Particle-tracking microrheology probes the rheology of soft materials by accurately tracking an ensemble of embedded colloidal tracer particles. One-particle analysis, which focuses on the trajectory of individual tracers is ideal for homogeneous materials that do not interact with the particles. By contrast, the characterization of heterogeneous, micro-structured materials or those where particles interact directly with the medium requires a two-particle analysis that characterizes correlations between the trajectories of distinct particle pairs. Here, we propose an optical-flow image analysis as an alternative to the tracking-based algorithms to extract one and two-particle microrheology information from video microscopy images acquired using diverse imaging contrast modalities. This technique, termed optical-flow microrheology (OFM), represents a high-throughput, operator-free approach for the characterization of a broad range of soft materials, making microrheology accessible to a wider scientific community.

Membrane-free complex coacervate microdroplets are compelling models for primitive compartmentalisation with the ability to form from biological molecules. However, understanding how molecular interactions can influence physicochemical properties and catalytic activity of membrane-free compartments is still in its infancy. This is important for defining the function of membrane-free compartments during the origin of life as well as in modern biology. Here, we use RNA-peptide coacervate microdroplets prepared with prebiotically relevant amino acids and a minimal hammerhead ribozyme. This is a model system to probe the relationship between coacervate composition, its properties and ribozyme activity. We show that ribozyme catalytic activity is inhibited within the coacervate compared to buffer solution, whilst variations in peptide sequence can modulate rates and yield of the ribozyme within the coacervate droplet by up to 15-fold. The apparent ribozyme rate constant is anti-correlated with its concentration and correlated to its diffusion coefficient within the coacervates. Our results provide a relationship between the physicochemical properties of the coacervate microenvironment and the catalytic activity of the ribozyme where membrane-free compartments could provide a selection pressure to drive molecular evolution on prebiotic earth.

MEX-5 regulates the formation and dissolution of P granules in Caenorhabditis elegans embryos, yet the thermodynamic basis of its activity remains unclear. Here, using a time-resolved in vitro reconstitution system, we show that MEX-5 dissolves preassembled liquid-like PGL-3/RNA condensates by altering RNA availability and shifting the phase boundary. We develop a microfluidic assay to systematically analyze how MEX-5 influences phase separation. By measuring the contribution of PGL-3 to phase separation, we show that MEX-5 reduces the free energy of PGL-3, shifting the equilibrium toward dissolution. Our findings provide a quantitative framework for understanding how RNA-binding proteins modulate condensate stability and demonstrate the power of microfluidics in precisely mapping phase transitions.

Many subcellular compartments are biomolecular condensates made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively because they either require fluorescent labels, which can perturb composition, or can distinguish only one or two components. Here we describe a label-free method based on quantitative phase imaging and analysis of tie-lines and refractive index to measure the composition of reconstituted condensates with multiple components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex ageing dynamics for condensates composed of full-length proteins. We then use analysis of tie-lines and refractive index to simultaneously resolve the concentrations of five macromolecular solutes in multicomponent condensates containing RNA and constructs of multiple RNA-binding proteins. Our measurements reveal an unexpected decoupling of density and composition, highlighting the need to determine molecular stoichiometry in multicomponent condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions 1 . Reciprocally, macromolecules restrict the movement of ‘structured’ water molecules within their hydration layers, reducing the available ‘free’ bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales 2 , 3 ; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

The kinetics of chemical reactions are determined by the law of mass action, which has been successfully applied to homogeneous, dilute mixtures. At nondilute conditions, interactions among the components can give rise to coexisting phases, which can significantly alter the kinetics of chemical reactions. Here, we derive a theory for chemical reactions in coexisting phases at phase equilibrium. We show that phase equilibrium couples the rates of chemical reactions of components with their diffusive exchanges between the phases. Strikingly, the chemical relaxation kinetics can be represented as a flow along the phase equilibrium line in the phase diagram. A key finding of our theory is that differences in reaction rates between coexisting phases stem solely from phase-dependent reaction rate coefficients. Our theory is key to interpreting how concentration levels of reactive components in condensed phases control chemical reaction rates in synthetic and biological systems.

Abstract The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. To test this idea, we focused our study on nuclear condensates reported in cell lines, demonstrated they occur in normal human and malignant breast cancer, and developed assays to test clinically active antineoplastic small molecule drugs relative to these condensates.To study the behavior of drugs within condensates, these were modeled in vitro with purified proteins and visualized by fluorescent confocal microscopy. We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. For each drug, the small molecule partitioned into the same condensate in vitro in which its established target resides in vivo. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration.Alkylating agents are a class of commonly used antineoplastic compounds, of which cisplatin is a prominent example. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Thus, we conclude that cisplatin preferentially modifies transcriptional condensate-associated DNA in cells, and that this causes selective condensate disruption. The mechanisms that produce drug resistance can provide clues to drug activity in the clinical setting. Investigating the behavior of tamoxifen within ER transcriptional condensates demonstrated that it disrupts these condensates in vitro and on oncogenes in cells; hormonal therapy resistant ESR1 mutations render these condensates resistant. MED1 overexpression, a poorly understood mechanism of tamoxifen resistance, increased the size of ER-MED1 condensates, thereby rending tamoxifen more dilute and ineffective when concentrated therein. This suggest that altering the size and nature of transcription condensates in breast cancer can mediate drug resistance in the clinical setting.Our results show that antineoplastic drugs partition selectively into condensates, that this can occur through physicochemical properties independent of their molecular targets, and that resistance to drugs may occur through condensate altering mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment. Citation Format: Isaac Klein, Ann Boija, Lena Afeyan, Susana Wilson Hawken, Mengyang Fan, Alessandra Dall'Agnese, Ozgur Oksuz, Jonathan Henninger, Krishna Shrinivas, Benjamin Sabari, Ido Sagi, Victoria Clark, Jesse Platt, Mrityunjoy Kar, Patrick McCall, Alicia Zamudio, John Mantiega, Eliot Coffey, Charles Li, Nancy Hannett, Yang Guo, Tim-Michael Decker, Tong Lee, Tinghu Zhang, Jing-Ke Weng, Dylan Taatjes, Arup Chakraborty, Phillip Sharp, Young Tae Chang, Anthony Hyman, Nathanael Gray, Richard Young. Partitioning of cancer therapeutics in nuclear condensates [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-10.

ABSTRACT Biomolecular condensates are membrane-less organelles made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which we show can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase microscopy to measure the composition of condensates with an arbitrarily large number of components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. In simplified multi-component protein/RNA condensates, we uncover a regime of constant condensate density and a large range of protein:RNA stoichiometry when varying average composition. The unexpected decoupling of density and composition highlights the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how non-equilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.

Networks of filamentous actin (F-actin) are important for the mechanics of most animal cells. These cytoskeletal networks are highly dynamic, with a variety of actin-associated proteins that control cross-linking, polymerization and force generation in the cytoskeleton. Inspired by recent rheological experiments on reconstituted solutions of dynamic actin filaments, we report a theoretical model that describes stress relaxation behavior of these solutions in the presence of severing proteins. We show that depending on the kinetic rates of assembly, disassembly, and severing, one can observe both length-dependent and length-independent relaxation behavior.

Biomolecules exist and function in cellular microenvironments that control their spatial organization, local concentration, and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate, and control the local physico-chemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to ∼30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.

Computational simulations of polymerizing actin filaments indicate that competition for a limiting pool of building blocks is not sufficient to control their length.