Long-term potentiation reconstituted with an artificial TARP/PSD-95 complex.

The critical role of AMPA receptor (AMPAR) trafficking in long-term potentiation (LTP) of excitatory synaptic transmission is now well established, but the underlying molecular mechanism is still uncertain. Recent research suggests that PSD-95 captures AMPARs via an interaction with the AMPAR auxiliary subunits-transmembrane AMPAR regulatory proteins (TARPs). To determine if such interaction is a core minimal component of the AMPAR trafficking and LTP mechanism, we engineered artificial binding partners, which individually were biochemically and functionally dead but which, when expressed together, rescue binding and both basal synaptic transmission and LTP. These findings establish the TARP/PSD-95 complex as an essential interaction underlying AMPAR trafficking and LTP.

O-GlcNAcylation modulates liquid-liquid phase separation of SynGAP/PSD-95.

Liquid-liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O-GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex. We identified multiple O-GlcNAc modification sites for the endogenous SynGAP isolated from rat brain and the recombinantly expressed protein. Protein semisynthesis was used to generate site-specifically O-GlcNAcylated forms of SynGAP, and in vitro and cell-based LLPS assays demonstrated that T1306 O-GlcNAc of SynGAP blocks the interaction with PSD-95, thus inhibiting LLPS. Furthermore, O-GlcNAcylation suppresses SynGAP/PSD-95 LLPS in a dominant-negative manner, enabling sub-stoichiometric O-GlcNAcylation to exert effective regulation. We also showed that O-GlcNAc-dependent LLPS is reversibly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). These findings demonstrate that OGT- and OGA-catalysed O-GlcNAc cycling may serve as an LLPS-regulating post-translational modification.

CaMKIIα-driven, phosphatase-checked postsynaptic plasticity via phase separation.

Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca2+ 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 Ca2+ 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 Ca2+ concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca2+ removal. Protein phosphatases control the Ca2+-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, Ca2+-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.

Shank3 Binds to and Stabilizes the Active Form of Rap1 and HRas GTPases via Its NTD-ANK Tandem with Distinct Mechanisms.

Shank1/2/3, major scaffold proteins in excitatory synapses, are frequently mutated in patients with psychiatric disorders. Although the Shank N-terminal domain and ankyrin repeats domain tandem (NTD-ANK) is known to bind to Ras and Rap1, the molecular mechanism underlying and functional significance of the bindings in synapses are unknown. Here, we demonstrate that Shank3 NTD-ANK specifically binds to the guanosine triphosphate (GTP)-bound form of HRas and Rap1. In addition to the canonical site mediated by the Ras-association domain and common to both GTPases, Shank3 contains an unconventional Rap1 binding site formed by NTD and ANK together. Binding of Shank3 to the GTP-loaded Rap1 slows down its GTP hydrolysis by SynGAP. We further show that the interactions between Shank3 and HRas/Rap1 at excitatory synapses are promoted by synaptic activation. Thus, Shank3 may be able to modulate signaling of the Ras family proteins via directly binding to and stabilizing the GTP-bound form of the enzymes.

Phase Separation-Mediated TARP/MAGUK Complex Condensation and AMPA Receptor Synaptic Transmission.

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.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation.

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.

Phase separation as a mechanism for assembling dynamic postsynaptic density signalling complexes.

The postsynaptic density (PSD) is an electron dense, semi-membrane bound compartment that lies beneath postsynaptic membranes. This region is densely packed with thousands of proteins that are involved in extensive interactions. During synaptic plasticity, the PSD undergoes changes in size and composition along with changes in synaptic strength that lead to long term potentiation (LTP) or depression (LTD). It is therefore essential to understand the organization principles underlying PSD assembly and rearrangement. Here, we review exciting new findings from recent in vitro reconstitution studies and propose a hypothesis that liquid-liquid phase separation mediates PSD formation and regulation. We also discuss how the properties of PSD formed via phase separation might contribute to the biological functions observed from decades of researches. Finally, we highlight unanswered questions regarding PSD organization and how in vitro reconstitution systems may help to answer these questions in the coming years.

Anchoring high concentrations of SynGAP at postsynaptic densities via liquid-liquid phase separation.

