Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through ...increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid-liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate K
. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and changes in substrate K
, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them.
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GEOZS, IJS, IMTLJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Eukaryotic cells possess numerous dynamic membrane-less organelles, RNP granules, enriched in RNA and RNA-binding proteins containing disordered regions. We demonstrate that the disordered regions of ...key RNP granule components and the full-length granule protein hnRNPA1 can phase separate in vitro, producing dynamic liquid droplets. Phase separation is promoted by low salt concentrations or RNA. Over time, the droplets mature to more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with formation of fibrous structures. Different disordered domains can co-assemble into phase-separated droplets. These biophysical properties demonstrate a plausible mechanism by which interactions between disordered regions, coupled with RNA binding, could contribute to RNP granule assembly in vivo through promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation could contribute to diseases involving aberrant RNA granules.
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•Intrinsically disordered regions (IDRs) of RNA-binding proteins can phase separate•RNA, crowding agents, and low salt promote IDR phase separation•Phase-separated droplets stabilize over time by formation of amyloid-like fibers•Multiple IDRs can be recruited to phase-separated droplets
Lin et al. show that numerous intrinsically disordered regions of RNA-binding proteins can phase separate to form liquid droplets, which become less dynamic over time through formation of amyloid-like fibers. This defines a process by which cells could establish and maintain RNP granules with different physical properties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing ...molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.
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GEOZS, IJS, IMTLJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between ...physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones.
Biomolecular condensates are two- and three-dimensional compartments in eukaryotic cells that concentrate specific collections of molecules without an encapsulating membrane. Many condensates behave ...as dynamic liquids and appear to form through liquid–liquid phase separation driven by weak, multivalent interactions between macromolecules. In this review, we discuss current models and data regarding the control of condensate composition, and we describe our current understanding of the composition of representative condensates including PML nuclear bodies, P-bodies, stress granules, the nucleolus, and two-dimensional membrane localized LAT and nephrin clusters. Specific interactions, such as interactions between modular binding domains, weaker interactions between intrinsically disorder regions and nucleic acid base pairing, and nonspecific interactions, such as electrostatic interactions and hydrophobic interactions, influence condensate composition. Understanding how specific condensate composition is determined is essential to understanding condensates as biochemical entities and ultimately discerning their cellular and organismic functions.
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•Composition plays an essential role in the function of biomolecular condensates.•Condensate components can be roughly classified as scaffolds or clients.•Some aspects of client recruitment by scaffolds are known, but much remains unknown.•Composition is increasingly understood through proteomics and mechanistic studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Cell surface transmembrane receptors often form nanometer- to micrometer-scale clusters to initiate signal transduction in response to environmental cues. Extracellular ligand oligomerization, ...domain-domain interactions, and binding to multivalent proteins all contribute to cluster formation. Here we review the current understanding of mechanisms driving cluster formation in a series of representative receptor systems: glycosylated receptors, immune receptors, cell adhesion receptors, Wnt receptors, and receptor tyrosine kinases. We suggest that these clusters share properties of systems that undergo liquid-liquid phase separation and could be investigated in this light.
Biomolecular condensates concentrate macromolecules into foci without a surrounding membrane. Many condensates appear to form through multivalent interactions that drive liquid-liquid phase ...separation (LLPS). LLPS increases the specific activity of actin regulatory proteins toward actin assembly by the Arp2/3 complex. We show that this increase occurs because LLPS of the Nephrin-Nck-N-WASP signaling pathway on lipid bilayers increases membrane dwell time of N-WASP and Arp2/3 complex, consequently increasing actin assembly. Dwell time varies with relative stoichiometry of the signaling proteins in the phase-separated clusters, rendering N-WASP and Arp2/3 activity stoichiometry dependent. This mechanism of controlling protein activity is enabled by the stoichiometrically undefined nature of biomolecular condensates. Such regulation should be a general feature of signaling systems that assemble through multivalent interactions and drive nonequilibrium outputs.
Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. ...Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.
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•Disordered Nephrin intracellular domain (NICD) forms phase-separated nuclear bodies•NICD phase separates via complex coacervation•Aromatic/hydrophobic residues and high (−) charge density promote phase separation•Disordered regions with NICD-like sequence features are common in human proteome
Pak et al. describe cellular liquid-liquid phase separation of a negatively charged intrinsically disordered protein, the Nephrin intracellular domain. Phase separation is driven by co-assembly with positively charged partners, a process termed complex coacervation. Disordered regions with NICD-like sequence features are common in the human proteome, suggesting complex coacervation may be widespread.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phase transitions of linear multivalent proteins control the reversible formation of many intracellular membraneless bodies. Specific non-covalent crosslinks involving domains/motifs lead to ...system-spanning networks referred to as gels. Gelation transitions can occur with or without phase separation. In gelation driven by phase separation multivalent proteins and their ligands condense into dense droplets, and gels form within droplets. System spanning networks can also form without a condensation or demixing of proteins into droplets. Gelation driven by phase separation requires lower protein concentrations, and seems to be the biologically preferred mechanism for forming membraneless bodies. Here, we use coarse-grained computer simulations and the theory of associative polymers to uncover the physical properties of intrinsically disordered linkers that determine the extent to which gelation of linear multivalent proteins is driven by phase separation. Our findings are relevant for understanding how sequence-encoded information in disordered linkers influences phase transitions of multivalent proteins.
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