An EHBP-1-SID-3-DYN-1 Axis Promotes Membranous Tubules Fission During Endocytic Recycling

Background: After endocytic uptake,recycling transport is deployed to deliver endocytosed macromolecules,fluid,membranes,and membrane proteins back to the cell surface,which is essential for a series of biological processes such as cytokinesis,cell migration,maintenance of cell polarity,and synaptic plasticity.Recycling endosomes consist of membrane tubules and vesicles.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 the membrane fission remains elusive.To identify additional recycling regulators,a large-scale RNAi genetic screen was performed using rab-10(RNAi)as the positive control to define candidate genes whose expression knockdown can lead to the hTAC-GFP overaccumulation phenotype.After the initial screen and the follow-up validation,RNAi-mediated knockdown of 54 candidates was found to lead to intracellular hTAC-GFP aggregation,including SID-3,WIP-1,DBN-1,HUM-2,and LET-313.Among them,ACK family tyrosine kinase SID-3 has been shown to be involved in the endocytic uptake of dsRNA.Objective: To study how SID-3 acts together with EHBP-1,NCK-1,and DYN-1 to regulate the membrane transport from early endosomes to recycling endosomes Methods: This project mainly utilizes cell biology and biochemical techniques,supplemented with genetic experiments to study the subject.Results: In C.elegans polarized intestinal cells,we recognized SID-3 as a previously unappreciated recycling regulator.RAB-10 effector EHBP-1 is required for the endosomal localization of SID-3.Accordingly,loss of SID-3 phenocopied the recycling defects observed in ehbp-1 or rab-10 mutants.Subsequently,we detected a protein interactive cascade among EHBP-1,SID-3,NCK-1,and DYN-1.In the absence of SID-3,DYN-1 failed to localize at the tubular recycling endosomes,and the membrane tubules break-away from endosomes was mostly absent,intimating that SID-3 synergizes with the downstream DYN-1 to promote endosomal tubules fission.In agreement with these observations,overexpression of DYN-1 significantly revived the 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,suggestive of the implication of basolateral recycling in the transport of RNA silencing signals.Conclusions: In C.elegans intestinal epithelia,there is a protein interactive cascade among EHBP-1,SID-3,NCK-1,and DYN-1.SID-3 acts downstream of EHBP-1 to direct recycling endosomal tubules fission in concert with NCK-1 and DYN-1.The dsRNA is internalized from the intestinal lumen via SID-2-mediated endocytosis.Basolateral recycling could be involved in the transport of RNA silencing signals,facilitating the release of apically endocytosed dsRNA into the body cavity.