Multiphasic Organizationand Differential Dynamicsof Proteins within Protein–DNA Biomolecular Condensates

Biomolecular condensates formed through liquid-liquid phase separation are increasingly recognized as critical regulators of genome organization and gene expression. While the role of proteins in driving phase separation is well-established, how DNA modulates the structure and dynamics of protein-DNA condensates remains less well understood. Here, we employ a minimalist coarse-grained model to investigate the interplay between homotypic protein-protein and heterotypic protein-DNA interactions in governing condensate formation, composition, and internal dynamics. Our simulations reveal that DNA chain length and flexibility critically influence condensate morphology, leading to the emergence of multiphasic and core-shell organizations under strong heterotypic interactions. We find that DNA recruitment into the condensate significantly alters protein mobility, giving rise to differential dynamics of proteins within the condensate. By analyzing the distribution profiles of protein displacements, we identify up to five distinct diffusion modes, including proteins bound to DNA, confined within the dense phase, or freely diffusing. These results provide a mechanistic framework for interpreting spatially heterogeneous protein dynamics observed in chromatin condensates and emphasize the direct role of DNA in tuning condensate properties. Our findings provide new insights into how biophysical parameters may control the functional architecture of protein-DNA condensates in biological systems.

Networking and Dynamic Switches in Biological Condensates

Liquid Liquid Phase Separation (LLPS) has emerged as a mechanism for the assembly of membraneless organelles in eukaryotes, but little is known about this process in bacteria. LLPS refers to the ability of macromolecules to demix into a dilute phase and a dense phase, called a ‘biomolecular condensate’, which can be observed as clusters or foci in the cell. The major challenge for the study of LLPS in bacteria is the poor spatial resolution of foci in such tiny cells. As a result, it is difficult to demonstrate the liquid‐like nature of a focus in bacterial cells using the conventional approaches for studying large condensates in eukaryotic cells. Here, we developed a rigorous experimental framework for the characterization of LLPS in bacteria, using Escherichia coli as the host organism and the intrinsically disordered protein McdB, which robustly forms liquid‐like droplets in vitro. McdB is a protein that coats a bacterial organelle called the carboxysome. This coating demarcates the carboxysome as cargo for its positioning system, which equally distributes carboxysomes along the cell length of rod‐shaped cyanobacteria. We developed a suite of experiments to investigate the LLPS activity of McdB in vivo, based on the ability of biomolecular condensates to tune their size and shape, fuse, dissolve, and transition between phase states. We used both overexpression and tunable promoters to express fluorescent fusions of McdB and cIEP8, a well‐known aggregator protein. We found that fluorescent fusions of McdB formed nucleoid‐excluded foci in E. coli, but also maintained a soluble phase in the cytoplasm, consistent with LLPS theory. The aggregator protein cIEP8, on the other hand, lacked a soluble fraction in the cytoplasm. Condensates form at a saturation concentration threshold, called Csat. A hallmark of LLPS is that condensates will dissolve if the concentration drops below Csat, while insoluble aggregates should remain as stable foci even after dilution. We decreased protein concentration in vivo by increasing cell volume and by generational dilution via cell division. In both methods, McdB foci dissolved while cIEP8 foci remained intact as insoluble aggregates in response to decreased concentration in the cell. Finally, we also discovered that a well‐established marker for insoluble protein aggregates in vivo, IbpA, does not colocalize with McdB foci. The result suggests that the colocalization of IbpA foci can be used as a broad‐use sensor for the material state of protein complexes in bacterial cells. Our results provide multiple lines of evidence in support of LLPS of McdB in vivo. More broadly, our experimental framework for studying LLPS in bacteria overcomes current limitations in the field and can be used to assess the LLPS activity of other proteins of interest in bacterial cells.

