Cortical granules are unique to oocytes and secrete their contents at fertilization to form a permanent block to polyspermy. Our long-term goal is to understand conserved mechanisms of fertilization from a perspective of the functional contribution of cortical granules. Our oocyte of choice for this goal is the sea urchin, where approximately 15,000 cortical granules are synchronous in biogenesis, translocation to the surface, docking to the plasma membrane, and secretion at fertilization. We will make use of the sea urchin oocyte for 1) the millions available from each female; 2) the cDNA clones in hand that encode proteins specific to the contents and membranes of cortical granules; 3) the ability to isolate cortical granules and reconstitute function; and 4) the amenable culture and maturation of oocytes in vitro and direct visualization of cortical granules. Here we propose three major goals: 1. Determine the functional contribution of the cortical granule protease to fertilization. This protease cleaves plasma membrane proteins of the egg, modifies the egg extracellular matrix, and alters other cortical granule content proteins. We will identify the proteolytic targets, enabling us to identify and functionally characterize proteins important for fertilization. 2. Biogenesis and translocation of cortical granules. We will dissect the mechanisms used by cortical granules in vivo to get to the cell surface (translocation), to make a near perfect monolayer of cortical granules at the plasma membrane, and the function of cortical granule membrane proteins in this regulation. 3. Identify the molecular basis for regulated exocytosis at fertilization. We will exploit the pre-docked status of cortical granules in this egg to identify proteins that regulate their exocytosis. We will focus on proteins that interact with SNARE homologues and of rab 3, and address both the mechanism for stimulating exocytosis, as well as the molecular clamp that blocks exocytosis until fertilization.
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