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• W2891048636 abstract "•Over 500 cerebral cavernous malformation (CCM) modifier genes discovered in C. elegans•Bioinformatics methods predict 29 conserved genes in core CCM-3/CCM3 network•Many genes exhibit ccm-3 phenotypes, including mop-25.2•Loss of mop-25.2 homolog MO25 causes stress fiber formation in endothelial cells Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by mutations in one of three genes (CCM1–3). Loss of CCM3 causes the poorest prognosis, and little is known about how it regulates vascular integrity. The C. elegans ccm-3 gene regulates the development of biological tubes that resemble mammalian vasculature, and in a genome-wide reverse genetic screen, we identified more than 500 possible CCM-3 pathway genes. With a phenolog-like approach, we generated a human CCM signaling network and identified 29 genes in common, of which 14 are required for excretory canal extension and membrane integrity, similar to ccm-3. Notably, depletion of the MO25 ortholog mop-25.2 causes severe defects in tube integrity by preventing CCM-3 localization to apical membranes. Furthermore, loss of MO25 phenocopies CCM3 ablation by causing stress fiber formation in endothelial cells. This work deepens our understanding of how CCM3 regulates vascular integrity and may help identify therapeutic targets for treating CCM3 patients. Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by mutations in one of three genes (CCM1–3). Loss of CCM3 causes the poorest prognosis, and little is known about how it regulates vascular integrity. The C. elegans ccm-3 gene regulates the development of biological tubes that resemble mammalian vasculature, and in a genome-wide reverse genetic screen, we identified more than 500 possible CCM-3 pathway genes. With a phenolog-like approach, we generated a human CCM signaling network and identified 29 genes in common, of which 14 are required for excretory canal extension and membrane integrity, similar to ccm-3. Notably, depletion of the MO25 ortholog mop-25.2 causes severe defects in tube integrity by preventing CCM-3 localization to apical membranes. Furthermore, loss of MO25 phenocopies CCM3 ablation by causing stress fiber formation in endothelial cells. This work deepens our understanding of how CCM3 regulates vascular integrity and may help identify therapeutic targets for treating CCM3 patients. The nematode worm Caenorhabditis elegans contains several biological tubes, including multicellular tubes such as the intestine and germline as well as the unicellular excretory canals. Defects in the development or maintenance of these organs are often lethal to the organism or compromise its fitness. We recently developed C. elegans models of the neurovascular disease cerebral cavernous malformation (CCM) and discovered key roles for the worm CCM3 gene, ccm-3, in promoting the extension and maintenance of excretory canals (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar) and germline development (Pal et al., 2017Pal S. Lant B. Yu B. Tian R. Tong J. Krieger J.R. Moran M.F. Gingras A.C. Derry W.B. CCM-3 promotes C. elegans germline development by regulating vesicle trafficking cytokinesis and polarity.Curr. Biol. 2017; 27: 868-876Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The function of CCM-3 is dependent on its protein binding partners within the striatin interacting phosphatase and kinase (STRIPAK) complex, and loss of ccm-3 or STRIPAK genes causes defects in endocytic recycling and cytoskeletal organization (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar, Pal et al., 2017Pal S. Lant B. Yu B. Tian R. Tong J. Krieger J.R. Moran M.F. Gingras A.C. Derry W.B. CCM-3 promotes C. elegans germline development by regulating vesicle trafficking cytokinesis and polarity.Curr. Biol. 2017; 27: 868-876Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, we lack a comprehensive understanding of how CCM-3/STRIPAK regulates these biological processes. Because