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. Recycling endosomes also contribute to macroautophagy, the process of cellular self-eating required for cell survival under adverse conditions. Thus understanding the endocytic recycling process and its role in cellular physiology is of fundamental importance to cell biology and has broad relevance to many areas of biomedicine including cancer and type II diabetes. 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. We then extend these findings to mammalian cells and to in vitro analysis of the relevant C. elegans proteins and their mammalian homologs. For many of these studies we focused on a system that we pioneered, the C. elegans intestine, a very simple model that allows facile analysis of endocytic membrane transport pathways within intact polarized epithelia. During the previous granting period we gained new understanding of how the early acting Rab GTPase RAB-5 is down regulated by its GAP TBC-2. We found that three recycling regulators directly interact with TBC-2 and contribute to TBC-2 endosomal recruitment. These include RAB-10, a key GTPase acting after RAB-5 in endocytic recycling; the Rho GTPase CED-10/Rac1 that we also show regulates recycling, and the F-BAR protein AMPH-1/ amphiphysin. This novel pathway represents an inhibitory cascade required to terminate the activity of RAB-5 as cargo transitions from early to recycling endosomes. We also elucidated molecular links of another RAB-10 effector EHBP-1 to membranes and the cytoskeleton, regulating the tubular architecture of endosomes. In new preliminary work we have identified key new links of the RAB-10 GTPase to the exocyst membrane tethering complex and propose to test mechanistic models for how RAB-10 influences exocyst during recycling and autophagy. We further identify another key recycling regulator, F-BAR protein SDPN-1/ Syndapin, and identify new SDPN-1 interaction partners important for recycling function. We propose extensive new in vivo and in vitro analysis to define how Syndapin family protein functions on endosomes. Given the high level of phylogenetic conservation of such pathways from worms to mammals, and our parallel analysis in human cells, we expect that our work will provide extensive insight into key conserved elements relevant across species. This work is important for understanding disease etiology and in identifying therapeutic targets for disease intervention.
Our proposed research focuses on the return of internalized macromolecules to the cell surface from endosomes (endocytic recycling), and autophagy (cellular self-eating), basic cell biological processes that are of fundamental importance to many areas of biomedicine. Endocytic recycling is a key control point in the insulin-stimulated movement of glucose transporters (Glut4) from endosomes to the plasma membrane, a process that goes awry in type II diabetes. Autophagy is also important in type II diabetes, as well as cancer and aging.
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