Germ cells depend on specialized post-transcriptional regulation for proper development and function, much of which is mediated by dynamic RNA granules. These membrane-less organelles form through the condensation of RNA and proteins, governed by multivalent biomolecular interactions. RNA granules compartmentalize cellular components, selectively enriching specific factors and modulating biochemical reactions. Over recent decades, various types of RNA granules have been identified in germ cells across species, with extensive studies uncovering their molecular roles and developmental significance. This review explores the mRNA regulatory mechanisms mediated by RNA granules in germ cells. We discuss the distinct spatial organization of specific granule components and the variations in material states of germ granules, which contribute to the regulation of mRNA storage and translation. Additionally, we highlight emerging research on how changes in these material states, during developmental stages, reflect the dynamic nature of germ granules and their critical role in development.

Cellular differentiation requires highly coordinated action of all three transcriptional systems to produce rRNAs, mRNAs and various 'short' and 'long' non-coding RNAs by RNA Polymerase I, II and III systems, respectively. RNA Polymerase I catalyzes transcription of about 400 copies of mammalian rDNA genes, generating 18S, 5.8S and 28S rRNA molecules. Lens fiber cell differentiation is a unique process to study transcriptional mechanisms of individual crystallin genes as their very high transcriptional outputs are directly comparable only to globin genes in erythrocytes. Importantly, both terminally differentiated lens fiber cells and mammalian erythrocytes degrade their nuclei through different mechanisms. In lens, the generation of the organelle-free zone (OFZ) includes the degradation of mitochondria, endoplasmic reticulum, Golgi apparatus and nuclei. Here, using RNA fluorescence in situ hybridization (FISH), we evaluated nascent rRNA transcription, located in the nucleoli, during the process of mouse lens fiber cell differentiation. Lens fiber cell nuclei undergo morphological changes including chromatin condensation prior to their denucleation. Remarkably, nascent rRNA transcription persists in all nuclei that are in direct proximity of the OFZ. Additionally, changes in both nuclei and nucleoli shape were evaluated via immunofluorescence detection of fibrillarin, nucleolin, UBF and other proteins. These studies demonstrate for the first time that highly condensed lens fiber cell nuclei have the capacity to support nascent rRNA transcription. Thus, we propose that 'late' production of rRNA molecules and consequently of ribosomes increases crystallin protein synthesis machinery within the mature lens fibers.

Coacervates have become the focus of great recent attention due to their observation in living systems in the form of membraneless organelles. One of the interesting features observed for both cellular and in vitro coacervates is the immiscibility among some of them, leading to formation of so-called multiphase coacervates. This study addresses the issue as to whether two coacervates formed by a polyanion (polyacrylate, PAA) with a polycation (poly(diallyldimethylammonium, PDDMA+) or a cationic surfactant (CnTA+) would be miscible in bulk or confined in complex core coacervate micelles, C3Ms, using an EO-PAA block copolymer for the latter.

Their mixtures were evaluated visually for bulk miscibility, or with DLS or cryo-TEM analyses, for their C3Ms. SAXS analyses were employed for characterization of the liquid crystalline structures, in both systems.

Our primary observation is that, although these coacervate systems exhibit similar interaction strengths, they remain immiscible when a surfactant liquid crystalline phase is present, both in bulk and within the micellar core. However, when this surfactant mesophase is disrupted, such as through the addition of salt, both systems become miscible. These findings emphasize the critical role of molecular ordering in the miscibility of coacervates, consistent with recent evidence highlighting the differences between liquid-liquid and liquid-liquid crystalline phase separation.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) play a crucial role in organizing biochemical processes within living cells. The phase transition of these condensates from a functional liquid-like state to a pathological gel-like or solid-like state is believed to be linked to cellular dysfunction and various diseases. Here, we present a biomimetic model to demonstrate that endogenous enzyme-catalyzed crosslinking within condensate-mimicked coacervate microdroplets can promote a liquid-to-gel phase transition. We identify the transformation in physical characteristics of the densely packed microdroplets including reduced internal mobility, increased storage modulus, selective blocking of large nanoparticles, and enhanced salt resistance. The reversible dynamics of gel-like microdroplets mediated by ionic strength exhibited a limited release and recapture of sequestered positively charged guest molecules. Furthermore, we validate that the phase transition contributes to a restricted biochemical process through an enzymatic cascade. Overall, this work represents an adaptive in vitro platform for exploring the phase transitions associated with the physiological functions of biomolecular condensates and offers chemical insights and perspectives for investigating potential mechanisms involved in phase transitions.

The size of a macromolecule in solution is strongly influenced by the size and concentration of added colloidal particles. Previous experimental and computer simulation studies have shown conflicting results regarding the influence of colloid size on coil compaction. We suggest the coil size depends on the Kuhn segment / nanoparticle size ratio and argue its subtle influence on the shrinking and expansion of a polymer chain.

Based upon the work of van der Schoot (1998) [42] we propose theory that predicts how the colloid size mediates the compaction of macromolecules in crowded environments. The theoretical predictions are compared to self-consistent field (SCF) lattice computations and scattering experiments on polymer solutions exposed to crowders.

The theoretical approach predicts that the shrinking of a polymer coil upon adding colloidal particles varies with the size of the colloids. We find coil shrinking is weakest when the colloidal particles are approximately the same size as the Kuhn segment length. The extent of coil shrinking passes a minimum at a specific colloid size relative to the Kuhn segment length, which is confirmed by SCF computations. Comparison with scattering experiments reveals that these experiments corroborate the extent of polymer shrinking at a given volume fraction of colloids. Our theoretical approach reproduces the functional dependence of the collapse on the crowder volume fraction.

