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Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles. Here, we demonstrate the design of pure RNA condensates from nanostructured, star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via linear overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through systematic sequence design, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful co-transcriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles for the task of compartmentalizing molecules and biochemical reactions.

Artificial biomolecular condensates are emerging as a versatile approach to organize molecular targets and reactions without the need for lipid membranes. Here we ask whether the temporal response of artificial condensates can be controlled via designed chemical reactions. We address this general question by considering a model problem in which a phase separating component participates in reactions that dynamically activate or deactivate its ability to self-attract. Through a theoretical model we illustrate the transient and equilibrium effects of reactions, linking condensate response and reaction parameters. We experimentally realize our model problem using star-shaped DNA motifs known as nanostars to generate condensates, and we take advantage of strand invasion and displacement reactions to kinetically control the capacity of nanostars to interact. We demonstrate reversible dissolution and growth of DNA condensates in the presence of specific DNA inputs, and we characterize the role of toehold domains, nanostar size, and nanostar valency. Our results will support the development of artificial biomolecular condensates that can adapt to environmental changes with prescribed temporal dynamics.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on-and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are dynamic membraneless organelles that compartmentalize proteins and RNA molecules to regulate key cellular processes. Diverse RNA species exert their effects on the cell by their roles in condensate formation and function. RNA abnormalities such as overexpression, modification, and mislocalization can lead to pathological condensate behaviors that drive various diseases, including cancer, neurological disorders, and infections. Here, we review RNA's role in condensate biology, describe the mechanisms of RNA-induced condensate dysregulation, note the implications for disease pathogenesis, and discuss novel therapeutic strategies. Emerging approaches to targeting RNA within condensates, including small molecules and RNA-based therapies that leverage the unique properties of condensates, may revolutionize treatment for complex diseases. [Display omitted] Han et al. review the role of RNA in biomolecular condensates, highlighting how RNA-induced dysregulation contributes to disease pathogenesis. They discuss emerging therapeutic strategies targeting RNA within condensates, which could revolutionize treatment for complex diseases like cancer and neurological disorders. [ABSTRACT FROM AUTHOR]

The coacervation of alpha‐synuclein (αSyn) into cytotoxic oligomers and amyloid fibrils are considered pathological hallmarks of Parkinson's disease. While aggregation is central to amyloid diseases, liquid–liquid phase separation (LLPS) and its interplay with aggregation have gained increasing interest. Previous work shows that factors promoting or inhibiting aggregation have similar effects on LLPS. This study provides a detailed scanning of a wide range of parameters, including protein, salt and crowding concentrations at multiple pH values, revealing different salt dependencies of aggregation and LLPS. The influence of salt on aggregation under crowding conditions follows a non‐monotonic pattern, showing increased effects at medium salt concentrations. This behavior can be elucidated through a combination of electrostatic screening and salting‐out effects on the intramolecular interactions between the N‐terminal and C‐terminal regions of αSyn. By contrast, this study finds a monotonic salt dependence of LLPS due to intermolecular interactions. Furthermore, it observes time evolution of the two distinct assembly states, with macroscopic fibrillar‐like bundles initially forming at medium salt concentration but subsequently converting into droplets after prolonged incubation. The droplet state is therefore capable of inhibiting aggregation or even dissolving aggregates through heterotypic interactions, thus preventing αSyn from its dynamically arrested state. [ABSTRACT FROM AUTHOR]

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) have become an extensive mechanism of macromolecular metabolism and biochemical reactions in cells. Large molecules like proteins and nucleic acids will spontaneously aggregate and assemble into droplet-like structures driven by LLPS when the physical and chemical properties of cells are altered. LLPS provides a mature molecular platform for innate immune response, which tightly regulates key signaling in liver immune response spatially and physically, including DNA and RNA sensing pathways, inflammasome activation, and autophagy. Take this, LLPS plays a promoting or protecting role in a range of liver diseases, such as viral hepatitis, non-alcoholic fatty liver disease, liver fibrosis, hepatic ischemia-reperfusion injury, autoimmune liver disease, and liver cancer. This review systematically describes the whole landscape of LLPS in liver innate immunity. It will help us to guide a better-personalized approach to LLPS-targeted immunotherapy for liver diseases. [ABSTRACT FROM AUTHOR]

The purpose of this review is to highlight transformative advances that have been made in the field of biomolecular condensates, with special emphasis on condensate material properties, physiology, and kinases, using the With-No-Lysine (WNK) kinases as a prototypical example. To convey how WNK kinases illustrate important concepts for biomolecular condensates, we start with a brief history, focus on defining features of biomolecular condensates, and delve into some examples of how condensates are implicated in cellular physiology (and pathophysiology). We then highlight how WNK kinases, through the action of "WNK droplets" that ubiquitously regulate intracellular volume and kidney-specific "WNK bodies" that are implicated in distal tubule salt reabsorption and potassium homeostasis, exemplify many of the defining features of condensates. Finally, this review addresses the controversies within this emerging field and questions to address. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid–liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated. [ABSTRACT FROM AUTHOR]

