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Ectodermal organ development, including lacrimal gland, is characterized by an interaction between an epithelium and a mesenchyme. Murine lacrimal gland is a good model to study non-stereotypical branching morphogenesis. In vitro cultures allow the study of morphogenesis events with easy access to high-resolution imaging. Particularly, embryonic lacrimal gland organotypic 3D cell cultures enable the follow-up of branching morphogenesis thanks to the analysis of territories organization by immunohistochemistry. In this chapter, we describe a method to culture primary epithelial fragments together with primary mesenchymal cells, isolated from embryonic day 17 lacrimal glands., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) more...

Changes in tissue geometry during developmental processes are associated with collective migration of cells. Recent experimental and numerical results suggest that these changes could leverage on the coexistence of nematic and hexatic orientational order at different length scales. How this multiscale organization is affected by the material properties of the cells and their substrate is presently unknown. In this study, we address these questions in monolayers of Madin-Darby canine kidney cells having various cell densities and molecular repertoires. At small length scales, confluent monolayers are characterized by a prominent hexatic order, independent of the presence of E-cadherin, monolayer density, and underlying substrate stiffness. However, all three properties affect the meso-scale tissue organization. The length scale at which hexatic order transits to nematic order, the "hexanematic" crossover scale, strongly depends on cell-cell adhesions and correlates with monolayer density. Our study demonstrates how epithelial organization is affected by mechanical properties, and provides a robust description of tissue organization during developmental processes., (© 2023. Springer Nature Limited.) more...

The simple light isotope metabolic-labeling technique relies on the in vivo biosynthesis of amino acids from U-[ 12 C]-labeled molecules provided as the sole carbon source. The incorporation of the resulting U-[ 12 C]-amino acids into proteins presents several key advantages for mass-spectrometry-based proteomics analysis, as it results in more intense monoisotopic ions, with a better signal-to-noise ratio in bottom-up analysis. In our initial studies, we developed the simple light isotope metabolic (SLIM)-labeling strategy using prototrophic eukaryotic microorganisms, the yeasts Candida albicans and Saccharomyces cerevisiae , as well as strains with genetic markers that lead to amino-acid auxotrophy. To extend the range of SLIM-labeling applications, we evaluated (i) the incorporation of U-[ 12 C]-glucose into proteins of human cells grown in a complex RPMI-based medium containing the labeled molecule, considering that human cell lines require a large number of essential amino-acids to support their growth, and (ii) an indirect labeling strategy in which the nematode Caenorhabditis elegans grown on plates was fed U-[ 12 C]-labeled bacteria ( Escherichia coli ) and the worm proteome analyzed for 12 C incorporation into proteins. In both cases, we were able to demonstrate efficient incorporation of 12 C into the newly synthesized proteins, opening the way for original approaches in quantitative proteomics. more...

Epithelia act as a barrier against environmental stress and abrasion and in vivo they are continuously exposed to environments of various mechanical properties. The impact of this environment on epithelial integrity remains elusive. By culturing epithelial cells on 2D hydrogels, we observe a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. Through active nematic modelling, we find that spontaneous half-integer defect formation underpinning large isotropic stress fluctuations initiate hole opening events. Our data show that monolayer rupture due to high tensile stress is promoted by the weakening of cell-cell junctions that could be induced by cell division events or local cellular stretching. Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure, with potential application towards a mechanistic understanding of compromised epithelial integrity during immune response and morphogenesis. more...

Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.) more...

Biological tissues are composed of various cell types working cooperatively to perform their respective function within organs and the whole body. During development, embryogenesis followed by histogenesis relies on orchestrated division, death, differentiation and collective movements of cellular constituents. These cells are anchored to each other and/or the underlying substrate through adhesion complexes and they regulate force generation by active cytoskeleton remodelling. The resulting contractility related changes at the level of each single cell impact tissue architecture by triggering changes in cell shape, cell movement and remodelling of the surrounding environment. These out of equilibrium processes occur through the consumption of energy, allowing biological systems to be described by active matter physics. 'Active nematics' a subclass of active matter encompasses cytoskeleton filaments, bacterial and eukaryotic cells allowing them to be modelled as rod-like elements to which nematic liquid crystal theories can be applied. In this review, we will discuss the concept of active nematics to understand biological processes across subcellular and multicellular scales, from single cell organization to cell extrusion, collective cell movements, differentiation and morphogenesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.) more...

