Molecular mechanisms of exocytotic vesicle fusion and release.

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Fusion of membrane bound vesicles with a target membrane is of ubiquitous importance for cellular function from intracellular trafficking to exocytotic release of various mediators from a wide range of different cell types. The mechanisms of exocytosis and their regulation are the central topic of the research program in my laboratory. Exocytotic release occurs from the interior of secretory vesicles to the outside of the cell via formation of a fusion pore. The SNARE (Soluble NSF Attachment REceptor) complex, which in mammalian neurons and neuroendocrine cells is composed of the proteins synaptobrevin-2, syntaxin-1, and SNAP-25, plays a key role in vesicle fusion. My laboratory has investigated vesicle fusion mechanisms in mast cells, which release histamine and heparin; in neutrophils, which are part of the innate immune system and release antimicrobial proteins; and in eosinophils, which release cytotoxic proteins to combat parasites. At present we are mostly focused on the investigation of the exocytotic fusion mechanism in chromaffin cells, which we have chosen for their specific advantages for experimental approaches and because their neuronal molecular fusion machinery is better known than those of other cell types. Over the next 5 years, the function of the neuronal SNARE proteins synaptobrevin2, syntaxin 1, SNAP25, and the accessory proteins synaptotagmin, complexin, Munc18 and Munc13 in vesicle fusion and priming will be investigated as a model system to understand the general mechanisms vesicle docking, priming and fusion. For these studies we will combine highly innovative experimental and computational approaches developed in my laboratory to elucidate the nanomechanical motions and interactions that lead to vesicle fusion and exocytotic release. Event Correlation Microscopy using microfabricated Electrochemical Detector Arrays will be used to experimentally interrogate specific molecular interactions and conformational changes related to fusion pore formation in cells and reconstituted systems. The experimental research will be complemented by molecular dynamics simulations to interpret the experimental data and to guide the experimental design. Various crystal structures of parts of the machinery have been determined, which provide very high atomistic resolution, but they represent static structures. To understand the functions of these protein complexes, their dynamic structural changes need to be elucidated. The long term goal of my research is to achieve a true understanding of the nanomechanical mechanisms of vesicle fusion docking, priming, fusion, and release. Our ultimately vision is to obtain realistic molecular movies of the actions of fusion machine, providing deep insight into the mechanisms of vesicle priming and fusion pore formation. This research will also advance our understanding of the related intracellular trafficking fusion events as well as viral entry, which employ closely related fusion mechanisms. .