Native postsynaptic density is a functional condensate formed via phase separation

Phase separation is emerging as a prime mechanism in organizing dynamic subsynaptic compartments. However, studying phase separation in synapses of living neurons is challenging due to the small size of synapses. In this study, we leverage native postsynaptic densities (PSDs) purified from the mouse brain to investigate their organization. Unlike reconstituted PSDs, which form liquid-like droplets, native PSDs exhibit a gel-like morphology with defined molecular composition. Despite their morphological rigidity, native PSDs retain full molecular plasticity, manifested by selectively recruiting or excluding synaptic proteins and undergoing Ca²⁺-dependent structural reorganization. Notably, CaMKII in purified PSDs can be rapidly activated by Ca²⁺, leading to sustained phosphorylation of GluA1 and other PSD proteins. Actin polymerization further enlarges PSD clusters, mirroring structural changes during synaptic potentiation. Thus, native PSDs are functional condensates formed via phase separation. The purified PSDs also serve as an easily accessible platform for studying dynamic regulation of synapses in test tubes.