Biophysical Journal

A subset of Transient Receptor Potential (TRP) channels display very steep temperature dependences and play key roles in thermosensation. To characterize the properties of these thermoTRP channels, two-state close-open gating models were developed for TRPM8, TRPV1, TRPM4, TRPM5, TRPA1 and TRPM3. In this study, we met the recurrent challenge of finding an initial set of model parameters enabling effective convergence during data fitting procedures. We performed algebraic calculations to derive equations for all gating model parameters as functions of key features of thermoTRP channel data obtained from patch-clamp experiments. We used a minimal set of experimental data: the steady-state open probability and time constant of current relaxation as functions of the membrane potential determined at two temperatures. Specifically, we could express the electric distance of the gating charge and the enthalpy and entropy changes associated with the gating transitions, as functions of the voltages for half-maximal activation, the voltages for maximal time constant of current relaxation and the maximal time constant. Our results provide a method to analytically estimate an initial set thermoTRP thermodynamic parameters enabling robust subsequent nonlinear global data fitting. This approach facilitates quantitative analysis of channel thermodynamics, and has potential applications to more complex gating models, and to the study of permeation, block and other ion channel gating mechanisms.

The ParABS system orchestrates chromosome segregation in many bacterial species. The centromere-like parS sites serve as nucleation points for the initial binding of the ParB protein. Subsequent diffusion on adjacent, non-specific DNA regions (spreading) in the presence of CTP and binding of more ParB molecules along with DNA looping via ParB-ParB interactions bring distal parts of the chromosome into proximity. ParB interaction with the ParA-ATPase motor protein, then, drives genomic segregation. It has been shown that in some bacterial species, the ParB-parS complex undergoes phase separation into a condensate. However, the physico-chemical properties of such condensates and their response to forces, such as those they may face in the cell, have not yet been characterized. Performing turbidity measurements in the presence of CTP and various concentrations of DNA and physiologically relevant mono and divalent salt It was shown that Mg2+ facilitates, while K+ concentrations higher than [~]20 mM disfavors, condensate formation. Microrheology measurements showed that condensates of ParB and DNA including parS sites (ParB-parS DNA) in the presence of CTP, are viscoelastic with a viscosity at Troom of [~]5 Pa{middle dot}s and able to quickly respond to deformations with a network relaxation time of 0.1 s. Additionally, fluorescence combined with force spectroscopy showed that mechanical disruption of ParB-DNA condensates in the presence of CTP requires [~] 5-7.5 pN of tension in the DNA, which is lower than the force required to stall a molecular motor such as RNA polymerase, but higher than the force required for the relocation of chromosomes and plasmids during segregation. These results support the idea that ParB-parS condensates dynamically rearrange at the molecular level while maintaining the cohesion necessary to sustain the drag force of segregation without interfering with genomic transactions. This physical mechanism could be the basis for the critical role of ParB-parS condensates in organizing and partitioning bacterial chromosomes. Statement of SignificanceCondensates formed by liquid-liquid phase separation enable cellular compartmentalization without the energy-costly production of a membrane enclosure and have been implicated in a wide array of cellular functions. Here, we analyze their chemo-physical properties in relation to their activity in bacterial chromosome segregation using the C. crescentus ParB protein and DNA containing parS specific binding sites. We find that the cohesion of condensates of ParB parS-containing DNA confers the mechanical stability necessary to pull genetic material towards the cell pole without interfering with genomic functions. We propose a mechanism to explain the function of ParB-parS DNA in chromosomal segregation.

