Dynamic Structural Properties of Synapses

点击翻译按钮获取中文摘要

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.