Promyelocytic leukemia nuclear bodies (PML NBs) are membraneless organelles (0.1-1 μm) integral to numerous fundamental cellular processes. Recent advances in cryo-EM and liquid-liquid phase separation (LLPS) research substantially advanced our understanding of PML NBs biophysical features and structural organization. Here, we identify Sp100-HMG, an Sp100 isoform, as a driver of PML NBs formation via LLPS, recruiting PML and accessory proteins (DAXX, ATRX). Dissection of this assembly process uncovered a hierarchical mechanism orchestrated by three distinct yet cooperative processes: I. Multimerization domain- and intrinsically disordered region (IDR)-mediated LLPS of Sp100-HMG, nucleating the initial core; II. C-terminal-dependent protein-protein interactions that enrich client components; and III. SUMOylation-directed PML recruitment, facilitating the formation of a stabilizing peripheral shell. Notably, this assembly paradigm extends beyond Sp100-HMG, as evidenced by ZBTB16—a PML NB-associated oncoprotein implicated in acute promyelocytic leukemia—adopting an analogous mechanism to organize PML-positive condensates. Functional validation further revealed that Sp100-HMG positive PML NBs exert dual regulatory control over transcriptional programs and cell cycle progression, highlighting their pleiotropic roles. Critically, this work redefines the canonical PML NB assembly model by demonstrating that Sp100-HMG, rather than PML, acts as a primary scaffold, with PML polymerization relegated to a secondary, shell-forming stabilizer. By correlating the unique spatial architecture of Sp100-HMG positive PML NBs with their functional outputs, our findings establish a mechanistic framework for understanding how PML condensate biogenesis dictates transcriptional and cell cycle regulation, offering new avenues for exploring PML NB function in physical and disease contexts. Key findings LLPS of Sp100-HMG directs de novo formation of PML NBs Polymerization, IDR and SUMOylation collectively contribute to PML NBs formation Sp100-HMG organized PML NBs in proximity to promoters strengthens local transcriptional regulation Sp100-HMG regulates cell cycle progression by regulating DAXX levels in the nucleoplasm Significance This study fundamentally redefines our understanding of PML NBs assembly by identifying Sp100-HMG -driven phase separation as a primary scaffold mechanism, challenging/complementing the canonical PML-centric model. We demonstrate that Sp100-HMG undergoes LLPS via its multimerization domain and IDR, forming an initial condensate core that recruits PML as a peripheral shell. Notably, this mechanism extends to ZBTB16, an oncoprotein linked to APL, suggesting broad biological relevance. Functionally, Sp100-HMG – PML NBs regulate transcription and cell cycle progression, acting as dynamic "protein sponges" that modulate nucleoplasmic or regional protein concentrations through their LLPS-driven breathing effect—expansion and contraction constrained by the PML shell. This work unveils a novel architectural paradigm for PML NBs and provides a mechanistic framework for future investigations.

Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit.

Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors.IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.

Targeting of biomolecular condensates to the autophagy pathway.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10μl.

Nuclear Protein Condensates and Their Properties in Regulation of Gene Expression

Biomolecular condensates are a promising platform for synthetic cell formation and constitute a potential missing link between the chemical and cellular stage of the origins of life. However, it has proven challenging to integrate complex reaction networks into biomolecular condensates, such as a cell-free in vitro transcription-translation (IVTT) system. Integrating IVTT into biomolecular condensates successfully is one precondition for condensation-based synthetic cell formation. Moreover, it would provide a proof of concept that biomolecular condensates are in principle compatible with the central dogma, one of the hallmarks of cellular life. Here, we have systemically investigated the compatibility of eight different (bio)molecular condensates with IVTT incorporation. Of these eight candidates, we have found that a green fluorescent protein-labeled, intrinsically disordered cationic protein (GFP-K72) and single-stranded DNA (ssDNA) can form biomolecular condensates that are compatible with up to μM fluorescent protein expression. This shows that biomolecular condensates can indeed integrate complex reaction networks, confirming their use as synthetic cell platforms and hinting at a possible role in the origin of life.

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.

