Properties Of Condensate Research Articles

C-terminal frameshift variant of TDP-43 with pronounced aggregation-propensity causes rimmed vacuole myopathy but not ALS/FTD

Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. Using genome-wide linkage analysis and whole exome sequencing in an extended five-generation family with an autosomal dominant rimmed vacuole myopathy, we identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43p.Trp385IlefsTer10) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43Trp385IlefsTer10 does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43p.Trp385IlefsTer10 behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43p.Trp385IlefsTer10 showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43p.Trp385IlefsTer10 is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. Our study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology.

Advances in Nucleotide Repeat Expansion Diseases: Transcription Gets in Phase.

Unstable DNA repeat expansions and insertions have been found to cause more than 50 neurodevelopmental, neurodegenerative, and neuromuscular disorders. One of the main hallmarks of repeat expansion diseases is the formation of abnormal RNA or protein aggregates in the neuronal cells of affected individuals. Recent evidence indicates that alterations of the dynamic or material properties of biomolecular condensates assembled by liquid/liquid phase separation are critical for the formation of these aggregates. This is a thermodynamically-driven and reversible local phenomenon that condenses macromolecules into liquid-like compartments responsible for compartmentalizing molecules required for vital cellular processes. Disease-associated repeat expansions modulate the phase separation properties of RNAs and proteins, interfering with the composition and/or the material properties of biomolecular condensates and resulting in the formation of abnormal aggregates. Since several repeat expansions have arisen in genes encoding crucial players in transcription, this raises the hypothesis that wide gene expression dysregulation is common to multiple repeat expansion diseases. This review will cover the impact of these mutations in the formation of aberrant aggregates and how they modify gene transcription.

Surfactants or scaffolds? RNAs of varying lengths control the thermodynamic stability of condensates differently

Biomolecular condensates, thought to form via liquid-liquid phase separation of intracellular mixtures, are multicomponent systems that can include diverse types of proteins and RNAs. RNA is a critical modulator of RNA–protein condensate stability, as it induces an RNA concentration-dependent reentrant phase transition—increasing stability at low RNA concentrations and decreasing it at high concentrations. Beyond concentration, RNAs inside condensates can be heterogeneous in length, sequence, and structure. Here, we use multiscale simulations to understand how different RNA parameters interact with one another to modulate the properties of RNA–protein condensates. To do so, we perform residue/nucleotide resolution coarse-grained molecular dynamics simulations of multicomponent RNA–protein condensates containing RNAs of different lengths and concentrations, and either FUS or PR25 proteins. Our simulations reveal that RNA length regulates the reentrant phase behavior of RNA–protein condensates: increasing RNA length sensitively rises the maximum value that the critical temperature of the mixture reaches, and the maximum concentration of RNA that the condensate can incorporate before beginning to become unstable. Strikingly, RNAs of different lengths are organized heterogeneously inside condensates, which allows them to enhance condensate stability via two distinct mechanisms: shorter RNA chains accumulate at the condensate’s surface acting as natural biomolecular surfactants, while longer RNA chains concentrate inside the core to saturate their bonds and enhance the density of molecular connections in the condensate. Using a patchy particle model, we additionally demonstrate that the combined impact of RNA length and concentration on condensate properties is dictated by the valency, binding affinity, and polymer length of the various biomolecules involved. Our results postulate that diversity on RNA parameters within condensates allows RNAs to increase condensate stability by fulfilling two different criteria: maximizing enthalpic gain and minimizing interfacial free energy; hence, RNA diversity should be considered when assessing the impact of RNA on biomolecular condensates regulation.

Electron-phonon correlations inducing excitonic excitations in semimetal and semiconducting materials

The influence of phonons on the low-energy excitonic excitations at zero temperature in the extended Falicov-Kimball model has been investigated. In the framework of the unrestricted Hartree-Fock approximation, a set of self-consistent equations for the excitonic condensate order parameter and a lattice distortion is derived when both electron-phonon coupling and electron-hole Coulomb interaction are treated on an equal footing. The low-energy excitation properties of the excitonic condensate are addressed in signatures of the optical conductivity and the dynamical excitonic susceptibility function. The real part of the optical conductivity is evaluated by the Kubo linear response theory and the imaginary part of the dynamical excitonic susceptibility is found by adapting the random phase approximation. In the semimetal state, one always finds a sharp peak in the optical conductivity spectrum indicating the stability of the excitonic condensation in the BCS type if the correlation between electrons and phonons becomes significant. In contrast, the peak is smeared out on the semiconducting side indicating the stability of the BEC-type excitonic condensate. In this semiconducting side, the sharp peak signature appears and the system turns to the BCS-type excitonic condensation state by increasing the electron-phonon correlations. In either the semimetal or the semiconducting normal state, increasing the electron-phonon correlations always reinforces a low-energy sharp peak in the dynamical excitonic susceptibility spectrum, indicating the existence of the tightly bound excitonic excitations before the condensation state. Specifically, on the semiconducting side, the ``halo'' phase with the preformed excitons exiting outside of the BEC-excitonic condensation state has been specified. The halo phase becomes more recognizable by raising the electron-phonon correlations.

