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 (which could account for up to 100 CaMKII holoenzymes). CaMKII at the PSD is in the phosphorylated form, and reversibility in its association with the PSD is inhibited by specific phosphatases, suggesting that addition of CaMKII depends on phosphorylation. Most recently, we have shown that the reversibility is blocked by chemical LTP (long term potentiation, thought to be a key step in establishing memory). The thickening and addition of CaMKII persists even when the conditions leading to LTP are reversed, suggesting that the induced association with the PSD is a key step leading to LTP. We have developed a method to affinity purify PSDs from other components of the PSD fraction, thereby allowing independent measurement of CaMKII content, as well as proteomic analysis by mass spectroscopy. These analyses are also identifying multiple new phosphorylation sites at synapses. After high pressure freezing of cultures at rest and after NMDA stimulation the cultures can be freeze-substituted and examined by post embedding immunogold and tomography of thin sections. Immunogold labeling shows the distribution PSD molecules while tomography is revealing a filamentous meshwork at the core structure of the PSD. Much effort during the last year has been devoted to improving methodology for visualizing the structure of these filaments and associated molecules, and labeling them with tomographic markers, aimed at deriving a dynamic molecular architecture of the PSD.
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