Determinants of viscoelasticity and flow activation energy in biomolecular condensates.

The form and function of biomolecular condensates are intimately linked to their material properties. Here, we integrate microrheology with molecular simulations to dissect the physical determinants of condensate fluid phase dynamics. By quantifying the timescales and energetics of network relaxation in a series of heterotypic viscoelastic condensates, we uncover distinctive roles of sticker motifs, binding energy, and chain length in dictating condensate dynamical properties. We find that the mechanical relaxation times of condensate-spanning networks are determined by both intermolecular interactions and chain length. We demonstrate, however, that the energy barrier for network reconfiguration, termed flow activation energy, is independent of chain length and only varies with the strengths of intermolecular interactions. Biomolecular diffusion in the dense phase depends on a complex interplay between viscoelasticity and flow activation energy. Our results illuminate distinctive roles of chain length and sequence-specific multivalent interactions underlying the complex material and transport properties of biomolecular condensates.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates.

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart active motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced active motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates.

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart active motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with reentrant phase behavior, the gradient-induced active motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and active transport of biomolecular condensates.

Sequence-specific interactions determine viscoelasticity and aging dynamics of protein condensates.

Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.

Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation of protein and RNA complexes into biomolecular condensates has emerged as a ubiquitous phenomenon in living systems. These protein-RNA condensates are thought to be involved in many biological functions in all forms of life. One of the most sought-after properties of these condensates is their dynamical properties, as they are a major determinant of condensate physiological function and disease processes. Measurement of the diffusion dynamics of individual components in a multicomponent biomolecular condensate is therefore routinely performed. Here, we outline the experimental procedure for performing in-droplet fluorescence correlation spectroscopy (FCS) measurements to extract the diffusion coefficient of individual molecules within a biomolecular condensate in vitro. Unlike more common experiments such as fluorescence recovery after photobleaching (FRAP), where data interpretation is not straightforward and strictly model dependent, FCS offers a robust and more accurate way to quantify biomolecular diffusion rates in the dense phase. The small observation volume allows FCS experiments to report on the local diffusion coefficient within a spatial resolution of <1 µm, making it ideal for probing spatial inhomogeneities within condensates as well as variable dynamics within subcompartments of multiphasic condensates.

RNA chain length and stoichiometry govern surface tension and stability of protein-RNA condensates.

Proteomic studies have shown that cellular condensates are frequently enriched in diverse RNA molecules, which is suggestive of mechanistic links between phase separation and transcriptional activities. Here, we report a systematic experimental and computational study of thermodynamic landscapes and interfacial properties of protein-RNA condensates. We have studied the affinity of protein-RNA condensation as a function of variable RNA sequence length and RNA-protein stoichiometry under different ionic environments and external crowding. We have chosen the PolyU sequences for RNA and arginine/glycine-rich intrinsically disordered peptide (RGG) for proteins as a model system of RNA-protein condensates, which we then investigate through in vitro microscopy measurements and coarse-grained molecular dynamics simulations. We find that crowding and RNA chain length can have a major stabilizing effect on the condensation. We also find that the RNA-protein charge ratio is a crucial variable controlling stability, interfacial properties, and the reentrant phase behavior of RGG-RNA mixtures.

Temperature-dependent reentrant phase transition of RNA-polycation mixtures.

Liquid-liquid phase separation (LLPS) of multivalent biopolymers is a ubiquitous process in biological systems and is of importance in bio-mimetic soft matter design. The phase behavior of biomolecules, such as proteins and nucleic acids, is typically encoded by the primary chain sequence and regulated by solvent properties. One of the most important physical modulators of LLPS is temperature. Solutions of proteins and/or nucleic acids have been shown to undergo liquid-liquid phase separation either upon cooling (with an upper critical solution temperature, UCST) or upon heating (with a lower critical solution temperature, LCST). However, many theoretical frameworks suggest the possibility of more complex temperature-dependent phase behaviors, such as an hourglass or a closed-loop phase diagram with concurrent UCST and LCST transitions. Here, we report that RNA-polyamine mixtures undergo a reentrant phase separation with temperature. Specifically, at low temperatures, RNA-polyamine mixtures form a homogenous phase. Increasing the temperature leads to the formation of RNA-polyamine condensates. A further increase in temperature leads to the dissolution of condensates, rendering a reentrant homogenous phase. This dual-response phase separation of RNA is not unique to polyamines but also observed with short cationic peptides. The immiscibility gap is controlled by the charge of the polycation, salt concentration, and mixture composition. Based on the existing theories of complex coacervation, our results point to a complex interplay between desolvation entropy, ion-pairing, and electrostatic interactions in dictating the closed-loop phase behavior of RNA-polycation mixtures.

Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides.

Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.

Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies.

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases-homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

Quantifying viscosity and surface tension of multicomponent protein-nucleic acid condensates.

Living cells organize their internal space into dynamic condensates through liquid-liquid phase separation of multivalent proteins in association with cellular nucleic acids. Here, we study how variations in nucleic acid (NA)-to-protein stoichiometry modulate the condensed phase organization and fluid dynamics in a model system of multicomponent heterotypic condensates. Employing a multiparametric approach comprised of video particle tracking microscopy and optical tweezer-induced droplet fusion, we show that the interfacial tension, but not viscosity, of protein-NA condensates is controlled by the NA/protein ratio across the two-phase regime. In parallel, we utilize fluorescence correlation spectroscopy to quantify protein and NA diffusion in the condensed phase. Fluorescence correlation spectroscopy measurements reveal that the diffusion of the component protein and NA within the condensate core is governed by the viscosity, and hence, also remains insensitive to the changes in NA-to-protein stoichiometry. Collectively, our results provide insights into the regulation of multicomponent heterotypic liquid condensates, reflecting how the bulk mixture composition affects their core versus surface organization and dynamical properties.

Methods for characterizing the material properties of biomolecular condensates.

Biomolecular condensates are membrane-less sub-cellular compartments that perform a plethora of important functions in signaling and storage. The material properties of biomolecular condensates such as viscosity, surface tension, viscoelasticity, and macromolecular diffusion play important roles in regulating their biological functions. Aberrations in these properties have been implicated in various neurodegenerative disorders and certain types of cancer. Unraveling the molecular driving forces that control the fluid structure and dynamics of biomolecular condensates across different length- and time-scales necessitates the application of innovative biophysical methodologies. In this chapter, we discuss major experimental techniques that are widely used to study the material states and dynamics of biomolecular condensates as well as their practical and conceptual limitations. We end this chapter with a discussion on more advanced tools that are currently emerging to address the complex fluid dynamics of these condensates.

Phase transition of RNA-protein complexes into ordered hollow condensates.

Liquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Divalent cations can control a switch-like behavior in heterotypic and homotypic RNA coacervates.

Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model system comprised of an arginine-rich peptide and RNA, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a general mechanism for modulating the biochemical environment of RNA coacervates in a cellular context.

Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes.

In eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a windowlike phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using ribonucleoprotein-inspired polypeptides with low-complexity RNA-binding sequences as well as an archetypal disordered RNP, fused in sarcoma, as model systems, we investigate the molecular driving forces underlying this nonmonotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC behavior of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, control the two-phase coexistence regime, regulate material properties of polypeptide-RNA condensates, and oppose condensate reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloidlike cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

Molecular Crowding Tunes Material States of Ribonucleoprotein Condensates.

Ribonucleoprotein (RNP) granules are membraneless liquid condensates that dynamically form,dissolve, and mature into a gel-like state in response to a changing cellular environment. RNP condensation islargely governed by promiscuous attractive inter-chain interactions mediated by low-complexity domains(LCDs). Using an archetypal disordered RNP, fused in sarcoma (FUS), here we study how molecular crowdingimpacts the RNP liquid condensation. We observe that the liquid⁻liquid coexistence boundary of FUS islowered by polymer crowders, consistent with an excluded volume model. With increasing bulk crowderconcentration, the RNP partition increases and the diffusion rate decreases in the condensed phase.Furthermore, we show that RNP condensates undergo substantial hardening wherein protein-dense dropletstransition from viscous fluid to viscoelastic gel-like states in a crowder concentration-dependent manner.Utilizing two distinct LCDs that broadly represent commonly occurring sequence motifs driving RNP phasetransitions, we reveal that the impact of crowding is largely independent of LCD charge and sequence patterns.These results are consistent with a thermodynamic model of crowder-mediated depletion interaction, whichsuggests that inter-RNP attraction is enhanced by molecular crowding. The depletion force is likely to play akey role in tuning the physical properties of RNP condensates within the crowded cellular space.