Biology subjects

Charras, G.

Publications and source records attributed to Charras, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanical Tension Actively Triggers RhoA-Mediated Cell Extrusion

Cell extrusion is a fundamental process in tissue homeostasis, morphogenesis, and cancer progression, facilitating the removal of cells either alive or through apoptosis. While biochemical signaling pathways are known to regulate extrusion, recent advances have underscored the importance of mechanical forces in this process. Here, using optogenetic control of RhoA activation in epithelial monolayers combined with Bayesian Inversion Stress Microscopy (BISM) and three-dimensional cell-based modeling, we uncover a counterintuitive mechanism whereby elevated tension, instead of stabilizing the monolayer, actively drives extrusion in highly contractile cells. We show that local RhoA activation enhances myosin II-dependent contractility and F-actin reorganization, which promotes cell stiffening, resulting in localized tension buildup. The ensuing tensile stress amplifies vertical mechanical fluctuations, which in turn trigger cell extrusion. Remarkably, these tension-induced extrusions occur both apically and basally. Furthermore, our findings show that RhoA-mediated contractility is not merely an effector of extrusion but also an active promoter of basal extrusion, independently of caspase activation. Our study demonstrates that tensile stress can directly initiate extrusion events and bias their outcome toward apical or basal fates. By identifying tension as a driver rather than a suppressor of extrusion, this work revises current models of epithelial homeostasis and highlights mechanical control as a targetable axis in disease and regeneration.

cell biology↗

bioRxiv2025

Control of cellular cortical tension and shape by RhoGTPase signalling

Shape changes are ubiquitous in biology, from cytokinesis at the single cell scale to tissue-scale morphogenesis involving coordinated changes in hundreds of cells. In all cases, morphogenesis is powered by gradients in mechanical tension that arise downstream of signalling. Many pathways converge on RhoGTPases that modulate the cytoskeleton and cell contractility to control cell mechanics and, subsequently, shape. Despite their physiological importance, we lack a quantitative understanding of how changes in signalling alter cortical mechanics to drive cell shape change. Here, we use optogenetics to quantitatively characterise the relationship between the amount of RhoGEF localised to the membrane, the downstream myosin recruitment, and the subsequent mechanical changes. We then show that cortical myosin amount and cortical tension increase linearly with the amount of membranous RhoGEF signalling. Based on these data, we develop a predictive model of the temporal evolution of RhoGEF membrane localisation, cortical myosin enrichment, and cortical tension in response to a pulse of light. Using this model together with an active surface model of the cell cortex, we show that the cellular shape changes induced by localised optogenetic recruitment of RhoGEF signalling can be predicted, directly linking gradients in signalling to shape change. Significance statementShape changes are ubiquitous in biology, during division in single cells and in tissue during embryogenesis. In all cases, shape change is powered by gradients in mechanical tension that arise downstream of changes in biochemical signals. Despite their importance, we lack a quantitative understanding of how changes in signals alter cell mechanics to drive cell shape change. Here, we control the location and amount of biochemical signal using light to quantitatively characterise the relationship between signals and their resulting biological and mechanical changes. We show that mechanical change scales linearly with the amount of biochemical signal. Based on this, we develop a mathematical model that predicts cell mechanical and shape changes from the location and amount of biochemical signals.

biophysics↗

bioRxiv2025

Emergent Epithelial Resilience under Cyclic Loading

Epithelial tissues are often exposed to cyclic deformations in their physiological environment. The mechanical integrity of epithelial tissues relies on intercellular adhesion proteins which link neighbouring cells and transmit forces across the cellular network. The stability of the intercellular adhesive bonds is critical. Under sustained stress, bond failure leads to cumulative damage that progressively weakens the material and ultimately causes failure at the tissue scale. Although the collective behaviour of adhesion bonds under static loading has been characterised to some extent, their response to dynamic loading remains largely unknown, despite its physiological importance. Here we combine quantitative experiments on MDCK monolayers with analytical and stochastic modeling of slip-bond intercellular linkers to investigate tissue resilience under cyclic loading. We find that cyclic loading significantly prolongs tissue lifetime and deformation before failure compared to constant tension, due to intervals of low stress facilitating bond repair. Our model identifies intrinsic rupture and repair timescales governing adhesion stability, revealing three regimes of tissue behavior: rupture, slow damage accumulation and stable equilibrium. Normalizing loading parameters by the intrinsic molecular timescales collapses experimental and simulated data into universal stability maps. These findings demonstrate that epithelial resilience emerges from stochastic adhesion bond dynamics, providing a predictive framework linking molecular adhesion turnover to macroscopic tissue mechanics under physiological cyclic forces. SIGNIFICANCEEpithelial tissues, the bodys primary mechanical barriers, experience continuous mechanical stress from physiological movements such as breathing or peristalsis. Through experiments and modeling, we show that a tissues ability to resist and repair damage under cyclic loading comes from the natural binding and unbinding of the molecules that link neighboring cells. We identify fundamental timescales governing rupture and recovery in cell-cell junctions, showing that tissue stability can be universally predicted from molecular adhesion kinetics. This work establishes a quantitative framework connecting microscopic adhesion kinetics to macroscopic tissue mechanics. It provides a tool for studying resilience in diverse biophysical soft matter systems and for understanding how living materials maintain integrity under fluctuating forces.