Biomaterials can play a crucial role in facilitating tissue regeneration, but their application is often limited by that they induce scarring rather than complete tissue restoration. Hydrogels with microporous architectures, engineered via 3D printing techniques or particle packing (granular hydrogels), have shown promise in providing a conducive microenvironment for cellular infiltration and favorable immune response. Nonetheless, there is a notably lacking in studies that demonstrate scarless regeneration solely through pore structure engineering. In this study, we demonstrate that optimizing micropore structure of injectable granular hydrogels via controlled liquid-liquid phase separation facilitates scarless wound healing. The building block particles are fabricated by precisely controlling the separation kinetics of two immiscible aqueous phases (gelling and porogenic) and timely arresting phase separation, to generate bicontinuous, hollow or closed porous structure. Employing a murine model, we reveal that the optimized pore structure significantly facilitates mature vascular network boosts pro-regenerative macrophage polarization (M2/M1) and CD4+/Foxp3+ regulatory T cells, culminating in scarless skin regeneration enriched with hair follicles. Moreover, our hydrogels outperform the clinical gold-standard collagen/proteoglycan scaffolds in a porcine model, showcasing superior cell infiltration, epidermal integration, and dermal regeneration. Micropore structure engineering of biomaterials presents a promising and biologics free pathway for tissue regeneration.

Reflecting on the diversity of the natural world, Darwin famously observed that "from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved." However, the examples that we are able to observe in nature are a consequence of chance, constrained by selection, drift, and epistasis. Here we explore how the efforts of synthetic biology to build new living systems can expand our understanding of the fundamental design principles that allow life to self-organize biological form, from cellular to organismal levels. We suggest that the ability to impose a length or timescale onto a biological activity is an essential strategy for self-organization in evolved systems and a key design target that is now being realized synthetically at all scales. By learning to integrate these strategies together, we are poised to expand on evolution's success and realize a space of synthetic forms not only beautiful but with diverse applications and transformative potential.

The nucleolus is essential for the efficient and accurate production of ribosomal subunits, which are crucial for assembling ribosomes-the cellular machinery responsible for protein synthesis. Emerging insights into its liquid-like nature have shed new light on the role of its unique biophysical properties in the activity and regulation of this organelle. In this perspective, we examine recent insights into nucleolar biophysical homeostasis, with a focus on its regulation as an acidic biomolecular condensate. We review current evidence on how nucleolar composition and biochemical activities could generate and maintain a proton gradient. Additionally, we propose an integrative model explaining how nucleolar acidity contributes to homeostasis at a molecular level, providing a unified framework for its role in health and disease.

We present an idea of protein molecules that challenges the traditional view of proteins as simple molecular machines and suggests instead that they exhibit a basic form of "intelligence". The idea stems from suggestions coming from Integrated Information Theory (IIT), network theory, and allostery to explore how proteins process information, adapt to their environment, and even show memory-like behaviors. We define protein intelligence using IIT and focus on how proteins integrate information (in terms of the parameter Φ coming from IIT) and balance their core (stable, ordered regions) and periphery (flexible, disordered regions). This balance allows proteins to remain stable while adapting to changes and operating in a critical state where order and disorder coexist. We summarize recent findings on conformational memory, allosteric regulation, protein intrinsic disorder, liquid-liquid phase separation, and critical transitions, and compare protein behavior to other complex systems like ecosystems and neural networks. While our perspective offers a unified framework to understand proteins, it also raises questions about applying intelligence concepts to molecular systems. We discuss how this understanding could advance protein engineering, drug design, and synthetic biology, while at the same time acknowledging the challenges of creating adaptive, "intelligent" proteins. This concept bridges the gap between mechanistic and systems-level views of proteins and offers a comprehensive understanding of their dynamic and adaptive nature. We have tried to redefine the traditionally metaphorical concept of "intelligence" in biochemistry as a measurable property while simultaneously establishing the material foundation of protein intelligence through the identification of fundamental elements such as memory and learning in molecular systems.

Among the pervasive transcripts from eukaryotic genomes, a novel subset, referred to as architectural RNAs (arcRNAs), has an essential role in assembling membraneless organelles (MLOs). These arcRNAs sequester specific RNA-binding proteins (RBPs) and promote phase separation through multivalent interactions. NEAT1_2, an archetypal arcRNA, serves as a blueprint for paraspeckle architecture, characterized by a shell-and-core micelle-like configuration and immiscibility with other MLOs, relying on the cooperative contributions of distinct modular RNA domains. arcRNAs regulate gene expression through three of MLO action modes (crucible, sponge, and hub), guided by the functional blueprints embedded in arcRNA sequences. Advanced high-throughput analyses have identified thousands of arcRNA candidates, underscoring their potential in organizing transient intracellular compartments and driving dynamic cellular processes.

This study investigates the use of spectral phasor analysis, hyperspectral imaging, and 6-acetyl-2-dimethylaminonaphthalene (ACDAN) fluorescence to explore key protein transitions: unfolding, amyloid aggregation, and liquid-liquid phase separation. We show that ACDAN fluorescence can sensitively detect subtle conformational changes before the complete protein unfolds, revealing early microenvironmental shifts. During amyloid formation, ACDAN identifies solvent dipolar relaxation events undetectable by conventional thioflavin T, providing critical insight into early aggregation events. Additionally, we map the physicochemical properties of protein biocondensates and highlight distinct microenvironments within these condensates, emphasizing the significance of dipolar relaxation in phase-separated systems. The approach provides a flexible and user-friendly toolkit for studying protein transitions, which can be easily implemented in commercial spectrofluorometers and microscopes.

