Deposited footprints let cells switch between confined, oscillatory, and exploratory migration

Emiliano Perez Ipina, Joseph d'Alessandro, Benoit Ladoux, Brian A. Camley

For eukaryotic cells to heal wounds, respond to immune signals, or metastasize, they must migrate, often by adhering to extracellular matrix (ECM). Cells may also deposit ECM components, leaving behind a footprint that influences their crawling. Recent experiments showed that some epithelial cell lines on micropatterned adhesive stripes move persistently in regions they have previously crawled over, where footprints have been formed, but barely advance into unexplored regions, creating an oscillatory migration of increasing amplitude. Here, we explore through mathematical modeling how footprint deposition and cell responses to footprint combine to allow cells to develop oscillation and other complex migratory motions. We simulate cell crawling with a phase field model coupled to a biochemical model of cell polarity, assuming local contact with the deposited footprint activates Rac1, a protein that establishes the cell's front. Depending on footprint deposition rate and response to the footprint, cells on micropatterned lines can display many types of motility, including confined, oscillatory, and persistent motion. On two-dimensional (2D) substrates, we predict a transition between cells undergoing circular motion and cells developing an exploratory phenotype. Small quantitative changes in a cell's interaction with its footprint can completely alter exploration, allowing cells to tightly regulate their motion, leading to different motility phenotypes (confined vs. exploratory) in different cells when deposition or sensing is variable from cell to cell. Consistent with our computational predictions, we find in earlier experimental data evidence of cells undergoing both circular and exploratory motion.

Clustering and ordering in cell assemblies with generic asymmetric aligning interactions

Thibault Bertrand, Joseph d'Alessandro, Ananyo Maitra, Shreyansh Jain, Barbara Mercier, Rene-Marc Mege, Benoît Ladoux, Raphael Voituriez

Collective cell migration plays an essential role in various biological processes, such as development or cancer proliferation. While cell-cell interactions are clearly key determinants of collective cell migration, the physical mechanisms that control the emergence of cell clustering and collective cell migration are still poorly understood. In particular, observations have shown that binary cell-cell collisions generally lead to antialignment of cell polarities and separation of pairs-a process called contact inhibition of locomotion (CIL), which is expected to disfavor the formation of large-scale cell clusters with coherent motion even though the latter is often observed in tissues. To solve this puzzle, we adopt a joint experimental and theoretical approach to determine the large-scale dynamics of cell assemblies from elementary pairwise cell-cell interaction rules. We quantify experimentally binary cell-cell interactions and show that they can be captured by a minimal equilibriumlike pairwise asymmetric aligning interaction potential that reproduces the CIL phenomenology. We identify its symmetry class, build the corresponding active hydrodynamic theory, and show on general grounds that such asymmetric aligning interaction destroys large-scale clustering and ordering, leading instead to a liquidlike microphase of cell clusters of finite size and short lived polarity or to a fully dispersed isotropic phase. Finally, this shows that CIL-like asymmetric interactions in cellular systems-or general active systems-control cluster sizes and polarity, and can prevent large-scale coarsening and long-range polarity, except in the singular regime of dense confluent systems.

A minimal physical model for curvotaxis driven by curved protein complexes at the cell's leading edge

Raj Kumar Sadhu, Marine Lucianob, Wang Xi, Cristina Martinez-Torres, Marcel Schroeder, Christoph Blum, Marco Tarantola, Stefano Villa, Samo Penic, et al.

Cells often migrate on curved surfaces inside the body, such as curved tissues, blood vessels, or highly curved protrusions of other cells. Recent in vitro experiments provide clear evidence that motile cells are affected by the curvature of the substrate on which they migrate, preferring certain curvatures to others, termed "curvotaxis." The origin and underlying mechanism that gives rise to this curvature sensitivity are not well understood. Here, we employ a "minimal cell" model which is composed of a vesicle that contains curved membrane protein complexes, that exert protrusive forces on the membrane (representing the pressure due to actin polymerization). This minimalcell model gives rise to spontaneous emergence of a motile phenotype, driven by a lamellipodia-like leading edge. By systematically screening the behavior of this model on different types of curved substrates (sinusoidal, cylinder, and tube), we show that minimal ingredients and energy terms capture the experimental data. The model recovers the observed migration on the sinusoidal substrate, where cells move along the grooves (minima), while avoiding motion along the ridges. In addition, the model predicts the tendency of cells to migrate circumferentially on convex substrates and axially on concave ones. Both of these predictions are verified experimentally, on several cell types. Altogether, our results identify the minimization of membrane -substrate adhesion energy and binding energy between the membrane protein complexes as key players of curvotaxis in cell migration.

A mechanosensitive caveolae-invadosome interplay drives matrix remodelling for cancer cell invasion

Pedro Monteiro, David Remy, Eline Lemerle, Fiona Routet, Anne-Sophie Mace, Chloe Guedj, Benoit Ladoux, Stephane Vassilopoulos, Christophe Lamaze, et al.

Invadosomes and caveolae are mechanosensitive structures that are implicated in metastasis. Here, we describe a unique juxtaposition of caveola clusters and matrix degradative invadosomes at contact sites between the plasma membrane of cancer cells and constricting fibrils both in 2D and 3D type I collagen matrix environments. Preferential association between caveolae and straight segments of the fibrils, and between invadosomes and bent segments of the fibrils, was observed along with matrix remodelling. Caveola recruitment precedes and is required for invadosome formation and activity. Reciprocally, invadosome disruption results in the accumulation of fibril-associated caveolae. Moreover, caveolae and the collagen receptor beta 1 integrin co-localize at contact sites with the fibrils, and integrins control caveola recruitment to fibrils. In turn, caveolae mediate the clearance of beta 1 integrin and collagen uptake in an invadosome-dependent and collagen-cleavage-dependent mechanism. Our data reveal a reciprocal interplay between caveolae and invadosomes that coordinates adhesion to and proteolytic remodelling of confining fibrils to support tumour cell dissemination.<br> Monteiro et al. show an alternating distribution of caveolae and invadosomes along collagen fibrils, whereby caveolae enhance integrin-mediated collagen uptake in an invadosome-activity-dependent manner to coordinate adhesion and extracellular matrix remodelling.

Inhomogeneous mechanotransduction defines the spatial pattern of apoptosis-induced compensatory proliferation

Takumi Kawaue, Ivan Yow, Yuping Pan, Anh Phuong Le, Yuting Lou, Mavis Loberas, Murat Shagirov, Xiang Teng, Jacques Prost, et al.

The number of cells in tissues is controlled by cell division and cell death, and its misregulation could lead to pathological conditions such as cancer. To maintain the cell numbers, a cell-elimination process called apoptosis also stimulates the proliferation of neighboring cells. This mechanism, apoptosis-induced compensatory proliferation, was originally described more than 40 years ago. Although only a limited number of the neighboring cells need to divide to compensate for the apoptotic cell loss, the mechanisms that select cells to divide have remained elusive. Here, we found that spatial inhomogeneity in Yes-associated protein (YAP)-mediated mechanotransduction in neighboring tissues determines the inhomogeneity of compensa-tory proliferation in Madin-Darby canine kidney (MDCK) cells. Such inhomogeneity arises from the non -uni-form distribution of nuclear size and the non-uniform pattern of mechanical force applied to neighboring cells. Our findings from a mechanical perspective provide additional insight into how tissues precisely maintain homeostasis.

Polarization and motility of one-dimensional multi-cellular trains

Jonathan E. Ron, Joseph d'Alessandro, Victor Cellerin, Raphael Voituriez, Benoit Ladoux, Nir S. Gov

Collective cell migration, whereby cells adhere to form multi-cellular clusters that move as a single entity, play an important role in numerous biological processes, such as during development and cancer progression. Recent experimental work focused on migration of one-dimensional cellular clusters, confined to move along adhesive lanes, as a simple geometry in which to systematically study this complex system. One-dimensional migration also arises in the body when cells migrate along blood vessels, axonal projections, and narrow cavities between tissues. We explore here the modes of one-dimensional migration of cellular clusters ("trains") by implementing cell-cell interactions in a model of cell migration that contains a mech-anism for spontaneous cell polarization. We go beyond simple phenomenological models of the cells as self-propelled particles by having the internal polarization of each cell depend on its interactions with the neighboring cells that directly affect the actin polymerization activity at the cell's leading edges. Both contact inhibition of locomotion and cryptic lamellipodia interactions be-tween neighboring cells are introduced. We find that this model predicts multiple motility modes of the cell trains, which can have several different speeds for the same polarization pattern. Compared to experimental data, we find that Madin-Darby canine kidney cells are poised along the transition region where contact inhibition of locomotion and cryptic lamellipodia roughly balance each other, where collective migration speed is most sensitive to the values of the cell-cell interaction strength.

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies

Nicolas Rose, Berenice Estrada Chavez, Surabhi Sonam, Thao Nguyen, Gianluca Grenci, Anne Bigot, Antoine Muchir, Benoit Ladoux, Bruno Cadot, et al.

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic pro-cedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a sub-stantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with suc-cessful development of diseased 3D myotubes displaying reduced contraction. The miniaturized myotube tech-nology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should substantially improve drug screening capability.

Cells, the "Little Thumbs" of living tissue

Joseph d'Alessandro, Alex Barbier-Chebbah, Raphael Voituriez, Benoit Ladoux

Mechanical stress driven by rigidity sensing governs epithelial stability

Surabhi Sonam, Lakshmi Balasubramaniam, Shao-Zhen Lin, Ying Ming Yow Ivan, Irina Pi-Jauma, Cecile Jebane, Marc Karnat, Yusuke Toyama, Philippe Marcq, et al.

Epithelia act as barriers against environmental stresses. They are continuously exposed to various mechanical stress and abrasion, which impact epithelial integrity. The impact of the environment on epithelial integrity remains elusive. By culturing epithelial cells on two-dimensional hydrogels, we observe a loss of epithelial monolayer integrity on soft substrates through spontaneous hole formation. These monolayer ruptures are associated with local cellular stretching and cell-division events. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from compressive stress on stiff substrates to highly tensile on soft, favouring hole formation. In agreement with an active nematic model, hole-opening events occur preferentially near spontaneous half-integer topological defects, which underpin large isotropic stress fluctuations triggering stochastic mechanical failure. Our results thus show that substrate stiffness provides feedback on the mechanical state of epithelial monolayers with potential application towards a mechanistic understanding of compromised epithelial integrity during normal and pathological human ontogenesis.<br> On soft substrates, epithelial tissues are under high tension and form holes that spontaneously heal. Thus, mechanical stress directly impacts the integrity of epithelia.

Tissue mechanics

Alexandre Kabla, Benoit Ladoux, Jean-Marc Di Meglio

Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Serena Petracchini, Daniel Hamaoui, Anne Doye, Atef Asnacios, Florian Fage, Elisa Vitiello, Martial Balland, Sebastien Janel, Frank Lafont, et al.