biophysics↗

bioRxiv2025

A keratin bundling transition uncages the nucleus in stretchedepithelia

There is broad consensus that intermediate filaments play a key role in protecting cells and tissues from large deformations. However, little is known about how they fulfil this function. Here we demonstrate that epithelial cells undergo a slow adaptation to stretch that couples a star- bundling transition of keratin filaments and the escape of the nucleus from its keratin cage. The bundling transition begins with a depletion of keratin filaments at tri-cellular junctions followed by a progressive accumulation in thick bundles that bisect cell-cell junctions. Bundling is a cooperative process that initiates in a few scattered cells and propagates to their neighbours through desmosomes, leading to the growth of multicellular clusters that contain a percolated network of thick keratin bundles. Bundling dynamics are slow and strongly influenced by the interaction between actin and keratin. Informed by a computational model, we provide evidence that keratin bundling generates a compressive stress on the nucleus, which is relaxed by nuclear escape from the keratin cage. The topological transitions identified here provide epithelia with a multiscale mechanism to adapt to sustained stretch.

biophysics↗

bioRxiv2025

Boundary constraints can determine pattern emergence

The robust patterning of cell fates during embryonic development requires precise coordination of signalling gradients within defined spatial constraints. Using a geometrically confined in vitro system derived from human embryonic stem cells, we demonstrate that patterning of neuromesodermal progenitors (NMPs) during axial elongation is driven by boundary-dependent mechanisms. Despite extensive work on radial fate patterning in confined 2D systems, the quantitative role of boundary conditions in shaping spatiotemporal dynamics remains unclear. Here, we show that a minimal reaction-diffusion model coupled with a simplified gene regulatory network accurately predicts spatial patterns across diverse geometries. Guided by its predictions, we identify Wnt signalling as a key component of the activator signal. Inhibition of Wnt secretion preserved initiation of patterning but disrupted subsequent morphogenesis, indicating distinct mechanisms govern pattern establishment versus maintenance. Our findings reveal how geometry encodes positional information that directs molecular patterning, providing insight into how spatial constraints and signalling dynamics guide robust tissue self-organisation during development.

systems biology↗

bioRxiv2024

Spatial and temporal signatures of cell competition revealed by K-function analysis

It is generally hypothesised that in MDCK wild-type versus ScrKD co-cultures mechanical competition leads to the physical elimination of ScrKD cells, while in MDCK wild-type versus RasV12 co-cultures biochemical competition results in the non-apoptotic extrusion of RasV12 cells. However, the exact spatiotemporal signatures of competitive interactions in these systems remains largely unknown. Utilising advanced single-cell image analysis, this research quantifies the spatiotemporal dynamics of these competitive interactions. Time-lapse microscopy, combined with cellular segmentation, tracking, cell-state classification, and K-function clustering analysis provide detailed insights into how wild-type cell proliferation relates to the elimination of mutant cells. We find striking differences in the underlying processes of each competition type. In the ScrKD competition, elimination seems driven by diffuse proliferative forces unrelated to the immediate elimination site. In contrast, RasV12 cell elimination is closely linked to local increases in wild-type cell mitoses prior to the RasV12 extrusion. Both competitions trigger an increase in local wild-type cell proliferation post-elimination, although the timing of these responses vary. This study not only sheds light on the diverse mechanisms of cell competition but also underscores the complexity of cellular interactions in tissue dynamics, providing new perspectives on cellular quality control and a new quantitative approach to characterise these interactions.

biophysics↗

bioRxiv2024

The actin cortex acts as a mechanical memory of morphology in confined migrating cells

