The European Physical Journal E

Cell states are increasingly conceptualized as attractors of high-dimensional dynamical systems, yet quantitative approaches for integrating phenotypic information into this framework remain limited. Here, we take an image-based approach that combines unsupervised machine learning (ML) with timelapse imaging to extract and characterize the temporal dynamics of morphological features. Using a cell line with endogenously tagged VE-cadherin, we acquired brightfield and fluorescence timelapse images of human induced pluripotent stem cell-derived endothelial cell (hiPSC-EC) monolayers, which adopt distinct phenotypes at two different magnitudes of shear stress in terms of their morphology, behavior, and VE-cadherin organization. To quantify these phenotypic cell states without segmentation, we trained a diffusion autoencoder to predict VE-cadherin signal from brightfield images. We identified interpretable ML-based features representing cell orientation, elongation, and local density. Treating these variables as dimensions of a morphological state space, we estimated a data-driven vector field and found that the two observed phenotypic cell states correspond to stable fixed points of the inferred dynamical system. Mapping measured cell migration coherence onto this space further distinguished the states. Imaging cells across intermediate shear stresses revealed a regime of bistability in which both states coexist, indicating that the shear-stress-dependent transition between endothelial cell states occurs as a bifurcation of the inferred dynamical system. Finally, we applied this method to study an N-terminal truncation of VE-cadherin, finding that mutated cells preserve alignment and coherent migration, but exhibit altered morphology and increased migration speed. This work demonstrates the applicability of a dynamical systems approach to quantitatively characterize morphological aspects of cell state from interpretable ML-based features.

Cell shape and mechanics are intricately connected and tightly regulated by mechanochemical events including biochemical signaling, cytoskeletal remodeling, and plasma membrane mechanics. While experimental advances in microscopy have shed light on the intricate coordination involved in cell shape change in response to different cues, the ability to conduct three-dimensional simulations in realistic geometries remains an open computational challenge. In this work, we develop a finite-element framework that incorporates advection-diffusion-reaction equations coupled with equations governing the kinematics of a deformable interface representing the cell membrane. We applied this framework to three distinct coupled mechanochemical systems, each governed by geometric partial differential equations, resulting in large deformations of the interface. In all three examples, our simulations revealed the emergence of feedback between cellular signaling, cytoskeletal organization, and cell shape. In our first two sets of simulations, we observed that cell migration and neutrophil protrusion were regulated by membrane tension-mediated feedback. In our final application, we predicted shape changes of a dendritic spine starting from a realistic geometry, and found that the complex shape of the spine gives rise to localized regimes of actin cytoskeleton remodeling not previously observed with idealized geometries. Thus, our finite-element framework allows us to generate new mechanistic insights for biophysical problems.

Animals navigating complex vertical environments must secure stable footholds, a challenge for species without feet. While arboreal climbing has evolved repeatedly in snakes, the physical mechanisms they use to scale broad, nearly flat surfaces remain poorly understood. By measuring three-dimensional body kinematics and per-contact forces on a smooth vertical wall with protruding posts, we show that cornsnakes climb by dynamically balancing forces across a highly redundant network of 5 to 16 simultaneous contacts--far exceeding the three contacts minimally required for physical stability. Using a computational model and a robotic climber, we demonstrate that while simple body undulations and passive friction are mechanically sufficient to climb this terrain, snakes systematically deviate from this passive baseline. While downward climbing relies primarily on friction, ascending snakes actively generate positive mechanical work at their contacts to propel themselves. Furthermore, we found that whenever a snake engages a new contact, it triggers a stereotyped, system-wide redistribution of force that seamlessly integrates the new foothold without disrupting whole-body balance. These results reveal how a continuous, flexible body can transform sparse environmental features into a robust, fault-tolerant network. This mechanism provides a biomechanical framework for understanding the repeated evolution of limbless climbing and offers physical principles for designing agile robots for unstructured terrain.

