Our goal is to identify the cellular elements responsible for mediating Ca++- triggered exocytosis. As an experimental system, we have chosen to use a cortex fraction derived from the sea urchin egg. When bathed in micromolar concentrations of free Ca++, egg cortex preparations have been shown to undergo an in vitro reaction that morphologically and biochemically approximates exocytosis. There is no other in vitro model for exocytosis that shares this essential feature. We have devised sensitive biochemical assays for this reaction and have identified a number of potentially useful inhibitors. Recently, we have developed a reconstituted exocytotic system based on the recombination of purified cortical secretory vesicle (CV) and egg plasma membrane (PM) fractions. Although we have not yet completed characterization of this preparation the evidence obtained, to date, strongly suggests that we have succeeded in reconstituting cortical exocytosis. We plan to continue our characterization: 1) by utilizing both electron microscopy and an immunofluorescent protection assay to demonstrate vectorial transport of CV contents across the egg plasma membrane, and 2) by utilizing a binding assay, based on the quantitation of a CV enzyme, to analyze the specificity of CV binding to the plasma membrane. We propose to use our reconstitution technology to investigate the proteins required for exocytosis and those that comprise the CV-plasma membrane junction. Two general approaches will be followed: 1) a biochemical approach in which we will a) test the ability of cytoskeletal components to enhance CV binding, b) attempt to inhibit CV binding with cytoskeletal toxins, and c) attempt to subfractionate, then reconstitute, the binding activity of the CV and PM fractions themselves; and 2) an immunological approach in which polyclonal and monoclonal antibodies (raised to cytoskeletal proteins, and to the CV and PM fractions) will be used to inhibit CV binding and/or exocytosis. Although we will utilize sea urchin eggs for this study, the fundamental similarity of Ca++-triggered exocytosis in all species suggests that it will be possible to extrapolate the results obtained to higher species. Elucidation of the molecular mechanism of exocytosis will be invaluable in the diagnosis and treatment of secretory dysfunction.
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