Protein Components of the Synaptic Adhesive Scaffold

描述(由申请人提供)：在最新的观点中，中央 神经系统突触可以被认为是由两个离散的亚域组成的 它们在结构和功能上重叠。第一个区域是突触 在电子显微镜下观察到的“支架”，由反式， 严格平行的突触前和突触后质膜增厚 通过跨越突触裂隙的“十字桥”连接在一起。脚手架是 即使在大力分馏和洗涤剂提取后仍保留 突触体，而且看起来很清楚它是通过粘连连接在一起的 分子，其身份目前尚不清楚。第二个子域是 神经传递机制，突触通过它来调节其 主要的生理功能。此子域名叠加在 并通过我们尚不了解的分子作用力与之相互作用。 很多工作都集中在分析细胞的生理成分上。 Synapse；然而，支架的主要成分及其如何 细胞间黏附成分之间的相互作用仍然难以捉摸。这是 因为没有足够的分馏技术来提纯完整的 中枢神经系统突触连接，以及令人困惑的候选蛋白质阵列 可能在不同的突触上运作。很明显，承认和消除贫困的努力 评估退变过程中突触蛋白的变化 必须首先依赖于参与的结构蛋白的完整目录 突触的发育和维持，以及它们如何相互作用；以及 第二，了解这些物质的细胞内结合伙伴 在突触信号现象中起作用的支架分子，以及在 依附于潜在的突触下层组件。 长期而言，我们想要确切地了解分子粘附力是如何 组织和稳定突触前到突触后的支架 中枢神经系统中的连接复合体。我们建议：i)使用一种新的细胞分级 我们设计的高产率纯化突触连接复合体的程序，以及 然后使用新开发的质谱学方法来鉴定成分 黏附和黏附相关分子，以及ii)开始研究 这些分子之间的相互作用导致突触的组装 中枢神经系统中的连接复合体。

Description (Provided by applicant): In the most contemporary view, the central nervous System synapse may be thought of as comprising 2 discrete subdomains which overlap structurally and functionally. The first domain is the synaptic "scaffold" which is observed by electron microscopy, consisting of apposed, rigorously parallel presynaptic and postsynaptic plasma membrane thickenings bound together by "crossbridges" that span the synaptic cleft. The scaffold is retained even after vigorous fractionation and detergent extraction of synaptosomes, and it seems clear that it is held together by adhesion molecules, whose identities remain unknown at present. The second subdomain is the neurotransmissional machinery through which the synapse mediates its primary physiological functions. This subdomain is superimposed upon the scaffold and interacts with it via molecular forces we don't understand as yet. Much effort has been focused on analyzing the physiological components of the synapse; however, the major constituents of the scaffold and how its intercellular adhesive components interact have remained elusive. This is because of inadequate fractionation techniques for the purification of intact CNS synaptic junctions, and the bewildering array of candidate proteins that may operate at different synapses. It is clear that efforts to recognize and evaluate changes in synaptic proteins which occur during degenerative processes must rely first on a complete catalog of the structural proteins involved in synaptic development and maintenance and how they interact with each other; and second, on an understanding of the intracellular binding partners for these scaffolding molecules which function in synaptic signaling phenomena, and in attachment to the underlying subsynaptic components. Long range, we want to understand exactly how molecular adhesive forces organize and stabilize the pre-to post-synaptic scaffold of the synaptic junctional complex in the CNS. We propose to: I) use a novel cell fractionation procedure we devised to purify synaptic junctional complexes in high yield, and then use newly developed methods in mass spectrometry to identify the component adhesion and adhesion associated molecules, and II) begin studies on the interactions between these molecules which lead to the assembly of the synaptic junctional complex in the CNS.