Architecture and control of vesicle fusion in excitable cells

要求1 经典神经科学提出了两种相互竞争的膜融合模型。在第一种情况下，囊泡完全与质膜融合，分散其全部内容物。这种胞吐作用的完全融合模型预测囊泡内容物将溢出到膜中并扩散远离融合位点。在第二种情况下，囊泡与质膜短暂连接，仅释放其组分的一部分。这个吻和运行模型预测，囊泡内容物将保持在囊泡腔内，然后将被重新捕获到细胞中，大部分是完整的。 为了确定这两种模型中的哪一种发生在神经内分泌细胞中，我们用全内反射荧光显微镜（TIRF）在活的PC12细胞中成像了单个荧光标记的囊泡。这种方法使我们能够在活细胞中真实的时间内跟踪和测量单个分泌囊泡的行为。通过观察囊泡成分在融合前、融合中和融合后的扩散行为，我们将确定两种经典的融合模型是否（或哪一种）适合触发PC12细胞中囊泡的胞吐作用。此外，我们将测量单个囊泡的行为，以确定囊泡融合行为的异质性，它们的拓扑结构，关系，以及通过细胞信号传导途径和病理学的调节。 目的2 几十种蛋白质控制着可兴奋细胞中囊泡的对接、融合和重新捕获。许多这些蛋白质的身份和功能作用已经通过遗传学、生物化学和电生理学的结合被发现。然而，这些蛋白质及其复合物的架构、结构和结构动力学尚未确定。 在这个目标中，我们已经开始映射的位置，架构和动力学的许多蛋白质提出的作用在胞吐和胞吞。为了实现这一目标，我们正在使用活细胞成像，超分辨率和电子显微镜的组合。通过这种多模式方法，单个蛋白质的位置与组织胞吐和胞吞位点的潜在细胞结构进行比较。这将使我们能够绘制质膜的结构沿着与负责囊泡运输的机器组件。这些研究将确定完整哺乳动物细胞中胞吐和胞吞机制的复杂三维结构。

Aim 1 Classical neuroscience has proposed two competing models for membrane fusion. In the first, vesicles completely merge with the plasma membrane, dispersing the entirety of their contents. This full fusion model of exocytosis predicts that vesicle contents will spill into the membrane and diffuse away from the site of fusion. In the second, vesicles transiently connect with the plasma membrane and release only a subset of their components. This kiss-and-run model predicts that the vesicle contents will remain within a vesicle cavity and then will be recaptured into the cell mostly intact. To determine which of these two models occurs in neuroendocrine cells, we have imaged single fluorescently-tagged vesicles in living PC12 cells with total internal reflection fluorescent microscopy (TIRF). This method allows us to track and measure the behavior of individual secretory vesicles in real time in living cells. By watching the diffusive behavior of vesicle components before, during, and after fusion, we will determine if (or which of) the two classical models of fusion fit triggered exocytosis of vesicles in PC12 cells. Furthermore, we will measure the behavior of individual vesicles to determine the heterogeneity of vesicle fusion behaviors, their topology, relationships, and regulation by cellular signaling pathway and pathologies. Aim 2 Dozens of proteins control the docking, fusion, and then recapture of vesicles in excitable cells. The identity and functional roles of many of these proteins have been discovered through a combination of genetics, biochemistry, and electrophysiology. However, the architecture, structure, and structural dynamics of these proteins and their complexes have yet to be determined. In this aim we have begun to map the location, architecture, and dynamics of many proteins proposed to act during exocytosis and endocytosis. To accomplish this, we are using a combination of live cell imaging, super-resolution, and electron microscopy. Through this multi-modal approach, the locations of individual proteins are being compared to the underlying cellular architecture that organizes exocytic and endocytic sites. This will allow us to map the architecture of the plasma membrane along with components of the machinery responsible for vesicle trafficking. These studies will determine the complex three dimensional structure of the exocytic and endocytic machinery in intact mammalian cells.