of its genetic tractability and conservation of core CCM genes we took advantage of C. elegans to interrogate the CCM gene network. CCM affects ∼1 in 250 individuals and is caused by weak junctions in blood capillaries, which can leak blood into the brain parenchyma. This causes symptoms that range from mild headaches and seizures to hemorrhagic stroke, and it is not possible to predict when a lesion will bleed. The only treatment presently available for these patients is surgical resection, which can have devastating effects depending on lesion location. CCM can arise sporadically or, in approximately 40% of cases, by inheritance of mutations in one of the three CCM genes (CCM1/KRIT1, CCM2/OSM, and CCM3/PDCD10) (Fischer et al., 2013Fischer A. Zalvide J. Faurobert E. Albiges-Rizo C. Tournier-Lasserve E. Cerebral cavernous malformations: from CCM genes to endothelial cell homeostasis.Trends Mol. Med. 2013; 19: 302-308Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). These genes encode three distinct scaffold proteins that form a hetero-trimeric complex, in which loss of any one can cause activation of the small GTPase RhoA and stimulation of Rho kinase (ROCK) (Borikova et al., 2010Borikova A.L. Dibble C.F. Sciaky N. Welch C.M. Abell A.N. Bencharit S. Johnson G.L. Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.J. Biol. Chem. 2010; 285: 11760-11764Crossref PubMed Scopus (105) Google Scholar). This causes actin stress fiber formation, which compromises the integrity of cell-cell junctions. Although CCM1 is the most commonly mutated gene, mutations in CCM3 cause the earliest onset and most aggressive form of this disease (Shenkar et al., 2015Shenkar R. Shi C. Rebeiz T. Stockton R.A. McDonald D.A. Mikati A.G. Zhang L. Austin C. Akers A.L. Gallione C.J. et al.Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations.Genet. Med. 2015; 17: 188-196Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). A possible reason for the enhanced aggressiveness associated with CCM3 mutations may be that it functions in multiple complexes concurrently. Along with the hetero-trimeric CCM complex, CCM3 is also present in the STRIPAK complex (Kean et al., 2011Kean M.J. Ceccarelli D.F. Goudreault M. Sanches M. Tate S. Larsen B. Gibson L.C.D. Derry W.B. Scott I.C. Pelletier L. et al.Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.J. Biol. Chem. 2011; 286: 25065-25075Crossref PubMed Scopus (106) Google Scholar). This complex contains a striatin backbone to which CCM3 binds and provides both phosphorylation and de-phosphorylation activity through germinal center kinase class III (GCKIII) kinases (MST3, MST4, and MST20) and protein phosphatase 2 (PP2A/C), respectively (Kean et al., 2011Kean M.J. Ceccarelli D.F. Goudreault M. Sanches M. Tate S. Larsen B. Gibson L.C.D. Derry W.B. Scott I.C. Pelletier L. et al.Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.J. Biol. Chem. 2011; 286: 25065-25075Crossref PubMed Scopus (106) Google Scholar). Also bound to striatin is the MOB kinase activator (MOB3) and the striatin-interacting proteins (STRIP1/2), which themselves tether a number of proteins, including the sarcolemma-associated protein (SLMAP), IKK kinase suppressor (SIKE), and cortactin-binding protein (CTTNBP2) (Kean et al., 2011Kean M.J. Ceccarelli D.F. Goudreault M. Sanches M. Tate S. Larsen B. Gibson L.C.D. Derry W.B. Scott I.C. Pelletier L. et al.Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.J. Biol. Chem. 2011; 286: 25065-25075Crossref PubMed Scopus (106) Google Scholar). STRIPAK has been implicated in a number of human diseases (Hwang and Pallas, 2014Hwang J. Pallas D.C. STRIPAK complexes: structure, biological function, and involvement in human diseases.Int. J. Biochem. Cell Biol. 2014; 47: 118-148Crossref PubMed Scopus (155) Google Scholar) and uses CCM3 as a kinase adaptor (Ceccarelli et al., 2011Ceccarelli D.F. Laister R.C. Mulligan V.K. Kean M.J. Goudreault M. Scott I.C. Derry W.B. Chakrabartty A. Gingras A.C. Sicheri F. CCM3/PDCD10 heterodimerizes with germinal center kinase III (GCKIII) proteins using a mechanism analogous to CCM3 homodimerization.J. Biol. Chem. 2011; 286: 25056-25064Crossref PubMed Scopus (55) Google Scholar). CCM/GCKIII activity affects Golgi localization (Kean et al., 2011Kean M.J. Ceccarelli D.F. Goudreault M. Sanches M. Tate S. Larsen B. Gibson L.C.D. Derry W.B. Scott I.C. Pelletier L. et al.Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.J. Biol. Chem. 2011; 286: 25065-25075Crossref PubMed Scopus (106) Google Scholar) and cytoskeletal organization by directing the phosphorylation of a number of substrates, such as the actin-binding protein moesin (Zheng et al., 2010Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. et al.CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations.J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (118) Google Scholar). Furthermore, emerging evidence indicates that CCM3 is able to act independently of the other two CCM proteins (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar, Yoruk et al., 2012Yoruk B. Gillers B.S. Chi N.C. Scott I.C. Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease.Dev. Biol. 2012; 362: 121-131Crossref PubMed Scopus (64) Google Scholar, You et al., 2017You C. Zhao K. Dammann P. Keyvani K. Kreitschmann-Andermahr I. Sure U. Zhu Y. EphB4 forward signalling mediates angiogenesis caused by CCM3/PDCD10-ablation.J. Cell. Mol. Med. 2017; 21: 1848-1858Crossref PubMed Scopus (25) Google Scholar, Zhou et al., 2016aZhou H.J. Qin L. Zhang H. Tang W. Ji W. He Y. Liang X. Wang Z. Yuan Q. Vortmeyer A. et al.Erratum: Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation.Nat. Med. 2016; 22: 1502Crossref PubMed Scopus (11) Google Scholar). Although there have been intense efforts to understand the mechanisms by which CCM1 and CCM2 regulate vascular integrity, much less is known about the role of CCM3. Our aim in this study was to interrogate the C. elegans ccm-3 network by first conducting a whole-genome RNAi screen to identify a comprehensive set of genes that exhibit similar synthetic lethal interactions with kri-1/CCM1 as ccm-3 (Figure 1). Large-scale reverse genetics screens in C. elegans have many advantages for understanding poorly characterized genes, which often provide important insights into the biological functions of genes implicated in human diseases (Meier et al., 2014Meier B. Cooke S.L. Weiss J. Bailly A.P. Alexandrov L.B. Marshall J. Raine K. Maddison M. Anderson E. Stratton M.R. et al.C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency.Genome Res. 2014; 24: 1624-1636Crossref PubMed Scopus (117) Google Scholar, Silverman et al., 2009Silverman G.A. Luke C.J. Bhatia S.R. Long O.S. Vetica A.C. Perlmutter D.H. Pak S.C. Modeling molecular and cellular aspects of human disease using the nematode Caenorhabditis elegans.Pediatr. Res. 2009; 65: 10-18Crossref PubMed Scopus (70) Google Scholar, Sin et al., 2014Sin O. Michels H. Nollen E.A.A. Genetic screens in Caenorhabditis elegans models for neurodegenerative diseases.Biochim. Biophys. Acta. 2014; 1842: 1951-1959Crossref PubMed Scopus (41) Google Scholar). We uncovered more than 500 genes that exhibited strong negative genetic interactions with kri-1 and function in a range of cellular processes, including vesicle trafficking and cytoskeletal dynamics. By using a phenolog-like approach (McGary et al., 2010McGary K.L. Park T.J. Woods J.O. Cha H.J. Wallingford J.B. Marcotte E.M. Systematic discovery of nonobvious human disease models through orthologous phenotypes.Proc. Natl. Acad. Sci. U S A. 2010; 107: 6544-6549Crossref PubMed Scopus (223) Google Scholar), we cross-referenced these C. elegans genes with a network assembled from genes previously shown to function in human CCM signaling. The resultant 29 genes (tier 1 hits) were systematically knocked down by RNAi and evaluated for ccm-3-specific phenotypes, such as excretory canal truncations. Of the 29 tier 1 genes, 14 caused strong excretory canal