Phase separation at the synapse.pdf

Review ARticlehttps:/doi.org/10.1038/s41593-019-0579-91Division of Life Science,State Key Laboratory of Molecular Neuroscience,Hong Kong University of Science and Technology,Hong Kong,China.2Center of Systems Biology and Human Health,Hong Kong University of Science and Technology,Hong Kong,China.*e-mail:mzhangust.hkEukaryotic cells orchestrate numerous biochemical reactions spatiotemporally by segregating each cell into structurally and functionally distinct compartments.In addition to classical membrane-enclosed cellular compartments,increasing recent evi-dence reveals a diverse class of cellular compartments that either lack membranes or are not enclosed by membranes,formed by a physical process known as liquidliquid phase separation and fre-quently referred to as membraneless compartments or biological condensates(Fig.1 and Box 1).Observation of membraneless condensates may date back to more than 100 years ago when Ramon y Cajal observed dense spots in the nuclei of silver-stained neurons1.These are now known as Cajal bodies,mRNA processing machineries localized within the nucleus and enriched in proteins and RNAs.Some well-recognized examples of membraneless condensates include various nuclear bodies,P granules,stress granules and processing bodies25.Neurons take cellular compartmentalization to extremes due to their elaborate morphologies and high degree of polarity.In addi-tion to membrane-enclosed organelles and membraneless conden-sates common to other cell types,neurons contain a unique type of membrane-semi-enclosed compartments known as synapses,which are molecular apparatuses dictating signal processing and transmis-sions in all nervous systems(Fig.2).Neither pre-nor postsynaptic compartments are enclosed by membrane bilayers.Underneath the postsynaptic plasma membranes of each synapse is a condensed protein-rich sub-compartment known as the postsynaptic density(PSD),a structure responsible for receiving,amplifying and storing signals initiated by presynaptic cells.PSDs are composed of densely packed proteins forming mega-assemblies a few hundred nanome-ters in width and 3050 nm thick68(Fig.2b).In the presynap-tic compartments,a layer of electron-dense material beneath the plasma membranes,known as the active zone,can also be observed by electron microscope9,and these electron-dense materials are also composed of densely packed proteins10.Active zones play critical roles in docking and priming readily releasable synaptic vesicles(SVs)and in the clustering and positioning of voltage-gated Ca2+channels(VGCCs)at subregions of the presynaptic active zone membrane to regulate the speed and strength of neurotransmitter releases10,11.Additionally,the reserve pools of SVs,which account for up to 90%of total vesicles12,13,are clustered together with proteins such as Synapsin and Intersectin and situated distal to the readily releasable pool of SVs docked to active zones(Fig.2b).A series of recent studies have provided evidence that formations of the condensed PSDs,presynaptic active zones and the clustered reserve pool SVs may involve phase-separation-mediated molecu-lar assemblies1417.Here we review these recent findings.We try to align the concepts and findings of phase separations in synapses with years of research in synaptic signaling complex trafficking,organization and clustering;and we try to provide implications of phase separation on synaptic formation and plasticity,as well as on psychiatric disorders.This review will not cover phase-separa-tion-mediated formation of pathological aggregates that may cause various neurodegenerative diseases.Readers are referred to several reviews on this topic1820.Formation of PSD assembly via phase separationPSDs are composed of densely packed protein mixtures without membrane enclosures.When initially discovered 60 years ago21,22,PSDs were observed as electron-dense thickenings beneath postsyn-aptic plasma membranes and thus open to the cytoplasm of den-dritic spines.Subsequent biochemical purification of PSDs revealed that the vast majority of molecular components of isolated PSDs are proteins23.Extensive biochemistry and electron microscopy(EM)investigations in the past 60 years,together with more recent pro-teomic studies,have elucidated that PSDs are composed of hundreds of different proteins with a wide range of abundances2429,and these proteins form interconnected disc-shaped molecular assemblies6,7,30.Scaffold proteins such as PSD-9531,GKAP(also called SAPAP)32,Shank333 and Homer34 are major components of PSDs.A prominent feature of PSDs is that the dense assembly is attached on one side to the postsynaptic plasma membranes and on the other side is exposed to the dendritic spine cytoplasm.Thus,PSDs are a type of condensed subcellular compartments that are not enclosed by lipid membranes.PSDs are dynamic molecular assemblies.Dendritic spines of excitatory synapses are highly dynamic both during development and in adult animals,and