Cells and tissues establish and maintain their unique architectures in large part through the tight regulation of protein and membrane transport. One key aspect of this process is endocytic recycling, the selective return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling is of fundamental importance to cell biology and has broad relevance to many areas of biomedicine. Endocytic recycling is particularly critical to the maintenance of cell polarity, a defining and essential feature of epithelial tissues. Our general approach has been to exploit powerful features of C. elegans genetics to characterize proteins that are required for the recycling process in vivo. During the previous granting period we gained new understanding of how RME-1/Ehd1 family proteins, identified in our previous screens, function in recycling. We also identified new proteins (ALX-1/Alix, SDPN-1/Syndapin, and ARF-6/Arf6) that function with RME-1 in this process. We went on to establish the C. elegans intestine as a model for elucidating endocytic membrane transport pathways in polarized epithelia, and showed for the first time that the small GTPase RAB-10/Rab10 is required for basolateral recycling in the worm intestine and polarized mammalian MDCK cells. To gain mechanistic insight into how RAB-10 drives recycling we identified five proteins that specifically interact with RAB-10 in the active, GTP-bound, conformation. These new proteins are likely RAB-10 effectors, directly mediating RAB-10 driven transport. We propose three new aims to further elucidate the molecular mechanisms underlying endocytic recycling. First we propose to test key predictions of the hypothesis that our newly identified RAB-10 binding proteins are RAB-10 effectors. This will be accomplished by better defining their physical interactions with RAB-10, determining the effects on recycling when each candidate effector is knocked out, and testing the ability of engineered interaction defective forms of these proteins to rescue recycling defects in knockout animals. Second we propose to test a model for C. elegans orthologs of Alix and Syndapin, that we developed during the prior granting period, suggesting that they control recycling through the recruitment and activation of actin regulators on endosomes. Finally we propose to leverage a newly isolated knockout for the only C. elegans Arf6 ortholog, to test the role of this key GTPase in endocytic transport in polarized epithelia, and to understand how it is regulated in this complex environment. The experiments proposed here should provide significant new insights into how endocytic recycling works. Given the high level of phylogenetic conservation of such pathways from worms to mammals, our work should provide extensive predictive insight into equivalent pathways in human cells. Our research focuses on the molecular mechanisms controlling endocytic recycling - the return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling is of fundamental importance to many areas of biomedicine. For instance, endocytic recycling is a key control point in the insulin- stimulated movement of glucose transporters (Glut4) from endosomes to the plasma membrane of adipose and muscle cells. Failure in this recycling event is thought to be a major cause of type II diabetes, a disease that has recently reached epidemic proportions in the United States. A better understanding of how endocytic recycling functions will be profoundly important in identifying therapeutic targets to combat this and other diseases.
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