Biology subjects

Style, R. W.

Publications and source records attributed to Style, R. W..

10 recordsLinked to original sources

bioRxiv2026

Partition Coefficients Reveal Changes in Properties of Low-Contrast Biomolecular Condensates

Biomolecular condensates are domains within cells with distinct compositions, held together by intermolecular cohesion. They are implicated in a variety of cellular processes, and in vitro studies have revealed the molecular driving forces that underly their condensation. However, in vitro condensates do not capture essential features of cellular condensates. In particular, enrichment of proteins, quantified by partition coefficients, is often exaggerated in these simplified systems. We show that the addition of free amino acids and other small molecules to model condensates can bring their partition coefficients within physiological range. In this limit, where there is low biochemical contrast between condensates and their surroundings, we observe striking changes to condensate behavior. Such low-contrast condensates exhibit large fluctuations in shape and composition and show enhanced sensitivity to changes in their environment. These behaviors reflect dramatic shifts to their material properties, including interfacial tension, rheology, and chemical susceptibilities. We note remarkable similarities in these effects across seemingly unrelated two-phase fluid systems. To explain these trends, we reformulate classic models of critical phenomena in terms of partition coefficients. This framework simplifies application of theory to experiments with near-critical fluids and suggests new experimental approaches for assessing condensate physiology in live cells.

biophysics↗

bioRxiv2026

Metabolites Shift Equilibria of Biomolecular Condensates

Biomolecular condensates compartmentalize biochemistry in living cells. While in vitro models of condensates involve only a few components, the cytoplasm is a complex mixture with thousands of components, including many small molecules. While many macromolecular drivers of phase separation have been revealed, the contributions from small molecules have received little attention. To quantify the impact of solutes on biomolecular condensates, we introduce susceptibility, a dimensionless descriptor of condensate response to solute perturbations. We measured how three model condensates, assembled by distinct cohesive mechanisms, respond to diverse solutes including amino acids and nucleotides. Generically, solutes shift condensate phase equilibria, with susceptibilities spanning over four orders of magnitude. These values reflect underlying molecular interactions, consistent with theoretical descriptions including Flory-Huggins and polyphasic linkages. As one example of the predictive power of susceptibility, we exploit enzymatic activity to induce condensation and modulate material properties. Our work establishes susceptibility as an indicator of the sensitivity of biomolecular condensates to solutes, with implications for cell physiology and therapeutic design. SIGNIFICANCE STATEMENTCells compartmentalize biochemistry using biomolecular condensates formed through phase separation. Although cells contain thousands of small molecules, little is known about their influence on condensation. In such complex mixtures, mapping full phase diagrams is infeasible. Alternatively, we introduce susceptibility to characterize system response around a working composition. Using three distinct model condensates and over a dozen solutes, we observe susceptibilities varying over four orders of magnitude. We provide a general thermodynamic framework that clarifies the driving forces behind these responses, rationalizing their magnitude and their dependence on location in the phase diagram. Our work provides a framework for understanding and harnessing solutes to regulate biomolecular condensation, with implications for cell physiology and therapeutic design.

biophysics↗

bioRxiv2025

Dynamic Shape Modulation of Deflated and Adhered Lipid Vesicles

Lipid membrane-bounded organelles often possess intricate morphologies with spatially varying curvatures and large membrane surface areas relative to internal volume (small reduced volumes). These features are thought to be essential for protein sorting and vesicle trafficking, but challenging to reproduce in vitro. Here, we show that weakly adhered giant unilamellar vesicles (GUVs) can be osmotically deflated to reduced volumes as low as 0.1, similar to what is found in flattened, disc-shaped organelles such as Golgi cisternae and ER sheets. Using shape analysis with the Canham-Helfrich model, we determine mechanical parameters including adhesion strength, membrane tension, and pressure of individual vesicles. We find that the rate of shape flattening during deflation is governed by a normalized adhesion strength that combines vesicle size, adhesion energy, and bending rigidity. For highly flattened disc-like vesicles, we identify a geometric relationship that allows the adhesion strength to be estimated solely from the vesicles aspect ratio, size, and bending rigidity. These results provide a quantitative experimental platform for bottom-up studies of membrane shaping mechanisms and shape-dependent phenomena, such as curvature-mediated protein sorting. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/650387v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@f23d45org.highwire.dtl.DTLVardef@194a99borg.highwire.dtl.DTLVardef@10b1ca5org.highwire.dtl.DTLVardef@85bd2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Ice and air: Visualisation of freeze-thaw embolism and freezing spread in young L. tulipifera leaves

Spring freezing is an unforgiving stress for young leaves, often leading to death, with consequences for tree productivity and survival. While both the plant water transport system and living tissues are vulnerable to freezing, we do not know whether damage to one or both of these systems causes death in leaves exposed to freezing. Whole saplings of Liriodendron tulipifera were exposed to freezing and thawing trajectories designed to mimic natural spring freezes. We monitored the formation of freeze-thaw xylem embolism and damage to photosynthetic tissues to reveal a predictable progression of ice formation across the leaf surface that is strongly influenced by leaf vein architecture, notably the presence or absence of bundle sheath extensions. Our data also show that freeze-thaw embolism occurs only in the largest vein orders where mean vessel diameter exceeds 30{micro}m. With evidence of both freeze-thaw embolism and damage to photosynthetic tissue, we conclude that this dual-mode lethality may be common among other wide-vesseled angiosperm-leaves, potentially playing a role in limiting geographic distributions, and show that bundle sheath extensions may stall or even prevent freezing spread. HighlightBoth ice and air lead likely lead to death in young L.tulipfera leaves exposed to freezing, with the spread of both governed by physical characteristics of these leaves.