there are good correlations between the volume of a dendritic spine and its synaptic strength35,36.EM stud-ies revealed that the volume of a dendritic spine is nearly linearly correlated with its PSD area and with the number of vesicles in the Phase separation at the synapseXudong Chen1,Xiandeng Wu1,Haowei Wu1 and Mingjie Zhang 1,2*Emerging evidence indicates that liquidliquid phase separation,the formation of a condensed molecular assembly within another diluted aqueous solution,is a means for cells to organize highly condensed biological assemblies(also known as bio-logical condensates or membraneless compartments)with very broad functions and regulatory properties in different subcel-lular regions.Molecular machineries dictating synaptic transmissions in both presynaptic boutons and postsynaptic densities of neuronal synapses may be such biological condensates.Here we review recent developments showing how phase separation can build dense synaptic molecular clusters,highlight unique features of such condensed clusters in the context of synaptic development and signaling,discuss how aberrant phase-separation-mediated synaptic assembly formation may contribute to dysfunctional signaling in psychiatric disorders,and present some challenges and opportunities of phase separation in synaptic biology.Nature NeuroSCieNCe| ARticleNaturE NEurosciENcEpresynaptic bouton37.Enlargement or shrinkage of PSDs involves addition or removal of new proteins such as glutamate receptors and their downstream scaffolding proteins from the PSD assembly35,38.Additionally,AMPA receptors within and outside PSD are con-stantly undergoing exchange,and receptors within the condensed PSD are still mobile but with much slower diffusion rates3942.Scaffold proteins such as PSD-95,GKAP,Shank3 and Homer in synapses also undergo dynamic movements,forming distinct condensed nanoclusters4246.Therefore,the PSD assembly has key features of membraneless biological condensates(Box 1):proteins in PSDs are highly concentrated;PSD condensates can grow or shrink;components within the condensed PSDs are mobile and can Free energyacefVolume fraction()PhaseseparationTemperature(C)bdddGGd/c=0ccdccOne-phaseTwo-phaseDiluted phase volume fractionCondensed phase volume fractiondStronger surfacetensionWeaker surfacetensionOstwald ripening-mediated fusionDispersedSmallerSofterHarderLargerSurface tension-mediated fusionNo fusion orexchange of materialsMembrane-boundcompartmentsR1R3R4R2NEx_in=NEx_outFig.1|Basic principles of phase separation illustrated by a simple two-component system.a,Free energy diagram showing phase separation of a two-component system(for example,a protein indicated by blue dots in aqueous solution indicated by golden dots in c)under one condition.A uniformly mixed system can undergo phase separation by lowering the free energy(G)to its minimal level,resulting in a two-phase system:a dilute phase with a lower protein concentration(d,expressed as the fraction volume for the dilute phase)and a condensed phase with a higher protein concentration(c,the fraction volume for the condensed phase).b,Phase diagram of the two-component system constructed by plotting the free energy minima as the function of temperature.The curve indicates a sharp boundary(or the threshold concentration)of the system undergoing from a homogenous single-phase state to a two-phase state.c,Phase separation results in formation of a condensed phase enriched with the a particular molecular component(here represented as blue dots)and surrounded by the diluted phase with a much lower concentration of the blue dot molecules.d,After phase separation,the components between the two phases can freely exchange.However,there is no net flow of components between the two phases,as the number of molecules(N)exchanged in and out are equal.e,Left:the condensed phase droplets can fuse with each other into larger droplets,as such fusion can reduce the surface tensions of smaller droplets.Additionally,small droplets can spontaneously shrink,and released molecules can be absorbed by other,larger droplet sizes,a process known as Ostwald ripening.Right:for membrane-separated compartments,spontaneous compartment fusion or materials exchange do not occur.f,Biological condensates are highly plastic and can undergo different forms of changes with regulatory inputs(R).A droplet size can be made to shrink or enlarge by simply decreasing or increasing the amount of materials in the system(R1 and R2).A droplet can become softer or harder upon different regulatory modifications(R3 and R4;for example,formation of reversible gel-like protein aggregates or even some non-reversible solid-like protein deposits).Nature NeuroSCieNCe| ARticleNaturE NEurosciENcEexchange with corresponding molecules in the dilute cytoplasm of dendritic spines.PSD may form via phase separation:initial observation.The first hint suggesting that the PSD may form via phase separation came from a recent study of the interaction between PSD-95 