Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and Plasticity

Abstract

Synapses are semi-membraneless, protein-dense, sub-micron chemical reaction compartments responsible for signal processing in each and every neuron. Proper formation and dynamic responses to stimulations of synapses, both during development and in adult, are fundamental to functions of mammalian brains, although the molecular basis governing formation and modulation of compartmentalized synaptic assemblies is unclear. Here, we used a biochemical reconstitution approach to show that, both in solution and on supported membrane bilayers, multivalent interaction networks formed by major excitatory postsynaptic density (PSD) scaffold proteins led to formation of PSD-like assemblies via phase separation. The reconstituted PSD-like assemblies can cluster receptors, selectively concentrate enzymes, promote actin bundle formation, and expel inhibitory postsynaptic proteins. Additionally, the condensed phase PSD assemblies have features that are distinct from those in homogeneous solutions and fit for synaptic functions. Thus, we have built a molecular platform for understanding how neuronal synapses are formed and dynamically regulated.

Temperature, Hydrostatic Pressure, and Osmolyte Effects on Liquid–Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications

“…These condensates are sometimes associated with membrane functiona si nt he nuclear pore complex [17] and signaling puncta, [18] or found near cellular membranes as in the case of postsynaptic density. [19,20] Biomolecular condensatesc an be driven by the interactions among folded globularp roteins, [19,20] and/or intrinsically disordered proteins (IDPs) or disordered protein regions (IDRs), [21,22] and can be composed mostly of folded proteins, [19] IDRs alone, [21,22] or proteins with folded domains and disordered linkers. [18] LLPSl ikely provides an energetically efficient meansf or cells to concentrate certain proteins and RNAss uch that binding affinities of biomolecules and activities of specific biochemical reactions can be regulated, as exemplified by recent discoverieso fR NA polymerase [ clusteringa nd coactivator condensation in the cellular transcriptional apparatus [23,24] and the role of LLPS of an euronal granule component IDR in regulating mRNAt ranslation.…”

PDZ Domains as Drug Targets

“…[100] PSD-95 is highly expressed in the adult mouse brain, with highest expression levels in the hippocampus, cortex, and olfactory bulb. Together with Shank3, SAP-90/PSD-95-associated protein 1 (SAPAP1) and SynGAP, PSD-95 represents the core of synaptic scaffolding.…”

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

“…For example, interactions between SH3 domain and proline‐rich motif are involved in the LLPS of the nephrin–NCK–N‐WASP system and the RIM–RIM‐BP system . PDZ domain‐mediated binding is required for phase separation of PSD scaffold proteins . Multivalent arginine‐rich linear motifs interact with the NPM1 pentamer, leading to LLPS .…”

“…189,190 PDZ domain-mediated binding is required for phase separation of PSD scaffold proteins. 191,192 Multivalent arginine-rich linear motifs interact with the NPM1 pentamer, leading to LLPS. 193 Within the protein-rich droplets, non-native transient interactions are expected to become more populated than in dilute solution.…”