Dynamic Structural Properties of Synapses

PSD组件在活动期间的运动进行了研究，通过预包埋纳米金标记的分离的海马神经元在文化中，使用类似的策略，在以前的工作与CaMKII。 高K+去极化通常用于去极化和激活神经元培养物，但正在测试更接近生理刺激的其他方案，例如在不存在Mg 2+的情况下应用NMDA和用甘氨酸进行突触激活。 现在，几乎所有的主要成分的PSD的抗体已经获得和测试的兼容性免疫电子显微镜，活动诱导的重组的PSD在分子水平上的一般图片开始出现。 主要的PSD支架，包括PSD-95和GKAP，似乎是相对稳定的短期活动，而柄，特别是柄1，移动更接近PSD。 虽然我们以前已经描述了运动的CaMKII向PSD在激活过程中，我们现在表明，另一个主要的调节蛋白，SynGAP，远离PSD的刺激。 这些刺激诱导的变化与PSD处AMPA受体标记的显著增加一致，与CaMKII和SynGAP在PSD处调节AMPA受体运输中的酶促作用以及结构作用相容。 分离的PSD的EM断层扫描正在用于更精确地描述蛋白质在活动后在PSD处的重新定位。 两个技术突破，分离PSD组分从海马切片文化，和负染色方法兼容EM断层扫描，一直是必不可少的。 从去极化海马切片分离的PSD显示出升高的CaMK II水平，证实在PSD处的活性诱导的变化在分离期间被保留。 CaMKII缔合结构域表现为在断层图像中容易识别的环，允许PSD内的个体CaMKII分子的准确定位。 绘制CaMKII的分布图将准确显示静息时和具有限定生理后果的刺激方案后PSD中CaMKII结合的位置。 这是一个需要探索的基本问题，因为CaMKII在PSD内的多个位置具有多个磷酸化靶点。 在用高K+去极化后定位CaMK II，随后是已知诱导长期变化的刺激方案，如chemLTP和chemLTD。 定量质谱方案用于跟踪蛋白质组成和磷酸化的平行变化。

Movements of PSD components during activity were studied by pre-embedding Nanogold labeling of dissociated hippocampal neurons in culture, using strategies similar to those employed in previous work with CaMKII. Depolarization with high K+ is typically used to depolarize and activate neuronal cultures, but other protocols closer to physiological stimulation, such as application of NMDA and synaptic activation with glycine in the absence of Mg2+, are being tested. Now that antibodies for nearly all major components of the PSD have been obtained and tested for compatibility for immuno-electron microscopy, a general picture of activity-induced re-organization of the PSD at the molecular level begins to emerge. The main PSD scaffold, consisting of PSD-95 and GKAPs, appears to be relatively stable during short-term activity, while Shanks, especially Shank1, move closer to the PSD. While we have previously described movement of CaMKII towards the PSD during activation, we now show that another major regulatory protein, SynGAP, moves away from the PSD upon stimulation. These stimulation induced changes coincide with a marked increase of AMPA receptor labeling at the PSD, compatible with enzymatic as well as structural roles of CaMKII and SynGAP in regulating AMPA receptor trafficking at the PSD. EM tomography of isolated PSDs is being used to describe with more precision the re-localization of proteins at the PSD following activity. Two technical breakthroughs, the isolation of PSD fractions from hippocampal slice cultures, and a negative staining method compatibile with EM tomography, have been essential. PSDs isolated from depolarized hippocampal slices show elevated levels of CaMKII confirming that activity-induced changes at the PSD are preserved during isolation. CaMKII association domains appear as rings that are readily recognizable in tomograms, allowing accurate localization of individual CaMKII molecules within the PSD. Mapping the distribution of CaMKII will show exactly where in the PSD CaMKII is bound at rest, and after stimulation protocols with defined physiological consequences. This is an essential question to explore because CaMKII has multiple phosphorylation targets at multiple locations within the PSD. Localization of CaMKII after depolarization with high K+ will be followed by stimulation protocols known to induce long-term changes, such as chemLTP and chemLTD. Quantitative mass spectrometry protocols are in place to track parallel changes in protein composition and phosphorylation.