Epithelial sheets must maintain robust barrier function while enduring severe mechanical deformations across various physiological environments. While baseline actomyosin contractility is understood to stabilize intercellular junctions and hence cell-cell contact integrity, how cell-generated active forces interact with external physical strain to dictate contact integrity remains poorly understood. In this study, we investigated the biophysical trade-offs between actomyosin contractility and barrier resilience when Madin-Darby Canine Kidney (MDCK) cell islands are subject to large stretch. In contrast to a high concentration (50 M) of the non-muscle myosin II inhibitor blebbistatin that disrupted cell-cell contacts, we first identified a lower concentration (10 M) that maintained cell-cell contact integrity in the absence of any stretch. Such moderate inhibition of non-muscle myosin II reduced, but preserved some level of actin bundle organization. Remarkably, when challenged with a pathological 38% linear stretch using a custom-built biaxial stretching device, 10 M blebbistatin treated epithelial islands exhibited significantly fewer cell-cell contact ruptures than untreated controls, demonstrating a potent protective effect against mechanical strain. Traction force microscopy revealed diminished cell-generated strain energy by over 60% indicating a partial but significant reduction in contractility upon 10 M blebbistatin treatment. Nanoindentation measurements revealed that moderate contractility inhibition decreased the cellular Young's modulus by more than 40%. Consequently, moderate contractility inhibition safeguards epithelial junctions through a dual mechanical effect: it simultaneously reduces baseline active tensile stresses due to cell contractility and lowers the passive elastic forces generated within the softened cell island during external stretch. Our findings indicate that this systemic reduction in forces dominates over any loss of biochemical adhesion strength at cell-cell contacts. We propose that shifting the epithelium from a rigid, highly stressed continuum to a more compliant, relaxed state by moderate contractility inhibition can serve as a general biophysical mechanism to preserve barrier integrity under severe mechanical challenge.

AO_SCPLOWBSTRACTC_SCPLOWEarly tumor detection is critical for improving patient survival and recovery. Clinically, tissue palpation is routinely used to identify regions of abnormal stiffness, a hallmark of many pathological conditions. However, palpation is restricted to anatomically accessible sites and remains highly operator dependent. Here, we introduce a method for real-time quantitative stiffness mapping using an unmodified commercial endoscope, with the goal of enhancing diagnostic capabilities and restoring mechanical feedback during endoscopic procedures. Our approach combines shear wave elastography with speckle imaging and an innovative synchronization strategy that enables the measurement of shear wave propagation using an unmodified commercial endoscope. The resulting wave fields are analyzed with the noise-correlation-inspired (NCI) method[1], providing pixel-wise estimates of shear wave velocity and, consequently, quantitative maps of local tissue stiffness. The method demonstrated robust performance in both benchtop and endoscopic configurations. Validation was achieved on polymer phantoms as well as on ex vivo and in vivo biological tissues, highlighting its potential for minimally invasive biomechanical imaging and real-time tissue characterization.

The cytoplasm is a crowded and dynamic fluid within which cellular building blocks such as mRNA, proteins, or organelles undergo transport and mixing. Although small things like proteins can eventually mix through diffusion, the high viscosity of cytoplasm means that it should be difficult to obtain significant mixing for structures in the size range of mRNA, multi-protein complexes or organelles. In large amoeboid cells, the cytoplasm undergoes active streaming coupled to cell motility, but this streaming is laminar flow which should not be effective for mixing. In this work we used a combination of live cell tracking of injected beads and computational analysis of motion and mixing in giant amoeba Chaos carolinensis with the initial goal of testing the possibility that large-scale cellular deformations during pseudopod formation might implement chaotic mixing by a Baker-transform like process. Instead, we found that Chaos carolinensis accelerates cytoplasmic mixing using a novel cytoplasmic gel state capture and release strategy. While it was previously thought that the amoeba sol to gel state transitions only occur at the trailing and leading edge of the cell body, our work indicates that these transitions occur frequently throughout the mid-cell region, driving the cytoplasmic mixing of beads and organelles. These results indicate that amoeba achieves nearly complete mixing between 1 and 2 cytoplasmic stream/flow cycle, effectively approximating the Bernoulli mixing regime and thus representing one of the theoretically fastest possible mixers.

