Proteins in the synapse carry out four major cell biological processes: exocytosis, endocytosis, signal transduction and adhesion. The synaptic proteins involved in endocytosis have been identified by their ability to associate with dynamin, a protein linked genetically to synaptic vesicle recycling. In Drosophila we have identified eight new proteins that are dynamin-associated and have cloned and sequenced two, DAP16O and syndapin. The human form of DAP16O has recently been shown to be overexpressed in Down's Syndrome. Examination of the properties of DAP16O and syndapin have revealed an unexpected link between endocytosis and the actin cytoskeleton. Careful morphological examination of DAP16O's synaptic distribution revealed a novel but logical micro-organization of the nerve terminal plasma membrane into defined zones of exocytosis surrounded by zones of endocytosis. In this grant we propose completing the characterization of the remaining six dynamin-associated proteins and examining the consequences of mutating the new proteins as well as DAP16O and syndapin. This is done, in part, using a novel, directed mutagenesis procedure to generate temperature-sensitive mutations. We explore how the function and the micro-organization of the nerve terminal into endocytotic and exocytotic zones are modified by mutations in the endocytotic machinery. Identifying synapse-specific gene products and establishing their function is essential if we are to use genomic databases, Drosophila or human, to understand the cellular basis of neurological disease.
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