Transient crosslinking controls the condensate formation pathway within chromatin networks.

The network structure of densely packed chromatin within the nucleus of eukaryotic cells acts in concert with nonequilibrium processes. Using statistical physics simulations, we explore the control provided by transient crosslinking of the chromatin network by structural-maintenance-of-chromosome (SMC) proteins over (i) the physical properties of the chromatin network and (ii) condensate formation of embedded molecular species. We find that the density and lifetime of transient SMC crosslinks regulate structural relaxation modes and tune the sol-vs-gel state of the chromatin network, which imparts control over the kinetic pathway to condensate formation. Specifically, lower density, shorter-lived crosslinks induce sollike networks and a droplet-fusion pathway, whereas higher density, longer-lived crosslinks induce gellike networks and an Ostwald-ripening pathway.

Label-Free Techniques for Probing Biomolecular Condensates.

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time. In particular, we review recent years' exciting development of label-free techniques and methodologies. We broadly organize the set of tools into 3 categories: (1) imaging-based techniques, such as transmitted-light microscopy (TLM) and Brillouin microscopy (BM), (2) force spectroscopy techniques, such as atomic force microscopy (AFM) and the optical tweezer (OT), and (3) microfluidic platforms and emerging technologies. We point out the tools' key opportunities, challenges, and future perspectives and analyze their correlative potential as well as compatibility with other techniques. Additionally, we review emerging techniques, namely, differential dynamic microscopy (DDM) and interferometric scattering microscopy (iSCAT), that have huge potential for future applications in studying biomolecular condensates. Finally, we highlight how some of these techniques can be translated for diagnostics and therapy purposes. We hope this Review serves as a useful guide for new researchers in this field and aids in advancing the development of new biophysical tools to study biomolecular condensates.

Probing the surface charge of condensates using microelectrophoresis.

Biomolecular condensates play an important role in cellular organization. Coacervates are commonly used models that mimic the physicochemical properties of biomolecular condensates. The surface of condensates plays a key role in governing molecular exchange between condensates, accumulation of species at the interface, and the stability of condensates against coalescence. However, most important surface properties, including the surface charge and zeta potential, remain poorly characterized and understood. The zeta potential of coacervates is often measured using laser doppler electrophoresis, which assumes a size-independent electrophoretic mobility. Here, we show that this assumption is incorrect for liquid-like condensates and present an alternative method to study the electrophoretic mobility of coacervates and in vitro condensate models by microelectrophoresis and single-particle tracking. Coacervates have a size-dependent electrophoretic mobility, originating from their fluid nature, from which a well-defined zeta potential is calculated. Interestingly, microelectrophoresis measurements reveal that polylysine chains are enriched at the surface of polylysine/polyaspartic acid complex coacervates, which causes the negatively charged protein ɑ-synuclein to adsorb and accumulate at the interface. Addition of ATP inverts the surface charge, displaces ɑ-synuclein from the surface and may help to suppress its interface-catalyzed aggregation. Together, these findings show how condensate surface charge can be measured and altered, making this microelectrophoresis platform combined with automated single-particle tracking a promising characterization technique for both biomolecular condensates and coacervate protocells.

Emerging insights into transcriptional condensates.

Eukaryotic transcription, a fundamental process that governs cell-specific gene expression, has long been the subject of extensive investigations in the fields of molecular biology, biochemistry, and structural biology. Recent advances in microscopy techniques have led to a fascinating concept known as "transcriptional condensates." These dynamic assemblies are the result of a phenomenon called liquid‒liquid phase separation, which is driven by multivalent interactions between the constituent proteins in cells. The essential proteins associated with transcription are concentrated in transcriptional condensates. Recent studies have shed light on the temporal dynamics of transcriptional condensates and their potential role in enhancing the efficiency of transcription. In this article, we explore the properties of transcriptional condensates, investigate how they evolve over time, and evaluate the significant impact they have on the process of transcription. Furthermore, we highlight innovative techniques that allow us to manipulate these condensates, thus demonstrating their responsiveness to cellular signals and their connection to transcriptional bursting. As our understanding of transcriptional condensates continues to grow, they are poised to revolutionize our understanding of eukaryotic gene regulation.

Short-distance vesicle transport via phase separation.

In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.

Physiological and pathological effects of phase separation in the central nervous system.