Numerical simulation of two-phase evaporation and condensation

The evaporation and condensation mechanism of two-phase flow is studied. The Euler-Euler pattern is used to express the evaporation and condensation properties of two-phase flow. The research shows that the tracking program can accurately track the interface. The thermal resistance on the confluence of the condensation part and insulation part becomes larger. The working fluids have significant influence of two-phase.

Dimensional Reduction for Single-Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67.

Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, methods that provide the resolution needed for complex kinetic studies of multicomponent condensation are desired. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the two-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from three-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a time scale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length, and GC content affect the threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1α and Ki-67, have much lower threshold concentrations for condensation than charge-based condensing agents, with Ki-67 being the most effective, requiring as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1α and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color are used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67-mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions.

Isobaric storage of compressed air: Introduction of a novel concept based on phase change materials and pressure equalizing modules

In industrial compressed air systems (CAS), compressed air receivers have a significant impact on the overall energy efficiency. Isobaric compressed air receivers have the potential to increase the amount of stored air significantly and, hence, the energy efficiency can be improved compared to the usage of conventional isochoric compressed air receivers. In this work, we propose a novel concept of an isobaric compressed air receiver based on pressure equalizing modules (PEQM). These PEQM are filled with a condensable gas – in this work called phase change material (PCMl/g) – and are placed inside the receiver. During the charging and discharging of a compressed air receiver equipped with PEQM, the PCMl/g condenses or evaporates, respectively. Due to the large volume change during the phase change of the PCMl/g air can be stored isobarically. However, a proper selection of suitable PCMl/g is crucial for efficient and safe use. Therefore, we introduce a three-step selection process based on an adapted digital-logic method. In this selection process, qualitative (e.g. flammability) and quantitative (e.g., heat of condensation) properties are considered. As a result, R-1234ze(E) is selected as PCMl/g which owns a vapor pressure of 800 kPa at a temperature of 314.7 K. First experiments with the novel PEQM filled with R-1234ze(E) have been conducted in a lab-scale test plant and the results are shown in this work. The storage capacity of the test plant has been increased by 37.9 %. Additionally, the phase change of the PCMl/g during charging and discharging has been observed. However, the phase change is not isobaric yet. Temperature changes during the phase change inside the PEQM hinder the isobaric storing of the working gas. Therefore, proper heat management inside the PEQM is necessary, for example, by using a second phase change material for heat storage.

Phase separation of Hippo signalling complexes

The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs‐Expanded (Crb‐Ex) and/or Merlin‐Kibra (Mer‐Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6‐hexanediol treatment. Overexpressing Ex or Kib induces formation of micron‐scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low‐complexity domains and purified Hpo‐Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated “signalosomes” induced by clustering of upstream pathway components.

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1–3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6–8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Programmable synthetic biomolecular condensates for cellular control.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications.

Effects of alpha-synuclein and synaptic vesicles on the material properties of synapsin condensates.

Biomolecular condensates can recruit and segregate client molecules and are involved in various physiological processes. The material properties of a biomolecular condensate, as a mesoscale manifestation of intermolecular interactions, are crucial to its biological and pathological functions. Despite emerging reports about the changes in material properties of condensates in disease-related proteins, quantitative studies of the material properties of condensates are still rare. Here, we apply a calibration-free micropipette aspiration (MPA) assay to quantify the surface tension and viscosity of synapsin condensates. Furthermore, we investigated the effect of alpha-synuclein and synaptic vesicles (SVs) on the material properties of synapsin condensate. The results suggest that the recruitment of alpha-synuclein will increase the viscosity of synapsin condensate, and indicate the potential effects of SVs in mediating the material properties of synapsin condensates. Synapsin condensates are known to be essential for the formation of SV clusters. Therefore, our findings will help understand the regulation of SV dynamics during neurotransmission.

