Biology subjects

Dar, F.

Publications and source records attributed to Dar, F..

8 recordsLinked to original sources

bioRxiv2023

Biomolecular condensates form spatially inhomogeneous network fluids

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

biophysics↗

bioRxiv2023

Quantitative description of the phase separation behavior of the multivalent SLP65-CIN85 complex

Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are downstream B cell receptor (BCR)-signaling effectors, required for a proper immune response. Both proteins phase separate together with vesicles to form pre-signaling clusters. Within this tripartite system, six PRMs of SLP65 interact promiscuously with three SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modelling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions) we reproduced the observed phase separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow to model the phase separation behavior of the SLP65/CIN85 system faithfully.

biophysics↗

bioRxiv2023

Crowder titrations enable the quantification of driving forces for macromolecular phase separation

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. Additionally, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation. SIGNIFICANCEPhase separation has emerged as a process of significant relevance to sorting macromolecules into distinct compartments, thereby enabling spatial and temporal control over cellular matter. Considerable effort is being invested into uncovering the driving forces that enable the separation of macromolecular solutions into coexisting phases. At its heart, this process is governed by the balance of macromolecule-solvent, inter-macromolecule, and solvent-solvent interactions. We show that the driving forces for phase separation, including the coefficients that measure interaction strengths between macromolecules, can be extracted by titrating the concentrations of crowders that enable macromolecules to phase separate at lower concentrations. Our work paves the way to leverage specific categories of measurements for quantitative characterizations of driving forces for phase separation.

biophysics↗

bioRxiv2023

Dynamical control enables the formation of demixed biomolecular condensates

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

biophysics↗

bioRxiv2022

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation, especially at low endogenous concentrations found in cells, are thought to follow the tenets of classical nucleation theory describing a sharp transition between a dense phase and a dilute phase characterized by dispersed monomers. Here, we used in vitro biophysical studies to study subsaturated solutions of phase separating RNA binding proteins with intrinsically disordered prion like domains (PLDs) and RNA binding domains (RBDs). Surprisingly, we find that subsaturated solutions are characterized by heterogeneous distributions of clusters comprising tens to hundreds of molecules. These clusters also include low abundance mesoscale species that are several hundreds of nanometers in diameter. Our results show that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. Interestingly, however, cluster formation and phase separation can be decoupled from one another using solutes that impact the solubilities of phase separating proteins. They can also be decoupled by specific types of mutations. Overall, our findings implicate the presence of distinct, sequence-specific energy scales that contribute to the overall phase behaviors of RNA binding proteins. We discuss our findings in the context of theories of associative polymers. Significance StatementMembraneless biomolecular condensates are molecular communities with distinct compositional preferences and functions. Considerable attention has focused on phase separation as the process that gives rise to condensates. Here, we show that subsaturated solutions of RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions. The formation of clusters in subsaturated solutions and condensates in supersaturated solution are coupled through sequence-specific interactions. Given the low endogenous concentrations of phase separating proteins, our findings suggest that clusters in subsaturated conditions might be of functional relevance in cells.

biophysics↗

bioRxiv2022

Structure-function relationships in mitochondrial transcriptional condensates

Phase separation organizes many membraneless structures in cells. The functional consequences of concentrating cellular machinery into biomolecular condensates, however, are largely unclear. One fundamental cellular function that has been linked to condensate formation is transcription. Here, we have reconstituted mitochondrial transcription in condensates from purified components. We find that the core components of the mttranscriptional machinery form multi-phasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than equivalent reactions in bulk solution. These condensate-mediated decreases in transcriptional rates are associated with the formation of dynamically arrested vesicular structures that are driven by the production and accumulation of RNA during transcription. Using coarse-grained, equilibrium simulations, we show that the generation of RNA alters the phase behavior and the organization of transcriptional components within condensates and that the in vitro mtcondensates are non-equilibrium structures. Together, our in vitro and in silico approaches shed light on how proteins and (ribo)nucleic acids biophysically self-assemble within mitochondria in vivo. Our results highlight the complex morphologies of transcribing, multicomponent condensates and they illustrate the interdependent structure-function relationships in condensates. Significance StatementMitochondria condense their genome into transcriptionally active mt-nucleoids. These structures fit the definition of biomolecular condensates that form via macromolecular phase separation. We take advantage of the ability to reconstitute mitochondrial transcriptional condensates in vitro from minimal components. We find that the production and accumulation of RNA alters the phase behavior of transcriptional condensates. The altered phase behavior is linked to the formation of arrested, non-equilibrium vesicular structures. Similar changes to phase behavior of proteins and (ribo)nucleic acids can be recapitulated in live mitochondria through knockdown of mt-nucleoid core components. Computer simulations help identify biophysical mechanisms that are needed to maintain the steady-state structures of transcriptional condensates.

