Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a dodecameric serine/threonine kinase that plays an essential role in synaptic plasticity. CaMKIIα is highly abundant in the post-synaptic density (PSD), comparable to the cytoskeletal proteins. However, it is unclear how CaMKIIα oligomers interact with each other at such a high density and whether they can eventually form well-ordered clusters. Here, we used high-speed AFM (HS-AFM), which enables visualization of protein dynamics in solution at the single-molecule level, to observe the dynamics of CaMKIIα at high-density conditions mimicking the PSD. Starting with HS-AFM observations for bare mica substrate, purified CaMKIIα was added to the buffer, which allowed us to observe the adsorption process of CaMKIIα from the solution onto the substrate and their subsequent diffusion on the mica substrate. CaMKIIα in the basal (inactivated) state formed small clusters consisting of 2–3 oligomers through intermolecular interactions as their density increased. In contrast, Ca 2+ /CaM-bound (activated) CaMKIIα formed statistically significantly larger clusters than those in the basal state when the density exceeded about 30% of PSD density. Then, CaMKIIα was incubated on the substrate before observation to visualize steady-state clusters at about 60% of PSD density. At this density, it was found that CaMKIIα in the basal state formed well-ordered worm-like chain clusters through the surface of the kinase domains. Even at 60% of PSD density, worm-like clusters in the activated state were larger than those in the basal state. This cluster growth was independent of CaMKIIα autophosphorylation. We also developed a mathematical model simulating the HS-AFM experiments to discuss the molecular mechanisms of cluster growth. Our results suggest that CaMKIIα has a structural function by cluster formation in synaptic plasticity in addition to its well-known kinase activity.
Suzuki et al. (Sun,) studied this question.