An EHBP-1-SID-3-DYN-1 axis promotes membranous tubule fission during endocytic recycling.

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-3-deficient 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. Author summary After endocytic uptake, a recycling transport system is deployed to deliver endocytosed macromolecules, fluid, membranes, and membrane proteins back to the cell surface. This process is essential for a series of biological processes such as cytokinesis, cell migration, maintenance of cell polarity, and synaptic plasticity. Recycling endosomes mainly consist of membrane tubules and often undergo membrane fission to generate vesicular carriers, which mediates the delivery of cargo proteins back to the plasma membrane. Previous studies suggested that RAB-10 and its effector protein EHBP-1 function jointly to generate and maintain recycling endosomal tubules. However, the mechanism coupling recycling endosomal tubulation and membrane fission remains elusive. Here, we identified SID-3 as a new interactor of EHBP-1. EHBP-1 is required for the endosomal localization of SID-3 and initiates a protein interaction cascade involving SID-3, NCK-1, and DYN-1/dynamin. We also found that SID-3 functions downstream of EHBP-1 to encourage membrane scission, and that ectopic expression of DYN-1 improves recycling transport in SID-3-depleted cells. Our findings revealed EHBP-1 as a point of convergence between RAB-10-mediated endosomal tubulation and SID-3-assisted membrane tubule fission during endocytic recycling.