Our long-term aim is to elucidate molecular mechanisms of neural signaling using a genetic approach in Drosophila. Here we focus on synaptic development and plasticity of the larval neuromuscular junction (NMJ). We have discovered several mutations among our collection of ts-paralytics, including nwk and 9-76, that cause striking defects in NMJ development and arborization, providing us with novel starting points to dissect regulatory mechanisms that control synaptic growth and plasticity. Our goals are to determine the in vivo functions of these genes using genetic, molecular, biochemical, and confocal microscopic techniques to analyze how defects in particular proteins result in the observed synaptic phenotypes. nwk causes NMJ overgrowth with an increase in bouton number and hyperbranching. It encodes an SH3-domain containing adaptor protein conserved from yeast to humans that binds directly to WASP, a regulator of actin assembly. It localizes to presynaptic periactive zones, a region specialized for endocytosis and regulation of synaptic growth. We have found that Nwk interacts genetically and biochemically with known endocytic proteins. Conversely, NMJ overgrowth in Nwk is suppressed by decreasing TGFp signaling. We hypothesize that Nwk normally functions to integrate actin assembly with endocytosis to downregulate TGFp signaling to sculpt synaptic growth. Genetic epistasis, yeast two-hybrid, and immunocytochemical experiments are proposed to test this hypothesis. 9-76 also causes NMJ overgrowth and hyperbranching. We found that it corresponds with the clumsy locus, which encodes a kainate-type glutamate receptor (GluR). Rescue experiments show that clumsy* is required presynaptically rather than postsynaptically for normal NMJ growth. Strong presynaptic overexpression of c/umsy+ mimics the 9-76 mutant phenotype and our results suggest that 9-76 encodes a mutant subunit that is overactive. We hypothesize that the Clumsy GluR is a component of a presynaptic signaling mechanism through which boutons self-monitor glutamate release enabling NMJ growth regulation in response to synaptic activity. We will test this hypothesis and dissect the affected mechanism by immunolocalization, structure-function, overexpression, and genetic epistasis experiments. Additional novel mechanisms regulating synaptic growth will be studied by phenotypic and molecular analysis of new mutants we have discovered that cause NMJ overgrowth or undergrowth. Synaptic growth and plasticity are of fundamental importance to neural communication, learning, and memory and are disrupted in many human neurological diseases. In particular, mutations of human homologs of Nwk have been associated with severe mental retardation. Consequently, our proposed analysis of nwk and other mutations affecting synaptic growth should have broad biological and medical significance by contributing important new information that will advance our understanding of the underlying molecules and mechanisms that regulate synaptic growth and plasticity. ? ?
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