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的突触后密度(PSD)是一个复杂的分子机器，是信息存储的关键部位。探索其结构的新方法表明，PSD-95的晶格状骨架标记形成了其核心，而其他结构成分--如激酶CaMKII--在晶格中占据了不同的位置。然而，完整神经元中的PSD在活动过程中迅速且可逆地改变大小。免疫标记显示，CaMKII是增加的质量的主要成分，涉及多达100个CaMKII全酶。小脑和前脑的PSD动态比较表明，细胞内α-CaMKII的丰度与PSD的增厚程度有关。CaMKII在PSD处聚集，以其磷酸化的形式存在，似乎CaMKII的添加依赖于磷酸化。我们还证明了化学LTP(长时程增强，被认为是建立记忆的关键步骤)阻止了可逆性。CaMKII的增厚和添加在LTP中持续存在，这表明诱导与PSD的结合是导致LTP的关键步骤。为了探索PSD的详细分子结构，我们开发了一种冷冻替代海马区培养的方法，然后用薄层扫描进行检查。断层扫描显示PSD的核心结构为丝状网状结构。我们已经开发了可视化这些细丝和相关分子结构的新方法。PSD中识别了几类细丝，并对它们的大小、数量和方向进行了编目。下一步是识别每一类灯丝。使用我们的旋转阴影技术，正在进行的隔离PSD的补充研究旨在绘制PSD特定支架分子的分布，包括PSD-93和SAP-97在PSD中的分布。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，从而允许独立测量CaMKII含量，以及通过质谱学(与S.Markey)进行蛋白质组学分析。这项研究是首次对纯化的PSD进行蛋白质组学分析，从而消除了PSD组分中大量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组分的质谱学数据的初步比较表明，在亲和纯化了专门的支架分子和谷氨酸受体，特别是AMPA类型的谷氨酸受体后，伴随着某些细胞骨架和突触前蛋白的显著减少。最终，我们计划使用我们最近描述的新的质量分析技术，以及通过对免疫金标记物的EM分析来确定PSD的每个主要组成部分的拷贝数。另一系列新的实验，使用选择性识别磷酸化的丝氨酸/苏氨酸残基的抗体，表明PSD含有激酶(S)和磷酸酶，负责调节几个内源蛋白质在Ser(Thr)-Pro基序的磷酸化。初步结果表明，1型或2A型磷酸酶参与了PSD-95的去磷酸化，而p38-γ参与了PSD-95的磷酸化。这些研究最终将有助于阐明活动诱导突触可塑性所涉及的一些分子机制。