The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80-500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.) more...

E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Because epithelial cells respond to rigidity and tension in tissue through E-cadherin, there must be active processes that test and respond to the mechanical properties of these adhesive contacts. Using submicrometer, E-cadherin-coated polydimethylsiloxane pillars, we find that cells generate local contractions between E-cadherin adhesions and pull to a constant distance for a constant duration, irrespective of pillar rigidity. These cadherin contractions require nonmuscle myosin IIB, tropomyosin 2.1, α-catenin, and binding of vinculin to α-catenin. Cells spread to different areas on soft and rigid surfaces with contractions, but spread equally on soft and rigid without. We further observe that cadherin contractions enable cells to test myosin IIA-mediated tension of neighboring cells and sort out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the mechanical characteristics of neighboring cells through cadherin contractions. more...

Biological tissues are composed of various cell types working cooperatively to perform their respective function within organs and the whole body. During development, embryogenesis followed by histogenesis relies on orchestrated division, death, differentiation and collective movements of cellular constituents. These cells are anchored to each other and/or the underlying substrate through adhesion complexes and they regulate force generation by active cytoskeleton remodeling. The resulting changes in contractility at the level of each single cell impact tissue architecture and remodeling by triggering changes in cell shape, cell movement and remodeling of the surrounding environment. These out of equilibrium processes occur through cellular energy consumption, allowing biological systems to be described by active matter physics. Cytoskeleton filaments, bacterial and eukaryotic cells can be considered as a sub-class of active matter termed "active nematics". These biological objects can be modelled as rod-like elements to which nematic liquid crystal theories can be applied. In this work, using an analogy from liquid crystal physics, we show that cell sorting and boundary formation can be explained using differences in nematic activity. This difference in nematic activity arises from a balance of inter- and intra-cellular activity. more...

Background Information: Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level., Results: Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces., Conclusions: Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces., Significance: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing., (© 2021 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.) more...

Collective cell migration is crucial to maintain epithelium integrity during developmental and repair processes. It requires a tight regulation of mechanical coordination between neighboring cells. This coordination embraces different features including mechanical self-propulsion of individual cells within cellular colonies and large-scale force transmission through cell-cell junctions. This review discusses how the plasticity of biomechanical interactions at cell-cell contacts could help cellular systems to perform coordinated motions and adapt to the properties of the external environment., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.) more...

Actomyosin machinery endows cells with contractility at a single-cell level. However, within a monolayer, cells can be contractile or extensile based on the direction of pushing or pulling forces exerted by their neighbours or on the substrate. It has been shown that a monolayer of fibroblasts behaves as a contractile system while epithelial or neural progentior monolayers behave as an extensile system. Through a combination of cell culture experiments and in silico modelling, we reveal the mechanism behind this switch in extensile to contractile as the weakening of intercellular contacts. This switch promotes the build-up of tension at the cell-substrate interface through an increase in actin stress fibres and traction forces. This is accompanied by mechanotransductive changes in vinculin and YAP activation. We further show that contractile and extensile differences in cell activity sort cells in mixtures, uncovering a generic mechanism for pattern formation during cell competition, and morphogenesis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.) more...

Living cells actively migrate in their environment to perform key biological functions-from unicellular organisms looking for food to single cells such as fibroblasts, leukocytes or cancer cells that can shape, patrol or invade tissues. Cell migration results from complex intracellular processes that enable cell self-propulsion, and has been shown to also integrate various chemical or physical extracellular signals. While it is established that cells can modify their environment by depositing biochemical signals or mechanically remodelling the extracellular matrix, the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here, we show that cells can retrieve their path: by confining motile cells on 1D and 2D micropatterned surfaces, we demonstrate that they leave long-lived physicochemical footprints along their way, which determine their future path. On this basis, we argue that cell trajectories belong to the general class of self-interacting random walks, and show that self-interactions can rule large scale exploration by inducing long-lived ageing, subdiffusion and anomalous first-passage statistics. Altogether, our joint experimental and theoretical approach points to a generic coupling between motile cells and their environment, which endows cells with a spatial memory of their path and can dramatically change their space exploration. more...