Poly-L-Lysine (PLL) mediates the non-specific adhesion of cells and is commonly used in Atomic Force Microscopy (AFM) measurements, to ensure that cells remain attached to the substrate. However, it is acknowledged that adhesion affects the measured mechanical properties, in particular in the case Red Blood Cells (RBCs). This results in a wide range of Youngs modulus E reported in the literature. The present study aims at providing a systematic approach to the impact of non-specific adhesion on the rheology of RBCs. It provides a correlation between the topography profile of adherent RBCs and their rheology, from weak (cPLL = 10-3 mg/mL) to strong-adhesion (cPLL = 100 mg/mL) regimes. Using RICM and AFM, we find that there is a continuum of RBC shapes promoted by adhesion, from concave to dome-shaped, as predicted by the theory of vesicle adhesion. Their elastic properties discriminate them into two populations depending on adhesion strength, where stiffer RBCs (E {gtrsim} 100 Pa) correlate with dome-shaped cells. These findings are supported by rheology measurements of the dynamic complex shear modulus G*(f): while the storage modulus increases with cell-substrate adhesion, reflective of an increased membrane shear modulus, the loss modulus remains unchanged. Finally, further analysis inspired by membrane theory shows that different deformation modes may be triggered during indentation of either weakly or strongly adhering RBCs, illustrating the limits of the Hertz model.

Scanning ion conductance microscopy (SICM) provides gentle, non-contact cell surface imaging, but it has not been used to investigate intracellular structures because the plasma membrane restricts nanopipette access. Here, we combined SICM with microsonication-based cell de-roofing to expose intracellular actin stress fibers (SFs) in U2OS cells for nanotopographical and -mechanical characterization. Importantly, the de-roofing conditions preserved actomyosin contractility, allowing analysis of SF structural and biomechanical changes during ATP-induced contraction. Resting SFs displayed an average height of 203{+/-}38 nm and width of 357{+/-}73 nm, and a complex surface architecture characterized by regularly spaced long-range height modulations (~500 nm periodicity; Wq ~25 nm) and smaller irregular corrugations (Ra ~19.2 nm). ATP stimulation reduced SF height and width by ~39% and ~15%, respectively, while largely preserving surface corrugation patterns. During contraction, some SFs separated into two longitudinal strands. High-resolution SICM imaging also revealed filamentous crosslinks mechanically coupling neighboring SFs, and nanomechanical measurements demonstrated local stiffening during contraction. These findings provide new insight into the structural and mechanical regulation of SF contraction and highlight the potential of SICM combined with cell de-roofing as a powerful platform for studying dynamic intracellular processes at nanometer resolution.

