BIOLOGY OF CORTICAL GRANULES

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Cortical granules are unique to oocytes and function during fertilization by secreting their contents to form a permanent block to polyspermy. We will study the biology of cortical granules with two major goals: l) to understand the mechanisms used by oocytes to regulate organelle biogenesis and protein storage; and 2) to understand their regulated secretion at fertilization. The oocyte of choice for these goals is from the sea urchin, where approximately 15,000 cortical granules are poised at the cell surface to exocytose in response to either sperm binding. It is the only oocyte for which l) cDNA clones have been isolated that encode content and membrane proteins specific to the cortical granules; 2) the cortical granules can be isolated in a functional form, and 3) in vitro culture, maturation and direct visualization of cortical granules are each possible. Cortical granules are different from most other secretory vesicles in that they remain docked at the cell surface for weeks, they are non recycling, and they contain over a dozen different proteins which are subcompartmentalized within the vesicle. Three specific aims are proposed: 1. We will identify determinants on the content proteins of cortical granule that are required for targeting into cortical granules. We will utilize cDNAs to the cortical granule specific proteins SFE 9, ovoperoxidase and hyalin in a protein reporter system to determine what minimal sequence is necessary for appropriate organelle targeting. Because the vesicle contains two morphologically and biochemically distinct regions, we will also examine the signals necessary for protein subcompartmentalization within the vesicle using proteins specifically compartmentalized in each region. 2. We will examine the mechanism and regulation of cortical granule translocation to the oocyte cell surface. We will determine the basis for selectivity of this process, i.e. selectivity for cortical granules, for their timing of translocation, and for the pathways used in organelle movement. In addition, we will examine the mechanism used to create a perfect monolayer of cortical granules at the oocyte cell surface. 3. We will identify the mechanism(s) of regulated secretion of cortical granules at fertilization. We will test the hypothesis that cortical granules contain a unique population of membrane proteins to direct the specialized biology of this vesicle by use of monoclonal antibodies against cortical granule membranes and by testing homologues of somatic cell proteins that are involved in regulated secretory function.