Apoptotic extrusion is crucial in maintaining epithelial homeostasis. Current literature supports that epithelia respond to extrusion by forming a supracellular actomyosin purse-string in the neighbors. However, whether other actin structures could contribute to extrusion and how forces generated by these structures can be integrated are unknown. Here, we found that during extrusion, a heterogeneous actin network composed of lamellipodia protrusions and discontinuous actomyosin cables, was reorganized in the neighboring cells. The early presence of basal lamellipodia protrusion participated in both basal sealing of the extrusion site and orienting the actomyosin purse-string. The co-existence of these two mechanisms is determined by the interplay between the cell-cell and cell-substrate adhesions. A theoretical model integrates these cellular mechanosensitive components to explain why a dual-mode mechanism, which combines lamellipodia protrusion and purse-string contractility, leads to more efficient extrusion than a single-mode mechanism. In this work, we provide mechanistic insight into extrusion, an essential epithelial homeostasis process. more...

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single cell behavior through a sustained memory of cell polarity., Competing Interests: Competing interests: Authors declare no competing interests. more...

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity., Competing Interests: The authors declare no competing interest. more...

The physical cues from the extracellular environment mediates cell signaling spatially and temporally. Cells respond to physical cues from their environment in a non-monotonic fashion. Despite our understanding of the role of substrate rigidity on single cell migration, how cells respond collectively to increasing extracellular matrix stiffness is not well established. Here we patterned multicellular epithelial Madin-Darby canine kidney (MDCK) islands on polyacrylamide gels of varying stiffness and studied their expansion. Our findings show that the MDCK islands expanded faster with increasing stiffness only up to an optimum stiffness, over which the expansion plateaued. We then focused on the expansion of the front of the assemblies and the formation of leader cells. We observed cell front destabilization only above substrate stiffness of a few kPa. The extension of multicellular finger-like structures at the edges of the colonies for intermediate and high stiffnesses from 6 to 60 kPa responded to higher substrate stiffness by increasing focal adhesion areas and actin cable assembly. Additionally, the number of leader cells at the finger-like protrusions increased with stiffness in correlation with an increase of the area of these multicellular protrusions. Consequently, the force profile along the epithelial fingers in the parallel and transverse directions of migration showed an unexpected relationship leading to a global force decrease with the increase of stiffness. Taken together, our findings show that epithelial cell colonies respond to substrate stiffness but in a non-trivial manner that may be of importance to understand morphogenesis and collective cell invasion during tumour progression. more...

Adherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodeling of AJ. Here, we investigated the role of non-muscle myosin II isoforms (NMIIA and NMIIB) in epithelial junction assembly. NMIIA and NMIIB differentially regulate biogenesis of AJ through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ., Competing Interests: MH, GS, JD, VC, TD, DW, JV, PM, RM, BL No competing interests declared, (© 2019, Heuzé et al.) more...

Morphological changes during development, tissue repair, and disease largely rely on coordinated cell movements and are controlled by the tissue environment. Epithelial cell sheets are often subjected to large-scale deformation during tissue formation. The active mechanical environment in which epithelial cells operate have the ability to promote collective oscillations, but how these cellular movements are generated and relate to collective migration remains unclear. Here, combining in vitro experiments and computational modeling, we describe a form of collective oscillations in confined epithelial tissues in which the oscillatory motion is the dominant contribution to the cellular movements. We show that epithelial cells exhibit large-scale coherent oscillations when constrained within micropatterns of varying shapes and sizes and that their period and amplitude are set by the smallest confinement dimension. Using molecular perturbations, we then demonstrate that force transmission at cell-cell junctions and its coupling to cell polarity are pivotal for the generation of these collective movements. We find that the resulting tissue deformations are sufficient to trigger osillatory mechanotransduction of YAP within cells, potentially affecting a wide range of cellular processes., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.) more...