A new heat transfer model based on non-equilibrium film theory for annular flow condensation of binary zeotropic mixtures

In recent years, zeotropic mixtures have been widely used as substitutes for pure fluids. Accordingly, their condensation properties have attracted increasing attention. However, it is a challenge to accurately describe the heat transfer performance of zeotropic mixtures in condensation, which is limited by the estimation of mixture effects, especially the mass transfer resistance therein. Focusing on this issue, the mass transfer due to the axial and radial concentration gradients in the vapor phase was considered, and a new heat transfer model for the annular flow of binary zeotropic mixtures in horizontal tubes was developed based on non-equilibrium film theory. In this model, the boiling point difference was introduced to approximate the effect of concentration gradients by combining with the vapor-liquid phase equilibrium properties of binary zeotropic mixtures. Secondly, equivalent specific heat capacities were defined for an analogy with single-phase heat transfer, which further simplifies the form of the model. The simplified model was evaluated with a consolidated database of 11 mixtures from 13 sources. The results show that the predictions of the model are in good agreement with the experimental results, with a mean absolute relative deviation (MARD) of 15.88 and 91.14% of points falling in the ±30% deviation band. Predictions were also performed for excluded data from other sources and satisfactory results were obtained with a MARD of 13.47%. Moreover, the mechanism of mixture effects is elaborated. It indicates that besides the commonly mentioned heat transfer degradation, there is also heat transfer enhancement for binary zeotropic mixtures condensation due to the opposite effects of the axial and radial concentration gradients.

Non-canonical interactions tune condensation and kinetically arrested material states of short RNA homopolymers.

Phase separation related processes have been frequently linked to biological compartmentalization and organization, and associated with cellular responses to stress, development, and disease. As a result, understanding biomolecular condensates and the parameters that modulate material and chemical properties has been an area of interest. Although much initial work focused on the role of proteins in phase separation, increasingly RNA-RNA interactions are being recognized for their potential to tune the condensate properties, influencing material properties, morphology, and dynamic organization. While many of these studies have focused on Watson-Crick base pairing as the primary networking interaction, previous work has also suggested a potential for non-canonical RNA-RNA interactions in long homopolymers to promote phase separation and modulate overall material properties. Here, we examine the phase separation of short RNA homopolymers in the presence of divalent cations and disordered peptides. Our studies reveal that particular short homopolymers form internally kinetically arrested droplets with limited monomer mixing despite continued fusion. We also probe the combined cooperative effects of various ions in modulating phase behavior and investigate the potential for homopolymer tracts in slightly more complex sequences to tune the material and phase separation propensities of different oligomers. Collectively, our results suggest a potential for tracts predominantly interacting through non-canonical interactions to tune the phase behavior and mesoscale properties of simplified condensates, as well as offer insight into the fundamental biophysics of RNA-based condensate systems and short homopolymer tracts in more complex biological systems.

Molecular features underlying phase separation of protein-nucleic acid condensates revealed by a new coarse-grained DNA model.

Recent advances in residue-level coarse-grained (CG) computational models for proteins have enabled molecular-level insights into the properties of protein condensates. However, most of the existing CG nucleic acid models include anisotropic potentials between bases that are computationally demanding and are not fully compatible with the existing CG protein models. Here, we present a new CG DNA model that utilizes isotropic potentials between bases and can be used for investigating protein-DNA complexes and nucleosome arrays, thereby enabling a mechanistic understanding of how molecular information is propagated up at the genome level in a computationally tractable manner. To demonstrate the suitability of our model to facilitate large-scale simulations with a molecular resolution, we simulate nucleosomes (mono, di, etc.) to generate equilibrium conformational ensembles. The simulation data provide essential insights into the role of chromatin in the liquid-liquid phase separation of HP1α protein. We find an extensive network of interactions between HP1α, DNA, and flexible histone tails in the condensates formed by these different biomolecules. The findings of this work illuminate the complex molecular framework that contributes to heterochromatin regulation and function.

A stickers-and-spacers framework explains the viscoelastic and aging properties of biomolecular condensates.

