PROJECT SUMMARY: Recycling endosomes, organelles intimately involved in the return of proteins and lipids to the plasma membrane after their endocytosis, are of fundamental importance to plasma membrane turnover and the control of plasma membrane composition. Advancing mechanistic understanding of recycling endosome formation and function thus holds broad relevance to many areas of biomedicine. The mechanisms that regulate recycling endosome biogenesis and function remain relatively poorly understood, lagging well behind our understanding of other trafficking pathways. Our studies take advantage of the enormous technical advantages in the simple animal model C. elegans that yield mechanistic insight into cell biological processes within the physiologically relevant context of an intact metazoan animal. We then extend these find ings to mammalian cells to expand understanding and define phylogenetic conservation. We focus on a system that we pioneered, the C. e/egans intestine, a simple model that allows facile analysis of endocytic membrane transport pathways within intact polarized epithelia. There is no cell division or cell replacement in the intestine after embryogenesis, further simplifying analysis of mutant phenotypes. During the previous granting period we greatly expanded our understanding of the essential endosomal regulator RME-8/DNAJC13. Our work elucidated autoregulatory mechanisms controlling the RME-8 protein that allow RME-8 to regulate the dynamics of endosomal coats and maintain separation of endosomal microdomains with conflicting functions. We also showed that RME-8 is required for autophagic lysosome reformation in C. elegans and mouse neurons, relevant to links between RME-8 to Parkinsonism. Our new studies during the last funding period also focused our attention on fundamental forces that drive endosomal fission, particularly of recycling tubules that must be severed from sorting endosomes to form recycling endosomes. Actin polymerization on endosomes has long been proposed to provide necessary membrane tension to promote fission, but a clear understanding of how actin contributes to tension, and how fission is regulated remain as large gaps in understanding that require more in-depth study. Our studies provided surprising new evidence that non-muscle myosin II (NMII) regulates endocytic recycling. We further discovered an endosomal signaling hub centered on the Syndapin protein that controls endosomal RhoA and actomyosin. Here we propose extensive studies in C. elegans to better define how the Syndapin pathway controls NMII and fission, extend this analysis into mammalian cells, and determine the role of specific NMII isoforms in the fission process. We also identified a connection of Syndapin-based RhoA-regulation that indicates signaling from endosomes to the nucleus for long-term regulation of recycling. Our studies will test key predictions of these models and gain new understanding of mechanisms that integrate acute ...