Phase-separated Droplets Research Articles

Mechanical Frustration of Phase Separation in the Cell Nucleus by Chromatin.

Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that cross-linked chromatin can mechanically suppress droplets' coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

SummaryNucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here, we use biophysical tools to study N-protein interactions with oligonucleotides of different lengths, examining the size, composition, secondary structure, and energetics of the resulting states. We observe the formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant change in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates.

Two timescales control the creation of large protein aggregates in cells

Protein aggregation is of particular interest because of its connection with many diseases and disorders. Many factors can alter the dynamics and result of this process, one of them being the diffusivity of the monomers and aggregates in the system. Here, we study experimentally and theoretically an aggregation process in cells, and we identify two distinct physical timescales that set the number and size of aggregates. The first timescale involves fast aggregation of small clusters freely diffusing in the cytoplasm, whereas in the second one, the aggregates are larger than the pore size of the cytoplasm and thus barely diffuse, and the aggregation process is slowed down. However, the process is not entirely halted, potentially reflecting a myriad of active but random forces that stir the aggregates. Such a slow timescale is essential to account for the experimental results of the aggregation process. These results could also have implications in other processes of spatial organization in cell biology, such as phase-separated droplets.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

Potential roles of G-quadruplex structures in RNA granules for physiological and pathological phase separation.

Cellular liquid-liquid phase separation is a physiologically inevitable phenomenon in molecularly crowded environments inside cells and serves to compartmentalize biomolecules to facilitate several functions, forming cytoplasmic and nuclear RNA granules. Abnormalities in the phase separation process in RNA granules are implicated in the onset of several neurodegenerative diseases; the initial liquid-like phase-separated droplets containing pathogenic proteins are prone to aberrantly mature into solid-like droplets. RNAs are involved in the maturation of physiological and pathological RNA granules and are essential for governing the fate of phase-transition processes. Notably, RNA G-quadruplex (G4RNA), which is the secondary structure of nucleic acids that are formed in guanine-rich sequences, appears to be an advantageous scaffold for RNA-derived phase separation because of its multivalent interactions with RNAs and RNA-binding proteins. Here, we summarize the properties of RNA granules in physiological and pathological phase separation and discuss the potential roles of G4RNA in granules.

Effects of liquid–liquid phase separation and relative humidity on the heterogeneous OH oxidation of inorganic–organic aerosols: insights from methylglutaric acid and ammonium sulfate particles

Abstract. Organic compounds residing near the surface of atmospheric aerosol particles are exposed to chemical reactions initiated by gas-phase oxidants, such as hydroxyl (OH) radicals. Aqueous droplets composed of inorganic salts and organic compounds can undergo phase separation into two liquid phases, depending on aerosol composition and relative humidity (RH). Such phase behavior can govern the surface characteristics and morphology of the aerosols, which in turn affect the heterogeneous reactivity of organic compounds toward gas-phase oxidants. In this work, we used an aerosol flow tube reactor coupled with an atmospheric pressure ionization source (direct analysis in real time) and a high-resolution mass spectrometer to investigate how phase separation in model aqueous droplets containing an inorganic salt (ammonium sulfate, AS) and an organic acid (3-methylglutaric acid, 3-MGA) with an organic-to-inorganic dry mass ratio (OIR) of 1 alters the heterogeneous OH reactivity. At high RH, 3-MGA/AS aerosols were aqueous droplets with a single liquid phase. When the RH decreased, aqueous 3-MGA/AS droplets underwent phase separation at ∼75 % RH. Once the droplets were phase-separated, they exhibited either a core–shell, partially engulfed or a transition from core–shell to partially engulfed structure, with an organic-rich outer phase and an inorganic-rich inner phase. The kinetics, quantified by an effective heterogenous OH rate constant, was found to increase gradually from 1.01±0.02×10-12 to 1.73±0.02×10-12 cm3 molec.−1 s−1 when the RH decreased from 88 % to 55 %. The heterogeneous reactivity of phase-separated droplets is slightly higher than that of aqueous droplets with a single liquid phase. This could be explained by the finding that when the RH decreases, higher concentrations of organic molecules (i.e., 3-MGA) are present at or near the droplet surface, which are more readily exposed to OH oxidation, as demonstrated by phase separation measurements and model simulations. This could increase the reactive collision probability between 3-MGA molecules and OH radicals dissolved near the droplet surface and secondary chain reactions. Even for phase-separated droplets with a fully established core–shell structure, the diffusion rate of organic molecules across the organic-rich outer shell is predicted to be fast in this system. Thus, the overall rate of reactions is likely governed by the surface concentration of 3-MGA rather than a diffusion limitation. Overall, understanding the aerosol phase state (single liquid phase versus two separate liquid phases) is essential to better probe the heterogenous reactivity under different aerosol chemical composition and environmental conditions (e.g., RH).