Prion-like low complexity domains (PLCDs) in RNA-binding proteins drive phase separation and contribute to stress granule assembly. Time-dependent changes, also known as aging, of biomolecular condensates, are implicated as a mechanism underlying the pathogenesis of neurodegenerative diseases. The sequence grammar encoding the driving forces for phase separation has come into focus through the development and application of a stickers-and-spacers model that classifies stickers as the segments of the polymers that form physical crosslinks with one another and spacers as the segments connecting the stickers and influencing the solubility of PLCDs. While condensates are often described as liquids, growing evidence suggests that they have viscoelastic properties, consistent with a network fluid structure deriving from the stickers-and-spacers architecture of PLCDs. Here we focus on determining how sequence features encode viscoelasticity and the time-dependent changes of condensates. We systematically titrated the interaction strength of the aromatic stickers in the PLCD of hnRNPA1. We used passive microrheology with optical tweezers to determine the sticker-dependent effects and find that viscosity, and elasticity scale with the inter-sticker interaction strength. We also find that increasing the sticker strength in the condensates lengthens the timescales where their rheological behavior is dominated by an elastic response. In aging condensates, an increasing fraction of molecules show sub-diffusive behavior, even in the absence of fibrillization. Increasing the effective solvation volume of spacers accelerates aging, while increasing sticker strength delays it. Our results reveal for the first time the sequence grammar underlying material properties and aging of PLCD condensates and provide new insights into the mechanisms of condensate aging.

RNA structure controls composition and network dynamics of condensates.

Biomolecular condensates can form by phase separation and likely serve myriad functions in cells. Many studies have characterized protein condensates, and sometimes these include model RNAs that are often nonspecific and/or homopolymeric, unlike native RNAs found in biomolecular condensates. How complex RNA sequences and structures control condensation with proteins remains poorly understood. We studied the influence of mRNA structure in phase separation using the Whi3 protein and a native mRNA binding partner, CLN3. We first created an evolutionary algorithm that designs shuffled CLN3 RNA sequences which maximize or minimize predicted free energies of folding while preserving mass, known Whi3 protein binding sites, encoded amino acid sequence, and nucleotide composition. RNAs with minimized free energies of folding contain many stable duplexes, while RNAs with maximized energies have unstable structures and long single-stranded regions. Using mass photometry, we show that RNAs with stable structures are mostly monomeric, while RNAs with unstable structures can multimerize. When mixed with protein, these differential RNA-RNA interactions affect dense phase compositions. Specifically, unstable RNAs form condensates with high RNA and low protein concentrations, while the converse is true for RNAs with stable structures. Using microbead rheology, we observe that unstable RNAs form condensates with long viscoelastic relaxation times that increase with condensate age, indicating elastic properties at many timescales. By contrast, stably-structured RNAs form less elastic condensates with shorter relaxation times that increase to a lesser degree with time. We hypothesize that such aging is controlled by the exchange of intra- for inter-molecular base pairing amongst RNAs whose folding free energies determine the timescale of these exchanges. Thus, RNA structure can have multi-scale consequences for biomolecular condensates, with impacts on condensate composition and material properties. This work underscores how specific RNA sequences can encode physical properties of condensates.

Revealing nanoscale structure and interfaces of polymer and protein condensates via cryo-electron microscopy.

Liquid-liquid phase separation of macromolecules is a ubiquitous process studied across biological and material sciences. In one instance, phase separation of proteins can be driven by low complexity intrinsically disordered regions that permit multivalent interactions between polypeptides. In synthetic systems, a complexation of oppositely charged polyelectrolytes or the immiscibility of two blocks can drive phase separation of polyelectrolytes and block copolymers respectively. The result of these processes are condensates that display liquid-like characteristics. The nature of these condensates has mainly been investigated through methods based on optical microscopy, such as assessing condensate morphology, fusion dynamics, and diffusion of molecules. Importantly, current methods used to study condensates cannot probe the nanoscale structure of the material. Here, we developed a method that applies cryo-electron microscopy (EM) to investigate the internal structure of condensates in vitro at the nanoscale. By bypassing the drying of material for conventional EM, we preserve the internal organization of molecules. We tested a range of material preparation protocols, including varying the temperature, concentration, and buffer conditions of the solution, along with vitrification procedures. We are able to produce both micron-sized spherical droplets and thin films of condensed material that are amenable to cryo-EM imaging. We applied this method to study condensates composed of intrinsically disordered proteins, polyelectrolytes and block copolymers. Our results reveal the internal organization of the different materials at the nanoscale. Furthermore, we relate the material properties of condensates composed of intrinsically disordered proteins to their nanoscale structure. We anticipate the use of cryo-EM beyond single particle analysis to understand phase separation of intrinsically disordered proteins and other macromolecules.

Single-chain and condensed-state behavior of hnRNPA1 from molecular simulations.

