Biology subjects

Weber, C. A.

Publications and source records attributed to Weber, C. A..

16 recordsLinked to original sources

bioRxiv2026

Theory for Biomolecular Catalysis in Phase-Separated Systems

Enzymatic reactions in biomolecular condensates are often assumed to be regulated through local enrichment of reactants. However, condensates also reshape molecular transport and reaction kinetics, making it unclear how phase separation controls catalysis in living cells. Here, we develop a quantitative theory of biomolecular catalysis in phase-separated systems and find that liquid condensates can act as tunable catalytic switches, transitioning between regimes of enhanced and suppressed enzymatic activity, exhibiting optimal responses at biologically relevant condensate sizes. We show that condensate-mediated catalysis cannot be understood from reactant enrichment alone, but instead emerges from the coupled interplay of molecular partitioning, diffusive transport, and phase-dependent reaction kinetics. The strongest regulatory effects occur under rapid interphase exchange, where the spatially heterogeneous catalytic network admits a system-level Michaelis-Menten description governed by system-averaged concentrations and reaction kinetics. Our framework predicts that micron-sized condensates can either enhance or suppress enzymatic activity by up to two orders of magnitude, and that optimal catalytic regulation can emerge at condensate sizes comparable to many biomolecular condensates. These results provide experimentally testable predictions for condensate-mediated catalysis and establish quantitative principles for understanding and engineering enzyme-catalysed reactions in biomolecular condensates.

biophysics↗

bioRxiv2026

Curvature-mediated prewetting organize mitochondrial nucleoid

The emergence of spatiotemporal order in diffusive cellular environments requires physical mechanisms to overcome the entropic drive toward disorder. While the organizing role of chemical signaling is well characterized, how complex membrane curvature regulate soluble molecular organization remains a fundamental open question. Here, we establish membrane curvature as a thermodynamic control parameter for the condensation of soluble proteins through prewetting. Using the mitochondrial transcription factor TFAM, we demonstrate that intrinsic membrane curvature locally drives a prewetting transitiona surface-mediated phase separation distinct from bulk condensation. By combining in vitro reconstitution, live-cell super-resolution imaging, cryo-electron tomography, and thermodynamic theory, we show that negatively high-curvature regions lower the nucleation energy barrier of Tfam, driving localized protein condensation at physiological concentrations. These results reveal that membrane curvature does not merely scaffold cellular machinery but manipulates the local free energy landscape, establishing a geometric principle for the spatial control of biological processes.

biophysics↗

bioRxiv2025

Enzyme-driven phase separation of synthetic condensates enables self-organizing compartments and protective microenvironments

Compartmentalization via phase separation is increasingly recognized as a dynamic regulator of cellular biochemistry, yet the consequences of coupling enzymatic reactions to compartmentalization remain poorly understood. We combine a minimal, cell-free expression system with kinetic modeling to show how coupling enzymatic activity to biomolecular condensates drives emergent self-regulation via droplet formation and dissolution. In-vitro mRNA transcription induces phase separation with an intrinsically disordered protein (mutant G3BP1), forming condensates that modulate transcription and degradation kinetics. Our kinetic model shows that phase separation reduces rate constants, with slower degradation within condensates than in the mRNA-protein-poor phase. This leads to more mRNA with a prolonged lifetime of mRNA relative to the case without condensates. Extending the model to sustained and oscillatory resource supply reveals that condensates elevate mean mRNA levels and buffer deviations from the mean compared to the non-condensate scenario. These findings provide a general mechanism of cross-regulation and feedback between phase separation and enzymatic networks, highlighting condensates as active regulators of biochemical flux rather than as passive organizers. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/696465v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d48750org.highwire.dtl.DTLVardef@ffc9eeorg.highwire.dtl.DTLVardef@1222de6org.highwire.dtl.DTLVardef@f36701_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Localized control of protein phase separation via membrane binding

