COMPONENTS AND KINETICS IN EXOCYTOSIS

我们继续研究膜融合， 分泌、病毒感染、受精和神经传递。 海 海胆卵皮质颗粒胞吐，钙触发的一个例子 膜融合，已经作为一个模型系统。 我们一直 研究某些蛋白质（NSF等人）可能会调节 卵皮质颗粒的胞吐作用。 我们确定 胞质溶胶不能恢复与分离的胞吐机制的融合 由NEM灭活。我们通过免疫印迹分析发现， 海胆卵的胞质中含有NSF，但这种蛋白质不存在 从一种体外制备物中， 的Ca2+。 然而，NSF和细胞之间的稳定和特异性相互作用 分离的胞吐机制可以在适当的条件下发生。 这些结果表明，NSF的作用可能是在建立 一个融合能力的安排，而不是在膜融合本身： 一种新的，不依赖于NSF的融合机制。 评估了失活和异质性的相对作用 进一步在来自海胆卵的分离的平面皮质中， 多种溶液交换方案和不同速率的灌注。 没有发现证据支持这一假设， 速率依赖性或非速率依赖性失活是导致 在产生次最大值的钙浓度下停止融合 皮质脱颗粒反应。 钙触发的胞吐作用是 异质性：在一定的钙浓度下，胞吐反应为次最大 浓度，因为所有能够在这些浓度下融合的颗粒 钙离子浓度已经融合。 秘密模特 钙依赖性失活不适合描述皮质 海胆卵的脱粒过程钙离子的最小数量 参与融合过程的估计，考虑到行为 一个单一的，阶梯状的过渡在阈值钙浓度。 这一分析表明，超过四个钙离子参与了 引发胞吐作用 我们的模型，它描述了动力学和 在海胆皮质颗粒胞吐中观察到的稳态特征， 已扩展，添加了第三个参数，表示 融合复合物的活化时间。 模型之间的差异 和胞吐作用的初始时间过程都被消除了 改性

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.