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.

兴奋性谷氨酸能突触处的突触密度（PSD）是一种复杂的分子机器（分子量大于十亿daltons），已知是信息处理和存储的关键位置。 为了探索PSD的详细分子组织，我们开发了一种新方法来冻结替代海马培养物，然后通过薄层中的EM层析成像检查它们。 断层扫描显示PSD的核心是包含PSD-95的大量垂直定向的细丝。 我们还确定了匹配AMPA和NMDA受体的两种主要类型的跨膜结构，这些结构均由包含垂直丝的PSD-95接触。 垂直丝的另一个（C末端）末端通过层次取向的细丝连接。 最近，我们发现，这些水平细丝的一类被命令形成六角形，将浓度浓缩在NMDA受体下的垂直细丝交联。 这些发现表明，PSD-95矩阵如何稳定谷氨酸受体，同时可以通过在PSD边缘上添加新受体及其结合伙伴来重塑。 现在，通过使用EM断层扫描来确定击倒PSD-95蛋白质家族的成员的效果，正在探索稳定PSD的脚手架的想法。 在将PSD-95击倒后，PSD的斑块竞争了，表明PSD-95脚手架的核心作用。 我们为分离的PSD的高分辨率EM层析成像开发了一种替代准备，该准备与免疫标记兼容以识别PSD的成分。 这一进展取决于发现与层析成像兼容的高分辨率负染色。 现在，我们已经表明，重要的激酶CaMKII可以在孤立的PSD中识别并映射，并且在NS003113-02 LN中概述了它们的动态方面。 一个使用质谱谱法的新项目旨在使用合成基因方法从高度纯化的制备中定量PSD成分，以产生重组人工蛋白。 还采用共价交联策略来识别PSD中最近的邻居分子。