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Show 70 Allen Farley college of science Eukaryotic intracellular membrane fusion is executed by a set of conserved and universal proteins called SNAREs, which form a four-helix bundle to catalyze fusion. In neurons, SNARE mediated synaptic vesicle fusion is regulated by complexin. The function of complexin is intensely debated, but current models suggest that it interacts with the SNARE complex in order to control calcium evoked fusion in cooperation with synaptotagmin. Protein crystallography has provided a high-resolution structure of complexin inter-acting with the SNARE four-helix bundle. This structure may illustrate a physiologically relevant interaction; however, it possibly describes an in vitro artifact. In this proposal we test the molecular predictions of this structure in vivo using the nematode C. elegans. Mutations to complexin have been shown to increase the rate of fusion of synaptic vesicles in C. elegans. In contrast, SNARE protein loss-of-function mutations decrease the rate of fusion. Using the crystal structure as a guide, we made mutations to the SNARE protein synaptobrevin that we predicted to disrupt com-plexin without compromising SNARE function. If complexin interacts with synaptobrevin as illustrated in the crystal structure, we predict a hyper-fusion phenotype similar to that of complexin. We have expressed these mutant forms of synaptobrevin in C. elegans lacking the native protein. We analyzed vesicle fusion in mutant strains using a pharmacological assay. Aldicarb inhibits acetylcholin-esterase such that strains with high rates of fusion are flooded with neurotransmitter and become para-lyzed. Strains with low rates of fusion are protected from the toxic affects of aldicarb. Using this approach, mutations interfering with complexin's function are expected to appear paralyzed due to hyper-fusion. In contrast, if mutations to synaptobrevin disrupt the function of the SNARE complex, then we predict a phe-notype that resists paralysis. Three of our mutant strains have a hyper-fusion phenotype. This phenotype is rare and therefore a very likely a result of disrupting the complexin interaction with synaptobrevin. These experiments strongly support the physiological relevance of the complexin-SNARE crystal struc-ture, and demonstrate that complexin's interaction with synaptobrevin is necessary for reducing synaptic vesicle fusion. COMPLEXIN INTERACTS WITH SYNAPTOBREVIN TO INHIBIT FUSION IN C. ELEGANS Allen Farley (Erik Jorgensen) Department of Biology University of Utah UNDERGRADUATE RESEARCH ABSTRACTS Erik Jorgensen |