In the liquid-liquid phase separation of proteins, dense liquid droplets often form within the dilute phase below the critical concentration. The resulting size distribution of these precritical droplets can be described by a scale-invariant distribution, which is characterized by an increasing average as the concentration approaches from below the critical value. This phenomenon can be leveraged for the quantitative estimation of the critical concentration. Here, to facilitate applications of this approach, we present the DropFit web server (https://www-cohsoftware.ch.cam.ac.uk/index.php/dropfit). DropFit can be used to estimate the critical concentration using experimental data on the length, area, or volume of the precritical droplets, which are taken away from the critical concentration and thus be more accurate and reproducible. We anticipate that the accurate value of the critical concentration under different experimental conditions will help understand the contributions from different macromolecules to the formation of protein condensates, and to investigate the perturbations that lead to pathological processes through the disruption of membraneless organelles.

The study of intrinsically disordered proteins (IDPs) and their role in biomolecular condensate formation has become a critical area of research, offering insights into fundamental biological processes and therapeutic development. Here, we present IPAMD (Intrinsically disordered Protein Aggregation Molecular Dynamics), a plugin-based software designed to simulate the formation dynamics of biomolecular condensates of IDPs. IPAMD provides a modular, efficient, and customizable simulation platform specifically designed for biomolecular condensate studies. It incorporates advanced force fields, such as HPS-based and Mpipi models, and employs optimization techniques for large-scale simulations. The software features a user-friendly interface and supports batch processing, making it accessible to researchers with varying computational expertise. Benchmarking and case studies demonstrate the ability of IPAMD to accurately simulate and analyze condensate structures and properties.

Proteins are the key molecular players of life, carrying out their functions through interactions. Microfluidic technologies have emerged as powerful tools for studying protein interactions with exquisite sensitivity, resolution, and throughput. In this review, we highlight recent advances in microfluidic approaches for protein interaction studies. We first explore continuous-flow microfluidics, which utilize diffusion-based techniques and electrophoretic methods, before examining the role of droplet microfluidics in probing protein interactions. We provide an overview of the diverse applications of these technologies in biophysical research, drug discovery, and clinical diagnostics. We conclude with a discussion of the potential of microfluidics for driving future innovations and emerging opportunities.

Biomolecular condensates are increasingly recognized as key regulators of chromatin organization, yet how their formation and properties arise from protein sequences remains incompletely understood. Cross-species comparisons can reveal both conserved functions and significant evolutionary differences. Here, we integrate in vitro reconstitution, molecular dynamics simulations, and cell-based assays to examine how Drosophila and human variants of Polyhomeotic (Ph)-a subunit of the PRC1 chromatin regulatory complex-drive condensate formation through their sterile alpha motif (SAM) oligomerization domains. We identify divergent interactions between SAM and the disordered linker connecting it to the rest of Ph. These interactions enhance oligomerization and modulate both the formation and properties of reconstituted condensates. Oligomerization influences condensate dynamics but minimally impacts condensate formation. Linker-SAM interactions also affect condensate formation in Drosophila and human cells and growth in Drosophila imaginal discs. Our findings show how evolutionary changes in disordered linkers can fine-tune condensate properties, providing insights into sequence-function relationships.

The nucleolus is a multiphasic biomolecular condensate that facilitates ribosome biogenesis, a complex process involving hundreds of proteins and RNAs. The proper execution of ribosome biogenesis likely depends on the material properties of the nucleolus. However, these material properties remain poorly understood due to the challenges of in vivo measurements. Here, we use micropipette aspiration (MPA) to directly characterize the viscoelasticity and interfacial tensions of nucleoli within transcriptionally active Xenopus laevis oocytes. We examine the major nucleolar subphases, the outer granular component (GC) and the inner dense fibrillar component (DFC), which itself contains a third small phase known as the fibrillar center (FC). We show that the behavior of the GC is more liquid-like, while the behavior of the DFC/FC is consistent with that of a partially viscoelastic solid. To determine the role of ribosomal RNA in nucleolar material properties, we degrade RNA using RNase A, which causes the DFC/FC to become more fluid-like and alters interfacial tension. Together, our findings suggest that RNA underlies the partially solid-like properties of the DFC/FC and provide insights into how material properties of nucleoli in a near-native environment are related to their RNA-dependent function.

Necroptosis is an inflammatory programmed cell death pathway triggered by RIPK3 activation through one of the upstream RHIM-domain-containing proteins including RIPK1, TRIF, and ZBP1. Whether necroptosis can be activated independent of the upstream signaling pathways leading to inflammatory pathogenesis remains ambiguous. Here, we revealed a mechanism in which a viral protein mediates direct RIPK3 activation resulting in severe inflammatory pathogenesis in patients. The nonstructural protein NSs of a pathogenic hemorrhagic virus, SFTSV, interacts with the RIPK3 kinase domain and forms biocondensate to promote RIPK3 autophosphorylation and necroptosis activation in an RHIM-independent manner. In parallel, sequestration of RIPK3 within the NSs-RIPK3 condensate inhibited RIPK3-mediated apoptosis and promoted viral replication. Infection with an SFTSV NSs mutant virus not forming NSs condensate triggered pronounced apoptosis resulting in reduced viral replication and decreased fatality in vivo. Blocking SFTSV-triggered necroptosis through depletion of MLKL or treatment with a RIPK3-kinase inhibitor reduced viral inflammatory pathogenesis and fatality in vivo. In contrast, blocking SFTSV-triggered apoptosis through depletion of RIPK3 resulted in enhanced viral replication and increased fatality in vivo. The virus-triggered necroptosis correlated with severe inflammatory pathogenesis and lethality in virus-infected patients. The NSs-RIPK3 condensate may represent a necroptosis activation mechanism that promotes viral pathogenesis.

