Dynamic Structural Properties of Synapses

突触后密度(PSD)是控制和稳定突触上受体部署的蛋白质的大集合。我们正在通过使用从培养中分离的海马神经元的嵌入前纳米金标记来绘制它们在PSD中的分布，来研究不同的蛋白质是如何对这一功能做出贡献的。这个项目关注的是PSD中更深的一层或大脑皮层。而在PSD较浅层组织电子致密核的支架是由MAGUK组成，主要是PSD-95，大脑皮层中的支架蛋白是Shank和Hmer。大脑皮层由排列在PSD核心层的GKAP分子划定。这些GKAP分子可以通过它们的N-末端和C-末端分别与MAGUK和Shanks结合，从而将PSD的核心层和大脑皮层连接在一起。使用包含其各自结合位点的表位抗体比较了Shank和GKAP的分布和动态。当Shank和GKAP分子相互结合时，两种蛋白质的标记分布应该一致。然而，Shank和GKAP上相互结合的标记在分布上有所不同，GKAP的标记分布较窄，靠近突触后膜，而Shank的标记分布较宽，深入PSD的细胞质。 在高K+去极化或NMDA作用下，PSD处有较多的Shank聚集，但GKAP标记的强度和分布无明显变化。去除培养液中的钙离子会导致PSD大脑皮层远端Shank标记的优先丢失。因此，在PSD复合体有两个Shank池，一个相对稳定的池沿着大脑皮层和内核之间的边界与GKAP结合，另一个在大脑皮层更深的层中有更动态的池。兴奋条件促进胫骨远端池的优先增加。 CaMKII的一种抑制剂证明，兴奋条件下Shank的积累是由CaMKII介导的。在兴奋性条件下，CaMKII介导另一种PSD组分--去泛素酶CyLD在大脑皮层的积聚和激活。在基础条件下，另一种存在于PSD的激酶IKK将CyLD磷酸化以促进CyLD活性，尽管与CaMKII相比水平较低。总之，这些数据表明，在基础和兴奋条件下，CyLD移位到PSD都会导致其激活，因此移位到大脑皮层可能对突触功能有调节作用。总之，这项工作揭示了大脑皮层，第二个不同的层，位于PSD核心层的深处，并通过GKAP-Shank相互作用附着在核心层上，参与了突触活动中蛋白质的调节。

The post synaptic density (PSD) is a large assemblage of proteins that control and stabilize deployment of receptors at synapses. We are investigating how different proteins contribute to this function by mapping their distributions in the PSD using pre-embedding Nanogold labeling of hippocampal neurons disassociated from cultures. This project focuses on a deeper layer or pallium in the PSD. While the scaffold organizing the electron dense core in the more superficial layer of the PSD is composed of MAGUKs, mainly PSD-95, the scaffold proteins in the pallium are Shank and Homer. The pallium is delimited by the GKAP molecules lining the core layer of the PSD. These GKAP molecules can bind both MAGUKs and Shanks through their N- and C-termini respectively to peg together the core and pallium layers of the PSD. The distribution and dynamics of Shank and GKAP were compared using antibodies against epitopes that include their respective binding sites. Where Shank and GKAP molecules are bound to each other, distribution of label for the two proteins should coincide. However, labels for the mutual binding sites on Shank and GKAP differed in distribution, with a narrower label distribution for GKAP located closer to the postsynaptic membrane, and a wider label distribution for Shank extending deeper into the cytoplasmic side of the PSD. Upon depolarization with high K+ or application of NMDA, more Shank accumulated at the PSD, preferentially in the deeper layers but there were no accompanying changes in the intensity or distribution of GKAP label. Removal of Ca2+from the medium resulted in a preferential loss of Shank label in the distal layer of the PSD pallium. Thus, there are two pools of Shank at the PSD complex, one relatively stable pool bound to GKAP along the border between the pallium and the inner core, and another more dynamic pool in a layer deeper in the pallium. Excitatory conditions promote a preferential increase in the distal pool of Shank. An inhibitor of CaMKII demonstrated that accumulation of Shank under excitatory conditions is mediated by CaMKII. Under excitatory conditions, CaMKII mediates accumulation and activation of another PSD component, the deubiquitinase CYLD, in the pallium. Under basal conditions, IKK, another kinase present at the PSD, phosphorylates CYLD to promote CYLD activity, although at a lower level compared than by CaMKII. Altogether these data indicate that translocation of CYLD to the PSD results in its activation both under basal and excitatory conditions, and thus translocation to the pallium may have a regulatory role in synaptic function. In sum, this work reveals that the pallium, a second distinct layer lying deep to the core layer of the PSD and attached to it by GKAP-Shank interactions, is involved in regulation of proteins during synaptic activity.