We have used components of the sea urchin egg cortical reaction to study the mechanism of calcium triggered exocytosis. Using reversible inhibitors of membrane fusion we have shown that the molecular steps which respond to free calcium, and are involved in triggering membrane fusion, are distinct from subsequent steps that are involved in the actual membrane merger. Studies of the calcium dependence of exocytosis under conditions where we progressively destroy the fusion machinery with mild NEM treatment (under conditions where we still achieve 100% vesicle fusion) show a reduction in the rate of fusion as well as an increase in the concentration of calcium where we observe the onset of fusion. This is consistent with predictions based on a computer simulation which we have written to model the calcium dependence of exocytosis as a function of the number and apparent Kd of fusion proteins per granule. Our finding that isolated cortical granules can fuse with liposomes, argues that the proteins mediating membrane fusion reside on the exocytotic granules and do not require ancillary proteins on the plasma membrane for fusion. Comparative inhibition of exocytosis in vitro and in vivo reveal that short wavelength UV light inhibits exocytosis at steps prior and subsequent to membrane fusion and suggest that the UV inhibition prior to fusion can be reversed by components present in the intact egg. In most cells exocytosis is followed by endocytosis to maintain membrane homeostasis. We have developed assays to measure endocytosis in sea urchin eggs so that we can study the coupling between exocytosis and endocytosis. Our preliminary studies show that endocytosis can be triggered by sperm or calcium ionophores, and inhibition of exocytotic granule fusion forestalls endocytosis.
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