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。通过这些研究，我们希望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，关系以及细胞信号传导途径和病理学的调节。 使用双色全内反射显微镜，我们已经表明，SLMV在PC 12细胞中的融合的主导模式是完全融合模式。因此，囊泡转运蛋白，包括囊泡乙酰胆碱转运蛋白，在几秒钟内扩散到质膜中。然而，这项工作的一个令人惊讶的发现是，离开囊泡的材料被迅速捕获在细胞表面上的预制簇上。这些由内吞蛋白网格蛋白和参与内吞作用的其他蛋白组成的簇抑制转运蛋白穿过质膜的自由扩散。 为了进一步研究负责捕获细胞表面上的VAChT的结构的密度和拓扑结构，我们使用了三种形式的超高分辨率成像：1）光活化定位显微镜，2）基态耗尽（GSD）超分辨率成像，和2）电子显微镜。这些方法的组合已经表明，PC12细胞中内吞网格蛋白包被结构的密度非常高。密度接近每平方微米2个结构。这些结构随机分布在细胞表面，并产生一个内吞纳米陷阱网络，能够快速捕获从胞吐囊泡逃逸的物质。我们认为，这一系统可以解释在高度兴奋的细胞中囊泡物质的快速回收，这是神经和神经内分泌系统持续发挥功能所必需的。 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结构，结构和结构动力学尚未确定。 在这个目标中，我们已经绘制了在胞吐和胞吞过程中起作用的蛋白质的位置、结构和动力学。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，单个蛋白质的位置和动力学正在与在纳米尺度上组织胞吐和胞吞位点的潜在细胞结构进行比较。这使我们能够绘制结构的质膜沿着蛋白质成分负责囊泡运输。这些研究确定了完整细胞中胞吐和胞吞蛋白质机制的复杂三维结构。 我们一直在使用双色TIRF显微镜和高通量图像分析的形式来检测和表征超过70种与内吞和外吞囊泡相关的蛋白质。这些研究已经快速绘制了这些蛋白质在质膜上的占有率和分布。我们的研究正在开发一个活的神经内分泌细胞质膜的内吞和胞吐机制的一般地形图。这些研究将提供一个系统水平的网络分析的机器负责囊泡融合和神经系统细胞中的夺回。

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. Through these studies we hope to 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. Using two-color total internal reflection microscopy we have shown that the dominant mode of fusion for SLMV in PC12 cells is the full fusion model. As such, vesicle transporters, including the vesicular acetylcholine transporter, diffuse into the plasma membrane within seconds. A surprising finding of this work, however, is that the material that exits vesicles is rapidly captured on preformed clusters on the cells surface. These clusters composed of the endocytic protein clathrin and other proteins involved in endocytosis inhibit the free diffusion of the transporter across the plasma membrane. To further investigate the density and topology of the structures responsible for capturing VAChT on the cell surface, we used three forms of ultra-high resolution imaging: 1) photo-activation localization microscopy, 2) ground state depletion (GSD) super-resolution imaging, and 2) electron microscopy. The combinations of these methods have shown that the density of endocytic clathrin-coated structures in PC12 cells is very high. The density approaches 2 structures per square micron. The structures are randomly distributed across the surface of the cell, and produce a network of endocytic nano-traps capable of rapidly capturing material that escapes from exocytic vesicles. We propose that this system can account for the rapidly recycling of vesicle material in highly excitable cells necessary for the continued function of the nervous and neuroendocrine system. 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 maped the location, architecture, and dynamics of 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 location, and dynamics of individual proteins are being compared to the underlying cellular architecture that organizes exocytic and endocytic sites at the nanometer scale. This allows us to map the architecture of the plasma membrane along with protein components responsible for vesicle trafficking. These studies are determining the complex three dimensional structure of the exocytic and endocytic protein machinery in intact cells. We have been using two-color TIRF microscopy and a form of high-throughput image analysis to detect and characterize over 70 proteins that are associated with endocytic and exocytic vesicles. These studies have rapidly mapped the occupancy and distribution of these proteins at the plasma membrane. Our studies are developing a general topographic map of the endocytic and exocytic machinery in living neuroendocrine cells at the plasma membrane. These studies will provide a systems level network analysis of the machinery responsible for vesicle fusion and recapture in cells of the nervous system.