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology.<br> Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.

The emergence of spontaneous coordinated epithelial rotation on cylindrical curved surfaces

Alexandros Glentis, Carles Blanch-Mercader, Lakshmi Balasubramaniam, Thuan Beng Saw, Joseph d'Alessandro, Sebastien Janel, Audrey Douanier, Benedicte Delaval, Frank Lafont, et al.

Three-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morpho-genetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1-dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order. Commons NonCommercial License

Local contractions regulate E-cadherin rigidity sensing

Yi-An Yang, Emmanuelle Nguyen, Gautham Hari Narayana Sankara Narayana, Melina Heuze, Chaoyu Fu, Hanry Yu, Rene-Marc Mege, Benoit Ladoux, Michael P. Sheetz

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-cadherincoated 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, alpha-catenin, and binding of vinculin to.-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.

Active nematics across scales from cytoskeleton organization to tissue morphogenesis

Lakshmi Balasubramaniam, Rene-Marc Mege, Benoît Ladoux

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.

Active forces modulate collective behaviour and cellular organization

Lakshmi Balasubramaniam, Rene-Marc Mege, Benoit Ladoux

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.

Physics of liquid crystals in cell biology

Amin Doostmohammadi, Benoit Ladoux

The past decade has witnessed a rapid growth in understanding of the pivotal roles of mechanical stresses and physical forces in cell biology. As a result, an integrated view of cell biology is evolving, where genetic and molecular features are scrutinised hand in hand with physical and mechanical characteristics of cells. Physics of liquid crystals has emerged as a burgeoning new frontier in cell biology over the past few years, fuelled by an increasing identification of orientational order and topological defects in cell biology, spanning scales from subcellular filaments to individual cells and multicellular tissues. Here, we provide an account of the most recent findings and developments, together with future promises and challenges in this rapidly evolving interdisciplinary research direction.

Direct measurement of near-nano-Newton forces developed by self-organizing actomyosin fibers bound α-catenin

Surabhi Sonam, Clemence Vigouroux, Antoine Jegou, Guillaume Romet-Lemonne, Christophe Le Clainche, Benoit Ladoux, Rene Marc Mege

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 alpha-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 alpha-catenin molecules allowed to identify alpha-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-alpha-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified alpha-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 alpha-catenin patches can apply near-nano-Newton forces. Significance: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing.

Mechanical plasticity in collective cell migration

Shreyansh Jain, Benoit Ladoux, Rene-Marc Mege

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.

EpCAM promotes endosomal modulation of the cortical RhoA zone for epithelial organization

Cecile Gaston, Simon De Beco, Bryant Doss, Meng Pan, Estelle Gauquelin, Joseph D'Alessandro, Chwee Teck Lim, Benoit Ladoux, Delphine Delacour

At the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity. The organization and force-generating property of actomyosin dictate the plasticity and behaviour of cells but the spatio-temporal regulation of this network is unclear. Here, the authors show that coupling between EpCAM/RhoA co-trafficking and actomyosin rearrangement is pivotal during cell spreading and polarization.

Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

Lakshmi Balasubramaniam, Amin Doostmohammadi, Thuan Beng Saw, Gautham Hari Narayana Sankara Narayana, Romain Mueller, Tien Dang, Minnah Thomas, Shafali Gupta, Surabhi Sonam, et al.

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.<br> It is now revealed, using cell cultures and in silico models, that weakening intercellular contacts is a fundamental process essential for switching from extensile to contractile tissue behaviour.

Adhesion-mediated heterogeneous actin organization governs apoptotic cell extrusion

Anh Phuong Le, Jean-Francois Rupprecht, Rene-Marc Mege, Yusuke Toyama, Chwee Teck Lim, Benoit Ladoux

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. Cell extrusion regulates monolayer cell density and is critical in maintaining epithelia integrity, which has implications in homeostasis, development, and cancer progression. Here the authors describe how monolayer integrate mechanical signals from tissue mechanics, cell-cell adhesion, cell-substrate adhesion and cytoskeleton coordinate cell extrusion.

Living proof of effective defects

M. -A. Fardin, B. Ladoux

A class of biological matter including elongated cells and filaments can be understood in the framework of active nematic liquid crystals. Within these systems, topological defects emerge and give rise to remarkable collective behaviours.

Migrating Epithelial Monolayer Flows Like a Maxwell Viscoelastic Liquid

S. Tlili, M. Durande, C. Gay, B. Ladoux, F. Graner, H. Delanoe-Ayari

We perform a bidimensional Stokes experiment in an active cellular material: an autonomously migrating monolayer of Madin-Darby canine kidney epithelial cells flows around a circular obstacle within a long and narrow channel, involving an interplay between cell shape changes and neighbor rearrangements. Based on image analysis of tissue flow and coarse-grained cell anisotropy, we determine the tissue strain rate, cell deformation, and rearrangement rate fields, which are spatially heterogeneous. We find that the cell deformation and rearrangement rate fields correlate strongly, which is compatible with a Maxwell viscoelastic liquid behavior (and not with a Kelvin-Voigt viscoelastic solid behavior). The value of the associated relaxation time is measured as tau = 70 +/- 15 min, is observed to be independent of obstacle size and division rate, and is increased by inhibiting myosin activity. In this experiment, the monolayer behaves as a flowing material with a Weissenberg number close to one which shows that both elastic and viscous effects can have comparable contributions in the process of collective cell migration.

The role of single-cell mechanical behaviour and polarity in driving collective cell migration

Shreyansh Jain, Victoire M. L. Cachoux, Gautham H. N. S. Narayana, Simon de Beco, Joseph D'Alessandro, Victor Cellerin, Tianchi Chen, Melina L. Heuze, Philippe Marcq, et al.

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 such as 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 genital or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after establishing 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-rear 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 behaviour through a sustained memory of cell polarity. Collective cell migration is usually attributed to large-scale transmission of signals through cell junctions. Here, the authors confine cells into a ring-shaped pattern and show that collective cell migration can arise at the single-cell level.

Channeling Effect and Tissue Morphology in a Perfusion Bioreactor Imaged by X-Ray Microtomography

Claire C. Beauchesne, Morgan Chabanon, Benjamin Smaniotto, Benoit Ladoux, Benoit Goyeau, Bertrand David

Background: Perfusion bioreactors for tissue engineering hold great promises. Indeed, the perfusion of culture medium enhances species transport and mechanically stimulates the cells, thereby increasing cell proliferation and tissue formation. Nonetheless, their development is still hampered by a lack of understanding of the relationship between mechanical cues and tissue growth. Methods: Combining tissue engineering, three-dimensional visualization and numerical simulations, we analyze the morphological evolution of neo-tissue in a model bioreactor with respect to the local flow pattern. NIH-3T3 cells were grown under perfusion for one, two and three weeks on a stack of 2 mm polyacetal beads. The model bioreactor was then imaged by X-ray micro-tomography and local tissue morphology was analyzed. To relate experimental observations and mechanical stimulii, a computational fluid dynamics model of flow around spheres in a canal was developed and solved using the finite element method. Results: We observe a preferential tissue formation at the bioreactor periphery, and relate it to a channeling effect leading to regions of higher flow intensity. Additionally, we find that circular crater-like tissue patterns form in narrow channel regions at early culture times. Using computational fluid dynamic simulations, we show that the location and morphology of these patterns match those of shear stress maxima. Finally, the morphology of the tissue is qualitatively described as the tissue grows and reorganizes itself. Conclusion: Altogether, our study points out the key role of local flow conditions on the tissue morphology developed on a stack of beads in perfusion bioreactors and provides new insights for effective design of hydrodynamic bioreactors for tissue engineering using bead packings.

Desmosomal Junctions Govern Tissue Integrity and Actomyosin Contractility in Apoptotic Cell Extrusion

Minnah Thomas, Benoit Ladoux, Yusuke Toyama

During apoptosis, or programmed cell death, a dead cell could be expelled from the tissue by coordinated processes between the dying cell and its neighbors. Apoptotic cell extrusion is driven by actomyosin cable formation and its contraction and lamellipodial crawling of the neighboring cells [1-4]. Throughout cell extrusion, the mechanical coupling of epithelia needs to be maintained in order to preserve tissue homeostasis [1]. Although much is known about the regulation of adherens junctions (AJs) in apoptotic cell extrusion [4-7], the role and dynamics of desmosomal junctions (DJs) during this process remain poorly understood. Here, we show that DJs stay intact throughout and are crucial for cell extrusion. Pre-existing DJs between the apoptotic cell and neighboring cells remain intact, even during the formation of de novo DJs between non-dying cells, suggesting the neighboring cells possess two DJs in the middle of apoptotic cell extrusion. We further found that an actomyosin cable formed in the vicinity of DJs upon apoptosis and subsequently deviated from DJs during its constriction. Interestingly, the departure of the actomyosin cable from DJs coincided with the timing when DJs lost their straightness, suggesting a release of junctional tension at DJs and a mechanical coupling between DJs and actomyosin contractility. The depletion of desmoplakin resulted in defective contractility and an inability to form de novo DJs, leading to a failure of apoptotic cell extrusion. Our study provides a framework to explain how desmosomes play pivotal roles in maintaining epithelial sheet integrity during apoptotic cell extrusion.

A subtle relationship between substrate stiffness and collective migration of cell clusters

Hayri E. Balcioglu, Lakshmi Balasubramaniam, Tomita Vasilica Stirbat, Bryant L. Doss, Marc-Antoine Fardin, Rene-Marc Mege, Benoit Ladoux

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.

Myosin II isoforms play distinct roles in adherens junction biogenesis

Melina L. Heuze, Gautham Hari Narayana Sankara Narayana, Joseph D 'Alessandro, Victor Cellerin, Tien Dang, David S. Williams, Jan C. M. Van Hest, Philippe Marcq, Rene-Marc Mege, et al.

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.

Sustained Oscillations of Epithelial Cell Sheets

Gregoire Peyret, Romain Mueller, Joseph d'Alessandro, Simon Begnaud, Philippe Marcq, Rene-Marc Mege, Julia M. Yeomans, Amin Doostmohammadi, Benoit Ladoux

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.

Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins

Shiqiong Hu, Hanna Grobe, Zhenhuan Guo, Yu-Hsiu Wang, Bryant L. Doss, Meng Pan, Benoit Ladoux, Alexander D. Bershadsky, Ronen Zaidel-Bar

Contractile arrays of actin and myosin II filaments drive many essential processes in nonmuscle cells, including migration and adhesion. Sequential organization of actin and myosin along one dimension is followed by expansion into a two-dimensional network of parallel actomyosin fibers, in which myosin filaments are aligned to form stacks. The process of stack formation has been studied in detail. However, factors that oppose myosin stack formation have not yet been described. Here, we show that tropomyosins act as negative regulators of myosin stack formation. Knockdown of any or all tropomyosin isoforms in rat embryonic fibroblasts resulted in longer and more numerous myosin stacks and a highly ordered actomyosin organization. The molecular basis for this, we found, is the competition between tropomyosin and alpha-actinin for binding actin. Surprisingly, excessive order in the actomyosin network resulted in smaller focal adhesions, lower tension within the network, and smaller traction forces. Conversely, disordered actomyosin bundles induced by alpha-actinin knockdown led to higher than normal tension and traction forces. Thus, tropomyosin acts as a check on alpha-actinin to achieve intermediate levels of myosin stacks matching the force requirements of the cell.

