Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 7324549
• 负责人: Thomas S Reese
• 金额: --
• 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/7324549
• 关键词: Macromolecular; Architecture; Synapse

The post synaptic density (PSD) at excitatory glutamatergic synapses is a complex molecular machine which appears to be a key site of information storage. New methods to probe its structure show that a lattice-like backbone labeling for PSD-95 forms its core, while other structural components-such as the kinase CaMKII-occupy various locations in the lattice. The PSDs in intact neurons, however, change size rapidly and reversibly during activity. Immunolabeling shows that CaMKII is a major component of the added mass, involving up to 100 CaMKII holoenzymes. Comparison of PSD dynamics in the cerebellum and forebrain indicate a correlation between cellular alpha-CaMKII abundance and degree of PSD thickening. CaMKII is aggregated at the PSD is in its phosphorylated form, and it appears that addition of CaMKII depends on phosphorylation. We have also shown that reversibility is blocked by chemical LTP (long term potentiation, thought to be a key step in establishing memory). Both the thickening and addition of CaMKII persists in LTP, suggesting that the induced association with the PSD is a key step leading to LTP. In order to explore the detailed molecular organization of the PSD, we have developed a method to freeze-substitute hippocampal cultures and then examine them by tomography of thin sections. Tomography reveals a filamentous meshwork as the core structure of the PSD. We have developed new methods for visualizing the structure of these filaments and associated molecules. Several classes of filaments are recognized in the PSD, and their sizes, numbers, and orientations have been catalogued. The next step is to identify eachclass of filament. Ongoing complementary studies with isolated PSDs, using our rotary shadowing technique, are aimed at mapping the distributions of PSD-specific scaffolding molecules, including PSD-93 and SAP-97 within the PSD. We have also developed a method to affinity- purify PSDs from other components of the PSD fraction, thereby allowing independent measurement of CaMKII content, as well as proteomic analysis by mass spectroscopy (with S. Markey). This study represents the first proteomic analysis of purified PSDs, thereby eliminating the contributions from abundant contaminating cytoskeletal elements and other proteins in the PSD fraction. Initial comparison of mass spectrometric data for the conventional and affinity-purified PSD fractions reveals enrichment following affinity purification of specialized scaffolding molecules and glutamate receptors, especially of the AMPA type, accompanied by a notable decrease in certain cytoskeletal and presynaptic proteins. Ultimately we plan to determine the number of copies of each major component of the PSD using new mass analysis techniques we recently described, as well as by EM analysis of immunogold labeled replicas. Another new series of experiments, using an antibody that selectively recognizes phosphorylated Ser/Thr residues followed by prolines, suggests that PSDs contain the kinase(s) and phosphatases responsible for the regulation of the phosphorylation of several endogenous proteins at Ser(Thr)-Pro motifs. Preliminary results indicate involvement of phosphatases of type 1 or 2A in dephosphorylation and involvement of p38-gamma in the phosphorylation of PSD-95. These studies will ultimately help clarify some of the molecular mechanisms involved in activity-induced synaptic plasticity.

兴奋性谷氨酸能突触处的突触密度（PSD）是一款复杂的分子机器，似乎是信息存储的关键位置。探测其结构的新方法表明，PSD-95的晶格样骨架标记形成了其核心，而其他结构组件如激酶Camkii-ocupupy在晶格中的各个位置。但是，完整神经元中的PSD在活动期间会迅速和可逆地改变大小。免疫标记表明CaMKII是增加质量的主要组成部分，涉及多达100个CAMKII Holoenzymes。小脑和前脑中PSD动力学的比较表明细胞α-Camkii丰度与PSD增厚程度之间的相关性。 CAMKII在PSD处汇总为其磷酸化形式，看来CaMKII的添加取决于磷酸化。我们还表明，可逆性被化学有限元（长期增强，被认为是建立记忆的关键步骤）所阻断。 LTP中CaMKII的增厚和添加持续存在，这表明与PSD的诱导关联是导致LTP的关键步骤。为了探索PSD的详细分子组织，我们开发了一种冻结亚植物海马培养物的方法，然后通过薄切片的层析成像进行检查。断层扫描揭示了丝状网状作品作为PSD的核心结构。我们开发了可视化这些丝和相关分子的结构的新方法。 PSD中识别出几类细丝，它们的大小，数字和方向已被分类。下一步是识别细丝的每个阶级。使用我们的旋转阴影技术的隔离PSD进行的互补研究旨在绘制PSD特异性脚手架分子的分布，包括PSD中PSD-93和SAP-97。我们还开发了一种方法，可以从PSD分数的其他组件中纯化PSD，从而可以独立地测量CAMKII含量，以及通过质谱（与S. Markey）进行蛋白质组学分析。这项研究代表了纯化的PSD的首次蛋白质组学分析，从而消除了PSD馏分中丰富的污染细胞骨架元素和其他蛋白质的贡献。常规和亲和力纯化的PSD级分的质谱数据的初步比较揭示了专门脚手架分子和谷氨酸受体的亲和力纯化后的富集，尤其是AMPA类型的富集，伴随着某些细胞骨架和突触前蛋白的显着降低。最终，我们计划使用我们最近描述的新质量分析技术以及通过IMMUNOGOLD标记的复制品的EM分析来确定PSD的每个主要组成部分的副本数量。使用一种选择性识别磷酸化的SER/THR残基的抗体随后是脯氨酸，这是另一个新的实验，这表明PSD含有激酶和磷酸酶，负责调节血清（THR）-PRO基序的几种内源性蛋白质磷酸化的磷酸化。初步结果表明1型或2A的磷酸酶参与p38-gamma的去磷酸化以及PSD-95磷酸化的参与。这些研究最终将有助于阐明参与活性引起的突触可塑性的一些分子机制。