Phase separation in aqueous solutions of macromolecules underlies the generation of biomolecular condensates in cells. Condensates are membraneless bodies, representing dense, macromolecule-rich phases that coexist with the dilute, macromolecule-deficient phases. In cells, condensates comprise hundreds of different macromolecular and small molecule solutes. How do different solutes contribute to the driving forces for phase separation? To answer this question, we introduce a formalism we term energy dominance analysis. This approach rests on analysis of shapes of the dilute phase boundaries, slopes of tie lines, and changes to dilute phase concentrations in response to perturbations of concentrations of different solutes. The framework is based solely on conditions for phase equilibria in systems with arbitrary numbers of macromolecules and solution components. Its practical application relies on being able to measure dilute phase concentrations of the components of interest. The dominance framework is both theoretically facile and experimentally applicable. We present the formalism that underlies dominance analysis and establish its accuracy and flexibility by deploying it to analyze phase diagrams probed in simulations and in experiments. [ABSTRACT FROM AUTHOR]

Cleavage and polyadenylation specificity factor subunit 6 (CPSF6, also known as CFIm68) is a 68 kDa component of the mammalian cleavage factor I (CFIm) complex. CPSF6 plays a role in maturation of the 3′ untranslated regions (UTR) of pre-mRNAs modulating mRNA alternative polyadenylation (APA), which determines the length of the 3′ UTR. HIV-1 induces translocation of CPSF6 and CPSF5 from nuclear paraspeckles to speckles, which are biomolecular condensates. Since depletion of CPSF6 modestly affects the ability of HIV-1 to replicate in primary cells, we hypothesize that the translocation of CPSF6 to speckles is a viral mechanism used to affect APA, which results in the modulation of cellular expression in favor of the virus, and may have important consequences in the identification of latent viral reservoirs. HIV-1-induced translocation of CPSF6 to biomolecular condensates may induce a change in the biochemical properties of the protein. Cleavage and polyadenylation specificity factor subunit 6 (CPSF6, also known as CFIm68) is a 68 kDa component of the mammalian cleavage factor I (CFIm) complex that modulates mRNA alternative polyadenylation (APA) and determines 3′ untranslated region (UTR) length, an important gene expression control mechanism. CPSF6 directly interacts with the HIV-1 core during infection, suggesting involvement in HIV-1 replication. Here, we review the contributions of CPSF6 to every stage of the HIV-1 replication cycle. Recently, several groups described the ability of HIV-1 infection to induce CPSF6 translocation to nuclear speckles, which are biomolecular condensates. We discuss the implications for CPSF6 localization in condensates and the potential role of condensate-localized CPSF6 in the ability of HIV-1 to control the protein expression pattern of the cell. [ABSTRACT FROM AUTHOR]

Abstract Biomolecular condensates formed by liquid-liquid phase separation (LLPS) have become an extensive mechanism of macromolecular metabolism and biochemical reactions in cells. Large molecules like proteins and nucleic acids will spontaneously aggregate and assemble into droplet-like structures driven by LLPS when the physical and chemical properties of cells are altered. LLPS provides a mature molecular platform for innate immune response, which tightly regulates key signaling in liver immune response spatially and physically, including DNA and RNA sensing pathways, inflammasome activation, and autophagy. Take this, LLPS plays a promoting or protecting role in a range of liver diseases, such as viral hepatitis, non-alcoholic fatty liver disease, liver fibrosis, hepatic ischemia-reperfusion injury, autoimmune liver disease, and liver cancer. This review systematically describes the whole landscape of LLPS in liver innate immunity. It will help us to guide a better-personalized approach to LLPS-targeted immunotherapy for liver diseases.

Plants, as sessile organisms, face the crucial challenge of adjusting growth and development with ever-changing environmental conditions. Protein synthesis is the fundamental process that enables growth of all organisms. Since elevated temperature presents a substantial threat to protein stability and function, immediate adjustments of protein synthesis rates are necessary to circumvent accumulation of proteotoxic stress and to ensure survival. This review provides an overview of the mechanisms that control translation under high-temperature stress by the modification of components of the translation machinery in plants, and compares them to yeast and metazoa. Recent research also suggests an important role for cytoplasmic biomolecular condensates, named stress granules, in these processes. Current understanding of the role of stress granules in translational regulation and of the molecular processes associated with translation that might occur within stress granules is also discussed. [ABSTRACT FROM AUTHOR]

