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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 M . 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 M , probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them. A chemically induced dimerization strategy was used to recruit SUMOylation enzymes into condensates, enabling quantification of the effect of phase separation on the activity of a SUMOylation enzyme cascade reaction.

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.Biomolecular condensates are membraneless molecular assemblies formed via liquid–liquid phase separation. They have a plethora of roles, ranging from controlling biochemical reactions to regulating cell organization and cell function. This article provides a framework for the study of condensate functions across these cellular length scales, offering to bring new understanding of biological processes.

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. The membrane that surrounds a cell is made up of a mixture of lipid molecules and proteins. Membrane proteins perform a wide range of roles, including transmitting signals into, and out of, cells and helping neighboring cells to stick together. To perform these tasks, these proteins commonly need to bind to other molecules—collectively known as ligands—that are found either inside or outside the cell. Membrane proteins are able to move around within the membrane, and in many systems, ligand binding causes the membrane proteins to cluster together. Although this clustering has been seen in many different systems, no general principles that describe how clustering occurs had been found. Now, Banjade and Rosen have constructed an artificial cell membrane to investigate the clustering of a membrane protein called Nephrin, which is essential for kidneys to function correctly. When it is activated, Nephrin interacts with protein ligands called Nck and N-WASP that are found inside cells and helps filaments of a protein called actin to form. These filaments perform a number of roles including enabling cells to adhere to each other and to move. In Banjade and Rosen's artificial system, when a critical concentration of ligands was exceeded, clusters of Nephrin, Nck and N-WASP suddenly formed. This suggests that the clusters form through a physical process known as ‘phase separation’. Banjade and Rosen found that this critical concentration depends on how strongly the proteins interact and the number of sites they possess to bind each other. Within the clusters, the three proteins formed large polymer chains. The clusters were mobile and, over time, small clusters coalesced into larger clusters. Even though the clusters persisted for hours, individual proteins did not stay in a given cluster for long and instead continuously exchanged back-and-forth between the cluster and its surroundings. When actin and another protein complex that interacts with N-WASP were added to the artificial membrane system, actin filaments began to form at the protein clusters. Banjade and Rosen suggest that such clusters act as ‘signaling zones’ that coordinate the construction of the actin filaments. Regions that are also found in many other signaling proteins mediate the interactions between Nephrin, Nck and N-WASP. Banjade and Rosen therefore suggest that phase separation and protein polymer formation could explain how many different types of membrane proteins form clusters.

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.

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. Our cells contain a variety of structures called organelles that perform specific roles within a cell. Some organelles are surrounded by a membrane, while others float inside the cell as spherical droplets made of proteins. These proteins contain several sticky regions, which are connected by flexible linker proteins. It is thought that the level of stickiness and the number of sticky regions, or domains, determine whether a protein will form a membraneless organelle. Often, proteins with similar sticky domains have different linkers, and until now, it was assumed that the linkers do not have any other purpose than stringing the domains together. To test this further, Harmon et al. used a combination of computer simulations and physics-based theory. In these simulations, the domains were kept the same, but the properties of linkers were changed to see if this would influence how the membraneless organelles are formed. The results showed that depending on the physical properties of the linkers, the proteins could huddle together and form dense spherical gel-like droplets similar to the membraneless organelles, or form open non-spherical gels. When the linkers were short, the proteins do not easily form droplets. Linkers that were sufficiently long but too bulky, lead to non-spherical gels. Compact linkers, however, enabled proteins to huddle and form spherical gels. The spherical droplet-spanning gels required much less protein compared to the open non-spherical gels. This suggests that proteins important for forming membraneless organelles can be distinguished from those that are not based on the properties of their linkers – even when their domains are similar. These findings further scientists’ knowledge of how specific types of proteins form membraneless organelles and will help to understand how membraneless organelles control many key aspects of how a cell works.

P bodies are archetypal biomolecular condensates that concentrate proteins and RNA without a surrounding membrane. While dozens of P body proteins are known, the concentrations of components in the compartment have not been measured. We used live cell imaging to generate a quantitative inventory of the major proteins in yeast P bodies. Only seven proteins are highly concentrated in P bodies (5.1–15µM); the 24 others examined are appreciably lower (most ≤ 2.6µM). P body concentration correlates inversely with cytoplasmic exchange rate. Sequence elements driving Dcp2 concentration into P bodies are distributed across the protein and act synergistically. Our data indicate that P bodies, and probably other condensates, are compositionally simpler than suggested by proteomic analyses, with implications for specificity, reconstitution and evolution.

A major energy carrier of the cell also promotes protein solubility Adenosine triphosphate (ATP) is the canonical energy carrier that powers cellular machines, drives metabolic reactions, and serves as a precursor to nucleic acids. On page 753 of this issue, Patel et al. ( 1 ) propose a previously unknown function for ATP in maintaining protein solubility and preventing macromolecular aggregation. They show that at physiologic concentrations (2 to 8 mM), the physical properties of ATP alone enable it to dissolve liquid-liquid phase-separated droplets and amyloid fibers. These findings suggest an additional way for cells to use ATP to maintain proteostasis in the crowded cytoplasm and also fine-tune the material properties of nonmembrane-bound organelles and the cell interior in general.

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells 1 – 3 . Assembly of mitotic chromosomes involves DNA looping by condensin 4 – 8 and chromatin compaction by global histone deacetylation 9 – 13 . Although condensin confers mechanical resistance to spindle pulling forces 14 – 16 , it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells. Histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division.

The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins.

Nuclear DNA in eukaryotes is wrapped around histone proteins to form nucleosomes on a chromatin fiber. Dynamic folding of the chromatin fiber into loops and variations in the degree of chromatin compaction regulate essential processes such as transcription, recombination, and mitotic chromosome segregation. Our understanding of the physical properties that allow chromatin to be dynamically remodeled even in highly compacted states is limited. Previously, we reported that chromatin has an intrinsic capacity to phase separate and form dynamic liquid-like condensates, which can be regulated by cellular factors [B. A. Gibson et al., Cell 179, 470–484.e421 (2019)]. Recent contradictory reports claim that a specific set of solution conditions is required for fluidity in condensates that would otherwise be solid [J. C. Hansen, K. Maeshima, M. J. Hendzel, Epigenetics Chromatin 14, 50 (2021); H. Strickfaden et al., Cell 183, 1772–1784.e1713 (2020)]. We sought to resolve these discrepancies, as our ability to translate with confidence these biophysical observations to cells requires their precise characterization. Moreover, whether chromatin assemblies are dynamic or static affects how processes such as transcription, loop extrusion, and remodeling will engage them inside cells. Here, we show in diverse conditions and without specific buffering components that chromatin fragments form phase separated fluids in vitro. We also explore how sample preparation and imaging affect the experimental observation of chromatin condensate dynamics. Last, we describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained chromatin movement observed in cells.