Trajectories And Vesicle Components Of A Glycosphingolipid-dependent Trafficking Pathway For Apical Polarity In The C. Elegans Intestinal Epithelia

Multicellular tubes are the fundemantal units of all internal organs.Their morphogenesis defects or dysfunctions often associated with diseases,such as colon cancer and polycystic kidney disease.Multicellular tubes are composed of polarized epithelial cells with their apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix.Although multiple conserved polarity cues have been identified,the underlying principles of their interaction and integration in the process of epithelial polarity establishment,polarized membrane biogenesis and tissue morphogenesis are not yet understood.Current thinking posits that,after an extracellular cue,apicobasal membrane domains of epithelial cells are established by membrane-and junction-associated polarity determinants(e.g.the canonical PAR-,Crumbs-and Scribble-related polarity complex)and subsequently maintained by directional trafficking.However,this theory cannot resolve the issue on how the polarity determinants themselves arrive at different membrane domains.Moreover,it has been challenged by the study in 3D in vitro and in vivo lumenogenesis models.Recent in vitro and in vivo studies of lumenogenesis in the 3D MDCK(Madin-Darby canine kidney)cultured cells and the expanding C.elegans intestine,respectively,have shown that trafficking is required to allocate the apical/lumenal domain,suggesting that intracellular trafficking itself might define membrane domain identities,in part by recruiting and targeting the canonical polarity determinants to their respective membrane domains during de novo membrane biogenesis.Our lab have previously shown that depleting vesicle-associated glycosphingolipids/GSLs,clathrin and its AP-1 adaptor suffices to mislocalize multiple apical membrane components,including the polarity determinants,to basolateral membrane domains and cause ectopic lumen formation on expanding C.elegans intestinal membranes,suggested that GSLs,clathrin and AP-1 are involved in the sorting process of apical membrane components and demonstrated the criticalfunction of GSLs in the previous lipid raft theory on polarity.Moreover,clathrin and AP-1 were found to genetically interact with GSLs in apical domain and lumen positioning in the C.elegans intestine.The results suggested that these vesicle-carrier-associated molecules are in vivo intrinsic-cues for apical polarity in the C.elegans tubular epithelia,and supported an alternate model for a putative vesicle-sorting function of GSLs in apical membrane biogenesis,namely their cooperative engagement with vesicle coats and adaptors in the sorting and directional routing of apical vesicles to the apical membrane domains.However,how GSLs/clathrin/AP-1 dependent apical sorting pathway directs the establishment of apical membrane domains,including its vesicle components and trajectories,are still unresolved.To identify molecular components of putative GSLs-dependent apical vesicles and to investigate the mode of regulation of their directionality,we here use the C.elegans single post-mitotic intestinal cell as an in vivo model.We first collect 50 trafficking-associated molecules-including the vesicle coat complex COPI/COPII components,small GTPase,vacuolar H+-ATPases components and SNARE proteins – identified from different genome-wide RNAi screens for apical-membranebiogenesis/tubulogenesis of the C.elegans multicellular intestine and unicellular excretory canal.These molecules characterize trafficking functions span a broad range from vesicle biogenesis,trafficking,docking to fusion.Since their loss-of-function cause the mislocaliztion of apical membrane proteins,they are all required for apical membrane biogenesis.Next,to explore whether any of these 50 trafficking molecules is part of a putative GSL-dependent apical sorting pathway,we perform genetic interaction screens between these 50 trafficking-related molecules and an easily tractable GSL biosynthetic enzyme,let-767.These genetic interaction screens identify twelve enhancers(enhancers are genes whose depletion cause de novo appearance of polarity conversion in let-767;s Dp3 mutant/RNAi larval intestines)and five suppressors(suppressors are genes whose depletion cause lower incidence of let-767(-/-)mutant/RNAi larval intestines with polarity conversion phenotype)of GSLs-depletion polarity conversion phenotype.Among them,enhancers are highly enriched for molecules previously implicated in ER-to-Golgi trafficking and thus the secretory/biosynthetic pathway,whereas suppressors contain post-Golgi molecules implicated in endocytosis,the degradative/lysosomal pathway and retrograde trafficking/recycling.In addition,enhancers themselves are required for apicobasal sorting,suggested that the secretory/biosynthetic pathway concomitantly expands and positions the apical membrane domain in the expanding epithelium.Through the analysis of the mechanism of suppression,we conclude that each suppressor knockdown,rather than inducing the polarity reversion,reduces basolateral misrouting of apical cargo(and subsequent basolateral lumen formation).Finally,RNAi-based in vivo imaging and loss-of-function studies of three suppressors-DAB-1/Disabled,RAB-7,and VHA-6(a V-ATPase component)– assign novel function in apical and basolateral membrane biogenesis to these endoand plasma-membrane-associated molecules,placing them upstream of GSL sorting site,and suggesting that they regulate the GSLs-dependent apical sorting pathway through endocytic recycling of apical and basolateral membrane proteins.Therefore,we here identify 17 vesicular components of the GSLs-dependent apical sorting pathway(including 12 enhancers and 5 suppressors).Among them,enhancers are distinguished as components of secretory/biosynthetic pathway,regulating the transport of newly synthetic apical membrane components to the GSLs-based sorting site.In contrast,suppressors are distinguished as components of endocytic recycling pathway,regulating the trafficking of endocytic apical and basolateral membrane components to the GSLs-based sorting site.Our findings confirm the endomembrane-based sorting nature of a GSL-regulated apical sorting pathway and support the earlier polarity models that proposed an inherent directionality of biosynthetic vesicular trafficking specifying membrane polarity.Futhermore,these findings provide important evidences for further investigate the role of vesicular trafficking in polarized membrane biogenesis and epithelial polarity establishment and also provide references for further study on the mechanisms and treating of related diseases.