Enhanced cell-cell contact stability and decreased N-cadherin-mediated migration upon fibroblast growth factor receptor-N-cadherin cross talk

Nguyen Thao, Laurence Duchesne, Gautham Hari Narayana Sankara Narayana, Nicole Boggetto, David D. Fernig, Chandrashekhar Uttamrao Murade, Benoit Ladoux, Rene-Marc Mege

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.

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity

Minerva Bosch-Fortea, Alejo E. Rodriguez-Fraticelli, Gonzalo Herranz, Mariam Hachimi, Maria D. Barea, Joanne Yong, Benoit Ladoux, Fernando Martin-Belmonte

Tubulogenesis in epithelial organs often initiates with the acquisition of apicobasal polarity, giving rise to the formation of small lumens that expand and fuse to generate a single opened cavity. In this study, we present a micropattern-based device engineered to generate epithelial tubes through a process that recapitulates in vivo tubule morphogenesis. Interestingly, tubulogenesis in this device is dependent on microenvironmental cues such as cell confinement, extracellular matrix composition, and substrate stiffness, and our set-up specifically allows the control of these extracellular conditions. Additionally, proximal tubule cell lines growing on micropattems express higher levels of drug transporters and are more sensitive to nephrotoxicity. These tubes display specific morphological defects that can be linked to nephrotoxicity, which would be helpful to predict potential toxicity when developing new compounds. This device, with the ability to recapitulate tube formation in vitro, has emerged as a powerful tool to study the molecular mechanisms involved in organogenesis and, by being more physiologically relevant than existing cellular models, becomes an innovative platform to conduct drug discovery assays.

FBXW5 Promotes Tumorigenesis and Metastasis in Gastric Cancer via Activation of the FAK-Src Signaling Pathway

Mei Shi Yeo, Vinod Vijay Subhash, Kazuto Suda, Hayri Emrah Balcioglu, Siqin Zhou, Win Lwin Thuya, Xin Yi Loh, Sriganesh Jammula, Praveen C. Peethala, et al.

F-box/WD repeat-containing protein 5 (FBXW5) is a member of the FBXW subclass of F-box proteins. Despite its known function as a component of the Skpl-Cullin-F-box (SCF) ubiquitin ligase complex, the role of FBXW5 in gastric cancer tumorigenesis and metastasis has not been investigated. The present study investigates the role of FBXW5 in tumorigenesis and metastasis, as well as the regulation of key signaling pathways in gastric cancer; using in-vitro FBXW5 knockdown/overexpression cell line and in-vivo models. In-vitro knockdown of FBXW5 results in a decrease in cell proliferation and cell cycle progression, with a concomitant increase in cell apoptosis and caspase-3 activity. Furthermore, knockdown of FBXW5 also leads to a down regulation in cell migration and adhesion, characterized by a reduction in actin polymerization, focal adhesion turnover and traction forces. This study also delineates the mechanistic role of FBXW5 in oncogenic signaling as its inhibition down regulates RhoA-ROCK 1 (Rho-associated protein kinase 1) and focal adhesion kinase (FAK) signaling cascades. Overexpression of FBXW5 promotes in-vivo tumor growth, whereas its inhibition down regulates in-vivo tumor metastasis. When considered together, our study identifies the novel oncogenic role of FBXW5 in gastric cancer and draws further interest regarding its clinical utility as a potential therapeutic target.

Influence of proliferation on the motions of epithelial monolayers invading adherent strips

Estelle Gauquelin, Sham Tlili, Cyprien Gay, Gregoire Peyret, Rene-Marc Mege, Marc A. Fardin, Benoit Ladoux

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.

Large-scale curvature sensing by directional actin flow drives cellular migration mode switching

Tianchi Chen, Andrew Callan-Jones, Eduard Fedorov, Andrea Ravasio, Agusti Brugues, Hui Ting Ong, Yusuke Toyama, Boon Chuan Low, Xavier Trepat, et al.

Cell migration over heterogeneous substrates during wound healing or morphogenetic processes leads to shape changes driven by different organizations of the actin cytoskeleton and by functional changes including lamellipodial protrusions and contractile actin cables. Cells distinguish between cell-sized positive and negative curvatures in their physical environment by forming protrusions at positive curvatures and actin cables at negative curvatures; however, the cellular mechanisms remain unclear. Here, we report that concave edges promote polarized actin structures with actin flow directed towards the cell edge, in contrast to well-documented retrograde flow at convex edges. Anterograde flow and contractility induce a tension anisotropy gradient. A polarized actin network is formed, accompanied by a local polymerization-depolymerization gradient, together with leading-edge contractile actin cables in the front. These cables extend onto non-adherent regions while still maintaining contact with the substrate through focal adhesions. The contraction and dynamic reorganization of this actin structure allows forward movements enabling cell migration over non-adherent regions on the substrate. These versatile functional structures may help cells sense and navigate their environment by adapting to external geometric and mechanical cues.

Cell shape and substrate stiffness drive actin-based cell polarity

Mukund Gupta, Bryant L. Doss, Leyla Kocgozlu, Meng Pan, Rene-Marc Mege, Andrew Callan-Jones, Raphael Voituriez, Benoit Ladoux

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.

Mechanical forces in cell monolayers

Tianchi Chen, Thuan Beng Saw, Rene-Marc Mege, Benoit Ladoux

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 a-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.

The irruption of mechanics in the chemistry of life

Rene Marc Mege, Benoit Ladoux

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.

Material approaches to active tissue mechanics

Wang Xi, Thuan Beng Saw, Delphine Delacour, Chwee Teck Lim, Benoit Ladoux

Communities of epithelial cells communicate through intercellular interactions, allowing them to coordinate their motility, which plays a key role in homeostasis, morphogenesis and cancer metastasis. Each cell in the epithelium is a constitutive energy-consuming agent, which can generate forces and interact with other cells through cell-cell junctions. Forces applied through external stimuli or endogenous cellular events are balanced by the cells within the epithelium, resulting in the adjustment of internal tissue contractile stresses and tissue reorganization. Materials science and microengineering techniques can be combined to create controllable environments to study epithelial movement and mechanics. By modulating the cell-material interface and by applying principles of active matter, key aspects of epithelial dynamics and mechanosensing mechanisms can be investigated. In this Review, we discuss epithelial tissues as active materials with particular rheological properties and active behaviours at different length scales. We highlight 2D and 3D materials for the study of epithelial dynamics and summarize key methods for the probing of epithelial mechanics. Tissue responses to mechanical stimuli are examined from the molecular level to the tissue level, and the effects of the shape, architecture and stiffness of the microenvironment are discussed.

Active superelasticity in three-dimensional epithelia of controlled shape

Ernest Latorre, Sohan Kale, Laura Casares, Manuel Gomez-Gonzalez, Marina Uroz, Leo Valon, Roshna Nair V, Elena Garreta, Nuria Montserrat, et al.

Fundamental biological processes are carried out by curved epithelial sheets that enclose a pressurized lumen. How these sheets develop and withstand three-dimensional deformations has remained unclear. Here we combine measurements of epithelial tension and shape with theoretical modelling to show that epithelial sheets are active superelastic materials. We produce arrays of epithelial domes with controlled geometry. Quantification of luminal pressure and epithelial tension reveals a tensional plateau over several-fold areal strains. These extreme strains in the tissue are accommodated by highly heterogeneous strains at a cellular level, in seeming contradiction to the measured tensional uniformity. This phenomenon is reminiscent of superelasticity, a behaviour that is generally attributed to microscopic material instabilities in metal alloys. We show that in epithelial cells this instability is triggered by a stretch-induced dilution of the actin cortex, and is rescued by the intermediate filament network. Our study reveals a type of mechanical behaviour-which we term active superelasticity -that enables epithelial sheets to sustain extreme stretching under constant tension.

Kalman Inversion Stress Microscopy

Vincent Nier, Gregoire Peyret, Joseph d'Alessandro, Shuji Ishihara, Benoit Ladoux, Philippe Marcq

Although mechanical cues are crucial to tissue morphogenesis and development, the tissue mechanical stress field remains poorly characterized. Given traction force time-lapse movies, as obtained by traction force microscopy of in vitro cellular sheets, we show that the tissue stress field can be estimated by Kalman filtering. After validation using numerical data, we apply Kalman inversion stress microscopy to experimental data. We combine the inferred stress field with velocity and cell-shape measurements to quantify the rheology of epithelial cell monolayers in physiological conditions, found to be close to that of an elastic and active material.

Biological Tissues as Active Nematic Liquid Crystals

Thuan Beng Saw, Wang Xi, Benoit Ladoux, Chwee Teck Lim

Live tissues can self-organize and be described as active materials composed of cells that generate active stresses through continuous injection of energy. In vitro reconstituted molecular networks, as well as single-cell cytoskeletons show that their filamentous structures can portray nematic liquid crystalline properties and can promote nonequilibrium processes induced by active processes at the microscale. The appearance of collective patterns, the formation of topological singularities, and spontaneous phase transition within the cell cytoskeleton are emergent properties that drive cellular functions. More integrated systems such as tissues have cells that can be seen as coarse-grained active nematic particles and their interaction can dictate many important tissue processes such as epithelial cell extrusion and migration as observed in vitro and in vivo. Here, a brief introduction to the concept of active nematics is provided, and the main focus is on the use of this framework in the systematic study of predominantly 2D tissue architectures and dynamics in vitro. In addition how the nematic state is important in tissue behavior, such as epithelial expansion, tissue homeostasis, and the atherosclerosis disease state, is discussed. Finally, how the nematic organization of cells can be controlled in vitro for tissue engineering purposes is briefly discussed.

Collective cell migration without proliferation: density determines cell velocity and wave velocity

Sham Tlili, Estelle Gauquelin, Brigitte Li, Olivier Cardoso, Benoit Ladoux, Helene Delanoe-Ayari, Francois Graner

Collective cell migration contributes to embryogenesis, wound healing and tumour metastasis. Cell monolayer migration experiments help in understanding what determines the movement of cells far from the leading edge. Inhibiting cell proliferation limits cell density increase and prevents jamming; we observe long-duration migration and quantify space-time characteristics of the velocity profile over large length scales and time scales. Velocity waves propagate backwards and their frequency depends only on cell density at the moving front. Both cell average velocity and wave velocity increase linearly with the cell effective radius regardless of the distance to the front. Inhibiting lamellipodia decreases cell velocity while waves either disappear or have a lower frequency. Our model combines conservation laws, monolayer mechanical properties and a phenomenological coupling between strain and polarity: advancing cells pull on their followers, which then become polarized. With reasonable values of parameters, this model agrees with several of our experimental observations. Together, our experiments and model disantangle the respective contributions of active velocity and of proliferation in monolayer migration, explain how cells maintain their polarity far from the moving front, and highlight the importance of strain-polarity coupling and density in long-range information propagation.