Despite the widespread use of lipid nanodiscs for structure-function studies of membrane proteins, little is known about how lipids are spatially organized within nanodiscs and how this organization influences embedded proteins. The activity and conformational equilibria of the human A2A adenosine receptor, a class A G protein-coupled receptor, are highly sensitive to anionic lipids that directly interact with the receptor. We leverage this lipid-dependent sensitivity to probe the accessibility of anionic lipids across nanodiscs of varying sizes. We identify a threshold concentration of anionic lipids required to fully populate active receptor conformations that is higher for POPS than for POPG. Computational simulations reveal that POPS and POPG each form lipid clusters reducing the effective availability of anionic lipids to interact with the receptor. This effect is stronger for POPS and scales with increasing nanodisc size, correlating with experimental biophysical and biochemical measurements. Simulations further identify positively charged residues within the membrane scaffold protein that coordinate anionic lipid headgroups. Targeted protein engineering reduces the threshold concentration of anionic lipids required for receptor activation, supporting strategies to control lipid accessibility within nanodiscs. Because membrane scaffold proteins are derived from apolipoprotein A-I, similar lipid-protein interactions may also influence lipid organization within biological systems such as HDL particles.

Integral membrane proteins remodel the surrounding lipid bilayer, but quantifying the resulting deformations and linking them to protein density in the membrane has remained challenging. Here, we introduce an integrative methodology that combines all-atom molecular dynamics (MD) simulations with multiscale small-angle X-ray scattering (SAXS) analysis to connect membrane strain to the protein/lipid ratio in proteoliposomes. Using outer membrane phospholipase A (OmpLA) reconstituted into lipid bilayers with both increased and decreased hydrophobic thickness, we systematically probe the effects of positive and negative hydrophobic mismatch.MD simulations demonstrate that OmpLA causes anisotropic, oscillatory thickness deformations extending up to eight times the radius of the first lipid shell surrounding the protein, yet the net change in average membrane thickness remains below 1%. Through our multiscale SAXS analysis, we quantitatively extract structural parameters, ranging from proteoliposome size to internal membrane architecture, using constrained Bayesian inference, with priors derived from MD findings. Specifically, we determine the protein/lipid molar ratio and average membrane strain, revealing excellent agreement between experiment and simulation. In thinner bilayers, substantial protein loss limits the analysis, highlighting the role of bilayer stability in sample preparation. Moreover, the predominance of OmpLA monomers in the thicker membranes is consistent with weak, membrane-mediated repulsive interactions between protein inclusions. Collectively, this integrative approach establishes a framework for quantifying protein-lipid interactions across molecular and mesoscale dimensions.

Weak magnetic fields affect many biological processes across the tree of life, though the precise molecular sensors and pathways involved in such magnetoresponses remain mostly uncharacterized. Fluorescence is a useful tool for investigating magnetic field effects in flavoproteins, as their chromophores fluorescence intensity can be shown to depend on the spin states of electronic radical pairs. Here, we describe a four-state ordinary differential equation model to understand what parameter sets result in fluorescence contrast between spin states in photocycles with singlet and triplet radical pairs. We conclude that only certain sets of parameters result in the fluorescence intensity being a good proxy measurement for singlet yield. In particular, we observe that the illumination intensity required to obtain fluorescence contrast depends on the rate of the slow spin-independent radical termination reactions that recover ground-state oxidized fluorophores. Moreover, to observe a magnetic field effect in fluorescence intensity when an external magnetic field modulates the singlet yield, the illumination intensity must be strong enough such that photoexcitation is not the rate-limiting step. This understanding suggests that flavoproteins that do not exhibit magnetic field effects in their fluorescence emission under certain experimental setups may still be sensitive to weak magnetic fields in terms of function, as magnetosensitivity in fluorescence depends strongly on illumination conditions.

Recent genetic and bioinformatic studies have led to the discovery that many bacterial species encode the genes required to produce sphingolipids. Shotgun lipidomic studies have identified numerous sphingolipid species with novel structures that do not exist in eukaryotic organisms. The impacts of these lipids on the biophysical properties of bacterial membranes have not yet been determined. In this study, we purify a novel anionic bacterial sphingolipid, ceramide phosphoglycerate (CPG), and investigate its effect on membrane zeta potential and bending stiffness. CPG and its precursor, ceramide 1-phosphate (C1P), are shown to increase the magnitude of the membrane zeta potential. These sphingolipids also increase the stiffness of these membranes, with CPG increasing rigidity more than C1P or ceramide. This work provides experimental and computational methods of lipid isolation and characterization that may be broadly applicable to a variety of uncharacterized bacterial sphingolipids. SIGNIFICANCEThe diversity of bacterial sphingolipids far exceeds those found in eukaryotes. However, the function and biophysical properties of these lipids are unknown. Characterization of these lipids is a challenge as they are not commercially available. In this study, we developed experimental methods to purify the anionic sphingolipid ceramide phosphoglycerate and incorporate it into liposomes for analysis. Furthermore, we built computational tools to determine the bending stiffness of sphingolipid-containing vesicles from thermal fluctuation data.

