The long term goal of this research is to elucidate molecular mechanisms involved in neurotransmitter release at mature chemical synapses.
The specific aims of the proposed research are to study, using electrophysiological, novel cell biological and traditional molecular genetic methods, mutations in four Drosophila genes which probably affect synaptic vesicle recycling, a little-studied process vital for presynaptic function. There are four major aims: 1) To characterize genetically four genes, chc encoding a clathirin heavy chain, stoned, e(shi)A and e(shi)B, which have been identified in the investigator's laboratory, and are likely involved in synaptic vesicle recycling. 2) To investigate the involvement of clathirin heavy chain and other novel clathrin associated proteins in presynaptic function. 3) To determine the molecular and physiological role of the e(shi)A gene in synaptic vesicle recycling. 4) To develop new assays for analyzing different intermediate steps or pathways in synaptic vesicle recycling. The experiments will use electrophysiological and quantitative cell biological assays to study potential defects in synaptic vesicle fusion and recycling in identified mutant synapses. Morphological methods will be used to examine the disposition of synaptic vesicle membrane in mutant nerve terminals. Recombinant DNA techniques will be used to identify molecules affected by the mutations, in order to examine their phylogenetic conservation, their subcellular localization and their intracellular traffic during the cycling of synaptic vesicle membrane. If successful, these experiments will constitute the first in vivo analysis of molecular mechanisms in synaptic vesicle recycling.
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