CaMKIIα holoenzymes self-organize into chain-like mesoscale clusters.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) is highly enriched in dendritic spines at concentrations comparable to those of cytoskeletal proteins and plays a central role in synaptic plasticity. During long-term potentiation, CaMKIIα further accumulates in spines. However, the mechanisms governing its higher-order organization remain poorly understood. Here, we use high-speed atomic force microscopy to visualize interholoenzyme interaction of CaMKIIα at mesoscopic scales (5 to 500 nanometers). Under freely diffusible conditions, CaMKIIα holoenzymes do not form stable clusters. In contrast, when spatially confined, they assemble into chain-like clusters mediated by kinase-domain interactions. These clusters expand upon activation, concomitant with the dissociation of the regulatory segment. Notably, the CaMKIIα Pro212→Leu (P212L) mutant associated with neurodevelopmental disorders forms extensive clusters even in the basal state. Together, our findings demonstrate that CaMKIIα-CaMKIIα interactions drive mesoscale cluster formation and that precise regulation of cluster size and activation-dependent growth might be critical for synaptic signaling.