At the earliest development of prebiotic chemistry, bacterial cells were primarily viewed as "bags of molecules." This longstanding viewpoint shaped and biased early research about life's origins, setting an initial target when considering the path from prebiotic chemistry to modern life. The two fields of systems chemistry and bacterial cell biology seem like oil and water, but each brings their own perspectives and methods to consider "what is life?". Here, we review the most recent discoveries in bacterial cell biology, focusing on biomolecular condensates to consider how they may impact our thinking of matter‐to‐life transitions. The presence of condensate compartments in the bacterial domain of life strengthens the hypothesis that condensates play roles in coordinating chemical systems in life's origins. Bacterial condensates have been shown to enhance enzymatic reactions, tune substrate specificity, and be responsive to environmental conditions and metabolites. Systems chemistry studies have further illuminated the unique chemical environment within condensates and strategies for logically tying chemical processes to the formation and dissolution of condensates. We consider the potential of biomolecular condensates to provide "incubator spaces" where new chemistries can develop and examine future challenges regarding the capability of condensates to yield emergent chemical systems capable of selection. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, gene transcription typically occurs in discrete periods of promoter activity, interspersed with intervals of inactivity. This pattern deviates from simple stochastic events and warrants a closer examination of the molecular interactions that activate the promoter. Recent studies have identified transcription factor (TF) clusters as key precursors to transcriptional bursting. Often, these TF clusters form at chromatin segments that are physically distant from the promoter, making changes in chromatin conformation crucial for promoter–TF cluster interactions. In this review, I explore the formation and constituents of TF clusters, examining how the dynamic interplay between chromatin architecture and TF clustering influences transcriptional bursting. Additionally, I discuss techniques for visualizing TF clusters and provide an outlook on understanding the remaining gaps in this field. [ABSTRACT FROM AUTHOR]

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid‐liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion‐induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL‐3 and Ddx4. Uniquely accessible to this method, we find an unexpected re‐entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α‐synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS‐modifying compounds can be screened highly efficiently. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is an extremely aggressive primary brain tumor with poor prognosis, short survival time post-diagnosis and high recurrence. Currently, no cure for GBM exists. The identification of an effective therapeutic modality for GBM remains a high priority amongst medical professionals and researches. In recent studies, inhalant cannabidiol (CBD) has demonstrated promise in effectively inhibiting GBM tumor growth. However, exactly how CBD treatment affects the physiology of these tumor cells remains unclear. Stress granules (SG) (a sub-class of biomolecular condensates (BMC)) are dynamic, membrane-less intracellular microstructures which contain proteins and nucleic acids. The formation and signaling of SGs and BMCs plays a significant role in regulating malignancies. This study investigates whether inhaled CBD may play an intervening role towards SGs in GBM tumor cells. Integrated bioinformatics approaches were preformed to gain further insights. This includes use of Immunohistochemistry and flow cytometry to measure SGs, as well as expression and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The findings of this study reveal that CBD receptors (and co-regulated genes) have the potential to play an important biological role in the formation of BMCs within GBM. In this experiment, CBD treatment significantly increased the volume of TIAR-1. This increase directly correlated with elevation in both eIF2α expression and p-eIF2α in CBD treated tissues in comparison to the placebo group (p < 0.05). These results suggest that inhalant CBD significantly up-regulated SGs in GBM, and thus support a theory of targeting BMCs as a potential therapeutic substrate for treating GBM. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are highly versatile membraneless organelles involved in a plethora of cellular processes. Recent years have witnessed growing evidence of the interaction of these droplets with membrane-bound cellular structures. Condensates' adhesion to membranes can cause their mutual molding and regulation, and their interaction is of fundamental relevance to intracellular organization and communication, organelle remodeling, embryogenesis, and phagocytosis. In this article, we review advances in the understanding of membrane–condensate interactions, with a focus on in vitro models. These minimal systems allow the precise characterization and tuning of the material properties of both membranes and condensates and provide a workbench for visualizing the resulting morphologies and quantifying the interactions. These interactions can give rise to diverse biologically relevant phenomena, such as molecular-level restructuring of the membrane, nano- to microscale ruffling of the condensate–membrane interface, and coupling of the protein and lipid phases. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are reversible compartments that form through a process called phase separation. Post-translational modifications like ADP-ribosylation can nucleate the formation of these condensates by accelerating the self-association of proteins. Poly(ADP-ribose) (PAR) chains are remarkably transient modifications with turnover rates on the order of minutes, yet they can be required for the formation of granules in response to oxidative stress, DNA damage, and other stimuli. Moreover, accumulation of PAR is linked with adverse phase transitions in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In this review, we provide a primer on how PAR is synthesized and regulated, the diverse structures and chemistries of ADP-ribosylation modifications, and protein-PAR interactions. We review substantial progress in recent efforts to determine the molecular mechanism of PAR-mediated phase separation, and we further delineate how inhibitors of PAR polymerases may be effective treatments for neurodegenerative pathologies. Finally, we highlight the need for rigorous biochemical interrogation of ADP-ribosylation in vivo and in vitro to clarify the exact pathway from PARylation to condensate formation.

