze ) and upward in the opposite arm , and was measured with TPI 575 anemometer hotwire probe and vane , which each gave similar results .", "These systems were soldered to two sets of 12V DC wires which extend throughout the growth chamber , and are powered by a digital automated timer system .", "12V DC power was generated from 120 V 60 Hz AC power by a Winkeyes power supply ( ASIN# B018G3ABWY ) .", "On testing day , during which plants must grow into one arm or the other , plants were further subdivided into control and experimental plants .", "All plants which had already grown into one maze arm were disqualified .", "Control plants were given no stimuli and left in the dark , experimental plants had the fan moved to the opposite arm from its last position and given the standard fan regimen without light .", "The day after testing day fans and lights were removed , plants were moved from growth chambers to a countertop and order-randomized before being scored by an independent observer with the experimenter in a separate room .", "Each plant was marked as either right , left , or neither .", "The criteria for was growth >5 mm above the decision point in only one maze arm .", "In the vast majority of cases , growth was in only one maze arm or neither , but two plants were disqualified and marked neither for growing into both maze arms .", "Once each plant’s growth direction was recorded , plant numerical IDs were matched back to their experimental condition and enantiomeric pattern of stimulus exposure ( with light beginning on either the left or the right maze arm ) , and scored one if their growth direction matched the direction of most recent light exposure and 0 if it was opposite the most recent light exposure .", "Scores were combined for each experimental condition and the proportion of plants growing according to the phototropic expectation was established and graphed .", "Fisher’s exact test was used to determine different ratios between binary outcome between two conditions and is appropriate for small samples .", "This is the same analysis performed by Gagliano et al . All tests were performed with GraphPad QuickCalcs software .", "Gagliano et al . do not mention attrition or exclusion criteria in their report , but attrition in this study was quite high at several stages , the most problematic being due to variable growth rates resulting in plants reaching the decision point prior to the test day or failing to reach it 24 hr afterwards , both of which resulted in plants being disqualified .", "A smaller number of plants were disqualified because their individual light or fan system was found to have disconnected during movement from one maze arm to the other , therefore interrupting their training by missing part or all of a stimulus .", "In these cases , plants were disqualified by the experimenter during training phases and not scored .", "Number of plants trained and tested vs plants successfully scored as growing left or right are shown in Table 1 .", "The controlled growth chamber used for training and testing plants had the capacity for 120 plants with mazes attached , and the experiment was performed four times in series to gather data .", "For each replication , approximately 350 seeds were germinated , of which approximately 250 would be planted , 120 transferred into the Y maze apparatus , and an average of 70 . 75 successfully scored as growing either left or right .", "Methods adapted from Gagliano et al . , with minor modifications , mostly due to differences in product availability between the United States and Australia .", "Gagliano et al . used Pisum sativum cv Massey Gem ( source unspecified ) , which was not available for my lab .", "Green Arrow ( Botanical Interests , USA ) was selected as a closely related cultivar with similar growth habit .", "I believe this difference should not affect the results , however , because associative learning must be a complex multigenic trait which is extremely unlikely to exist within one cultivar but not be present in a very closely related cultivar .", "Green Arrow , like Massey Gem , is a full sun cultivar , and thus should have similar propensity for phototropism .", "Similarly , Gagliano et al . used Osmocote seed raising and cutting mix , Scotts Australia ) , whereas I used Hoffman seed starter potting and planting mix ( Good Earth Inc , NY ) , though its composition closely matches the soil used in the Gagliano et al . experiment .", "Instead of one 5 . 3 m2 controlled environment room ( source unspecified ) , two controlled growth chambers ( PGR15 Growth Chamber , Conviron ) were used , one for the initial growth and soil emergence stage , one for the training and testing stage .", "Importantly , the light , temperature , and humidity conditions in my chambers are the same as those of Gagliano et al . ( see Germination Conditions and Growth Conditions ) , and the doors to the growth chambers were only opened with the outside room darkened to prevent the interference of external light ." ] ]

[ "Gagliano et al . ( Learning by association in plants , 2016 ) reported associative learning in pea plants .", "Associative learning has long been considered a behavior performed only by animals , making this claim particularly newsworthy and interesting .", "In the experiment , plants were trained in Y-shaped mazes for 3 days with fans and lights attached at the top of the maze .", "Training consisted of wind consistently preceding light from either the same or the opposite arm of the maze .", "When plant growth forced a decision between the two arms of the maze , fans alone were able to influence growth direction , whereas the growth direction of untrained plants was not affected by fans .", "However , a replication of their protocol failed to demonstrate the same result , calling for further verification and study before mainstream acceptance of this paradigm-shifting phenomenon .", "This replication attempt used a larger sample size and fully blinded analysis ." ]

[ "Associative learning is a simple learning ability found in most animals , which involves linking together two different cues .", "For example , the dogs in Pavlov’s famous experiment were trained to associate sound with the arrival of food , and eventually started salivating upon hearing the sound alone .", "Plants , like animals , are capable of complex behaviors .", "The snapping leaves of a Venus fly trap or the sun-tracking abilities of sunflowers are examples of instinctive responses to environmental cues that have evolved over many generations .", "Whether or not plants can learn during their lifetimes has remained unknown .", "A handful of studies have tested for associative learning in plants , the most convincing of which was published in 2016 .", "In this study , pea plants were exposed to two signals: light , the plant version of dog food , and wind , equivalent to the sound in Pavlov’s experiment .", "Just as dogs salivate in response to food , plants instinctively grow towards light , whereas air flow does not affect the direction of growth .", "The plants were grown inside Y-shaped mazes and their ‘selection’ of one particular arm was used as a ‘read-out’ of learned behavior .", "The experiments trained growing plants by exposing them to wind and light from either the same direction or opposite directions .", "Once the plants were at the point of ‘choosing’ between the two arms , they were exposed to wind in the absence of light .", "Wind by itself appeared to influence the direction the trained plants took , with wind attracting plants trained with wind and light together and repelling plants trained with wind and light apart .", "Untrained plants remained unaffected , making random selections .", "These observations were interpreted as the strongest evidence of associative learning in plants and if true would have great scientific and philosophical significance .", "Kasey Markel therefore set out to confirm and expand on these findings by replicating the 2016 study .", "As many conditions as possible were kept identical , such as the training regime .", "The new experiments also used more plants and , most importantly , were done ‘blind’ meaning the people recording the data did not know how the plants had been trained .", "This ensured the expectations of the researcher would not influence the final results .", "The new study found no evidence for associative learning , but did not rule it out altogether .", "This is because some experimental details in the first study remained unknown , such as the exact model of lights and fans originally used .", "This work demonstrates the importance of replicating scientific experiments .", "In the future , Markel hopes their results will pave the way for further , rigorous testing of the hypothesis that plants can learn ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Correlates of decision variables are routinely found in prefrontal cortex ( PFC ) during value-guided decision making ( Clithero and Rangel , 2014; Kennerley and Walton , 2011 ) .", "They have been richly described using static , economic models of choice ( Glimcher and Fehr , 2014 ) .", "Neuroeconomic accounts explain firing rates of single neurons or human neuroimaging data in terms of experimental variables that motivate choice behaviour .", "These include the magnitude or likelihood of available reward , and the costs involved in obtaining those rewards .", "By forming neural representations of such quantities , it is argued that the brain can use these representations to guide selection of the most valuable alternative .", "Our present understanding of economic decision formation has been founded upon careful study of neural representations of value and how they differ across PFC subregions ( Glimcher and Fehr , 2014; Rushworth et al . , 2012; Rangel and Hare , 2010 ) .", "However , an alternative perspective on the origins of neural representations during decision making stems from choice models from mathematical psychology ( Busemeyer and Townsend , 1993 ) .", "Such models seek to explain decisions in terms of their temporal evolution or dynamics .", "Whilst originally accounting for temporally varying features of behaviour , such as reaction times and eye gaze ( Busemeyer and Townsend , 1993; Ratcliff and Rouder , 1998; Krajbich et al . , 2010 ) , they have also successfully captured temporally varying features of neural activity , most notably in the lateral intraparietal cortex ( LIP ) during perceptual choice ( Shadlen and Newsome , 2001; Gold and Shadlen , 2007 ) .", "These psychological models can also be related to other dynamical models ( Bogacz et al . , 2006 ) such as nonlinear attractor network models firmly rooted in neurobiology ( Wang , 2002 ) .", "The common feature of both psychological and neurobiological accounts is that they focus on the temporal evolution of a decision signal across time , rather than the strength with which it represents decision variables at a particular fixed point in time .", "Contrasting the two perspectives , it becomes apparent that even if neuronal activity underwent the exact same physiological process on different trials ( such as a ramp-to-threshold ) , it might nevertheless appear to represent certain economic features of a decision .", "Put simply , as the decision unfolds , neural activity will be higher on trials that ramp quickly than on those that ramp slowly .", "It will therefore correlate with any variable that affects the decision speed , and this effect will appear most prominent at timepoints in the trial when the ramping process , or rate of change , is maximal .", "Even in more complex dynamical situations , it is easy to see how economic variables that predict the rate of change would appear to be represented in neural activity if analysed without knowledge of the underlying dynamics .", "This might particularly be true of the value of the chosen item ( ‘chosen value’ ) , which strongly affects behavioural reaction times in economic choice tasks .", "This should then influence how we interpret the meaning of such activity ( Hunt , 2014; O’Doherty , 2014 ) .", "The neuroeconomic perspective often labels chosen value representations as a ‘post-decision’ signal , arguing that chosen value signals do not reflect the decision competition itself but instead the outcome of the comparison process ( Cai and Padoa-Schioppa , 2012; Blanchard and Hayden , 2014 ) .", "One interpretation of such representations is that they are needed for subsequent computation of a reward prediction error ( Rangel and Hare , 2010 ) .", "Yet the dynamical perspective proposes that they may in fact originate as a consequence of time evolving decision processes .", "Rather than casting a chosen value representation as signaling ‘pre-decision’ or ‘post-decision’ variables to downstream brain areas , it argues that such correlates inevitably emerge as a decision is made .", "In its most extreme form , this hypothesis might contend that chosen value signals would be fully accounted for by variation in underlying decision rates .", "To assess this proposal more carefully , it becomes critical to index how neural dynamics unfold on individual trials .", "In PFC , tackling this problem provides unique challenges .", "Several recent studies have recovered single-trial dynamical information in structures close to motor output , based upon neuronal spiking data ( Thura and Cisek , 2014; Kaufman et al . , 2015; Murakami et al . , 2014; Bollimunta et al . , 2012; Kiani et al . , 2014; Carnevale et al . , 2015 ) .", "Yet the distance of PFC from either sensory input or motor output produces neural activity that is poorly aligned with simple features of the experimental task ( Rigotti et al . , 2013 ) .", "Attempts to understand PFC activity should therefore extract its dynamical state based on neural information alone , rather than first sorting spike trains by motor output or experimental variables as is common in other approaches .", "It is also unclear whether neuronal firing rates are in fact the most reliable source of single-trial information .", "The esotericism of PFC neuronal responses is compounded by a high degree of stochasticity in neuronal spike trains , delivering statistical challenges to obtaining single-trial information ( Churchland et al . , 2007; Park et al . , 2014 ) .", "Current techniques sometimes overcome this problem by examining tens or hundreds of simultaneously recorded single neurons ( Churchland et al . , 2007 ) .", "Whilst this can be fruitful , such data is rarely available in humans , where the contribution of PFC to value-guided choice is most critical .", "In the present study , we therefore sought an alternative index of single-trial neural dynamics that overcame these limitations .", "We focussed on observations at the mesoscopic scale of the local field potential ( LFP ) .", "One advantage of this approach is that it can be easily related to simultaneously recorded neuronal spike trains on a trial-by-trial basis in animals , but also underlies the magnetoencephalography ( MEG ) signal observable in humans ( Buzsáki et al . , 2012 ) .", "As such , we could recover dynamical information from electrophysiology recordings in macaque monkeys and also from non-invasive magnetoencephalography ( MEG ) recordings in humans .", "This provides an important bridge between observations at microscopic ( cellular ) and macroscopic ( whole-brain ) scales .", "Importantly , our index provides information about the speed of a local internal neural decision process that goes beyond a simple behavioural measure of reaction time .", "Using this index , we could draw several new conclusions about PFC activity during choice .", "We first demonstrate a temporal evolution from action value difference to chosen action signals that are selectively present in dorsolateral prefrontal cortex ( DLPFC ) neurons , but not anterior cingulate cortex ( ACC ) or orbitofrontal cortex ( OFC ) .", "This contrasts against the ubiquitous encoding across all three subregions of ‘chosen value’ .", "However , we then demonstrate that correlates of chosen value may arise as a consequence of dynamical processing , as opposed to being purely represented as a neuroeconomic post-decision variable .", "Next , by simultaneously indexing decision formation across multiple PFC subregions , we show that functional interactions can be reshaped in a task-dependent manner .", "We find that OFC and ACC dynamics selectively influence dorsolateral DLPFC activity on delay- and effort-based decisions respectively .", "Finally , our observations can be related to predictions from a dynamical neural network model of choice .", "This provides formal links to established dynamical mechanisms underlying perceptual decision-making ." ], [ "We first adopted a classical approach to analysing our data , examining features of the task represented by neural activity .", "Using multiple regression on single neuron firing rates , we observed a dynamic ( time-varying ) signature of value correlates in the DLPFC ( n=303 neurons ) ( Figure 