CaMKII Drives Synaptic Maturation by Coordinating Spine Remodeling and Receptor Segregation via Liquid–Liquid Phase Separation

A hallmark of mature excitatory synapses is their localization on dendritic spines, which increase in number and enlarge during development. Recent super-resolution studies have uncovered another key feature of mature synapses-an intricate synaptic nanoarchitecture. Trans-synaptic nanocolumns align AMPA-type glutamate receptor (AMPAR) nanodomains with presynaptic release sites, ensuring efficient synaptic transmission as neurons mature. However, the mechanism by which these key features of synaptic maturation emerge remains unclear. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule implicated in regulating excitatory synaptic transmission. Recent findings show CaMKII, beyond its function as a kinase, serves as a structural element through its ability to undergo liquid-liquid phase separation (LLPS). Upon activation in vitro, it segregates AMPAR from NMDA-type glutamate receptors (NMDAR), forming biphasic condensates. Given that CaMKII expression increases during development, we hypothesized that it serves as the driving force behind synaptic maturation. Using super-resolution microscopy and primary hippocampal cultures prepared from embryonic rat pups of both sexes, we found that immature neurons, which express lower levels of CaMKII, exhibit smaller spine density and size and less-developed AMPAR and NMDAR nanodomain segregation compared with mature neurons. Remarkably, overexpressing CaMKII in immature neurons was sufficient to recapitulate the features of mature synapses, by increasing spine density, size, and receptor nanodomain segregation. Conversely, a single CaMKII mutation (I205K), which prevents LLPS, abolished these effects. Our findings support that CaMKII-mediated LLPS is the driving force shaping the mature synaptic landscape, suggesting a previously overlooked mechanistic link between dendritic spine formation, enlargement, and receptor nanodomain organization.