Force-dependent binding of vinculin to α-catenin regulates cell-cell contact stability and collective cell behavior

Rima Seddiki, Gautham Hari Narayana Sankara Narayana, Pierre-Olivier Strale, Hayri Emrah Balcioglu, Gregoire Peyret, Mingxi Yao, Phuong Anh Le, Chwee Teck Lim, Jie Yan, et al.

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, alpha-catenin, and F-actin as a function of stiffness, as well as the dynamics of GFP-tagged wildtype and mutated alpha-catenins, altered for their binding capability to vinculin, we demonstrate that the force-dependent binding of vinculin stabilizes alpha-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 alpha-catenin/vinculin interaction, manipulated here by mutagenesis and mechanical control, is a core regulator of AJ mechanics and long-range cell-cell interactions.

Emergent patterns of collective cell migration under tubular confinement

Wang Xi, Surabhi Sonam, Thuan Beng Saw, Benoit Ladoux, Chwee Teck Lim

Collective epithelial behaviors are essential for the development of lumens in organs. However, conventional assays of planar systems fail to replicate cell cohorts of tubular structures that advance in concerted ways on out-of-plane curved and confined surfaces, such as ductal elongation in vivo. Here, we mimic such coordinated tissue migration by forming lumens of epithelial cell sheets inside microtubes of 1-10 cell lengths in diameter. We show that these cell tubes reproduce the physiological apical-basal polarity, and have actin alignment, cell orientation, tissue organization, and migration modes that depend on the extent of tubular confinement and/or curvature. In contrast to flat constraint, the cell sheets in a highly constricted smaller microtube demonstrate slow motion with periodic relaxation, but fast overall movement in large microtubes. Altogether, our findings provide insights into the emerging migratory modes for epithelial migration and growth under tubular confinement, which are reminiscent of the in vivo scenario.

Mechanobiology of collective cell behaviours

Benoit Ladoux, Rene-Marc Mege

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.

Modeling collective cell migration in geometric confinement

Victoria Tarle, Estelle Gauquelin, S. R. K. Vedula, Joseph D'Alessandro, C. T. Lim, Benoit Ladoux, Nir S. Gov

Monolayer expansion has generated great interest as a model system to study collective cell migration. During such an expansion the culture front often develops 'fingers', which we have recently modeled using a proposed feedback between the curvature of the monolayer's leading edge and the outward motility of the edge cells. We show that this model is able to explain the puzzling observed increase of collective cellular migration speed of a monolayer expanding into thin stripes, as well as describe the behavior within different confining geometries that were recently observed in experiments. These comparisons give support to the model and emphasize the role played by the edge cells and the edge shape during collective cell motion.

Topological defects in epithelia govern cell death and extrusion

Thuan Beng Saw, Amin Doostmohammadi, Vincent Nier, Leyla Kocgozlu, Sumesh Thampi, Yusuke Toyama, Philippe Marcq, Chwee Teck Lim, Julia M. Yeomans, et al.

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling(1), oncogenic transformation(2,3) and overcrowding of cells(4-6). Despite the important linkage of cell extrusion to developmental(7), homeostatic(5) and pathological processes(2,8) such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments(9,10) in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity(11), caspase-3-mediated cell death, and extrusions. Importantly, the defectdriven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an a-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

Julie Salomon, Cecile Gaston, Jeremy Magescas, Boris Duvauchelle, Danielle Canioni, Lucie Sengmanivong, Adeline Mayeux, Gregoire Michaux, Florence Campeotto, et al.

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.

Nanoscale architecture of cadherin-based cell adhesions

Cristina Bertocchi, Yilin Wang, Andrea Ravasio, Yusuke Hara, Yao Wu, Talgat Sailov, Michelle A. Baird, Michael W. Davidson, Ronen Zaidel-Bar, et al.

Multicellularity in animals requires dynamic maintenance of cell-cell contacts. Intercellularly ligated cadherins recruit numerous proteins to form supramolecular complexes that connect with the actin cytoskeleton and support force transmission. However, the molecular organization within such structures remains unknown. Here we mapped protein organization in cadherin-based adhesions by super-resolution microscopy, revealing a multi -compartment nanoscale architecture, with the plasma -membrane-proximal cadherin-catenin compartment segregated from the actin cytoskeletal compartment, bridged by an interface zone containing vinculin. Vinculin position is determined by alpha-catenin, and following activation, vinculin can extend similar to 30 nm to bridge the cadherin-catenin and actin compartments, while modulating the nanoscale positions of the actin regulators zyxin and VASP. Vinculin conformational activation requires tension and tyrosine phosphorylation, regulated by Abl kinase and PTP1B phosphatase. Such modular architecture provides a structural framework for mechanical and biochemical signal integration by vinculin, which may differentially engage cadherin-catenin complexes with the actomyosin machinery to regulate cell adhesions.

Coordination between Intra- and Extracellular Forces Regulates Focal Adhesion Dynamics

Bibhu Ranjan Sarangi, Mukund Gupta, Bryant L. Doss, Nicolas Tissot, France Lam, Rene-Marc Mege, Nicolas Borghi, Benoit Ladoux

Focal adhesions (FAs) are important mediators of cell substrate interactions. One of their key functions is the transmission of forces between the intracellular acto-myosin network and the substrate. However, the relationships between cell traction forces, FA architecture, and molecular forces within FAs are poorly understood. Here, by combining Forster resonance energy transfer (FRET)-based molecular force biosensors with micropillar-based traction force sensors and high-resolution fluorescence microscopy, we simultaneously map molecular tension across vinculin, a key protein in FAs, and traction forces at FAs. Our results reveal strong spatiotemporal correlations between vinculin tension and cell traction forces at FAs throughout a wide range of substrate stiffnesses. Furthermore, we find that molecular tension within individual FAs follows a biphasic distribution from the proximal (toward the cell nucleus) to distal end (toward the cell-edge). Using super-resolution imaging, we show that such a distribution relates to that of FA proteins. On the basis of our experimental data, we propose a model in which FA dynamics results from tension changes along the FAs.

Epithelial Cell Packing Induces Distinct Modes of Cell Extrusions

Leyla Kocgozlu, Thuan Beng Saw, Phuong Anh Le, Ivan Yow, Murat Shagirov, Eunice Wong, Rene-Marc Mege, Chwee Teck Lim, Yusuke Toyama, et al.

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.

Mechanics of epithelial tissues during gap closure

Simon Begnaud, Tianchi Chen, Delphine Delacour, Rene-Marc Mege, Benoit Ladoux

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 'pursestring' and cell crawling mechanisms regulated by cell-substrate and cell-cell interactions, cellular mechanics and physical constraints from the environment.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

Wangsun Choi, Bipul R. Acharya, Gregoire Peyret, Marc-Antoine Fardin, Rene-Marc Mege, Benoit Ladoux, Alpha S. Yap, Alan S. Fanning, Mark Peifer

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.

Single cell rigidity sensing: A complex relationship between focal adhesion dynamics and large-scale actin cytoskeleton remodeling

Mukund Gupta, Bryant Doss, Chwee Teck Lim, Raphael Voituriez, Benoit Ladoux

Many physiological and pathological processes involve tissue cells sensing the rigidity of their environment. In general, tissue cells have been shown to react to the stiffness of their environment by regulating their level of contractility, and in turn applying traction forces on their environment to probe it. This mechanosensitive process can direct early cell adhesion, cell migration and even cell differentiation. These processes require the integration of signals over time and multiple length scales. Multiple strategies have been developed to understand force- and rigidity-sensing mechanisms and much effort has been concentrated on the study of cell adhesion complexes, such as focal adhesions, and cell cytoskeletons. Here, we review the major biophysical methods used for measuring cell-traction forces as well as the mechanosensitive processes that drive cellular responses to matrix rigidity on 2-dimensional substrates.

Inference of Internal Stress in a Cell Monolayer

Vincent Nier, Shreyansh Jain, Chwee Teck Lim, Shuji Ishihara, Benoit Ladoux, Philippe Marcq

We combine traction force data with Bayesian inversion to obtain an absolute estimate of the internal stress field of a cell monolayer. The method, Bayesian inversion stress microscopy, is validated using numerical simulations performed in a wide range of conditions. It is robust to changes in each ingredient of the underlying statistical model. Importantly, its accuracy does not depend on the rheology of the tissue. We apply Bayesian inversion stress microscopy to experimental traction force data measured in a narrow ring of cohesive epithelial cells, and check that the inferred stress field coincides with that obtained by direct spatial integration of the traction force data in this quasi one-dimensional geometry.

Mechanical confinement triggers glioma linear migration dependent on formin FHOD3

Pascale Monzo, Yuk Kien Chong, Charlotte Guetta-Terrier, Anitha Krishnasamy, Sharvari R. Sathe, Evelyn K. F. Yim, Wai Hoe Ng, Beng Ti Ang, Carol Tang, et al.

Glioblastomas are extremely aggressive brain tumors with highly invasive properties. Brain linear tracks such as blood vessel walls constitute their main invasive routes. Here we analyze rat C6 and patient-derived glioma cell motility in vitro using micropatterned linear tracks to mimic blood vessels. On laminin-coated tracks (3-10 mu m), these cells used an efficient saltatory mode of migration similar to their in vivo migration. This saltatory migration was also observed on larger tracks (50-400 mu m in width) at high cell densities. In these cases, the mechanical constraints imposed by neighboring cells triggered this efficient mode of migration, resulting in the formation of remarkable antiparallel streams of cells along the tracks. This motility involved microtubule-dependent polarization, contractile actin bundles and dynamic paxillin-containing adhesions in the leading process and in the tail. Glioma linear migration was dramatically reduced by inhibiting formins but, surprisingly, accelerated by inhibiting Arp2/3. Protein expression and phenotypic analysis indicated that the formin FHOD3 played a role in this motility but not mDia1 or mDia2. We propose that glioma migration under confinement on laminin relies on formins, including FHOD3, but not Arp2/3 and that the low level of adhesion allows rapid antiparallel migration.

Front-Rear Polarization by Mechanical Cues: From Sincle Cells to Tissues

Benoit Ladoux, Rene-Marc Mege, Xavier Trepat

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.