1B ) .", "Early in the trial , value correlates were found in the reference frame of action value difference ( i . e . left minus right saccade value influenced neural firing; Figure 1B , blue ) , but late in the trial these evolved into correlates of the eventual chosen saccade ( left vs . right chosen saccade influenced neural firing; Figure 1B , red ) ( Kim et al . , 2008; Louie and Glimcher , 2010 ) .", "DLPFC neurons that showed strong selectivity for left minus right saccade option value 300 ms after decision onset also showed strong selectivity for left minus right choices 700 ms after decision onset ( Figure 1C ) .", "This temporal evolution from action value difference to categorical choice was selectively present in DLPFC , but not ACC ( n = 321 neurons ) or OFC ( n = 212 neurons ) ( Figure 1—figure supplement 1 ) .", "Notably , however , all three regions showed correlates of chosen value with a similar timecourse ( Figure 1B , Figure 1—figure supplement 1 ) .", "We then applied the same approach to consider dynamics of LFP value correlates ( electrodes: DLPFC , n=208; ACC , n=207; OFC , n=146 ) .", "The overall shape of the choice phase evoked LFP was surprisingly consistent across PFC subregions ( Figure 1D ) and subjects ( Figure 1—figure supplement 2 ) .", "All areas contained a fast biphasic component shortly after sensory input , and a slower component lasting several hundred milliseconds after this .", "Because of this similarity , we collapsed across regions ( see Figure 1—figure supplement 3 for regions separately ) , and regressed LFP amplitude onto both chosen value and unchosen value across time .", "This revealed two timepoints at which correlates of value emerged ( Figure 1E ) .", "Early in the trial ( ~250 ms ) value correlates were positive for both chosen and unchosen value , but later in the trial ( ~450 ms ) value correlates for unchosen value flipped to become negatively signed .", "This implies a temporal evolution from an early representation of value sum ( =chosen+unchosen value ) to a later representation of value difference ( =chosen-unchosen value ) .", "This progression is notably similar to observations made in human PFC during value-guided choice using MEG ( Hunt et al . , 2012 ) .", "Crucially , these value correlates were maximal when the event-related LFP was ramping , not peaking ( that is , when the temporal derivative of the LFP was large [Figure 1F] ) .", "This suggests chosen and unchosen value influence the rate at which LFP dynamics unfold on each trial , rather than being explicitly represented by the amplitude of an evoked response .", "As revealed by these classical analyses , value correlates vary across time at both microscopic ( single unit ) and mesoscopic ( LFP ) scales .", "Yet it remains unclear how we might bridge these dynamics at different scales .", "Below , we demonstrate that forming such bridges is of fundamental importance , especially if it can be achieved at the level of individual trials .", "It allows us to interrogate the relationship between neural dynamics and behaviour , the encoding of neuroeconomic variables , and how brain regions interact during choice .", "Moreover , by applying to the same approach to human data , it links observations in human MEG recordings with their underlying neuronal counterparts .", "One approach to bridging across data scales would be to extract single-trial information about choice-related dynamics from one scale , and use it to explain variance in the other .", "The evoked LFP appeared consistent across different recording electrodes and sessions ( Figure 2—figure supplement 1 ) , and possessed a high single-trial signal to noise ratio ( Figure 2—figure supplement 2 ) .", "We therefore pursued a data-driven index of single-trial dynamics from LFP data .", "The approach we adopted was to apply principal components analysis ( PCA ) across trials ( see Methods ) .", "For each subject , the input data for macaque PCA was decision-locked raw LFP data , stacked across all electrodes from all recording sessions to form a large matrix X . X has dimensions nSingleTrials by nTimebins ( Figure 2A ) .", "PCA decomposes X into temporal principal components V , with dimensions nTimepoints by nComponents , and component weights U , with dimensions nSingleTrials by nComponents ( Figure 2A ) .", "The principal components ( PCs ) in V provide a set of temporal basis functions ( Figure 2B ) that capture the principal modes of variation in the shape of the waveform across trials .", "The single-trial weights in U tell us how much of each PC is present in each single-trial response .", "They are returned separately for each electrode .", "Because data are stacked , however , the decomposition has the same meaning across different electrodes and recording sessions . 10 . 7554/eLife . 11945 . 007Figure 2 . Extraction of internal dynamics from LFP data via principal components analysis ( PCA ) .", "( A ) A large matrix of single-trial data is formed by stacking data across all recordings .", "This was performed separately within each subject .", "Consistent evoked potentials were found across recording electrodes ( Figure 2—figure supplement", "1 ) and trials ( Figure 2—figure supplement 2 ) .", "The single trial-weights U are returned separately for each trial on each electrode , but their interpretation is determined by the shape of the components in V , and so is common to all electrodes .", "( B ) The first and second principal components of this matrix , for example macaque subject N . ( C ) The shapes of PC1 and PC2 are consistent across subjects .", "( D ) Left panel: the effect of adding/subtracting PC1 to the main ERP shape in example subject N , modulating its amplitude; right panel: the effect of adding/subtracting PC2 to PC1 , modulating its latency .", "As shown in Figure 2—figure supplement 3 , this shift in latency can also be related to changes in low-frequency oscillatory phase during the decision period .", "( E ) The influence of chosen value , unchosen value and error trials on PC2 scores ( i . e . the second column of matrix V ) , estimated via multiple regression .", "Bars show regression coefficients ( a . u . ) , mean /- s . e . across electrodes ( macaque ) .", "** denotes p<0 . 01 , one-sample T-test .", "Effects are shown separately for monkey K ( who saccaded freely during choice epoch , and indicated response using joystick ) in Figure 2—figure supplement 4 .", "Influence of variables on PC1 scores shown in Figure 2—figure supplement 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 00710 . 7554/eLife . 11945 . 008Figure 2—figure supplement 1 . Consistency of evoked potentials across different recording electrodes/recording sessions , in an example subject ( monkey N ) .", "The similarity between evoked potentials allows a common dimensionality reduction to be applied across all recorded sessions , eliminating the problem of matching principal components across sessions . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 00810 . 7554/eLife . 11945 . 009Figure 2—figure supplement 2 . High signal to noise ratio in single-trial LFP responses . All trials , unfiltered , from a single session are stacked for an example electrode .", "The evoked potentials plotted in ( Figure 1—figure supplement", "2 ) and ( Figure 2—figure supplement", "1 ) can clearly be seen at the single trial level , allowing single trial dynamics to be readily estimated . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 00910 . 7554/eLife . 11945 . 010Figure 2—figure supplement 3 . The relationship between single-trial PC2 weights and single-trial time-frequency decomposition ( estimated across all electrodes in DLPFC ) .", "The top panel shows the circular-linear correlation ( Berens , 2009 ) between the phase of the LFP in each frequency band for each timebin , and the PC2 weight for the corresponding trial .", "Note that this measure is non-negative by definition .", "This indicates that , as expected if PC2 alters the latency of a slow evoked potential , single-trial PC2 weights correlate with the oscillation phase in the theta range .", "The bottom panel shows the correlation coefficient between the power in each frequency band for each timebin , and the PC2 weight for the corresponding trial .", "This suggests a positive relationship between single-trial PC2 weights and high beta/low gamma oscillations late in the decision period . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 01010 . 7554/eLife . 11945 . 011Figure 2—figure supplement 4 . Regression of decision variables onto PC2 ( left ) and PC1 ( right ) in monkey K ( free saccade ) .", "Monkey K was free to saccade during the 1s choice period , and made a joystick response rather than a saccade response after 1s had elapsed .", "Because of the different shape of the evoked response in monkey K ( Figure 1—figure supplement", "2 ) and the differences in PC1/2 timecourses as a consequence ( Figure 2C ) , we here plot the effects of decision variables on PC weights separately for monkey K . ( Note that PC1 still controlled waveform amplitude , and PC2 still controlled waveform latency , in this subject . ) The effects of chosen value and error trials are similar for PC2 ( left panel ) as for other subjects ( Figure 2E ) , but only reach significance in OFC and ACC .", "There is also a negative effect of unchosen value on PC2 in all regions , which is not clearly seen in other subjects ( Figure 2E ) .", "However , the effects of chosen and unchosen value on PC1 ( right panel ) are highly significant , but not in the same direction as for other subjects ( Figure 2 . – figure supplement 5 ) .", "We hypothesise that this is because in monkey K , PC1 is near zero during the early phase of the decision , but strongly modulated during the later phase ( Figure 2C ) , which might imply a modulation by value difference as opposed to overall value . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 01110 . 7554/eLife . 11945 . 012Figure 2—figure supplement 5 . Regression of decision variables onto PC1 . The influence of chosen value , unchosen value and error trials on PC1 scores , estimated via multiple regression .", "Bars show mean /- s . e . across electrodes .", "** denotes p<0 . 01; * denotes p<0 . 05 , one sample T-test . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 012 The top two PCs were strikingly consistent across all four macaque subjects ( Figure 2C ) .", "Importantly , they were readily interpretable in terms of their effects on LFP dynamics .", "PC1 ( Figure 2B , left panel ) resembled the basic shape of the event-related field potential ( Figure 1D ) .", "Adding or subtracting PC1 therefore captures variation across trials in response amplitude ( Figure 2D , left panel ) .", "More notable , however , was PC2 , which was relatively flat at the beginning of the decision , but from 200 ms onwards resembled the temporal derivative of PC1 ( Figure 2B , right panel ) .", "Adding or subtracting the temporal derivative of a waveform controls the latency at which it peaks ( Figure 2D , right panel ) ( Friston et al . , 1998; Mayhew et al . , 2006 ) .", "Knowledge about PC2 weights therefore provides a parsimonious description of single-trial ERP latencies , a key feature of the dynamical ERP response .", "Consistent with this idea , PC2 weights were also found to correlate with the phase of low frequency ( theta frequency [4-–8 Hz] ) oscillations during the decision period ( Figure 2—figure supplement 3 ) .", "In light of this , we hypothesised that factors modulating reaction time in value-based choice studies ( Busemeyer and Townsend , 1993 ) would affect the weight of PC2 , controlling the latency of the LFP waveform .", "We found chosen value had a strong and consistent effect on PC2 across all regions studied .", "When chosen value was higher , PC2 was more positive , implying the waveform peaked earlier in time ( Figure 2E; Figure 2—figure supplement 4 ) .", "On ‘error trials’ , where the subject chose the less valuable option , PC2 weights were more positive than on correct ( Figure 2E ) .", "Under the assumption that higher value trials , and error trials , were associated with faster decision dynamics ( Busemeyer and Townsend , 1993; Ratcliff and Rouder , 1998 ) , PC2 weights therefore provide a neurally-derived index of the speed of the decision on each trial .", "Crucially , however , they are obtained separately for each individual electrode .", "They therefore provide a local measurement of neural dynamics , beyond a simple behavioural measure of reaction time .", "PC1 weights , capturing response amplitude rather than latency , were primarily influenced by value sum ( same sign for both chosen and unchosen value , with the exception of ACC ) ( Figure 2—figure supplement 5 ) .", "We next considered whether our internal LFP-derived index might explain features of neural firing that we could not previously explain using external experimentally-derived variables .", "We explored this idea using multiple regression .", "We regressed experimental factors onto neural firing rates ( see Supplementary file 1 for full list ) to capture variance explained by these factors , as in Figure 1B .", "However , we also included as a coregressor the single-trial PC2 weight for that trial , estimated from the decomposition of the LFP .", "This allows us to examine the influence of PC2 weights on neuronal firing , having controlled for the contribution of all external decision variables .", "To avoid contamination between spikes and LFP , we used LFP data recorded simultaneously from a neighbouring electrode in the same cortical area .", "( Note that reaction time is not included as an additional measure of trial-by-trial decision dynamics in our task , as the imposed 1s choice delay prior to response led to a floor effect in subjects’ response times . )", "Figure 3A and B shows two individual neurons from DLPFC that exemplify the action value-to-choice transformation depicted in Figure 1B/C .", "As can be seen , variance in their firing is explained by the action value difference early ( ~200-–400 ms ) , and the chosen action late ( ~600–800 ms ) in the decision process .", "In both cases , the LFP-derived PC2 single trials weights explain additional variance as this value-to-choice transformation occurs ( Figure 3A/B ) .", "Across the population , PC2 had a similar effect at this point in time ( Figure 3C ) .", "44 . 9% of neurons in DLPFC were found to have a significant modulation by LFP-derived PC2 weights during the choice epoch ( 23 . 4% positively modulated , 21 . 5% negatively modulated , p<0 . 01 in a 250 ms–750 ms window , corrected for multiple comparisons across time ) .", "This finding forms a link between choice dynamics at mesoscopic and microscopic scales , over and above that which can be obtained from examining time-varying value correlates ( as in Figure 1 ) .", "Additional analyses confirmed that this relationship could not be explained as a simple consequence of first-order correlations between LFP amplitude and firing rate ( Whittingstall and Logothetis , 2009 ) . 10 . 7554/eLife . 11945 . 013Figure 3 . Single unit firing in DLPFC explained by simultaneously recorded LFP dynamics , over and above contribution from experimental variables .", "( A ) / ( B ) Two example neurons from DLPFC each showing a transition from encoding action value difference ( blue ) to later encoding the selected action ( red ) , and influenced by the LFP-derived PC2 single trial weights ( cyan ) in the intervening period .", "The coefficient of partial determination ( CPD ) is plotted for all three factors ( see Methods for full list of other task-related variables included in regression model ) .", "( C ) Timecourse of population CPD explained by LFP-derived PC2 weights ( for n=205 DLPFC neurons that had a simultaneously recorded LFP on a separate DLPFC electrode ) .", "Lines show mean /- s . e . across neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 013 Having formed this link between our measure of single-trial LFP dynamics and neural firing , we could then address several questions concerning the roles of these dynamics in value-guided decision making .", "The representation of chosen value is found in many neural structures during choice , but its interpretation has remained unclear .", "It has been pointed out that this is a ‘post-decision’ variable , and several suggestions have been offered for why this might need to be encoded ( Rangel and Hare , 2010 ) .", "In our study , the timecourse of this signal ( found across all three cortical areas , Figure 1—figure supplement", "1 ) appears similar to the timecourse of influence of the LFP PC2 weights on neuronal firing ( Figure 3C ) .", "We sought to explore the relationship between chosen value correlates identified in single unit firing and our single-trial indices of ERP amplitude ( PC1 ) and latency ( PC2 ) .", "To address this , we reanalysed the findings in Figure 1B , but asked whether including the top two LFP principal components in our regression model selectively reduced the variance explained by chosen value , but not by action value difference or chosen action ( see Methods ) .", "As a control , we compared this model to one where two noise components ( PC101 and PC102 ) from the LFP PCA were included as coregressors instead of PC1 and PC2 .", "We found that including the LFP-derived PC1/PC2 as coregressors caused a significant reduction in chosen value coding , but not of action value difference or chosen action coding ( Figure 4; Figure 4—figure supplement 1 ) .", "Similar results could also be obtained via an alternative approach , in which instead of using noise components , principal components were shuffled across trials in a fashion that preserved their underlying correlation with chosen value , or by examining the contribution of PC1 or PC2 alone ( Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 11945 . 