Enhancer-promoter communication is fundamental to gene regulation in metazoans, yet the mechanisms underlying these interactions remain debated. Two primary models have been proposed: the structural bridge model, in which enhancers and promoters come into close proximity through stable, protein-mediated interactions, and the hub model, in which dynamic clusters of transcription-associated proteins facilitate communication over variable distances. Emerging evidence suggests that although enhancer-promoter pairs do come into close proximity during transcriptional activation, these interactions are highly transient, and the precise distances remain challenging to measure. Moving forward, resolving the distinctions between these models will require novel techniques to more precisely measure the spatial and temporal dynamics of enhancer-promoter interactions. Understanding how enhancers interact with promoters will deepen our understanding of the regulation of gene expression and the molecular underpinnings of transcriptional control.

Our genome is exposed to thousands of DNA lesions every day, posing a significant threat to cellular viability. To deal with these lesions, cells have evolved sophisticated repair mechanisms collectively known as the DNA damage response. DNA double-strand breaks (DSBs) are very cytotoxic damages, and their repair requires the precise and coordinated recruitment of multiple repair factors to form nuclear foci. Recent research highlighted that these repair structures behave as biomolecular condensates, i.e. membraneless compartments with liquid-like properties. The formation of condensates is driven by weak, multivalent interactions among proteins and nucleic acids, and recent studies highlighted the roles of poly(ADP-ribose) (PAR) and RNA in regulating DSBs-related condensates. Additionally, the FET family of RNA-binding proteins (including FUS, EWS and TAF15), has emerged as a critical player in the DNA damage response, with recent evidence suggesting that FET proteins support the formation and dynamics of repair condensates. Notably, phase separation of FET proteins is implicated also in their pathological functions in cancer biology, highlighting the pervasive role of condensation. This review will provide an overview of biomolecular condensates at DSBs, focusing on the interplay among PAR and RNA in the spatiotemporal regulation of FET proteins at repair complexes. We will also discuss the role of FET condensates in cancer biology and how they are targeted for therapeutic purposes. The study of biomolecular condensates holds great promise for advancing our understanding of key cellular processes and developing novel therapeutic strategies, but requires careful consideration of potential challenges.

Transcription factors (TFs) contribute to organismal development and function by regulating gene expression. Despite decades of research, the factors determining the specificity and speed at which eukaryotic TFs detect their target binding sites remain poorly understood. Recent studies have pointed to intrinsically disordered regions (IDRs) within TFs as key regulators of the process by which TFs find their target sites on DNA (the TF target search). However, IDRs are challenging to study because they can confer specificity despite low sequence complexity and can be functionally conserved despite rapid sequence divergence. Nevertheless, emerging computational and experimental approaches are beginning to elucidate the sequence-function relationship within the IDRs of TFs. Additional insights are informing potential mechanisms underlying the IDR-directed search for the DNA targets of TFs, including incorporation into biomolecular condensates, facilitating TF co-localization, and the hypothesis that IDRs recognize and directly interact with specific genomic regions.

Cytoskeletal structures shape and confer resistance to cells. The intermediate filament protein vimentin forms versatile structures that play key roles in cytoskeletal crosstalk, in the integration of cellular responses to a variety of external and internal cues, and in the defense against stress. Such multifaceted roles can be fulfilled thanks to the vast variety of vimentin proteoforms, which in turn arise from the combinations of a myriad of tightly regulated posttranslational modifications. Diverse vimentin proteoforms will differentially shape its polymeric assemblies, underlying vimentin ability to organize in filaments, bundles, squiggles, droplets, cell surface-bound and/or various secreted forms. Interestingly, certain vimentin dots or droplets have been lately categorized as biomolecular condensates. Biomolecular condensates are phase-separated membraneless structures that are critical for the organization of cellular components and play important roles in pathophysiology. Recent findings have unveiled the importance of low complexity sequence domains in vimentin filament assembly. Moreover, several oxidants trigger the transition of vimentin filaments into phase-separated biomolecular condensates, a reversible process that may provide clues on the role of condensates as seeds for filament formation. Revisiting previous results in the light of recent knowledge prompts the hypothesis that vimentin condensates could play a role in traffic of filament precursors, cytoskeletal crosstalk and cellular responses to stress. Deciphering the "vimentin posttranslational modification code", that is, the structure-function relationships of vimentin proteoforms, constitutes a major challenge to understand the regulation of vimentin behavior and its multiple personalities. This will contribute to unveil essential cellular mechanisms and foster novel opportunities for drug discovery.

The cytoplasm is a dense and complex milieu in which a plethora of biochemical reactions occur. Its structure is not understood so far, albeit being central to cellular functioning. In this review, we highlight a novel perspective in which the physical properties of the cytoplasm are regulated in space and time and actively contribute to cellular function. Furthermore, we underscore recent findings that the dynamic formation of local assemblies within the cytoplasm, such as condensates and polysomes, serves as a key regulator of mesoscale cytoplasmic dynamics.

