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是增加肿块的主要成分。对缺血性脑psd的质量测量显示，其质量的增加足以允许添加大约100个CaMKII全酶。PSD上的CaMKII呈磷酸化形式，其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。我们开发了一种从PSD组分中亲和纯化PSD的方法，从而可以独立测量CaMKII的含量。静息培养物经过高压冷冻和NMDA刺激后，可以冷冻替代，并通过包埋后免疫金和薄片断层扫描检查。免疫金标记显示PSD分子的分布，断层扫描显示PSD的核心结构为丝状网。

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. Measurements of the mass of PSDs from ischemic brains shows an increase in mass sufficient to allow for the addition of approximately 100 CaMKII holoenzymes. CaMKII at the PSD is in the phosphorylated form, and reversibility is inhibited by specific phosphatases, suggesting that addition of CaMKII depends on phosphorylation. We have developed a method to affinity purify PSDs from other components of the PSD fraction, thereby allowing independent measurement of CaMKII content. After high pressure freezing of cultures at rest and after NMDA stimulation they can be freeze-substituted and examined by post embedding immunogold and tomography of thin sections. Imunnogold labeling shows the distribution PSD molecules while tomography is showing a filamentous meshwork as the core structure of the PSD.