014Figure 4 . Chosen value , but not action value difference or chosen action , is explained away by neural dynamics . CPD for chosen value ( green ) is reduced by including PC1/PC2 from the LFP decomposition as coregressors in the decision model .", "The reduction in CPD ( n=205 DLPFC neurons ) for each of the decision variables in Figure 1C as a consequence of including PC1/2 is shown , by subtracting it from a control model that included two noise components ( PC101/102 ) .", "Note that the maximal value that this reduction can take is determined by the CPD for each regressor at each point in time ( shown in Figure 1B ) .", "Lines show mean /- s . e . across neurons .", "Dots denote timepoints with a significant ( p<0 . 05 , permutation test ) change in CPD across the DLPFC population .", "See also Figure 4 .", "– figure supplement 1 for OFC/ACC , and Figure 4—figure supplement 2 for the contribution of PC2 alone .", "Figure 4—figure supplement 3 compares the effect of local DLPFC PC weights with respect to those from a distal , simultaneously recorded brain region ( i . e . either OFC or ACC ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 01410 . 7554/eLife . 11945 . 015Figure 4—figure supplement 1 . CPD for chosen value is reduced by including PC1/PC2 as coregressors in the decision model across all three regions . The reduction in CPD for chosen value as a consequence of including PC1/2 is shown , relative to a control model that included two noise components ( PC101/102 ) .", "Lines show mean /- s . e . across all neurons with simultaneously recorded LFP on another electrode in the same brain region . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 01510 . 7554/eLife . 11945 . 016Figure 4—figure supplement 2 . CPD for chosen value is reduced by including PC1/PC2 alone as coregressors . Whilst the main figure shows the reduction in chosen value by the inclusion of both PC1 and PC2 as coregressors in the regression model , this figure shows the independent influence of adding/removing PC1 and PC2 alone on the coefficient of partial determination ( CPD ) for chosen value in neurons across all areas .", "Across all three regions , PC1 reduces chosen value CPD to a greater extent than PC2 .", "This is particularly the case in ACC , where PC1 showed a different relationship to chosen value than in DLPFC/OFC ( see Figure 2—figure supplement 5 ) .", "Lines show mean /- s . e . across neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 01610 . 7554/eLife . 11945 . 017Figure 4—figure supplement 3 . Local PC1/2 ( controlling for larger-scale global influences ) reduces chosen value CPD more than global PC1/2 ( controlling for local influences ) .", "( A ) Underlying correlation structure in principal component weights ( PC2 ) across simultaneously electrodes , both within region ( on-diagonal ) and across region ( off-diagonal ) .", "Each histogram shows the distribution of correlation coefficients across PC2 weights from simultaneously recorded electrodes .", "As might be expected , PC2 weights are correlated within region but also across regions .", "However , the median correlation ( vertical line ) is generally lower across-regions than within-region .", "This suggests that PC2 weights reflect local dynamics .", "( B ) Greater reduction in CPD of chosen value in DLPFC neurons by local , rather than distal , PC1/2 .", "Local PC1/2 weights ( another electrode in the same region ) are first orthogonalised with respect to PC1/2 weights from simultaneously recorded electrodes in another distal brain region ( either OFC or ACC ) .", "These are then included as a coregressor in the regression onto decision variables , as in Figure 4 .", "The CPD for chosen value is then subtracted from that of a separate model , where the converse analysis has been performed: PC1/2 weights from the distal region are included , having orthogonalised those weights with respect to local PC1/2 weights .", "As a consequence , positive values indicate that local PC1/2 weights cause a greater reduction in chosen value coding than distal PC1/2 weights . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 017 We then asked whether chosen value coding was reduced more by local ( within-region ) neural dynamics , compared to global ( whole-brain ) dynamics .", "We found a smaller but significant reduction could also still be found even if these ‘local’ principal components ( i . e . from the same brain region ) were orthogonalised with respect to those of another , simultaneously recorded brain region , when compared to performing the same analysis in reverse ( Figure 4—figure supplement 3 ) .", "This implies that some of the neuronal variance attributed to chosen value correlates originates as a consequence of the speed and amplitude at which dynamics unfold locally within a particular cortical area , and demonstrates the utility of our single-trial index for capturing these local dynamics .", "ACC and OFC have established roles in effort- and delay-based decisions respectively ( Rudebeck et al . , 2006; Prevost et al . , 2010; Croxson et al . , 2009; Kurniawan et al . , 2013; Kable and Glimcher , 2007; Parvizi et al . , 2013 ) .", "Because our LFP decomposition indexes decision dynamics locally , we hypothesised that the internal dynamics of ACC and OFC might preferentially influence activity in DLPFC on different trial types .", "Specifically , we predicted that firing rates in DLPFC would be preferentially affected by ACC internal dynamics on effort-based trials , but by OFC internal dynamics on delay-based trials .", "We could test this hypothesis by examining sessions in which DLPFC , ACC and OFC were all recorded simultaneously .", "We built a regression model in which LFP-derived PC2 weights from OFC and ACC competed for variance in explaining neural firing in DLPFC ( see Methods ) .", "We estimated this separately on delay and effort trials .", "Both areas were found to influence DLPFC activity ( Figure 5—figure supplement 1 ) , but strikingly , ACC explained more variance in DLPFC firing on effort trials than delay trials ( Figure 5A , magenta ) , whereas the converse was true for OFC ( Figure 5A , black ) .", "This was not found to be true for analyses performed in the reverse direction ( using DLPFC/ACC PC2 weights to explain OFC firing , or DLPFC/OFC PC2 weights to explain ACC firing ) . 10 . 7554/eLife . 11945 . 018Figure 5 . ACC and OFC local dynamics explain greater DLPFC neural firing on effort-based and delay-based decisions , respectively .", "( A ) The relative CPD for DLPFC firing explained by PC2 on effort trials minus delay trials , where PC2 is derived from simultaneously recorded ACC LFP ( magenta ) and OFC LFP ( black ) ( n=124 DLPFC units which had simultaneous recordings in both OFC and ACC ) .", "ACC explains more variance on effort than delay trials ( positive-going values ) , whereas the converse is true for OFC ( negative-going values ) .", "See also Figure 5—figure supplement 1 .", "Lines show mean /- s . e . across neurons .", "Dots denote timepoints with a significant ( p<0 . 05 , permutation test ) change in CPD across the DLPFC population .", "( B ) The same analysis as in Figure 5A , having first performed a median split for chosen value-selectivity .", "Neurons with high chosen value selectivity ( top panel , n=62 DLPFC units ) show the effect found in Figure 5A , whereas neurons with low chosen value selectivity ( bottom panel , n= 62 DLPFC units ) do not . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 01810 . 7554/eLife . 11945 . 019Figure 5—figure supplement 1 . Cross-regional interactions , separately for delay versus effort trials . CPD for DLPFC firing explained by PC2 on effort trials ( left ) and delay trials ( right ) , where PC2 is derived from simultaneously recorded ACC LFP ( magenta , top row ) and OFC LFP ( black , bottom row ) ( n=124 DLPFC units , which had simultaneous recordings in both OFC and ACC ) .", "OFC and ACC PC2 weights compete for variance to explain neural activity in the same regression model ( see Methods ) .", "Lines show mean /- s . e . across neurons .", "Dots denote timepoints with a significant ( p<0 . 05 , permutation test ) change in CPD across the DLPFC population . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 019 Notably , OFC and ACC PC2 weights modulated DLPFC neuron firing from around 200 ms after choice onset ( Figure 5—figure supplement 1 ) , consistent with the time at which DLPFC neurons first begin to encode choice values ( Figure 1B ) .", "These effects on DLPFC firing were maintained for several hundred milliseconds .", "Critically however , just prior to when response coding in DLPFC was peaking ( around 800 ms , see Figure 1B ) , the contribution of ACC and OFC PC2 to DLPFC neuron firing changed in a cost-specific way .", "OFC PC2 began to selectively explain spiking variance on delay trials , whilst ACC began to explain spiking variance on effort trials ( Figure 5—figure supplement 1 ) .", "We also found that when we performed a median split on DLPFC neurons by the degree to which they encoded chosen value in the analysis shown in Figure 1B , neurons with high chosen value selectivity were also those preferentially influenced by other regions’ dynamics ( Figure 5B ) .", "If DLPFC chosen value coding is interpreted as reflecting the speed at which a decision process unfolds , then these findings imply that the influence of one region’s internal dynamics on another region’s dynamics can be flexibly reshaped according to current task demands .", "This was not true , by contrast , when a median split was performed based upon the response selectivity of the DLPFC neuronal population .", "The LFP value correlates in Figure 1E showed similar temporal profiles to a previous study in which human subjects made binary value-guided choices whilst undergoing MEG ( Hunt et al . , 2012 ) .", "We therefore asked whether the MEG signal from this study , in the PFC subregion where this temporal profile had been observed ( ventromedial prefrontal cortex , MNI 6 , 28 , –8 mm ) , could also be subjected to a similar single-trial PCA decomposition as our LFP data .", "Each subject provides a single ‘virtual electrode’ from which observations are made , and so data were stacked to form the matrix X with dimensions nSingleTrials ( [=nTrials*nSubjects] ) by nTimebins ( see Methods ) .", "This was then decomposed as for the macaque data .", "We found a similar relationship between PC1 and PC2 ( Figure 6A ) controlling waveform amplitude and latency ( Figure 6B ) .", "Moreover , PC2 had a similar relationship to chosen value and error trials as in macaques ( Figure 6C ) .", "Finally , in this experiment , we also had a direct behavioural readout of decision formation on every trial as subjects could respond at any time after decision onset .", "Subject reaction times were strongly negatively predictive of PC2 , over and above any contribution of chosen value , unchosen value or errors ( Figure 6C ) .", "Our observations here provide a link between the dynamics of choice at the mesoscopic scale in macaques and its macroscopic counterpart in humans . 10 . 7554/eLife . 11945 . 020Figure 6 . PCA decomposition of human MEG data shows similar characteristics to decomposition of monkey LFP data .", "( A ) Averaged evoked response from data beamformed to ventromedial prefrontal cortex ( MNI coordinate ( 6 , 28 , -8mm ) ) , from a previous study of value-guided decision making ( Hunt et al . , 2012; 2013 ) .", "Lines show mean /- s . e . across trials .", "( B ) PCA decomposition of human MEG data yields two principal components similar to those found in macaque LFP data ( cf . Figure 2C ) .", "( C ) The influence of chosen value , unchosen value and error trials on PC2 scores , estimated via multiple regression , in humans .", "Effects are similar to those found in macaque PFC ( cf . Figure 2E ) .", "Also shown , in yellow , is the additional effect of reaction time ( orthogonalised with respect to chosen value , unchosen value and error trials ) .", "Bars show mean /- s . e . across trials; ** denotes p<0 . 01 , one-sample T-test . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 020 The transition in value correlates observed at the macroscopic scale in human MEG can be explained with reference to a class of recurrent neural network models displaying competition via mutual inhibition ( Hunt et al . , 2012 ) .", "Such models have success in explaining many features of neural data in perceptual choice ( Shadlen and Newsome , 2001; Wang , 2002 ) , focusing on how a decision might be implemented locally within a single cortical area of interest .", "We therefore asked whether these models might explain some of our observations at the microscopic ( single-neuron ) level , and whether a similar decomposition approach can be applied to mesoscopic ( LFP ) predictions from the network model as to our data .", "We simulated a spiking attractor network model of choice , configured with the same connectivity structure and parameters as had been used in a previous study of perceptual decision-making ( Wang , 2002 ) ( Figure 7A; see also ‘Supplementary details of network modelling’ ) .", "We analysed the model predictions as we had the data , regressing action value difference , chosen value and chosen action onto single-neuron firing rates .", "Strikingly , value correlates in the network model possessed the same temporal profile as in DLPFC neurons ( compare Figure 1B with Figure 7B ) .", "One discrepancy was the more sustained chosen value signal in the network model than in the data ( which may result from inputs to the model persisting even after an attractor basin has been reached ) .", "We then ran a similar dimensionality reduction on summed network activity , a proxy for the model’s LFP predictions , as we had done on both macaque LFP and human MEG data .", "By contrast with the LFP data , we obtained a single principal component that controlled both waveform amplitude and latency ( see ‘Supplementary details of network modelling’ , and Figure 7—figure supplement 1 ) .", "This suggests that within the network model there is covariation between these two features of the simulated ERP waveform , whereas in the data they are orthogonal .", "However , this principal component correlated with value in a similar fashion to both macaque and human data ( compare Figure 2E/6C with Figure 7C ) .", "We then regressed the principal component back onto single unit firing rates , as in Figure 3 .", "We found a similar timecourse of influence of the model’s principal component on single unit firing to that found in the data ( compare Figure 3C with Figure 7D , cyan ) .", "Moreover , we found that the internal dynamics of the model extracted from the PCA explained chosen value coding in a similar fashion to that observed in data ( Figure 4 ) , causing a marked reduction ( compare Figure 7B with Figure 7D , green ) .", "Our observations in this study may therefore be explained mechanistically via a simple attractor network model of choice , which similarly captured LFP dynamics in our previous human MEG study ( Hunt et al . , 2012 ) .", "Further details of network modelling are provided in Supplementary Information . 10 . 7554/eLife . 11945 . 021Figure 7 . Relationship between value , LFP and single units explained by competition via mutual inhibition .", "( A ) Model schematic .", "A and B units receive value-related inputs , integrate these via recurrent excitation , and competition via mutual inhibition .", "See ‘Supplementary Details of Network Modelling’ for details .", "( B ) Correlates of decision variables in attractor network model single unit activity .", "Regression coefficient for option value difference ( blue line ) , chosen value ( green line ) and chosen option ( red line ) onto firing rates of ‘A’ selective units in the network model .", "Compare with Figure 1B: in DLPFC , ‘A’ selective units are hypothesised to correspond to left selective units , and ‘B’ units to right selective units .", "( C ) Regression of decision variables onto PC1 from PCA-decomposed LFP model predictions , as in figure 2e/f .", "Note that in model , PC1 captures variability in decision latencies , not PC2 as in data ( see Figure 7—figure supplement 1 ) .", "( D ) Correlates of decision variables when model LFP PC1 is included as coregressor .", "LFP PC1 from model explains firing rates with a similar timecourse to LFP PC2 from data ( Figure 3C ) , but explains away much of the contribution of chosen value to single unit firing ( Figure 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 02110 . 