Phase separation, also known as biomolecule condensate, participates in physiological processes such as transcriptional regulation, signal transduction, gene expression, and DNA damage repair by creating a membrane-free compartment. Phase separation is primarily caused by the interaction of multivalent non-covalent bonds between proteins and/or nucleic acids. The strength of molecular multivalent interaction can be modified by component concentration, the potential of hydrogen, posttranslational modification, and other factors. Notably, phase separation occurs frequently in the cytoplasm of mitochondria, the nucleus, and synapses. Phase separation in vivo is dynamic or stable in the normal physiological state, while abnormal phase separation will lead to the formation of biomolecule condensates, speeding up the disease progression. To provide candidate suggestions for the clinical treatment of nervous system diseases, this review, based on existing studies, carefully and systematically represents the physiological roles of phase separation in the central nervous system and its pathological mechanism in neurodegenerative diseases.

RNA-binding properties orchestrate TDP-43 homeostasis through condensate formation in vivo.

Insoluble cytoplasmic aggregate formation of the RNA-binding protein TDP-43 is a major hallmark of neurodegenerative diseases including Amyotrophic Lateral Sclerosis. TDP-43 localizes predominantly in the nucleus, arranging itself into dynamic condensates through liquid-liquid phase separation (LLPS). Mutations and post-translational modifications can alter the condensation properties of TDP-43, contributing to the transition of liquid-like biomolecular condensates into solid-like aggregates. However, to date it has been a challenge to study the dynamics of this process in vivo. We demonstrate through live imaging that human TDP-43 undergoes nuclear condensation in spinal motor neurons in a living animal. RNA-binding deficiencies as well as post-translational modifications can lead to aberrant condensation and altered TDP-43 compartmentalization. Single-molecule tracking revealed an altered mobility profile for RNA-binding deficient TDP-43. Overall, these results provide a critically needed in vivo characterization of TDP-43 condensation, demonstrate phase separation as an important regulatory mechanism of TDP-43 accessibility, and identify a molecular mechanism of how functional TDP-43 can be regulated.

Sequestration of translation initiation factors in p62 condensates.

Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.

Self-demixing of mRNA copies buffers mRNA:mRNA and mRNA:regulator stoichiometries.

Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.

The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

HIV-Induced CPSF6 Condensates.

Viruses are obligate parasites that rely on their host's cellular machinery for replication. To facilitate their replication cycle, many viruses have been shown to remodel the cellular architecture by inducing the formation of membraneless organelles (MLOs). Eukaryotic cells have evolved MLOs that are highly dynamic, self-organizing microenvironments that segregate biological processes and increase the efficiency of reactions by concentrating enzymes and substrates. In the context of viral infections, MLOs can be utilized by viruses to complete their replication cycle. This review focuses on the pathway used by the HIV-1 virus to remodel the nuclear landscape of its host, creating viral/host niches that enable efficient viral replication. Specifically, we discuss how the interaction between the HIV-1 capsid and the cellular factor CPSF6 triggers the formation of nuclear MLOs that support nuclear reverse transcription and viral integration in favored regions of the host chromatin. This review compiles current knowledge on the origin of nuclear HIV-MLOs and their role in early post-nuclear entry steps of the HIV-1 replication cycle.

A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

Liquid-liquid Phase Separation in Viral Function.

An emerging set of results suggests that liquid-liquid phase separation (LLPS) is the basis for the formation of membrane-less compartments in cells. Evidence is now mounting that various types of virus-induced membrane-less compartments and organelles are also assembled via LLPS. Specifically, viruses appear to use intracellular phase transitions to form subcellular microenvironments known as viral factories, inclusion bodies, or viroplasms. These compartments - collectively referred to as viral biomolecular condensates - can be used to concentrate replicase proteins, viral genomes, and host proteins that are required for virus replication. They can also be used to subvert or avoid the intracellular immune response. This review examines how certain DNA or RNA viruses drive the formation of viral condensates, the possible biological functions of those condensates, and the biophysical and biochemical basis for their assembly.

CASC3 Biomolecular Condensates Restrict Turnip Crinkle Virus by Limiting Host Factor Availability.