SynGAP, encoded by SYNGAP1, is a Ras/Rap GTPase activator specifically expressed in the nervous systems. SynGAP is one of the most abundant proteins in the postsynaptic densities (PSDs) of excitatory synapses and acts as a critical synaptic activity brake by tuning down synaptic GTPase activities. Mutations of SYNGAP1 have been frequently linked to brain disorders including intellectual disability, autisms, and seizure. SynGAP has been shown to undergo fast dispersions from synapses in response to stimulations, a strategy that neurons use to control the specific activities of the enzyme within the tiny, semi-open compartments in dendritic spines. However, the mechanism governing the activity-dependent synaptic localization modulations of SynGAP is poorly understood. It has been shown recently that SynGAP α1, via specifically binding to PSD-95, can undergo liquid-liquid phase separation forming membraneless, condensed protein-rich sub-compartments. This phase transition-mediated, PSD-95-dependent synaptic enrichment of SynGAP α1 not only suggests a dynamic anchoring mechanism of the protein within the PSD, but also implies a new model for the PSD formation in living neurons.

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

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.

Neuronal Synapses: Microscale Signal Processing Machineries Formed by Phase Separation?

The organization principles underlying non-membrane-bound organelles have started to unravel in the past 10 years. A new biophysical model known as biomolecular condensates has been proposed to explain many aspects of membraneless organelle assembly and regulation. Neurons are extremely complex, and each neuron can contain tens of thousands of synapses, building an extensive neuronal circuit. Intriguingly, neuronal synapses are characterized by specialized compartmentalization, where highly enriched supramolecular complexes are semi-membrane-enclosed into submicrometer-sized signal processing compartments. Recent findings have demonstrated that this postsynaptic density may be driven by phase separation, and an increasing number of studies of membraneless compartments have shed light on the important molecular features shared by these organelles. Here, we discuss the unique morphology and composition of synapses and consider how synaptic assembly might be driven by phase separation. Understanding the molecular behavior of this semi-membrane-bound compartment could ultimately help to explain the mechanistic details underlying synaptic transmission and plasticity, as well as the numerous brain disorders caused by synaptic defects.

Basal condensation of Numb and Pon complex via phase transition during Drosophila neuroblast asymmetric division.

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.

Mechanisms of MAGUK-mediated cellular junctional complex organization.

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.

PDZ Ligand Binding-Induced Conformational Coupling of the PDZ-SH3-GK Tandems in PSD-95 Family MAGUKs.

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.

Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity.

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.

A binding site outside the canonical PDZ domain determines the specific interaction between Shank and SAPAP and their function.

Shank and SAPAP (synapse-associated protein 90/postsynaptic density-95-associated protein) are two highly abundant scaffold proteins that directly interact with each other to regulate excitatory synapse development and plasticity. Mutations of SAPAP, but not other reported Shank PDZ domain binders, share a significant overlap on behavioral abnormalities with the mutations of Shank both in patients and in animal models. The molecular mechanism governing the exquisite specificity of the Shank/SAPAP interaction is not clear, however. Here we report that a sequence preceding the canonical PDZ domain of Shank, together with the elongated PDZ BC loop, form another binding site for a sequence upstream of the SAPAP PDZ-binding motif, leading to a several hundred-fold increase in the affinity of the Shank/SAPAP interaction. We provide evidence that the specific interaction afforded by this newly identified site is required for Shank synaptic targeting and the Shank-induced synaptic activity increase. Our study provides a molecular explanation of how Shank and SAPAP dosage changes due to their gene copy number variations can contribute to different psychiatric disorders.

Mice with Shank3 Mutations Associated with ASD and Schizophrenia Display Both Shared and Distinct Defects.

Genetic studies have revealed significant overlaps of risk genes among psychiatric disorders. However, it is not clear how different mutations of the same gene contribute to different disorders. We characterized two lines of mutant mice with Shank3 mutations linked to ASD and schizophrenia. We found both shared and distinct synaptic and behavioral phenotypes. Mice with the ASD-linked InsG3680 mutation manifest striatal synaptic transmission defects before weaning age and impaired juvenile social interaction, coinciding with the early onset of ASD symptoms. On the other hand, adult mice carrying the schizophrenia-linked R1117X mutation show profound synaptic defects in prefrontal cortex and social dominance behavior. Furthermore, we found differential Shank3 mRNA stability and SHANK1/2 upregulation in these two lines. These data demonstrate that different alleles of the same gene may have distinct phenotypes at molecular, synaptic, and circuit levels in mice, which may inform exploration of these relationships in human patients.

Rapid dispersion of SynGAP from synaptic spines triggers AMPA receptor insertion and spine enlargement during LTP.

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.