7554/eLife . 11945 . 022Figure 7—figure supplement 1 . Dimensionality reduction of LFP predictions from attractor network model of competition via mutual inhibition .", "( A ) Averaged evoked LFP response from the model .", "( B ) Model-derived PC1 and PC2 , obtained via similar dimensionality reduction as applied to LFP and MEG data in main text .", "( C ) PC1 captures variation in final attractor state , but also captures variation in rise time of model latency ( see ‘Supplementary Details of Network Modelling’ for explanation ) .", "As such , PC1 captures the rate at which model reaches an attractor state , and is analogous to a PC2 in data .", "It is used for regression in Figure 7C . DOI: http://dx . doi . org/10 . 7554/eLife . 11945 . 022" ], [ "Many previous studies ( Kim et al . , 2008; Daw et al . , 2006; Padoa-Schioppa , 2013; Hampton et al . , 2006; Padoa-Schioppa and Assad , 2006 ) , including our own ( Kennerley et al . , 2011 ) , note the representation of chosen value in multiple brain structures during choice .", "Why this quantity is encoded so widely has been a matter of debate ( Rangel and Hare , 2010 ) .", "It is important to remember that this encoding comes from the viewpoint of the experimenter , seeking to describe variability across trials at a particular timepoint in the trial or decision process .", "As we have shown here , and would be predicted from previous behavioural studies ( Busemeyer and Townsend , 1993; Ratcliff and Rouder , 1998; Krajbich et al . , 2010 ) , decision processes are dynamic and decision formation inherently exhibits variability across trials .", "This means that what appears to the experimenter as encoding of a decision related-signal may be a consequence of another ongoing process .", "To paraphrase a recent observation ( Cisek , 2006 ) , “the role of a decision-making system is to produce decisions , not to describe them . ”", "Decision-making systems accumulate evidence in favour of different alternatives across time , and generate a categorical choice .", "This has been most carefully considered in integrate-to-bound models of perceptual choice and their neural correlates ( Gold and Shadlen , 2007 ) .", "Crucially , the time at which sensory evidence is maximally encoded in a neural integrator is not at the beginning of the decision ( when no integration has occurred ) , nor at the end ( when the bound has been reached on all trials ) , but in the middle of the decision process – when on some trials the bound has been reached , whereas on others little net evidence has been accumulated for each alternative .", "It can be very difficult in practice to infer when a decision begins or ends , and this poses difficulties when labelling a neural correlate as ‘pre-decision’ or ‘post-decision’ .", "The dynamical perspective instead explains certain correlates as emerging as the decision is formed .", "These signals may still remain of functional significance for other computations ( such as subsequent computation of the reward prediction error [Rangel and Hare , 2010] ) , but our perspective on how they are generated is changed .", "Value-guided choices are , like perceptual decisions , a dynamical process ( Busemeyer and Townsend , 1993; Krajbich et al . , 2010; Summerfield and Tsetsos , 2012 ) .", "This brings into focus the potentially important role played by decision dynamics in generating correlates of chosen value .", "In the present study , we found that in DLPFC correlates of chosen value occurred maximally during the transformation from an initial representation of evidence ( action value difference ) to an eventual representation of choice ( chosen action ) ( Figure 1B ) .", "A similar temporal progression could also be found in the network model of competition via mutual inhibition ( Figure 7B ) .", "Crucially , however , a portion of the variance captured by chosen value was then explained away by including the speed and amplitude of the local LFP response on that trial as a coregressor , estimated via PCA decomposition of the LFP ( Figure 4 ) .", "It is important to note that there are some caveats to this interpretation .", "Firstly , only some , not all , of the variance was removed by including PCA components as coregressors .", "As such , it may be the case that there is coding of chosen value during the decision that is not explained by the amplitude and speed of the evoked response during decision formation .", "However , despite the reasonable signal-to-noise ratio observed in LFP data , single-trial PC weights will likely contain a degree of observation noise .", "Hence our estimates likely form a lower bound on how much of the chosen value signal might be explained away by LFP dynamics .", "To address this question further , future studies might seek to estimate the degree of observation noise in estimating dynamics , and the theoretical limit that this imposes on how much variance could be explained in neural firing .", "A further important caveat is that chosen value correlates may be explained by different mechanisms at different points in the trial .", "LFPs are often interpreted as measurements of local synaptic input , and this may imply that our principal components mediate chosen value representations at the level of single units .", "It is also clear from Figure 1—figure supplement 1 that correlates of chosen value are present in single unit firing until the end of the decision epoch .", "Future work may investigate the origin and functional significance of this persistent chosen value coding , perhaps for use at later task stages .", "Our single-trial index also provides critical information about how dynamics simultaneously unfold at different speeds in different brain areas .", "This was evidenced most clearly by examining the interaction between OFC and ACC principal component weights , and DLPFC neuronal firing .", "Previous work has suggested a functional specialisation of OFC and ACC for delay- and effort-based decisions , respectively .", "In rats , lesions made to OFC lead to impulsive choices ( of a small , immediate reward over a larger , delayed reward ) whereas lesions made to ACC lead to less effortful choices ( of a small , easily obtained reward over a larger reward demanding more work ) ( Rudebeck et al . , 2006 ) .", "In humans , functional imaging activations yield a similar double dissociation between effortful and delay-based decisions ( Prevost et al . , 2010; Croxson et al . , 2009; Kurniawan et al . , 2013; Kable and Glimcher , 2007 ) , and further evidence can be drawn from the effects of electrical stimulation of ACC ( Parvizi et al . , 2013 ) .", "Our results extend these findings by suggesting that the rate at which dynamics unfold in OFC preferentially influences a decision signal in DLPFC on delay-based trials relative to effort-based trials , whereas the converse is true for ACC’s influence on DLPFC ( Figure 5 ) .", "This supports the view that the effective influence of one brain region on another is modulated by the type of decision currently being made ( Hunt et al . , 2014; Tauste Campo et al . , 2015 ) .", "We note , however , that our analysis does not test the possibility is that a third ( unobserved ) variable could jointly affect both regions , doing so differentially on effort vs . delay trials .", "Variation in ERP latency also corresponds to a shift in the phase of an oscillation ( at low frequencies , present in the evoked response ) .", "Consistent with this , we found PC2 weights correlated with the phase of oscillations in the theta range ( 4-–8 Hz ) as the decision was made ( Figure 2—figure supplement 3 ) .", "This suggests a possible way in which future studies may link our measure of LFP latency to spike-LFP coupling ( Koralek et al . , 2013; Canolty et al . , 2010 ) or inter-regional LFP coherence ( Nacher et al . , 2013 ) during cognitive tasks .", "The network model attempts to capture the dynamics of a single cortical region , but not interactions between regions .", "In our study , competitive dynamics similar to the model ( saccadic action value difference transforming into chosen action ) were clearly visible in DLPFC ( Figure 1B/7B ) , but not in other regions ( Figure 1—figure supplement 1 ) .", "However , at the mesoscopic scale , dynamics of chosen value correlates were relatively indistinguishable across regions ( Figure 2E , Figure 1—figure supplement 3 ) .", "One explanation for these phenomena is that competition proceeds in a distributed fashion across multiple areas ( Rushworth et al . , 2012; Hunt et al . , 2014; Cisek , 2012 ) .", "Competition in DLPFC might occur selectively in saccadic action space , but this might be complemented by parallel competitions in other regions in other reference frames .", "Examples of such competitions might include those over particular decision attributes ( e . g . OFC for delay , ACC for effort/physical action value ) , abstract goods ( Padoa-Schioppa , 2013; Padoa-Schioppa and Assad , 2006 ) , internal state variables ( Bouret and Richmond , 2010 ) , attentional allocation ( Lim et al . , 2011 ) , goal or task-relevant prioritisation ( Hunt et al . , 2014; Hare et al . , 2009 ) or other reference frames critical for the decision at hand ( Hunt et al . , 2013 ) .", "Though lesion evidence clearly implicates areas like ACC , OFC and ventromedial PFC as critical and dissociable in value-based decision-making ( Kennerley et al . , 2006; Rudebeck et al . , 2008; Camille et al . , 2011; Noonan et al . , 2010 ) , our results indicate that identifying ‘chosen value’ signals is not sufficient to understand the functional dissociations of these areas in the decision-making process .", "Further studies are needed to fully understand the reference frame in which different decision areas contribute to decision-making ( Hunt et al . , 2013; 2014; Boorman et al . , 2013 ) , with a careful consideration of the relationship between underlying choice dynamics and neuronal correlates of value .", "Future recurrent network models of choice involving hierarchical competitions across multiple areas ( Hunt et al . , 2014; Chaudhuri et al . , 2015 ) might also capture this distributed approach , and seek to explain our observations concerning the selective effort- and delay-based interactions across areas ( Figure 5 ) .", "A distributed account would predict that in other experimental paradigms , single neurons would show similar competitive dynamics to our DLPFC neurons but in complementary frames of reference ( Rustichini and Padoa-Schioppa , 2015; Strait et al . , 2014 ) .", "PCA is one of many possible approaches to obtaining a useful set of temporal basis functions to describe variation in ERP waveforms , and it may be improved upon by future investigations .", "We selected it for its simplicity , and its known properties of returning a temporal derivative when capturing evoked responses of variable durations ( Friston et al . , 1998; Mayhew et al . , 2006; Woolrich et al . , 2004 ) .", "Directly computing the ERP waveform and its temporal derivative would also be a valid approach to derive temporal basis functions .", "However , this approach may face problems if different components of the evoked response do not covary with each other .", "For example , the large , fast evoked response observed across all regions within 200 ms of stimulus onset ( Figure 1D ) was comparatively small in the first two principal components ( Figure 2B ) .", "This implies that cross-trial variation in this early sensory-evoked component occurred largely orthogonal to cross-trial variation in the later ( putatively decision-related ) component .", "This feature of the data is identified automatically using PCA .", "The LFP decomposition returned by PCA shows a notable similarity to that observed in decompositions of trial-averaged waveforms from multi-electrode recordings in motor cortex during movement ( Churchland et al . , 2012 ) .", "This provides links to dynamical systems perspectives on such activity .", "Unlike previous studies , however , our approach leverages mesoscopic dynamics containing high signal to noise ratio and reproducibility to extract single-trial information .", "This dispenses any requirement to first relate neural activity to experimental variables by averaging or regression ( Churchland et al . , 2012; Mante et al . , 2013 ) .", "We contend that this is particularly important in studying PFC and other areas distant from sensory input or motor output , whose relationship to experimental variables may often be considerably more complex than the experimenter envisaged ( Rigotti et al . , 2013 ) ." ], [ "Full details of neurophysiological recording procedures are detailed in ( Hosokawa et al . , 2013 ) and ( Kennerley et al . , 2009 ) , and precise recording locations in ( Hosokawa et al . , 2013 ) .", "In brief , four male rhesus macaques served as subjects .", "Arrays of 10-–24 tungsten microelectrodes ( FHC Instruments ) were lowered acutely each day .", "Recordings were made from dorsolateral prefrontal cortex ( DLPFC; primarily the dorsal bank of the principal sulcus ) , orbitofrontal cortex ( OFC; primarily areas 11 and 13 between the medial and lateral orbital sulci ) and anterior cingulate cortex ( ACC; primarily area 24c in the dorsal bank of the cingulate sulcus ) .", "Recordings were also made from two subjects in the cingulate motor area , but this region is not considered in the present study .", "We excluded 5 sessions in monkey B that contained exclusively effort-based or delay-based decisions , 1 further session in monkey B where LFP data was not recorded , and 2 sessions in monkey E where the LFP data was heavily artifact-contaminated .", "Any electrode which did not contain a well-isolated neuron , and so might not lie within grey matter , was not included in the LFP analysis .", "From visual inspection of the evoked data , we further excluded from further analysis ~4% of individual electrodes that contained a high degree of artifact in the LFP recording .", "The numbers of recording electrodes included from each subject , after all exclusion criteria were applied , are shown in Figure 1—figure supplement", "2 . ( As multiple units could occasionally be isolated from the same electrode , the total number of neurons exceeded the total number of electrodes for each region . ) All procedures were in accord with the National Institute of Health guidelines and the recommendations of the University of California Berkeley Animal Care and Use Committee .", "Full details of the experimental task are detailed in ( Hosokawa et al . , 2013 ) .", "In brief , subjects were well-trained on the expected value of a set of 32 pictures , 16 of which predicted a quantity of reward ( fruit juice ) and associated effort to obtain reward , and 16 of which predicted a quantity of reward and an associated delay to reward ( Figure 1A ) .", "The costs and benefits were titrated such that choice probabilities were approximately equally ( and linearly ) affected by both cost and benefit ( Hosokawa et al . , 2013 ) .", "Subjects performed a cost-benefit decision task where they chose between two pictures on each trial .", "On half of the trials ( ‘effort’ trials ) they chose between a pseudorandomly selected pair of the ‘effort’-associated pictures .", "On half of the trials ( ‘delay’ trials ) they chose between a pseudorandomly selected pair of the ‘delay’-associated pictures .", "Effort and delay trials were interleaved .", "On each trial , subjects fixated a central fixation point for 1 s , followed by the appearance of the two pictures on left and right sides of the screen for 1 s ( ‘choice phase’ ) .", "Throughout this choice phase , the monkey held fixation .", "Importantly , this meant that the time period when the monkey could respond ( after the onset of the go cue , at 1000 ms ) was outside of the window of our analyses , and so there was no potential behavioural ( motoric ) confound in neural analyses .", "This was with the exception of monkey K , who could not be sufficiently well-trained to fixate and so was free to saccade during the 1 s period .", "After the appearance of the go cue , the monkey saccaded to the preferred picture ( except monkey K , who made a joystick response to the side with the preferred picture ) .", "Subjects received juice reward after the ‘cost’ ( effort/delay ) was delivered .", "Only successfully completed trials are included in our analysis .", "Full details of the experimental task , MEG data acquisition and analysis protocols are provided in ( Hunt et al . , 2013 ) .", "Briefly , 18 subjects chose between two risky prospects consisting of differential levels of monetary reward