and SynGAP14.SynGAP is a negative regulator of synaptic strength by stimulating the GAP activities of small G proteins such as Ras and Rap4752.SynGAP mutations are associated with epilepsy,intellectual disability and autism51,53.SynGAP exists at a very high abundance with a near stoichiometric ratio to PSD-95 in PSD24.The interac-tion between SynGAP and PSD-95 is highly specific and stable14.Apparently,synaptic activities are highly sensitive to the dosage of SynGAP,as SynGAP haploinsufficiency is highly penetrant in caus-ing intellectual disability and autism54.Thus,in addition to func-tioning as a GTPase activating enzyme,SynGAP may play certain nonenzymatic roles in PSD via specifically binding to PSD-95.PSD-95 and SynGAP,either as purified proteins mixed in test tubes or as proteins co-expressed in heterologous cells,formed spherical droplets via phase separation14.Phase separation of the PSD-95SynGAP complex requires homotrimer formation of SynGAP as well as SynGAP-binding-induced PSD-95 multimer-ization14,55,56,showing that the PSD-95SynGAP complex phase separation is governed by the specific and multivalent interaction between the two proteins57.A SynGAP mutant,which retained the same binding affinity to PSD-95 but was a monomer,was com-pletely incapable of undergoing phase separation with PSD-9514.It should be noted that the PSD-95 and SynGAP used in the study by Zeng etal.14 were not full-length proteins.Including the first two PDZ domains in PSD-95 further increases interaction valency between PSD-95 and SynGAP58 and thus should enhance phase separation of the complex57.Similarly,a large segment of intrinsi-cally disordered region between the catalytic domain and the coiled coil domain of SynGAP may also modulate phase separation of the PSD-95SynGAP complex2,3.Hippocampal neurons with wild-type SynGAP replaced by the monomeric SynGAP mutant showed defective PSD targeting of SynGAP,presumably due to the impaired co-enrichment with PSD-95 via phase separation.Neurons expressing this monomeric SynGAP mutant were hypersensitive to weaker stimulations14,which provides a possible mechanistic explanation for SynGAP mutation-induced hyperexcitation in patient brains54.Phase-separation-mediated autonomous co-condensation of PSD-95 and SynGAP led the authors to suggest that highly condensed PSD assemblies observed under EM may form via phase separation14.PSD formation via phase separation:a biochemical reconstitu-tion approach.Based on the initial observation of the PSD-95SynGAP complex phase separation,Zeng etal.tried to reconstitute the PSD assembly first by using four purified PSD scaffolding proteins:PSD-95,GKAP,Shank and Homer15.These four scaf-fold proteins serve to connect the transmembrane ion channels and receptors on the postsynaptic plasma membranes to the actin cytoskeleton at the interface between PSD and spine cytoplasm5961 by forming a large protein network via highly specific and strong interactions(Fig.3a).To obtain high quality recombinant proteins,the authors used fragments of GKAP and Shank instead of the full-length proteins for the reconstitution experiments(Fig.3a).When mixed at a 1:1:1:1 ratio,the mixture underwent phase separation,forming spherical droplets with all four proteins co-condensed (Fig.3b).Importantly,the phase separation of the four-scaffold-pro-tein mixture occurred at individual protein concentrations as low as 1 M,which is well below the concentrations of these proteins in synapses28,62,63.The specific and multivalent interactions connect-ing the PSD scaffold proteins into a large protein network is critical for the phase separation and co-condensation of the four-protein PSD mixture.A GKAP mutant with its PDZ-binding motif(PBM)removed was still able to form phase separation with PSD-95,and a Shank3 and Homer3 mixture could also form condensed droplets via phase separation.However,the PSD-95GKAPPBM phase was found to demix from the Shank3Homer3 phase(Fig.3b).The condensates formed by the four scaffold proteins can recruit and co-condensate the NR2B tail and SynGAP(Fig.3c).Thus,PSD scaffold condensates appear to be able to cluster receptors and concentrate enzymes,two hallmark features of scaffold proteins in synapses64,65.Since PSDs are formed right beneath the synaptic plasma membranes,the reconstitution of PSD assemblies was also tested by tethering the NR2B tail to supported lipid bilayers(SLB)and then adding the synaptic scaffold proteins and SynGAP to the SLB15.Interestingly,the reconstituted PSD assembly on the SLB also underwent phase separation via spinodal decomposition (Fig.3d).The concentration threshold for the PSD protein mixture to form phase separation on a two-dimensional SLB is lower than in a three-dimensional solution.The formed PSD condensates have a web-like structure,reminiscent of perforated PSDs observed by EM.Box 1|Phase-separation-mediated formation of membraneless compartmentsWhen biomolecules are dissolved in water,these molecules tend to mix homogenou