N-cadherin adhesion has been reported to enhance cancer and neuronal cell migration either by mediating actomyosin-based force transduction or initiating fibroblast growth factor receptor (FGFR)-dependent biochemical signalling. Here we show that FGFR1 reduces N-cadherin-mediated cell migration. Both proteins are co-stabilised at cell-cell contacts through direct interaction. As a consequence, cell adhesion is strengthened, limiting the migration of cells on N-cadherin. Both the inhibition of migration and the stabilisation of cell adhesions require the FGFR activity stimulated by N-cadherin engagement. FGFR1 stabilises N-cadherin at the cell membrane through a pathway involving Src and p120. Moreover, FGFR1 stimulates the anchoring of N-cadherin to actin. We found that the migratory behaviour of cells depends on an optimum balance between FGFR-regulated N-cadherin adhesion and actin dynamics. Based on these findings we propose a positive feed-back loop between N-cadherin and FGFR at adhesion sites limiting N-cadherin-based single-cell migration. more...

Biological systems integrate dynamics at many scales, from molecules, protein complexes and genes, to cells, tissues and organisms. At every step of the way, mechanics, biochemistry and genetics offer complementary approaches to understand these dynamics. At the tissue scale, in vitro monolayers of epithelial cells provide a model to capture the influence of various factors on the motions of the tissue, in order to understand in vivo processes from morphogenesis, cancer progression and tissue remodelling. Ongoing efforts include research aimed at deciphering the roles of the cytoskeleton, of cell-substrate and cell-cell adhesions, and of cell proliferation-the point we investigate here. We show that confined to adherent strips, and on the time scale of a day or two, monolayers move with a characteristic front speed independent of proliferation, but that the motion is accompanied by persistent velocity waves, only in the absence of cell divisions. Here we show that the long-range transmission of physical signals is strongly coupled to cell density and proliferation. We interpret our results from a kinematic and mechanical perspective. Our study provides a framework to understand density-driven mechanisms of collective cell migration. more...

A general trait of living cells is their ability to exert contractile stresses on their surroundings and thus respond to substrate rigidity. At the cellular scale, this response affects cell shape, polarity, and ultimately migration. The regulation of cell shape together with rigidity sensing remains largely unknown. In this article we show that both substrate rigidity and cell shape contribute to drive actin organization and cell polarity. Increasing substrate rigidity affects bulk properties of the actin cytoskeleton by favoring long-lived actin stress fibers with increased nematic interactions, whereas cell shape imposes a local alignment of actin fibers at the cell periphery. more...

In various physiological processes, the cell collective is organized in a monolayer, such as seen in a simple epithelium. The advances in the understanding of mechanical behavior of the monolayer and its underlying cellular and molecular mechanisms will help to elucidate the properties of cell collectives. In this Review, we discuss recent in vitro studies on monolayer mechanics and their implications on collective dynamics, regulation of monolayer mechanics by physical confinement and geometrical cues and the effect of tissue mechanics on biological processes, such as cell division and extrusion. In particular, we focus on the active nematic property of cell monolayers and the emerging approach to view biological systems in the light of liquid crystal theory. We also highlight the mechanosensing and mechanotransduction mechanisms at the sub-cellular and molecular level that are mediated by the contractile actomyosin cytoskeleton and cell-cell adhesion proteins, such as E-cadherin and α-catenin. To conclude, we argue that, in order to have a holistic understanding of the cellular response to biophysical environments, interdisciplinary approaches and multiple techniques - from large-scale traction force measurements to molecular force protein sensors - must be employed., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.) more...

Mechanical constraints are recognized as a key regulator of biological processes, from molecules to organisms, throughout embryonic development, tissue regeneration and in situations of physiological regulation and pathological disturbances. The study of the influence of these physical constraints on the living, in particular on the cells and the organisms of the animal kingdom, has been the object for a decade of important work carried out at the interface between biology, physics and mechanics, constituting a new discipline: mechanobiology. Here we briefly describe the remarkable advances in understanding how cells and tissues both generate and perceive mechanical stresses, and how these constrains dictate cell shape, migration, cell differentiation and finally adaptation of tissues to their environment during morphogenesis, injury and repair., (© 2018 médecine/sciences – Inserm.) more...

