Membraneless Organelles Research Articles

Membraneless organelles in health and disease: exploring the molecular basis, physiological roles and pathological implications

AbstractOnce 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.

Chromatin compaction during confined cell migration induces and reshapes nuclear condensates

AbstractCell migration through small constrictions during cancer metastasis requires significant deformation of the nucleus, with associated mechanical stress on the nuclear lamina and chromatin. However, how mechanical deformation impacts various subnuclear structures, including protein and nucleic acid-rich biomolecular condensates, is largely unknown. Here, we find that cell migration through confined spaces gives rise to mechanical deformations of the chromatin network, which cause embedded nuclear condensates, including nucleoli and nuclear speckles, to deform and coalesce. Chromatin deformations exhibit differential behavior in the advancing vs. trailing region of the nucleus, with the trailing half being more permissive for de novo condensate formation. We show that this results from increased chromatin heterogeneity, which gives rise to a shift in the binodal phase boundary. Taken together, our findings show how chromatin deformation impacts condensate assembly and properties, which can potentially contribute to cellular mechanosensing.

Diverse effects of fluorescent labels on alpha-synuclein condensate formation during liquid-liquid phase separation.

Liquid-liquid phase separation is an emerging field of study, dedicated to understanding the mechanism and role of biomolecule assembly into membraneless organelles. One of the main methods employed in studying protein and nucleic acid droplet formation is fluorescence microscopy. Despite functioning as an excellent tool for monitoring biomolecule condensation, a few recent reports have presented possible drawbacks of using fluorescently labeled particles. It was observed that fluorescent tags could alter the process of protein liquid-liquid phase separation and even promote their aggregation. In this study, we examined the influence of three different protein labels on alpha-synuclein phase separation in vitro and determined that the changes in droplet formation were related to both the type, as well as concentration of the fluorescently tagged alpha-synuclein. Both protein-based labels (mCherry and eGFP) induced the formation of significantly larger droplets, while fluorescein-tagged alpha-synuclein generated an abundance of small condensates or noticeably inhibited the process of LLPS. The study also revealed that alpha-synuclein with protein-based labels could self-associate at much lower concentrations than its untagged counterpart, forming either large droplets or protein aggregates.

Novel insights into RNA polymerase II transcription regulation: transcription factors, phase separation, and their roles in cardiovascular diseases.

Transcription factors (TFs) are specialized proteins that bind DNA in a sequence-specific manner and modulate RNA polymerase II (Pol II) in multiple steps of the transcription process. Phase separation is a spontaneous or driven process that can form membrane-less organelles called condensates. By creating different liquid phases at active transcription sites, the formation of transcription condensates can reduce the water content of the condensate and lower the dielectric constant in biological systems, which in turn alters the structure and function of proteins and nucleic acids in the condensate. In RNA Pol II transcription, phase separation formation shortens the time at which TFs bind to target DNA sites and promotes transcriptional bursting. RNA Pol II transcription is engaged in developing several diseases, such as cardiovascular disease, by regulating different TFs and mediating the occurrence of phase separation. This review aims to summarize the advances in the molecular mechanisms of RNA Pol II transcriptional regulation, in particular the effect of TFs and phase separation. The role of RNA Pol II transcriptional regulation in cardiovascular disease will be elucidated, providing potential therapeutic targets for the management and treatment of cardiovascular disease.

Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes.

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the combined effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.

Nonspecific Yet Selective Interactions Contribute to Small Molecule Condensate Binding.

Biomolecular condensates are essential in various cellular processes, and their misregulation has been demonstrated to underlie disease. Small molecules that modulate condensate stability and material properties offer promising therapeutic approaches, but mechanistic insights into their interactions with condensates remain largely lacking. We employ a multiscale approach to enable long-time, equilibrated all-atom simulations of various condensate-ligand systems. Systematic characterization of the ligand binding poses reveals that condensates can form diverse and heterogeneous chemical environments with one or multiple chains to bind small molecules. Unlike traditional protein-ligand interactions, these chemical environments are dominated by nonspecific hydrophobic interactions. Nevertheless, the chemical environments feature unique amino acid compositions and physicochemical properties that favor certain small molecules over others, resulting in varied ligand partitioning coefficients within condensates. Notably, different condensates share similar sets of chemical environments but at different populations. This population shift drives ligand selectivity toward specific condensates. Our approach can enhance the interpretation of experimental screening data and may assist in the rational design of small molecules targeting specific condensates.

