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

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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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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. ll ll

Ca 2+ /calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca 2+ 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 2+ 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 2+ concentration induces GluN2Bmediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca 2+ removal. Protein phosphatases control the Ca 2+ -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 2+ -induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.

Highlights d CASK CaM kinase domain binds to Mint1 with a nanomolar affinity d An elongated Mint1 fragment wraps around the back side of CaMK d Ca 2+ /CaM does not affect CASK-CaMK binding to Mint1 d The CASK/Mint1 structure explains some CASK variants found in patients

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