Solubility Product Constant Governs Multivalent Protein Phase Separations

Liquid-liquid phase separation (LLPS) has emerged as an intriguing phenomenon inside living cells. The phase-separated droplets, where specific biochemical events can take place and which also buffer cytosolic concentrations, are composed of weakly binding multivalent proteins or nucleic acids. Despite the explosion of this field of study, a general purpose quantitative principle to predict phase separation for multicomponent (“heterotypic”) binders has yet to emerge. In this study, we used non-spatial (NFSim) and spatial (SpringSaLaD) network-free rule-based modeling to determine the concentration threshold for LLPS as a function of molecular valencies. Remarkably, the simulations suggest that the phase separation behavior conforms to the solubility product constant (Ksp), familiar for ions in solution in equilibrium with solid salts: as long as the product of monomer concentrations exceeded the Ksp, the system showed phase separation - irrespective of the individual monomer concentrations. The solubility product constant decreases as the molecular valency of binding partners increases, shifting the concentration threshold to lower values. When we mixed molecules with different valencies, penta-valent and tri-valent, for example, Ksp followed an expression determined by ideal stoichiometries, similar to that of a mixed-valent salt like Al2(SO4)3. Spatial simulations revealed the importance of molecular flexibility and inter binding sites distance of the multivalent molecules. When the inter-site distances of a heterotypic tetravalent molecular pair are exactly matched, a prevalence of dimer (“dimeric trap”) was observed. A rigid molecular pair, with equal inter-site distances, produced this “dimeric trap” more prominently, while unequal inter-site distances disrupted the trap and formed large clusters that displayed a Ksp similar to that found in the non-spatial simulations. Thus our study establishes a general principle underlying the concentration threshold for phase separations, the Ksp, and how molecular structure can shape it. (Support: NIH grants R24GM137787 and R01GM132859)

Investigation of Dynamics along Divergent Tau Aggregation Pathways

Intrinsically disordered proteins (IDPs) are ubiquitous and perform a range of functions vital to cell signaling, cytoskeletal architecture, and cellular physiology. IDP function depends on their ability to dynamically reconfigure themselves to interact with other proteins, lipids, and biopolymer binding partners. Microtubule-associated protein tau has been reported to form phase-separated droplets as well as a variety of amyloid fibril morphologies. This behavioral complexity makes tau an interesting model for studying the molecular recognition features, sequence-dependence, dynamic factors, and structural intricacies associated with IDP behaviors. Studies of tau in vitro rely on polyanions to induce aggregation, but recent evidence suggests that the morphology of fibrils formed in vitro is distinct from those formed in vivo and may be inducer dependent. While these observations indicate a tunable self-assembly landscape, we do not understand the structural or kinetic mechanisms by which a given inducer drives tau aggregation into one morphology or another. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments probe the accessibility of backbone amides throughout a protein and can track local secondary structure associated with tau self-assembly. However, the rapid fluctuations exhibited by tau and other IDPs can make them challenging to interrogate using conventional HDX-MS. We describe the application of a microfluidic mixing apparatus that extends the dynamic range of HDX-MS into the millisecond regime, revealing the conformational dynamics of monomeric tau. We also examine changes in HDX behavior observed over the course of tau aggregation reactions, complemented by fluorescence-based kinetic measurements. Taken together, these approaches could advance our understanding of the tau self-assembly landscape and demonstrate the value of HDX-MS in studying similarly challenging IDP systems.

