Dynamic Structural Properties of Synapses

突触后致密物（PSD）是控制和稳定突触处受体部署的蛋白质的大集合。 我们正在研究不同的蛋白质如何通过使用从培养物中分离的海马神经元的预嵌入纳米金标记来绘制它们在PSD中的分布来促进这一功能。 这个项目的重点是在PSD更深的一层或paleums。 虽然在PSD的更表层中组织电子致密核心的支架由MAGUK（主要是PSD-95）组成，但在palpatient中的支架蛋白是Shank和Homer。 该palcipine是由GKAP分子内衬的PSD的核心层界定。 这些GKAP分子可以分别通过其N-和C-末端结合MAGUK和柄两者，以将PSD的核心层和棕榈层钉在一起。 使用针对包括其各自结合位点的表位的抗体比较柄和GKAP的分布和动力学。 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，标签的相互结合位点的柄和GKAP的分布不同，与较窄的标签分布GKAP位于更接近突触后膜，和较宽的标签分布柄延伸到更深的PSD的细胞质侧。 在去极化与高K+或应用的NMDA，更多的柄积累在PSD，优先在更深的层，但没有伴随的变化的强度或分布的GKAP标签。 从培养基中去除Ca 2+导致PSD paleum远端层中的Shank标签的优先损失。 因此，在PSD复合体中存在两个柄部池，一个相对稳定的池沿软腭和内核之间的边界沿着与GKAP结合，另一个更动态的池在软腭中更深的层中。 兴奋性条件促进优先增加的远端池的小腿。 CaMKII的抑制剂表明，在兴奋性条件下，Shank的积累是由CaMKII介导的。 在兴奋性条件下，CaMKII介导的另一个PSD组件，去泛素化酶CYLD，在palatum的积累和激活。 在基础条件下，IKK，另一种激酶存在于PSD，磷酸化CYLD，以促进CYLD活性，虽然在较低的水平相比，由CaMK II。 总之，这些数据表明，易位的CYLD PSD的结果在其激活基础和兴奋性条件下，因此易位到paladium可能有调节作用的突触功能。 总之，这项工作表明，palpalm，第二个不同的层深躺在PSD的核心层，并通过GKAP-Shank相互作用连接到它，参与调节突触活动期间的蛋白质。

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.