Neurogenetics of TS-Paralytic Mutants in Drosophila

描述(申请人提供)：我们的长期目标是用遗传方法阐明果蝇神经信号的分子机制。在这里，我们集中在幼虫神经肌肉接头(NMJ)的突触发育和可塑性。我们在我们收集的ts-麻痹药中发现了几个突变，包括NWK和9-76，它们导致NMJ发育和树枝形成的显著缺陷，为我们剖析控制突触生长和可塑性的调节机制提供了新的起点。我们的目标是使用遗传、分子、生化和共聚焦显微镜技术来确定这些基因的体内功能，以分析特定蛋白质的缺陷是如何导致观察到的突触表型的。NWK导致NMJ过度生长，导致Bouton数增加和超分支。它编码一个含有SH3结构域的接头蛋白，该接头蛋白从酵母到人类保守，直接与肌动蛋白组装的调节因子WASP结合。它定位于突触前周围活动区，这是一个专门用于内吞和调节突触生长的区域。我们已经发现NWK与已知的内吞蛋白在基因和生化上相互作用。相反，NWK中NMJ的过度生长可以通过减少TGFp信号来抑制。我们假设NWK的正常功能是整合肌动蛋白组装和内吞作用，从而下调TGFp信号，从而塑造突触生长。遗传上位性、酵母双杂交和免疫细胞化学实验被用来验证这一假说。9-76也会导致NMJ过度生长和过度分枝。我们发现它对应于编码海人酸型谷氨酸受体(Glur)的笨拙基因座。救援实验表明，对于正常的NMJ生长，笨拙*是突触前而不是突触后所必需的。突触前c/umsy+的强烈过度表达模仿了9-76突变的表型，我们的结果表明9-76编码了一个过度活跃的突变亚基。我们假设，笨拙的GluR是突触前信号机制的一个组成部分，通过该机制，Boutons自我监控谷氨酸的释放，从而使NMJ能够调节生长以响应突触活动。我们将通过免疫定位、结构-功能、过度表达和遗传上位性实验来检验这一假说并剖析其影响机制。此外，还将通过对我们发现的导致NMJ过度生长或生长不足的新突变体的表型和分子分析来研究调节突触生长的其他新机制。突触的生长和可塑性对神经交流、学习和记忆至关重要，在许多人类神经疾病中都被破坏。特别是，人类NWK同源物的突变与严重的精神发育迟滞有关。因此，我们对NWK和其他影响突触生长的突变的分析应该具有广泛的生物学和医学意义，因为它提供了重要的新信息，将促进我们对调节突触生长和可塑性的潜在分子和机制的理解。

DESCRIPTION (provided by applicant): 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.