Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 6843035
• 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/6843035
• 关键词: affinity labeling; calmodulin dependent protein kinase; cell component structure /function; cerebral ischemia /hypoxia; electron microscopy; freezing; nerve /myelin protein; neurons; phosphorylation; protein isoforms; spectrin; structural biology; synapses; tomography

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.

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。对来自缺血脑的PSD质量的测量显示，质量的增加足以允许添加大约100个CaMKII全酶。PSD处的CaMKII以磷酸化形式存在，可逆性被特定的磷酸酶抑制，提示CaMKII的添加依赖于磷酸化。我们开发了一种从PSD组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。静息和NMDA刺激后的培养物经高压冷冻后，可进行冷冻替代，并进行包埋后的免疫金标记法和薄层摄影检查。免疫金标记法显示了PSD分子的分布，而断层扫描显示PSD的核心结构是丝状网状结构。