The aim of this application is the elucidation of the role of the synaptic vesicle protein, Synaptotagmin, in neurotransmitter release. This will be accomplished by molecularly identifying and phenotypically characterizing mutations in the synaptotagmin (syt) gene of Drosophila. Synaptotagmin has been identified as a potential docking and fusion protein based on its calcium dependent lipid binding and potential association with presynaptic proteins, the latrotoxin receptor and voltage-gated calcium channel. As such, mutations in Synaptotagmin should directly affect docking and fusion in vivo. In order to test this, we have cloned the Drosophila Synaptotagmin cDNA and generated antibodies specific for Drosophila Synaptotagmin. We have also investigated the developmental expression of message and protein. syt message is neuron specific and present in all PNS and most if not all CNS neurons of the embryo. Protein expressed coincident with axonogenesis and is rapidly transported to the synapses of CNS and muscles. We have localized the syt gene to cytological bands 23A6-A7-B1. In addition, we have identified and mapped four essential genes to 23A4-7-23B1-2. Mutations in one complementation group do not complement the Curly (Cy) mutation (cy maps at 23 B1-2). Embryos homozygous for Cy mutations are morphologically normal and some lack muscle contractions suggesting a direct defect in neurotransmission. We will generate addition syt mutations using EMS, P-element enhancer detectors and gamma-rays to revert Cy. We will molecularly characterize syt mutations using genomic Southerns, Northerns and direct sequencing of PCRed exons. We will phenotypically characterize syt mutants by immunocytochemical and electron microscopy localization of Synaptotagmin, and by electrophysiological recordings of the excitatory junctional potential in larval muscle. Together these two approaches should allow determination of specific defects of neurotransmission. Finally we will initiate studies to identify Drosophila proteins that interact with Synaptotagmin in order to expand this genetic and phenotypic approach to other proteins involved in neurotransmitter release.