magnitude and probability .", "Subjective values for each option were estimated using Prospect theory .", "We analysed data from ‘comparison’ trials ( where both options appeared simultaneously , and subjects were free to respond at any time after decision onset ) .", "The analysed data were beamformed to a region of ventromedial prefrontal cortex ( MNI coordinates = 6 , 28 , −6 mm ) previously found to contain dynamics analogous to those of the biophysical network model ( Hunt et al . , 2012 ) .", "All subjects provided informed consent in accordance with local ethical guidelines .", "Well-isolated single units ( see [Hosokawa et al . , 2013] for details ) were timelocked to the onset of the choice epoch of successfully completed trials to create rasters ( lasting from 1 s prior to choice onset to 2 s after choice onset ) .", "Each trial’s data was then convolved with a boxcar to estimate the local average firing rate of the neuron on each trial in 200 ms sliding bins .", "These binned data were then regressed , across trials , against five variables using ordinary linear regression: a constant term for effort trials , a constant term for delay trials , the value difference between left and right options , whether the subject chose left or right , and the value of the chosen option .", "For each neuron , we calculated the coefficient of partial determination for each factor as in ( Kennerley et al . , 2011 ) : CPD ( Xi ) = [SSE ( X~i ) - SSE ( X ) ]/SSE ( X~i ) where SSE ( X ) refers to the sum of squared errors in a regression model that includes a set of regressors X , and X~i is a set of all the regressors included in the full model except Xi .", "In Figure 1B/Figure 1—figure supplement 1 , we plot the mean /- s . e . across all neurons recorded in a given brain area ( for sessions that were also included in the LFP analysis ) .", "In Figure 1C , we calculated the Z-statistic for action value difference at 300 ms and chosen action at 700 ms for each neuron from the regression model , and plotted the relationship between these Z-statistics across all DLPFC neurons .", "LFP data were downsampled from 1 KHz to 100 Hz , and timelocked from 500 ms before to 1000 ms after the onset of the choice phase .", "The average event-related waveform was computed for each electrode , and the grand mean /- s . e . of all electrodes within each brain region was plotted ( Figure 1D ) .", "Based on previous behavioural modelling of the task ( Hosokawa et al . , 2013 ) , value for each option was defined as reward level ( scaled from 1 to 4 ) minus cost level ( scaled from 1 to 4 ) .", "For Figure 1E , we estimated the influence of chosen value and unchosen value on the evoked response at each timepoint using linear regression across trials , plotting the mean /- s . e . of the Z-scored regression coefficient across electrodes .", "In Figure 1—figure supplement 3 , we split this regression into chosen and unchosen reward and cost .", "For Figure 1F , we estimated the local temporal derivative of the grand mean event-related potential , averaged over a local sliding window of 80 ms . To extract single trial information from the local field potential ( LFP ) , we used principal components analysis ( PCA ) of event-related data .", "Single-trial LFP data were timelocked from 200 ms before to 1000 ms after the onset of the choice phase .", "Separately for each subject , all data from all electrodes in OFC , ACC and DLPFC were stacked to form a large matrix X . The dimensions of X are [nRecordedElectrodes*nTrials] by nTimepoints .", "Each row of X corresponds to the LFP data collected from a single trial , on a single electrode .", "To extract single trial information from the human data , a similar ‘stacked’ matrix was formed , but here single-trial data across all subjects were stacked , to make a matrix X with dimensions [nSubjects*nTrials] by nTimepoints .", "Each row of X corresponds to the beamformed data collected from a single trial , in a single subjects .", "( One difficulty faced by this approach is that beamforming contains a singular value decomposition ( SVD ) step to determine the orientation of sources , and when beamforming is run separately on each subject , then the meaning of a positive-going and negative-going deflection can be different across subjects because of an arbitrary sign flip induced by SVD .", "To overcome this , we used an iterated procedure that selected a sign for each subject that maximised the correlation coefficient in the evoked response , between subjects , and then multiplied each subject’s data by the appropriate sign .", "Note that this sign-flipping correction does not produce changes in the PCA decomposition of the data , but instead simply ensures that the principal component weights can be interpreted in a consistent manner across subjects .", "It would not be a necessary step were a similar analysis to be run on a scalp potential , for example , whose sign is already consistent across subjects .", ") The mean timecourse of the data was removed prior to running PCA , such that the principal components then captured cross-trial variability in the shape of the event-related waveform .", "We also removed trials that potentially contained non-neuronal ( e . g . movement , electrical ) artefacts .", "To achieve this , within-trial variability was indexed by taking the square root of the standard deviation of the event-related waveform across time .", "We excluded any trials whose within-trial variability index lay more than 2 . 32 standard deviations above the mean ( >99th percentile ) .", "In macaque data , most trials occur more than once in X , as in each recording session multiple LFP electrodes are recorded simultaneously and so there is a separate row for each electrode .", "Simultaneous multielectrode recording is not fundamental to the analysis approach of extracting single-trial dynamics .", "However , it becomes important when relating LFP and cellular data , below .", "In human data , each trial occurs only once , as there is one ‘virtual electrode’ per subject .", "PCA was performed using the svd function in MATLAB .", "To ensure components had similar interpretations across subjects , we constrained PC1 to be positive 190 ms after stimulus onset , and PC2 to be negative 530 ms after stimulus onset .", "This constraint can be reasonably imposed by flipping the sign of both the principal component and its corresponding PC weights where necessary ( as the sign of components returned by singular value decomposition is arbitrary ) .", "After this constraint was applied , highly similar components were observed across subjects ( Figure 2C ) .", "In all subjects , PC1 controlled waveform amplitude , and PC2 controlled waveform latency .", "However , the timecourse of monkey K’s principal components were notably different in latency from other subjects’ waveforms ( Figure 2C ) , and also had somewhat different value correlates , particularly in PC1 , likely due to an inability to sustain fixation .", "For this reason , Figure 2E and Figure 2—figure supplement 4 excludes data from monkey K , and monkey K’s PCA value correlates are plotted separately in Figure 2—figure supplement", "3 . Importantly , this does not affect the interpretation of PC2 , whose effects remain similar in monkey K as in other monkeys .", "We note that it would have equally been possible to run dimensionality reduction on the data from a single electrode , in a single session .", "However , the difficulty faced with such an approach is how then to match components between different components and sessions , such that the components from the different PCAs have the same interpretation .", "The stacked PCA approach here is analogous to that used in 'dual regression' of functional MRI data , where resting state networks are matched across subjects by stacking all subjects into a large matrix before running dimensionality reduction ( Filippini et al . , 2009 ) .", "It is appropriate due to the high degree of consistency of ERP waveforms ( Figure 1—figure supplement 2 and Figure 2—figure supplements 1/2 ) .", "Single neuron data were rasterised and binned as for the regression analysis of decision variables .", "For main Figure 3C , a regression model was estimated containing 19 coregressors to model out the effect of all experimental variables ( see Supplementary file 1 for a full list ) , plus the regressor of interest ( trial-by-trial weights of PC2 , derived from PCA decomposition of LFP from a simultaneously recorded electrode in another part of DLPFC ) .", "The CPD for the LFP-derived PC2 on single neuron firing was then estimated from this model .", "To calculate the percentage of individual significant neurons ( in main text ) , we estimated a significance criterion that controlled for multiple comparisons across time empirically from the data , by repeating the regression model but permuting the design matrix ( Nichols and Holmes , 2002 ) .", "Within a time window of 250 ms-–750 ms after choice onset , this yielded a p<0 . 01 significance criterion of Z>2 . 99 for significant positive responses , and Z<-3 . 05 for significant negative responses .", "Any neurons exceeding these thresholds at any point during this time window are reported as significant .", "We found the CPD for PC2 remained similar when a reduced model of experimental variables was used , containing 6 coregressors rather than 19 – a constant term for each trial type , an indicator variable for the chosen action ( L-R ) , the action value difference between left and right options , and the chosen value .", "For Figure 3A/B , we used this model and plotted CPD for action value difference , LFP-derived PC2 and chosen action in two single-neuron examples .", "For Figure 4 and associated figure supplements , we repeated this model , but subtracted the CPD for chosen value , action value difference and chosen action for a model that included PC1 and PC2 as coregressors , versus a model that included PC101 and PC102 ( to account for any potential general reduction in CPD by including coregressors ) .", "Positive values on this figure therefore indicate a reduction in explained variance by experimental factors when PC1 and PC2 are included as coregressors in the model .", "Note that these analyses required simultaneously recorded LFP from another electrode in the same brain region , but this was only available for some recording sessions .", "The number of recorded neurons in each analysis was therefore smaller than in Figure 1/Figure 1—figure supplement 1 ( DLPFC: n=205 neurons; OFC: n=114; ACC: n=194 ) .", "For Figure 4—figure supplement 3 , we sought to explore whether local LFP principal components provide greater contributions to the reduction in chosen value variance than those recorded from other areas .", "We therefore repeated the same analysis as in main Figure 4 , but first orthogonalised local ( DLPFC ) PC1/2 weights with respect to PC1/2 weights from another simultaneously recorded area ( either OFC/ACC ) .", "However , because both electrodes will contain observation noise on every trial , this analysis alone may not fully control for distal brain region effects .", "To provide a fairer comparison , we therefore performed the same analysis in reverse: examining the effect of the distal OFC/ACC PC1/2 weights , orthogonalised with respect to the local DLPFC PC1/2 weights .", "Figure 4—figure supplement 3B shows the latter analysis ( distal orthogonalised with respect to local ) subtracted from the former ( local orthogonalised with respect to distal ) .", "For Figure 5 and associated figure supplement , we examined the 40 recorded sessions where DLPFC , OFC and ACC recordings were made simultaneously .", "Single neuron firing rates from the 124 DLPFC units were explained using a model that contained separate 6 terms: separate constant terms for effort and delay trials , separate LFP-derived PC2 weights from OFC for delay and effort trials , and separate LFP-derived PC2 weights from ACC for delay and effort trials .", "Figure 5—figure supplement 1 shows the separate effects of PC2 from ACC and OFC for delay and effort trials respectively , on DLPFC firing; Figure 5 shows the difference in CPD for effort minus delay trials for each region .", "Significance for neuronal populations in Figures 4 , 5 was assessed using a non-parametric permutation test ( Nichols and Holmes , 2002 ) .", "At each timepoint , for each neuron , we compared the CPD for the relevant variable of interest to the CPD for the same variable in a 500 ms pre-choice baseline period .", "Across the neuronal population , we tested whether this difference was significantly greater than zero , by comparing the mean difference to that of a null distribution , generated by randomly sign-flipping each neuron’s effect and averaging across this permuted data .", "10 , 000 permutations were used .", "We elected to use a non-parametric test as the difference in CPD will yield a non-Gaussian distribution; in practice , however , similar results could be obtained with parametric statistics ( Student’s T-test ) ." ] ]

[ "Activity in prefrontal cortex ( PFC ) has been richly described using economic models of choice .", "Yet such descriptions fail to capture the dynamics of decision formation .", "Describing dynamic neural processes has proven challenging due to the problem of indexing the internal state of PFC and its trial-by-trial variation .", "Using primate neurophysiology and human magnetoencephalography , we here recover a single-trial index of PFC internal states from multiple simultaneously recorded PFC subregions .", "This index can explain the origins of neural representations of economic variables in PFC .", "It describes the relationship between neural dynamics and behaviour in both human and monkey PFC , directly bridging between human neuroimaging data and underlying neuronal activity .", "Moreover , it reveals a functionally dissociable interaction between orbitofrontal cortex , anterior cingulate cortex and dorsolateral PFC in guiding cost-benefit decisions .", "We cast our observations in terms of a recurrent neural network model of choice , providing formal links to mechanistic dynamical accounts of decision-making ." ]

[ "In 1848 , a railroad worker named Phineas Gage suffered an accident that was to secure him a place in neuroscience lore .", "While constructing a new railway line , a mistimed explosion propelled an iron bar into the base of his skull , where it passed behind his left eye before exiting through the top of his head .", "Gage survived the accident , but those who knew him reported significant changes in his personality and behaviour .", "Gage’s ability to make decisions was particularly impaired by his injury .", "Decision-making involves weighing up the costs and benefits associated with alternative courses of action .", "It entails looking into the future to decide whether an anticipated reward will justify the effort or expense necessary to obtain it .", "This process is dependent on a region of the brain called the prefrontal cortex , the area that sustained the most damage in Phineas Gage .", "While many studies have shown correlations between activity in particular parts of prefrontal cortex and the outcome of decisions , little is known about how this activity evolves over time as a decision is made .", "To explore this process , Hunt et al . trained macaque monkeys to choose between pairs of images that were associated with specific rewards ( quantities of fruit juice ) and costs ( either amounts of work or fixed delays ) .", "Electrode recordings revealed changes in prefrontal activity that varied over time as the monkeys deliberated over each pair of images , choosing for example between a large reward after a long delay versus a smaller reward immediately .", "This activity was consistent with a mathematical model of decision-making , which also explains data from brain imaging experiments in humans .", "This provides an important link between human data and electrode recordings in animals .", "However , some of the patterns of activity observed in both macaques and humans appeared to reflect the speed at which decisions were made , rather than the outcome of the decisions themselves .", "By extracting information about decision speed on each decision from each region , it was shown that communication between regions of prefrontal cortex changes when choices are between two different amounts of work , as opposed to two different delays .", "Further experiments are needed to explore this phenomenon and to determine how other brain regions interact with the prefrontal cortex to support the decision-making process ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "computational and systems biology", "cancer biology" ]