Early Passage Dependence of Mesenchymal Stem Cell Mechanics Influences Cellular Invasion and Migration

Stephen T. Spagnol, Wei-Chun Lin, Elizabeth A. Booth, Benoit Ladoux, Hillard M. Lazarus, Kris Noel Dahl

The cellular structures and mechanical properties of human mesenchymal stem cells (hMSCs) vary significantly during culture and with differentiation. Previously, studies to measure mechanics have provided divergent results using different quantitative parameters and mechanical models of deformation. Here, we examine hMSCs prepared for clinical use and subject them to mechanical testing conducive to the relevant deformability associated with clinical injection procedures. Micropipette aspiration of hMSCs shows deformation as a viscoelastic fluid, with little variation from cell to cell within a population. After two passages, hMSCs deform as viscoelastic solids. Further, for clinical applicability during stem cell migration in vivo, we investigated the ability of hMSCs to invade into micropillar arrays of increasing confinement from 12 to 8 mu m spacing between adjacent micropillars. We find that hMSC samples with reduced deformability and cells that are more solid-like with passage are more easily able to enter the micropillar arrays. Increased cell fluidity is an advantage for injection procedures and optimization of cell selection based on mechanical properties may enhance efficacy of injected hMSC populations. However, the ability to invade and migrate within tight interstitial spaces appears to be increased with a more solidified cytoskeleton, likely from increased force generation and contractility. Thus, there may be a balance between optimal injection survival and in situ tissue invasion.

Protrusive waves guide 3D cell migration along nanofibers

Charlotte Guetta-Terrier, Pascale Monzo, Jie Zhu, Hongyan Long, Lakshmi Venkatraman, Yue Zhou, PeiPei Wang, Sing Yian Chew, Alexander Mogilner, et al.

In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electro-spun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Racl/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.

Regulation of epithelial cell organization by tuning cell-substrate adhesion

Andrea Ravasio, Phuong Anh Le, Thuan Beng Saw, Victoria Tarle, Hui Ting Ong, Cristina Bertocchi, Rene-Marc Mege, Chwee Teck Lim, Nir S. Gov, et al.

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.

Celebrating Soft Matters 10th Anniversary: Cell division: a source of active stress in cellular monolayers

Amin Doostmohammadi, Sumesh P. Thampi, Thuan B. Saw, Chwee T. Lim, Benoit Ladoux, Julia M. Yeomans

We introduce the notion of cell division-induced activity and show that the cell division generates extensile forces and drives dynamical patterns in cell assemblies. Extending the hydrodynamic models of lyotropic active nematics we describe turbulent-like velocity fields that are generated by the cell division in a confluent monolayer of cells. We show that the experimentally measured flow field of dividing Madin-Darby Canine Kidney (MDCK) cells is reproduced by our modeling approach. Division-induced activity acts together with intrinsic activity of the cells in extensile and contractile cell assemblies to change the flow and director patterns and the density of topological defects. Finally we model the evolution of the boundary of a cellular colony and compare the fingering instabilities induced by cell division to experimental observations on the expansion of MDCK cell cultures.

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity

Pierre-Olivier Strale, Laurence Duchesne, Gregoire Peyret, Lorraine Montel, Nguyen Thao, Evelyn Png, Robert Tampe, Sergey Troyanovsky, Sylvie Henon, et al.

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell cell contact fluidity.

Gap geometry dictates epithelial closure efficiency

Andrea Ravasio, Ibrahim Cheddadi, Tianchi Chen, Telmo Pereira, Hui Ting Ong, Cristina Bertocchi, Agusti Brugues, Antonio Jacinto, Alexandre J. Kabla, et al.

Closure of wounds and gaps in tissues is fundamental for the correct development and physiology of multicellular organisms and, when misregulated, may lead to inflammation and tumorigenesis. To re-establish tissue integrity, epithelial cells exhibit coordinated motion into the void by active crawling on the substrate and by constricting a supracellular actomyosin cable. Coexistence of these two mechanisms strongly depends on the environment. However, the nature of their coupling remains elusive because of the complexity of the overall process. Here we demonstrate that epithelial gap geometry in both in vitro and in vivo regulates these collective mechanisms. In addition, the mechanical coupling between actomyosin cable contraction and cell crawling acts as a large-scale regulator to control the dynamics of gap closure. Finally, our computational modelling clarifies the respective roles of the two mechanisms during this process, providing a robust and universal mechanism to explain how epithelial tissues restore their integrity.

Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing

Mukund Gupta, Bibhu Ranjan Sarangi, Joran Deschamps, Yasaman Nematbakhsh, Andrew Callan-Jones, Felix Margadant, Rene-Marc Mege, Chwee Teck Lim, Raphael Voituriez, et al.

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.

The mechanotransduction machinery at work at adherens junctions

B. Ladoux, W. J. Nelson, J. Yan, R. M. Mege

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 Ca2+-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein beta-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein alpha-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 alpha-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that alpha-catenin is the core mechanosensor that allows cells to locally sense, transduce and adapt to environmental mechanical constrains.

Mechanical link between durotaxis, cell polarity and anisotropy during cell migration

D. Aubry, M. Gupta, B. Ladoux, R. Allena

Cell migration, a fundamental mechanobiological process, is highly sensitive to the biochemical and mechanical properties of the environment. Efficient cell migration is ensured by the intrinsic polarity of the cell, which triggers a transition from an isotropic to an anisotropic configuration of the actomysion filaments responsible for the protrusion-contraction movement of the cell. Additionally, polarity may be highly influenced by the substrate rigidity, which results in a phenomenon called durotaxis. In the present work, we propose a two-dimensional finite element model able to capture three main features of cell migration: durotaxis, cell polarity and anisotropy. The cell is modelled as a continuum able to develop cyclic active strains regulated by the polymerization and depolymerization of the acto-myosin filaments and synchronized with the adhesion forces between the cell and the substrate underneath. A generalized Maxwell model is used to describe the viscoelastic behaviour of the cell constituted by a solid anisotropic branch with active strains (i.e. the acto-myosin filaments) and a fluid viscoelastic branch (i.e. the cytoplasm). Several types of substrate have been tested which are homogeneously soft or stiff or include both regions. The numerical results have been qualitatively compared with experimental observations showing a good agreement and have allowed us to find the mechanical link between durotaxis, cell polarity and anisotropy.

Mechanics of epithelial closure over non-adherent environments

Sri Ram Krishna Vedula, Gregoire Peyret, Ibrahim Cheddadi, Tianchi Chen, Agusti Brugues, Hiroaki Hirata, Horacio Lopez-Menendez, Yusuke Toyama, Luis Neves de Almeida, et al.

The closure of gaps within epithelia is crucial to maintain its integrity during biological processes such as wound healing and gastrulation. Depending on the distribution of extracellular matrix, gap closure occurs through assembly of multicellular actin-based contractile cables or protrusive activity of border cells into the gap. Here we show that the supracellular actomyosin contractility of cells near the gap edge exerts sufficient tension on the surrounding tissue to promote closure of non-adherent gaps. Using traction force microscopy, we observe that cell-generated forces on the substrate at the gap edge first point away from the centre of the gap and then increase in the radial direction pointing into the gap as closure proceeds. Combining with numerical simulations, we show that the increase in force relies less on localized purse-string contractility and more on large-scale remodelling of the suspended tissue around the gap. Our results provide a framework for understanding the assembly and the mechanics of cellular contractility at the tissue level.

Micropillar substrates: A tool for studying cell mechanobiology

Mukund Gupta, Leyla Kocgozlu, Bibhu Ranjan Sarangi, Felix Margadant, Mohammed Ashraf, Benoit Ladoux

Increasing evidence has shown that mechanical cues from the environment play an important role in cell biology. Mechanotransduction or the study of how cells can sense these mechanical cues, and respond to them, is an active field of research. However, it is still not clear how cells sense and respond to mechanical cues. Thus, new tools are being rapidly developed to quantitatively study cell mechanobiology. Particularly, force measurement tools such as micropillar substrates have provided new insights into the underlying mechanisms of mechanosensing. In this chapter, we provide detailed protocol for fabrication, characterization, functionalization, and use of the micropillar substrates.

Actomyosin bundles serve as a tension sensor and a platform for ERK activation

Hiroaki Hirata, Mukund Gupta, Sri Ram Krishna Vedula, Chwee Teck Lim, Benoit Ladoux, Masahiro Sokabe

Tensile forces generated by stress fibers drive signal transduction events at focal adhesions. Here, we report that stress fibers per se act as a platform for tension-induced activation of biochemical signals. The MAP kinase, ERK is activated on stress fibers in a myosin II-dependent manner. In myosin II-inhibited cells, uniaxial stretching of cell adhesion substrates restores ERK activation on stress fibers. By quantifying myosin II- or mechanical stretch-mediated tensile forces in individual stress fibers, we show that ERK activation on stress fibers correlates positively with tensile forces acting on the fibers, indicating stress fibers as a tension sensor in ERK activation. Myosin II-dependent ERK activation is also observed on actomyosin bundles connecting E-cadherin clusters, thus suggesting that actomyosin bundles, in general, work as a platform for tension-dependent ERK activation.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer

Andrea Ravasio, Sree Vaishnavi, Benoit Ladoux, Virgile Viasnoff

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Collective migration: a task- sharing between cells leaders and supercellular coordination

Myriam Reffay, Maria-Carla Parrini, Olivier Cochet-Escartin, Benoit Ladoux, Axel Buguin, Sylvie Coscoy, Francois Amblard, Jacques Camonis, Pascal Silberzan

Forces driving epithelial wound healing

Agusti Brugues, Ester Anon, Vito Conte, Jim H. Veldhuis, Mukund Gupta, Julien Colombelli, Jose J. Munoz, G. Wayne Brodland, Benoit Ladoux, et al.

A fundamental feature of multicellular organisms is their ability to self-repair wounds through the movement of epithelial cells into the damaged area. This collective cellular movement is commonly attributed to a combination of cell crawling and 'purse-string' contraction of a supracellular actomyosin ring. Here we show by direct experimental measurement that these two mechanisms are insufficient to explain force patterns observed during wound closure. At early stages of the process, leading actin protrusions generate traction forces that point away from the wound, showing that wound closure is initially driven by cell crawling. At later stages, we observed unanticipated patterns of traction forces pointing towards the wound. Such patterns have strong force components that are both radial and tangential to the wound. We show that these force components arise from tensions transmitted by a heterogeneous actomyosin ring to the underlying substrate through focal adhesions. The structural and mechanical organization reported here provides cells with a mechanism to close the wound by cooperatively compressing the underlying substrate.

Force-dependent conformational switch of α-catenin controls vinculin binding

Mingxi Yao, Wu Qiu, Ruchuan Liu, Artem K. Efremov, Peiwen Cong, Rima Seddiki, Manon Payre, Chwee Teck Lim, Benoit Ladoux, et al.