Protein oligomerization equilibria are central to many biological processes and are often highly sensitive to environmental conditions such as ionic strength, pH, and ligand binding. Quantitative characterization of these equilibria remains experimentally challenging because stable protein complexes frequently dissociate only at concentrations that are difficult to access with conventional biophysical methods. Fluorescence correlation spectroscopy (FCS) is uniquely suited to this problem, as it provides direct access to diffusion coefficients of fluorescently labeled proteins at nanomolar concentrations. However, the quantitative interpretation of FCS data from oligomeric systems requires a rigorous mathematical framework and careful experimental practice that have not previously been described in sufficient detail to guide implementation. Here, we provide a comprehensive description of the experimental workflow and analytical framework for determining dissociation equilibrium constants by FCS, covering instrument calibration, sample preparation, data quality control, after-pulse correction, and nonlinear least-squares fitting. We discuss common sources of error and provide practical guidance on critical experimental considerations including surface passivation, buffer preparation, equilibration time, and the role of labeling efficiency. Using the homotrimeric sliding clamp PCNA as a model system, we demonstrate the complete workflow under a range of KCl concentrations and show that moderate ionic strength stabilizes the PCNA trimer while very high salt partially destabilizes the complex. The approach is general and applicable to any reversible protein self-association reaction accessible by fluorescence detection at low protein concentrations.

Single-stranded DNA-binding (SSB) proteins play a crucial role in DNA replication by binding to single-stranded DNA (ssDNA) in multiple binding modes, depending on conditions such as salt and protein concentrations. The coverage-dependent effects on the kinetics of these binding modes remain incompletely understood. In particular, the bimodal binding kinetics and the further SSB-ssDNA shortening observed when SSB is removed from the media. Here, we develop a kinetic model extending the Tonks-McGhee-von Hippel framework to incorporate ligand crowding and mode transformations, capturing the inhibition of SSB binding and transitions to higher binding modes as coverage increases. This model quantitatively reproduces experimental binding kinetics and coverage-dependent behaviors observed for human mitochondrial SSB (HmtSSB) and E. coli SSB (EcoSSB). Our findings elucidate the impact of ligand crowding on SSB-ssDNA interactions and provide a generalizable framework for studying multimode ligand binding to polymers, with implications for understanding genome maintenance mechanisms.

Reaction diffusion (RD) systems play a fundamental role in numerous biochemical and biophysical processes. Here, we present a novel analytical framework for solving RD equations by applying the Wentzel Kramers Brillouin Jeffreys (WKBJ) formalism to Ca nanodomains generated by individual membrane channels, a widely used paradigm for intracellular Ca signaling. Previous models have primarily focused on stationary Ca nanodomains while neglecting diffusion and saturation of intracellular Ca buffers and sensors. In contrast, we derive analytical solutions without these simplifying assumptions. Our analysis demonstrates that sustained Ca influx generates continuously expanding distributions of free Ca, whereas Ca bound buffers and sensors propagate as traveling waves. These predictions are supported experimentally by measurements of one-dimensional fluorescence profiles produced by single-channel activity and two-dimensional profiles generated by whole cell Ca currents. The analytical framework developed here readily extends Michaelis Menten type kinetics to reaction diffusion systems and may therefore be broadly applicable to biochemical and biophysical processes in which diffusion cannot be neglected.

