EXOCYTOTIC MEMBRANE FUSION

胞吐膜融合

• 批准号: 3857165
• 负责人: J ZIMMERBERG
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3857165
• 关键词: calcium; cell membrane; egg /ovum; exocytosis; fluorescent dye /probe; measurement; membrane activity; membrane fusion; membrane lipids; nephelometry; sea urchins; technology /technique development

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.

我们已经开发了简单的光散射分析和荧光染料 转移测定以跟随膜融合反应。 使用这些测定， 已经研究了介导这种疾病的分子机制的定位， 海胆卵中钙离子引发的胞吐作用。 我们发现， 胞吐囊泡能够相互融合， 微摩尔浓度的钙。 该反应是特异性的， 钙，10 mM镁不支持融合。 囊泡-囊泡融合 不需要添加任何细胞质蛋白或有机因子。 在 此外，我们发现，钙触发的融合可以抑制 用胰蛋白酶或NEM预处理囊泡。 因此我们相信 至少部分融合机制由蛋白质组成，和 这些蛋白质必须存在于囊泡本身。 我们还 开始测量海胆皮层融合反应的速度 颗粒与卵质膜，发现我们可以观察到融合 在出现钙触发的10毫秒内 此外，我们发现， 颗粒-质膜融合反应可被以下任一种抑制 聚氧乙烯脱水山梨醇单油酸酯或聚氧乙烯脱水山梨醇单月桂酸酯 而不是脱水山梨糖醇单油酸酯，这表明聚氧乙烯可以相互作用 在胞吐囊泡表面附近有一个亲水位点， 抑制融合。 最后我们发现溶血磷脂酰胆碱， 磷脂酰胆碱或油酸可以抑制钙触发的融合， 孤立的平面皮质 这种抑制作用，就像吐温洗涤剂一样， 是可逆的。