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.
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•Biochemical reconstitution reveals PSD assembly formation via phase separation•The ePSD condensates cluster NMDA receptor and promote actin bundle formation•The ePSD condensates selectively enrich SynGAP and actively exclude gephyrin•The ePSD condensates can be modulated by activity-dependent protein modifications
Phase transition-mediated formation of excitatory postsynaptic density condensates revealed by biochemical reconstitutions.
Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how ...active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.
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•RIM and RIM-BP mixture forms liquid-liquid phase-separation-mediated condensates•Specific multivalent interaction between RIM and RIM-BP is essential for the LLPS•RIM and RIM-BP condensates cluster Ca2+ channels in solution and on membrane surface•RIM and RIM-BP are plausible organizers of presynaptic active zones
Clustering of Ca2+ channels at presynaptic active zones is critical for precise control of neurotransmitter release. Wu et al. show that the presynaptic active zone scaffold proteins RIM and RIM-BP form self-assembled condensates via liquid-liquid phase separations capable of clustering voltage-gated Ca2+ channels on lipid membrane bilayers.
Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous ...cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD.
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•SynGAP forms a coiled-coil trimer, and each binds to two molecules of PSD-95•SynGAP/PSD-95 complex undergoes liquid-liquid-phase separation•SynGAP/PSD-95 phase separation suggests a possible PSD formation mechanism•Phase-separation-mediated SynGAP PSD enrichment is correlated with synaptic activity
The interaction between two major components of postsynaptic densities induces phase separation of the newly formed complex into liquid-like droplets, suggesting a mechanism for the formation and activity-dependent modulation of synaptic complexes.
SynGAP is a Ras-GTPase activating protein highly enriched at excitatory synapses in the brain. Previous studies have shown that CaMKII and the RAS-ERK pathway are critical for several forms of ...synaptic plasticity including LTP. NMDA receptor-dependent calcium influx has been shown to regulate the RAS-ERK pathway and downstream events that result in AMPA receptor synaptic accumulation, spine enlargement, and synaptic strengthening during LTP. However, the cellular mechanisms whereby calcium influx and CaMKII control Ras activity remain elusive. Using live-imaging techniques, we have found that SynGAP is rapidly dispersed from spines upon LTP induction in hippocampal neurons, and this dispersion depends on phosphorylation of SynGAP by CaMKII. Moreover, the degree of acute dispersion predicts the maintenance of spine enlargement. Thus, the synaptic dispersion of SynGAP by CaMKII phosphorylation during LTP represents a key signaling component that transduces CaMKII activity to small G protein-mediated spine enlargement, AMPA receptor synaptic incorporation, and synaptic potentiation.
Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate ...receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3–GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3–GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3–GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3–GK is a common feature for other MAGUKs including CASK and PALS1.
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•PSD-95 PDZ3 conformationally couples with SH3–GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization.•PDZ and SH3–GK conformational coupling is specific to PSD-95 and PSD-93 and does not occur in SAP102.•PSD-95 and PSD-93 can form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-NMDA receptor synaptic signaling complex.•Ligand binding-induced conformational coupling between PDZ and SH3–GK is a common feature for other MAGUKs including CASK and PALS1.
•The GK domain of MAGUKs has evolved from a nucleotide kinase into a versatile protein binding module.•MAGUK GKs can bind to both phosphorylated and non-phosphorylated target proteins.•The PDZ-SH3-GK ...tandem of MAGUKs forms a structural and functional supramodule.•MAGUKs are capable of organizing very large signalsomes via multimerization of the PDZ-SH3-GK tandems.
Membrane-associated guanylate kinases (MAGUKs) are a family of scaffold proteins that are enriched in cellular junctions and essential for tissue development and homeostasis. Mutations of MAGUKs are linked to many human diseases including cancers, psychiatric disorders, and intellectual disabilities. MAGUKs share a common PDZ-SH3-GK tandem domain organization at the C-terminal end. In this review, we summarize the mechanistic basis governing target recognition and regulations of this binding by the PDZ-SH3-GK tandem of various MAGUKs. We also discuss recent discoveries showing unique folding features of MAGUK PDZ-SH3-GK tandems that facilitate ligand-induced oligomerization of MAGUKs and phase transition of MAGUK-assembled synaptic signaling complexes.
Ca
/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca
oscillations. Despite decades of extensive research, new mechanisms underlying ...CaMKIIα's function in synapses are still being discovered. Here, we discover that Shank3 is a specific binding partner for autoinhibited CaMKIIα. We demonstrate that Shank3 and GluN2B, via combined actions of Ca
and phosphatases, reciprocally bind to CaMKIIα. Under basal condition, CaMKIIα is recruited to the Shank3 subcompartment of postsynaptic density (PSD) via phase separation. Rise of Ca
concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca
removal. Protein phosphatases control the Ca
-dependent shuttling of CaMKIIα between the two PSD subcompartments and PSD condensate formation. Activation of CaMKIIα further enlarges the PSD assembly and induces structural LTP. Thus, Ca
-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.
Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) modulate AMPAR synaptic trafficking and transmission via disc-large (DLG) subfamily of membrane-associated guanylate kinases (MAGUKs). ...Despite extensive studies, the molecular mechanism governing specific TARP/MAGUK interaction remains elusive. Using stargazin and PSD-95 as the representatives, we discover that the entire tail of stargazin (Stg_CT) is required for binding to PSD-95. The PDZ binding motif (PBM) and an Arg-rich motif upstream of PBM conserved in TARPs bind to multiple sites on PSD-95, thus resulting in a highly specific and multivalent stargazin/PSD-95 complex. Stargazin in complex with PSD-95 or PSD-95-assembled postsynaptic complexes form highly concentrated and dynamic condensates via phase separation, reminiscent of stargazin/PSD-95-mediated AMPAR synaptic clustering and trapping. Importantly, charge neutralization mutations in TARP_CT Arg-rich motif weakened TARP’s condensation with PSD-95 and impaired TARP-mediated AMPAR synaptic transmission in mice hippocampal neurons. The TARP_CT/PSD-95 interaction mode may have implications for understanding clustering of other synaptic transmembrane proteins.
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•The entire tail of stargazin binds to PSD-95 with high affinity and specificity•Stargazin/PSD-95 complex form condensed assembly via phase separation•Other TARPs and MAGUKs interact with each other like stargazin/PSD-95 does•Stargazin/PSD-95 phase separation is required for AMPAR synaptic transmission
Zeng et al. report that stargazin uses its entire C-terminal tail to bind to PSD-95 via a specific and multivalent interaction mode that governs the formation of condensed stargazin/PSD-95 assembly via liquid-liquid phase separation, a process critical for AMPAR synaptic targeting and transmission.
Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) ...asymmetric cell divisions and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentiation. Here we discover that Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. The multivalent Numb-Pon interaction leads to high binding specificity and liquid-liquid phase separation of the complex. Perturbations of the Numb/Pon complex phase transition impair the basal localization of Numb and its subsequent suppression of Notch signaling during NB asymmetric divisions. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes.
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