-Synuclein (Syn) is an intrinsically disordered protein that preferentially binds anionic membranes with lipid packing defects. Cholesterol is an abundant membrane component that regulates packing and organization within membranes, yet its effect on Syn binding remains unclear as prior studies report both cholesterol-mediated enhancement and suppression. Here, we investigated whether these conflicting effects reflect differences in the intrinsic packing state of the phospholipid bilayer. Using a quantitative fluorescence microscopy-based binding assay, we measured Syn binding preferences among reconstituted phosphatidylcholine/phosphatidylserine membranes with varied cholesterol content, lipid tail chemistry, and vesicle curvature. We found that cholesterols effect depended on the underlying packing regime of the membrane. In defect-rich membranes, cholesterol reduced Syn binding, consistent with cholesterol tightening lipid packing and reducing Syn-accessible defects. In membranes with intermediate defect content, cholesterol enhanced binding, whereas tightly packed membranes remained largely insensitive to cholesterol except when high cholesterol content was combined with high membrane curvature. Curvature further shaped these responses, with high curvature compressing cholesterol-dependent differences between membrane compositions. These results show that cholesterol does not universally promote or inhibit Syn binding. Instead, cholesterol regulates Syn-membrane interactions through a packing-regime-dependent mechanism shaped by both lipid tail chemistry and membrane curvature. This framework helps reconcile opposing reports in the literature and highlights membrane physical state as a key determinant of how cholesterol modulates Syn binding. SignificanceSyn is a membrane-binding protein associated with Parkinsons disease, but the role of cholesterol in regulating its membrane interactions has remained unclear. Some studies report that cholesterol enhances Syn association with membranes, whereas others show that cholesterol suppresses it. This work helps explain why both outcomes can occur. We show that cholesterols effect depends on the membranes underlying packing state: cholesterol can reduce, enhance, or have little effect on Syn binding depending on the membrane environment. These findings shift the question from whether cholesterol is generally pro- or anti-binding to how cholesterol reshapes the membrane physical states that control Syn association.

Budding is a fundamental membrane-remodeling process central to many cellular functions and is exploited by numerous enveloped viruses to acquire their lipid envelopes. Despite extensive molecular characterization, the physical mechanisms that determine whether budding proceeds to completion or becomes stalled remain unclear. Here, we develop a theoretical model based on the Helfrich elastic formalism to investigate how membrane geometry and boundary conditions regulate the elastic energy of viral budding. We analyze two representative cases: budding from a flat membrane, characteristic of HIV-1 and alphaviruses, and budding from a vesicle, as observed for SARS-CoV-2 in the ER-Golgi intermediate compartment (ERGIC). Our results reveal distinct energetic pathways: vesicle-like geometries exhibit a stronger energetic bias toward closure, whereas flat membranes develop extended low-slope regions in the energy landscape that can hinder completion. Relaxing far-field boundary constraints reduces the energetic cost associated with membrane area conservation and renders the flat-membrane case energetically comparable to the vesicle case, providing a physical explanation for why viruses frequently bud adjacent to one another or within pre-curved membrane regions. Comparison with thin-section TEM images of alphavirus budding shows results consistent with the theoretical membrane profiles. Together, these findings establish how curvature coupling, boundary flexibility, and local membrane geometry cooperate to control the efficiency and completion of membrane budding.

