Molecular Biology of the Synapse

描述(由申请人提供)： 该项目的长期目标是为确定神经传递的分子机制做出贡献。突触传递是外吞和内吞的循环。在之前的项目期间，我们已经确定，即使在低生理刺激率的情况下，笼蛋白通路对于突触小泡的内吞作用也是必不可少的。此外，我们还阐明了体内突触小泡循环所需的一些分子相互作用。为了使这项工作更上一层楼，我们认为需要两种方法。一种是利用生物物理方法，包括X射线结晶学、表面等离子体共振、定点突变和溶液生物化学，来阐明这些复杂的相互作用导致囊泡组装和脱涂层的机制。另一种是利用电生理学方法来确定这种大分子复合体在活突触中组装的时间顺序。因此，该提案围绕以下具体目标进行组织：目标1：确定活突触中神经内吞的一组核心蛋白质-蛋白质相互作用的时间进程。目的2：研究网状蛋白包膜囊泡组装的机制及其调控。我们将评估适配器的大亚基包含多个网状蛋白结合位点的假设，评估有争议的假设，即参与与AP-2直接相互作用的FxDxF和DPF序列基序也参与与网状蛋白的直接相互作用，确定各种不同类型的网状蛋白结合位点在网状蛋白TD上的哪里相互作用，并评估Eps15是在组装的网状蛋白晶格中设置顶点-顶点/边-边距离的尺子。目的3：研究网状蛋白包衣囊泡脱膜的机理。我们将确定Hsc70的ATPase结构域中的ATP水解如何将构象变化传递到底物结合域，从而导致它结合或释放底物，确定Axin与Hsc70之间相互作用的性质，阐明J-结构域刺激Hsc70的ATPase活性的机制，并确定‘Lid’的打开和关闭在Hsc70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.