Molecular Biology of the Synapse

描述（由申请人提供）： 该项目的长期目标是有助于确定神经传递的分子机制。突触传递是一个胞吞和胞吐的循环。在之前的项目期间，我们确定了网格蛋白通路对于突触囊泡内吞作用是必不可少的，即使在低生理速率的刺激下。此外，我们阐明了一些突触囊泡在体内回收所需的分子相互作用。为了使这项工作更上一层楼，我们认为需要采取两种方法。一种是利用生物物理方法，包括X射线晶体学，表面等离子体共振，定点诱变和溶液生物化学来阐明这些复杂的相互作用导致囊泡组装和脱壳的机制。另一种是利用电生理学方法来确定这种大分子复合物在活突触中组装的时间顺序。因此，该提案是围绕以下具体目标组织的：目标1：确定一组核心的蛋白质-蛋白质相互作用的时间过程，在活突触神经元内吞作用的基础。目的2：研究网格蛋白包被囊泡的组装及其调控机制。我们将评估衔接子的大亚基含有多个网格蛋白结合位点的假设，评估参与与AP-2的直接相互作用的FxDxF和DPF序列基序也参与与网格蛋白的直接相互作用的有争议的假设，确定网格蛋白TD上各种不同类型的网格蛋白结合位点相互作用的位置，并评估Eps 15是在组装网格蛋白晶格中设置顶点-顶点/边-边距离的标尺的假设。目的3：表征网格蛋白包被的囊泡脱膜的机制。我们将确定如何ATP水解在Hsc 70的ATP酶结构域传输的构象变化的底物结合结构域，使其结合或释放底物，确定生长素和Hsc 70之间的相互作用的性质，阐明的机制，其中J-结构域刺激Hsc 70的ATP酶活性，并确定的“盖子”打开和关闭的Hsc 70机制中的作用。这些研究的成功完成将使我们能够更深入地了解内吞作用和神经传递的基本过程。

DESCRIPTION (provided by applicant): The long-term goals of this project are to contribute to the determination of the molecular mechanisms which underlie neurotransmission. Synaptic transmission is a cycle of exo- and endocytosis. During the previous project period, we established that the clathrin pathway is essential for synaptic vesicle endocytosis, even during low physiological rates of stimulation. Furthermore, we elucidated a number of the molecular interactions which are required for synaptic vesicle recycling in vivo. In order to take this work to the next level, we believe two types of approaches are required. One is to utilize biophysical methods including X-ray crystallography, surface plasmon resonance, site-directed mutagenesis and solution biochemistry to elucidate mechanisms by which these complex interactions lead to vesicle assembly and uncoating. The other is to utilize electrophysiological methods to determine the temporal order of assembly of this macromolecular complex in living synapses. Therefore the proposal is organized around the following specific aims: Aim 1: Determine the time-course of a core set of protein-protein interactions that underlie neuronal endocytosis in living synapses. Aim 2: Characterize mechanisms of clathrin coated vesicle assembly and its regulation. We will evaluate the hypothesis that the large subunits of the adaptors contain multiple clathrin binding sites, evaluate the controversial hypothesis that the FxDxF and DPF sequence motifs that are involved in direct interactions with AP-2 are also involved in direct interactions with clathrin, determine where on clathrin TD the various different types of clathrin binding sites interact, and evaluate the hypothesis that Eps15 is a ruler that sets vertex-vertex/edge-edge distances in an assembling clathrin lattice. Aim 3: Characterize mechanisms of clathrin coated vesicle uncoating. We will determine how ATP hydrolysis in the ATPase domain of Hsc70 transmits a conformational change to the substrate binding domain which causes it to either bind or release substrate, determine the nature of the interaction between auxilin and Hsc70, elucidate the mechanism by which the J-domain stimulates the ATPase activity of Hsc70, and determine the role of the 'lid' opening and closing in the Hsc70 mechanism. Successful completion of these studies will allow us to achieve a deeper level of understanding of the fundamental processes of endocytosis and neurotransmission.