The shaping of a multicellular body and repair of adult tissues require fine--tuning of cell adhesion, cell mechanics, and intercellular transmission of mechanical load. Adherens junctions (AJs) are the major intercellular junctions by which cells sense and exert mechanical force on each other. However, how AJs adapt to mechanical stress and how this adaptation contributes to cell-cell cohesion and eventually to tissue-scale dynamics and mechanics remains largely unknown. Here, by analyzing the tension-dependent recruitment of vinculin, α-catenin, and F-actin as a function of stiffness, as well as the dynamics of GFP-tagged wild-type and mutated α-catenins, altered for their binding capability to vinculin, we demonstrate that the force-dependent binding of vinculin stabilizes α-catenin and is responsible for AJ adaptation to force. Challenging cadherin complexes mechanical coupling with magnetic tweezers, and cell-cell cohesion during collective cell movements, further highlight that tension-dependent adaptation of AJs regulates cell-cell contact dynamics and coordinated collective cell migration. Altogether, these data demonstrate that the force-dependent α-catenin/vinculin interaction, manipulated here by mutagenesis and mechanical control, is a core regulator of AJ mechanics and long-range cell-cell interactions., (© 2018 Seddiki et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).) more...

The way in which cells coordinate their behaviours during various biological processes, including morphogenesis, cancer progression and tissue remodelling, largely depends on the mechanical properties of the external environment. In contrast to single cells, collective cell behaviours rely on the cellular interactions not only with the surrounding extracellular matrix but also with neighbouring cells. Collective dynamics is not simply the result of many individually moving blocks. Instead, cells coordinate their movements by actively interacting with each other. These mechanisms are governed by mechanosensitive adhesion complexes at the cell-substrate interface and cell-cell junctions, which respond to but also further transmit physical signals. The mechanosensitivity and mechanotransduction at adhesion complexes are important for regulating tissue cohesiveness and thus are important for collective cell behaviours. Recent studies have shown that the physical properties of the cellular environment, which include matrix stiffness, topography, geometry and the application of external forces, can alter collective cell behaviours, tissue organization and cell-generated forces. On the basis of these findings, we can now start building our understanding of the mechanobiology of collective cell movements that span over multiple length scales from the molecular to the tissue level. more...

We present a phenomenological model intended to describe at the protein population level the formation of cell-cell junctions by the local recruitment of homophilic cadherin adhesion receptors. This modeling may have a much wider implication in biological processes since many adhesion receptors, channel proteins and other membrane-born proteins associate in clusters or oligomers at the cell surface. Mathematically, it consists in a degenerate reaction-diffusion system of two partial differential equations modeling the time-space evolution of two cadherin populations over a surface: the first one represents the diffusing cadherins and the second one concerns the fixed ones. After discussing the stability of the solutions of the model, we perform numerical simulations and show relevant analogies with experimental results. In particular, we show patterns or aggregates formation for a certain set of parameters. Moreover, perturbing the stationary solution, both density populations converge in large times to some saturation level. Finally, an exponential rate of convergence is numerically obtained and is shown to be in agreement, for a suitable set of parameters, with the one obtained in some in vitro experiments. more...

The cadherin-catenin adhesion complex is the key component of the intercellular adherens junction (AJ) that contributes both to tissue stability and dynamic cell movements in epithelial and nonepithelial tissues. The cadherin adhesion complex bridges neighboring cells and the actin-myosin cytoskeleton, and thereby contributes to mechanical coupling between cells which drives many morphogenetic events and tissue repair. Mechanotransduction at cadherin adhesions enables cells to sense, signal, and respond to physical changes in their environment. Central to this process is the dynamic link of the complex to actin filaments (F-actin), themselves structurally dynamic and subject to tension generated by myosin II motors. We discuss in this review recent breakthroughs in understanding molecular and cellular aspects of the organization of the core cadherin-catenin complex in adherens junctions, its association to F-actin, its mechanosensitive regulation, and dynamics., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.) more...