truncations and membrane defects when knocked down in wild-type animals. The most severe canal defects were caused by ablation of mop-25.2, a homolog of mouse embryo scaffolding protein 25 (MO25), which is involved in mediating AMPK signaling (Boudeau et al., 2003Boudeau J. Baas A.F. Deak M. Morrice N.A. Kieloch A. Schutkowski M. Prescott A.R. Clevers H.C. Alessi D.R. MO25α/β interact with STRADalpha/β enhancing their ability to bind, activate and localize LKB1 in the cytoplasm.EMBO J. 2003; 22: 5102-5114Crossref PubMed Scopus (355) Google Scholar, Boudeau et al., 2004Boudeau J. Scott J.W. Resta N. Deak M. Kieloch A. Komander D. Hardie D.G. Prescott A.R. van Aalten D.M.F. Alessi D.R. Analysis of the LKB1-STRAD-MO25 complex.J. Cell Sci. 2004; 117: 6365-6375Crossref PubMed Scopus (125) Google Scholar). Our analysis reveals a role for MOP-25.2, independent of its canonical binding partners, in the localization of both CCM-3 and GCK-1 to apical membranes necessary for biological tube integrity. Finally, we show that loss of MO25 phenocopies CCM3 ablation in endothelial cells by causing actin stress fiber formation. The combination of unbiased screening and bioinformatics analysis in C. elegans affords a powerful and efficient method for understanding how CCM proteins regulate biological tube development and may even uncover therapeutic targets. We previously observed that, although loss of neither kri-1 nor ccm-3 on its own affects the survival of the worm, ablation of ccm-3 in kri-1 mutants results in synthetic lethality (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar). We reasoned that other genes that cause similar negative genetic interactions with kri-1 may act in the ccm-3 pathway or cooperate with its in vivo functions. Therefore, we conducted a full-genome RNAi screen using the Source BioScience RNAi Library, which covers ∼96% of annotated genes in the C. elegans genome, and identified 562 genes that reduced fitness of kri-1 mutants but not wild-type worms (Figure 1). These phenotypes, which include lethality at all stages of development, slow growth, and reduced fertility, all represent negative genetic interactions (Figure S1). Using the GeneMANIA application in Cytoscape, we constructed a network on the basis of these interactions to evaluate connectivity between our screen hits and genes known to be involved in CCM signaling (Figures S1 and S2, blue circles). Of the 562 genes identified, 237 had annotated interactions (genetic, physical, and/or predicted) with one another, while the remaining 325 “orphan” genes had only genetic interactions with kri-1 (Figure S1). For this study we focused on the 237 genes with documented interactions. Using Gene Ontology (GO) classification, these genes were found to be associated with a number of cellular processes, with a notable enrichment in cell localization and migration categories (including cell migration, cell motility, localization of the cell, and inductive cell migration) (Figure S2). Analysis of their functional roles indicate that a number fall into well-defined signaling cascades or cell process categories (Figure 2; Table S1). For example, we identified several cytoskeletal genes (i.e., actin, tubulin, interfilament organizer, and chaperones) and genes involved in small G protein signaling (i.e., small GTPases, GEF, and RHO-activating proteins). This is in line with previously described roles of CCM proteins in the regulation of actin polymerization and cell junction stability through Rho signaling (Borikova et al., 2010Borikova A.L. Dibble C.F. Sciaky N. Welch C.M. Abell A.N. Bencharit S. Johnson G.L. Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.J. Biol. Chem. 2010; 285: 11760-11764Crossref PubMed Scopus (105) Google Scholar, Stockton et al., 2010Stockton R.A. Shenkar R. Awad I.A. Ginsberg M.H. Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity.J. Exp. Med. 2010; 207: 881-896Crossref PubMed Scopus (262) Google Scholar, Whitehead et al., 2009Whitehead K.J. Chan A.C. Navankasattusas S. Koh W. London N.R. Ling J. Mayo A.H. Drakos S.G. Jones C.A. Zhu W. et al.The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases.Nat. Med. 2009; 15: 177-184Crossref PubMed Scopus (283) Google Scholar). Consistent with observations in mammalian systems (Draheim et al., 2015Draheim K.M. Li X. Zhang R. Fisher O.S. Villari G. Boggon T.J. Calderwood D.A. CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.J. Cell Biol. 2015; 208: 987-1001Crossref PubMed Scopus (37) Google Scholar, Zhou et al., 2015Zhou Z. Rawnsley D.R. Goddard L.M. Pan W. Cao X.J. Jakus Z. Zheng H. Yang J. Arthur J.S.C. Whitehead K.J. et al.The cerebral cavernous malformation pathway controls cardiac development via regulation of endocardial MEKK3 signaling and KLF expression.Dev. Cell. 2015; 32: 168-180Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, Zhou et al., 2016bZhou Z. Tang A.T. Wong W.-Y. Bamezai S. Goddard L.M. Shenkar R. Zhou S. Yang J. Wright A.C. Foley M. et al.Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.Nature. 2016; 532: 122-126Crossref PubMed Scopus (178) Google Scholar) we also identified a number of MAPK signaling genes, such as a MEKK3 kinase, MAPK phosphatases, and a KLF transcription factor. Finally, several vesicle-trafficking proteins (including the exocyst complex, endosome-associated GTPase, and components of the adaptin complex) were identified, consistent with our recent observations on CCM-3/STRIPAK function in biological tube development (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar, Pal et al., 2017Pal S. Lant B. Yu B. Tian R. Tong J. Krieger J.R. Moran M.F. Gingras A.C. Derry W.B. CCM-3 promotes C. elegans germline development by regulating vesicle trafficking cytokinesis and polarity.Curr. Biol. 2017; 27: 868-876Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). To refine the primary network into a set of genes that are evolutionarily conserved, we used a “phenolog”-like approach (McGary et al., 2010McGary K.L. Park T.J. Woods J.O. Cha H.J. Wallingford J.B. Marcotte E.M. Systematic discovery of nonobvious human disease models through orthologous phenotypes.Proc. Natl. Acad. Sci. U S A. 2010; 107: 6544-6549Crossref PubMed Scopus (223) Google Scholar). We reasoned that filtering the primary screen hits to identify nematode genes with human homologs would enrich for genes relevant to CCM biology. We selected 35 genes (Table S4) previously shown to function in human CCM signaling (Borikova et al., 2010Borikova A.L. Dibble C.F. Sciaky N. Welch C.M. Abell A.N. Bencharit S. Johnson G.L. Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.J. Biol. Chem. 2010; 285: 11760-11764Crossref PubMed Scopus (105) Google Scholar, Fischer et al., 2013Fischer A. Zalvide J. Faurobert E. Albiges-Rizo C. Tournier-Lasserve E. Cerebral cavernous malformations: from CCM genes to endothelial cell homeostasis.Trends Mol. Med. 2013; 19: 302-308Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, Glading and Ginsberg, 2010Glading A.J. Ginsberg M.H. Rap1 and its effector KRIT1/CCM1 regulate beta-catenin signaling.Dis. Model. Mech. 2010; 3: 73-83Crossref PubMed Scopus (93) Google Scholar, Storkebaum et al., 2011Storkebaum E. Quaegebeur A. Vikkula M. Carmeliet P. Cerebrovascular disorders: molecular insights and therapeutic opportunities.Nat. Neurosci. 2011; 14: 1390-1397Crossref PubMed Scopus (67) Google Scholar, Zhang et al., 2013aZhang M. Dong L. Shi Z. Jiao S. Zhang Z. Zhang W. Liu G. Chen C. Feng M. Hao Q. et al.Structural mechanism of CCM3 heterodimerization with GCKIII kinases.Structure. 