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Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence. The design principles underlying biomolecular phase separation of membrane-less organelles remain poorly understood. Using model homopolymers, Fisher et al. show that the formation kinetics of coexisting liquid phases can be tuned by exploiting differences between arginine and lysine residues.

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA–protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA–protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties. Significance Phase transitions have recently emerged as a key mechanism for intracellular organization. However, the underlying molecular interactions and nature of the resulting condensed phases are poorly understood. Here, we identify a role for LAF-1 in the liquid phase separation of P granules—RNA/protein assemblies implicated in germ-line maintenance. We adapt microrheology techniques to measure precise viscoelastic properties of LAF-1 liquid droplets. Our experiments reveal that electrostatic disordered protein interactions give rise to droplets with tunable material properties. RNA can fluidize protein droplets by decreasing the viscosity and increasing internal molecular dynamics. Our results provide insight into the mechanism by which molecular level interactions can give rise to liquid phase organelles with tunable material properties, potentially underlying biologically adaptable functions.

Liquid—liquid phase separation of tropoelastin has long been considered to be an important early step in the complex process of elastin fiber assembly in the body and has inspired the development of elastin-like peptides with a wide range of industrial and biomedical applications. Despite decades of study, the material state of the condensed liquid phase of elastin and its subsequent maturation remain poorly understood. Here, using a model minielastin that mimics the alternating domain structure of full-length tropoelastin, we examine the elastin liquid phase. We combine differential interference contrast (DIC), fluorescence, and scanning electron microscopy with particle-tracking microrheology to resolve the material transition occurring within elastin liquids over time in the absence of exogenous cross-linking. We find that this transition is accompanied by an intermediate stage marked by the coexistence of insoluble solid and dynamic liquid phases giving rise to significant spatial heterogeneities in material properties. We further demonstrate that varying the length of the terminal hydrophobic domains of minielastins can tune the maturation process. This work not only resolves an important step in the hierarchical assembly process of elastogenesis but further contributes mechanistic insight into the diverse repertoire of protein condensate maturation pathways with emerging importance across biology.

The realization that the cell is abundantly compartmentalized into biomolecular condensates has opened new opportunities for understanding the physics and chemistry underlying many cellular processes 1 , fundamentally changing the study of biology 2 . The term biomolecular condensate refers to non-stoichiometric assemblies that are composed of multiple types of macromolecules in cells, occur through phase transitions, and can be investigated by using concepts from soft matter physics 3 . As such, they are intimately related to aqueous two-phase systems 4 and water-in-water emulsions 5 . Condensates possess tunable emergent properties such as interfaces, interfacial tension, viscoelasticity, network structure, dielectric permittivity, and sometimes interphase pH gradients and electric potentials 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 – 14 . They can form spontaneously in response to specific cellular conditions or to active processes, and cells appear to have mechanisms to control their size and location 15 , 16 – 17 . Importantly, in contrast to membrane-enclosed organelles such as mitochondria or peroxisomes, condensates do not require the presence of a surrounding membrane.

Tau is an intrinsically disordered protein with an important role in maintaining the dynamic instability of neuronal microtubules. Despite intensive study, a detailed understanding of the functional mechanism of tau is lacking. Here, we address this deficiency by using intramolecular single-molecule Förster Resonance Energy Transfer (smFRET) to characterize the conformational ensemble of tau bound to soluble tubulin heterodimers. Tau adopts an open conformation on binding tubulin, in which the long-range contacts between both termini and the microtubule binding region that characterize its compact solution structure are diminished. Moreover, the individual repeats within the microtubule binding region that directly interface with tubulin expand to accommodate tubulin binding, despite a lack of extension in the overall dimensions of this region. These results suggest that the disordered nature of tau provides the significant flexibility required to allow for local changes in conformation while preserving global features. The tubulin-associated conformational ensemble is distinct from its aggregation-prone one, highlighting differences between functional and dysfunctional states of tau. Using constraints derived from our measurements, we construct a model of tubulin-bound tau, which draws attention to the importance of the role of tau’s conformational plasticity in function.

