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.
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