Protein misfolding and aggregation drive some of the most prevalent and lethal disorders of our time, including Alzheimer's and Parkinson's diseases, now affecting tens of millions of people worldwide. The complexity of these diseases, which are often multifactorial and related to age and lifestyle, has made it challenging to identify the causes of the accumulation of aberrant protein deposits. An insight into the origins of these deposits comes from reports of a widespread presence of protein aggregates even under normal cellular conditions. This observation is best accounted for by the thermodynamic hypothesis of protein aggregation. According to this hypothesis, many proteins are expressed at levels close to their supersaturation limits, so that their native states are metastable against aggregation. Here we integrate the evidence behind this hypothesis and outline actionable therapeutic strategies that could halt protein aggregation at its source.

Heart transplantation is the gold standard treatment for patients with end-stage heart failure. However, there is a shortage of donor hearts available. The short tolerable cold ischemic time for delivering donor hearts to matching recipients is closely responsible for this shortage. Here we uncover the phenomenon of mineralocorticoid receptor (MR) phase separation, which exacerbates injury to the murine and human donor heart during cold storage and can be modulated with pharmacological inhibition to improve preservation quality. Interestingly, donor cardiomyocytes strongly expressed MR, which undergoes preservation-related phase separation. The phenomenon of macromolecular phase separation is not limited to the heart or MR during preservation. Cold preservation of the lung, liver and kidney also displays phase separation of other transcriptional regulators including histone deacetylase 1 (HDAC1), bromodomain-containing 4 (BRD4) and MR. Our results reveal an understudied area of preservation biology that may be further exploited to improve the preservation of multiple solid organs.

Membraneless organelles (MLOs) are fundamental to cellular organization, enabling biochemical processes by concentrating biomolecules and regulating reactions within confined environments. While micrometer-scale synthetic droplets are extensively studied as models of MLOs, submicron droplets remain largely unexplored despite their potential to uniquely regulate biomolecular processes. Here, submicron droplets are generated by a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a model to investigate their effect on DNA assembly in crowded environments. The findings reveal that submicron droplets exhibit distinct advantages over microdroplets by acting as submicron catalytic centers that concentrate DNA and accelerate assembly kinetics. This enhancement is driven by a cooperative mechanism wherein global crowding from PEG induces an excluded volume effect, while local crowding from dextran provides weak but nonspecific interactions with DNA. By exploiting both the confinement and phase properties of submicron droplets, a rapid and sensitive assay is developed for miRNA detection using confined fluorescent readouts. These findings highlight the unique ability of submicron droplets to amplify biomolecular assembly processes, provide new insights into the interplay between global and local crowding effects in cellular-like environments, and present a platform for biomarker detection and visualization.

Protein-RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein-RNA condensation model, we determined the sequence of events that starts with sub-second formation of a protomer with two RNAs per protein dimer. Association of additional RNA molecules to weaker secondary binding sites in this protomer kickstarts crystallization-like assembly of a molecular condensate. Primary nucleation is faster than the sum of secondary nucleation and growth, which is a multistep process. Protein-RNA nuclei grow over hundreds of seconds into filaments and subsequently into nanoclusters with approximately 600 nm diameter. Cryoelectron microscopy reveals an internal structure formed by incoming layers of protein-RNA filaments made of ribonucleoprotein oligomers, reminiscent of genome packing of a nucleocapsid. These nanoclusters progress to liquid condensate droplets that undergo further partial coalescence to yield typical hydrogel-like protein-RNA coacervates that may represent the scaffold of large viral factory condensates in infected cells. Our integrated experimental kinetic investigation exposes rate-limiting steps and structures along a key biological multistep pathway present across life kingdoms.

Understanding the emergence of biological compartmentalization in the context of the primordial soup is essential for unraveling the origin of life on Earth. This study revisits the classical coacervate theory, examining its historical development, supporting evidence, and major criticisms. Building upon Alexandr Oparin's foundational ideas, we propose an updated perspective in which the first biological compartments emerged through the formation of ribonucleoprotein (RNP) condensates-complexes of intrinsically disordered peptides and RNAs-via liquid-liquid phase separation (LLPS). Drawing on contemporary insights into how LLPS mediates intracellular organization, we argue that such membraneless RNP-based aggregates could have facilitated biochemical reactions in the aqueous environments of early Earth. By reinterpreting Oparin's coacervates through the lens of modern molecular biology, this study offers a renewed framework for understanding the origin of biological compartmentalization within the RNP-world hypothesis.

Phase separation is fundamental for cellular organization and function, profoundly impacting a range of biological processes from gene expression to cellular signaling pathways, pivotal in stem cell biology. This review explores the primary types of phase separation and their mechanisms, emphasizing how phase separation is integral to maintaining cellular integrity and its significant implications for disease progression. It elaborates on current insights into how phase separation influences stem cell biology, discussing the challenges in translating these insights into practical applications. These challenges stem from the complex dynamics of phase separation, the need for advanced imaging techniques, and the necessity for real-time, in situ analysis within living systems. Addressing these challenges through innovative methodologies and gaining a deeper understanding of the molecular interactions that govern phase separation in stem cells are essential for developing precise, targeted therapies. Ultimately, advancing our understanding of phase separation could transform stem cell-based therapeutic approaches, opening up novel strategies for disease treatment and advancements in regenerative medicine.

