Emerging evidence indicates that liquid-liquid 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 biological condensates or membraneless compartments) with very broad functions and regulatory properties in different subcellular 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.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Liquid–liquid phase separation (LLPS) facilitates the formation of condensed biological assemblies with well-delineated physical boundaries, but without lipid membrane barriers. LLPS is increasingly ...recognized as a common mechanism for cells to organize and maintain different cellular compartments in addition to classical membrane-delimited organelles. Membraneless condensates have many distinct features that are not present in membrane-delimited organelles and that are likely indispensable for the viability and function of living cells. Malformation of membraneless condensates is increasingly linked to human diseases. In this review, we summarize commonly used methods to investigate various forms of LLPS occurring both in 3D aqueous solution and on 2D membrane bilayers, such as LLPS condensates arising from intrinsically disordered proteins or structured modular protein domains. We then discuss, in the context of comparisons with membrane-delimited organelles, the potential functional implications of membraneless condensate formation in cells. We close by highlighting some challenges in the field devoted to studying LLPS-mediated membraneless condensate formation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles ...(SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.
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•Synaptic vesicles coat active zone condensate but coacervate with synapsin condensate•SV/synapsin co-condensates encapsulate SV-coated active zone condensates•A presynaptic bouton-like assembly with reserve and tethered SV pools is reconstituted•Membraneless condensates and membrane organelles can interact with distinct modes
Each presynaptic bouton contains reserve pool synaptic vesicles (SVs) and a few active zone-tethered SVs. Wu et al. show that SVs coat the surface of protein condensates formed by active zone proteins, such as RIM, RIM-BP, and ELKS, but coacervate with synapsin, forming a multi-phase organization with the docked pool SVs surrounded by the bulk reserve pool SVs as observed in presynaptic boutons.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
CASK forms an evolutionarily conserved tripartite complex with Mint1 and Veli critical for neuronal synaptic transmission and cell polarity. The CASK CaM kinase (CaMK) domain, in addition to ...interacting with Mint1, can also bind to many different target proteins, although the mechanism governing CASK-CaMK/target interaction selectivity is unclear. Here, we demonstrate that an extended sequence in the N-terminal unstructured region of Mint1 binds to CASK-CaMK with a dissociation constant of ∼7.5 nM. The high-resolution crystal structure of CASK-CaMK in complex with this Mint1 fragment reveals that the C-lobe of CASK-CaMK binds to a short sequence common to known CaMK targets and the N-lobe of CaMK engages an α helix that is unique to Mint1. Biochemical experiments together with structural analysis reveal that the CASK and Mint1 interaction is not regulated by Ca2+/CaM. The CASK/Mint1 complex structure provides mechanistic explanations for several CASK mutations identified in patients with brain disorders and cancers.
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•CASK CaM kinase domain binds to Mint1 with a nanomolar affinity•An elongated Mint1 fragment wraps around the back side of CaMK•Ca2+/CaM does not affect CASK-CaMK binding to Mint1•The CASK/Mint1 structure explains some CASK variants found in patients
Wu et al. discover that an elongated fragment of Mint1/X11α binds to both the N- and C-lobes of CASK-CaMK domain and reveal the mechanism underlying a new mode of highly specific and stable scaffolding role of CASK-CaMK.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Formation of biomolecular condensates that are not enclosed by membranes via liquid-liquid phase separation (LLPS) is a general strategy that cells adopt to organize membraneless subcellular ...compartments for diverse functions. Neurons are highly polarized with elaborate branching and functional compartmentalization of their neurites, thus, raising additional demand for the proper subcellular localization of both membraneless and membrane-based organelles. Recent studies have provided evidence that several protein assemblies involved in the establishment of neuronal stem cell (NSC) polarity and in the asymmetric division of NSCs form distinct molecular condensates via LLPS. In synapses of adult neurons, molecular apparatuses controlling presynaptic neurotransmitter release and postsynaptic signaling transmission are also likely formed via LLPS. These molecular condensates, though not enclosed by lipid bilayers, directly associate with plasma membranes or membrane-based organelles, indicating that direct communication between membraneless and membrane-based organelles is a common theme in neurons and other types of cells.
Phase separation-mediated formation of biological condensates are a powerful means of concentrating and localizing various molecular assemblies to specific subcellular regions in cells. Wu et al. review recent progresses of phase separation in forming membraneless biological condensates that regulate neuronal stem cell development as well as synaptic signaling in developed neurons.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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•Presynaptic bouton contains multiple synaptic vesicle-containing sub-compartments.•SVs and proteins in different sub-compartments phase separate with distinct modes.•Phase separation ...of presynaptic protein complexes is critical for each step of SV cycling.•Presynaptic bouton is a mesoscale structure formed by membraneless and membranous organelles.
Action potential-induced neurotransmitter release in presynaptic boutons involves coordinated actions of a large list of proteins that are associated directly or indirectly with membrane structures including synaptic vesicles and plasma membranes. These proteins are often highly abundant in different synaptic bouton sub-compartments, and they rarely act alone. Instead, these proteins interact with each other forming intricate and distinct molecular complexes. Many of these complexes form condensed clusters on membrane surfaces. This review summarizes findings in recent years showing that many of presynaptic protein complex assemblies are formed via phase separation. These protein condensates extensively interact with lipid membranes via distinct modes, forming various mesoscale structures by different mode of organizations between membraneless condensates and membranous organelles. We discuss that such mesoscale interactions could have deep implications on mobilization, exocytosis, and retrieval of synaptic vesicles.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Neuronal synapses encompass three compartments: presynaptic axon terminal, synaptic cleft, and postsynaptic dendrite. Each compartment contains densely packed molecular machineries that are involved ...in synaptic transmission. In recent years, emerging evidence indicates that the assembly of these membraneless substructures or assemblies that are not enclosed by membranes are driven by liquid-liquid phase separation. We review here recent studies that suggest the phase separation-mediated organization of these synaptic compartments. We discuss how synaptic function may be linked to its organization as biomolecular condensates. We conclude with a discussion of areas of future interest in the field for better understanding of the structural architecture of neuronal synapses and its contribution to synaptic functions.
This article is part of the Neuropharmacology Special Issue on ‘Glutamate Receptors - The Glutamatergic Synapse’.
•Phase separation governs structural organization of glutamatergic synapses.•Distinct interaction modes between synaptic vesicles and presynaptic condensates.•Nanodomain organization in the postsynaptic densities.•Assembly of nanocolumnar structures across the synaptic cleft.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZAGLJ, ZRSKP
In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular ...compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.
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•Scaffold protein Pclo extracts SVs from reserve pool condensate in response to Ca2+•Pclo enriches into the active zone condensate via binding to RIMBP and ELKS1•Pclo promotes vesicle tethering on the surface of active zone condensate•Ca2+ induces SV transport from reserve pool condensate to active zone condensate
Using the reconstituted presynaptic terminal local vesicle transport as a paradigm, Qiu and Wu et al. demonstrate that short-distance and directional vesicle transport can be achieved via regulated phase separation of vesicles with different protein condensates without involving molecular motors.
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