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.

Highlights d RIM and RIM-BP mixture forms liquid-liquid phaseseparation-mediated condensates d Specific multivalent interaction between RIM and RIM-BP is essential for the LLPS d RIM and RIM-BP condensates cluster Ca 2+ channels in solution and on membrane surface d RIM and RIM-BP are plausible organizers of presynaptic active zones

Highlights d The entire tail of stargazin binds to PSD-95 with high affinity and specificity d Stargazin/PSD-95 complex form condensed assembly via phase separation d Other TARPs and MAGUKs interact with each other like stargazin/PSD-95 does d Stargazin/PSD-95 phase separation is required for AMPAR synaptic transmission

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Edited by Paul E. FraserLiquid-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.In eukaryotic cells, reaction components are spatiotemporally compartmentalized so that materials are concentrated and activities are localized and protected from damaging activities, such as proteolysis, changes in pH, and undesired covalent modifications. Classical organelles are membrane-enclosed where the lipid bilayer provides a physical barrier to separate their interior contents from the exterior environment. Examples include Golgi apparatus, mitochondria, and endoplasmic reticulum (ER). 3 However, many organelles are not membrane-enclosed (often referred to as membraneless compartments in the literature), and such organelles include but are not limited to germ granules, stress granules, nucleoli, centrosomes, and synapses in neurons. In these membraneless compartments, due to the lack of physical separation, molecules can freely exchange with their counterparts in the surrounding bulk solution. Sharp concentration gradients are maintained between the proteinaceous (and sometimes protein and nucleic acid mixtures) interior and the much more diluted exterior. Reaction machineries can reversibly assemble and disassemble within a short time window, as fast as a few seconds. Reaction constituents can be integrated or removed to control specific activities. While recognized for many years, the mechanisms governing the formation of membraneless organelles have remained unclear until about 10 years ago. The first direct experimental evidence came from the study of P granules in germ cells of Caenorhabditis elegans (1). P granule is a collection of RNA and RNA-binding proteins (RBPs) localized at the posterior cortex of a dividing embryo. P granules appear as spherical droplets with liquid-like properties, and they fuse with one another, deform under shear stress, and flow off the surface of the nucleus. Fluorescence recovery afte...

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Biomolecular condensates consisting of proteins and nucleic acids can serve critical biological functions, so that some condensates are referred as membraneless organelles. They can also be disease‐causing, if their assembly is misregulated. A major physicochemical basis of the formation of biomolecular condensates is liquid–liquid phase separation (LLPS). In general, LLPS depends on environmental variables, such as temperature and hydrostatic pressure. The effects of pressure on the LLPS of a binary SynGAP/PSD‐95 protein system mimicking postsynaptic densities, which are protein assemblies underneath the plasma membrane of excitatory synapses, were investigated. Quite unexpectedly, the model system LLPS is much more sensitive to pressure than the folded states of typical globular proteins. Phase‐separated droplets of SynGAP/PSD‐95 were found to dissolve into a homogeneous solution already at ten‐to‐hundred bar levels. The pressure sensitivity of SynGAP/PSD‐95 is seen here as a consequence of both pressure‐dependent multivalent interaction strength and void volume effects. Considering that organisms in the deep sea are under pressures up to about 1 kbar, this implies that deep‐sea organisms have to devise means to counteract this high pressure sensitivity of biomolecular condensates to avoid harm. Intriguingly, these findings may shed light on the biophysical underpinning of pressure‐related neurological disorders in terrestrial vertebrates.

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.