Chronic infections persist in large part thanks to protection that biofilms afford their bacterial creators. The extracellular polymeric substance of biofilms is a hydrated matrix of DNA, polysaccharides, and structural proteins, amongst other components, through which nutrients, signalling molecules, and antimicrobial agents must diffuse to reach the bacteria within. Quantitative measurement of transport on the nanoscale within in vivo biofilms remains challenging due to optical heterogeneity, autofluorescence, active remodelling of biofilms and the ambiguity in trajectory reconstruction during single-particle tracking (SPT). Here, we present a methodological framework for measuring molecular transport in defined minimal extracellular matrix models using quantum dots as fluorescent nanoscale probes imaged with high-speed SlimVar microscopy. To establish conditions in which high-diffusivity particle trajectories can be reliably reconstructed, upper limits to quantum dot concentrations were estimated from Brownian motion. The 99th-percentile inter-frame jump distance was estimated from the three-dimensional Brownian jump distance distribution and used to define a target average nearest neighbour distance, and therefore a per-particle volume, used for calculating a concentration which minimises the probability of trajectory collision during data acquisition. Quantum dot movement was imaged at sub-millisecond frame rates and diffusion coefficients were calculated in a 20% glycerol control and in DNA nanostar hydrogels modelling minimal extracellular matrix scaffolds assembled at 250 M and 500 M. Median diffusion coefficients decreased from 94.9 m2*s-1 in glycerol to 15.9 m2*s-1 and 8.3 m2*s-1 in the 250 M and 500 M hydrogels, respectively. More broadly, this work establishes a workflow for quantitative SPT in minimal biofilm models. Rather than attempting to reproduce the full biological complexity of native biofilms, this approach provides the basis of a modular experimental framework in which individual extracellular matrix components can be incorporated sequentially and their effects on molecular transport quantified.

Defining a body axis is a central aspect of animal morphogenesis. During regeneration from excised Hydra tissue pieces, the newly formed body axis typically preserves the orientation of the parent body axis and aligns with the inherited nematic organization of the supracellular actomyosin fibers. Here we show that this inherited orientation can be overridden by geometric confinement. Tissue spheroids confined in narrow cylindrical channels in a frustrating configuration, with the inherited axis initially perpendicular to the channel, regenerate with their body axis aligned along the channel. Using high-resolution live imaging we show that this reorientation is accompanied by remodeling of the nematic fiber organization. New fibers form parallel to the channel axis in the initially disordered closure regions, creating sharp domain boundaries with the inherited transverse fibers. These domain boundaries subsequently propagate, with perpendicular fibers dissolving and new fibers forming along the channel axis. The confined tissue behaves as a solid-like active nematic material, storing anisotropic strain over long timescales while allowing nematic reorganization relative to the material frame. Our results suggest that coupling between tissue strain and nematic alignment contributes to fiber reorientation and body-axis patterning, highlighting how external mechanical constraints can redirect the body axis during morphogenesis.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

Precise, non-invasive manipulation of individual living cells remains a central challenge in biomedical science, with far-reaching implications for single-cell analysis, tissue engineering, and the study of cell-cell interactions. Here, we report the first demonstration of single-cell control using bulk acoustic standing-wave acoustofluidics with closed-loop feedback. We introduce VeLO (Vector-based Local Optimization), a model-free, reinforcement learning-inspired algorithm that enables programmable two-dimensional manipulation of individual cells using a single piezoelectric transducer. Without prior calibration or physical modeling, VeLO learns system dynamics online from acoustically induced cell displacements and automatically adapts to nonlinear, time-varying conditions. We achieve robust control across multiple cell types (DU-145, Jurkat, K-562) and independent manipulation of multiple cells, including controlled cell-cell contact. By combining simplicity of hardware with autonomous, adaptive control, this approach establishes multimodal acoustofluidics as a versatile tool for label-free, high-precision single-cell manipulation.

Mechanical forces have a major effect on cell behavior. Most cells in vitro are grown under static conditions on hard tissue culture plastic, conditions that do not accurately reflect living tissues. The ability of cells to sense and respond to mechanical forces is essential for key biological processes, including development, proliferation, and migration. Disruption of the ability to respond to mechanical forces are known to be a critical factor in many diseases, including cardiovascular disease, progeria, and cancer. Here, we present the design, fabrication, and biological testing of a custom-built cell-stretching device that applies controlled biaxial strain to cells cultured on a polydimethylsiloxane (PDMS) membrane. We then used this device to examine how cells respond to strain. In response to biaxial strain, MCF-7 cells activated the mechanosensitive immediate early gene (IEX-1), with its expression increasing significantly after 1 and 3 hours of stretching. Cells exposed to mechanical strain also remodeled their cytoskeleton in a direction-dependent manner. Under uniaxial strain, actin filaments reoriented perpendicular to the stretch direction, whereas biaxially stretched cells do not promote directional reorientation, but instead appear to reinforce actin at the cell periphery. Similarly, cells under uniaxial strain exhibited changes in nuclear orientation and shape that were not observed under biaxial strain. Nuclear area remained unchanged in either strain condition. These results highlight that the biaxial stretcher can be used to apply strain to cells, and that cells respond differently to biaxial strain compared to what has been reported for uniaxial strain.