The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 μM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation.

Autophagosomes get wet.

Agudo-Canalejo et al. now report in Nature that the process of wetting, whereby a liquid establishes a contact with a surface, underlies interactions of phase-separated droplets with autophagic membranes.

Controlled Formation of All-Aqueous Janus Droplets by Liquid-Liquid Phase Separation of an Aqueous Three-Phase System.

Janus droplets have been demonstrated in a wide range of applications, ranging from drug delivery, to biomedical imaging, to bacterial detection. However, existing fabrication strategies often involve nonaqueous solvents, such as organic solvent or oil, which largely limits their use in fields that require a high degree of biocompatibility. Here, we present a method to achieve all-aqueous Janus droplets by liquid-liquid phase separation of an aqueous three-phase system (A3PS). An aqueous droplet containing two initially miscible polymers is first injected into an aqueous solution of another concentrated polymer, and then it spontaneously phase-separates into a Janus droplet due to the diffusive mass exchange between the drop and bulk phases during equilibration. To achieve continuous generation of the Janus droplets, the A3PS is further integrated with microfluidics and electrospray. The size and shape of the phase-separated Janus droplets can be easily controlled by tuning the operation parameters, such as the flow rate and/or the initial composition of the drop phases. Dumbbell-shaped and snowman-shaped Janus droplets with average sizes between 100 and 400 μm can be generated by both coflow microfluidics and electrospray. In particular, the phase-separated Janus droplets can simultaneously load two different liposomes into each compartment, which are promising carriers for combination drugs. The obtained Janus droplets are superior templates for biocompatible materials, which can serve as building blocks such as high-order droplet patterns for constructing advanced biomaterials.

Nuclear Condensates of p300 Formed Though the Structured Catalytic Core Can Act as a Storage Pool of p300 with Reduced HAT Activity

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core, representing a rare example of biomolecular phase separation occurred through structured regions - the catalytic HAT domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

Self-programmed enzyme phase separation and multiphase coacervate droplet organization.

Membraneless organelles are phase-separated droplets that are dynamically assembled and dissolved in response to biochemical reactions in cells. Complex coacervate droplets produced by associative liquid–liquid phase separation offer a promising approach to mimic such dynamic compartmentalization. Here, we present a model for membraneless organelles based on enzyme/polyelectrolyte complex coacervates able to induce their own condensation and dissolution. We show that glucose oxidase forms coacervate droplets with a cationic polysaccharide on a narrow pH range, so that enzyme-driven monotonic pH changes regulate the emergence, growth, decay and dissolution of the droplets depending on the substrate concentration. Significantly, we demonstrate that time-programmed coacervate assembly and dissolution can be achieved in a single-enzyme system. We further exploit this self-driven enzyme phase separation to produce multiphase droplets via dynamic polyion self-sorting in the presence of a secondary coacervate phase. Taken together, our results open perspectives for the realization of programmable synthetic membraneless organelles based on self-regulated enzyme/polyelectrolyte complex coacervation.

Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.

HP1β carries an acidic linker domain and requires H3K9me3 for phase separation

ABSTRACT Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1β which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1β mutants defective in H3K9me3 binding were less efficient in phase separationin vitro and failed to accumulate at heterochromatin in vivo. We propose that multivalent interactions of HP1β with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments in vivo.

Modeling Elastically Mediated Liquid-Liquid Phase Separation.