Abstract The coacervation of alpha‐synuclein (αSyn) into cytotoxic oligomers and amyloid fibrils are considered pathological hallmarks of Parkinson's disease. While aggregation is central to amyloid diseases, liquid–liquid phase separation (LLPS) and its interplay with aggregation have gained increasing interest. Previous work shows that factors promoting or inhibiting aggregation have similar effects on LLPS. This study provides a detailed scanning of a wide range of parameters, including protein, salt and crowding concentrations at multiple pH values, revealing different salt dependencies of aggregation and LLPS. The influence of salt on aggregation under crowding conditions follows a non‐monotonic pattern, showing increased effects at medium salt concentrations. This behavior can be elucidated through a combination of electrostatic screening and salting‐out effects on the intramolecular interactions between the N‐terminal and C‐terminal regions of αSyn. By contrast, this study finds a monotonic salt dependence of LLPS due to intermolecular interactions. Furthermore, it observes time evolution of the two distinct assembly states, with macroscopic fibrillar‐like bundles initially forming at medium salt concentration but subsequently converting into droplets after prolonged incubation. The droplet state is therefore capable of inhibiting aggregation or even dissolving aggregates through heterotypic interactions, thus preventing αSyn from its dynamically arrested state.

A hallmark of biomolecular condensates formed via liquid-liquid phase separation is that they dynamically exchange material with their surroundings, and this process can be crucial to condensate function. Intuitively, the rate of exchange can be limited by the flux from the dilute phase or by the mixing speed in the dense phase. Surprisingly, a recent experiment suggests that exchange can also be limited by the dynamics at the droplet interface, implying the existence of an ‘interface resistance’. Here, we first derive an analytical expression for the timescale of condensate material exchange, which clearly conveys the physical factors controlling exchange dynamics. We then utilize sticker-spacer polymer models to show that interface resistance can arise when incident molecules transiently touch the interface without entering the dense phase, i.e., the molecules ‘bounce’ from the interface. Our work provides insight into condensate exchange dynamics, with implications for both natural and synthetic systems.

Liquid-liquid phase separation (LLPS) is integral to various biological processes, facilitating signal transduction by creating a condensed, membrane-less environment that plays crucial roles in diverse physiological and pathological processes. Recent evidence has underscored the significance of LLPS in human health and disease. However, its implications in respiratory diseases remain poorly understood. This review explores current insights into the mechanisms and biological roles of LLPS, focusing particularly on its relevance to respiratory diseases, aiming to deepen our understanding and propose a new paradigm for studying phase separation in this context.

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

A recent study in our group reports a new “condensates to VPS41-associated phagic vacuole (VAPVs) conversion pathway” that is essential for macroautophagy/autophagy degradation in plant cells. Here, we compare the autophagy process between plants and other eukaryotic systems and discuss the potential roles of biomolecular condensates and synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in plant autophagy. [ABSTRACT FROM AUTHOR]

Abstract The phenomenon of phase separation is quite common in cells, and it is involved in multiple processes of life activities. However, the current research on the correlation between protein modifications and phase separation and the interference with the tendency of phase separation has some limitations. Here we focus on several post-translational modifications of proteins, including protein phosphorylation modification at multiple sites, methylation modification, acetylation modification, ubiquitination modification, SUMOylation modification, etc., which regulate the formation of phase separation and the stability of phase separation structure through multivalent interactions. This regulatory role is closely related to the development of neurodegenerative diseases, tumors, viral infections, and other diseases, and also plays essential functions in environmental stress, DNA damage repair, transcriptional regulation, signal transduction, and cell homeostasis of living organisms, which provides an idea to explore the interaction between novel protein post-translational modifications and phase separation. Video Abstract

Abstract Phase separation is a cellular phenomenon where macromolecules aggregate or segregate, giving rise to biomolecular condensates resembling "droplets" and forming distinct, membrane-free compartments. This process is pervasive in biological cells, contributing to various essential cellular functions. However, when phase separation goes awry, leading to abnormal molecular aggregation, it can become a driving factor in the development of diseases, including tumor. Recent investigations have unveiled the intricate connection between dysregulated phase separation and tumor pathogenesis, highlighting its potential as a novel therapeutic target. This article provides an overview of recent phase separation research, with a particular emphasis on its role in tumor, its therapeutic implications, and outlines avenues for further exploration in this intriguing field.

