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...

The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of non-proteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes – of these compounds- findings that are usually dismissed, as these typically synthesize multiple products. Here, we provide a method using follow-on biosynthetic-pathway screens to identify the endpoint biosynthetic enzyme and thus the compound through which they act. The approach is based on the principle that all subsequently identified downstream biosynthetic enzymes contribute to the synthesis of at least this one end product. We describe how to: systematically target lipid biosynthetic pathways; optimize targeting conditions; take advantage of pathway branchpoints; and validate results by genetic assays and biochemical analyses. This approach extends the power of unbiased genetic/RNAi screens to identify in vivo functions of non-nucleic-acid-based metabolites beyond their metabolic roles.

Biological tubes consist of polarized epithelial cells with apical membranes building the central lumen and basolateral membranes contacting adjacent cells or the extracellular matrix. Cellular polarity requires distinct inputs from outside the cell, e.g., the matrix, inside the cell, e.g., vesicular trafficking and the plasma membrane and its junctions. 1Many highly conserved polarity cues have been identified, but their integration during the complex process of polarized tissue and organ morphogenesis is not well understood. It is assumed that plasmamembrane-associated polarity determinants, such as the partitioning-defective (PAR) complex, define plasma membrane domain identities, whereas vesicular trafficking delivers membrane components to these domains, but lacks the ability to define them. In vitro studies on lumenal membrane biogenesis in mammalian cell lines now indicate that trafficking could contribute to defining membrane domains by targeting the polarity determinants, e.g., the PARs, themselves. 2This possibility suggests a mechanism for PARs' asymmetric distribution on membranes and places vesicle-associated polarity cues upstream of membraneassociated polarity determinants. In such an upstream position, trafficking might even direct multiple membrane components, not only polarity determinants, an original concept of polarized plasma membrane biogenesis 3,4 that was largely abandoned due to the failure to identify a molecularly defined intrinsic vesicular sorting mechanism. Our two recent studies on C. elegans intestinal tubulogenesis reveal that glycosphingolipids (GSLs) and Vesicular sorting controls the polarity of expanding membranes in the C. elegans intestine Hongjie Zhang, Ahlee Kim, Nessy Abraham, Liakot A. Khan and Verena Göbel* Department of Pediatrics; Massachusetts General Hospital; Harvard Medical School; Boston, MA USA the well-recognized vesicle components clathrin and its AP-1 adaptor are required for targeting multiple apical molecules, including polarity regulators, to the expanding apical/lumenal membrane. 5,6These findings support GSLs' long-proposed role in in vivo polarized epithelial membrane biogenesis and development and identify a novel function in apical polarity for classical post-Golgi vesicle components. They are also compatible with a vesicle-intrinsic sorting mechanism during membrane biogenesis and suggest a model for how vesicles could acquire apical directionality during the assembly of the functionally critical polarized lumenal surfaces of epithelial tubes.

Reference 48 was omitted in the Methods section. This reference should be cited as follows: "GST-MPS1 (ref. 48) was expressed in E. coli BL21 (DE3)plysS cells", under the heading 'Recombinant production, in vitro phosphorylation and phospho-mapping of Cnn1' . The original reference 48 has been re-numbered as reference 49, and the new reference 48 is as follows:

In the version of this article initially published, the text detailing the HPLC elution protocol in the Equipment Setup section (a 2-min column pre-equilibration in 9:1 A:B (vol/vol); sample injection; 2-min wash with 100% A; 14-min linear gradient to 100% B; and 15-min post-run column equilibration) was incorrect and has been changed to read: a 2-min column preequilibration in 9:1 A:B (vol/vol); sample injection; 2-min wash with 100% A; 12-min linear gradient to 100% B; and 1-min post-run column equilibration. The error has been corrected in the HTML and PDF versions of the article.