[ [ "‘Precision medicine’ promotes the molecular characterization of a patient’s tumor with genomic approaches , which requires tissue extraction usually obtained via biopsy .", "A number of examples demonstrate successful translation of genomic information obtained from biopsies into clinical applications ( Doroshow and Kummar , 2014 ) , but these approaches also have inherent limitations , such as their invasive nature or sampling artifacts caused by intra-tumor heterogeneity ( Sottoriva et al . , 2013; Fisher et al . , 2013; Gerlinger et al . , 2012 ) .", "These limitations can be addressed by medical imaging that has served as crucial diagnostic tool and treatment guidance in clinical oncology .", "In contrast to biopsies , medical imaging is usually noninvasive , can be applied longitudinally , and provides information about the entire visible tumor volume .", "In this way , medical imaging has the potential to characterize phenotypic information of tumors and thus complement molecular interrogation ( Choi et al . , 2016 ) .", "As imaging is already used routinely throughout the course of treatment this facilitates ready access to this type of data .", "Therefore , imaging has the potential to serve as valuable diagnostic tool in clinical decision making by complementing biological interrogation or serving as a surrogate in settings where biospecimen-derived diagnostics is not feasible , such as in longitudinal monitoring .", "Radiomics is an emerging field that translates these medical images into mineable data by extracting a large number of quantitative imaging features that objectively define tumor intensity , shape , size , and texture ( Gillies et al . , 2016; Aerts , 2016; Lambin et al . , 2012; Kumar et al . , 2012 ) in a robust and reproducible way ( Zhao et al . , 2016; Fried et al . , 2014; Balagurunathan et al . , 2014; Leijenaar et al . , 2013 ) .", "As this approach is applied to existing standard of care images , radiomics can be cost-effectively integrated with genomics or serve as surrogate in cases where biopsies are not feasible ( O'Connor et al . , 2015 ) .", "Hence , such strategies can be of value for the development of clinical biomarkers for diagnosis , prognosis , and prediction of response to specific treatments ( Choi et al . , 2016; Huang et al . , 2016a , 2016b; Aerts et al . , 2016; Nicolasjilwan et al . , 2015; Parmar et al . , 2015a , Parmar et al . , 2015b; Aerts et al . , 2014; Chong et al . , 2014; Coroller et al . , 2015; Gevaert et al . , 2012; Ganeshan et al . , 2012; Win et al . , 2013; Mattonen et al . , 2016; Grossmann et al . , 2017 ) .", "Due to the enormous potential for precision medicine , an increasing number of studies have investigated associations between imaging and tumor biology in different cancer types ( Aerts et al . , 2014; Gevaert et al . , 2012; Diehn et al . , 2008; Grossmann et al . , 2016; Gutman et al . , 2015; Segal et al . , 2007; Li et al . , 2016; Yoon et al . , 2015 ) .", "However , these studies focused on specific genetic associations , or tended to be underpowered due to a limited number of available samples and lacked validation via independent datasets .", "Here , we present a broad radiomic-genomic analysis in independent and large cohorts of patients with lung cancer .", "We rigorously investigated the mechanistic connections between imaging phenotypes and underlying molecular pathways .", "Furthermore , we validated key associations via immunohistochemical staining and related these associations to clinical factors .", "In addition , we developed and validated radiomic predictors of pathway activation status , and investigated the prognostic value of combining radiomic biomarkers with genetic and clinical data .", "In this study , we aimed at uncovering whether radiomic approaches have the potential to predict both molecular and clinical characteristics of tumors noninvasively and therefore have the potential to augment clinical decision-making using data extracted from standard of care medical images ." ], [ "To investigate the main associations of radiomics and underlying molecular pathways , we developed association modules describing radiomic-pathway coherency .", "Bi-clustering allowed simultaneous grouping of coherently expressed features and pathways into a single module , thereby reducing dimensionality .", "Using this approach , we identified thirteen radiomic-pathway modules in Dataset1 that were independently validated in Dataset2 ( FDR < 0 . 05 ) .", "Figure 3A and Table 2 summarize these modules , while a detailed version of every module is given in Figure 3—source data 1 . 10 . 7554/eLife . 23421 . 008Figure 3 . Radiomic-pathway-clinical modules .", "( A ) Clustering of significantly validated radiomic-pathway association modules ( FDR < 0 . 05 ) .", "Normalized enrichment scores ( NESs ) have been biclustered to coherently expressed modules .", "Every heatmap in this figure corresponds to a module ( M1 - M13 ) with radiomic features in columns and pathways in rows .", "Heatmap sizes are proportional to module sizes .", "Elements are NESs given in Z-scores across features , and are displayed in blue when positive and green when negative .", "Horizontal color bars above every module indicate radiomic feature groups ( black = first order statistics , orange = texture , purple = shape , red = wavelet , and pink = Laplace of Gaussian ) .", "Representative molecular pathways are displayed .", "( B ) Clinical module network .", "We investigated if modules were associated with overall survival ( red ) , stage ( yellow ) , histology ( purple ) , or no clinical factor ( white ) .", "Relationships of modules based on their number of shared radiomic features ( thickness of blue lines ) are displayed by a network .", "While we found that most modules yield clinical information , overlaps of modules did not indicate relationships to similar clinical factors . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 00810 . 7554/eLife . 23421 . 009Figure 3—source data 1 . Enlarged heatmaps of every module depicting normalized enrichment scores ( NESs ) of every pair of radiomic feature and molecular pathway clustered in a module . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 00910 . 7554/eLife . 23421 . 010Figure 3—figure supplement 1 . Predictive capabilities of representative radiomic features from every module for genetic mutations in KRAS , EGFR , and TP53 in a subset of the discovery cohort . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 01010 . 7554/eLife . 23421 . 011Figure 3—figure supplement 2 . Association of representative features with smoking history in a subset of the discovery cohort . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 01110 . 7554/eLife . 23421 . 012Table 2 . Summary of common themes in all of the identified radiomic-pathway association modules .", "Columns 1–3 display the module name , the number of radiomic features ( nr ) , and pathways ( np ) , respectively .", "Columns 4–5 hold the radiomic and pathway themes present in each module . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 012ModulenrnpRadiomicPathway M167Wavelet texture gray-level runsLipid and lipoprotein metabolism , Notch signaling , circadian clock M2585Wavelet intensity entropy; Laplace of Gaussian intensity standard deviationImmune system , p53 M3417Wavelet minimum intensityNeural system , axon guidance M42514Intensity variance and mean; wavelet minimum intensity minBiological oxidations , signaling by insulin receptor , signaling by GPCR , neuronal system M5588Wavelet texture gray-level runs; wavelet intensity range and median; ( wavelet ) texture information correlation and cluster tendencyAxon guidance and synaptic transmission , lipoprotein metabolism , cell type determination M6647Laplace of Gaussian standard deviation; wavelet texture gray-level runs; wavelet texture cluster tendencyCircadian clock , signaling by Notch M7398Laplace of Gaussian intensity entropy; wavelet intensity variance; Laplace of Gaussian texture information correlationMitochondria , Pol III transcription M82017Laplace of Gaussian standard deviationTCA cycle and electron transport , TGF-beta receptor signaling , response to stress , transcription regulation , protein synthesis , M9830Intensity variance; wavelet intensity varianceImmune system , p53 , cell cycle regulation checkpoints , cell-cell interaction , circadian clock M10583Shape surface ( SH ) ; wavelet texture gray-level runsAxon guidance , neuronal system , ( innate ) immune system , hemostasis , FGFR signaling , TGF-beta receptor signaling , Notch signaling , circadian clock M111766Wavelet intensity range; wavelet texture information correlationHemostasis , neural system M123227Wavelet texture entropy; intensity variance; wavelet texture cluster tendencyP53 , immune system M133926Intensity entropyGene expression regulation , Pol II/III transcription In general , we found that distinct radiomic features were associated with distinct biological processes .", "For example , texture entropy and cluster features , as well as voxel intensity variance features were associated with the immune system , the p53 pathway , and other pathways involved in cell cycle regulation in modules M2 , M9 , and M12 ( Table 2 and Figure 3A ) .", "In another module ( M8 ) , we found those features to also be associated with transforming growth factor beta ( TGF-β ) receptor signaling .", "Further examples for radiomic-pathway links included two modules ( M13 and M7 ) that were highly enriched for pathways involved in mitochondrial pathways , transcription , translation , and RNA regulatory mechanisms; with only one exception , all features in the larger module ( M13 ) were voxel intensity entropy features .", "In addition to this feature type , the smaller module ( M7 ) contained mainly textural variance and information correlation features .", "For every module , we assessed prognostic association to overall survival ( OS ) and associations to stage and histology based on the radiomic features of a module ( Figure 3B and Table 3 ) .", "Three modules ( M2 , M9 , and M12 ) were significantly prognostic for OS ( p<0 . 02 ) , ten modules ( M2 , M4-8 , and M10-13 ) were significantly associated with stage ( p<0 . 01 ) , and five modules ( M5 , M6 , and M10-12 ) were significantly associated with histology ( p<0 . 05 ) .", "The exact p-values of all modules are given in Supplementary file 2 . 10 . 7554/eLife . 23421 . 013Table 3 . Pathway prediction and clinical association .", "For every module , the independent validation performance of the strongest radiomic based pathway predictors is indicated per module by the area under the curve ( AUC ) of the receiver operator characteristic .", "In addition , we highlight whether a module was significantly associated with overall survival ( OS ) , TNM stage ( ST ) , or pathologic histology ( HI ) ( p<0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 01310 . 7554/eLife . 23421 . 014Table 3—source data 1 . Radiomic pathway predictors . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 014ModuleStrongest radiomic based pathway predictionAUCOSSTHI M1Wavelet ( HHH ) texture ( GLCM ) correlation → Cholesterol biosynthesis0 . 64 , p=0 . 014 M2Laplace of Gaussian intensity standard deviation → Autodegration of the E3 Ubiquitin ligase COP10 . 69 , p=8e-4xx M3Wavelet minimum intensity → Trafficking of GLUR2 containing AMPA receptors0 . 67 , p=0 . 003 M4Wavelet intensity minimum → Glutathione conjugation0 . 68 , p=9e-4x M5Texture information correlation → Trafficking of GLUR2 containing AMPA receptors0 . 69 , p=7e-4xx M6Wavelet texture cluster prominence → Notch1 intracellular domain regulation of transcription0 . 66 , p=0 . 007xx M7Laplace of Gaussian intensity entropy → RNA polymerase III transcription0 . 62 , p=0 . 031x M8Laplace of Gaussian intensity standard deviation → Pyruvate metabolism and citric acid TCA cycle0 . 72 , p=6e-5x M9Wavelet intensity variance → Trafficking of GLUR2 containing AMPA receptors0 . 64 , p=0 . 020x M10Shape compactness and shape sphericity → TRAF6 mediated NFkB activation0 . 66 , p=0 . 003xx M11Wavelet texture cluster tendency → Platelet aggregation plug formation0 . 69 , p=6e-4xx M12Wavelet texture entropy → G0 and early G10 . 65 , p=0 . 007xxx M13Laplace of Gaussian intensity entropy → RNA polymerase II transcription initiation and promoter opening0 . 68 , p=0 . 001x We examined and summarized the relationships of clinical status , module size , and overlap of modules in a network ( Figure 3B and Table 3 ) .", "We found that smaller modules tended not to be associated with the tested clinical factors .", "The total number of shared features or pathways was generally low ( mean Jaccard index 0 . 22 , range [0 . 01 , 0 . 59] ) .", "Interestingly , certain modules with higher overlap still showed different clinical associations .", "To test whether radiomic features can predict if a pathway is activated or deleted in individual patients , we fitted univariate models of radiomic features on Dataset1 and selected for every module the strongest predictor in Dataset1 according to the area under the curve ( Fawcett , 2006 ) ( AUC ) for validation in Dataset2 .", "As shown in Table 3 and Table 3—source data 1 , the overall biological and radiomic themes in a module were well represented by these individual predictors .", "For example , a Laplace of Gaussian intensity standard deviation feature was predictive of the autodegration pathway of the E3 ubiquitin ligase COP1 ( AUC = 0 . 69 , p<10−4 ) in module M2 , which was also associated with p53 .", "Importantly , COP1 mediates p53 and may interact with autophagy ( Rabbani et al . , 2014; Kobayashi et al . , 2013 ) , which are known drivers of tumorigenesis .", "Indeed , this module M2 was associated with OS .", "We found further examples of this radiomic-genomic-clinical link to be important: For example , a texture feature ( information correlation ) predicted trafficking of GLUR2 containing AMPA receptors ( AUC = 0 . 69 , p<10−4 ) in module 5 , which was associated with lipoprotein metabolism and stage .", "Further , two shape features ( sphericity and compactness ) predicted TRAF6 mediated NFkB activation ( AUC = 0 . 66 , p=0 . 003 ) in module 10 , which was also associated with axon guidance and histology .", "Furthermore , we assessed these representative features in terms of their predictive value for driver mutations in the discovery cohort; based on a subset of 60 patients whose tumors were profiled with Sanger sequencing , we estimate that the prevalence of mutated EGFR , KRAS , and TP53 are 15% , 35% , and 20% , respectively .", "In particular , we found strong performance for mutations in EGFR and KRAS by several features , but only one considerable performance for TP53 ( Figure 3—figure supplement 1 ) .", "Interestingly , predictive value for EGFR and KRAS were selective in that features had relatively high performance for one gene but not both .", "Predictive power for smoking history was low to moderate ( Figure 3—figure supplement 2 ) .", "To further investigate putative connections between radiomics , immune response pathways , and OS we performed immunohistochemical staining of 22 tumors for CD3 , a T-cell co-receptor .", "These tumors were predicted to show relatively high or low immune response by a radiomics feature selected from the three modules ( M2 , M9 , and M12 ) that were associated with OS .", "As represented in Figure 4 , we found agreement between radiomics and pathology; cases that were pathologically scored to have high CD3 enrichment also expressed significantly higher radiomic values ( one-sided Wilcoxon rank sum test , p=0 . 008 ) .", "Furthermore , we tested the extent to which radiomic predictors of inflammation can be reproduced immunohistochemically .", "We built on our previous results suggesting that the radiomic shape feature sphericity predicts NFkB activation ( module 10 ) and analyzed 24 stained tumors that were predicted to have relatively high or low NFkB activity for RelA , the p65 subunit of NFkB ( Figure 4—figure supplement 1 ) .", "Pathological assessment of enrichment for RelA revealed that those cases that indicated high RelA enrichment on average also had higher radiomic feature scores ( one-sided Wilcoxon rank sum test , p=0 . 06 ) . 10 . 7554/eLife . 23421 . 015Figure 4 . Test for agreement between radiomic and pathological immune response assessment . Two representative cases are shown where radiomic predictions of immune response were confirmed by immunohistochemical staining for nuclear CD3 highlighting lymphocytes in brown .", "Each case is displayed in 0 . 6X and 2 . 0X magnification of the tumor slides , and an axial slice of the corresponding diagnostic CT scan and the total tumor volume is given for comparison .", "Automated quantifications of lymphocytes are displayed in addition to the radiomics score incorporated to classify into high and low responders . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 01510 . 7554/eLife . 23421 . 016Figure 4—figure supplement 1 . Representative cases of immunohistochemical staining for RelA . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 016 To build on previously published results , we investigated prognostic value of an existing radiomic signature for survival of lung cancer .", "We fitted a Cox proportional-hazards model of this signature on Dataset1 and observed significant validation by the concordance-index ( CI ) on Dataset2 ( CI = 0 . 