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Phase separation of biomolecules into condensates has emerged as a mechanism for intracellular organization and affects many intracellular processes, including reaction pathways through the clustering of enzymes and pathway intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here we show that both native and synthetic condensates display an exponential size distribution, which is captured by Monte Carlo simulations of fast nucleation followed by coalescence. In contrast, pathological aggregates exhibit a power-law size distribution. These distinct behaviours reflect the relative importance of nucleation and coalescence kinetics. We demonstrate this by utilizing a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. The appearance of exponential distributions for abrupt nucleation versus power-law distributions under continuous nucleation may reflect a general principle that determines condensate size distributions.

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timings of gene induction during the earliest phases of embryogenesis. Here we employ high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate, Ciona intestinalis with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by derepression of the ERF repressor by ERK activity, which works in concert with localized activators such as Foxa.a. Absence of ERF results in derepression of target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the 8- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also employed by other vertebrates.

Endogenous biomolecular condensates, comprised of a multitude of proteins and RNAs, can organize into multiphasic structures, with compositionally-distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here, we utilizein vivocondensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein region (IDR) results in enhanced immiscibility and multiphase formation. Interestingly, we found that oligomerization tunes the miscibility of IDRs in an asymmetric manner, with the effect being more pronounced when the IDR exhibiting stronger homotypic IDR interactions is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism which cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

SummaryBiomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce VECTOR (ViscoElasticChromatinTethering andORganization), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture.

The mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes collectively represent one of the most frequently mutated cellular entities in cancer, second only to TP53. Mutations across the 29 human genes that encode mSWI/SNF complex subunits occur in over 20% of human cancers, with mutations affecting ARID1A and ARID1B, the largest BAF subunits, being the most frequent. However, the functional contributions of these subunits, particularly their commonly mutated N-terminal intrinsically disordered regions (IDRs) and a highly conserved ARID DNA-binding domain, to BAF function remain poorly understood. Here, we demonstrate that the IDRs of ARID1A/B, coupled with the ARID domain, drive biomolecular condensate formation and BAF chromatin localization in cells. We define ARID1A/B IDRs as two-part systems, facilitating homotypic BAF complex interactions (i.e., valence generated by localized condensation) and heterotypic complex interactions, that together establish a highly specific, sequence-encoded protein interaction network within condensates. Both types of interactions are required for appropriate genome-wide targeting of BAF complexes, DNA accessibility generation, and appropriate gene expression. Replacement of the ARID1A N-terminal IDR with IDRs derived from two unrelated proteins FUS and DDX4, rescues generic condensation of BAF but not chromatin occupancy, DNA accessibility, and heterotypic interactions, highlighting the sequence-specificity embedded in the IDR of ARID1A. Taken together, these data establish a role for the largest and most frequently perturbed IDRs within a major chromatin remodeler and explain how biomolecular condensate formation enables both genomic localization and functional partner recruitment. Furthermore, these findings lay the groundwork for mapping IDR sequence specificity or “grammar”, that dictates the co-condensation network formation, and suggests that targeted disruption of these mechanisms may represent new targeted therapeutic opportunities across multiple cancers. Citation Format: Ajinkya Patil, Amy R. Strom, Clayton K. Collings, Joao A. Paulo, Tobias Wauer, Akshay Sankar, Jessica D. St.Laurent, Kasey S. Cervantes, Steven P. Gygi, Clifford P. Brangwynne, Cigall Kadoch. Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3485.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

