Our long term goal is to understand the molecular and genetic bases of regulated neurotransmitter vesicular release, one of the principal processes in synaptic transmission. Neurotransmitter vesicular release is likely to result from a complex set of interactions among proteins, some of which have yet to be identified and characterized. Using synaptic transmission in the soil nematode Caenorhabditis elegans (C. elegans) as a model system, we propose to characterize the interactions among the products of seven genes known to participate in synaptic transmission in order to describe the genetic pathway in which they function. We also propose to identify new proteins involved in neurotransmitter vesicular release through the isolation of mutations in the genes which encode them. We will use as our entry point for the analysis the gene unc-11, a gene that has been cloned in our laboratory and that is strongly implicated in synaptic neurotransmitter vesicular release in C. elegans. The project involves two types of analysis. One, molecular analysis, will help to characterize the structure, function, and spatial distribution of the protein encoded by the gene unc-11. A related approach, the use of the yeast two-hybrid system, will help to identify other proteins that interact with the UNC-1 protein. The second type of analysis is genetic. It will help to characterize unc-11's interactions with other (novel and known) genes involved in neurotransmitter release in C. elegans through the isolation and characterization of second-site extragenic suppressors. A related approach to isolate regulatory functions will be the isolation of non-allelic mutations that alter the expression of reporter genes driven by unc-11's promoter sequences. Our results will provide insight into the components and mechanisms of regulated neurotransmitter vesicular release in C. elegans. Since neurotransmitter vesicular release is an excellent example of vesicle- mediated regulated exocytosis, our findings will be relevant to ongoing research in an important process in cell biology. Furthermore, our results will provide tools for the identification of homologous proteins in humans and the potential for understanding human neurological diseases resulting from abnormal or defective mechanisms of neurotransmitter release.
