Postsynaptic protein assembly in three- and two-dimensions studied by mesoscopic simulations

Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D) or beneath the membrane (2D), the underlying difference between the two has not been well clarified. To compare the phase behaviors in 3D and 2D, postsynaptic density (PSD) serves as a model system. PSD is a protein condensate located under the postsynaptic membrane that influences the localization of glutamate receptors and thus contributes to synaptic plasticity. Recent in vitro studies have revealed the formation of droplets of various soluble PSD proteins via liquid-liquid phase separation. However, it is unclear how these protein condensates are formed beneath the membrane and how they specifically affect the localization of glutamate receptors in the membrane. In this study, focusing on the mixture of a glutamate receptor complex, AMPAR-TARP, and a ubiquitous scaffolding protein, PSD-95, we constructed a mesoscopic model of protein-domain interactions in PSD and performed comparative molecular simulations. The results showed a sharp contrast in the phase behaviors of protein assemblies in 3D and those under the membrane (2D). A mixture of a soluble variant of the AMPAR-TARP complex and PSD-95 in the 3D system resulted in a phase-separated condensate, which was consistent with the experimental results. However, with identical domain interactions, AMPAR-TARP embedded in the membrane formed clusters with PSD-95, but did not form a stable separated phase. Thus, the cluster formation behaviors of PSD proteins in the 3D and 2D systems were distinct. The current study suggests that, more generally, stable phase separation can be more difficult to achieve in and beneath the membrane than in 3D systems. SIGNIFICANCESynaptic plasticity is a key factor in memory and learning. Upon learning, protein condensates that form beneath the postsynaptic membrane are known to change their nature. Recent studies have suggested that condensate formation is related to liquid-liquid phase separation based on in vitro experiments of soluble parts. However, the phase behavior can be strongly dependent on physical dimensions. The mechanism by which condensate grows beneath the membrane is not well characterized. Taking advantage of the ease of systematic comparison using computer simulations, we investigated the phase behaviors of postsynaptic protein assemblies in 3D and 2D systems. The results revealed that even when a 3D system exhibited clear phase separation, the corresponding 2D system did not exhibit it stably.