Macromolecular Architecture Of The Synapse

兴奋性谷氨酸能突触的突触后密度（PSD）是一个复杂的分子机器（分子量大于10亿道尔顿），是信息加工和存储的关键位点。为了探索PSD的详细分子结构，我们开发了一种新的方法来冷冻替代海马培养物，然后在薄片上进行EM断层扫描。断层扫描显示PSD的核心是一个包含PSD-95的垂直定向细丝的大阵列。我们还发现了两种主要类型的psd跨膜结构，它们与AMPA和NMDA受体相匹配，它们都被含有垂直丝的PSD-95接触。垂直丝的其他（c端）端由水平取向丝连接。最近，我们发现一类水平丝与集中在NMDA受体下的垂直丝形成六边形交联。这些发现表明PSD-95基质可以稳定谷氨酸受体，同时可以通过在PSD边缘添加新的受体及其结合伙伴来重塑。支架稳定PSD的想法现在正在通过使用EM断层扫描来确定敲除PSD-95蛋白家族成员的影响。在PSD-95被敲除后，PSD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可替代的制备方法，用于分离的PSD的高分辨率EM断层扫描，该制备方法与免疫标记兼容，以识别PSD的成分。这一进展取决于与断层扫描兼容的高分辨率阴性染色的发现。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，并在NS003113-02 LN中概述了它们的动态方面。一个新的项目是利用质谱技术，利用合成基因的方法，从高纯度的制剂中定量分析PSD成分，以产生重组人工蛋白。共价交联策略也被应用于鉴定PSD中最近邻的分子。

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.