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.

兴奋性突触的突触后致密物（PSD）是一个复杂的分子机器，是信息储存的关键部位。探测其结构的新方法表明，PSD-95的晶格样骨架标记形成其核心，而其他结构组分（如激酶CaMKII）占据晶格中的不同位置。然而，完整神经元中的PSD在活动期间迅速且可逆地改变大小。免疫标记表明，CaMKII是一个主要组成部分的质量增加，涉及多达100个CaMKII全酶。小脑和前脑中PSD动力学的比较表明细胞α-CaMKII丰度和PSD增厚程度之间的相关性。CaMKII以磷酸化形式聚集在PSD处，并且似乎CaMKII的添加取决于磷酸化。我们还发现可逆性被化学LTP（长时程增强，被认为是建立记忆的关键步骤）阻断。在LTP中，CaMKII的增厚和增加都持续存在，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，我们开发了一种方法，冷冻替代海马培养物，然后通过薄切片断层扫描检查它们。断层扫描显示PSD的核心结构为丝状网状结构。我们已经开发了新的方法来可视化这些细丝和相关分子的结构。几类细丝在PSD中被识别，并且它们的尺寸、数量和方向已经被编目。下一步是识别每一类灯丝。正在进行的补充研究与孤立的PSD，使用我们的旋转阴影技术，旨在映射PSD特异性支架分子的分布，包括PSD-93和SAP-97内的PSD。我们还开发了一种从PSD级分的其他组分中亲和纯化PSD的方法，从而允许独立测量CaMKII含量，以及通过质谱法进行蛋白质组学分析（用S. Markey）。这项研究代表了第一次蛋白质组学分析纯化的PSD，从而消除了丰富的污染细胞骨架元素和其他蛋白质的PSD馏分的贡献。常规和亲和纯化的PSD馏分的质谱数据的初步比较显示富集后的亲和纯化的专门的支架分子和谷氨酸受体，特别是AMPA型，伴随着显着减少某些细胞骨架和突触前蛋白。最终，我们计划使用我们最近描述的新质量分析技术以及免疫金标记副本的EM分析来确定PSD的每个主要组分的拷贝数。另一系列新的实验，使用选择性识别磷酸化的Ser/Thr残基，然后是脯氨酸的抗体，表明PSD含有负责调节Ser（Thr）-Pro基序处几种内源性蛋白质磷酸化的激酶和磷酸酶。初步结果表明参与磷酸酶1或2A型在去磷酸化和参与的p38-γ在磷酸化的PSD-95。这些研究将最终有助于阐明活动诱导的突触可塑性的分子机制。