Cells adjust their internal circuits in response to changes in their environment. Hence, exposing cells to changing conditions provides a way to probe the intrinsic dynamics of cellular internal circuits. Metabolic networks are examples of such circuits since metabolic fluxes dynamically adjust when environmental conditions are transiently altered. Most existing theoretical frameworks focus on cellular metabolic steady states and do not consider the dynamics of changes in metabolic fluxes. In this work, we applied transfer function analysis from control theory to analyze the changes of NADH oxidative fluxes in the mitochondria and cytoplasm in mouse oocytes in response to dynamical perturbations of oxygen depletion and recovery. We observed an overshoot of NADH oxidative flux in the cytoplasm upon oxygen recovery which is absent in the mitochondrial NADH oxidative flux. Metabolic perturbation experiments and transfer function analysis indicate that this cytoplasmic NADH overshoot results from the coupling of the mitochondrial and cytoplasmic NADH cycles. The degree of overshoot is determined by competing timescales associated with the exchange rates of lactate and pyruvate with the media and their interconversion rates catalyzed by lactate dehydrogenase. Applying control theory to the data enables the inference of the exchange and conversion rates of pyruvate and lactate, allowing predictions of the contribution of lactate to mitochondrial respiration. Our work indicates that the oocytes maintain a homeostatic respiration rate across nutrient conditions by modulating the contribution of lactate to mitochondrial respiration.

Cetaceans reduce the net cost of sustained swimming through intermittent locomotion, alternating active fluking with unpowered gliding. The energy balance of this strategy is central to understanding survival rates, population sustainability, and the effects of anthropogenic and environmental pressures. While active-phase energetics have been characterized extensively, the glide phase remains largely unexplored. Here we derive the optimal glide duration (Topt) and the maximum glide duration beyond which energy savings vanish (Tzero) for three odontocetes spanning a 20-fold range in body mass, using high-fidelity CAD models and wall-modeled large eddy simulations. We show analytically that speed retention at Topt and mass-specific peak energy savings are both fully determined by the active-to-passive drag ratio, propulsive efficiency, and swimming speed, independently of body morphometry and drag coefficient, and are therefore invariant across species at any given speed. These passive-phase optima extend the known size-independent active-phase invariants to the glide phase, towards a scale-independent energetic framework for burst-and-glide locomotion in small cetaceans.

Auxetic metamaterials exhibit negative Poisson's ratio behaviors due to their architecture of periodically arranged unit cells. Although mechanical metamaterials are well established at the macroscale, programmable auxetic units remain scarce at the nanoscale. DNA origami offers a promising platform to bridge this gap, but design principles for dynamically deformable 3D auxetic nanostructures remain largely unexplored. Here, we develop design strategies for such 3D auxetic metastructures built from wireframe DNA origami. As a model system, we use a 3D re-entrant triangular unit composed of double-stranded DNA (dsDNA) bundle edges connected by single-stranded DNA (ssDNA) joints. Using coarse-grained molecular dynamics (MD) and umbrella-sampling free-energy simulations, we examine how edge design and joint-connection scheme govern auxetic responses and the energetics of the structural transformation. Our results show that auxetic performance and deformation energetics emerge from the coupled effects of DNA bundle rigidity and connector mechanics at the joints. This study provides mechanistic insights and design guidelines for programmable auxetic motion and energetics in 3D DNA origami metamaterials, advancing the development of stimuli-responsive nanomechanical devices.