60 , Noether p=0 . 04 ) .", "Furthermore , we tested combinations of clinical , genetic , and radiomic data and observed that the combinations of data types tended to result in higher performances than given by the individual data alone ( Figure 5 ) .", "In particular , the performance of a clinical model increased from CI = 0 . 65 ( Noether p=0 . 001 ) to CI = 0 . 73 ( p=2×10−9 ) when adding the radiomic and an existing gene signature ( Hou et al . , 2010 ) ; this increase was significant at p=0 . 001 by permutation test .", "This combined radiomic-genetic-clinical model also performed significantly better than the combined radiomic-clinical model ( p=0 . 007 ) and the clinical-genetic model ( p=0 . 01 ) .", "Adding radiomics to clinical data alone did not result in a significant increase ( p=0 . 3 ) .", "We repeated this analysis with a novel radiomic survival signature and other published gene signatures ( Yuan et al . , 2004; Chen et al . , 2007; Hsu et al . , 2009 ) , and found that the clinical-genetic-radiomic models consistently yielded the highest performances in nearly all cases ( Figure 5—figure supplement 1 and Figure 5—figure supplement 2 ) . 10 . 7554/eLife . 23421 . 017Figure 5 . Combining prognostic signatures for overall survival . We tested combinations of clinical , genomic , and radiomic signatures .", "To a clinical Cox proportional-hazards regression model with stage and histology , we first added a published gene signature and next a published radiomic signature .", "These models were fitted on Dataset1 and evaluated with the C-index ( CI ) on Dataset2 .", "An asterisk indicates significance ( p<0 . 05 ) .", "Combining different data types resulted in increased prognostic performances .", "By adding radiomic and genomic information , the initial performance of the clinical model was increased from CI = 0 . 65 ( Noether p=0 . 001 ) to CI = 0 . 73 ( p=2×10−9 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 01710 . 7554/eLife . 23421 . 018Figure 5—figure supplement 1 . Prognostic performance of two radiomic signatures ( i . e . , a previously published and a novel signature ) combined with genetic and clinical information . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 01810 . 7554/eLife . 23421 . 019Figure 5—figure supplement 2 . Prognostic performance of two radiomic signatures combined with different gene signatures and clinical information . DOI: http://dx . doi . org/10 . 7554/eLife . 23421 . 019" ], [ "Medical imaging plays a crucial role in cancer diagnosis , treatment , and response monitoring .", "Radiomics allows quantification of the radiographic phenotype of a tumor ( Kuo and Jamshidi , 2014; Gillies et al . , 2010; Rutman and Kuo , 2009 ) , but the underlying connections of radiomics to tumor biology and clinical factors have not been elucidated yet .", "In this study , we identified novel and consistent associations between radiomic phenotype data , underlying molecular pathways , and clinical factors of patients with lung cancer in a North American cohort , and validated our findings in a European cohort and with immunohistochemical staining .", "In addition , we presented radiomic predictors for pathway activations , and demonstrated the complementary prognostic value of combining radiomic , genetic , and clinical information .", "Preliminary studies have previously investigated associations between imaging features , clinical factors , and molecular data for a number of cancer types as outlined in recent reviews ( Gillies et al . , 2016; Kuo and Jamshidi , 2014; Gillies et al . , 2010; Rutman and Kuo , 2009; Cook et al . , 2013 ) .", "Our analysis builds on these studies in that we performed a rigorous classification of a comprehensive set of radiomic features in terms of underlying molecular pathways on a genome-wide scale and clinical factors in large and independent cohorts .", "Although the long-term vision is to augment clinical decision making , the current goal of our study is to satisfy the need of the radiomic and oncological community to better understand the underlying biological rationale of radiomic predictions .", "Furthermore , we are the first to publicly share all study data and analysis code with the growing radiomic and biomedical community to enable further translational research .", "We identified and independently validated thirteen radiomic-pathway modules with coherent expression patterns , eleven of which were significantly associated with OS , stage , or histology .", "By basing these clinical associations exclusively on radiomic features , we could demonstrate that the associated molecular pathways robustly matched radiomic- based hypotheses .", "For example , based on radiomic features modules M2 , M9 , and M12 were prognostic and also associated with stage .", "These modules were highly enriched for immune system , p53 , and cell-cycle regulation pathways , biological processes that are widely recognized to play key roles in lung cancer .", "For example , it has been established that cell cycle regulation is of utmost importance in lung cancer ( Baldi et al . , 2011 ) .", "Furthermore , the status of p53 is reported to be a predictor of survival in lung cancer patients ( Ahrendt et al . , 2003 ) and a recent review has laid out how p53 can modulate innate immune system responses ( Menendez et al . , 2013 ) .", "Radiomic features in these prognostic modules M2 , M9 , and M12 quantified textural entropy and dispersion image intensity values suggesting associations between textural heterogeneity , cell cycling , and prognosis .", "Therefore , these results suggest that noninvasive radiomic surrogates may benefit diagnostic methods in assessing cell cycling and immune system states of tumors .", "We aimed at confirming our statistical results indicating connections between radiomics , immune response , and survival by immunohistochemical staining of lymphocytes in cases for which a relatively high or low immune response was predicted according to a radiomics score .", "We generally found high agreement between pathology and radiomics , especially in cases where immune response was predicted to be high .", "In cases of predicted low responders that showed high pathological immune response , the cause of disagreement may be a heavy distribution of CD3 clusters in the extreme periphery of the tumor with very little staining in the bulk of the tumor .", "In cases of predicted high responders that showed little to no immune response , this could be due to the lack of normal tissue margin around the edge of the tumor section or a sampling effect .", "Similarly , we stained tissue for RelA , the p65 subunit of NFkB , to validate radiomic predictions of inflammation .", "Overall , we found high agreement between pathology and radiomics , although at lower statistical significance .", "Future studies with whole mount sections stained with multi-plex phenotyping can help determine the relationship between a radiomic and a genetic immune or inflammation signature , and the gold standard .", "A variety of textural features were also associated with stage and histology ( module M5 ) .", "Similar associations have been reported by Ganeshan et al . ( 2010 ) , who suggested that 2D texture features of lung cancer CT scans could predict if tumor stage was II or above .", "Here , we found that texture features were enriched for axon guidance and lipoprotein metabolism .", "Furthermore , we observed strong associations between image intensity entropy features and pathways involved in gene expression , transcription regulation , and mitochondrial processes ( M13 and M7 ) .", "Previous research has suggested that imaging can detect consequences of an increase in the hypoxia-inducible factor as a result of absence of oxygen ( Gillies et al . , 2010 ) .", "Hence , if extracting quantitative information about mitochondrial pathways from medical images leads to assessment of hypoxia status of a tumor , this may ultimately aid in clinical decision-making as alternative therapies for hypoxic tumor areas are being developed ( Denny , 2010; Bryant et al . , 2014 ) .", "Indeed , previous work has indicated that CT pixel intensities correlate with hypoxia markers such as Glut-1 and pimonidazole ( Ganeshan et al . , 2013 ) .", "Those two modules ( M1 and M3 ) that were not associated with any of the tested clinical factors were relatively small modules; these modules suggested radiomic associations to circadian clock and neural system .", "The impact of these pathways to the clinical factors we tested is not apparent from current lung cancer literature , which could explain why these modules did not show clinical associations .", "Our results further suggest that radiomic approaches could have the potential to predict molecular states of pathways .", "We found that the highest predictors of every module was also a suitable representative of the overall biological and radiomic themes of that module .", "Amongst these examples of pathways that showed high predictability in terms of radiomics , we found various pathways essential for tumorigenesis such as cell cycle pathways ( e . g . , G0 and early G1 ) , signaling pathways ( e . g . , Notch and NfKB ) , and tumor suppressor pathways ( e . g . , COP1 autodegration and p53 ) .", "Furthermore , we tested those radiomic pathway predictors for predictive value of driver mutations .", "Thereby , the highest performances were found for mutations in EGFR and KRAS , which is in line with current radiomic-genetic literature ( Aerts et al . , 2016; Gutman et al . , 2015; Liu et al . , 2016; Rizzo et al . , 2016 ) .", "Interestingly , however , the highest performance for the tumor suppressor and cell cycle regulator TP53 we found was given by a textural entropy feature that also predicted G0 and early G1 ( module M12 ) .", "In addition , features expressed selectivity for predicting mutations , which was suggested previously ( Gutman et al . , 2015 ) .", "These results highlight the diagnostic potential , as ready information on pathway and mutation status may permit advanced patient stratification .", "Previous studies have indicated that gene expression can be predicted by imaging features ( Gevaert et al . , 2012; Segal et al . , 2007; Gevaert et al . , 2014 ) .", "To our knowledge , however , no study has examined and independently validated radiomic models for specific pathways , including biological validation such as immunohistochemical staining .", "Finally , we verified a previously described prognostic radiomic signature and observed that the best performance is achieved when combining radiomic , genetic , and clinical data .", "These results strongly suggest that radiomic data contain complementary prognostic information and are robust , as the published radiomic signature ( Aerts et al . , 2014 ) has not been tested on our data before .", "Notably , these prognostic improvements were relatively stable to substitution of radiomic or gene signatures .", "A related indication of improved survival predictions by combining imaging features and molecular data has been recently given for glioblastoma , however without validation ( Nicolasjilwan et al . , 2015 ) .", "It is worth noting that for the first time we also demonstrate that radiomic prognostication generalizes across cohorts from different continents .", "Three research tracks have recently been proposed for clinical translation of such imaging biomarkers ( O'Connor et al . , 2017 ) , including biological validation , technical validation , and evaluation of cost-effectiveness .", "Our study conforms with several of these roadmap recommendations by advancing results on a previously proposed radiomic signature ( Aerts et al . , 2014 ) with additional biological validation and investigations on how genetic data and clinical factors impact this signature .", "Fixing a radiomic signature for technical validation and cost-effectiveness verification should be considered in subsequent studies to overcome additional translational gaps .", "Although the long-term vision would be to augment clinical decision making , the current goal of our study is to contribute in satisfying the need of the radiomic and oncological community to understand the underlying biology of radiomic predictions .", "Our study is limited by its retrospective nature .", "Imaging protocols are not standardized and hence variability in CT acquisition and reconstruction parameters is inherent in clinical practice .", "However , despite this , no corrections by cohort or scanner type were made in this study illustrating the translational aspect of our results that generalized across institutions .", "Hence , we expect that the performance of radiomics will further improve , as imaging data are becoming more standardized .", "In fact , multiple studies have already documented the robustness of radiomic feature extractions in terms of reproducibility and repeatability in test/re-test settings ( Fried et al . , 2014; Balagurunathan et al . , 2014; Leijenaar et al . , 2013; Parmar et al . , 2015a; Aerts et al . , 2014; Grove et al . , 2015 ) .", "Another limitation of this study is that the current cohorts mainly focused on early stage ( I - III ) tumors , hence generalization of radiomic-genomic associations to late stage tumors should be drawn with precaution only .", "However , most radiomic applications do focus on early stage tumors as the current radiomic approach requires segmentation of tumors which for late stage tumors remains to be of particular complexity .", "Furthermore , although our study provides multiple facets of validation , immunohistochemical validation was restricted to considerably smaller sample sizes as compared to our statistical validations due to limited availability of frozen tissue .", "Prospective protocols can ensure availability of sufficient tissue for additional validation .", "Biological material investigated in this study has been acquired by single-needle biopsies , thus the interpretation of our genomic data is limited due to heterogeneity of lung cancer tumors .", "However , as our results validated in independent data and because known drivers of tumorigenesis were among the main pathways found to be associated with radiomic features , this suggests that these associations have been established in an early evolutionary step in tumorigenesis and are therefore reasonable representatives of the overall tumor .", "Prospective studies with defined spatial matchings of biopsies and/or single cell analyses could provide deeper insight into whether the strengths of these associations can be further increased .", "Prospective studies will also be required to assess clinical utility of combining radiomic , genomic , and clinical data into prognostic models .", "In conclusion , this study presented novel and consistent associations between radiomics , molecular pathways , and clinical factors .", "We applied an independent discovery and validation design on large patient cohorts from different continents with enough variability that allowed confidence in the generalization of our results .", "Furthermore , we performed biological validation and demonstrated that radiomics predicts molecular pathway status and thus improves the prognostic performances of clinical and gene signatures .", "The clinical impact of our results is illustrated by the fact that it advances the molecular knowledge of automated radiomic characterization of tumors , information currently not used clinically .", "This may provide opportunities to improve decision-support at low additional cost as imaging is routinely used in clinical practice as standard of care ." ], [ "Data underlying this study is made publically available with this article; Dataset1 can be downloaded as Figure 2—source data 1 and Dataset2 can be downloaded as Figure 2—source data", "2 . We analyzed two cohorts of patients with non-small cell lung cancer ( NSCLC ) , Dataset1 and Dataset2 , each consisting of pretreatment diagnostic computed tomography ( CT ) scans , gene expression profiles , and clinical data .", "While the larger cohort Dataset1 ( North American ) is novel and served as a discovery cohort , Dataset2 ( European ) has been previously published with CT scans and gene expression data ( Aerts et al . , 2014 ) , and was used for independent validation of our findings .", "Patients in Dataset1 were treated in the Thoracic Oncology Program at the H . Lee Moffitt Cancer Center , Tampa , Florida , USA; we included patients with diagnosed primary tumors who underwent surgical resection and collected contrast-enhanced CT scans obtained within 60 days of the diagnosis between years 2006 and 2009 .", "Patients in Dataset2 were treated at MAASTRO clinical , Maastricht , NL; we included patients with confirmed primary tumors who received surgery .", "Further details of Dataset2 are given by Aerts et al . ( 2014 ) .", "The majority of CT scans were recorded to be contrast-enhancing ( 89% and 71% of patients in Dataset1 and Dataset2 , respectively ) .", "For analyses involving CT scans and gene expression data , 262 and 89 patients were available for Dataset1 and Dataset2 , respectively .", "In addition , clinical data were available for 224 and 87 patients , respectively .", "Clinical outcomes investigated were overall survival ( OS ) , pathologic TNM stage ( combined T , N , and M stages , according to the latest version 7 of the IASLC guideline for lung cancer [Mirsadraee et al . , 2012] ) , and pathologic histology ( grouped into adenocarcinoma , squamous carcinoma , and others ) .", "Clinical stage and histology were used when pathologic information was not available .", "Tumors in these cohorts were mainly early stage; in Dataset1 among the 224 clinically annotated cases 26 were stage IIIB or IV and in Dataset2 among the 87 clinically annotated cases 3 cases were stage IV .", "These late stages have been grouped into ‘other’ for analysis .", "Further clinical cohort characteristics are given in Table", "1 . For tumors in both cohorts , expression of 60 , 607 probes was measured on a custom Rosetta/Merck Affymetrix 2 . 