Cell migration in narrow microenvironments is a hallmark of numerous physiological processes, involving successive cycles of confinement and release that drive significant morphological changes. However, it remains unclear whether migrating cells can retain a memory of their past morphological states, which could potentially enhance their navigation through confined spaces. By combining cell migration assays on standardized microsystems with biophysical modeling and biochemical perturbations, we demonstrate that local geometry governs these morphological switches, thereby facilitating cell passage through long and narrow gaps. We uncovered a long-term memory of past confinement events in migrating cells, with morphological states correlated across transitions through actin cortex remodeling. These findings suggest that mechanical memory in migrating cells plays an active role in their migratory potential in confined environments.

biophysics↗

bioRxiv2024

Sustained Strain Applied at High Rates Drives Dynamic Tensioning in Epithelial Cells

Epithelial cells experience long lasting loads of different magnitudes and rates. How they adapt to these loads strongly impacts tissue health. Yet, much remains unknown about the evolution of cellular stress in response to sustained strain. Here, by subjecting cell pairs to sustained strain, we report a bimodal stress response, where in addition to the typically observed stress relaxation, a subset of cells exhibits a dynamic tensioning process with significant elevation in stress within 100s, resembling active pulling-back in muscle fibers. Strikingly, the fraction of cells exhibiting tensioning increases with increasing strain rate. The tensioning response is accompanied by actin remodeling, and perturbation to actin abrogates it, supporting cell contractilitys role in the response. Collectively, our data show that epithelial cells adjust their tensional states over short timescales in a strain-rate dependent manner to adapt to sustained strains, demonstrating that the active pulling-back behavior could be a common protective mechanism against environmental stress.

biophysics↗

bioRxiv2024

The cytoplasm of living cells can sustain transient and steady intracellular pressure gradients

Understanding the physical basis of cellular shape change in response to both internal and external mechanical stresses requires characterisation of cytoplasmic rheology. At subsecond time-scales and micron length-scales, cells behave as fluid-filled sponges in which shape changes necessitate intracellular fluid redistribution. However, whether these cytoplasmic poroelastic properties play an important role in cellular mechanical response over length- scales and time-scales relevant to cell physiology remains unclear. Here, we investigated whether and how a localised deformation of the cell surface gives rise to transient intracellular flows spanning several microns and lasting seconds. Next, we showed that pressure gradients induced in the cytoplasm can be sustained over several minutes. We found that stable pressure gradients can arise from the combination of cortical tension, cytoplasmic poroelasticity and water flows across the membrane. Overall our data indicate that intracellular cytosolic flows and pressure gradients may play a much greater role than currently appreciated, acting over time- and length-scales relevant to mechanotransduction and cell migration, signifying that poroelastic properties need to be accounted for in models of the cell. Significance statementUnderstanding how cells change shape dynamically under the influence of external and internal forces requires characterisation of the mechanical response of the cytoplasm, the viscous material that fills their interior. The cytoplasm consists of a porous solid phase bathed in a fluid, the cytosol. As the cytoplasm is incompressible, any cellular shape change necessitates redistribution of the cytosol within the cell and its flow rate sets the time-scale for deformation. How the cytoplasmic mechanical response affects cell physiology remains poorly understood. We show that the unique physical properties of the cytoplasm allow cells to sustain cellular-scale pressure gradients over minute time-scales. As a consequence, pressure-driven mechanisms may play a much greater role in cell physiology than currently appreciated.

biophysics↗

bioRxiv2023

Frictiotaxis underlies adhesion-independent durotaxis

Cells move directionally along gradients of substrate stiffness, a process called durotaxis. The current consensus is that durotaxis relies on cell-substrate focal adhesions to sense stiffness and transmit forces that drive directed motion. Therefore, focal adhesion-independent durotaxis is thought to be impossible. Here, we show that confined cells can perform durotaxis despite lacking strong or specific adhesions. This durotactic migration depends on asymmetric myosin distribution and actomyosin retrograde flow. We show that the mechanism of this adhesion-independent durotaxis is that stiffer substrates offer higher friction. We propose a physical model that predicts that non-adherent cells polarise and migrate towards regions of higher friction - a process that we call frictiotaxis. We demonstrate frictiotaxis in experiments by showing that cells migrate up a friction gradient even when stiffness is uniform. Our results broaden the potential of durotaxis to guide any cell that contacts a substrate and reveal a new mode of directed migration based on friction, with implications for immune and cancer cells, which commonly move with non-specific interactions.