The control of tissue growth, which is a key to maintain the protective barrier function of the epithelium, depends on the balance between cell division and cell extrusion rates [1, 2]. Cells within confluent epithelial layers undergo cell extrusion, which relies on cell-cell interactions [3] and actomyosin contractility [4, 5]. Although it has been reported that cell extrusion is also dependent on cell density [6, 7], the contribution of tissue mechanics, which is tightly regulated by cell density [8-12], to cell extrusion is still poorly understood. By measuring the multicellular dynamics and traction forces, we show that changes in epithelial packing density lead to the emergence of distinct modes of cell extrusion. In confluent epithelia with low cell density, cell extrusion is mainly driven by the lamellipodia-based crawling mechanism in the neighbor non-dying cells in connection with large-scale collective movements. As cell density increases, cell motion is shown to slow down, and the role of a supracellular actomyosin cable formation and its contraction in the neighboring cells becomes the preponderant mechanism to locally promote cell extrusion. We propose that these two distinct mechanisms complement each other to ensure proper cell extrusion depending on the cellular environment. Our study provides a quantitative and robust framework to explain how cell density can influence tissue mechanics and in turn regulate cell extrusion mechanisms., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...

Cell migrations are diverse. They constitutemajor morphogenetic driving forces during embryogenesis, but they contribute also to the loss of tissue homeostasis and cancer growth. Capabilities of cells to migrate as single cells or as collectives are controlled by internal and external signalling, leading to the reorganisation of their cytoskeleton as well as by the rebalancing of cell-matrix and cell-cell adhesions. Among the genes altered in numerous cancers, cadherins and growth factor receptors are of particular interest for cell migration regulation. In particular, cadherins such as N-cadherin and a class of growth factor receptors, namely FGFRs cooperate to regulate embryonic and cancer cell behaviours. In this review, we discuss on reciprocal crosstalk between N-cadherin and FGFRs during cell migration. Finally, we aim at clarifying the synergy between N-cadherin and FGFR signalling that ensure cellular reorganization during cell movements, mainly during cancer cell migration and metastasis but also during developmental processes., (Copyright © 2016 Elsevier GmbH. All rights reserved.) more...

The closure of gaps is crucial to maintaining epithelium integrity during developmental and repair processes such as dorsal closure and wound healing. Depending on biochemical as well as physical properties of the microenvironment, gap closure occurs through assembly of multicellular actin-based contractile cables and/or protrusive activity of cells lining the gap. This review discusses the relative contributions of 'purse-string' and cell crawling mechanisms regulated by cell-substrate and cell-cell interactions, cellular mechanics and physical constraints from the environment., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...

Directed cell migration is a complex process that involves front-rear polarization, characterized by cell adhesion and cytoskeleton-based protrusion, retraction, and contraction of either a single cell or a cell collective. Single cell polarization depends on a variety of mechanochemical signals including external adhesive cues, substrate stiffness, and confinement. In cell ensembles, coordinated polarization of migrating tissues results not only from the application of traction forces on the extracellular matrix but also from the transmission of mechanical stress through intercellular junctions. We focus here on the impact of mechanical cues on the establishment and maintenance of front-rear polarization from single cell to collective cell behaviors through local or large-scale mechanisms., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...

Morphogenesis requires dynamic coordination between cell-cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell-cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions., (© 2016 Choi et al.) more...

Collective migration of cells is of fundamental importance for a number of biological functions such as tissue development and regeneration, wound healing and cancer metastasis. The movement of cell groups consisting of multiple cells connected by cell-cell junctions depends on both extracellular and intercellular contacts. Epithelial cell assemblies are thus regulated by a cross-talk between cell-substrate and cell-cell interactions. Here, we investigated the onset of collective migration in groups of cells as they expand from a few cells into large colonies as a function of extracellular matrix (ECM) protein coating. By varying the amount of ECM presented to the cells, we observe that the mode of colony expansion, as well as their overall geometry, is strongly dependent on substrate adhesiveness. On high ECM protein coated surfaces, cells at the edges of the colonies are well spread exhibiting large outward-pointing protrusive activity, whereas cellular colonies display more circular and convex shapes on less adhesive surfaces. Actin structures at the edge of the colonies also show different organizations with the formation of lamellipodial structures on highly adhesive surfaces and a pluricellular actin cable on less adhesive ones. The analysis of traction forces and cell velocities within the cellular assemblies confirm these results. By increasing ECM protein density, cells exert higher traction forces together with a higher outward motility at the edges. Furthermore, tuning cell-cell adhesion of epithelial cells modified the mode of expansion of the colonies. Finally, we used a recently developed computational model to recapitulate the emergent experimental behaviors of expanding cell colonies and extract that the main effect of the different cell-substrate interactions is on the ability of edge cells to form outward lamellipodia-driven motility. Overall, our data suggest that switching behaviors of epithelial cell assemblies result in a tug-of-war between friction forces at the cell-substrate interface and cell-cell interactions. more...