ABSTRACTIn higher eukaryotes, the nucleolus harbors at least three sub-phases that facilitate multiple functionalities including ribosome biogenesis. The three prominent coexisting sub-phases are the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). Here, we review recent efforts in profiling sub-phase compositions that shed light on the types of physicochemical properties that emerge from compositional biases and territorial organization of specific types of macromolecules. We highlight roles played by molecular grammars which refers to protein sequence features including the substrate binding domains, the sequence features of intrinsically disordered regions, and the multivalence of these distinct types of domains / regions. We introduce the concept of a barcode of emergent physicochemical properties of nucleoli. Although our knowledge of the full barcode remains incomplete, we hope that the concept prompts investigations into undiscovered emergent properties and engenders an appreciation for how and why unique microenvironments control biochemical reactions

Cells contain numerous membrane-less (MLOs) and multiple membrane-bound organelles. Most of the MLOs have a spherical shape except for a few organelles (that have a filamentous appearance, e.g. FXR1 condensates, L-bodies, and TIS granules). Some MLOs form associations with the membrane-bound organelles, such as the endoplasmic reticulum (ER). This fact was recently revealed by the discovery of a new, unique filamentous membrane-less organelle called ''TIS granule'' that was directly interlinked and twisted with the endoplasmic reticulum (ER). The most interesting insight of the TIS granules is that the transcribed genetic information carried by 3′ UTR of mRNA is translated by them, which makes them one of the most crucial sub-cellular organelles. Multiple TIS granules form a reticular meshwork with the endoplasmic reticulum (ER), ultimately creating an inter-organelle space or subcellular compartment known as the ''TIGER Domain'' that is enriched with membrane proteins-encoding mRNAs, some intermediate proteins (e.g. HuR) and a few chaperones. TIGER domain enables 3′ UTR-mediated protein–protein interactions (PPIs) between specific & functionally relevant proteins, such as membrane and 3′ UTR dependent proteins. For more than six decades, only ribosomes were credited as solo membrane-less organelles involved in the cell's translation process. However, this review acknowledges and highlights another less familiar translation companion of the ribosome (i.e. TIS granule) and elaborates on its composition, assembly, morphology and complete functionality in an easily understandable way.

Cell fate is precisely modulated by complex but well-tuned molecular signaling networks, whose spatial and temporal dysregulation commonly leads to hazardous diseases. Biomolecular condensates (BCs), as a newly emerging type of biophysical assemblies, decipher the molecular codes bridging molecular behaviors, signaling axes, and clinical prognosis. Particularly, physical traits of BCs play an important role; however, a panoramic view from this perspective toward clinical practices remains lacking. In this review, we describe the most typical five physical traits of BCs, and comprehensively summarize their roles in molecular signaling axes and corresponding major determinants. Moreover, establishing the recent observed contribution of condensate physics on clinical therapeutics, we illustrate next-generation medical strategies by targeting condensate physics. Finally, the challenges and opportunities for future medical development along with the rapid scientific and technological advances are highlighted. [ABSTRACT FROM AUTHOR]

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity—the condensates in oral epithelium disassembled within 1–2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4–7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic "macromolecular crowding") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm—the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid–liquid phase separation (LLPS) in cells of the oral mucosa. [ABSTRACT FROM AUTHOR]

Microtubule plus-end tracking protein (+TIP) networks from various species undergo liquid biomolecular condensation in vitro. The mesoscopic size of microtubule tip-localized +TIP networks in cells presents challenges in categorizing them as canonical biomolecular condensates; however, the recent alignment of correlative in vitro and in vivo evidence, particularly in budding yeast, along with the model's explanatory power, lends substantial support to this notion. The liquid condensate model of +TIP networks, the '+TIP-body model', offers a promising framework to advance our understanding of microtubule specialization. To fulfill their actual cellular role, individual microtubules become functionally specialized through a broad range of mechanisms. The 'search and capture' model posits that microtubule dynamics and functions are specified by cellular targets that they capture (i.e., a posteriori), independently of the microtubule-organizing center (MTOC) they emerge from. However, work in budding yeast indicates that MTOCs may impart a functional identity to the microtubules they nucleate, a priori. Key effectors in this process are microtubule plus-end tracking proteins (+TIPs), which track microtubule tips to regulate their dynamics and facilitate their targeted interactions. In this review, we discuss potential mechanisms of a priori microtubule specialization, focusing on recent findings indicating that +TIP networks may undergo liquid biomolecular condensation in different cell types. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) accompany RNA from synthesis to decay, mediating every aspect of RNA metabolism and impacting diverse cellular and developmental processes in eukaryotes. Many RBPs undergo phase separation along with their bound RNA to form and function in dynamic membraneless biomolecular condensates for spatiotemporal coordination or regulation of RNA metabolism. Increasing evidence suggests that phase-separating RBPs with RNA-binding domains and intrinsically disordered regions play important roles in plant development and stress adaptation. Here, we summarize the current knowledge about how dynamic partitioning of RBPs into condensates controls plant development and enables sensing of experimental changes to confer growth plasticity under stress conditions, with a focus on the dynamics and functional mechanisms of RBP-rich nuclear condensates and cytoplasmic granules in mediating RNA metabolism. We also discuss roles of multiple factors, such as environmental signals, protein modifications, and N 6-methyladenosine RNA methylation, in modulating the phase separation behaviors of RBPs, and highlight the prospects and challenges for future research on phase-separating RBPs in crops. This review discusses the phase separation dynamics of RNA-binding protein (RBP)-rich condensates and the mechanisms by which they control plant development and sense experimental changes to confer growth plasticity under stress conditions. The roles of multiple factors in modulating the phase separation behaviors of RBPs are also discussed, and the prospects and challenges for future research on phase-separating RBPs in crops are highlighted. [ABSTRACT FROM AUTHOR]

