PROJECT SUMMARY/ABSTRACT The internalization and recycling of receptors is a key biological process in all eukaryotic cells. The early/sorting endosome is the initial destination of receptors internalized from the plasma membrane (PM). This endosome serves as a major sorting station from which receptors are shunted to late endosomes and lysosomes for degradation, or are recycled back to the PM through a transitory network of vesicular and tubular recycling endosomes. Whereas a decade ago most researchers thought that active sorting directed proteins to the degradation pathways, targeting to the recycling pathway was thought to be largely a passive process that occurs by default. However, recent evidence supports active sorting to the recycling pathways by specific sorting nexin (SNX) and other proteins that bind to the cytoplasmic tails of receptors and specifically target them for recycling. Although recycling is an essential process for all mammalian cells, its complex regulation is poorly understood including the sorting of receptors on endosomal membranes, the budding and fission of vesicles and tubules from the endosome, and the transport of receptors back to the PM. As such, our knowledge of endosomal function lags substantially behind that of receptor internalization mechanisms. A key group of regulatory proteins that controls sorting and trafficking at the endosome is the retromer complex. Originally identified in the retrieval of biosynthetic cargo from endosomes to the Golgi complex, the retromer has recently been implicated in the regulation of a variety of key cellular pathways both within and beyond the scope of endocytic trafficking including endocytic recycling, mitochondrial homeostasis, the centrosome cycle and ciliogenesis. The retromer complex also interacts with other key endocytic regulatory proteins, including the tubular endosome scaffold MICAL-L1; its interaction partner and endosomal fission modulator, EH domain containing 1 (EHD1); and a host of SNX proteins that mediate endosomal cargo sorting. The retromer also links to the actin cytoskeleton via the WASH complex. Our laboratory has been focusing on an overall understanding of the mechanisms by which endocytic regulatory proteins function both in endocytic pathways and in non-endocytic trafficking. Our primary expertise is in biochemistry and molecular cell biology coupled with advanced light microscopy, but we recognize the need to incorporate in vivo components into our approach and have ongoing collaborations with other groups to examine these processes in whole organisms, including zebrafish and worms. In our studies, we will address significant and as-yet-unresolved biological problems such as: 1) how endosomal fission is regulated and linked to sorting and recycling and 2) how key endocytic proteins mediate the biogenesis of the primary cilium.