EXOCYTOTIC MEMBRANE FUSION

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We have developed simple light scattering assays and fluorescent dye transfer assays to follow membrane fusion reactions. Using these assays we have studied the localization of the molecular machinery that mediates calcium-triggered exocytosis in sea urchin eggs. We find that isolated exocytic vesicles are capable of fusing with each other in response to micromolar concentrations of calcium. This reaction is specific for calcium, 10 mM magnesium does not support fusion. Vesicle-vesicle fusion does not require any added cytoplasmic proteins or organic factors. In addition we find that calcium-triggered fusion can be inhibited by pre-treatment of the vesicles with either trypsin or NEM. Thus we believe that at least part of the fusion mechanism is comprised of proteins, and that these proteins must reside on the vesicles themselves. We have also begun to measure the speed of the fusion reaction of sea urchin cortical granules with the egg plasma membrane and find that we can observe fusion within 10 msec of presenting a calcium trigger. In addition we find that the granule-plasma membrane fusion reaction can be inhibited by either polyoxyethylene sorbitan monooleate or polyoxyethylene sorbitan monolaurate but not by sorbitan monooleate suggesting that polyoxyethylene can interact with a hydrophilic site, near the surface of the exocytic vesicle to inhibit fusion. Finally we find that lysophosphatidylcholine but not phosphatidylcholine or oleic acid can inhibit calcium triggered fusion in isolated planar cortices. This inhibition, like that of tween detergents, is reversible.