Phase separation of protein-rich condensates is key for the spatial organization of cells. Multivalent proteins can phase separate in the bulk cytoplasm to form 3D condensates or at the membrane surface to form 2D condensates. How cells control 2D versus 3D phase transitions is not well understood. Here, we combine an in vitro model system of membrane surface phase separation with thermodynamic modelling to explore the relation of 2D and 3D phase transitions. We engineered the phase-separating protein FUS to bind to membranes and quantified 2D and 3D phase separation as a function of total FUS concentration and salt concentration. We find that membrane binding induces the formation of 2D surface condensates far below the bulk saturation concentration. In addition to direct FUS-membrane binding, surface condensates sequester more FUS molecules from the bulk via protein-protein interactions, resulting in a substantial deviation from the classical binding isotherm. By changing the protein-protein interaction strength via salt titration, we find that the saturation concentrations for 2D and 3D phase separation are coupled. By extending the classical theory of condensate wetting via membrane binding, we recapitulate the experimental results and show that tuning the membrane-binding strength of a phase-separating protein provides a robust way to control 2D condensation at the membrane over a wide concentration range without entering the 3D condensation regime. Taken together, our results provide a simple framework to understand how cells tune protein interactions and membrane binding to control 2D and 3D phase transitions.

biophysics↗

bioRxiv2025

The environmental stress response controls the biophysical properties of the cytoplasm and is critical for survival in quiescence

All organisms have evolved survival strategies to cope with changes in environmental conditions. Nutrient deprivation, one of the most frequently encountered stresses in nature, causes haploid budding yeast to enter a reversible state of non-proliferation known as quiescence, which entails extensive remodeling of gene expression, metabolism and the cellular biophysical properties. Yeast cells can adapt to and survive long periods of time in glucose starvation-induced quiescence, provided they are able to respire in the early stages of glucose withdrawal. When respiration is blocked during glucose withdrawal, cells prematurely age and exhibit markedly reduced survival and cytoplasmic diffusion. We find here that respiration is required to induce a quiescence-related gene expression program. Induction of this program prior to withdrawing glucose in respiration-inhibited cells bypasses the need for respiration and rescues survival and biophysical properties to levels seen in glucose-starved but respiration-competent cells. This rescue effect relies on proteomic adaptation, which partially occurs through inactivation of Ras/PKA signaling and activation of the environmental stress response via the transcription factors Msn2/4. This signaling cascade triggers the expression of stress response genes and modulates the cytoplasmic diffusion state of cells, ensuring long-term survival in quiescence even in the absence of respiration. Our results highlight the importance of stress adaptation in quiescence and aging, integrating gene expression control and modulation of cytoplasmic properties to maintain cell fitness.

cell biology↗

bioRxiv2025

Theory of non-dilute binding and surface phase separation applied to membrane-binding proteins

Surface binding and surface phase separation of cytosolic scaffold proteins on lipid membranes are involved in many cellular processes, such as cell signaling, cell adhesion, and cortex regulation. However, the interplay between surface binding and surface phase separation is poorly understood. In this work, we study this interplay by deriving a general thermodynamic model and applying it to in vitro reconstitution experiments of membrane-binding proteins involved in tight junction initiation. Our theory extends the classical surface binding isotherm to account for non-dilute and heterogeneous conditions where components can phase separate. We use our theory to demonstrate how surface phase separation is governed by the interaction strength among membrane-bound scaffold proteins and their binding affinity to the membrane surface. Comparing the theory to reconstitution experiments, we show that tuning the oligomerization state of the adhesion receptors in the membrane controls surface phase transition and patterning of the scaffold protein ZO1. These findings suggest a fundamental role of the interplay between non-dilute surface binding and surface phase separation in forming the tight junction. More broadly, our work highlights non-dilute surface binding and surface phase separation as a common organizational principle for membrane-associated structures in living cells.

biophysics↗

bioRxiv2024

Transport kinetics across interfaces between coexisting liquid phases

Biomolecular condensates provide compartments that organize biological processes in the cell. Spatial organization of condensates relies on transport across phase boundaries. We investigate the kinetics of molecule transport across interfaces in phase-separated mixtures. Using non-equilibrium thermodynamics we derive the interfacial kinetics, describing the movement of the interface, and of molecular transport across the interface. In the limit of a thin interface, we show that breaking local equilibrium at the interface introduces a local transport resistance. Subsequently, using a continuum approach, we explore two physical scenarios where such a resistance emerges: a mobility minimum and a potential barrier at the interface. These scenarios lead to the same effective transport model in the sharp interface limit, characterized by a single interface resistance parameter. We discuss experimental conditions for which interface resistance could be observed and how these scenarios could be distinguished.