Liquid-liquid phase separation of proteins has been observed to occur on biological membranes, where it is thought to play a role in diverse cellular behaviors. Recent work has demonstrated colocalization between protein condensates on opposing leaflets of the bilayer, suggesting that protein phase separation may be coupled across the bilayer. However, the mechanism behind this coupling phenomenon remains poorly understood. Here we seek to understand the protein-protein and protein-membrane interactions that give rise to transbilayer coupling of protein condensates. We perform coarse-grained molecular dynamics simulations of a bilayer with a disordered protein condensate tethered to each leaflet surface. In this system, we observe stable, coupled diffusion of the condensates across the membrane. We find that increasing the protein-protein interaction strength leads to decoupling, driven by competing membrane curvatures induced by each condensate. However, by applying membrane tension we suppress curvature and restore coupling even at higher protein interaction strengths. Under coupling conditions, we find that lipid entropy is reduced upon direct contact with proteins, but this effect is not transferred to the opposing leaflet. Interestingly, further analysis reveals increased transverse lipid packing (interdigitation) beneath the condensates relative to protein-free regions. Based on these observations, we propose that enhanced lipid interdigitation mediates interleaflet communication and serves as the primary mechanism driving transbilayer coupling of condensates in this system. This work provides insight into a potential physical mechanism for transmembrane communication in cellular contexts and suggests directions for future investigation. Significance StatementLiquid-like condensates are active participants at cellular membranes, where they act as organizers and catalysts for various cellular processes. Recent work has demonstrated that protein condensates can couple across the bilayer; however, the molecular mechanism of this transbilayer coupling remained unknown. Here, we investigate the molecular basis of transmembrane condensate coupling through detailed analysis and propose a mechanism for the phenomenon. This work advances our understanding of how information is transmitted across the bilayer, with implications in cellular requiring coordination across the membrane, such as signaling, and more broadly in the field of membrane biophysics.

Verapamil, a clinically used calcium channel blocker, enhances the activity of several tuberculosis antibiotics, but its mechanism of action and physiological effects on bacteria remain unresolved. A central debate concerns whether verapamil primarily inhibits efflux pumps or disrupts membrane energetics. Here, we use Escherichia coli as a model system to quantify single-cell and population-level physiological responses to verapamil with high temporal resolution. Real-time measurements of the rotational speed of individual flagellar motors, a single-cell proxy for the proton motive force (PMF), reveal a heterogeneous response to verapamil: treated cells exhibit either a dose-dependent gradual decrease in PMF, or a rapid collapse of PMF. Although loss of the outer-membrane efflux channel TolC increases growth inhibition by verapamil, it does not alter the rapid PMF disruptions observed at the single-cell level, suggesting that efflux contributes to long-term susceptibility but not to the initial PMF disruption. Independent assays of population-level motility, pH, and membrane-integrity suggest that verapamil may selectively dissipate the electrical component of PMF while leaving intracellular pH largely unchanged. A minimal electrical circuit model captures both steady-state and dynamic behavior. Together, these findings demonstrate that verapamil rapidly and reversibly perturbs bacterial membrane energetics through a mechanism distinct from classical protonophores, helping to reconcile conflicting interpretations of its activity and clarifying how membrane effects may interact with efflux inhibition during antibiotic potentiation.

Budding yeast septins assemble into filamentous networks bound to the inner plasma membrane. In situ or in vitro, septins are implicated in membrane deformations. We therefore suspected that septins might alter membrane mechanical properties both directly or indirectly. To decipher whether septins directly tune the rigidity of membranes, we used a cell free in vitro approach. To this end, using AFM, we measured the mechanical response of reconstituted GUVs pre-incubated with septins. Unexpectedly, we find that large GUVs (typically tens of {micro}m diameter size) are more deformable in the presence of septins. Theoretical modeling suggests that this peculiar behavior is likely due to initial micrometer membrane "wrinkled" deformations imposed by septins. Conversely, small GUVs (1 to 2 microns in diameter) cannot undergo any micrometric deformations and are thereby less deformable with septin filaments bound. Our findings suggest that, in specific cellular context, septins could provide a membrane reservoir and eventually facilitate membrane deformations. Significance statementFilamentous cytoskeletal septins, interacting with membranes would be expected to enhance membrane rigidity. Upon mechanical stress, GUVs larger than tens of microns appear, more deformable in the presence of septins. Septins initial membrane reshaping is responsible for this unexpected behavior, as shown by theoretical modeling. However smaller non deformable vesicles are more rigid, with septins bound.