2013; 21: 680-688Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) to generate a predicted “CCM-ome.” This CCM-ome gene list, generated using GeneMANIA and STRING algorithms, was converted to their nematode orthologs, yielding 653 genes (see STAR Methods for more details). Comparing this list with the 562 genes identified from the unbiased RNAi screen revealed 29 genes in common, which we refer to as “tier 1” hits (Figure 2, triangles, and Figure 3A; Table 1). We reasoned that genes identified in the RNAi screen that are orthologous to human genes in the CCM-ome would predict roles in CCM3 biology.Table 1Tier 1 Hits and Their Human HomologsGene NameWB Gene IDTruncation RatingHuman Gene HomologHuman Gene IDapl-1WBGene00000149no truncationAPLP1ENSG00000105290cct-2WBGene00000378truncationCCT2ENSG00000166226cdc-25.1WBGene00000386truncationCDC25AENSG00000164045cdc-42WBGene00000390strong truncationCDC42ENSG00000070831dbr-1WBGene00000937no truncationDBR1ENSG00000138231ephx-1WBGene00019487truncationARHGEF16ENSG00000130762erfa-1WBGene00020269no truncationETF1ENSG00000120705glp-1WBGene00001609truncationNOTCH2ENSG00000134250H28G03.1WBGene00019249truncationHNRNPA1ENSG00000135486his-13WBGene00001887no truncationHIST2H3DENSG00000183598icd-1WBGene00002045truncationBTF3ENSG00000145741lin-23WBGene00003009no truncationFBXW11ENSG00000072803mop-25.2WBGene00013140strong truncationCAB39ENSG00000135932nhr-23WBGene00003622no truncationRORAENSG00000069667nhr-69WBGene00003659truncationHNF4AENSG00000101076pab-1WBGene00003902truncationPABPC4ENSG00000090621ppfr-2WBGene00017064no truncationPPP4R2ENSG00000163605rab-5WBGene00004268truncationRAB5BENSG00000111540rrc-1WBGene00009800no truncationARHGAP30ENSG00000186517scc-1WBGene00004737truncationRAD21ENSG00000164754sdn-1WBGene00004749no truncationSDC1ENSG00000115884sec-15WBGene00016188truncationEXOC6ENSG00000138190sec-3WBGene00018703strong truncationEXOC1ENSG00000090989sek-6WBGene00012162no truncationMAP2K4ENSG00000065559skr-21WBGene00004827no truncationSKP1ENSG00000113558T02G5.7WBGene00020166no truncationACAT1ENSG00000075239T04G9.4WBGene00020215no truncationAASDHPPTENSG00000149313ubl-1WBGene00006725no truncationRPS27AENSG00000143947unc-112WBGene00006836no truncationFERMT2ENSG00000073712Many C. elegans genes are the single homologs for multiple human genes or gene isoforms. Listed above are the human homologs on the basis of the BLASTP matches per Wormbase.org. For an expanded list of possible human homologs, see Table S4. Open table in a new tab Many C. elegans genes are the single homologs for multiple human genes or gene isoforms. Listed above are the human homologs on the basis of the BLASTP matches per Wormbase.org. For an expanded list of possible human homologs, see Table S4. To investigate their in vivo functions, we used the C. elegans excretory canal as a model. This unicellular tube extends bi-directionally along both sides of the worm and functions as a renal system for the animal (Nelson and Riddle, 1984Nelson F.K. Riddle D.L. Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.J. Exp. Zool. 1984; 231: 45-56Crossref PubMed Scopus (146) Google Scholar) (Figure 3B). Loss of ccm-3 causes significant truncations and “cyst” formation in excretory canals (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar). We systematically ablated the 29 tier 1 genes by RNAi in wild-type animals and quantified excretory canal lengths. This was compared with a list of 29 randomly selected “control set” genes (Table S2). We found that the tier 1 set was significantly enriched (p = 6.9 × 10−36) for genes that caused canal truncations compared with the control set (Figures 3C, S3A, and S3B). Using the distribution of canal lengths under control conditions (Figure 3B), we denote “truncations” as canals that are <80% the length of those in wild-type animals. For a gene to cause a biologically relevant truncation when ablated, it must cause at least 5 times more canal truncations in the population of worms compared with control RNAi; a population of worms on control RNAi exhibits ∼7% truncation (33 of 500), so for a gene to cause truncation, ≥35% of animals must have truncated canals. We defined a strong truncation to be similar to ccm-3 mutants (≥80% of animals with truncated canals). Analysis of individual genes within each set (Figures S3A and S3B; Tables 1 and S2) revealed that the control set had 26 genes with no truncation, 3 genes