Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 8158186
• 负责人: Thomas S Reese
• 金额: $ 180.08万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158186
• 关键词: Affinity; Architecture; Binding; Development; Filament; Freezing; Glutamates; Goals; Hippocampus (Brain); Link; Maps; Methods; Microtomy; Molecular; Molecular Weight; Phosphotransferases; Preparation; Protein Family; Proteins; Recombinants; Resolution; Role; Site; Synapses; Synthetic Genes; calmodulin-dependent protein kinase II; crosslink; density; insight; scaffold

The post synaptic density (PSD) at excitatory glutamatergic synapses is a complex molecular machine (molecular weight greater than one billion Daltons) which is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we have developed a new method to freeze-substitute hippocampal cultures and then examine them by EM tomography in thin sections. Tomography reveals that the core of the PSD is a large array of vertically oriented filaments that contain PSD-95. We also identify two major type of transmembrane structures at PSDs matching AMPA and NMDA receptors, which are both contacted by the PSD-95 containing vertical filaments. The other (c-terminus) ends of the vertical filaments are linked by horizonatally oriented filaments. Recently, we have discovered that one class of these horizontal filaments is ordered to form hexagons cross-linking the vertical filaments concentrated under the NMDA receptors. These findings show how the PSD-95 matrix can stabilize glutamate receptors, and at the same time could be remodeled by the addition of new receptors and their binding partners at the edges of the PSD. The idea that the scaffold stabilizes the PSD is now being explored by using EM tomography to determine the effects of knocking down members of the PSD-95 family of proteins. After PSD-95 is knocked down, patches of PSD competely unravel, demonstrating the central role of the PSD-95 scaffold. We have developed an alternative preparation for high resolution EM tomography of isolated PSDs that is compatible with immunolabeling to identify components of the PSD. This advance depends on the discovery of a high resolution negative stain compatible with tomography. We have now shown that the important kinase, CaMKII, can be recognized and mapped in isolated PSDs, and their dynamic aspects investigated as outlined in NS003113-02 LN. A new project using mass spectroscometry is aimed at the quantification of PSD components from the highly purified preparation using a synthetic gene approach to produce recombinant artificial proteins. A covalent cross-linking strategy is also being applied to identify nearest neighbor molecules in the PSD.

兴奋性谷氨酸突触的突触后密度（PSD）是一个复杂的分子机器（分子量大于十亿道尔顿），被认为是信息处理和存储的关键部位。 为了探索 PSD 的详细分子组织，我们开发了一种新方法来冷冻替代海马培养物，然后通过薄切片中的 EM 断层扫描对其进行检查。 断层扫描显示 PSD 的核心是大量垂直方向的细丝，其中含有 PSD-95。 我们还确定了与 AMPA 和 NMDA 受体匹配的 PSD 上的两种主要类型的跨膜结构，这两种结构都通过包含垂直丝的 PSD-95 接触。 垂直丝的另一端（C 末端）由水平定向的丝连接。 最近，我们发现一类水平丝被有序地形成六边形，交联集中在 NMDA 受体下方的垂直丝。 这些发现表明 PSD-95 基质如何稳定谷氨酸受体，同时可以通过在 PSD 边缘添加新受体及其结合伙伴来重塑。 现在正在通过使用 EM 断层扫描来探索支架稳定 PSD 的想法，以确定击倒 PSD-95 蛋白质家族成员的效果。 PSD-95 被击倒后，PSD 斑块完全解开，证明了 PSD-95 支架的核心作用。 我们开发了一种替代制剂，用于分离 PSD 的高分辨率 EM 断层扫描，该制剂与免疫标记兼容，可识别 PSD 的成分。 这一进展取决于与断层扫描兼容的高分辨率负染色的发现。 我们现在已经证明，重要的激酶 CaMKII 可以在分离的 PSD 中被识别和定位，并且如 NS003113-02 LN 中概述的那样研究了它们的动态方面。 一个使用质谱法的新项目旨在使用合成基因方法生产重组人工蛋白，对高度纯化的制剂中的 PSD 成分进行定量。 共价交联策略也被应用于识别 PSD 中最近的邻居分子。