Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single postmitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity via sphingolipid synthesis, and reveal ceramideglucosyltransferases (CGTs) as endpoint biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids (GSLs), CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and suggest they sort new components to the expanding apical membrane.

SUMMARYMany unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell C.elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1 overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1-AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.

SUMMARYClathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1-mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1 function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path. (Belfiore et al., 2002). The temperature-sensitive strain chc-1(b1025) was maintained at 16°C unless indicated otherwise. RNAi and screensA systematic C. elegans tubulogenesis RNAi screen was designed and carried out as previously described, using animals carrying an erm-1::gfp transgene, outlining the lumens of the intestine, the excretory canal and the gonad (Zhang et al., 2011). RNAi was performed by feeding (Timmons et al., 2001).Standard RNAi conditions (used in the screen) were defined as dsRNA induction by 2 mM IPTG. Mild RNAi conditions were empirically determined for specific genes after testing serial concentrations of IPTG and/or dilutions with mock RNAi bacteria: for chc-1, IPTG was titrated down to 2 nM; for aps-1, RNAi bacteria were diluted 1:10 with mock RNAi bacteria. For double RNAi, equal amounts of RNAi bacteria of two clones were mixed. RNAi initiated after completion of embryogenesis involved placing eggs or larvae on RNAi plates for evaluating the same generation. DsRed feedingchc-1(b1025ts) animals were fed on plates containing DsRed RNAi bacteria for at least 12 hours. The DsRed bacterial feeding strain contains a DsRed plasmid in HT115 bacteria that constitutively produces a faint red color. Phenotype reversalchc-1(b1025ts) mutant hermaphrodites were allowed to lay eggs for 1 hour (at 16°C) and subsequently removed. The plates with eggs were transferred to 22°C for 5 hours, then returned to 16°C. Animals were singled the next day and phenotype development and reversal were observed for 6 days. Lipid labeling and assessment of vesicle associationFor lipid labeling, 150 l E. coli OP50 or HT115 were spiked with 2 l 5 mM labeled lipid stock solutions (NBD-C6-glucosylceramide stock was 100 M), for a feeding period of ~8 hours. The same amounts were used f...