Inclusion bodies (IBs) of respiratory syncytial virus (RSV) are formed by liquid-liquid phase separation (LLPS) and contain internal structures termed "IB-associated granules" (IBAGs), where anti-termination factor M2-1 and viral mRNAs are concentrated. However, the mechanism of IBAG formation and the physiological function of IBAGs are unclear. Here, we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein (mRNP) condensates formed by secondary LLPS. Mechanistically, the RSV nucleoprotein (N) and M2-1 interact with and recruit PABP to IBs, promoting PABP to bind viral mRNAs transcribed in IBs by RNA-recognition motif and drive secondary phase separation. Furthermore, PABP-eIF4G1 interaction regulates viral mRNP condensate composition, thereby recruiting specific translation initiation factors (eIF4G1, eIF4E, eIF4A, eIF4B and eIF4H) into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment. Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase. [ABSTRACT FROM AUTHOR]

Phase separation is a cellular phenomenon where macromolecules aggregate or segregate, giving rise to biomolecular condensates resembling "droplets" and forming distinct, membrane-free compartments. This process is pervasive in biological cells, contributing to various essential cellular functions. However, when phase separation goes awry, leading to abnormal molecular aggregation, it can become a driving factor in the development of diseases, including tumor. Recent investigations have unveiled the intricate connection between dysregulated phase separation and tumor pathogenesis, highlighting its potential as a novel therapeutic target. This article provides an overview of recent phase separation research, with a particular emphasis on its role in tumor, its therapeutic implications, and outlines avenues for further exploration in this intriguing field. [ABSTRACT FROM AUTHOR]

Activator of G-protein signaling 3 (AGS3; also known as GPSM1), a receptor-independent activator of G-protein signaling, oscillates among defined subcellular compartments and biomolecular condensates (BMCs) in a regulated manner that is likely related to the functional diversity of the protein. We determined the influence of cell stress on the cellular distribution of AGS3 and core material properties of AGS3 BMCs. Cellular stress (oxidative, pHi and thermal) induced the formation of AGS3 BMCs in HeLa and COS-7 cells, as determined by fluorescent microscopy. Oxidative stress-induced AGS3 BMCs were distinct from G3BP1 stress granules and from RNA processing BMCs defined by the P-body protein Dcp1a. Immunoblots indicated that cellular stress shifted AGS3, but not the stress granule protein G3BP1 to a membrane pellet fraction following cell lysis. The stress-induced generation of AGS3 BMCs was reduced by co-expression of the signaling protein Gai3, but not the AGS3-binding partner DVL2. Fluorescent recovery following photobleaching of individual AGS3 BMCs indicated that there are distinct diffusion kinetics and restricted fluidity for AGS3 BMCs. These data suggest that AGS3 BMCs represent a distinct class of stress granules that serve as a previously unrecognized signal processing node. [ABSTRACT FROM AUTHOR]

The phenomenon of phase separation is quite common in cells, and it is involved in multiple processes of life activities. However, the current research on the correlation between protein modifications and phase separation and the interference with the tendency of phase separation has some limitations. Here we focus on several post-translational modifications of proteins, including protein phosphorylation modification at multiple sites, methylation modification, acetylation modification, ubiquitination modification, SUMOylation modification, etc., which regulate the formation of phase separation and the stability of phase separation structure through multivalent interactions. This regulatory role is closely related to the development of neurodegenerative diseases, tumors, viral infections, and other diseases, and also plays essential functions in environmental stress, DNA damage repair, transcriptional regulation, signal transduction, and cell homeostasis of living organisms, which provides an idea to explore the interaction between novel protein post-translational modifications and phase separation. 4w-q829MkFQ4xisZd8YwRd Video Abstract [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation has been recognized as universal mechanisms in living cells for the formation of RNA-protein condensates and ordered lipid domains. These biomolecular condensates or domains nucleate, diffuse and interact with each other across physical dimensions to perform their biological functions. Here we summarize key features of biophysical principles underlying the multi-dimensional condensation of RNA-protein condensates and ordered lipid domains, which are related to nuclear transcription, and signaling on cell membranes. Uncovering physicochemical factors that govern the spatiotemporal coupling of those condensates presents a new avenue in their functions and associated human diseases. [ABSTRACT FROM AUTHOR]

