Unconventional protein secretion (UPS) pathways are conserved across species. However, the underlying mechanisms that regulate Golgi-bypassing UPS of integral proteins remain elusive. In this study, we show that RAB-8 and SMGL-1/NBAS are required for the UPS of integral proteins in C. elegans intestine. SMGL-1 resides in the ER-Golgi intermediate compartment and adjacent RAB-8-positive structures, and NRZ complex component CZW-1/ZW10 is required for this residency. Notably, SMGL-1 acts as a guanine nucleotide exchange factor for RAB-8, ensuring UPS of integral proteins by driving the activation of RAB-8. Furthermore, we show that Pseudomonas aeruginosa infection elevated the expression of SMGL-1 and RAB-8. Loss of SMGL-1 or RAB-8 compromised resistance to environmental colchicine, arsenite, and pathogenic bacteria. These results suggest that the SMGL-1/RAB-8-mediated UPS could integrate environmental signals to serve as a host defense response. Together, by establishing the C. elegans intestine as a multicellular model, our findings provide insights into RAB-8-dependent Golgi-bypassing UPS, especially in the context of epithelia in vivo.

The ACK family tyrosine kinase SID-3 is involved in the endocytic uptake of double-stranded RNA. Here we identified SID-3 as a previously unappreciated recycling regulator in the Caenorhabditis elegans intestine. The RAB-10 effector EHBP-1 is required for the endosomal localization of SID-3. Accordingly, animals with loss of SID-3 phenocopied the recycling defects observed in ehbp-1 and rab-10 single mutants. Moreover, we detected sequential protein interactions between EHBP-1, SID-3, NCK-1, and DYN-1. In the absence of SID-3, DYN-1 failed to localize at tubular recycling endosomes, and membrane tubules breaking away from endosomes were mostly absent, suggesting that SID-3 acts synergistically with the downstream DYN-1 to promote endosomal tubule fission. In agreement with these observations, overexpression of DYN-1 significantly increased recycling transport in SID-3deficient cells. Finally, we noticed that loss of RAB-10 or EHBP-1 compromised feeding RNAi efficiency in multiple tissues, implicating basolateral recycling in the transport of RNA silencing signals. Taken together, our study demonstrated that in C. elegans intestinal epithelia, SID-3 acts downstream of EHBP-1 to direct fission of recycling endosomal tubules in concert with NCK-1 and DYN-1.

Cargo sorting and the subsequent membrane carrier formation require a properly organized endosomal actin network. To better understand the actin dynamics during endocytic recycling, we performed a genetic screen in C. elegans and identified RTKN-1/Rhotekin as a requisite to sustain endosome-associated actin integrity. Loss of RTKN-1 led to a prominent decrease in actin structures and basolateral recycling defects. Furthermore, we showed that the presence of RTKN-1 thwarts the actin disassembly competence of UNC-60A/cofilin. Consistently, in RTKN-1–deficient cells, UNC-60A knockdown replenished actin structures and alleviated the recycling defects. Notably, an intramolecular interaction within RTKN-1 could mediate the formation of oligomers. Overexpression of an RTKN-1 mutant form that lacks self-binding capacity failed to restore actin structures and recycling flow in rtkn-1 mutants. Finally, we demonstrated that SDPN-1/Syndapin acts to direct the recycling endosomal dwelling of RTKN-1 and promotes actin integrity there. Taken together, these findings consolidated the role of SDPN-1 in organizing the endosomal actin network architecture and introduced RTKN-1 as a novel regulatory protein involved in this process.

There has been a consensus that actin plays an important role in scission of the clathrin-coated pits (CCPs) together with large GTPases of the dynamin family in metazoan cells. However, the recruitment, regulation and functional interdependence of actin and dynamin during this process remain inadequately understood. Here, based on small-scale screening and in vivo live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism Caenorhabditis elegans. We found that loss of Wiskott−Aldrich syndrome protein (WASP)-interacting protein (WIP-1) impaired CCP scission in a manner that is independent of the C. elegans homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin. Moreover, the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1 (also known in other organisms as Abp1), another actin-binding protein. We demonstrate that the interaction between DBN-1 and F-actin is essential for Dynamin-1 (DYN-1) recruitment at endocytic sites. In addition, the recycling regulator RME-1, a homolog of mammalian Eps15 homology (EH) domain-containing proteins, is increasingly recruited at the arrested endocytic intermediates induced by F-actin loss or DYN-1 inactivation, which further stabilizes the tubular endocytic intermediates. Our study provides new insights into the molecular network underlying F-actin participation in the scission of CCPs. This article has an associated First Person interview with the first author of the paper.

Highlights d Loss of LET-502/ROCK affects endocytic recycling in C. elegans intestine d LET-502 interacts with RAB-5(GDP) and promotes RABX-5 GEF activity toward RAB-5 d RABX-5 and RME-6 activate RAB-5 in spatially distinct endosome subpopulations d LET-502-RABX-5 replenishes RAB-5 in a deep cytosolic subset of endosomes