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.

兴奋性突触的突触后致密物（postsynaptic density，PSD）是一个复杂的分子机器（分子量大于10亿道尔顿），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EM断层扫描在薄切片中对其进行检查。 断层扫描显示PSD的核心是含有PSD-95的垂直取向细丝的大阵列。 我们还确定了两种主要类型的跨膜结构在PSD匹配AMPA和NMDA受体，这都是由PSD-95含有垂直丝接触。 垂直细丝的另一端（C-末端）通过水平取向的细丝连接。 最近，我们已经发现，这些水平丝的一类是有序的，形成六边形交联的垂直丝集中在NMDA受体。 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伴侣来重塑。 支架稳定PSD的想法现在正在通过使用EM断层扫描来确定敲低PSD-95蛋白质家族成员的影响进行探索。 在PSD-95被敲除后，PSD的斑块完全解开，证明了PSD-95支架的核心作用。 我们已经开发了一种替代制备的高分辨率EM断层扫描分离PSD是兼容的免疫标记，以确定组件的PSD。 这一进展取决于发现一种与断层扫描兼容的高分辨率负染剂。 我们现在已经表明，重要的激酶，CaMKII，可以识别和定位在孤立的PSD，和他们的动态方面的研究概述在NS 003113 -02 LN。 一个新的项目，使用质谱仪的目的是定量PSD成分的高度纯化的制剂，使用合成基因的方法来生产重组人工蛋白。 共价交联策略也被应用于识别PSD中的最近邻分子。