DNA is organized into chromatin, a complex polymeric material that stores information and controls gene expression. An emerging mechanism for biological organization, particularly within the crowded nucleus, is biomolecular phase separation into condensed droplets of protein and nucleic acids. However, the way in which chromatin impacts the dynamics of phase separation and condensate formation is poorly understood. Here we utilize a powerful optogenetic strategy to examine the interplay of droplet coarsening with the surrounding viscoelastic chromatin network. We demonstrate that droplet growth dynamics are directly inhibited by the chromatin-dense environment, which gives rise to an anomalously slow coarsening exponent, β ≈ 0.12, contrasting with the classical prediction of β = 1/3. Using scaling arguments and simulations, we show how this arrested growth can arise due to subdiffusion of individual condensates, predicting β ≈ α/3, where α is the diffusive exponent. Tracking the fluctuating motion of condensates within chromatin reveals a subdiffusive exponent, α ≈ 0.5, which explains the anomalous coarsening behaviour and is also consistent with Rouse-like dynamics arising from the entangled chromatin. Our findings have implications for the biophysical regulation of the size and shape of biomolecular condensates and suggest that condensate emulsions can be used to probe the viscoelastic mechanical environment within living cells. Biomolecules in the cell nucleus form condensates at a rate slower than that predicted by the theory of droplet growth. Experiments on living cells attribute this anomalous coarsening behaviour to subdiffusive dynamics in the crowded nucleus.

Membraneless organelles or condensates form through liquid–liquid phase separation1–4, which is thought to underlie gene transcription through condensation of the large-scale nucleolus5–7 or in smaller assemblies known as transcriptional condensates8–11. Transcriptional condensates have been hypothesized to phase separate at particular genomic loci and locally promote the biomolecular interactions underlying gene expression. However, there have been few quantitative biophysical tests of this model in living cells, and phase separation has not yet been directly linked with dynamic transcriptional outputs12,13. Here, we apply an optogenetic approach to show that FET-family transcriptional regulators exhibit a strong tendency to phase separate within living cells, a process that can drive localized RNA transcription. We find that TAF15 has a unique charge distribution among the FET family members that enhances its interactions with the C-terminal domain of RNA polymerase II. Nascent C-terminal domain clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA polymerase II to drive transcriptional output. These results suggest that positive feedback between interacting transcriptional components drives localized phase separation to amplify gene expression. Wei et al. show that clusters of unphosphorylated RNA polymerase II seed the nucleation of phase-separated condensates of TAF15, which further recruit RNA polymerase II to amplify transcriptional activation.

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid–liquid phase separation (LLPS)1,2. Biomolecular interactions—particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions—are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3–6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7–9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates—including the nucleolus, Cajal bodies, stress granules and P-bodies—implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates. Heterotypic multicomponent interactions are shown to dominate the liquid–liquid phase separation that enables the formation of intracellular condensates.

I.ABSTRACTA hallmark of biomolecular condensates formed via liquid-liquid phase separation is the dynamic exchange of condensate components with the surroundings. Such material exchange can be key to condensate function, as the rate of component exchange can impact biochemical reaction rates in the condensates, the speed of response of condensates to a changing environment, and their number and sizes. How is this exchange rate controlled? Intuitively, this rate can be limited by the flux of materials from the dilute phase or by the speed of mixing inside the dense phase. Surprisingly, recent experiments suggest that this rate can also be limited by the dynamics of molecules at the droplet interface, implying the existence of an “interface resistance”. Here, we combined theory and simulation to show that an interface resistance can arise when incident molecules contact the interface without entering the dense phase. We find that such “bouncing” occurs for incident molecules that are capable of self-collapse into a non-sticking conformation. Our work highlights the underappreciated role of interface resistance in condensate exchange dynamics, with implications for natural and synthetic systems.

Phase separation of biomolecules into condensates has emerged as a ubiquitous mechanism for intracellular organization and impacts many intracellular processes, including reaction pathways through clustering of enzymes and their intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here, we utilize a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. We find that both native nuclear speckles and FUS condensates formed with the synthetic Corelet system obey an exponential size distribution, which can be recapitulated in Monte Carlo simulations of fast nucleation followed by coalescence. By contrast, pathological aggregation of cytoplasmic Huntingtin polyQ protein exhibits a power-law size distribution, with an exponent of −1.41 ± 0.02. These distinct behaviors reflect the relative importance of nucleation and coalescence kinetics: introducing continuous condensate nucleation into the Monte Carlo coarsening simulations gives rise to polyQ-like power-law behavior. We demonstrate that the emergence of power-law distributions under continuous nucleation reflects a “rich get richer” effect, whose extent may play a general role in the determination of condensate size distributions.

