Novel Molecular Tools for Imaging Synaptic Dynamics

描述（由申请人提供）：本项目的目标是分析突触发育和可塑性过程中细胞和分子水平上发生的动态变化。这些事件将在一个充分表征的遗传模型系统，果蝇神经肌肉接头（NMJ）进行检查。我们将产生一个工具集的光活化绿色荧光蛋白（PA-GFP）融合的面板前和突触后蛋白。将制备多个转基因系，包括启动子融合体和UAS效应子系。分子探针包括泡状SNARE蛋白突触结合蛋白、PSD95/MAGUK衔接蛋白大碟（Dlg）和谷氨酸受体亚基dGluRIIA的PA-GFP融合物。此外，我们将融合TGF-β信号级联的分子成分，被认为是参与突触发育的逆行控制。这些包括Smad蛋白Mad和II型BMP受体wishful thinking（wit）。将进行广泛的遗传学和生理学验证实验以证明PA-GFP融合报告基因忠实地定位于正确的突触位点，是功能性的并且可以拯救突变表型，并且当在体内表达时不赋予显性表型。在第二个具体的目标，我们建议使用光激活探针研究突触发育和可塑性的几个突出的问题。这些实验包括对突触的活性依赖性生长、参与NMJ生长的逆行生长因子的贩运、释放位点之间的囊泡组分的易位以及经历可塑性的突触的标记的分析。选择拟议的实验是因为问题的重要性和它们对后续研究的潜力。表达分子探针的细胞系将广泛用于研究突触发育和可塑性的研究人员，并将免费共享。我们将研究的问题是研究突触发育和可塑性的研究人员普遍感兴趣的，结果将具有广泛的相关性。公共卫生相关性：在这个项目中，我们将产生新的分子工具，用于研究突触如何修改其结构，并部署分子组件作为神经活动的函数。我们将为基础研究人员提供各种有价值的工具，用于成像神经元连接，因为它们的发展和作为先前经验的函数进行修改。这些工具将使研究人员能够研究特定蛋白质是如何部署和调节的。由于有许多神经系统疾病涉及突触生长，维持和/或可塑性的缺陷，这些研究将有助于这些神经系统疾病的合理分析。

DESCRIPTION (provided by applicant): The goal of this project is to analyze the dynamic changes that occur at a cellular and molecular level during synaptic development and plasticity. These events will be examined in a well characterized genetic model system, the Drosophila neuromuscular junction (NMJ). We will generate a toolset of photoactivatable green fluorescent protein (PA-GFP) fusions made to a panel of pre- and postsynaptic proteins. Multiple transgenic lines will be made, including both promoter fusions and UAS effector lines. The molecular probes include PA- GFP fusions of the vesicular SNARE protein synaptotagmin, the PSD95/MAGUK adaptor protein disks large (Dlg) and the glutamate receptor subunit dGluRIIA. In addition, we will make fusion constructs of molecular components of the TGF-beta signaling cascade that is believed to be involved in the retrograde control of synaptic development. These include the Smad protein Mad, and the type II BMP receptor wishful thinking (wit). Extensive genetic and physiological validation experiments will be performed to demonstrate that the PA- GFP fusion reporters faithfully localize to the correct synaptic sites, are functional and can rescue mutant phenotypes, and do not confer dominant phenotypes when expressed in vivo. In the second specific aim, we propose to use the photoactivatable probes to investigate several outstanding questions in synaptic development and plasticity. The experiments include an analysis of activity-dependent growth of the synapse, the trafficking of retrograde growth factors involved in NMJ growth, the translocation of vesicular components between release sites, and the tagging of synapses undergoing plasticity. The proposed experiments were selected both for the importance of the question and for their potential for follow-on studies. The lines expressing the molecular probes will be of widespread use to researchers studying synaptic development and plasticity, and will be freely shared. The problems we will investigate are of universal interest to researchers studying synaptic development and plasticity, and the results will be of broad relevance. PUBLIC HEALTH RELEVANCE: In this project we will generate new molecular tools for examining how synapses modify their structures and deploy molecular components as a function of neural activity. We will make available to basic researchers a variety of valuable tools for imaging neuronal connections as they develop and as they are modified as a function of prior experience. These tools will allow researchers to study how specific proteins are deployed and regulated. As there are numerous neurological disorders that involve defects in synaptic growth, maintenance, and/or plasticity, these studies will contribute to a rational analysis of these disorders of the nervous system.