Optical characterization of molecular interaction strength in protein condensates.

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

Chromatin inspired bio-condensation between biomass DNA and guanosine monophosphate produces all-nucleic hydrogel as a hydrotropic drug carrier.

The integration of biomolecules into supramolecular nanostructures forms the basis of the natural world. Naturally occurring liquid-liquid phase separation resulting in biomolecular condensates has inspired the formation of biomolecule-based smart materials with multi-dimensional applications. A non-covalent bio-condensation between biomass DNA and guanosine monophosphate (GMP) has been described, mimicking chromatin folding and creating a unique "all-nucleic" DNA-GMP condensates. These condensates initiate the formation of G-quadruplex-based superstructures, assembling into super-helical fibres driven by synergistic hydrogen bonding and stacking, which have been thoroughly investigated. This simple, one-step method for the bio-condensation of biomass DNA leads to an "all-nucleic" hydrogel with higher-order self-assembly and excellent mechanical properties. While most of the reported DNA based biomaterials, including hydrogels, require precisely sequenced and molecularly architectured DNA building blocks, we have developed a simple, universal, and facile bio-condensation method that utilizes biomass DNA acquired from any bio-resource to fabricate DNA hydrogels. The hydrogel efficiently encapsulates and sustains the release of both hydrophilic and hydrophobic drugs, demonstrating its competency as a drug carrier. We believe this energy-efficient and low-cost method represents a new technique for using biomass DNA as building blocks for the next generation of soft materials.

Wnt signalosomes: What we know that we do not know.

Signaling through the Wnt/β-catenin pathway is relayed through three multiprotein complexes: (1) the membrane-associated signalosome, which includes the activated Wnt receptors, (2) the cytoplasmic destruction complex that regulates turnover of the transcriptional coactivator β-catenin, and (3) the nuclear enhanceosome that mediates pathway-specific transcription. Recent discoveries have revealed that Wnt receptor activities are tightly regulated to maintain proper tissue homeostasis and that aberrant receptor upregulation enhances Wnt signaling to drive tumorigenesis, highlighting the importance of signalosome control. These studies have focused on the detailed process by which Wnt ligands engage their coreceptors, LRP5/6 and Frizzled. However, the components that constitute the signalosome and the regulation of their assembly remain undefined. In this review, we discuss Wnt/β-catenin signalosome composition and the mechanisms that regulate signalosome assembly, including the role of biomolecular condensates and ubiquitylation. We also summarize the evidence for the presence of Wnt ligand-independent signalosome formation.

Transcription regulation by biomolecular condensates.

Biomolecular condensates regulate transcription by dynamically compartmentalizing the transcription machinery. Classic models of transcription regulation focus on the recruitment and regulation of RNA polymerase II by the formation of complexes at the 1-10 nm length scale, which are driven by structured and stoichiometric interactions. These complexes are further organized into condensates at the 100-1,000 nm length scale, which are driven by dynamic multivalent interactions often involving domain-ligand pairs or intrinsically disordered regions. Regulation through condensate-mediated organization does not supersede the processes occurring at the 1-10 nm scale, but it provides regulatory mechanisms for promoting or preventing these processes in the crowded nuclear environment. Regulation of transcription by transcriptional condensates is involved in cell state transitions during animal and plant development, cell signalling and cellular responses to the environment. These condensate-mediated processes are dysregulated in developmental disorders, cancer and neurodegeneration. In this Review, we discuss the principles underlying the regulation of transcriptional condensates, their roles in physiology and their dysregulation in human diseases.

Enzyme-Responsive DNA Condensates.

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remains elusive. Here, we demonstrate that the activity of nucleic-acid cleaving enzymes can be localized within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organize different substrates in concentric subcompartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides expanding our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.

Protein condensates unfold G-quadruplex resembling a helicase activity.

