COMPONENTS AND KINETICS IN EXOCYTOSIS

胞吐作用的组成部分和动力学

• 批准号: 2575691
• 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/2575691
• 关键词: calcium; cell membrane; egg /ovum; exocytosis; granule; membrane fusion; sea urchins

We have continued our research on membrane fusion, the fundamental step in secretion, viral infection, fertilization and neuro-transmission. Sea urchin egg cortical granule exocytosis, an example of calcium triggered membrane fusion, has served as a model system. We have been investigating how certain proteins (NSF et al.) might regulate the exocytosis of the egg's cortical granules. We have established that cytosol is unable to restore fusion to the isolated exocytotic machinery inactivated by NEM. We have found using immunoblot analysis that sea urchin eggs contain NSF in their cytosol but that this protein is absent from an in vitro preparation which undergoes fusion upon the provision of Ca2+. However, a stable and specific interaction between NSF and the isolated exocytotic machinery can occur under the appropriate conditions. These results suggest that the role of NSF might be in the establishment of a fusion-competent arrangement rather than in membrane fusion per se: a novel, NSF-independent mechanism of fusion. The relative roles of inactivation and heterogeneity were evaluated further in the isolated planar cortex from the sea urchin eggs using multiple solution exchange protocols and perfusion at different rates. No evidence was found to support the hypothesis that either rate-dependent or rate-independent inactivation is responsible for the cessation of fusion at calcium concentrations which produced sub-maximal responses in cortical degranulation. Calcium-triggered exocytosis was heterogeneous: exocytotic responses were sub-maximal at certain calcium concentrations because all the granules capable of fusing at those concentrations of calcium had fused. Secretory models featuring calcium-dependent inactivation are inappropriate for describing cortical degranulation in the sea urchin egg. The minimum number of calcium ions involved in the fusion process was estimated by considering the behavior of a single, step-like transition at a threshold calcium concentration. This analysis suggests that more than four calcium ions are involved in triggering exocytosis. Our model, which describes both the kinetic and steady-state features observed in sea urchin cortical granule exocytosis, has been extended with the addition of a third parameter representing the activation time for fusion complexes. Discrepancies between the model and the initial time course of exocytosis have been eliminated with this modification.

我们继续研究膜融合，这是基本步骤 分泌、病毒感染、受精和神经传递。 海 海胆卵皮质颗粒胞吐作用，钙触发的一个例子 膜融合已作为模型系统。 我们曾经 研究某些蛋白质（NSF 等）如何调节 卵皮质颗粒的胞吐作用。 我们已经确定 胞质溶胶无法恢复与孤立的胞吐机制的融合 被 NEM 灭活。我们通过免疫印迹分析发现，海 海胆卵的细胞质中含有 NSF，但这种蛋白质不存在 来自在提供后进行融合的体外制剂 Ca2+。 然而，NSF 与 在适当的条件下可以发生孤立的胞吐机制。 这些结果表明 NSF 的作用可能在于建立 具有融合能力的排列而不是膜融合本身： 一种新颖的、独立于 NSF 的融合机制。 评估失活和异质性的相对作用 进一步在从海胆卵中分离出的平面皮层中使用 多种溶液交换方案和不同速率的灌注。 没有发现证据支持以下假设： 速率依赖性或速率无关的失活是造成 在产生次最大钙浓度时融合停止 皮质脱粒反应。 钙触发的胞吐作用是 异质性：胞吐反应在某些钙条件下低于最大 浓度，因为所有颗粒都能够在这些浓度下融合 钙浓度已融合。 分泌模型具有 钙依赖性失活不适合描述皮质 海胆卵脱粒。钙离子最少数量 通过考虑行为来估计融合过程中所涉及的 在阈值钙浓度下的单一阶梯状转变。 该分析表明超过四个钙离子参与 触发胞吐作用。 我们的模型描述了动力学和 在海胆皮质颗粒胞吐作用中观察到的稳态特征， 已通过添加代表的第三个参数进行了扩展 融合复合物的激活时间。 模型之间的差异 并且胞吐作用的初始时间过程已被消除 修改。