Components And Kinetics In Exocytosis

本项目主要研究钙触发胞吐作用的机制，这是一种普遍存在的真核生物过程，通过这种过程，囊泡融合到质膜上并释放其内容物。诸如VAMP、SNAP-25和syntaxin等SNAREs对于细胞内运输至关重要，但它们的确切分子作用是什么？它们如何与其他蛋白质相互作用？利用SNAREs对外源蛋白酶的不同敏感性，我们量化了从天然分泌囊泡中选择性去除鉴定的SNAREs而不丧失融合能力。使用先前建立的融合实验和高灵敏度免疫印迹法，我们分析了这些SNARE蛋白与Ca2+触发的膜融合之间的关系。每个囊泡的融合程度和膜间融合复合物的数量都与鉴定的卵皮质囊泡（CV） SNAREs的测量密度无关。没有syntaxin， CVs仍然具有融合能力。令人惊讶的是，对于一种（而不是另一种）蛋白酶，融合的Ca2+依赖性与CV SNARE密度相关，这表明与SNAREs相关的天然蛋白复合物，其结构确保了高Ca2+敏感性。由于SNAREs可能在体内CV对接过程中发挥作用，并且在去除SNARE后进一步的蛋白水解最终会破坏融合，因此我们假设，受调节融合的触发步骤（Ca2+敏感性以及融合的催化和执行）需要在SNAREs下游发挥作用的其他蛋白质。

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.