Clathrin and AP-1 regulate apical polarity and lumen formation duringC. eleganstubulogenesis

Abstract

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...

AP-1 is required for the maintenance of apico-basal polarity in theC. elegansintestine

“…Several hypotheses can be proposed for AP-1 function in apical trafficking. First, AP-1 could be implicated solely in basolateral targeting followed by transcytosis of apical proteins; we do not favour this hypothesis because preventing endocytosis by targeting dynamin should allow us to see apical cargos at the basolateral membrane, something we did not observe [see also accompanying paper (Zhang et al, 2012)]. Alternatively, AP-1 could target a factor to the basolateral membrane, which would be required to exclude apical proteins; in the early embryo, the RhoGAP PAC-1 excludes CDC-42 and PAR-6 from cell-cell contact and its absence leads to a lateral localisation of these two proteins (Anderson et al, 2008).…”

Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells

“…We therefore propose that a SOAP-1/AP-1/clathrin module controls E-cad apical sorting in the epidermis. In the intestine, AP-1 is also required for apical sorting but triggers a very different phenotype in which the lateral membrane is converted into an apical membrane (Shafaq-Zadah et al, 2012;Zhang et al, 2012). Interestingly, we observed that E-cad is lateral in the intestine of larvae and not missorted upon AP-1 depletion, whereas the loss of soap-1 had no effect on the localisation of PAR-6 (Shafaq-Zadah et al, 2012) or CDC-42 (G.G.…”

“…The AP-1 functions identified in neurons (Dwyer et al, 2001) or in the intestine (Shafaq-Zadah et al, 2012;Zhang et al, 2012) cannot account for this lethality. At the developmental level, embryonic elongation requires seam cell elongation driven by actin tension in epidermal cells and functional muscles attached to the epidermis from twofold stage (Chisholm and Hardin, 2005).…”

“…The first observations that AP-1 and clathrin were implicated in apical sorting were made in the C. elegans intestine (Shafaq-Zadah et al, 2012;Zhang et al, 2012). To test the role of these factors on E-cad localisation in another epithelial tissue we carefully examined the apico-basal distribution of E-cad in the intestine.…”

Noncentrosomal microtubules in C. elegans epithelia

“…Second, the AP-1 clathrin adaptor (known as APS-1 in C. elegans) is required for actin organization and apical targeting of several proteins including the polarity factor PAR-6 and E-cadherin (Gillard et al, 2015;Shafaq-Zadah et al, 2012;Zhang et al, 2012). Third, loss of APS-1 induces ectopic mini-intestinal lumen also observed after the loss of the cytosolic chaperonin CCT-1 (also known as TCP-1), which affects both the actin and MT filaments (Saegusa et al, 2014;Shafaq-Zadah et al, 2012;Zhang et al, 2012).…”