The nucleolus facilitates transcription, processing, and assembly of ribosomal RNA (rRNA), the most abundant RNA in cells. Nucleolar function is facilitated by its multiphase liquid properties, but nucleolar fluidity and its connection to ribosome biogenesis remain unclear. Here, we used quantitative imaging, mathematical modeling, and pulse-chase nucleotide labelling to map nucleolar rRNA dynamics. Inconsistent with a purely diffusive process, rRNA steadily expands away from the transcriptional sites, moving in a slow (~1Å/s), radially-directed fashion. This motion reflects the viscoelastic properties of a highly concentrated gel of entangled rRNA, whose constant polymerization drives steady outward flow. We propose a new viscoelastic rRNA release model, where nucleolar rRNA cleavage and processing reduce entanglement, fluidizing the nucleolar periphery to facilitate release of mature pre-ribosomal particles.

The cell nucleus can be thought of as a complex, dynamic, living material, which functions to organize and protect the genome and coordinate gene expression. These functions are achieved via intricate mechanical and biochemical interactions among its myriad components, including the nuclear lamina, nuclear bodies, and the chromatin itself. While the biophysical organization of the nuclear lamina and chromatin have been thoroughly studied, the concept that liquid–liquid phase separation and related phase transitions play a role in establishing nuclear structure has emerged only recently. Phase transitions are likely to be intimately coupled to the mechanobiology of structural elements in the nucleus, but their interplay with one another is still not understood. Here, we review recent developments on the role of phase separation and mechanics in nuclear organization and discuss the functional implications in cell physiology and disease states.

Eukaryotic cytoplasm organizes itself via both membrane-bound organelles and membrane-less biomolecular condensates (BMCs). Known BMCs exhibit liquid-like properties and are typically visualized on the scale of ~1 μm. They have been studied mostly by microscopy, examining select individual proteins. Here, we investigate the global organization of native cytoplasm with quantitative proteomics, using differential pressure filtration, size exclusion, and dilution experiments. These assays reveal that BMCs form throughout the cytosplasm, predominantly at the mesoscale of ~100 nm. Our data indicate that at least 18% of the proteome is organized via such mesoscale BMCs, suggesting that cells widely employ dynamic liquid-like clustering to organize their cytoplasm, at surprisingly small length scales.

All structures within living cells must form at the right time and place. This includes condensates such as the nucleolus, Cajal bodies and stress granules, which form via liquid–liquid phase separation of biomolecules, particularly proteins enriched in intrinsically disordered regions (IDRs)1,2. In non-living systems, the initial stages of nucleated phase separation arise when thermal fluctuations overcome an energy barrier due to surface tension. This phenomenon can be modelled by classical nucleation theory (CNT), which describes how the rate of droplet nucleation depends on the degree of supersaturation, whereas the location at which droplets appear is controlled by interfacial heterogeneities3,4. However, it remains unknown whether this framework applies in living cells, owing to the multicomponent and highly complex nature of the intracellular environment, including the presence of diverse IDRs, whose specificity of biomolecular interactions is unclear5–8. Here we show that despite this complexity, nucleation in living cells occurs through a physical process similar to that in inanimate materials, but the efficacy of nucleation sites can be tuned by their biomolecular features. By quantitatively characterizing the nucleation kinetics of endogenous and biomimetic condensates in living cells, we find that key features of condensate nucleation can be quantitatively understood through a CNT-like theoretical framework. Nucleation rates can be substantially enhanced by compatible biomolecular (IDR) seeds, and the kinetics of cellular processes can impact condensate nucleation rates and specificity of location. This quantitative framework sheds light on the intracellular nucleation landscape, and paves the way for engineering synthetic condensates precisely positioned in space and time. Experiments using endogenous and biomimetic condensates in cells show that nucleation in cells resembles the physical process in inanimate materials, but is tuned by biomolecular features.