biophysics↗

bioRxiv2024

Size control and oscillations of active droplets in synthetic cells

Oscillations in the formation and dissolution of molecular assemblies inside living cells are pivotal in orchestrating various cellular functions and processes. However, designing such rhythmic patterns in synthetic cells remains a challenge. Here, we demonstrate the spontaneous emergence of spatio-temporal oscillations in the number of droplets, size, and position within a synthetic cell. The coacervate-based droplets in these synthetic cells sediment and fuse at the cells bottom. Through a newly discovered size control mechanism, the sedimented, large droplets shrink by expelling droplet material. The expelled molecules nucleate new droplets at the top of the synthetic cell, which grow and sediment again. These oscillations are sustained by converting chemical fuel into waste and can continue for hundreds of periods without evidence of fatigue. Strikingly, the period of the oscillation is in the minutes regime and tunable. The design of oscillating organelles in synthetic cells brings us closer to creating more life-like materials and de novo life.

biophysics↗

bioRxiv2023

The interplay between molecular assembly and phase separation

Many biological functions and dysfunctions rely on two fundamental processes, molecular assembly and the formation of condensed phases such as biomolecular condensates. Condensed phases generally form via phase separation, while molecular assemblies are clusters of molecules of various sizes, shapes, and functionality. We developed a theory that relies on thermodynamic principles to understand the interplay between molecular assembly and phase separation. We propose two prototypical classes of protein interactions and characterize their different equilibrium states and relaxation dynamics. We obtain results consistent with recent in vitro experimental observations of reconstituted proteins, including anomalous size distribution of assemblies, the gelation of condensed phases, and the change in condensate volume during ageing. Our theory provides the framework to unravel the mechanisms underlying physiological assemblies essential for cellular function, and aberrant assemblies which are associated with several neurodegenerative disorders.

biophysics↗

bioRxiv2023

Liquid spherical shells are a non-equilibrium steady state

Liquid-liquid phase separation is the process in which two immiscible liquids demix. This spontaneous phenomenon yields spherical droplets that eventually coarsen to one large, stable droplet governed by the principle of minimal free energy. In chemically fueled phase separation, the formation of phase-separating molecules is coupled to a fuel-driven, nonequilibrium reaction cycle. Chemically fueled phase separation yields dissipative structures sustained by a continuous fuel conversion. Such dissipative structures are ubiquitous in biology but poorly understood as they are governed by non-equilibrium thermodynamics. Here, we bridge the gap between passive, close-to-equilibrium, and active, dissipative structures with chemically fueled phase separation. We observe that spherical, active droplets can transition into a new morphology--a liquid, spherical shell of droplet material. A spherical shell would be highly unstable at equilibrium. Only by continuously converting chemical energy, this dissipative structure can be sustained. We demonstrate the transition mechanism, which is related to the activation of a product outside of the droplet, and the deactivation within the droplets leading to gradients of droplet material. We characterize how far out of equilibrium the spherical shell state is and the chemical power necessary to sustain it. Our work suggests new avenues for assembling complex stable morphologies, which might already be exploited to form membraneless organelles by cells.

biophysics↗

bioRxiv2021

Aggregation controlled by condensate rheology

Biomolecular condensates in living cells can exhibit a complex rheology including viscoelastic and glassy behaviour. This rheological behavior of condensates was suggested to regulate polymerisation of cytoskeletal filaments and aggregation of amyloid fibrils. Here, we theoretically investigate how the rheological properties of condensates can control the formation of linear aggregates. To this end, we propose a kinetic theory for linear aggregation in coexisting phases, which accounts for the aggregate size distribution and the exchange of aggregates between inside and outside of condensates. The rheology of condensates is accounted in our model via aggregate mobilities that depend on aggregate size. We show that condensate rheology determines whether aggregates of all sizes or dominantly small aggregates are exchanged between condensate inside and outside on the time-scale of aggregation. As a result, the ratio of aggregate numbers inside to outside of condensates differs significantly. Strikingly, we also find that weak variations in the rheological properties of condensates can lead to a switch-like change of the number of aggregates. These results suggest a possible physical mechanism for how living cells could control linear aggregation in a switch-like fashion through variations in condensate rheology. SIGNIFICANCEThe intracellular space can be organized through phase-separated condensates that often exhibit rheological properties reminiscent of complex fluids. These condensates can affect biochemical processes such as the formation of linear aggregates, in particular biofilaments or amyloids. Here, we propose a theoretical model for how condensate rheology can control the irreversible formation of linear aggregates. A key finding is that size and number of aggregates change in a switch-like fashion upon weak changes in condensate rheology. Our model paves the way to unravel the physiochemical mechanisms of how the rheology of condensates can control aberrant protein aggregation. Such mechanisms may explain how rheological changes, such as ageing or the transition to dormancy, give rise to diseases related to protein aggregation.

