EXOCYTOTIC MEMBRANE FUSION

我们已经开发了简单的光散射分析和荧光染料 转移法跟踪膜融合反应。使用这些化验方法，我们 研究了调节的分子结构的局部化 钙引发海胆卵的胞吐作用。我们发现与世隔绝 胞外囊泡能够相互融合以响应 钙的微摩尔浓度。这种反应是特定于 钙、10毫米镁不支持熔合。囊泡-囊泡融合 不需要添加任何细胞质蛋白质或有机因子。在……里面 此外，我们发现钙触发的融合可以被抑制 用胰酶或NEM对囊泡进行预处理。因此，我们相信 融合机制的至少一部分是由蛋白质组成的， 这些蛋白质必须存在于小泡本身。我们还有 开始测量海胆皮质融合反应的速度 颗粒与卵细胞质膜结合后发现可以观察到融合 在出现钙质触发器后的10毫秒内。另外，我们发现， 颗粒-质膜融合反应可被任何一种药物抑制 聚氧乙烯山梨醇单油酸酯或聚氧乙烯山梨醇单月桂酸酯 但不是通过山梨醇单油酸盐，这表明聚氧乙烯可以相互作用 具有亲水性的部位，靠近胞外小泡的表面以 抑制核聚变。最后我们发现溶血磷脂酰胆碱 磷脂酰胆碱或油酸可抑制钙触发的脑融合 孤立的平面皮质点。这种抑制作用，就像青少年洗涤剂一样， 是可逆的。

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.