cell biology↗

bioRxiv2023

Learning dynamic image representations for self-supervised cell cycle annotation

Time-based comparisons of single-cell trajectories are challenging due to their intrinsic heterogeneity, autonomous decisions, dynamic transitions and unequal lengths. In this paper, we present a self-supervised framework combining an image autoencoder with dynamic time series analysis of latent feature space to represent, compare and annotate cell cycle phases across singlecell trajectories. In our fully data-driven approach, we map similarities between heterogeneous cell tracks and generate statistical representations of single-cell trajectory phase durations, onset and transitions. This work is a first effort to transform a sequence of learned image representations from cell cycle-specific reporters into an unsupervised sequence annotation.

cell biology↗

bioRxiv2023

Dissecting the subcellular forces sculpting early C. elegans embryos

Embryo shape is determined by individual cell mechanics, intercellular interaction strength, and geometrical constraints. Models based on surface tensions at cell interfaces can predict 3D static cellular arrangements within aggregates. However, predicting the dynamics of such arrangements is challenging due to difficulties in measuring temporal changes in tensions. Here, we characterise the spatiotemporal changes in cellular tensions shaping the early nematode embryo using AFM, live microscopy, and tension inference. Using excoriated embryos, we validate a hybrid inference pipeline that calibrates relative inferred tensions temporally using cortical myosin enrichment and absolute tensions using AFM measurements. Applied to embryos within their native shell, we infer a spatiotemporal map of absolute tensions, revealing that ABa, ABp, and EMS compaction is driven by increased tension at free surfaces, while P2s initial exclusion is due to high tension at intercellular contacts. We uncover a direct and non-affine contribution of cadherins to cell-cell contact tension, comparable to cadherins indirect contribution via actomyosin regulation. HighlightsO_LIP lineage cells have lower cortical tensions than AB lineage cells C_LIO_LIEnrichment of Myosin-II at the cell cortex is a good predictor of cell-medium tension but is not sufficient to determine tension at cell-cell contacts. C_LIO_LIMyosin-informed tension inference allows determination of the spatiotemporal evolution of all surface tensions within the embryo. C_LIO_LIABa, ABp, and EMS compact due to high tensions at their cell-medium interfaces compared to their cell-cell interfaces, while P2 is initially excluded due to high cell-cell contact tensions. C_LIO_LICadherins contribute directly in a non-linear way by reducing cell-cell contact tension by nearly 50%. C_LI Open AccessFor the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

biophysics↗

bioRxiv2023

Fracture in Living Cell Monolayers

The ability of tissues to sustain mechanical stress and avoid rupture is a fundamental pillar of their function. Rupture in response to physiological levels of stress can be undesired, for example resulting from disease or genetic mutations, or be an integral part of developmental processes, such as during blastocoel formation in mouse or leg eversion in flies. Despite its importance, we know very little about rupture in cellularised tissues because it is a multi-scale phenomenon that necessitates comprehension of the interplay between mechanical forces and processes at the molecular and cellular scales. Using a combination of mechanical measurements, live imaging and computational modelling, we characterise rupture in epithelial monolayers. We show that, despite consisting of only a single layer of cells, monolayers can withstand surprisingly large deformations, often accommodating several-fold increases in their length before rupture. At large deformation, epithelia increase their stiffness multiple-fold in a process controlled by a supracellular network of keratin filaments. Perturbing keratin organisation fragilised monolayers and prevented strain stiffening. Using computational approaches, we show that, although the kinetics of adhesive bond rupture ultimately control tissue strength, tissue rheology and the history of deformation prior to failure set the strain and stress that the tissue reaches at the onset of fracture. Our data paint a picture of epithelia as versatile materials that combine resistance to shocks with deformability when subjected to low strain rates.

biophysics↗

bioRxiv2022

Force propagation between epithelial cells depends on active coupling and mechano-structural polarization

Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ("cell doublet"). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells shows an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depends on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue is key to maintain signal strength and leads to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

biophysics↗

bioRxiv2022

Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms

Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we have used a combination of optogenetics and computational modelling to better understand how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we find that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the torque balance exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

cell biology↗

bioRxiv2022

Tension at intercellular junctions is necessary for accurate orientation of cell division in the epithelium plane

