Project Summary Cytokinesis is essential for cell proliferation, cell differentiation, and tissue homeostasis. Most events and proteins of cytokinesis are conserved from yeast to humans. Majority of the proteins involved in cytokinesis have been identified, however, the mechanisms of actomyosin contractile-ring constriction, plasma-membrane expansion in the cleavage furrow, and extracellular matrix remodeling are still poorly understood; nor do we understand how cells coordinate these events for successful cell division. Here we propose to continue elucidating the molecular mechanisms of cytokinesis using fission yeast as a model system. Previous studies and our solid preliminary data led to the central hypothesis of this proposal that coordination between exocytosis and endocytosis is essential for successful plasma membrane deposition and septum formation during cytokinesis, and septins and Ync13 are among the crucial coordinators. We will use complementary genetic, cellular, microscopic (confocal, TIRFM, electron microscopy, electron tomography, and super-resolution), biochemical, structural, and computational approaches to test this hypothesis by investigating three specific aims: 1) Elucidate how septins define the sites of vesicle tethering by the exocyst and how they affect the sites of endocytosis; 2) Characterize how Ync13 coordinates exocytosis and endocytosis during cytokinesis; 3) Investigate molecular mechanisms of trafficking, anchoring, and regulation of glucan synthases during cytokinesis. Our studies on the relationships between septins, the exocyst, Munc13/UNC-13 protein Ync13, F- BAR protein Rga7, coiled-coil protein Rng10, and glucan synthases will provide molecular links among the main cytokinesis events. Our proposed studies are significant because they will advance the understanding of cytokinesis in three important ways: a) What are the roles of septins in exocytosis and endocytosis during cytokinesis; b) how Ync13 coordinates exocytosis and endocytosis for successful cytokinesis; c) how septum synthases are trafficked and regulated. The concepts learned from this project will be applicable to understand the coordination of septins, the exocyst, plasma-membrane deposition, and extracellular matrix remodeling in human cells because our three specific aims involve the most conserved aspects of cytokinesis. Because the fungal specific essential enzymes such as glucan synthases, which build the septum during cytokinesis, are targets of several antifungals, our studies on the regulators of these synthases proposed here may lead to novel targets for antifungal drugs. Thus, our discoveries on both conserved and fungal-specific aspects of cytokinesis may be harnessed to improve human health.