Components And Kinetics In Exocytosis

胞吐作用的组成和动力学

• 批准号: 6813720
• 负责人: JOSHUA 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/6813720
• 关键词: calcium flux; clathrin; endocytosis; exocytosis; intracellular transport; membrane fusion; membrane proteins; protein protein interaction; protein structure function; protein transport; syntaxin

This project is centered on the mechanisms of calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs. We have also proposed a new model for the cycle of exocytosis and endocytosis that can explain the regulation of transient fusion during exocytosis. With non-structural curving proteins such as epsin, which can change the spontaneous curvature (Js) of a membrane by insertion, it is possible to imagine a scenario by which cells regulate the activity of these curving proteins to effect local budding and flattening of membranes in endocytosis and exocytosis. For example, PIP2 accumulation may lead to epsin binding, thus increasing Js and the free energy of the patch of membrane (E). The lipids respond by budding into a vesicle with the same actual curvature (J), relieving the bending stress. Clathrin, which binds to epsin, can then polymerize into a cage around the vesicle, perhaps displacing epsin (which may remain at the neck to facilitate pinching off). When the vesicle is completely detached, it uncoats. Once epsin and clathrin are gone, the spontaneous curvature of the bilayer returns to 0, but as the membrane is actually curved, stress returns and the vesicle becomes metastable (E=500 kBT) This provides a stressed vesicle having a driving force for flattening out during exocytosis. For any vesicle about to fuse, by regulating the spontaneous curvature of the membrane, through proteins such as epsin, cells can regulate the degree to which fusion pores open and remain small, or repeatedly open and close "kiss and run" rather than flattening out in complete fusion.

这个项目是集中在钙触发的胞吐作用的机制，普遍存在的真核过程中，囊泡融合到质膜和释放其内容物。SNARE如VAMP、SNAP-25和syntaxin对于细胞内运输是必不可少的，但它们的确切分子作用是什么，它们与其他蛋白质的相互作用是如何表现的？利用SNARE对外源蛋白酶的不同敏感性，我们定量了从天然分泌囊泡中选择性去除鉴定的SNARE而不丧失融合能力。使用先前建立的融合试验和高灵敏度的免疫印迹协议，我们分析了这些陷阱蛋白和Ca 2+触发的膜融合之间的关系。无论是融合的程度，也不是每个囊泡的膜间融合复合物的数量与所测得的密度确定的蛋皮质囊泡（CV）SNARE。在没有突触融合蛋白的情况下，CV保持融合能力。令人惊讶的是，对于一种（而不是另一种）蛋白酶，融合的Ca 2+依赖性与CV SNARE密度相关，表明与SNARE相关的天然蛋白质复合物，其结构确保高Ca 2+敏感性。由于SNARE可能在体内CV对接期间起作用，并且由于SNARE去除后进一步的蛋白水解最终消融融合，我们假设调节融合的触发步骤（Ca 2+敏感性和融合的催化和执行）需要在SNARE下游起作用的额外蛋白质。 我们还提出了一个新的胞吐和胞吞循环模型，可以解释胞吐过程中瞬时融合的调节。对于非结构性弯曲蛋白质，如胰蛋白酶，它可以通过插入改变膜的自发曲率（Js），可以想象这样一种情况，即细胞调节这些弯曲蛋白质的活性，以实现内吞和胞吐中膜的局部出芽和扁平化。例如，PIP 2积累可导致胰蛋白酶结合，从而增加Js和膜片的自由能（E）。脂质的反应是出芽形成具有相同实际曲率（J）的囊泡，从而减轻弯曲应力。与胰蛋白酶结合的网格蛋白可以在囊泡周围形成一个笼子，可能取代胰蛋白酶（胰蛋白酶可能留在颈部以便于夹断）。当囊泡完全脱落时，它就脱去了外壳。一旦胰蛋白酶和网格蛋白消失，双层的自发曲率恢复为0，但由于膜实际上是弯曲的，应力恢复，囊泡变得亚稳态（E=500 kBT）。对于任何即将融合的囊泡，通过调节膜的自发曲率，通过蛋白质如胰蛋白酶，细胞可以调节融合孔打开和保持小的程度，或者反复打开和关闭“吻和运行”，而不是在完全融合中变平。