How do synaptic vesicles fuse with the plasma membrane? How are the components of the synaptic vesicle recycled? To answer these questions we propose to use genetic and cell biological techniques to identify and characterize proteins required for synaptic function in the nematode Caenorhabditis elegans. So far we have identified eleven genes that are required for neurotransmission in C. elegans. First, ric-5 and ric-7, two of these eleven genes, will be characterized at a molecular level. Null alleles will be isolated, these genes cloned, and the subcellular location of their gene products characterized. Finally, the synaptic morphology and the distribution of synaptic vesicle components in ric-5 and ric-7 mutants will be characterized. Second,new loci that function at the synapse will be identified. It is likely that mutations that eliminate all synaptic function will be lethal. We have devised a screen that can isolate both viable and lethal mutations that affect neurotransmission. This screen is even more powerful since it selects for such mutations by pharmacologically eliminating wild-type animals. Viable and lethal mutations from this screen that specifically disrupt nervous function will be genetically characterized. In summary, these genetic studies will complement existing biochemical studies of the synapse in two ways: (l) The proposed screens will identify new proteins that function in the synapse. These results will aid researchers using biochemical approaches because vertebrate homologs of these proteins can be identified. The activity of these proteins can then be tested in cell free assays that reconstitute vesicular exocytosis in vitro. (2) The analysis of the synaptic defects in mutants provides functional data about the role of particular proteins in neurotransmission in vivo. Together, these results will significantly advance our understanding of the molecular events involved in the exocytosis and endocytosis of synaptic vesicles. These basic studies may provide reagents that eventually can be used to alleviate the symptoms of myasthenia gravis, amyotrophic lateral sclerosis, and Alzheimer's Disease. In addition, we have found that at least three of the genes isolated in our screens seem to affect endocytosis at the nerve terminal. Endocytosis is the most common mechanism of viral entry into cells. The mechanisms of endocytosis elucidated in this proposal may provide a clearer understanding of the mechanisms of viral infection, particularly those that attack neurons or cause motor disease such as Herpes or HIV.
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