Iron is one of the most abundant elements on earth. The most recognized role of iron in living organisms is its incorporation in the heme-containing protein hemoglobin, which is abundantly found in the red blood cells that facilitate the oxygen transportation throughout the body. In fact, about 70% of organism's iron is found in hemoglobin. However, besides being essential for oxygen transport and serving as a crucial component of the molecular oxygen-carrying proteins hemoglobin and myoglobin, iron has a wide range of other biological functions. It is involved in numerous metabolic and regulatory processes and therefore is indispensable for almost all living organisms. Since iron enzymes are responsible for most of the redox metallo-catalysts, it is not surprising that 6.5% of all human enzymes are expected to be iron-dependent. Furthermore, iron-binding proteins account for about 2% of the entire proteome. The ironome encompasses heme-binding proteins, proteins binding individual iron ions, and iron-sulfur cluster-binding proteins. Although the structure-function relations of ordered iron-binding proteins are rather well understood, the prevalence and functionality of intrinsic disorder in iron-binding proteins remain to be evaluated. To fill this knowledge gap, in this study, we evaluate the intrinsic disorder of the human ironome. Our analysis revealed that the human ironome contains a noticeable level of functional intrinsic disorder, with most noticeable applications in protein-protein interactions, posttranslational modifications, and liquid-liquid phase separation.

Understanding how the genome is organized into multi-level chromatin structures within cells and how these chromatin structures regulate gene transcription influencing animal development and human diseases has long been a major goal in genetics and cell biology. Recent evidence suggests that chromatin structure formation and remodeling is regulated not only by chromatin loop extrusion but also by phase-separated condensates. Here, we discuss recent findings on the mechanisms of chromatin organization mediated by phase separation, with a focus on the roles of phase-separated condensates in chromatin structural dysregulation in human diseases. Indeed, these mechanistic revelations herald promising therapeutic strategies targeting phase-separated condensates-leveraging their intrinsic biophysical susceptibilities to restore chromatin structure dysregulated by aberrant phase separation.

Undruggable targets are those of therapeutical significance but challenging for conventional drug design approaches. Such targets often exhibit unique features, including highly dynamic structures, a lack of well-defined ligand-binding pockets, the presence of highly conserved active sites, and functional modulation by protein-protein interactions. Recent advances in computational simulations and artificial intelligence have revolutionized the drug design landscape, giving rise to innovative strategies for overcoming these obstacles. In this review, we highlight the latest progress in computational approaches for drug design against undruggable targets, present several successful case studies, and discuss remaining challenges and future directions. Special emphasis is placed on four primary target categories: intrinsically disordered proteins, protein allosteric regulation, protein-protein interactions, and protein degradation, along with discussion of emerging target types. We also examine how AI-driven methodologies have transformed the field, from applications in protein-ligand complex structure prediction and virtual screening to de novo ligand generation for undruggable targets. Integration of computational methods with experimental techniques is expected to bring further breakthroughs to overcome the hurdles of undruggable targets. As the field continues to evolve, these advancements hold great promise to expand the druggable space, offering new therapeutic opportunities for previously untreatable diseases.

Mitochondria are central metabolic organelles that control cell fate and the development of mitochondrial diseases. Traditionally, phase separation directly regulates cell functions by driving RNA, proteins, or other molecules to concentrate into lipid droplets. Recent studies show that phase separation regulates cell functions and diseases through the regulation of subcellular organelles, particularly mitochondria. In fact, phase separation is involved in various mitochondrial activities including nucleoid assembly, autophagy, and mitochondria-related inflammation. Here, we outline the key mechanisms through which phase separation influences mitochondrial activities and the development of mitochondrial diseases. Insights into how phase separation regulates mitochondrial activities and diseases will help us develop interventions for related diseases.

Liquid-liquid phase separation (LLPS) in proteins is essential for cellular organization and biomolecular condensation. However, current methods to induce phase separation often lack precise spatiotemporal control. This study introduces Ponceau S as a selective modulator and monitors phase separation in bovine serum albumin and lysozyme. We demonstrate that Ponceau S effectively promotes the protein complex into liquid droplets by binding to hydrophobic regions and driving intermolecular interactions. Notably, Ponceau S fluorescence increases within protein-rich phases, reflecting the restricted molecular motion in these environments. Furthermore, the phase separation induced by Ponceau S is finely tunable through salt and 1,6-hexanediol adjustments, which influence droplet fusion and dissolution dynamics. This work highlights the potential of small molecules like Ponceau S to precisely regulate and monitor protein phase separation, providing a new dimension of control for applications in biomolecular engineering, drug delivery, and synthetic biology.

The study of biomolecular condensates (BMCs) is of great current interest because of the proposed roles of these types of assemblies in biological function and disease. In living cells, BMCs form in a highly heterogeneous environment and are influenced by concentration gradients of various relevant species. Furthermore, the biological functionality of the BMCs requires precise spatial control of their formation in some cases. In recent years, a number of in vitro experimental approaches have emerged that allow the generation, study, and manipulation of BMCs through the creation of well-defined spatially heterogeneous solution conditions relevant for BMC formation. In this concept article, it is presented in what way such methods can contribute to improved understanding and control of BMCs.