Liquid-liquid phase separation and related phase transitions have emerged as generic mechanisms in living cells for the formation of membraneless compartments or biomolecular condensates. The surface between two immiscible phases has an interfacial tension, generating capillary forces that can perform work on the surrounding environment. Here we present the physical principles of capillarity, including examples of how capillary forces structure multiphase condensates and remodel biological substrates. As with other mechanisms of intracellular force generation, for example, molecular motors, capillary forces can influence biological processes. Identifying the biomolecular determinants of condensate capillarity represents an exciting frontier, bridging soft matter physics and cell biology.

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.

Cells compartmentalize their intracellular environment to orchestrate countless simultaneous biochemical processes. Many intracellular tasks rely on membrane-less organelles, multicomponent condensates that assemble by liquid-liquid phase separation. A decade of intensive research has provided a basic understanding of the biomolecular driving forces underlying the form and function of such organelles. Here we review the technologies enabling these developments, along with approaches to designing spatiotemporally actuated organelles based on multivalent low-affinity interactions. With these recent advances, it is now becoming possible both to modulate the properties of native condensates and to engineer entirely new structures, with the potential for widespread biomedical and biotechnological applications.

Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies., SCOPUS: ar.j, info:eu-repo/semantics/published

Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington's disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease.

Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction and chain length influence the phase behavior and regulate the formation of finite-size aggregates preempting macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a “soft” criterion for predicting the underlying phase transition of a model protein. Additionally, we find that this order parameter is strongly correlated to the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveals that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results predict that classical LLPS should be the dominant phase transition for disordered proteins and suggests a possible reason behind recent findings of widespread phase separation throughout living cells.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165Eindicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically-generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that crosslinked chromatin can mechanically suppress droplets’ coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

SummaryMany enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ∼6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.HighlightsCell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19Drug screen reveals critical role for membrane lipid composition in fusionSpike’s unusual membrane-proximal cysteines and aromatics are essential for fusionCholesterol tunes relative infectivity of SARS-CoV-2 viral particles

All structures within living cells must form at the right time and place. This includes condensates such as the nucleolus, Cajal bodies and stress granules, which form via liquid-liquid phase separation of biomolecules, particularly proteins enriched in intrinsically disordered regions (IDRs)

The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid–liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction. The nucleolus is a membraneless organelle involved in ribonucleoprotein assembly, including ribosome biogenesis. Recent evidence indicates that the nucleolus is a biomolecular condensate that forms via liquid–liquid phase separation (LLPS), and insights from studies within the LLPS framework are increasing our understanding of the relationship between nucleolar structure and function.

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed by several microtubule nucleation pathways. In the spindle center, Targeting Protein for XKlp2 (TPX2) promotes branching microtubule nucleation, where new microtubules are nucleated from pre-existing ones. Until the onset of spindle assembly, TPX2 is sequestered by importins-α/β, yet the molecular nature of this regulation remains unclear, particularly since TPX2 was recently found to undergo a liquid-liquid phase separation to execute its function. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity as a 1:1:1 mono-dispersed trimer. We identify a new nuclear localization sequence (NLS) on TPX2, which contributes to its high-affinity interaction with importin-α. Interestingly, importin-β alone can also associate with TPX2, and does so via dispersed, weak interactions. Interactions of both importin-α and importin-β with TPX2 each inhibit its propensity for phase separation, and consequently its ability to orchestrate branching microtubule nucleation. In sum, our study explains how TPX2 is regulated in order to facilitate spindle assembly, and provides novel insight into how a protein phase separation can be inhibited via weak biomolecular interactions.Significance StatementThe discovery that proteins can undergo phase separation is revolutionizing biology. Characterization of dozens of phase separating proteins in vitro over the past several years has mainly focused on how macromolecules undergo liquid-liquid phase separation (LLPS). The next, and possibly bigger challenge is to investigate how LLPS is regulated in the cell, namely how it is inhibited to spatiotemporally control a certain cellular function. Here, we addressed this challenge by identifying how the spindle assembly factor TPX2 is inhibited by importins from undergoing LLPS and thereby turning on spindle assembly.