physiology↗

bioRxiv2024

Tunable bicontinuous macroporous cell culture scaffolds via kinetically controlled phase separation

Three-dimensional (3D) scaffolds enable biological investigations with a more natural cell conformation. However, the porosity of synthetic hydrogels is often limited to the nanometer scale, which confines the movement of 3D encapsulated cells and restricts dynamic cell processes. Precise control of hydrogel porosity across length scales remains a challenge and the development of porous materials that allow cell infiltration, spreading, and migration in a manner more similar to natural ECM environments is desirable. Here, we present a straightforward and reliable method for generating kinetically-controlled macroporous systems using liquid-liquid phase separation between poly(ethylene glycol) (PEG) and dextran. Photopolymerization-induced phase separation resulted in macroporous hydrogels with tunable pore size. Varying light intensity and hydrogel composition controlled polymerization kinetics, time to percolation, and complete gelation, which defined the average pore diameter (O = 1- 300 m) and final gel stiffness of the formed hydrogels. Critically, for biological applications, macroporous hydrogels were prepared from aqueous polymer solutions at physiological pH and temperature using visible light, allowing for direct cell encapsulation. We encapsulated human dermal fibroblasts in a range of macroporous gels with different pore sizes. Porosity improved cell spreading with respect to bulk gels and allowed migration in the porous systems.

bioengineering↗

bioRxiv2023

Phase-Separated Droplets Swim to Their Dissolution

Biological macromolecules can condense into liquid domains. In cells, these condensates form membraneless organelles that can organize chemical reactions1,2. However, little is known about the physical consequences of chemical activity in and around condensates. Working with model bovine serum albumin (BSA) condensates3, we show that droplets swim along chemical gradients. Active BSA droplets loaded with urease swim toward each other. Passive BSA droplets show diverse responses to externally applied gradients of the enzymes substrate and products. In all these cases, droplets swim toward solvent conditions that favor their dissolution. We call this behavior dialytaxis, and expect it to be generic, as conditions which favor dissolution typically reduce interfacial tension, whose gradients are well-known to drive droplet motion4,5. These results suggest alternative physical mechanisms for active transport in living cells, and may enable the design of fluid micro-robots.

biophysics↗

bioRxiv2022

Keratin filament mechanics and energy dissipation are determined by metal-like plasticity

Cell mechanics is determined by an intracellular biopolymer network, including intermediate filaments that are expressed in a cell-type specific manner. A prominent pair of intermediate filaments are keratin and vimentin as the epithelial-to-mesenchymal transition is associated with a switch from keratin to vimentin. The transition coincides with a change in cellular mechanics, and thus dynamic properties of the cells. This observation raises the question of how the mechanical properties already differ on the single filament level. Here we use optical tweezers and a computational model to compare the stretching and dissipation behavior of the two filament types. We find that keratin and vimentin filaments behave in opposite ways: keratin filaments elongate, but retain their stiffness, whereas vimentin filaments soften, but retain their length. This finding is explained by fundamentally different ways to dissipate energy: viscous subunits sliding within keratin filaments and non-equilibrium helix unfolding in vimentin filaments.

biophysics↗

bioRxiv2022

A Phase-separating Thiol-ene Photoresin for Volumetric Bioprinting of Macroporous Hydrogels

Macroporous scaffolds facilitate solute transport and cell-cell communication, but materials allowing for in situ pore formation and 3D printing in aqueous solutions are scarce. Here, we introduce an efficient thiol-ene photoclick resin for light-assisted fabrication of cell-compatible macroporous hydrogels via photopolymerization-induced phase separation (PIPS). This resin consists of norbornene-functionalized polyvinyl alcohol, di-thiol crosslinker and dextran sulfate, which can rapidly form a hydrogel with interconnected pores by PIPS. The pore size is tunable in the range of 2-40 m as a function of light intensity, polymer composition and molecular charge. Unlike conventional methods to porous materials, PIPS uniquely allows in situ pore formation in the presence of living cells, thereby enabling 3D cell culture and bioprinting applications. We demonstrate fast 3D photoencapsulation of living cells, enhanced cell spreading in macroporous hydrogels, and tomographic volumetric bioprinting of cm-scale hydrogel constructs with hierarchical pores within 20 seconds. Collectively, this resin is cell-compatible, low-cost, easy-to-make and highly efficient for PIPS, offering promises for fast photofabrication of living tissues with complex porous structures.

bioengineering↗

bioRxiv2021

Non-specific adhesive forces between filaments and membraneless organelles

Membraneless organelles are liquid-like domains that form inside living cells by phase-separation. While standard physical models of their formation assume their surroundings to be a simple liquid, the cytoplasm is an active viscoelastic environment. To investigate potential coupling of phase separation with the cytoskeleton, we quantify structural correlations of stress granules and microtubules in a human-derived epithelial cell line. We find that microtubule networks are significantly perturbed in the vicinity of stress granules, and that large stress granules conform to the local pore-structure of the microtubule network. When microtubules are depolymerized by nocodazole, tubulin enrichment is localized near the surface of stress granules. We interpret these data using a thermodynamic model of partitioning of particles to the surface and bulk of droplets. This analysis shows that proteins generically have a non-specific affinity for droplet interfaces, which becomes most apparent when they weakly partition to the bulk of droplets and have a large molecular weight. In this framework, our data is consistent with a weak ([lsim] kbT) affinity of tubulin sub-units for stress granule interfaces. As microtubules polymerize their affinity for interfaces increases, providing sufficient adhesion to deform droplets and/or the network. We validate this basic physical phenomena in vitro through the interaction of a simple protein-RNA condensate with tubulin and microtubules.

biophysics↗

bioRxiv2021

Sustained Enzymatic Activity and Flow in Crowded Protein Droplets

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

biophysics↗