Ovarian cancer has the highest mortality rate among gynecologic tumors worldwide, with unclear underlying mechanisms of pathogenesis. RNA-binding proteins (RBPs) primarily direct post-transcriptional regulation through modulating RNA metabolism. Recent evidence demonstrates that RBPs are also implicated in transcriptional control. However, the role and mechanism of RBP-mediated transcriptional regulation in tumorigenesis remain largely unexplored. Here, we show that the RBP heterogeneous ribonucleoprotein L (hnRNPL) interacts with chromatin and regulates gene transcription by forming phase-separated condensates in ovarian cancer. hnRNPL phase separation activates PIK3CB transcription and glycolysis, thus promoting ovarian cancer progression. Notably, we observe that the PIK3CB promoter is transcribed to produce a non-coding RNA which interacts with hnRNPL and promotes hnRNPL condensation. Furthermore, hnRNPL is significantly amplified in ovarian cancer, and its high expression predicts poor prognosis for ovarian cancer patients. By using cell-derived xenograft and patient-derived organoid models, we show that hnRNPL knockdown suppresses ovarian tumorigenesis. Together, our study reveals that phase separation of the chromatin-associated RBP hnRNPL promotes PIK3CB transcription and glycolysis to facilitate tumorigenesis in ovarian cancer. The formed hnRNPL-PIK3CB-AKT axis depending on phase separation can serve as a potential therapeutic target for ovarian cancer.

Coacervates have been widely used to mimic membraneless organelles (MLOs). However, coacervates without a membrane or stabilizing surface do not feature the same level of stability as MLOs. This study shows that specifically engineered surface-active proteins can interact with the interface of polypeptide coacervates, conferring resistance to coacervate dissolution and fusion. Modulating the molecular characteristics of these coacervate stabilizing proteins highlighted that their dimerization aids in achieving effective interface stabilizers. Cryo-TEM imaging showed a densely packed protein monolayer at the coacervate-liquid interface, while single-molecule super-resolution microscopy captured the dynamic nature of this protein layer, with the proteins rapidly (un)docking and moving across the coacervate interface within milliseconds. These findings suggest a dynamic form of coacervate stabilization driven by transient protein interactions at the condensate interface. This unique form of coacervate stabilization not only provides a new approach to developing stable and dynamically exchanging synthetic condensate systems but, as model systems, can also significantly contribute to our understanding of the mechanisms underlying the temporal stability of MLOs in nature.

Biomolecular condensates (BCs) are phase-separated viscoelastic hubs within demixed solutions enriched in proteins and nucleic acids. Such condensates, also called membraneless organelles, are increasingly observed in cells and serve as transient hubs for spatial organization and compartmentalization of biomolecules. Along with the transiency of formation and dissolution, their ability to sequester molecules has inspired us to develop BCs as potential vehicles to transport and deliver molecular cargo. We recently reported the design of disulfide bond cross-linked phase-separating peptide (PSP) condensates that spontaneously dissolve in reducing conditions ( JACS, 2024, 146 , 255299 ). Based on the premise that the highly reducing cytoplasm could dissolve PSP condensates and release partitioned cargo, here, we demonstrate the ability of PSP condensates to deliver molecular cargo to the cytoplasm of HeLa cells efficiently. We show that PSP condensates deliver a variety of cargos that differ in their sizes and chemistries, including small molecules, peptides, GFP protein (31 kDa), DNA (1.7 kbp), and mRNA. The transfection efficiencies of PSP condensates for delivering DNA and mRNA were also significantly greater than those of a commercial transfection agent. With room to tailor the condensate properties based on cargo and cell types, these results showcase the potential of disulfide-cross-linked PSPs as effective and customizable cellular delivery vehicles, filling a critical demand gap for such delivery systems.

Multiple factors drive biomolecular condensate formation. In plants, condensation of the transcription factors AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 attenuates response to the plant hormone auxin. Here, we report that actin-mediated movement of cytoplasmic ARF condensates enhances condensation. Coarse-grained molecular simulations of active polymers reveal that applied forces drive the associations of macromolecules to enhance phase separation while giving rise to dense phases that preferentially accumulate motile molecules. Our study highlights how molecular motility can drive phase separation, with implications for motile condensates while offering insights into cellular mechanisms that can regulate condensate dynamics.

An increasing number of biomolecules have been shown to phase-separate into biomolecular condensates - membraneless subcellular compartments capable of regulating distinct biochemical processes within living cells. The speed with which they exchange components with the cellular environment can influence how fast biochemical reactions occur inside condensates and how fast condensates respond to environmental changes, thereby directly impacting condensate function. While Fluorescence Recovery After Photobleaching (FRAP) experiments are routinely performed to measure this exchange timescale, it remains a challenge to distinguish the various physical processes limiting fluorescence recovery and identify each associated timescale. Here, we present a reaction-diffusion model for condensate exchange dynamics and show that such exchange can differ significantly from that of conventional liquid droplets due to the presence of a percolated molecular network, which gives rise to different mobility species in the dense phase. In this model, exchange can be limited by diffusion of either the high- or low-mobility species in the dense phase, diffusion in the dilute phase, or the attachment/detachment of molecules to/from the network at the surface or throughout the bulk of the condensate. Through a combination of analytic derivations and numerical simulations in each of these limits, we quantify the contributions of these distinct physical processes to the overall exchange timescale. Demonstrated on a biosynthetic DNA nanostar system, our model offers insight into the predominant physical mechanisms driving condensate material exchange and provides an experimentally testable scaling relationship between the exchange timescale and condensate size. Interestingly, we observe a newly predicted regime in which the exchange timescale scales nonquadratically with condensate size.