causing truncation, and no genes causing strong truncation. Conversely, the tier 1 set contained only 15 genes with no truncation, 11 genes causing truncations, and 3 genes causing strong truncations, which was a significant enrichment in genes required for canal extension compared with the control set (p = 0.0031). Tier 1 genes causing canal truncations (Figure 3A, triangle- and diamond-outlined genes) represent a range of cellular processes, such as rab-5 (early endosome targeting GTPase), the Rho GEF ortholog ephx-1, the small GTPase cdc-42, and sec-3/sec-15 (exocyst). We also identified a number of genes that have no previously documented roles in excretory canal morphology, including the chaperonin complex subunit cct-2, the RNA lariat debranching enzyme dbr-1, polyadenylate-binding protein pab-1, and Rad21/Rec8-like cohesion protein scc-1. In addition, two uncharacterized genes, the RBM (RNA binding motif-containing) ortholog H28G03.1 and nuclear hormone receptor nhr-69, also caused canal truncations when ablated. Because the methodology of both the RNAi screen and the subsequent bioinformatic filtering do not bias against potential CCM1/kri-1 interactors, we asked whether any of the tier 1 hits would also cause kri-1 phenotypes. Loss of kri-1 causes resistance to ionizing radiation (IR)-induced germline apoptosis in the pachytene region (Ito et al., 2010Ito S. Greiss S. Gartner A. Derry W.B. Cell-nonautonomous regulation of C. elegans germ cell death by kri-1.Curr. Biol. 2010; 20: 333-338Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (Figure S3C, yellow asterisks). Conversely, loss of ccm-3 causes oocyte growth defects that leads to their death in the distal end of the germline (Figure S3D, red asterisk). We quantified germ cell corpses after ablating tier 1 genes by RNAi in wild-type worms exposed to 60 Gy of IR, using kri-1 and the anti-apoptotic Bcl-2 homolog ced-9 as controls. Although ablation of some genes (cct-2, cdc-25.1, cdc-42, erfa-1, T04G9.4, ubl-1, and unc-112) caused germline defects that made it impossible to quantify apoptotic corpses, five genes (Figure S3E; dbr-1, his-13, icd-1, lin-23, and nhr-23) caused suppression of apoptosis similar to kri-1 RNAi (n = 50, p < 0.05 from control RNAi, p > 0.05 from kri-1 RNAi). If we define suppression as less than or equal to the levels of physiological apoptosis, or two corpses per gonad arm (Gumienny et al., 1999Gumienny T.L. Lambie E. Hartwieg E. Horvitz H.R. Hengartner M.O. Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline.Development. 1999; 126: 1011-1022PubMed Google Scholar), ablation of kri-1 by RNAi caused ∼50% suppression of apoptosis (24 of 50 germline arms with two or fewer corpses). Only his-13 RNAi caused a greater suppression of apoptosis (29 of 50 germline arms) than kri-1. Of the tier 1 hits (Figure S3A), knockdown of MO25 ortholog mop-25.2 caused the most severe canal truncations (50 of 50 canals with canals <80% wild-type length) that were even more severe than knockdown of ccm-3 or gck-1 (p < 0.05 in both cases) (Figures 4A and 4B ). Consistent with loss of ccm-3 (Lant et al., 2015Lant B. Yu B. Goudreault M. Holmyard D. Knight J.D.R. Xu P. Zhao L. Chin K. Wallace E. Zhen M. et al.CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.Nat. Commun. 2015; 6: 6449Crossref PubMed Scopus (58) Google Scholar), we frequently observed distended lumen and distal canal tip cysts in animals depleted of mop-25.2 (Figure 4C, middle and bottom). Interestingly, RNAi to either gck-1 or mop-25.2 significantly exacerbated truncations in ccm-3 mutants (p = 1.089 × 10−9 a" @default.
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• W2891048636 date "2018-09-01" @default.
• W2891048636 modified "2023-10-18" @default.
• W2891048636 title "Interrogating the ccm-3 Gene Network" @default.
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• W2891048636 doi "https://doi.org/10.1016/j.celrep.2018.08.039" @default.
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