The shaping of a multicellular body, and the maintenance and repair of adult tissues require fine-tuning of cell adhesion responses and the transmission of mechanical load between the cell, its neighbors and the underlying extracellular matrix. A growing field of research is focused on how single cells sense mechanical properties of their micro-environment (extracellular matrix, other cells), and on how mechanotransduction pathways affect cell shape, migration, survival as well as differentiation. Within multicellular assemblies, the mechanical load imposed by the physical properties of the environment is transmitted to neighboring cells. Force imbalance at cell-cell contacts induces essential morphogenetic processes such as cell-cell junction remodeling, cell polarization and migration, cell extrusion and cell intercalation. However, how cells respond and adapt to the mechanical properties of neighboring cells, transmit forces, and transform mechanical signals into chemical signals remain open questions. A defining feature of compact tissues is adhesion between cells at the specialized adherens junction (AJ) involving the cadherin super-family of Ca(2+)-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein β-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein α-catenin, which can also recruit vinculin, making the mechanical connection between cell-cell adhesion proteins and the contractile actomyosin cytoskeleton. The cadherin-catenin adhesion complex is a key component of the AJ, and contributes to cell assembly stability and dynamic cell movements. It has also emerged as the main route of propagation of forces within epithelial and non-epithelial tissues. Here, we discuss recent molecular studies that point toward force-dependent conformational changes in α-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that α-catenin is the core mechanosensor that allows cells to locally sense, transduce and adapt to environmental mechanical constrains. more...

Matrix rigidity sensing regulates a large variety of cellular processes and has important implications for tissue development and disease. However, how cells probe matrix rigidity, and hence respond to it, remains unclear. Here, we show that rigidity sensing and adaptation emerge naturally from actin cytoskeleton remodelling. Our in vitro experiments and theoretical modelling demonstrate a biphasic rheology of the actin cytoskeleton, which transitions from fluid on soft substrates to solid on stiffer ones. Furthermore, we find that increasing substrate stiffness correlates with the emergence of an orientational order in actin stress fibres, which exhibit an isotropic to nematic transition that we characterize quantitatively in the framework of active matter theory. These findings imply mechanisms mediated by a large-scale reinforcement of actin structures under stress, which could be the mechanical drivers of substrate stiffness-dependent cell shape changes and cell polarity. more...

Force sensing at cadherin-mediated adhesions is critical for their proper function. α-Catenin, which links cadherins to actomyosin, has a crucial role in this mechanosensing process. It has been hypothesized that force promotes vinculin binding, although this has never been demonstrated. X-ray structure further suggests that α-catenin adopts a stable auto-inhibitory conformation that makes the vinculin-binding site inaccessible. Here, by stretching single α-catenin molecules using magnetic tweezers, we show that the subdomains MI vinculin-binding domain (VBD) to MIII unfold in three characteristic steps: a reversible step at ~5 pN and two non-equilibrium steps at 10-15 pN. 5 pN unfolding forces trigger vinculin binding to the MI domain in a 1:1 ratio with nanomolar affinity, preventing MI domain refolding after force is released. Our findings demonstrate that physiologically relevant forces reversibly unfurl α-catenin, activating vinculin binding, which then stabilizes α-catenin in its open conformation, transforming force into a sustainable biochemical signal. more...

