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.

兴奋性谷氨酸突触的突触后密度（PSD）是一个复杂的分子机器，似乎是信息存储的关键部位。探测其结构的新方法表明，PSD-95 的类似晶格的主链标记形成其核心，而其他结构成分（例如激酶 CaMKII）占据晶格中的不同位置。然而，完整神经元中的 PSD 在活动过程中会快速且可逆地改变大小。免疫标记显示 CaMKII 是增加质量的主要成分，涉及多达 100 个 CaMKII 全酶。小脑和前脑 PSD 动态的比较表明细胞 α-CaMKII 丰度与 PSD 增厚程度之间存在相关性。 CaMKII 以磷酸化形式在 PSD 处聚集，并且看来 CaMKII 的添加取决于磷酸化。我们还表明，可逆性被化学 LTP（长期增强作用，被认为是建立记忆的关键步骤）所阻断。 CaMKII 的增稠和添加在 LTP 中持续存在，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，我们开发了一种冷冻替代海马培养物的方法，然后通过薄片断层扫描对其进行检查。断层扫描揭示了丝状网状结构作为 PSD 的核心结构。我们开发了新方法来可视化这些细丝和相关分子的结构。 PSD 中识别了几类细丝，并且对它们的尺寸、数量和方向进行了分类。下一步是识别每一类灯丝。使用我们的旋转阴影技术，正在进行对分离 PSD 的补充研究，旨在绘制 PSD 特异性支架分子的分布图，包括 PSD 内的 PSD-93 和 SAP-97。我们还开发了一种从 PSD 组分的其他成分中亲和纯化 PSD 的方法，从而可以独立测量 CaMKII 含量，以及通过质谱法进行蛋白质组分析（与 S. Markey 合作）。这项研究代表了首次对纯化的 PSD 进行蛋白质组学分析，从而消除了 PSD 组分中丰富的污染细胞骨架元素和其他蛋白质的影响。对常规和亲和纯化的 PSD 级分的质谱数据的初步比较表明，在专门的支架分子和谷氨酸受体（尤其是 AMPA 类型）亲和纯化后出现富集，同时某些细胞骨架和突触前蛋白显着减少。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复制品的 EM 分析来确定 PSD 每个主要成分的副本数量。另一系列新的实验使用选择性识别磷酸化 Ser/Thr 残基和脯氨酸的抗体，表明 PSD 含有负责调节 Ser(Thr)-Pro 处几种内源蛋白磷酸化的激酶和磷酸酶主题。初步结果表明 1 型或 2A 型磷酸酶参与去磷酸化，p38-gamma 参与 PSD-95 磷酸化。这些研究最终将有助于阐明与活动诱导的突触可塑性有关的一些分子机制。