biophysics↗

bioRxiv2021

Stochastic dynamics of single molecules across phase boundaries

We discuss the stochastic trajectories of single molecules in a phase-separated liquid, when a dense and a dilute phase coexist. Starting from a continuum theory of macroscopic phase separation we derive a stochastic Langevin equation for molecular trajectories that takes into account thermal fluctuations. We find that molecular trajectories can be described as diffusion with drift in an effective potential, which has a steep gradient at phase boundaries. We discuss how the physics of phase coexistence affects the statistics of molecular trajectories and in particular the statistics of displacements of molecules crossing a phase boundary. At thermodynamic equilibrium detailed balance imposes that the distributions of displacements crossing the phase boundary from the dense or from the dilute phase are the same. Our theory can be used to infer key phase separation parameters from the statistics of single-molecule trajectories. For simple Brownian motion, there is no drift in the presence of a concentration gradient. We show that interactions in the fluid give rise to an average drift velocity in concentration gradients. Interestingly, under non-equilibrium conditions, single molecules tend to drift uphill the concentration gradient. Thus, our work bridges between single-molecule dynamics and collective dynamics at macroscopic scales and provides a framework to study single-molecule dynamics in phase-separating systems.

biophysics↗

bioRxiv2021

Quantitative Theory for the Diffusive Dynamics of Liquid Condensates

To unravel the biological functions of membraneless liquid condensates it is crucial to develop a quantitative understanding of the physics underlying their dynamics. Key processes within such condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date we lack a physics-based quantitative framework for the dynamics of labeled condensate components. Here, we derive the corresponding theory, building on the physics of phase separation, and quantitatively validate this framework via experiments. We show that using our theory we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Strikingly, our theory can also be used to determine the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This bypasses recently described quenching artefacts in the dense phase, which can underestimate partition coefficients by orders of magnitude. Our experimentally verified theory opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities and quantifying rates of biochemical reactions in liquid condensates.

biophysics↗

bioRxiv2021

Local thermodynamics governs the formation and dissolution of protein condensates in living cells

Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm C. elegans which play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams which are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intra-cellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the non-equilibrium nature of the cytoplasm is manifested on larger scales. SIGNIFICANCE STATEMENTLiving cells rely on a continuous flux of energy to spatially organize biochemical processes. It remained unclear whether cells can achieve this spatial organization via thermodynamic principles. Here, we report the striking behavior of a cold-blooded organism that reacts to environmental temperature changes similar to a thermodynamic system at local equilibrium. Our key finding is that protein-rich droplets form and dissolve reversibly with temperature due to changes in the organism?s entropy. We show that the organism uses a specific molecule to extend droplet stability to the natural temperature range of the organisms habitat. Due to the relevance of such protein droplets for the organism?s fertility, our works shed light on how molecular components could facilitate biological functions via thermodynamic principles.

biophysics↗

bioRxiv2019

Lipid digestion and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant - though lower - level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics, to characterize the cells response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP to ATP ratio strongly increases within 20-30 seconds. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, bulk autophagy, a process that frees amino acids, as well as lipid degradation via {beta}-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and {beta}-oxidation as important energy providers during acute glucose starvation.

systems biology↗

bioRxiv2018

Optimal strategies for inhibition of protein aggregation

Protein aggregation has been implicated in many diseases.1-7 Therapeutic strategies for these diseases propose the use of drugs to inhibit specific molecular events during the aggregation process.8-11 However, viable treatment protocols require balancing the efficacy of the drug with its toxicity while accounting for the underlying events of aggregation and inhibition at the molecular level. Here, we combine aggregation kinetics and control theory to determine optimal protocols which prevent protein aggregation via specific reaction pathways. We find that the optimal inhibition of primary and fibril-dependent secondary nucleation require fundamentally different drug administration protocols. We test the efficacy of our approach on experimental data for Amyloid-{beta} aggregation of Alzheimers disease in the model organism C. elegans. Our results pose and answer the question of the link between the molecular basis of protein aggregation and optimal strategies for inhibiting it, opening up new avenues for the design of rational therapies to control pathological protein aggregation.

biophysics↗