Synaptotagmins (Syts) are Ca{superscript 2}-sensing exocytosis regulators whose tandem C2 domains interact with phosphoinositides and membranes to trigger neurotransmitter and hormone release. Although Ca{superscript 2} binding is known to enhance C2 domain-membrane interactions, the sequence determinants governing lipid binding and membrane penetration across Syt isoforms remain incompletely understood. Here, we performed MARTINI coarse-grained molecular dynamics simulations of isolated C2A and C2B domains from eight Ca{superscript 2}-sensing Syt isoforms (Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9, and Syt10) interacting with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing plasma membranes. To systematically modulate electrostatic properties, we introduced partial and full charge-flip mutations at conserved acidic residues within the calcium-binding loops (CBLs). By integrating simulations across multiple isoforms and charge states, we sought to identify the dominant sequence determinants governing membrane interactions. We found that PIP2 binding to both, CBLs and polybasic patches (PBs), is associated with loop net charge, yielding correlations > 0.95 across all isoforms. However, membrane penetration is not sufficiently explained by loop net charge alone. The local phenylalanines additionally increase membrane penetration independent of loop net charge. Together, these findings establish a comprehensive electrostatic-aromatic framework where loop net charge governs PIP2 binding, whereas loop net charge and local phenylalanine enrichment jointly govern membrane penetration across Syt C2 domains.

Membrane-proximal (MP) actin represents the subset of cortical f-actin localized within 10 nm of the plasma membrane. Here, we describe a family of single-molecule MP actin (SM-MPAct) probes that diffuse within the plasma membrane and are transiently immobilized through binding to f-actin, enabling the localization of MP actin in both time and space. These probes quantify aspects of MP actin structure, dynamics, and remodeling by analyzing probe positions using a combined single-particle tracking and correlation-function approach. This is demonstrated using chemical and physical perturbations of actin and actin-binding proteins, and by interrogating MP actin organization and dynamics in early B cell receptor (BCR) activation. Upon crosslinking of the IgM BCR, MP actin transiently remodels to increase the size of actin corals, facilitating the efficient assembly of BCR clusters and the local accumulation of MP actin. Notably, analogous remodeling is not detected in measurements using total f-actin probes, indicating that SM-MPAct is uniquely sensitive to the f-actin pool that regulates signaling processes at the plasma membrane. STATEMENT OF SIGNIFICANCEThe actin cortex provides mechanical stability to the plasma membrane and contributes to the organization and dynamics of plasma membrane components. This report presents single-molecule probes and analytical methods to characterize the density, mesh size, motion, and turnover dynamics of the portion of the actin mesh in direct contact with the plasma membrane, enabling quantitative studies of actin remodeling in plasma membrane processes. This general framework is demonstrated through quantification of actin remodeling during early B cell receptor signaling and could be applied to a broad range of cell processes.

A major challenge in neuroscience is to predict how currents in nanodomains affect voltage and ionic concentrations. Cable and Rall theory provide analytic current-voltage relations by neglecting concentration gradients, and the impact of concentration gradients is usually studied numerically with the Poisson-Nernst-Planck (PNP) model. A precise quantitative understanding of the combined dynamics remains limited because analytic current-voltage-concentration relations are missing. In this work we derive such relations using a novel approach based on cross-diffusion equations. For narrow cylindrical domains, we derive time-dependent and steady-state expressions that explicitly show how currents affect voltage and ionic concentrations. We find that the influx of only one ion can significantly change the concentrations of all the other ions even if no channels for these ions are present. After a current injection we compute a biphasic voltage transient where the small-time asymptotic corresponds to the steady-state solution of the cable equation. We show that the accuracy of cable theory prediction for the voltage depends on how the current is distributed among the various ions. Finally, we develop an iterative method to accurately compute steady-state profiles for voltage and concentrations using first-order results by subdividing a cylinder into small segments.