RNA-protein (RNP) granules are fundamental components in cells, where they perform multiple crucial functions. Many RNP granules form via phase separation driven by protein-protein, protein-RNA, and RNA-RNA interactions. Notably, associated proteins frequently contain intrinsically disordered regions (IDRs) that can associate with multiple partners. Previously, we showed that synthetic RNA molecules containing multiple hairpin coat-protein binding sites can phase-separate, forming granules capable of selectively incorporating proteins inside. Here, we expand this platform by introducing a phage coat protein with a known IDR that facilitates protein-protein interactions. We show that the coat protein phase-separates on its own in vivo and that introduction of hairpin-containing RNA molecules can lead to dissolvement of the protein granules. We further demonstrate via multiple assays that RNA valency, determined by the number of hairpins present on the RNA, leads to distinctly different phase behaviors, effectively forming a polyphasic, programmable RNP granule. Moreover, by incorporating the gene for a blue fluorescent protein into the RNA, we demonstrate a phase-dependent boost of protein titer. These insights not only shed light on the behavior of natural granules but also hold profound implications for the biotechnology field, offering a blueprint for engineering cellular compartments with tailored functionalities.

We present a phase-field model based on the Cahn-Hilliard equation to investigate the properties of phase separation in DNA nanostar systems. Leveraging a realistic free-energy functional derived from Wertheim theory, our model captures the thermodynamic properties of self-assembling DNA nanostars under various conditions. This approach allows for the study of both one-component and multi-component systems, including mixtures of different nanostar species and cross-linkers. Through numerical simulations, we demonstrate the model's ability to replicate experimental observations, including liquid-liquid phase separation, surface tension variation, and the structural organization of multi-component systems. Our results highlight the versatility and predictive power of the Cahn-Hilliard framework, particularly for complex systems where detailed simulations are computationally prohibitive. This work provides a robust foundation for studying DNA-based materials and their potential applications in nanotechnology and biophysics, including liquid-liquid phase separation in cellular environments.

Liquid droplets recruit their relevant proteins and function together. Previous studies for a series of guest proteins clarified several rules of the recruitment and translational dynamics in the droplets; however, the other guest parameters such as structures, sizes, and amino-acid compositions might mask the single parameter effect. Here, we characterized the properties of GFP mutants with different charged compositions, but the same structure and size, in fused in sarcoma (FUS) droplets using single-molecule fluorescence microscopy. The recruitment of GFP mutants depended on their absolute net charge, whereas the diffusion did not. In the recruitment vs. diffusion plots, GFP mutants with large net charges were distinct from other proteins, demonstrating the importance of long-range electrostatic interaction on the recruitment.

The tumor suppressor p53 modulates the transcription of a variety of genes, constituting a protective barrier against anomalous cellular proliferation. High-frequency "hotspot" mutations result in loss of function by the formation of amyloid-like aggregates that correlate with cancerous progression. We show that full-length p53 undergoes spontaneous homotypic condensation at submicromolar concentrations and in the absence of crowders to yield dynamic coacervates that are stoichiometrically dissolved by DNA. These coacervates fuse and evolve into hydrogel-like clusters with strong thioflavin T binding capacity, which further evolve into fibrillar species with a clearcut branching growth pattern. The amyloid-like coacervates can be rescued by the human papillomavirus master regulator E2 protein to yield large regular droplets. Furthermore, we kinetically dissected an overall condensation mechanism, which consists of a nucleation-growth process by the sequential addition of p53 tetramers, leading to discretely sized and monodisperse early condensates followed by coalescence into bead-like coacervates that slowly evolve to the fibrillar species. Our results suggest strong similarities to condensation-to-amyloid transitions observed in neurological aggregopathies. Mechanistic insights uncover novel key early and intermediate stages of condensation that can be targeted for p53 rescuing drug discovery.

The coronavirus disease 2019 (COVID-19) pandemic has been linked to long-term neurological effects with multifaceted complications of neurodegenerative diseases. Several studies have found that pathological changes in transactive response DNA-binding protein of 43 kDa (TDP-43) are involved in these cases. This review explores the causal interactions between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and TDP-43 from multiple perspectives. Some viral proteins of SARS-CoV-2 have been shown to induce pathological changes in TDP-43 through its cleavage, aggregation, and mislocalization. SARS-CoV-2 infection can cause liquid-liquid phase separation and stress granule formation, which accelerate the condensation of TDP-43, resulting in host RNA metabolism disruption. TDP-43 has been proposed to interact with SARS-CoV-2 RNA, though its role in viral replication remains to be fully elucidated. This interaction potentially facilitates viral replication, while viral-induced oxidative stress and protease activity accelerate TDP-43 pathology. Evidence from both clinical and experimental studies indicates that SARS-CoV-2 infection may contribute to long-term neurological sequelae, including amyotrophic lateral sclerosis-like and frontotemporal dementia-like features, as well as increased phosphorylated TDP-43 deposition in the central nervous system. Biomarker studies further support the link between TDP-43 dysregulation and neurological complications of long-term effects of COVID-19 (long COVID). In this review, we presented a novel integrative framework of TDP-43 pathology, bridging a gap between SARS-CoV-2 infection and mechanisms of neurodegeneration. These findings underscore the need for further research to clarify the TDP-43-related neurodegeneration underlying SARS-CoV-2 infection and to develop therapeutic strategies aimed at mitigating long-term neurological effects in patients with long COVID.