The direction in which a cell divides is set by the orientation of its mitotic spindle and is important for determining cell fate, controlling tissue shape and maintaining tissue architecture. Division perpendicular to the plane of the substrate can promote tissue stratification during development or wound healing, but also metastasis when orientation is aberrant. Much is known about the molecular mechanisms involved in setting the spindle orientation. However, less is known about the contribution of mechanical factors, such as tissue tension, in ensuring spindle orientation in the plane of the epithelium, despite epithelia being continuously subjected to mechanical stresses. Here, we used suspended epithelial monolayers devoid of extracellular matrix and subjected to varying levels of tissue tension to study the orientation of division relative to the tissue plane. We found that decreasing tissue tension by compressing the monolayers or by inhibiting myosin contractility leads to a higher frequency of out-of-plane divisions. Reciprocally, accurate in-plane division can be restored by increasing tissue tension by increasing cell contractility or by tissue stretching. By considering the full three-dimensional geometry of the epithelium, we show that spindles are sensitive to tissue tension, independently of cell shape, through its impact on the tension at subcellular surfaces. Overall, our data suggest that accurate spindle orientation in the plane of the epithelium necessitates the presence of a sufficiently large tension at intercellular junctions. Significance statementIn growing epithelia, divisions are typically oriented in the plane of the tissue to drive expansion. In some organs, divisions are then re-oriented so that they occur perpendicular to the epithelium plane to drive tissue stratification and cell differentiation. When uncontrolled, this switch in orientation can lead to defects in tissue organisation and, in cancer, likely contribute to metastasis. While much is known about the molecular mechanisms controlling mitotic spindle orientation, less is known about the role of mechanical factors. Here we use mechanical and chemical perturbations to show that mechanics plays a role in controlling the plane of division. Overall, our data suggest that the orientation of spindles in the epithelium plane requires sufficient tension across intercellular junctions.

biophysics↗

bioRxiv2021

Learning the rules of cell competition without prior scientific knowledge

AO_SCPLOWBSTRACTC_SCPLOWDeep learning is now a powerful tool in microscopy data analysis, and is routinely used for image processing applications such as segmentation and denoising. However, it has rarely been used to directly learn mechanistic models of a biological system, owing to the complexity of the internal representations. Here, we develop an end-to-end machine learning model capable of learning the rules of a complex biological phenomenon, cell competition, directly from a large corpus of time-lapse microscopy data. Cell competition is a quality control mechanism that eliminates unfit cells from a tissue and during which cell fate is thought to be determined by the local cellular neighborhood over time. To investigate this, we developed a new approach ({tau}-VAE) by coupling a probabilistic encoder to a temporal convolution network to predict the fate of each cell in an epithelium. Using the{tau} -VAEs latent representation of the local tissue organization and the flow of information in the network, we decode the physical parameters responsible for correct prediction of fate in cell competition. Remarkably, the model autonomously learns that cell density is the single most important factor in predicting cell fate - a conclusion that is in agreement with our current understanding from over a decade of scientific research. Finally, to test the learned internal representation, we challenge the network with experiments performed in the presence of drugs that block signalling pathways involved in competition. We present a novel discriminator network that, using the predictions of the{tau} -VAE, can identify conditions which deviate from the normal behaviour, paving the way for automated, mechanism-aware drug screening.

cell biology↗

bioRxiv2020

Automated deep lineage tree analysis using a Bayesian single cell tracking approach

Single-cell methods are beginning to reveal the intrinsic heterogeneity in cell populations, which arises from the interplay or deterministic and stochastic processes. For example, the molecular mechanisms of cell cycle control are well characterised, yet the observed distribution of cell cycle durations in a population of cells is heterogenous. This variability may be governed either by stochastic processes, inherited in a deterministic fashion, or some combination of both. Previous studies have shown poor correlations within lineages when observing direct ancestral relationships but remain correlated with immediate relatives. However, assessing longer-range dependencies amid noisy data requires significantly more observations, and demands the development of automated procedures for lineage tree reconstruction. Here, we developed an open-source Python library, btrack, to facilitate retrieval of deep lineage information from live-cell imaging data. We acquired 3,500 hours of time-lapse microscopy data of epithelial cells in culture and used our software to extract 22,519 fully annotated single-cell trajectories. Benchmarking tests, including lineage tree reconstruction assessments, demonstrate that our approach yields high-fidelity results and achieves state-of-the-art performance without the requirement for manual curation of the tracker output data. To demonstrate the robustness of our supervision-free cell tracking pipeline, we retrieve cell cycle durations and their extended inter- and intra-generational family relationships, for up to eight generations, and up to fourth cousin relationships. The extracted lineage tree dataset represents approximately two orders of magnitude more data, and longer-range dependencies, than in previous studies of cell cycle heritability. Our results extend the range of observed correlations and suggest that strong heritable cell cycling is present. We envisage that our approach could be extended with additional live-cell reporters to provide a detailed quantitative characterisation of biochemical and mechanical origins to cycling heterogeneity in cell populations.

biophysics↗