-Synuclein (Syn) is an intrinsically disordered protein whose interactions with lipid membranes are central to both its physiological function and its role in synucleopathies. While membrane charge, phase, and curvature are each known to influence Syn binding, these properties are typically examined independently, leaving their combined effects on both equilibrium and dynamic membrane association unresolved. Here, we systematically investigate how membrane phase and charge jointly regulate Syn binding, curvature sensitivity, and exchange dynamics using fluorescence microscopy, circular dichroism spectroscopy, and fluorescence recovery after photobleaching (FRAP), complemented by coarse-grained molecular dynamics simulations. Under zwitterionic conditions, Syn preferentially binds highly curved gel-phase membranes, driven by curvature-dependent enrichment of packing defects arising from faceted vesicle morphologies. Incorporation of anionic lipids selectively enhances binding in liquid-phase membranes while attenuating curvature-dependent partitioning in gel-phase membranes. Dynamic measurements reveal that membrane phase and charge also govern the stability of membrane-associated Syn, with gel-phase membranes and anionic lipids promoting kinetically stabilized states. Simulations show that curvature-induced defect formation is strongly amplified in gel-phase membranes but largely insensitive to charge. These findings establish that Syn-membrane interactions are governed by a cooperative interplay between membrane phase, curvature, and charge and highlight the importance of resolving both thermodynamic and kinetic contributions to protein-membrane binding.

Genomic DNA is subject to forces and torsion. Some arise mechanically, while others can be entropic, such as those due to crowding within the nuclear environment. Indeed, about 30-40% of the cell is occupied by molecules other than water, and of these, the vast majority are macromolecules. Here, we explore both experimentally and theoretically the interplay between tension, torsion, and macromolecular crowding. Using pharmaceutically relevant crowders of different molecular weights, Dextran 70, and polyethylene glycol (PEG), we observed that macromolecular crowding of unwound, stretched DNA effectively opposed the tension and promoted the formation of plectonemes. A theoretical model representing the equilibrium between B- and L-form DNA fit to the experimental measurements indicates the contractile tension produced by macromolecular crowding of DNA. SIGNIFICANCE STATEMENTDistinct DNA conformers are involved in different cellular processes. Genomic DNA is both stretched and unwound by enzymes in a crowded intracellular medium. This can induce conformational changes between extended, twisted and more compact, plectonemic forms. This study explores the effect of macro-molecular crowding on the conformations of DNA subject to tension and torque. Fitting experimental data to a model for the right-to-left-handed DNA transition, we show that macromolecular crowding induces a contractile force that favors DNA writhe and that such force depends both on the concentration and molecular weight of the crowder.

GPCR oligomerization has been reported for decades, yet its extent and functional relevance in living cells remain unresolved because existing approaches, often done in bulk, are poorly account for local receptor density, a major determinant of intermolecular interactions. Here, we establish a generic quantitative imaging framework that links spatially resolved FRET measurements describing protein oligomerization to local membrane protein in living cells. Using automated high-throughput analysis of fluorescence images, the method generates large density-resolved datasets that enable direct quantification of receptor oligomerization parameters, including apparent affinity, oligomerization state, and monomer/dimer populations at the submicrometer scale. Applied to class A GPCRs in HEK293 cells, the approach reveals receptor-specific density-dependent equilibria between monomers and dimers over physiologically relevant expression ranges, with no evidence for stable higher-order oligomers under basal conditions. The receptors studied exhibit distinct apparent affinities for dimerization, ranging from predominantly monomeric to dynamic monomer-dimer equilibria, indicating that local membrane density strongly influences receptor organization and that it is receptor dependent. The agreement between our measurements and low-density single-molecule studies further suggests that previously reported higher-order oligomers may partly reflect density-driven receptor proximity effects. By bridging single-molecule and ensemble measurements within a unified quantitative framework, this work reconciles conflicting observations in the GPCR oligomerization literature and provides a broadly applicable strategy for investigating membrane protein organization in living cells. SignificanceGPCR oligomerization in living cells is strongly influenced by the local protein density, yet most approaches do not quantitatively account for this parameter. Here, we introduce a quantitative high-throughput imaging framework that directly relates membrane protein local density to local oligomerization state in living cells. Applied to distinct GPCRs over physiologically relevant density ranges, the method reveals distinct density-dependent monomer-dimer equilibrium and apparent affinities for self-association. These results help reconcile longstanding discrepancies, where distinct oligomerization states have been measured depending on experimental conditions. More broadly, this work establishes local membrane protein density as a key determinant of membrane protein organization, and provides a quantitative framework applicable to membrane protein complexes in their native cellular context.