The exon-junction complex (EJC) plays a role in post-transcriptional gene regulation and exerts antiviral activity towards several positive-strand RNA viruses. However, the spectrum of RNA viruses that are targeted by the EJC or the underlying mechanisms are not well understood. EJC components from Arabidopsis thaliana were screened for antiviral activity towards Turnip crinkle virus (TCV, Tombusviridae). Overexpression of the accessory EJC component CASC3 inhibited TCV accumulation > 10-fold in Nicotiana benthamiana while knock-down of endogenous CASC3 resulted in a > 4-fold increase in TCV accumulation. CASC3 forms cytoplasmic condensates and deletion of the conserved SELOR domain reduced condensate size 7-fold and significantly decreased antiviral activity towards TCV. Mass spectrometry of CASC3 complexes did not identify endogenous stress granule or P-body markers and CASC3 failed to co-localize with an aggresome-specific dye suggesting that CASC3 condensates are distinct from well-established membraneless compartments. Mass spectrometry and bimolecular fluorescence complementation assays revealed that CASC3 sequesters Heat shock protein 70 (Hsp70-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two host factors with roles in tombusvirus replication. Overexpression of Hsp70-1 or GAPDH reduced the antiviral activity of CASC3 2.1-fold and 2.8-fold, respectively, and suggests that CASC3 inhibits TCV by limiting host factor availability. Unrelated Tobacco mosaic virus (TMV) also depends on Hsp70-1 and CASC3 overexpression restricted TMV accumulation 4-fold and demonstrates that CASC3 antiviral activity is not TCV-specific. Like CASC3, Auxin response factor 19 (ARF19) forms poorly dynamic condensates but ARF19 overexpression failed to inhibit TCV accumulation and suggests that CASC3 has antiviral activities that are not ubiquitous among cytoplasmic condensates.

Biomolecular Condensation of the Human Papillomavirus E2 Master Regulator with p53: Implications in Viral Replication.

p53 exerts its tumour suppressor activity by modulating hundreds of genes and it can also repress viral replication. Such is the case of human papillomavirus (HPV) through targeting the E2 master regulator, but the biochemical mechanism is not known. We show that the C-terminal DNA binding domain of HPV16 E2 protein (E2C) triggers heterotypic condensation with p53 at a precise 2/1 E2C/p53 stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Interestingly, transfection experiments show that E2 co-localizes with p53 in the nucleus with a grainy pattern, and recruits p53 to chromatin-associated foci, a function independent of the DNA binding capacity of p53 as judged by a DNA binding impaired mutant. Depending on the length, DNA can either completely dissolve or reshape heterotypic droplets into irregular condensates containing p53, E2C, and DNA, and reminiscent of that observed linked to chromatin. We propose that p53 is a scaffold for condensation in line with its structural and functional features, in particular as a promiscuous hub that binds multiple cellular proteins. E2 appears as both client and modulator, likely based on its homodimeric DNA binding nature. Our results, in line with the known role of condensation in eukaryotic gene enhancement and silencing, point at biomolecular condensation of E2 with p53 as a means to modulate HPV gene function, strictly dependent on host cell replication and transcription machinery.

A condensate dynamic instability orchestrates actomyosin cortex activation.

A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.

Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

Screening membraneless organelle participants with machine-learning models that integrate multimodal features.

Protein self-assembly is one of the formation mechanisms of biomolecular condensates. However, most phase-separating systems (PS) demand multiple partners in biological conditions. In this study, we divided PS proteins into two groups according to the mechanism by which they undergo PS: PS-Self proteins can self-assemble spontaneously to form droplets, while PS-Part proteins interact with partners to undergo PS. Analysis of the amino acid composition revealed differences in the sequence pattern between the two protein groups. Existing PS predictors, when evaluated on two test protein sets, preferentially predicted self-assembling proteins. Thus, a comprehensive predictor is required. Herein, we propose that properties other than sequence composition can provide crucial information in screening PS proteins. By incorporating phosphorylation frequencies and immunofluorescence image-based droplet-forming propensity with other PS-related features, we built two independent machine-learning models to separately predict the two protein categories. Results of independent testing suggested the superiority of integrating multimodal features. We performed experimental verification on the top-scored proteins DHX9, Ki-67, and NIFK. Their PS behavior in vitro revealed the effectiveness of our models in PS prediction. Further validation on the proteome of membraneless organelles confirmed the ability of our models to identify PS-Part proteins. We implemented a web server named PhaSePred (http://predict.phasep.pro/) that incorporates our two models together with representative PS predictors. PhaSePred displays proteome-level quantiles of different features, thus profiling PS propensity and providing crucial information for identification of candidate proteins.