MOLECULAR GENETIC ANALYSIS OF DEVELOPING SYNAPSES

 描述(申请人提供)：神经细胞交流的主要方式是通过化学突触释放神经递质。大脑的功能以及存储和处理信息的能力依赖于这些准确而可靠地形成的突触连接。这项建议旨在发展一种分子，了解调节这一突触发生和突触维持过程的机制。我们建议结合遗传学、细胞生物学、分子生物学和活体成像的方法，通过研究一组神经-神经突触的特定组装来识别和表征信号通路中协调突触发育的分子成分的作用。在以前的工作中，我们通过可视化荧光标记成分对新形成的突触的招募来描述新生突触形成中细胞事件的顺序。此外，我们还分离了破坏这些突触形成的突变体。克隆其中的一个子集，鉴定了一个F-box蛋白，它选择性地针对泛素介导的降解蛋白，肌动蛋白和微管细胞骨架的调节因子，一个转录调节蛋白和一个新的保守蛋白。利用各种分子、遗传和蛋白质相互作用研究，我们现在建议在机制水平上确定这些蛋白质如何发挥调节突触组装的功能。此外，我们将使用遗传学方法分离和鉴定其他干扰线虫机械感觉神经元及其突触伙伴之间信号传递的基因，以扩大我们对这一过程的分子理解。总之，这些方法将有助于确定在突触形成和突触维持过程中细胞用来识别和相互交流的机制。尽管线虫的突触发生无疑没有脊椎动物那么复杂，但已经很明显，类似的途径在这两个系统中都发挥着作用。因此，对线虫中参与这一过程的分子的分析应该有助于确定一套普遍的和可能保守的原则，这些原则对于一般的突触发生机制是共同的。

 DESCRIPTION (provided by applicant): The primary means by which nerve cells communicate is via the release of neurotransmitter at chemical synapses. The function of the brain and the ability of the brain to store and process information depend on these synaptic connections forming precisely and reliably. This proposal is directed towards developing a molecular understanding the mechanisms that regulated this process of synaptogenesis and synapse maintenance. We propose to use a combination of genetics, cell biology, molecular biology and live imaging to identify and characterize the role of molecular components of the signaling pathways that coordinate synaptic development by studying the specific assembly of a set of nerve-nerve synapses. In previous work, we described the order of cellular events in nascent synapse formation by visualizing the recruitment of fluorescent-tagged components to newly forming synapses. Furthermore, we isolated mutants that disrupt the formation of these synapses. Cloning of a subset of these identified an F-box protein that selectively targets proteins for ubiquitin-mediated degradation, regulators of both the actin and microtubule cytoskeleton, a transcriptional regulatory protein and a novel conserved protein. Using a variety of molecular, genetic and protein interaction studies we now propose to determine at a mechanistic level how these proteins function to regulate synapse assembly. In addition, we will use genetic approaches to isolate and characterize additional genes that disrupt signaling between mechanosensory neurons and their synaptic partners in C. elegans to extend our molecular understanding of the process. Together these approaches will help define mechanisms that cells use to identify and communicate with one another during the process of synapse formation and synaptic maintenance. While synaptogenesis is undoubtedly less complex in C. elegans than in vertebrates, it is already clear that similar pathways operate in both systems. Thus, analysis of the molecules participating in the process in C. elegans should help define a set of general and likely conserved principles that are common to synaptogenesis mechanisms in general.