Force sensing at cadherin-mediated adhesions is critical for their proper function. alpha-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 alpha-catenin adopts a stable auto-inhibitory conformation that makes the vinculin-binding site inaccessible. Here, by stretching single alpha-catenin molecules using magnetic tweezers, we show that the subdomains M-I vinculin-binding domain (VBD) to M-III unfold in three characteristic steps: a reversible step at similar to 5pN and two non-equilibrium steps at 10-15 pN. 5 pN unfolding forces trigger vinculin binding to the M-I domain in a 1: 1 ratio with nanomolar affinity, preventing M-I domain refolding after force is released. Our findings demonstrate that physiologically relevant forces reversibly unfurl alpha-catenin, activating vinculin binding, which then stabilizes alpha-catenin in its open conformation, transforming force into a sustainable biochemical signal.

The Nanoscale Architecture of Force-Bearing Focal Adhesions

Hedde van Hoorn, Rolf Harkes, Ewa M. Spiesz, Cornelis Storm, Danny van Noort, Benoit Ladoux, Thomas Schmidt

The combination of micropillar array technology to measure cellular traction forces with super-resolution imaging allowed us to obtain cellular traction force maps and simultaneously zoom in on individual focal adhesions with single-molecule accuracy. We achieved a force detection precision of 500 pN simultaneously with a mean single-molecule localization precision of 30 nm. Key to the achievement was a two-step etching process that provided an integrated spacer next to the micropillar array that permitted stable and reproducible observation of cells on micropillars within the short working distance of a high-magnification, high numerical aperture objective. In turn, we used the technology to characterize the super-resolved structure of focal adhesions during force exertion. Live-cell imaging on MCF-7 cells demonstrated the applicability of the inverted configuration of the micropillar arrays to dynamics measurements. Forces emanated from a molecular base that was localized on top of the micropillars. What appeared as a single adhesion in conventional microscopy were in fact multiple elongated adhesions emanating from only a small fraction of the adhesion on the micropillar surface. Focal adhesions were elongated in the direction of local cellular force exertion with structural features of 100-280 nm in 3T3 Fibroblasts and MCF-7 cells. The combined measure of nanoscale architecture and force exerted shows a high level of stress accumulation at a single site of adhesion.

Adhesive interactions of N-cadherin limit the recruitment of microtubules to cell-cell contacts through organization of actomyosin

Charlotte Plestant, Pierre-Olivier Strale, Rima Seddiki, Nguyen Emmanuelle, Benoit Ladoux, Rene-Marc Mege

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.

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells

M. Reffay, M. C. Parrini, O. Cochet-Escartin, B. Ladoux, A. Buguin, S. Coscoy, F. Amblard, J. Camonis, P. Silberzan

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Epithelial bridges maintain tissue integrity during collective cell migration

Sri Ram Krishna Vedula, Hiroaki Hirata, Mui Hoon Nai, Agusti Brugues, Yusuke Toyama, Xavier Trepat, Chwee Teck Lim, Benoit Ladoux

The ability of skin to act as a barrier is primarily determined by the efficiency of skin cells to maintain and restore its continuity and integrity. In fact, during wound healing keratinocytes migrate collectively to maintain their cohesion despite heterogeneities in the extracellular matrix. Here, we show that monolayers of human keratinocytes migrating along functionalized micropatterned surfaces comprising alternating strips of extracellular matrix (fibronectin) and non-adherent polymer form suspended multicellular bridges over the non-adherent areas. The bridges are held together by intercellular adhesion and are subjected to considerable tension, as indicated by the presence of prominent actin bundles. We also show that a model based on force propagation through an elastic material reproduces the main features of bridge maintenance and tension distribution. Our findings suggest that multicellular bridges maintain tissue integrity during wound healing when cell-substrate interactions are weak and may prove helpful in the design of artificial scaffolds for skin regeneration.

Collective Cell Migration: A Mechanistic Perspective

Sri Ram Krishna Vedula, Andrea Ravasio, Chwee Teck Lim, Benoit Ladoux

Collective cell migration is fundamental to gaining insights into various important biological processes such as wound healing and cancer metastasis. In particular, recent in vitro studies and in silico simulations suggest that mechanics can explain the social behavior of multicellular clusters to a large extent with minimal knowledge of various cellular signaling pathways. These results suggest that a mechanistic perspective is necessary for a comprehensive and holistic understanding of collective cell migration, and this review aims to provide a broad overview of such a perspective.

Guidance of collective cell migration by substrate geometry

Kevin Doxzen, Sri Ram Krishna Vedula, Man Chun Leong, Hiroaki Hirata, Nir S. Gov, Alexandre J. Kabla, Benoit Ladoux, Chwee Teck Lim

Collective behavior refers to the emergence of complex migration patterns over scales larger than those of the individual elements constituting a system. It plays a pivotal role in biological systems in regulating various processes such as gastrulation, morphogenesis and tissue organization. Here, by combining experimental approaches and numerical modeling, we explore the role of cell density ('crowding'), strength of intercellular adhesion ('cohesion') and boundary conditions imposed by extracellular matrix (ECM) proteins ('constraints') in regulating the emergence of collective behavior within epithelial cell sheets. Our results show that the geometrical confinement of cells into well-defined circles induces a persistent, coordinated and synchronized rotation of cells that depends on cell density. The speed of such rotating large-scale movements slows down as the density increases. Furthermore, such collective rotation behavior depends on the size of the micropatterned circles: we observe a rotating motion of the overall cell population in the same direction for sizes of up to 200 mm. The rotating cells move as a solid body, with a uniform angular velocity. Interestingly, this upper limit leads to length scales that are similar to the natural correlation length observed for unconfined epithelial cell sheets. This behavior is strongly altered in cells that present a downregulation of adherens junctions and in cancerous cell types. We anticipate that our system provides a simple and easy approach to investigate collective cell behavior in a well-controlled and systematic manner.

Multiarray cell stretching platform for high-magnification real-time imaging

Yuli Huang, Nguyen Nam-Trung, Khoi Seng Lok, Peter Peng Foo Lee, Maohan Su, Min Wu, Leyla Kocgozlu, Benoit Ladoux

Aim: This article reports the development of a multiarray microchip with real-time imaging capability to apply mechanical strains onto monolayered cell cultures. Materials & methods: Cells were cultured on an 8-mu m thick membrane that was positioned in the microscope focal plane throughout the stretching process. Each stretching unit was assembled from three elastomeric layers and a glass coverslip. A programmable pneumatic control system was developed to actuate this platform. Multiple stretching experiments were conducted with various cell lines. Results: The platform provides a maximum uniform strain of 69%. Acute and long-term cell morphological changes were observed. The supreme imaging capability was verified by real-time imaging of transfected COS-7 stretching and poststretching imaging of immunofluorescence-stained PTK2. Conclusion: The platform reported here is a powerful tool for studying mechanically induced physiological changes in cells. Such a device could be used in tissue regeneration for maintaining essential cell growth conditions.

Hutchinson-Gilford progeria syndrome alters nuclear shape and reduces cell motility in three dimensional model substrates

Elizabeth A. Booth-Gauthier, Vicard Du, Marion Ghibaudo, Andrew D. Rape, Kris Noel Dahl, Benoit Ladoux

Cell migration through tight interstitial spaces in three dimensional (3D) environments impacts development, wound healing and cancer metastasis and is altered by the aging process. The stiffness of the extracellular matrix (ECM) increases with aging and affects the cells and cytoskeletal processes involved in cell migration. However, the nucleus, which is the largest and densest organelle, has not been widely studied during cell migration through the ECM. Additionally, the nucleus is stiffened during the aging process through the accumulation of a mutant nucleoskeleton protein lamin A, progerin. By using microfabricated substrates to mimic the confined environment of surrounding tissues, we characterized nuclear movements and deformation during cell migration into micropillars where interspacing can be tuned to vary nuclear confinement. Cell motility decreased with decreased micropillar (mu P) spacing and correlated with increased dysmorphic shapes of nuclei. We examined the effects of increased nuclear stiffness which correlates with cellular aging by studying Hutchinson-Gilford progeria syndrome cells which are known to accumulate progerin. With the expression of progerin, cells showed a threshold response to decreased mu P spacing. Cells became trapped in the close spacing, possibly from visible micro-defects in the nucleoskeleton induced by cell crawling through the mP and from reduced force generation, measured independently. We suggest that ECM changes during aging could be compounded by the increasing stiffness of the nucleus and thus changes in cell migration through 3D tissues.

α-Catenin and Vinculin Cooperate to Promote High E-cadherin-based Adhesion Strength

William A. Thomas, Cecile Boscher, Yeh-Shiu Chu, Damien Cuvelier, Clara Martinez-Rico, Rima Seddiki, Julie Heysch, Benoit Ladoux, Jean Paul Thiery, et al.

Maintaining cell cohesiveness within tissues requires that intercellular adhesions develop sufficient strength to support traction forces applied by myosin motors and by neighboring cells. Cadherins are transmembrane receptors that mediate intercellular adhesion. The cadherin cytoplasmic domain recruits several partners, including catenins and vinculin, at sites of cell-cell adhesion. Our study used force measurements to address the role of alpha E-catenin and vinculin in the regulation of the strength of E-cadherin-based adhesion. alpha E-catenin-deficient cells display only weak aggregation and fail to strengthen intercellular adhesion over time, a process rescued by the expression of alpha E-catenin or chimeric E-cadherin center dot alpha E-catenins, including a chimera lacking the alpha E-catenin dimerization domain. Interestingly, an alpha E-catenin mutant lacking the modulation and actin-binding domains restores cadherin-dependent cell-cell contacts but cannot strengthen intercellular adhesion. The expression of alpha E-catenin mutated in its vinculin-binding site is defective in its ability to rescue cadherin-based adhesion strength in cells lacking alpha E-catenin. Vinculin depletion or the overexpression of the alpha E-catenin modulation domain strongly decreases E-cadherin-mediated adhesion strength. This supports the notion that both molecules are required for intercellular contact maturation. Furthermore, stretching of cell doublets increases vinculin recruitment and alpha 18 anti-alpha E-catenin conformational epitope immunostaining at cell-cell contacts. Taken together, our results indicate that alpha E-catenin and vinculin cooperatively support intercellular adhesion strengthening, probably via a mechanoresponsive link between the E-cadherin beta-catenin complexes and the underlying actin cytoskeleton.