TAR DNA‐binding protein 43 (TDP‐43) is a multidomain protein involved in the regulation of RNA metabolism, and its aggregates have been observed in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Numerous studies indicate TDP‐43 can undergo liquid–liquid phase separation (LLPS) in vitro and is a component of biological condensates. Homo‐oligomerization via the folded N‐terminal domain (aa:1–77) and the conserved helical region (aa:319–341) of the disordered, C‐terminal domain is found to be an important driver of TDP‐43 phase separation. However, a comprehensive molecular view of TDP‐43 phase separation, particularly regarding the nature of heterodomain interactions, is lacking due to the challenges associated with its stability and purification. Here, we utilize all‐atom and coarse‐grained (CG) molecular dynamics (MD) simulations to uncover the network of interdomain interactions implicated in TDP‐43 phase separation. All‐atom simulations uncovered the presence of transient, interdomain interactions involving flexible linkers, RNA‐recognition motif (RRM) domains and a charged segment of disordered C‐terminal domain (CTD). CG simulations indicate these inter‐domain interactions which affect the conformational landscape of TDP‐43 in the dilute phase are also prevalent in the condensed phase. Finally, sequence and surface charge distribution analysis coupled with all‐atom simulations (at high salt) confirmed that the transient interdomain contacts are predominantly electrostatic in nature. Overall, our findings from multiscale simulations lead to a greater appreciation of the complex interaction network underlying the structural landscape and phase separation of TDP‐43. [ABSTRACT FROM AUTHOR]

Abstract Identification of environmental stress sensors is one of the most important research topics in plant abiotic stress research. Traditional strategies to identify stress sensors or early signaling components based on the cell membrane as a primary site of sensing and calcium signal as a second messenger have had only limited successes. Therefore, the current theoretical framework underlying stress sensing in plants should be reconsidered and additional mechanisms need to be introduced. Recently, accumulating evidence has emerged to suggest that liquid-liquid phase separation (LLPS) is a major mechanism for environmental stress sensing and response in plants. In this review, we briefly introduce LLPS regarding its concept, compositions, and dynamics, and then summarize recent progress of LLPS research in plants, emphasizing the contribution of LLPS to the sensing of various environmental stresses, such as dehydration, osmotic stress, and low and high temperatures. Finally, we propose strategies to identify key proteins that sense and respond to environmental stimuli on the basis of LLPS, and discuss the research directions of LLPS in plant abiotic stress responses and its potential application in enhancing stress tolerance in crops.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid–liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquidlike properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication. [ABSTRACT FROM AUTHOR]

The term "biomolecular condensates" is used to describe membraneless compartments in eukaryotic cells, accumulating proteins and nucleic acids. Biomolecular condensates are formed as a result of liquid–liquid phase separation (LLPS). Often, they demonstrate properties of liquid-like droplets or gel-like aggregates; however, some of them may appear to have a more complex structure and high-order organization. Membraneless microcompartments are involved in diverse processes both in cytoplasm and in nucleus, among them ribosome biogenesis, regulation of gene expression, cell signaling, and stress response. Condensates properties and structure could be highly dynamic and are affected by various internal and external factors, e.g., concentration and interactions of components, solution temperature, pH, osmolarity, etc. In this review, we discuss variety of biomolecular condensates and their functions in live cells, describe their structure variants, highlight domain and primary sequence organization of the constituent proteins and nucleic acids. Finally, we describe current advances in methods that characterize structure, properties, morphology, and dynamics of biomolecular condensates in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time. [ABSTRACT FROM AUTHOR]