Membrane-less organelles, formed by liquid-liquid phase separation, participate in many vital cellular processes and have received extensive attention recently. A notable form of noncanonical nucleic acid secondary structure, G-quadruplex (G4), interacts with the scaffolding proteins in these membrane-less organelles and becomes an integral part of this condensed phase. However, the structure and stability features of the integrated G4 remain poorly characterized. Herein, we employed NMR along with other biophysical methods to investigate the conformation of a G4 within condensates formed by a disordered protein known as DDX4N1. We discovered that the human telomeric sequence MHT24, which forms a G4 structure in a non-condensed phase solution of protein DDX4N1, unfolds when it is within DDX4N1 condensates due to phase separation. Our findings provide an instance of a protein acquiring new functionality through phase separation process, which deepen our understanding of how protein condensates regulate G4 structure and their functions.

Accelerated Simulations Reveal Physicochemical Factors Governing Stability and Composition of RNA Clusters.

Under certain conditions, RNA repeat sequences phase separate, yielding protein-free biomolecular condensates. Importantly, RNA repeat sequences have also been implicated in neurological disorders, such as Huntington's disease. Thus, mapping repeat sequences to their phase behavior, functions, and dysfunctions is an active area of research. However, despite several advances, it remains challenging to characterize the RNA phase behavior at a submolecular resolution. Here, we have implemented a residue-resolution coarse-grained model in LAMMPS─that incorporates both the RNA sequence and structure─to study the clustering propensities of protein-free RNA systems. Importantly, we achieve a multifold speedup in the simulation time compared to previous work. Leveraging this efficiency, we study the clustering propensity of all 20 nonredundant trinucleotide repeat sequences. Our results align with findings from experiments, emphasizing that canonical base-pairing and G-U wobble pairs play dominant roles in regulating cluster formation of RNA repeat sequences. Strikingly, we find strong entropic contributions to the stability and composition of RNA clusters, which is demonstrated for single-component RNA systems as well as binary mixtures of trinucleotide repeats. Additionally, we investigate the clustering behaviors of trinucleotide (odd) repeats and their quadranucleotide (even) counterparts. We observe that odd repeats exhibit stronger clustering tendencies, attributed to the presence of consecutive base pairs in their sequences that are disrupted in even repeat sequences. Altogether, our work extends the set of computational tools for probing RNA cluster formation at submolecular resolution and uncovers physicochemical principles that govern the stability and composition of the resulting clusters.

Nonspecific interactions in transcription regulation and organization of transcriptional condensates

Eukaryotic cells are characterized by a high degree of compartmentalization of their internal contents, which ensures precise and controlled regulation of intracellular processes. During many processes, including different stages of transcription, dynamic membrane-free compartments called biomolecular condensates are formed. Transcription condensates contain various transcription factors and RNA polymerase and are formed by high and low specificity interactions between protein factors, DNA and nearby RNA. This review discusses recent data demonstrating the important role of nonspecific multivalent protein-protein and RNA-protein interactions in the organization and regulation of transcription.

Spatiotemporal protein interactome profiling through condensation-enhanced photocrosslinking.

Resolving protein-protein interactions (PPIs) inside biomolecular condensates is crucial for elucidating their functions and regulation mechanisms. The transient nature of condensates and the multiple localizations of clients, however, have rendered it challenging to determine compartment-specific PPIs. Here we developed a condensation-enhanced, spatially directed, metabolic incorporation-assisted photocrosslinking strategy-termed DenseMAP-for spatiotemporally resolved dissection of the direct protein interactome within condensates. By leveraging our condensation-enhanced photocrosslinker and the spatially directed biotin tagging, DenseMAP enabled stress-granule-specific interactome mapping of the N6-methyladenosine readers YTHDF1 and YTHDF2, and uncovered the functional role of phosphorylation on the SARS-CoV-2 nucleocapsid protein in regulating virus replication. Further applying DenseMAP for direct interactome mapping of the subcompartmental scaffold protein NPM1 deciphered nucleolar granular component proteome, and unveiled the critical role of SUMOylation in controlling nucleolar proteome homeostasis. DenseMAP provides a platform technology for analysing functional PPI networks within subcellular and subcompartmental condensates under diverse physiological and/or pathological settings.

Unravelling the Molecular Dialogue of Beneficial Microbe-Plant Interactions.