Intrinsically disordered proteins (IDPs) are essential components for the formation of membraneless organelles, which play key functional and regulatory roles within biological systems. These complex assemblies form and dissolve spontaneously over time via liquid-liquid phase separation of IDPs. Mutations in their amino acid sequence can alter their phase behavior, which has been linked to the emergence of severe diseases. We study the conformation and phase behavior of a low-complexity domain of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), using coarse-grained implicit solvent molecular dynamics simulations. We systematically analyze how these properties are affected by the number of aromatic residues within the examined sequences. We find a significant compaction of the chains and an increase in the critical temperature with increasing number of aromatic residues. The local persistence length is determined in single-chain simulations, revealing strong sequence-specific variations along the chain contour. Comparing single-chain and condensed state simulations, we find much more collapsed polymer conformations in the dilute systems, even at temperatures near the estimated theta temperature of the solution. These observations strongly support the hypothesis that aromatic residues play a dominant role for condensation, which is further corroborated by a detailed analysis of the intermolecular contacts, and conversely that important properties of condensates are captured in coarse-grained simulations. Interestingly, we observe density inhomogeneities within the condensates near criticality, which are driven by electrostatic interactions. Finally, we find that the relatively small fraction of hydrophobic residues in the IDPs results in interfacial tensions which are significantly lower compared to typical combinations of immiscible simple liquids.

Mutual regulation between membrane rafts and protein condensates in T cell activation.

T cells are central effectors of adaptive immunity, mediating critical defenses against infections and cancer. T cell activation must be tightly regulated to integrate and transduce various signals detected at the plasma membrane and elicit proper immune responses. Despite decades of research, the regulatory mechanisms of normal and aberrant T cell activation are not fully understood. Recently, we reported that biomolecular condensates form at the T cell immunological synapse via liquid-liquid phase separation driven by multivalent macromolecular interactions. Specifically, the transmembrane protein Linker for Activation of T cells (LAT) is phosphorylated upon antigenic stimulation to multivalently interact with proteins called Grb2 and Sos1, forming condensates with fluid properties in vitro and in living cells. Notably, LAT is recruited to the ordered, cholesterol-rich membrane domains known as lipid rafts, and such recruitment has been implicated in T cell activation. Thus, we hypothesized that LAT may mediate interplay between protein and membrane lipid phase separation in T-cell signaling. Here, we report physical and functional coupling between membrane rafts and protein condensates via LAT using both in vitro reconstituted membrane systems and live cells. To study how LAT condensates and membrane rafts cooperatively regulate T-cell signaling, we perturbed membrane raft domains and observed a decrease in condensate density and a dampening of downstream signaling. Moreover, using fluorescent probes that are sensitive to local membrane lipid packing in living cells, we quantitatively measure changes in membrane biophysical properties during condensate assembly and see an increase in lipid packing in the local membrane environment surrounding LAT condensates. By evaluating effects of lipid remodeling on LAT condensate properties, and effect of condensate formation on membrane properties, we provide evidence that membrane rafts and LAT condensates mutually regulate T cell activation to tune immune signaling.

Tuning Material States and Functionalities of G-Quadruplex-Modulated RNA-Peptide Condensates

RNA encodes sequence- and structure-dependent interactions to modulate the assembly and properties of biomolecular condensates. RNA G-quadruplexes (rG4s) formed by guanine-rich sequences can trigger the formation of liquid- or solid-like condensates that are involved in many aberrant phase transitions. However, exactly how rG4 motifs modulate different phase transitions and impart distinct material properties to condensates is unclear. Here, using RNA oligonucleotides and cationic peptides as model systems, we show that RNA-peptide condensates exhibit tunability in material properties over a wide spectrum via interactions arising from rG4 folding/unfolding kinetics. rG4-containing oligonucleotides formed strong pairwise attraction with peptides and tended to form solid-like condensates, while their less-structured non-G4 mutants formed liquid-like droplets. We find that the coupling between rG4 dissociation and RNA-peptide complex coacervation triggers solid-to-liquid transition of condensates prior to the complete unfolding of rG4s. This coupling points to a mechanism that material states of rG4-modulated condensates can be finely tuned from solid-like to liquid-like by the addition of less-structured RNA oligonucleotides, which have weak but dominant binding with peptides. We further show that the tunable material states of condensates can enhance RNA aptamer compartmentalization and RNA cleavage reactions. Our results suggest that condensates with complex properties can emerge from subtle changes in RNA oligonucleotides, contributing ways to treat dysfunctional condensates in diseases and insights into prebiotic compartmentalization.