biophysics↗

bioRxiv2020

Ligand Effects on Phase Separation of Multivalent Macromolecules

Biomolecular condensates enable spatial and temporal control over cellular processes by concentrating biomolecules into non-stoichiometric assemblies. Many condensates form via reversible phase transitions of condensate-specific multivalent macromolecules known as scaffolds. Phase transitions of scaffolds can be regulated by changing the concentrations of ligands, which are defined as non-scaffold molecules that bind to specific sites on scaffolds. Here, we use theory and computation to uncover rules that underlie ligand-mediated control over scaffold phase behavior. We use the stickers-and-spacers model wherein reversible non-covalent crosslinks among stickers drive phase transitions of scaffolds, and spacers modulate the driving forces for phase transitions. We find that the modulatory effects of ligands are governed by: the valence of ligands; whether they bind directly to stickers versus spacers; and the relative affinities of ligand-scaffold versus scaffold-scaffold interactions. In general, all ligands have a diluting effect on the concentration of scaffolds within condensates. Whereas monovalent ligands destabilize condensates, multivalent ligands can stabilize condensates by binding directly to spacers or destabilize condensates by binding directly to stickers. Bipartite ligands that bind to stickers and spacers can alter the structural organization of scaffold molecules within condensates even when they have a null effect on condensate stability. Our work highlights the importance of measuring dilute phase concentrations of scaffolds as a function of ligand concentration in cells. This can reveal whether ligands modulate scaffold phase behavior by enabling or suppressing phase separation at endogeneous levels thereby regulating the formation and dissolution of condensates in vivo. SignificancePhase transitions of multivalent macromolecules known as scaffolds help drive the formation of functional biomolecular condensates in cells. The formation and dissolution of condensates is tightly regulated, as aberrant phase behavior is associated with disease. Here, we show that distinct types of ligands can exert control over the formation and dissolution of condensates by binding to distinct sites on scaffold molecules. We further show that the extent and direction of regulation can be inferred through direct measurements of how ligands impact scaffold phase boundaries. Our findings have broad implications for understanding and modeling ligand-mediated regulation of condensates in cells, and for designing novel molecules that exert regulatory control over condensates.

biophysics↗

bioRxiv2019

LASSI: A lattice model for simulating phase transitions of multivalent proteins

Biomolecular condensates form via phase transitions that combine phase separation or demixing and networking of key protein and RNA molecules. Proteins that drive condensate formation are either linear or branched multivalent proteins where multivalence refers to the presence of multiple protein-protein or protein-nucleic acid interaction domains or motifs within a protein. Recent work has shown that multivalent protein drivers of phase transitions are in fact biological instantiations of associative polymers. Such systems can be characterized by stickers-and-spacers architectures where stickers contribute to system-specific spatial hierarchies of directional interactions and spacers control the concentration-dependent inhomogeneities in densities of stickers around one another. The collective effects of interactions among stickers and spacers lead to the emergence of dense droplet phases wherein the stickers form percolated networks of polymers. To enable the calculation of system-specific phase diagrams of multivalent proteins, we have developed LASSI (LAttice simulations of Sticker and Spacer Interactions), which is an efficient open source computational engine for lattice-based polymer simulations built on the stickers and spacers framework. In LASSI, a specific multivalent protein architecture is mapped into a set of beads on the 3-dimensional lattice space with proper coarse-graining, and specific sticker-sticker interactions are modeled as pairwise anisotropic interactions. For efficient and broad search of the conformational ensemble, LASSI uses Monte Carlo methods, and we optimized the move set so that LASSI can handle both dilute and dense systems. Also, we developed quantitative measures to extract phase boundaries from LASSI simulations, using known and hidden collective parameters. We demonstrate the application of LASSI to two known archetypes of linear and branched multivalent proteins. The simulations recapitulate observations from experiments and importantly, they generate novel quantitative insights that augment what can be gleaned from experiments alone. We conclude with a discussion of the advantages of lattice-based approaches such as LASSI and highlight the types of systems across which this engine can be deployed, either to make predictions or to enable the design of novel condensates.\n\nAuthor SummarySpatial and temporal organization of molecular matter is a defining hallmark of cellular ultrastructure and recent attention has focused intensely on organization afforded by membraneless organelles, which are referred to as biomolecular condensates. These condensates form via phase transitions that combine phase separation and networking of condensate-specific protein and nucleic acid molecules. Several questions remain unanswered regarding the driving forces for condensate formation encoded in the architectures of multivalent proteins, the molecular determinants of material properties of condensates, and the determinants of compositional specificity of condensates. Building on recently recognized analogies between associative polymers and multivalent proteins, we have developed and deployed LASSI, an open source computational engine that enables the calculation of system-specific phase diagrams for multivalent proteins. LASSI relies on a priori identification of stickers and spacers within a multivalent protein and mapping the stickers onto a 3-dimensional lattice. A Monte Carlo engine that incorporates a suite of novel and established move sets enables simulations that track density inhomogeneities and changes to the extent of networking among stickers as a function of protein concentration and interaction strengths. Calculation of distribution functions and other nonconserved order parameters allow us to compute full phase diagrams for multivalent proteins modeled using a stickers-and-spacers representation on simple cubic lattices. These predictions are shown to be system-specific and allow us to rationalize experimental observations while also enabling the design of systems with bespoke phase behavior. LASSI can be deployed to study the phase behavior of multicomponent systems, which allows us to make direct contact with cellular biomolecular condensates that are in fact multicomponent systems.

biophysics↗