Structural organization of the genome into transcriptionally active euchromatin and silenced heterochromatin is essential for eukaryotic cell function. Heterochromatin is a more compact form of chromatin, and is associated with characteristic post-translational histone modifications and chromatin binding proteins. Phase-separation has recently been suggested as a mechanism for heterochromatin formation, through condensation of heterochromatin associated proteins. However, it is unclear how phase-separated condensates can contribute to stable and robust repression, particularly for heritable epigenetic changes. The Polycomb complex PRC1 is known to be key for heterochromatin formation, but the multitude of Polycomb proteins has hindered our understanding of their collective contribution to chromatin repression. Here, we take a quantitative live cell imaging approach to show that PRC1 proteins form multicomponent condensates through hetero-oligomerization. They preferentially seed at H3K27me3 marks, and subsequently write H2AK119Ub marks. Using optogenetics to nucleate local Polycomb condensates, we show that Polycomb phase separation can induce chromatin compaction, but phase separation is dispensable for maintenance of the compacted state. Our data are consistent with a model in which the time integral of historical Polycomb phase separation is progressively recorded in repressive histone marks, which subsequently drive chromatin compaction. These findings link the equilibrium thermodynamics of phase separation with the fundamentally non-equilibrium concept of epigenetic memory.

The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid-liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction.

There is emerging evidence for transcription condensates in the activation of gene expression1–3. However, there is considerably less information regarding transcriptional repression, despite its pervasive importance in regulating gene expression in development and disease. Here, we explore the role of liquid-liquid phase separation (LLPS) in the organization of the Groucho/TLE (Gro) family of transcriptional corepressors, which interact with a variety of sequence-specific repressors such as Hes/Hairy4. Gro-dependent repressors have been implicated in a variety of developmental processes, including segmentation of theDrosophilaembryo and somitogenesis in vertebrates. These repressors bind to specific recognition sequences, but instead of interacting with coactivators (e.g., Mediator) they recruit Gro corepressors5. Gro contains a series of WD40 repeats that are thought to mediate oligomerization6. How putative Hes/Gro oligomers repress transcription has been the subject of numerous studies5, 6. Here we show that Hes/Gro complexes form discrete puncta within nuclei of livingCionaembryos. These puncta rapidly dissolve during the onset of mitosis and reappear in the ensuing cell cycle. Modified Hes/Gro complexes that are unable to bind DNA exhibit the properties of viscous liquid droplets, similar to those underlying the biogenesis of P-granules inC. elegans7and nucleoli inXenopusoocytes8. These observations provide vivid evidence for LLPS in the control of gene expression and suggest a simple physical exclusion mechanism for transcriptional repression. WD40 repeats have been implicated in a wide variety of cellular processes in addition to transcriptional repression9. We suggest that protein interactions using WD40 motifs might be a common feature of processes reliant on LLPS.

Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine invivo biophysical interrogation and invitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA invitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors.We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence-dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction, and chain length impact the formation of finite-size aggregates, which can preempt macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a "soft" predictive criterion for distinguishing when a model protein undergoes macroscopic phase separation vs finite aggregation. Additionally, we find that this order parameter is strongly correlated with the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveal that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results suggest that classical LLPS should be the primary phase transition for disordered proteins when short-ranged attractive interactions dominate and suggest a possible reason behind recent findings of widespread phase separation throughout living cells.