Macromolecular Architecture Of The Synapse

兴奋性谷氨酸突触的突触后密度（PSD）是一个复杂的分子机制，是信息储存的关键部位。对PSD-95结构的新探测方法表明，PSD-95的晶格状主链标记形成了其核心，而其他结构成分（如激酶camkii）占据了晶格中的不同位置。然而，完整神经元的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。事实上，最近比较小脑和前脑PSD动态的结果表明，细胞α - camkii丰度与PSD增厚程度之间存在相关性。CaMKII以磷酸化形式聚集在PSD上，并且其与PSD关联的可逆性被特定的磷酸酶抑制，这表明CaMKII的添加依赖于磷酸化。最近，我们已经证明这种可逆性也被化学LTP（长期增强，被认为是建立记忆的关键步骤）所阻断。CaMKII的增厚和添加在LTP中持续存在，这表明与PSD的诱导关联是导致LTP的关键步骤。为了探索PSD的详细分子结构，我们开发了一种冷冻替代海马培养物的方法，然后通过薄切片断层扫描对其进行检查。断层扫描显示丝状网状结构为PSD的核心结构。在过去的一年中，人们一直致力于改进可视化这些细丝和相关分子结构的方法，并用层析标记物标记它们。正在进行的对分离的PSD的补充研究，使用我们的旋转阴影技术，旨在绘制PSD特异性支架分子的分布，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，以及通过质谱进行蛋白质组学分析（与S. Markey合作）。这项研究似乎是第一次对纯化的PSD进行蛋白质组学分析，从而消除了PSD部分中大量污染的细胞骨架元件和其他蛋白质的贡献。常规和亲和纯化的PSD组分的质谱数据的初步比较显示，亲和纯化后的特殊支架分子和谷氨酸受体，特别是AMPA型的富集，伴随着某些细胞骨架和突触前蛋白的显着减少。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免疫金标记复制品的EM分析来确定PSD的每个主要成分的拷贝数。另一系列新的实验，使用一种抗体选择性地识别磷酸化的丝氨酸/苏氨酸残基，然后是脯氨酸，表明psd含有负责调节Ser(Thr)-Pro基序上几种内源性蛋白磷酸化的激酶和磷酸酶。初步结果表明1型或2A型磷酸酶参与PSD-95的去磷酸化，p38- γ参与PSD-95的磷酸化。这些研究最终将有助于阐明活动诱导的突触可塑性的一些分子机制。

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. In fact, recent results comparing 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 reversibility in its association with the PSD is inhibited by specific phosphatases, suggesting that addition of CaMKII depends on phosphorylation. Most recently, we have shown that the reversibility is also 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. Much effort during the last year has been devoted to improving methodology for visualizing the structure of these filaments and associated molecules, and labeling them with tomographic markers. 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 appears to represent 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.