We propose a continuum theory of the liquid-liquid phase separation in an elastic network, where phase-separated microscopic droplets rich in one fluid component can form as an interplay of fluids mixing, droplet nucleation, network deformation, thermodynamic fluctuation, etc. We find that the size of the phase-separated droplets decreases with the shear modulus of the elastic network in the form of ∝[modulus]^{-1/3} and the number density of the droplet increases almost linearly with the shear modulus ∝[modulus], which are verified by the experimental observations. Phase diagrams in the space of (fluid constitution, mixture interaction, network modulus) are provided, which can help to understand similar phase separations in biological cells and also to guide fabrications of synthetic cells with desired phase properties.

The Effect of Curing Temperature on the Morphology and Electro-Optical Properties of a Flexible UV-Cured Polymer Dispersed Liquid Crystal (PDLC)

In this work, the experimental data on flexible PDLC films at the industrial scale and the effect of cure temperature (Tc) on the morphology and electro-optical properties of a UV-cured PDLC system via polymer-induced phase separation (PIPS) method were studied. Under constant UV radiation intebsity and thickness, the morphological parameters such as dimension, number density, and volume fraction of the phase-separated liquid crystal droplets as well as the optical transmissions and switching voltages as a function of cure temperature were determined. The effect of cure temperature on the morphology and electro-optical properties of UV-cured PDLC films were analyzed.

Modeling the Mechanisms by Which Coexisting Biomolecular RNA–Protein Condensates Form

Liquid-liquid phase separation is an emerging mechanism for intracellular organization. This work presents a mathematical model to examine molecular mechanisms that yield phase-separated droplets composed of different RNA-protein complexes. Using a Cahn-Hilliard diffuse interface model with a Flory-Huggins free energy scheme, we explore how multiple (here two, for simplicity) protein-RNA complexes (species) can establish a heterogeneous droplet field where droplets with single or multiple species phase separate and evolve during coarsening. We show that the complex-complex de-mixing energy tunes whether the complexes co-exist or form distinct droplets, while the transient binding kinetics dictate both the timescale of droplet formation and whether distinct species phase separate into droplets simultaneously or sequentially. For specific energetics and kinetics, a field of droplets driven by the formation of only one protein-RNA complex will emerge. Slowly, the other droplet species will accumulate inside the preformed droplets of the other species, allowing them to occupy the same droplet space. Alternatively, unfavorable species mixing creates a parasitic relationship: the slow-to-form protein-RNA complex will accumulate at the surface of a competing droplet species, siphoning off the free protein as it is released. Once this competing protein-RNA complex has sufficiently accumulated on the droplet surface, it can form a new droplet that is capable of sharing an interface with the first complex droplet but is not capable of mixing. These results give insights into a wide range of phase-separation scenarios and heterogeneous droplets that coexist but do not mix within the nucleus and the cytoplasm of cells.

Beyond Langmuir: surface-bound macromolecule condensates.

Macromolecule condensates, phase separation, and membraneless compartments have become an important area of cell biology research where new biophysical concepts are emerging. This article discusses the possibility that condensates assemble on multivalent surfaces such as DNA, microtubules, or lipid bilayers by multilayer adsorption. Langmuir isotherm theory conceptualized saturable surface binding and deeply influenced physical biochemistry. Brunauer-Emmett-Teller (BET) theory extended Langmuir’s ideas to multilayer adsorption. A BET-inspired biochemical model predicts that surface-binding proteins with a tendency to self-associate will form multilayered condensates on binding surfaces. These “bound condensates” are expected to assemble well below the saturation concentration for liquid–liquid phase separation, so they can compete subunits away from phase-separated droplets and are thermodynamically pinned to the binding surface. Tau binding to microtubules is an interesting test case. The nonsaturable binding isotherm is reminiscent of BET predictions, but assembly of Tau-rich domains at low concentrations requires a different model. Surface-bound condensates may find multiple biological uses, particularly in situations where it is important that condensate assembly is spatially constrained, such as gene regulation.

Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.