Plants are an intrinsic part of the soil community, which is comprised of a diverse range of organisms that interact in the rhizosphere through continuous molecular communications. The molecular dialogue within the plant microbiome involves a complex repertoire of primary and secondary metabolites that interact within different liquid matrices and biofilms. Communication functions are likely to involve membrane-less organelles formed by liquid-liquid phase separation of proteins and natural deep eutectic solvents that play a role as alternative media to water. We discuss the chemistry of inter-organism communication and signalling within the biosphere that allows plants to discriminate between harmful, benign and beneficial microorganisms. We summarize current information concerning the chemical repertoire that underpins plant-microbe communication and host-range specificity. We highlight how the regulated production, perception and processing of reactive oxygen species (ROS) is used in the communication between plants and microbes and within the communities that shape the soil microbiome.

Nonequilibrium thermodynamics of non-ideal reaction-diffusion systems: Implications for active self-organization.

We develop a framework describing the dynamics and thermodynamics of open non-ideal reaction-diffusion systems, which embodies Flory-Huggins theories of mixtures and chemical reaction network theories. Our theory elucidates the mechanisms underpinning the emergence of self-organized dissipative structures in these systems. It evaluates the dissipation needed to sustain and control them, discriminating the contributions from each reaction and diffusion process with spatial resolution. It also reveals the role of the reaction network in powering and shaping these structures. We identify particular classes of networks in which diffusion processes always equilibrate within the structures, while dissipation occurs solely due to chemical reactions. The spatial configurations resulting from these processes can be derived by minimizing a kinetic potential, contrasting with the minimization of the thermodynamic free energy in passive systems. This framework opens the way to investigating the energetic cost of phenomena, such as liquid-liquid phase separation, coacervation, and the formation of biomolecular condensates.

Chemical reactions regulated by phase-separated condensates

Phase-separated liquid condensates can spatially organize and thereby regulate chemical processes. However, the physicochemical mechanisms underlying such regulation remain elusive as the intramolecular interactions responsible for phase separation give rise to a coupling between diffusion and chemical reactions at nondilute conditions. Here, we derive a theoretical framework that decouples the phase separation of scaffold molecules from the reaction kinetics of diluted clients. As a result, phase volume and client partitioning coefficients become control parameters, which enables us to dissect the impact of phase-separated condensates on chemical reactions. We apply this framework to two chemical processes and show how condensates affect the yield of reversible chemical reactions and the initial rate of a simple assembly process. In both cases, we find an optimal condensate volume at which the respective chemical reaction property is maximal. Our work can be applied to experimentally quantify how condensed phases alter chemical processes in systems biology and unravel the mechanisms of how biomolecular condensates regulate biochemistry in living cells. Published by the American Physical Society 2024

Interplay between membranes and biomolecular condensates in the regulation of membrane-associated cellular processes.

Liquid‒liquid phase separation (LLPS) has emerged as a key mechanism for organizing cellular spaces independent of membranes. Biomolecular condensates, which assemble through LLPS, exhibit distinctive liquid droplet-like behavior and can exchange constituents with their surroundings. The regulation of condensate phases, including transitions from a liquid state to gel or irreversible aggregates, is important for their physiological functions and for controlling pathological progression, as observed in neurodegenerative diseases and cancer. While early studies on biomolecular condensates focused primarily on those in fluidic environments such as the cytosol, recent discoveries have revealed their existence in close proximity to, on, or even comprising membranes. The aim of this review is to provide an overview of the properties of membrane-associated condensates in a cellular context and their biological functions in relation to membranes.

Caspase-2 kills cells with extra centrosomes.

Centrosomes are membrane-less organelles that orchestrate a wide array of biological functions by acting as microtubule organizing centers. Here, we report that caspase-2-driven apoptosis is elicited in blood cells failing cytokinesis and that extra centrosomes are necessary to trigger this cell death. Activation of caspase-2 depends on the PIDDosome multi-protein complex, and priming of PIDD1 at extra centrosomes is necessary for pathway activation. Accordingly, loss of its centrosomal adapter, ANKRD26, allows for cell survival and unrestricted polyploidization in response to cytokinesis failure. Mechanistically, cell death is initiated upstream of mitochondria via caspase-2-mediated processing of the BCL2 family protein BID, driving BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Remarkably, BID-deficient cells enforce apoptosis by engaging p53-dependent proapoptotic transcriptional responses initiated by caspase-2. Consistently, BID and MDM2 act as shared caspase-2 substrates, with BID being kinetically favored. Our findings document that the centrosome limits its own unscheduled duplication by the induction of PIDDosome-driven mitochondrial apoptosis to avoid potentially pathogenic polyploidization events.