Cells build tissues through local exchanges of force and information, yet the rules governing these interactions are difficult to infer from sparse observations. Here, we introduce waxMorph, a differentiable cell-based framework for generating and reconstructing three-dimensional morphogenesis. In synthetic and biological data, waxMorph reproduced established mechanochemical shape programs, inferred continuous trajectories from static tissue volumes, and recovered spatially organized latent signals. In a developing mouse myocardium dataset, it reconstructed unobserved intermediate geometries more accurately than optimal-transport interpolation, while in forelimbs it distinguished related developmental trajectories. By varying the capacity and spatial organization of the latent cues available to cells, waxMorph also provides a model-based way to quantify the complexity of shape assembly. waxMorph is built within the spatial-computing ecosystem of NVIDIA Warp. It provides an open-source, Python-native, GPU-accelerated, hybrid physics-AI framework for learning how local cellular interactions give rise to biological form.

Microscopes are essential tools for discoveries on a scale invisible to the unaided human eye. The development of immuno-fluorescence followed by molecular biology techniques and fluorescent fusion proteins have revolutionised the use of optical microscopy in bioscience. The quality of the data produced is dependent upon the sample, its preparation and the instrument used. However, instruments can degrade over time without easily visible changes to the produced images and, in turn, negatively impacts results. By testing instruments and doing comparisons between results over time and between different instruments, problems can be highlighted and corrective action can be taken. Using small fluorescent beads the point spread function (PSF) of the microscope can be recorded and the image resolution measured. Beads were prepared in a concentration matched to the field of view size and dried onto coverslips and mounted on slides. The beads were then imaged as 3D Z-stacks of sufficient size to fully enclose the PSF of the system. This data was uploaded to OMERO and processed using OMERO-metrics, an OMERO plugin developed for this purpose. This paper summarizes the development of workflows and protocols to enable this process, presents the results obtained and demonstrates the detection of significant instrument issues.

There is a vast literature on the voltage gating of ion channels, with a fairly large fraction concerned with potassium channels, especially of the KV1 family, including Shaker. Experimental evidence derived from protein structure has been interpreted to give gating mechanisms that largely disregard water. We propose that the K+ ion, in order to pass through the gating region and enter the cavity pore, must be largely dehydrated. Competitive interactions of each single hydration shell water at the gate, with counterions, protein, or other water molecules, can remove one water at a time. There are several such interactions for the ion hydration shell; for the ion to pass through the gating region, there must be enough such interactions to leave the ion with at most two hydrating water molecules, in which case the gate is open. Protein conformational changes are secondary, small, and mostly unimportant. The hypothesis has a second part: protons, previously shown to be candidate carriers of the gating current (Kariev and Green, JPC B, 2019, Membranes, 2022, 2024) are capable of reaching the gate; adding four protons to the gate prevents dehydration, leaving the ion with at least three hydrating water molecules, enough to block passage. Quantum calculations presented here support the dehydration part of the hypothesis; they also mostly support the second part, concerning the protons, but further work will be required to fully confirm this. The hypothesis explains the experimental finding that the P475D mutant is essentially constitutively open, while the P475S mutant, with a wider gate opening, is closed at all relevant potentials; the computations presented here show the mechanism for this in detail, further confirming the first part of the hypothesis, and largely but not completely confirming the second part, concerning protons, while showing where further work is needed. This mechanism can also qualitatively account for flicker noise and fluctuations, and their consequences.

What drove nucleic acids (NA) to associate with proteins (PR) at the Origins of Life? We reason from polymer physics and the Central Dogma (CD) that the fitness value of cooperating through a division of labor - NA for replication fidelity and PR for functional fitness - is much higher than for either polymer alone. Our model shows a Pareto Front, where NA and PR can bootstrap each other to achieve autocatalytic cooperativity towards biology.