Adhesive interactions of cadherins induce crosstalk between adhesion complexes and the actin cytoskeleton, allowing strengthening of adhesions and cytoskeletal organization. The underlying mechanisms are not completely understood, and microtubules (MTs) might be involved, as for integrin-mediated cell-extracellular-matrix adhesions. Therefore, we investigated the relationship between N-cadherin and MTs by analyzing the influence of N-cadherin engagement on MT distribution and dynamics. MTs progressed less, with a lower elongation rate, towards cadherin adhesions than towards focal adhesions. Increased actin treadmilling and the presence of an actomyosin contractile belt, suggested that actin relays inhibitory signals from cadherin adhesions to MTs. The reduced rate of MT elongation, associated with reduced recruitment of end-binding (EB) proteins to plus ends, was alleviated by expression of truncated N-cadherin, but was only moderately affected when actomyosin was disrupted. By contrast, destabilizing actomyosin fibers allowed MTs to enter the adhesion area, suggesting that tangential actin bundles impede MT growth independently of MT dynamics. Blocking MT penetration into the adhesion area strengthened cadherin adhesions. Taken together, these results establish a crosstalk between N-cadherin, F-actin and MTs. The opposing effects of cadherin and integrin engagement on actin organization and MT distribution might induce bias of the MT network during cell polarization. more...

In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex. more...

Classical cadherins play a crucial role in establishing intercellular adhesion, regulating cortical tension, and maintaining mechanical coupling between cells. The mechanosensitive regulation of intercellular adhesion strengthening depends on the recruitment of adhesion complexes at adhesion sites and their anchoring to the actin cytoskeleton. Thus, the molecular mechanisms coupling cadherin-associated complexes to the actin cytoskeleton are actively being studied, with a particular focus on α-catenin and vinculin. We have recently addressed the role of these proteins by analyzing the consequences of their depletion and the expression of α-catenin mutants in the formation and strengthening of cadherin-mediated adhesions. We have used the dual pipette assay to measure the forces required to separate cell doublets formed in suspension. In this commentary, we briefly summarize the current knowledge on the role of α-catenin and vinculin in cadherin-actin cytoskeletal interactions. These data shed light on the tension-dependent contribution of α-catenin and vinculin in a mechanoresponsive complex that promotes the connection between cadherin and the actin cytoskeleton and their requirement in the development of adhesion strengthening. more...

N-cadherin is a major adhesion molecule involved in the development and plasticity of the nervous system. N-cadherin-mediated cell adhesion regulates neuroepithelial cell polarity, neuronal precursor migration, growth cone migration and synaptic plasticity. In vitro, it has been involved in signaling events regulating processes such as cell mobility, proliferation and differentiation. N-cadherin has also been implicated in adhesion-dependent protection against apoptosis in non-neuronal cells. In this study, we investigated if the engagement of N-cadherin participates to the control of neuronal cells survival/death balance. We observed that plating either primary mouse spinal cord neurons or primary rat hippocampal neurons on N-cadherin recombinant substrate greatly enhances their survival compared to non-specific adhesion on poly-L-lysine. We show that N-cadherin engagement, in the absence of other survival factors (cell-matrix interactions and serum), protects GT1-7 neuronal cells against apoptosis. Using this cell line, we then searched for the signaling pathways involved in the survival effect of N-cadherin engagement. The PI3-kinase/Akt survival pathway and its downstream effector Bad are not involved, as no phosphorylation of Akt or Bad proteins in response to N-cadherin engagement was observed. In contrast, N-cadherin engagement activated the Erk1/2 MAP kinase pathway. Moreover, N-cadherin ligation mediated a 2-fold decrease in the level of the pro-apoptotic protein Bim-EL whereas the level of the anti-apoptotic protein Bcl-2 was unchanged. Inhibition of Mek1/2 kinases with U0126, and the resulting inhibition of Erk1/2 phosphorylation, induced the increase of both the level of Bim-EL and apoptosis of cells seeded on the N-cadherin substrate, suggesting that Erk phosphorylation is necessary for cell survival. Finally, the overexpression of a phosphorylation defective form of Bim-EL prevented N-cadherin-engagement induced cell survival. In conclusion, our results show that N-cadherin engagement mediates neuronal cell survival by enhancing the MAP kinase pathway and down-regulating the pro-apoptotic protein Bim-EL. more...