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Description
Learning and memory rely on long-lasting yet adaptable changes in synaptic function. These changes require precise nanoscale organization of post-synaptic proteins, but the mechanisms that establish and regulate this organization remain unclear. Here we show that the multivalent dodecameric kinase CaMKII undergoes Ca²⁺-dependent liquid–liquid phase separation (LLPS) with the NMDA receptor subunit GluN2B, forming condensates that recruit and spatially reorganize post-synaptic components.
To examine condensate material properties, we assessed their response to the endogenous CaMKII inhibitor, Camk2n1, which acts as an LLPS condensate disruptor. We found that newly formed condensates exhibit partial disassembly, whereas aged condensates undergo rapid and complete dispersion, indicating a time-dependent loss of resistance to disruption. Phos-tag analysis further reveals phosphorylation changes within condensates, suggesting that LLPS provides a biochemical microenvironment for activity-dependent protein modification.
To evaluate how CaMKII–GluN2B LLPS influences synaptic organization, we next examined its structural consequences in reconstituted post-synaptic systems. Excitatory synapses contain multiple glutamate receptor types that assemble into functionally distinct nanoscale clusters, and the spatial arrangement of these clusters is critical for synaptic signaling efficiency. We found that CaMKII incorporation produces a phase-in-phase architecture in which Stargazin/AMPAR and Neuroligin-1 are enriched in an inner phase, while CaMKII–GluN2B forms an outer phase. This LLPS-driven compartmentalization provides a physical basis for the nanoscale separation of AMPAR- and NMDAR-associated clusters. Dual-color dSTORM imaging in neurons reveals corresponding spatial segregation, which collapses upon disruption of CaMKII–GluN2B binding, demonstrating in-cell relevance. Consistent with this mechanism, developmental and genetic perturbations of CaMKII–GluN2B interactions modulate this nanoscale organization, suggesting that LLPS-like organization emerges during synapse maturation.
Together, these results establish CaMKII–GluN2B LLPS as a biophysical mechanism for synaptic protein nanoscale organization and reveal that condensates display time-dependent material instability that may contribute to the flexibility and remodeling capacity of post-synaptic signaling assemblies.
