Architecture and control of vesicle fusion in excitable cells

目标1

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). 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 cell's surface. These clusters are composed of the endocytic protein clathrin and AP2 and 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 after exocytosis, we used three forms of ultra-high resolution imaging methods: 1) photo-activation localization microscopy, 2) ground state depletion (GSD) super-resolution imaging, and 2) electron microscopy. The combinations of these three methods have demonstrated that the density of endocytic clathrin-coated structures in PC12 cells is very high. The density approaches 2 structures per square micron. These 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 begun to map the location, architecture, and dynamics of the proteins proposed to act during exocytosis and endocytosis in PC12 cells. To accomplish this, we are using a combination of high-throughput 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. This will allow us to map the molecular architecture of the plasma membrane responsible for vesicle trafficking. These studies will determine the complex three dimensional structure of the exocytic and endocytic protein machinery in intact mammalian cells. Our studies are developing a general topographic map of the endocytic and exocytic machinery in living neuroendocrine cells at the plasma membrane. We hope that these studies will provide a network systems level analysis of the machinery reposing for vesicle fusion and recapture in cells of the nervous system.