Liquid-liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane-less organelles (MLOs). Cells have well-defined different membrane-surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs as stress granules, nucleoli and P bodies to cover vital functions and regulatory activities. However, the mechanism of intracellular molecule assembly into functional compartments within a living cell remains till now not fully understood. in vitro and in vivo investigations unveiled that MLOs emerge after preceding liquid-liquid, liquid-gel, liquid-semi-crystalline, or liquid-crystalline phase separations. Liquid-liquid and liquid-gel MLOs form the majority of cellular phase separation events, while the occurrence of micro-sized crystals in cells was only rarely observed, however can be considered as a result of a preceding protein phase separation event. In vivo, also known and termed as in cellulo crystals, are reported since 1853. In some cases, they have been linked to vital cellular functions, such as storage and detoxification. However, the occurrence of in cellulo crystals is also associated to diseases like cataract, hemoglobin C diseases, etc. Therefore, better knowledge about the involved molecular processes will support drug discovery investigations to cure diseases related to in cellulo crystallization. We summarize physical and chemical determinants known today required for phase separation initiation and formation and in cellulo crystal growth. In recent years it has been demonstrated that LLPS plays a crucial role in cell compartmentalization and formation of MLOs. Here we discuss potential mechanisms and potential crowding agents involved in protein phase separation and in cellulo crystallization.

Liquid-liquid phase separation of flexible biomolecules has been identified as a ubiquitous phenomenon underlying the formation of membraneless organelles that harbor a multitude of essential cellular processes. We use nuclear magnetic resonance (NMR) spectroscopy to compare the dynamic properties of an intrinsically disordered protein (measles virus NTAIL) in the dilute and dense phases at atomic resolution. By measuring 15N NMR relaxation at different magnetic field strengths, we are able to characterize the dynamics of the protein in dilute and crowded conditions and to compare the amplitude and timescale of the different motional modes to those present in the membraneless organelle. Although the local backbone conformational sampling appears to be largely retained, dynamics occurring on all detectable timescales, including librational, backbone dihedral angle dynamics and segmental, chainlike motions, are considerably slowed down. Their relative amplitudes are also drastically modified, with slower, chain-like motions dominating the dynamic profile. In order to provide additional mechanistic insight, we performed extensive molecular dynamics simulations of the protein under self-crowding conditions at concentrations comparable to those found in the dense liquid phase. Simulation broadly reproduces the impact of formation of the condensed phase on both the free energy landscape and the kinetic interconversion between states. In particular, the experimentally observed reduction in the amplitude of the fastest component of backbone dynamics correlates with higher levels of intermolecular contacts or entanglement observed in simulations, reducing the conformational space available to this mode under strongly self-crowding conditions.

Once considered unconventional cellular structures, membraneless organelles (MLOs), cellular substructures involved in biological processes or pathways under physiological conditions, have emerged as central players in cellular dynamics and function. MLOs can be formed through liquid-liquid phase separation (LLPS), resulting in the creation of condensates. From neurodegenerative disorders, cardiovascular diseases, aging, and metabolism to cancer, the influence of MLOs on human health and disease extends widely. This review discusses the underlying mechanisms of LLPS, the biophysical properties that drive MLO formation, and their implications for cellular function. We highlight recent advances in understanding how the physicochemical environment, molecular interactions, and post-translational modifications regulate LLPS and MLO dynamics. This review offers an overview of the discovery and current understanding of MLOs and biomolecular condensate in physiological conditions and diseases. This article aims to deliver the latest insights on MLOs and LLPS by analyzing current research, highlighting their critical role in cellular organization. The discussion also covers the role of membrane-associated condensates in cell signaling, including those involving T-cell receptors, stress granules linked to lysosomes, and biomolecular condensates within the Golgi apparatus. Additionally, the potential of targeting LLPS in clinical settings is explored, highlighting promising avenues for future research and therapeutic interventions.

Decision letter: Defining basic rules for hardening influenza A virus liquid condensates