RNA binding proteins (RBPs) are enriched in phase separated biomolecular assemblies across cell types. These RBPs often harbor arginine–glycine rich RGG motifs, which can drive phase separation, and can preferentially interact with RNA G-quadruplex (G4) structures, particularly in the neuron. Increasing evidence underscores the important role that RNA sequence and structure play in contributing to the form and function of protein condensates, however, less is known about the role of G4 RNAs and their interaction with RGG domains specifically. In this study we focused on the model protein, Fragile X mental retardation protein (FMRP), to investigate how G4-containing RNA sequences impact the phase behavior and material properties of condensates. FMRP is implicated in the development of Fragile X Syndrome, and is enriched in neuronal granules where it is thought to aid in mRNA trafficking and translational control. Here, we examined RNA sequences with increasing G content and G4 propensity in complex with the RGG-containing low complexity region (LCR) of FMRP. We found, that while increasing G content triggers aggregation of poly-arginine, all RNA sequences supported phase separation into liquid droplets with FMRP-LCR. Combining microrheology, and fluorescence recovery after photobleaching, we measured a moderate increase in viscosity and decrease in dynamics for increasing G-content, and detected no measurable increase in elasticity as a function of G4 structure. Additionally, we found that while methylation of FMRP decreased RNA binding affinity, this modification did not impact condensate material properties suggesting that RNA sequence/structure can play a greater role than binding affinity in determining the emergent properties of condensates. Together, this work lends much needed insight into the ways in which G-rich RNA sequences tune the assembly, dynamics and material properties of protein/RNA condensates and/or granules.

Tau is a microtubule binding protein that forms pathological aggregates in the brain in Alzheimer’s disease and other tauopathies. Disease etiology is thought to arise from loss of native interactions between tau and microtubules, as well as from gain of toxicity tied to tau aggregation, although neither mechanism is well understood. Here we investigate the link between function and disease using disease-associated and disease-motivated mutants of tau. We find that mutations to highly conserved proline residues in repeats 2 and 3 of the microtubule binding domain have differential effects on tau binding to tubulin and the capacity of tau to enhance tubulin polymerization. Notably, mutations to these residues result in an increased affinity for tubulin dimers while having a negligible effect on binding to stabilized microtubules. We measure conformational changes in tau on binding to tubulin that provide a structural framework for the observed altered affinity and function. Additionally, we find that these mutations do not necessarily enhance aggregation, which could have important implications for tau therapeutic strategies that focus solely on searching for tau aggregation inhibitors. We propose a model that describes tau binding to tubulin dimers and a mechanism by which disease-relevant alterations to tau impact its function. Together, these results draw attention to the interaction between tau and free tubulin as playing an important role in mechanisms of tau pathology.

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R 9 ) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality. Fluorescence-based oligonucleotide probes, also known as molecular beacons (MBs), are popular for detecting nucleic acids with high specificity. Here, the authors demonstrate self-sequestration of MB-based biosensors and target strands within peptide-based coacervates, increasing local concentrations and significantly increasing the sensitivity and kinetics of the DNA biosensors.

Phase separation of biomolecules into distinct material states is expanding our understanding of cellular processes, particularly nuclear chromatin organization linked to gene regulation. Heterochromatin, containing DNA, repressive histone marks, and HP1α protein, functions in gene silencing and telomeric regulation. While HP1α and DNA phase separation contributes to heterochromatin formation, underlying mechanisms remain unclear. Given telomeric RNAs’ proposed role in heterochromatin formation, we investigated interactions between telomeric RNA, DNA, and HP1α. We demonstrate that HP1α phase separates with both Telomeric Repeat containing RNA (TERRA) and DNA. When both nucleic acids are present, HP1α forms multiphase condensates where HP1α-TERRA clusters localize at the HP1α-DNA phase interface. This multiphase architecture results from sequence-specific TERRA-HP1α interactions. TERRA RNA clusters stabilize DNA condensates and modulate their interfacial properties. This work reveals a previously uncharacterized role for TERRA transcripts in regulating HP1α condensates and provides insights into how structured non-coding RNAs influence protein condensate material properties and organization. These findings advance understanding of complex biomolecular interactions governing nuclear compartmentalization through cooperative phase separation mechanisms, highlighting the sophisticated regulatory roles of non-coding RNAs in chromatin organization. HP1α forms multiphase condensates with telomeric RNA and DNA, providing new insights into the role of non-coding RNAs in heterochromatin organization at telomeres.

Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro . However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including ‘high concentration’ binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of ∼ 3–8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function. Ultrafast-scanning fluorescence correlation spectroscopy has now been used to measure the molecular interactions underlying the phase behaviour of disordered proteins. Sequence-encoded conformational fluctuations of these proteins are shown to give rise to phase-separated droplets of surprisingly low concentrations. These results provide insight into how the structural features of the droplets affect the properties of liquid-phase intracellular organelles.