Hox genes are a family of homeodomain transcription factors that regulate specialized morphological structures along the anterior-posterior axis of metazoans. Over the past few decades, researchers have focused on defining how Hox factors with similar in vitro DNA binding activities achieve sufficient target specificity to regulate distinct cell fates in vivo. In this review, we highlight how protein interactions with other transcription factors, many of which are also homeodomain proteins, result in the formation of transcription factor complexes with enhanced DNA binding specificity. These findings suggest that Hox-regulated enhancers utilize distinct combinations of homeodomain binding sites, many of which are low-affinity, to recruit specific Hox complexes. However, low-affinity sites can only yield reproducible responses with high transcription factor concentrations. To overcome this limitation, recent studies revealed how transcription factors, including Hox factors, use intrinsically disordered domains (IDRs) to form biomolecular condensates that increase protein concentrations. Moreover, Hox factors with altered IDRs have been associated with altered transcriptional activity and human disease states, demonstrating the importance of IDRs in mediating essential Hox output. Collectively, these studies highlight how Hox factors use their DNA binding domains, protein-protein interaction domains, and IDRs to form specific transcription factor complexes that yield accurate gene expression. [Display omitted] • Hox complex formation with other homeodomain factors increase DNA specificity. • Different Hox complexes have distinct DNA binding site requirements. • Low-affinity DNA sites increase Hox binding specificity. • Hox factors are concentrated within distinct nuclear sub-compartments. • Hox factor intrinsically disordered regions (IDRs) form biomolecular condensates. • Hox factors with altered IDRs have been associated with human diseases. [ABSTRACT FROM AUTHOR]

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High-risk neuroblastoma is a very aggressive pediatric cancer, accounting for ~15% of childhood cancer mortality. Therefore, novel therapeutic strategies for the treatment of neuroblastoma are urgently sought. Here, we focused on the potential implications of the Dual-specificity tYrosine-Regulated Kinase (DYRK) family and downstream signaling pathways. We used bioinformatic analysis of public datasets from neuroblastoma cohorts and cell lines to search correlations between patient survival and expression of DYRK kinases. Additionally, we performed biochemical, molecular, and cellular approaches to validate and characterize our observations, as well as an in vivo orthotopic murine model of neuroblastoma. We identified the DYRK3 kinase as a critical mediator of neuroblastoma cell proliferation and in vivo tumor growth. DYRK3 has recently emerged as a key regulator of several biomolecular condensates and has been linked to the hypoxic response of neuroblastoma cells. Our data suggest a role for DYRK3 as a regulator of the neuroblastoma-specific protein CAMKV, which is also required for neuroblastoma cell proliferation. CAMKV is a very understudied member of the Ca2+/calmodulin-dependent protein kinase family, originally described as a pseudokinase. We show that CAMKV is phosphorylated by DYRK3, and that inhibition of DYRK3 kinase activity induces CAMKV aggregation, probably mediated by its highly disordered C-terminal half. Importantly, we provide evidence that the DYRK3/CAMKV signaling module could play an important role for the function of the mitotic spindle during cell division. Our data strongly support the idea that inhibition of DYRK3 and/or CAMKV in neuroblastoma cells could constitute an innovative and highly specific intervention to fight against this dreadful cancer. [ABSTRACT FROM AUTHOR]

Identification of environmental stress sensors is one of the most important research topics in plant abiotic stress research. Traditional strategies to identify stress sensors or early signaling components based on the cell membrane as a primary site of sensing and calcium signal as a second messenger have had only limited successes. Therefore, the current theoretical framework underlying stress sensing in plants should be reconsidered and additional mechanisms need to be introduced. Recently, accumulating evidence has emerged to suggest that liquid-liquid phase separation (LLPS) is a major mechanism for environmental stress sensing and response in plants. In this review, we briefly introduce LLPS regarding its concept, compositions, and dynamics, and then summarize recent progress of LLPS research in plants, emphasizing the contribution of LLPS to the sensing of various environmental stresses, such as dehydration, osmotic stress, and low and high temperatures. Finally, we propose strategies to identify key proteins that sense and respond to environmental stimuli on the basis of LLPS, and discuss the research directions of LLPS in plant abiotic stress responses and its potential application in enhancing stress tolerance in crops. [ABSTRACT FROM AUTHOR]

Summary: Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood.Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle‐associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live‐cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells.We show that TYMaV IBs are phase‐separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication.Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation. [ABSTRACT FROM AUTHOR]

Transcription is the fundamental process of gene expression, which in eukaryotes occurs within the complex physicochemical environment of the nucleus. Decades of research have provided extreme detail in the molecular and functional mechanisms of transcription, but the spatial and genomic organization of transcription remains mysterious. Recent discoveries show that transcriptional components can undergo phase separation and create distinct compartments inside the nucleus, providing new models through which to view the transcription process in eukaryotes. In this review, we focus on transcriptional condensates and their phase separation-like behaviors. We suggest differentiation between physical descriptions of phase separation and the complex and dynamic biomolecular assemblies required for productive gene expression, and we discuss how transcriptional condensates are central to organizing the three-dimensional genome across spatial and temporal scales. Finally, we map approaches for therapeutic manipulation of transcriptional condensates and ask what technical advances are needed to understand transcriptional condensates more completely. [ABSTRACT FROM AUTHOR]