Physically based principles of cell adhesion mechanosensitivity in tissues

Benoit Ladoux, Alice Nicolas

The minimal structural unit that defines living organisms is a single cell. By proliferating and mechanically interacting with each other, cells can build complex organization such as tissues that ultimately organize into even more complex multicellular living organisms, such as mammals, composed of billions of single cells interacting with each other. As opposed to passive materials, living cells actively respond to the mechanical perturbations occurring in their environment. Tissue cell adhesion to its surrounding extracellular matrix or to neighbors is an example of a biological process that adapts to physical cues. The adhesion of tissue cells to their surrounding medium induces the generation of intracellular contraction forces whose amplitude adapts to the mechanical properties of the environment. In turn, solicitation of adhering cells with physical forces, such as blood flow shearing the layer of endothelial cells in the lumen of arteries, reinforces cell adhesion and impacts cell contractility. In biological terms, the sensing of physical signals is transduced into biochemical signaling events that guide cellular responses such as cell differentiation, cell growth and cell death. Regarding the biological and developmental consequences of cell adaptation to mechanical perturbations, understanding mechanotransduction in tissue cell adhesion appears as an important step in numerous fields of biology, such as cancer, regenerative medicine or tissue bioengineering for instance. Physicists were first tempted to view cell adhesion as the wetting transition of a soft bag having a complex, adhesive interaction with the surface. But surprising responses of tissue cell adhesion to mechanical cues challenged this view. This, however, did not exclude that cell adhesion could be understood in physical terms. It meant that new models and descriptions had to be created specifically for these biological issues, and could not straightforwardly be adapted from dead matter. In this review, we present physical concepts of tissue cell adhesion and the unexpected cellular responses to mechanical cues such as external forces and stiffness sensing. We show how biophysical approaches, both experimentally and theoretically, have contributed to our understanding of the regulation of cellular functions through physical force sensing mechanisms. Finally, we discuss the different physical models that could explain how tissue cell adhesion and force sensing can be coupled to internal mechanosensitive processes within the cell body.

Emerging modes of collective cell migration induced by geometrical constraints

Sri Ram Krishna Vedula, Man Chun Leong, Tan Lei Lai, Pascal Hersen, Alexandre J. Kabla, Chwee Teck Lim, Benoit Ladoux

The role of geometrical confinement on collective cell migration has been recognized but has not been elucidated yet. Here, we show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell-cell interactions. Using microfabrication techniques to allow epithelial cell sheets to migrate into strips whose width was varied from one up to several cell diameters, we identified the modes of collective migration in response to geometrical constraints. We observed that a decrease in the width of the strips is accompanied by an overall increase in the speed of the migrating cell sheet. Moreover, large-scale vortices over tens of cell lengths appeared in the wide strips whereas a contraction-elongation type of motion is observed in the narrow strips. Velocity fields and traction force signatures within the cellular population revealed migration modes with alternative pulling and/or pushing mechanisms that depend on extrinsic constraints. Force transmission through intercellular contacts plays a key role in this process because the disruption of cell-cell junctions abolishes directed collective migration and passive cell-cell adhesions tend to move the cells uniformly together independent of the geometry. Altogether, these findings not only demonstrate the existence of patterns of collective cell migration depending on external constraints but also provide a mechanical explanation for how large-scale interactions through cell-cell junctions can feed back to regulate the organization of migrating tissues.

E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

Audrius Jasaitis, Maruxa Estevez, Julie Heysch, Benoit Ladoux, Sylvie Dufour

The interplay between cadherin-and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (mu FSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on mFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior.

Locomotion Control of Caenorhabditis elegans through Confinement

Felix Lebois, Pascal Sauvage, Charlotte Py, Olivier Cardoso, Benoit Ladoux, Pascal Hersen, Jean-Marc Di Meglio

The model organism Caenorhabditis elegans shows two distinct locomotion patterns in laboratory situations: it swims in low viscosity liquids and it crawls on the surface of an agar gel. This provides a unique opportunity to discern the respective roles of mechanosensation (perception and proprioception) and mechanics in the regulation of locomotion and in the gait selection. Using an original device, we present what to our knowledge are new experiments where the confinement of a worm between a glass plate and a soft agar gel is controlled while recording the worm's motion. We observed that the worm continuously varied its locomotion characteristics from free swimming to slow crawling with increasing confinement so that it was not possible to discriminate between two distinct intrinsic gaits. This unicity of the gait is also proved by the fact that wild-type worms immediately adapted their motion when the imposed confinement was changed with time. We then studied locomotory deficient mutants that also exhibited one single gait and showed that the light touch response was needed for the undulation propagation and that the ciliated sensory neurons participated in the joint selection of motion period and undulation-wave velocity. Our results reveal that the control of maximum curvature, at a sensory or mechanical level, is a key ingredient of the locomotion regulation.

Cell crawling mediates collective cell migration to close undamaged epithelial gaps

Ester Anon, Xavier Serra-Picamal, Pascal Hersen, Nils C. Gauthier, Michael P. Sheetz, Xavier Trepat, Benoit Ladoux

Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 mu m was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.

Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness

Lea Trichet, Jimmy Le Digabel, Rhoda J. Hawkins, Ram Krishna Vedula, Mukund Gupta, Claire Ribrault, Pascal Hersen, Raphael Voituriez, Benoit Ladoux

Cell migration plays a major role in many fundamental biological processes, such as morphogenesis, tumor metastasis, and wound healing. As they anchor and pull on their surroundings, adhering cells actively probe the stiffness of their environment. Current understanding is that traction forces exerted by cells arise mainly at mechanotransduction sites, called focal adhesions, whose size seems to be correlated to the force exerted by cells on their underlying substrate, at least during their initial stages. In fact, our data show by direct measurements that the buildup of traction forces is faster for larger substrate stiffness, and that the stress measured at adhesion sites depends on substrate rigidity. Our results, backed by a phenomenological model based on active gel theory, suggest that rigidity-sensing is mediated by a large-scale mechanism originating in the cytoskeleton instead of a local one. We show that large-scale mechanosensing leads to an adaptative response of cell migration to stiffness gradients. In response to a step boundary in rigidity, we observe not only that cells migrate preferentially toward stiffer substrates, but also that this response is optimal in a narrow range of rigidities. Taken together, these findings lead to unique insights into the regulation of cell response to external mechanical cues and provide evidence for a cytoskeleton-based rigidity-sensing mechanism.

Micro-patterned porous substrates for cell-based assays

Fanny Evenou, Jean-Marc Di Meglio, Benoit Ladoux, Pascal Hersen

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (mu PM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. mPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.

Magnetic micropillars as a tool to govern substrate deformations

Jimmy le Digabel, Nicolas Biais, Jerome Fresnais, Jean-Francois Berret, Pascal Hersen, Benoit Ladoux

Magnetic actuated microdevices can be used to achieve several complex functions in microfluidics and microfabricated devices. For example, magnetic mixers and magnetic actuators have been proposed to help handling fluids at a small scale. Here, we present a strategy to create magnetically actuated micropillar arrays. We combined microfabrication techniques and the dispersion of magnetic aggregates embedded inside polymeric matrices to design micrometre scale magnetic features. By creating a magnetic field gradient in the vicinity of the substrate, well-defined forces were applied on these magnetic aggregates which in turn induced a deflection of the micropillars. By dispersing either spherical aggregates or magnetic nanowires into the gels, we can induce synchronized motions of a group of pillars or the movement of isolated pillars under a magnetic field gradient. When combined with microfabrication processes, this versatile tool leads to local as well as global substrate actuations within a range of dimensions that are relevant for microfluidics and biological applications.

BIOPHYSICS Push it, pull it

Pascal Hersen, Benoit Ladoux

During migration, cells interact with their environment by exerting mechanical forces on it. A combination of two techniques shows that they do so in all three dimensions by a push-pull mechanism.

Running Worms: C. elegans Self-Sorting by Electrotaxis

Xavier Maniere, Felix Lebois, Ivan Matic, Benoit Ladoux, Jean-Marc Di Meglio, Pascal Hersen

The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm's crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.

Mechanics of cell spreading within 3D-micropatterned environments

Marion Ghibaudo, Jean-Marc Di Meglio, Pascal Hersen, Benoit Ladoux

Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.

Velocity Fields in a Collectively Migrating Epithelium

L. Petitjean, M. Reffay, E. Grasland-Mongrain, M. Poujade, B. Ladoux, A. Buguin, P. Silberzan

We report quantitative measurements of the velocity field of collectively migrating cells in a motile epithelium. The migration is triggered by presenting free surface to an initially confluent monolayer by using a microstencil technique that does not damage the cells. To avoid the technical difficulties inherent in the tracking of single cells, the field is mapped using the technique of particle image velocimetry. The main relevant parameters, such as the velocity module, the order parameter, and the velocity correlation function, are then extracted from this cartography. These quantities are dynamically measured on two types of cells (collectively migrating Madin-Darby canine kidney (MDCK) cells and fibroblastlike normal rat kidney (NRK) cells), first as they approach confluence, and then when the geometrical constraints are released. In particular, for MDCK cells filling up the patterns, we observe a sharp decrease in the average velocity after the point of confluence, whereas the densification of the monolayer is much more regular. After the peeling off of the stencil, a velocity correlation length of similar to 200 mu m is measured for MDCK cells versus only similar to 40 mu m for the more independent NRK cells. Our conclusions are supported by parallel single-cell tracking experiments. By using the biorthogonal decomposition of the velocity field, we conclude that the velocity field of MDCK cells is very coherent in contrast with the NRK cells. The displacements in the fingers arising from the border of MDCK epithelia are very oriented along their main direction. They influence the velocity field in the epithelium over a distance of similar to 200 mu m.

Microfabricated substrates as a tool to study cell mechanotransduction

Jimmy le Digabel, Marion Ghibaudo, Lea Trichet, Alain Richert, Benoit Ladoux

Mechanical cell-substrate interactions affect many cellular functions such as spreading, migration, and even differentiation. These interactions can be studied by incorporating micro- and nanotechnology-related tools. The design of substrates based on these technologies offers new possibilities to probe the cellular responses to changes in their physical environment. The investigations of the mechanical interactions of cells and their surrounding matrix can be carried out in well-defined and near physiological conditions. In particular, this includes the transmission of forces as well as rigidity and topography sensing mechanisms. Here, we review techniques and tools based on nano- and micro-fabrication that have been developed to analyze the influence of the mechanical properties of the substrate on cell functions. We also discuss how microfabrication methods have improved our knowledge on cell adhesion and migration and how they could solve remaining problems in the field of mechanobiology.

Traction forces exerted by epithelial cell sheets

A. Saez, E. Anon, M. Ghibaudo, O. du Roure, J-M Di Meglio, P. Hersen, P. Silberzan, A. Buguin, B. Ladoux

Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

Strength Dependence of Cadherin-Mediated Adhesions

Benoit Ladoux, Ester Anon, Mireille Lambert, Aleksandr Rabodzey, Pascal Hersen, Axel Buguin, Pascal Silberzan, Rene-Marc Mege

Traction forces between adhesive cells play an important role in a number of collective cell processes Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands On the most rigid substrates, cells appear well-spread and present cadherin adhesions and cytoskeletal organization similar to those classically observed on cadherin-coated glass substrates However, when cells are cultured on softer substrates, a change in morphology is observed the cells are less spread, with a more disorganized actin network. A quantitative analysis of the cells adhering on the cadherin-coated surfaces shows that forces are correlated with the formation of cadherin adhesions. The stiffer the substrates, the larger are the average traction forces and the more developed are the cadherin adhesions. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechanosensitive regulation of cadherin-mediated intercellular junctions through the cellular contractile machinery.