0 microarray chipset ( HuRSTA_2a520709 . CDF , GEO accession number GPL15048 ) by the Moffitt Cancer Center .", "Gene expression of Dataset2 is available also at Gene Expression Omnibus ( GEO ) through accession number GSE58661 .", "Gene expression values were normalized with the robust multi-array average ( RMA ) algorithm ( Irizarry et al . , 2003 ) implemented in the ‘affy’ Bioconductor package ( Gautier et al . , 2004 ) .", "Probes have been curated by choosing the most variant representative among probes mapping to the same gene identifier ( Entrez Gene ) resulting in a total of 21 , 766 unique genes .", "We extracted 636 features grouped into I ) tumor intensity ( voxel statistics ) , II ) shape , III ) texture , IV ) wavelet , and V ) Laplace of Gaussian features .", "Group I-IV features have been defined as specified by Aerts et al . ( 2014 ) .", "In addition , we added new features to Group III ( see GLSZM below ) .", "Group I features are first-order statistics ( e . g . mean , skewness ) of all voxel intensity values in the tumor volume mask .", "Group II features describe the shape and size of a tumor ( e . g . compactness ) .", "Group III features quantify texture in tumor images describing clustering of voxels with similar appearance by means of a gray-level co-occurrence matrix ( GLCM ) , a run-length gray-level matrix ( RLGL ) , or a gray-level size-zone matrix ( GLSZM ) .", "These features quantify how frequent voxels of same gray-level are adjacent to each other ( GLCM ) , how many voxels of the same gray-level appear in a consecutive run ( RLGL ) , or the sizes of flat zones , areas of same gray-level in all directions ( GLSZM ) .", "Group IV features are Group I-III features ( except GLSZM ) assessed after a wavelet decomposition of the image , which highlights sharp transitions in the intensity frequency spectrum .", "Group V consists of Group I features that have been calculated after applying a Laplace of Gaussian transformation to the image , which highlights edge structures .", "Detailed description and analytical definitions of the features added to the Aerts et al . ( 2014 ) feature set ( n = 440 ) are given in Supplementary file", "1 . Features were calculated in 3D .", "For normalization , slice thicknesses of all scans were interpolated to a voxel sizes of 1 × 1×1 mm3 .", "To test if a radiomic feature was associated with a molecular pathway , Spearman’s rank correlation coefficient rho was calculated for the expression of every gene across all patients and weighted by -log10 ( p ) , where p is the p-value of rho .", "The resulting gene rank was input to a preranked gene set enrichment analysis ( GSEA ) algorithm ( Subramanian et al . , 2005 ) version 2 . 0 . 14 on the C2 collection version 4 of the Molecular Signature Database ( MSigDB ) ( Liberzon et al . , 2011 ) .", "This collection contains the expert-curated set of pathways from the Reactome database ( Joshi-Tope et al . , 2005 ) .", "Those 511 out of 674 pathways were considered that contained at least 15 and at most 500 genes .", "GSEA reports normalized enrichment scores ( NESs ) for every pathway , which we further analyzed .", "To identify coherently expressed expressed features and pathways , a matrix holding an NESs for every pair of radiomic feature and Reactome pathway was biclustered with the Iterative Signature Algorithm ( ISA ) using the ‘isa2’ and ‘eisa’ packages in R and Bioconductor ( Bergmann et al . , 2003; Csárdi et al . , 2010 ) .", "As a result , each bicluster contains a set of coherently expressed features and pathways and is referred to as module .", "Potential module redundancy was limited using the ‘isa . unique’ function in the ‘isa2’ package with a maximum correlation threshold of 0 . 3 .", "To avoid parameter sensitivity with ISA , row and column clustering seed thresholds were set to a liberal sequence of 1 . 5 to 2 . 5 by 0 . 5 to include all potential signals .", "This procedure yielded 20 putative modules .", "To validate these modules , we developed and applied a correlation based statistic r:=mean ( CX ) +mean ( CY ) , where CX and CY are the Spearman rank correlations of all pairs of features and pathways in a module , respectively .", "The true r was calculated for every module in Dataset1 and validated on Dataset2 with random permutation tests ( N = 1000 ) .", "After correcting for multiple-hypothesis testing with the false-discovery-rate ( FDR ) ( Benjamini and Hochberg , 1995 ) , the validation resulted in 13 significantly enriched modules ( FDR < 0 . 05 ) .", "In total , the modules captured the associations between 210 radiomic features and 206 pathways .", "Module size was defined as n/N + m/M , where n and m are the number of features and pathways in a module , respectively , and N = 636 and M = 511 are the total numbers of features and pathways across all modules , respectively .", "Overlap of two modules was defined by the Jaccard index ( Theodoridis and Koutroumbas , 2008 ) , which is the size of union of features divided by the size of intersection of features of two module .", "Hereby , same feature names under different transformations were considered equivalent .", "To test radiomic pathway predictors , we used gene set variation analysis ( GSVA ) in Bioconductor ( Hänzelmann et al . , 2013 ) to calculate pathway enrichment scores per patient .", "Next , we fitted univariate logistic regression models of every feature to predict the NES sign of pathways ( which corresponded to activation or deletion ) in Dataset1 .", "We assessed the concordance between the predicted probabilities of the pathway sign and the true sign with the area under the curve ( AUC ) of the receiver operator characteristic ( ROC ) ( Bradley , 1997 ) .", "The strongest predictor of each module according to the AUCs in Dataset1 was evaluated on Dataset2 for validation; significance of AUCs was calculated according to Noether for binary outcomes ( Pencina and D'Agostino , 2004 ) .", "Associations to OS were assessed by calculating the mean concordance-index ( Harrell et al . , 1982 ) of all features in a module univariately using the ‘survcomp’ package in Bioconductor ( Schröder et al . , 2011 ) , and by validating this statistic with repeated random permutation tests ( N = 1000 ) .", "Similarly , associations to stage and histology were assessed by the mean of Kruskal-Wallis chi square statistics and permutation tests .", "As clinical information was not part of the module identification process , a meta-analysis of the results in Dataset1 and Dataset2 was conducted to account for sample size differences and other dataset specific variations .", "For this , a Fisher Z-transformation ( Whitlock , 2005 ) of the independent p-values in both datasets was employed for every module with weights equal to the respective sample sizes in Dataset1 and Dataset2 .", "We tested additive prognostic effects of integrating radiomic , gene expression , and clinical data by combining in a Cox proportional-hazards model the predictions of ( I ) a clinical Cox model with stage and histology , ( II ) an NSCLC OS gene signature , and ( III ) an NSCLC OS radiomic signature .", "We tested five published gene signatures ( Hou et al . , 2010; Yuan et al . , 2004; Chen et al . , 2007; Hsu et al . , 2009 ) without inclusion of clinical and radiomic data and retained the strongest performing signature by Hou et al . ( 2010 ) to challenge potential performance increases .", "To test for generalizability of radiomics , we tested a published radiomic signature by Aerts et al . ( 2014 ) and a novel signature developed in the current study .", "We developed this novel radiomic signature using a supervised feature selection algorithm followed by a stepwise Cox regression approach on Dataset1: First , we employed the minimum-redundancy maximum-relevance ( mRMR ) algorithm implemented in the ‘mRMRe’ R package ( De Jay et al . , 2013 ) on all radiomic features with respect to OS to select a non-redundant , highly informative ranked set of complementary features .", "Next , we trained Cox models incrementally , adding features starting by the highest ranked feature .", "We performed repeated random cross-validation ( N = 1 , 000 ) to measure the performance of each model , and retained the model with the highest mean CI .", "Finally , these fitted models were tested on Dataset2 for validation .", "All statistical analyses were carried out using the R software ( R Development Core Team , 2013 ) version 3 . 1 . 0 on a Linux operating system .", "Details of version numbers of utilized packages are available in Supplementary file", "2 . We selected 25 cases each that were predicted to have high and low immune response by using the value of the radiomic feature in the prognostic modules M2 , M9 , and M12 that showed the highest absolute correlation to the mean expression of genes in the CTLA4 inhibitory pathway that is supported to be associated with immune activity ( Postow et al . , 2015; Pardoll , 2012; Wolchok and Saenger , 2008 ) .", "In total , 22 cases were available with enough tumor tissue and sufficient staining quality .", "Tumor cross section slides were stained using a Ventana Discovery XT automated system ( Ventana Medical Systems , Tucson , AZ ) as per manufacturer's protocol with recommended reagents .", "Briefly , slides were deparaffinized with EZ Prep solution ( Ventana ) and a heat-induced antigen retrieval method was used under mild cell conditioning using CC1 antigen retrieval buffer ( Ventana ) .", "A rabbit primary antibody for CD3 , ( 790–4341 , Ventana ) was used at supplied concentration and incubated for 16 min .", "Next a Ventana OmniMap Anti-Rabbit Secondary Antibody was applied to the samples for 16 min and the Ventana ChromoMap kit was used as the detection system .", "Slides were then counterstained with Hematoxylin and dehydrated .", "Finally , the slides were cover slipped as per normal laboratory protocol .", "We selected 25 cases each that were predicted to have high and low NFkB activity .", "The same procedure as for the CD3 staining was applied , with the exception that a standard cell conditioning was used with CC2 antigen retrieval buffer ( Ventana ) .", "Furthermore , a rabbit polyclonal primary antibody for RelA ( NFkB p65 ) , ( Spring Biosciences E2750 ) was used at 1:600 dilution* and incubated for 32 min .", "In total , 24 cases were available with enough tumor tissue and sufficient staining quality .", "The lymphocytes are highlighted by brown nuclear staining of CD3 .", "The staining pattern was analyzed by a board-certified pathologist ( MB ) and scored into low and high enrichment .", "The percentage and intensity ( weak 1+ , moderate 2+ and intense 3+ ) of staining were recorded as well as the number and size of clustering of CD3 positive cells .", "The pathologist also chose the appropriate area from each sample for image analysis .", "We observed that the tissue section that has a complete cross section of the tumor with a complete rim of adjacent benign lung parenchyma is most ideal for image analysis .", "This is because the lymphocytic infiltration is commonly present at the periphery of the tumor .", "In addition to this assessment by a pathologist , a computational system was implemented for automatic evaluation ( Supplementary file 3 ) ." ] ]

[ "Medical imaging can visualize characteristics of human cancer noninvasively .", "Radiomics is an emerging field that translates these medical images into quantitative data to enable phenotypic profiling of tumors .", "While radiomics has been associated with several clinical endpoints , the complex relationships of radiomics , clinical factors , and tumor biology are largely unknown .", "To this end , we analyzed two independent cohorts of respectively 262 North American and 89 European patients with lung cancer , and consistently identified previously undescribed associations between radiomic imaging features , molecular pathways , and clinical factors .", "In particular , we found a relationship between imaging features , immune response , inflammation , and survival , which was further validated by immunohistochemical staining .", "Moreover , a number of imaging features showed predictive value for specific pathways; for example , intra-tumor heterogeneity features predicted activity of RNA polymerase transcription ( AUC = 0 . 62 , p=0 . 03 ) and intensity dispersion was predictive of the autodegration pathway of a ubiquitin ligase ( AUC = 0 . 69 , p<10-4 ) .", "Finally , we observed that prognostic biomarkers performed highest when combining radiomic , genetic , and clinical information ( CI = 0 . 73 , p<10-9 ) indicating complementary value of these data .", "In conclusion , we demonstrate that radiomic approaches permit noninvasive assessment of both molecular and clinical characteristics of tumors , and therefore have the potential to advance clinical decision-making by systematically analyzing standard-of-care medical images ." ]

[ "Medical imaging covers a wide range of techniques that are used to look inside the body , including X-rays , MRI scans and ultrasound .", "A process called radiomics uses computer algorithms to process the data collected by these techniques to identify and precisely measure a large number of features that would not otherwise be quantifiable by human experts .", "By doing so , radiomics can automatically measure the radiographic characteristics of a tumor .", "For example , radiomics can establish the size , shape and texture of a tumor to help to diagnose cancer and guide its treatment .", "Research has suggested that radiomics can predict certain clinical characteristics of cancer , such as how far through the body the cancer has spread , how likely it is to respond to treatment , and how likely a patient is to survive .", "However , these radiomic characteristics have not yet been precisely linked to the biological processes that drive how cancer develops and spreads .", "Cancers develop as a result of genetic changes that activate “molecular pathways” in the cells and trigger processes such as cell division and inflammation .", "To work out exactly which changes are behind a particular tumor , a sample of the tumor from biopsy or surgery is analyzed using genomics techniques .", "Linking radiomics features to the molecular processes active in a tumor can generate further information that can complement the molecular data .", "Images are routinely collected on all cancer patients yet molecular data is not .", "Hence , in some cases , the images can be used to infer the molecular underpinnings of cancer in individual patients .", "Grossmann et al . have now analyzed radiomic , genomic and clinical data collected from approximately 350 patients with lung cancer .", "The analysis revealed links between biological processes normally detected by genomics – in particular , inflammatory responses – and radiomics features .", "Furthermore , these features could also be associated with clinical characteristics , such as tumor type and patient survival rates .", "These results were further validated by using a technique called immunohistochemical staining on tumor tissue obtained by surgery .", "Further investigation revealed that certain radiomics features can predict the state of molecular pathways that are key to cancer development ( such as the inflammatory response ) .", "Furthermore , Grossmann et al . found that combining data from radiomics , genomics and clinical parameters predicts how the cancer will progress better than any of these parameters can predict on their own .", "These results demonstrate the complementary value of radiomic data to genomic and clinical data .", "There are many different algorithms that can be used to process images for radiomics .", "Before radiomics can be used clinically to assess the biological processes underlying the tumors of patients , a specific algorithm needs to be decided upon and then tested in prospective clinical trials ." ]