Cytoskeletal coherence requires myosin-IIA contractility

Yunfei Cai, Olivier Rossier, Nils C. Gauthier, Nicolas Biais, Marc-Antoine Fardin, Xian Zhang, Lawrence W. Miller, Benoit Ladoux, Virginia W. Cornish, et al.

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

In the middle of it all: Mutual mechanical regulation between the nucleus and the cytoskeleton

Kris Noel Dahl, Elizabeth A. Booth-Gauthier, Benoit Ladoux

The nucleus is typically treated as the large phase-dense or easy-to-label structure at the center of the cell which is manipulated by the governing mechanical machinery inside the cytoplasm. However, recent evidence has suggested that the mechanical properties of the nucleus are important to cell fate. We will discuss many aspects of the structural and functional interconnections between nuclear mechanics and cellular mechanics in this review. There are numerous implications for the progression of many disease states associated with both nuclear structural proteins and cancers. The nucleus itself is a large organelle taking up significant volume within the cell, and most studies agree that nuclei are significantly stiffer than the surrounding cytoplasm. Thus when a cell is exposed to force, the nucleus is exposed to and helps resist that force. The nucleus and nucleoskeleton are interconnected with the cellular cytoskeleton, and these connections may aid in helping disperse forces within tissues and/or with mechanotransduction. During translocation and transmigration the nucleus can act as a resistive element. Understanding the role of mechanical regulation of the nucleus may aid in understanding cellular motility and crawling through confined geometries. Thus the nucleus plays a role in developing mechanical territories and niches, affecting rates of wound healing and allowing cells to transmigrate through tissues for developmental, repair or pathological means. (C) 2009 Elsevier Ltd. All rights reserved.

Substrate Topography Induces a Crossover from 2D to 3D Behavior in Fibroblast Migration

Marion Ghibaudo, Lea Trichet, Jimmy Le Digabel, Alain Richert, Pascal Hersen, Benoit Ladoux

In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are plated on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin 11 activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally, the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars.

Mechanical forces induced by the transendothelial migration of human neutrophils

Aleksandr Rabodzey, Pilar Alcaide, Francis W. Luscinskas, Benoit Ladoux

The mechanisms regulating neutrophil transmigration of vascular endothelium are not fully elucidated, but involve neutrophil firm attachment and passage through endothelial cell-cell junctions. The goal of this study was to characterize the tangential forces exerted by neutrophils during transendothelial migration at cell-cell junctions using an in vitro laminar shear flow model in which confluent activated endothelium is grown on a microfabricated pillar substrate. The tangential forces are deduced from the measurement of pillar deflection beneath the endothelial cell-cell junction as neutrophils transmigrate. The force diagram displays an initial force increase, which coincides with neutrophil penetration into the intercellular space and formation of a gap in VE-cadherin staining. This is followed by a rapid and large increase of traction forces exerted by endothelial cells on the substrate in response to the transmigration process and the disruption of cell-cell contacts. The average maximum force exerted by an actively transmigrating neutrophil is three times higher than the force generated by an adherent neutrophil that does not transmigrate. Furthermore, we show that substrate rigidity can modify the mechanical forces induced by the transmigration of a neutrophil through the endothelium. Our data suggest that the force induced by neutrophil transmigration plays a key role in the disruption of endothelial adherens junctions.

Social life of epithelial cells

Mathieu Poujade, Erwan Grasland-Mongroin, Laurence Petitjean, Myriam Reffay, Axel Buguin, Benoit Ladoux, Pascal Silberzan

Cooperative retraction of bundled type IV pili enables nanonewton force generation

Nicolas Biais, Benoit Ladoux, Dustin Higashi, Magdalene So, Michael Sheetz

The causative agent of gonorrhea, Neisseria gonorrhoeae, bears retractable filamentous appendages called type IV pili (Tfp). Tfp are used by many pathogenic and nonpathogenic bacteria to carry out a number of vital functions, including DNA uptake, twitching motility ( crawling over surfaces), and attachment to host cells. In N. gonorrhoeae, Tfp binding to epithelial cells and the mechanical forces associated with this binding stimulate signaling cascades and gene expression that enhance infection. Retraction of a single Tfp filament generates forces of 50 - 100 piconewtons, but nothing is known, thus far, on the retraction force ability of multiple Tfp filaments, even though each bacterium expresses multiple Tfp and multiple bacteria interact during infection. We designed a micropillar assay system to measure Tfp retraction forces. This system consists of an array of force sensors made of elastic pillars that allow quantification of retraction forces from adherent N. gonorrhoeae bacteria. Electron microscopy and fluorescence microscopy were used in combination with this novel assay to assess the structures of Tfp. We show that Tfp can form bundles, which contain up to 8 - 10 Tfp filaments, that act as coordinated retractable units with forces up to 10 times greater than single filament retraction forces. Furthermore, single filament retraction forces are transient, whereas bundled filaments produce retraction forces that can be sustained. Alterations of noncovalent protein - protein interactions between Tfp can inhibit both bundle formation and high-amplitude retraction forces. Retraction forces build over time through the recruitment and bundling of multiple Tfp that pull cooperatively to generate forces in the nanonewton range. We propose that Tfp retraction can be synchronized through bundling, that Tfp bundle retraction can generate forces in the nanonewton range in vivo, and that such high forces could affect infection.

Collective migration of an epithelial monolayer in response to a model wound

M. Poujade, E. Grasland-Mongrain, A. Hertzog, J. Jouanneau, P. Chavrier, B. Ladoux, A. Buguin, P. Silberzan

Using an original microfabrication-based technique, we experimentally study situations in which a virgin surface is presented to a confluent epithelium with no damage made to the cells. Although inspired by wound-healing experiments, the situation is markedly different from classical scratch wounding because it focuses on the influence of the free surface and uncouples it from the other possible contributions such as cell damage and/or permeabilization. Dealing with Madin-Darby canine kidney cells on various surfaces, we found that a sudden release of the available surface is sufficient to trigger collective motility. This migration is independent of the proliferation of the cells that mainly takes place on the fraction of the surface initially covered. We find that this motility is characterized by a duality between collective and individual behaviors. On the one hand, the velocity fields within the monolayer are very long range and involve many cells in a coordinated way. On the other hand, we have identified very active "leader cells" that precede a small cohort and destabilize the border by a fingering instability. The sides of the fingers reveal a pluricellular actin "belt" that may be at the origin of a mechanical signaling between the leader and the followers. Experiments performed with autocrine cells constitutively expressing hepatocyte growth factor (HGF) or in the presence of exogenous HGF show a higher average velocity of the border and no leader.

Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates

Alexandre Saez, Marion Ghibaudo, Axel Buguin, Pascal Silberzan, Benoit Ladoux

The physical properties of the cellular environment are involved in regulating the formation and maintenance of tissues. In particular, substrate rigidity appears to be a key factor dictating cell response on culture surfaces. Here we study the behavior of epithelial cells cultured on microfabricated substrates engineered to exhibit an anisotropic stiffness. The substrate consists of a dense array of micropillars of oval cross-section, so that one direction is made stiffer than the other. We demonstrate how such an anisotropic rigidity can induce directional epithelial growth and guide cell migration along the direction of greatest rigidity. Regions of high tractional stress and large cellular deformations within the sheets of cells are concentrated at the edges, in particular at the two poles of the islands along their long axis, in correlation with the orientation of actin stress fibers and focal adhesions. By inducing scattering activity of epithelial cells, we show that isolated cells also migrate along the direction of greatest stiffness. Taken together, these findings show that the mechanical interactions of cells with their microenvironment can be tuned to engineer particular tissue properties.

Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow

Yunfei Cai, Nicolas Biais, Gregory Giannone, Monica Tanase, Guoying Jiang, Jake M. Hofman, Chris H. Wiggins, Pascal Silberzan, Axel Buguin, et al.

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that similar to 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and similar to 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.

Traction forces exerted through N-cadherin contacts

Arthur Ganz, Mireille Lambert, Alexandre Saez, Pascal Silberzan, Axel Buguin, Rene Marc Mege, Benoit Ladoux

Background information. Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level.<br> Results. We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a mu FSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude.<br> Conclusions. These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechano-transduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.

Micro-actuators: When artificial muscles made of nematic liquid crystal elastomers meet soft lithography

A Buguin, MH Li, P Silberzan, B. Ladoux, P Keller

The production, via soft lithography, of micron-sized thermoresponsive pillars made of side-on nematic LCE leads to the formation of surface-responsive structures. The individual, monodisperse pillars, when cut out from the surface, behave as micro-actuators, showing contraction of around 35% at the nematic to isotropic phase transition

Is the mechanical activity of epithelial cells controlled by deformations or forces?

A Saez, A Buguin, P Silberzan, B. Ladoux

The traction forces developed by cells depend strongly on the substrate rigidity. In this letter, we characterize quantitatively this effect on MDCK epithelial cells by using a microfabricated force sensor consisting in a high-density array of soft pillars whose stiffness can be tailored by changing their height and radius to obtain a rigidity range from 2 nN/mu m up to 130 nN/mu m. We find that the forces exerted by the cells are proportional to the spring constant of the pillars meaning that, on average, the cells deform the pillars by the same amount whatever their rigidity. The relevant parameter may thus be a deformation rather than a force. These dynamic observations are correlated with the reinforcement of focal adhesions that increases with the substrate rigidity.

An array of microfabricated pillars to study cell migration

A Buguin, P Chavrier, B. Ladoux, O du Roure, A Saez, P Silberzan

Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels, These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells.

Force mapping in epithelial cell migration

O du Roure, A Saez, A Buguin, RH Austin, P Chavrier, P Silberzan, B. Ladoux

We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multi pie-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 mum) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior.

Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy

B. Ladoux, JP Quivy, P Doyle, O du Roure, G Almouzni, JL Viovy

Fluorescence videomicroscopy and scanning force microscopy were used to follow, in Peal time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays. Our data provide definite information on the forces involved (a few piconewtons) and on the reaction path. DNA compaction as a function of time revealed unique features of the assembly reaction in these extracts. They imply a sequential process with at least three steps, involving DNA wrapping as the final event. An absolute and quantitative measure of the kinetic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained.

Dynamics of a tethered polymer in shear flow

PS Doyle, B. Ladoux, JL Viovy

The dynamics of a single polymer tethered to a solid surface in a shear flow was observed using fluorescently labeled DNA chains. Dramatic shear enhanced temporal fluctuations in the chain extension were observed. The rate of these fluctuations initially decreased for increasing shear rate (gamma)overdot and increased above a critical (gamma)overdot. Simulations revealed that these anomalous dynamics arise from a continual recirculating motion of the chain or cyclic dynamics. These dynamics arise from a coupling of the chain velocity in the flow direction to thermally driven fluctuations of the chain in the shear gradient direction.