Border forces and friction control epithelial closure dynamics

Epithelization, the process whereby an epithelium covers a cell-free surface, is not only central to wound healing but also pivotal in embryonic morphogenesis, regeneration, and cancer. In the context of wound healing, the epithelization mechanisms differ depending on the sizes and geometries of the wounds as well as on the cell type while a unified theoretical decription is still lacking. Here, we used a barrier-based protocol that allows for making large arrays of well-controlled circular model wounds within an epithelium at confluence, without injuring any cells. We propose a physical model that takes into account border forces, friction with the substrate, and tissue rheology. Despite the presence of a contractile actomyosin cable at the periphery of the wound, epithelization was mostly driven by border protrusive activity. Closure dynamics was quantified by an epithelization coefficient $D = \sigma_p/\xi$ defined as the ratio of the border protrusive stress $\sigma_p$ to the friction coefficient $\xi$ between epithelium and substrate. The same assay and model showed a high sensitivity to the RasV12 mutation on human epithelial cells, demonstrating the general applicability of the approach and its potential to quantitatively characterize metastatic transformations.Comment: 44 pages, 17 figure

Un substrat de micropiliers pour étudier la migration cellulaire

Les propriétés mécaniques des cellules jouent un rôle prépondérant dans de nombreux événements de la vie cellulaire comme le développement embryonnaire, la formation des tissus ou encore le développement des métastases. La migration cellulaire est en partie caractérisée par des interactions mécaniques. Ainsi, les forces de traction qu’exercent les cellules sur leur environnement impliquent, en parallèle, une réorganisation dynamique des processus d’adhérence et du cytosquelette interne de la cellule. Pour évaluer ces forces, un substrat a été développé, constitué d’un réseau forte densité de micro-piliers déformables sur lequel se déplacent les cellules. Cette surface est fabriquée par des méthodes de lithographie empruntées à la micro-électronique. Les piliers mesurent environ un micromètre et sont en caoutchouc, donc suffisamment déformables pour fléchir sous l’effet des forces exercées par les cellules. L’analyse au microscope des déflexions individuelles de chaque pilier a permis de quantifier en temps réel les forces locales que des cellules exercent sur leur substrat lors de leurs processus d’adhérence et de dissociation.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

Local light-activation of the Src oncoprotein in an epithelial monolayer promotes collective extrusion

International audienceTransformed isolated cells are usually extruded from normal epithelia and subsequently eliminated. However, multicellular tumors outcompete healthy cells, highlighting the importance of collective effects. Here, we investigate this situation in vitro by controlling in space and time the activity of the Src oncoprotein within a normal Madin-Darby Canine Kidney (MDCK) epithelial cell monolayer. Using an optogenetics approach with cells expressing a synthetic light-sensitive version of Src (optoSrc), we reversibly trigger the oncogenic activity by exposing monolayers to well-defined light patterns. We show that small populations of activated optoSrc cells embedded in the non-transformed monolayer collectively extrude as a tridimensional aggregate and remain alive, while the surrounding normal cells migrate towards the exposed area. This phenomenon requires an interface between normal and transformed cells and is partially reversible. Traction forces show that Src-activated cells either actively extrude or are pushed out by the surrounding cells in a non-autonomous way

Mathematical description of bacterial traveling pulses

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on E. coli have shown precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at a macroscopic scale. This modeling task is complemented with numerical simulations in accordance with the experimental observations. Our model is derived from an accurate kinetic description of the mesoscopic run-and-tumble process performed by bacteria. This model can account for recent experimental observations with E. coli. Qualitative agreements include the asymmetry of the pulse and transition in the collective behaviour (clustered motion versus dispersion). In addition we can capture quantitatively the main characteristics of the pulse such as the speed and the relative size of tails. This work opens several experimental and theoretical perspectives. Coefficients at the macroscopic level are derived from considerations at the cellular scale. For instance the stiffness of the signal integration process turns out to have a strong effect on collective motion. Furthermore the bottom-up scaling allows to perform preliminary mathematical analysis and write efficient numerical schemes. This model is intended as a predictive tool for the investigation of bacterial collective motion

Mechanical cell competition kills cells via induction of lethal p53 levels.

Cell competition is a quality control mechanism that eliminates unfit cells. How cells compete is poorly understood, but it is generally accepted that molecular exchange between cells signals elimination of unfit cells. Here we report an orthogonal mechanism of cell competition, whereby cells compete through mechanical insults. We show that MDCK cells silenced for the polarity gene scribble (scrib(KD)) are hypersensitive to compaction, that interaction with wild-type cells causes their compaction and that crowding is sufficient for scrib(KD) cell elimination. Importantly, we show that elevation of the tumour suppressor p53 is necessary and sufficient for crowding hypersensitivity. Compaction, via activation of Rho-associated kinase (ROCK) and the stress kinase p38, leads to further p53 elevation, causing cell death. Thus, in addition to molecules, cells use mechanical means to compete. Given the involvement of p53, compaction hypersensitivity may be widespread among damaged cells and offers an additional route to eliminate unfit cells.This work was supported by a Cancer Research UK Programme Grant (EP and LW A12460), a Royal Society University Research fellowship to EP (UF0905080), a Wellcome Trust PhD studentship to I.K, a Cambridge Cancer Centre PhD studentship to MG and Core grant funding from the Wellcome Trust (092096) and CRUK (C6946/A14492).This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/ncomms1137

Mathematical description of bacterial traveling pulses

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on {\em E. coli} have shown precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at a macroscopic scale. This modeling task is complemented with numerical simulations in accordance with the experimental observations. Our model is derived from an accurate kinetic description of the mesoscopic run-and-tumble process performed by bacteria. This model can account for recent experimental observations with {\em E. coli}. Qualitative agreements include the asymmetry of the pulse and transition in the collective behaviour (clustered motion versus dispersion). In addition we can capture quantitatively the main characteristics of the pulse such as the speed and the relative size of tails. This work opens several experimental and theoretical perspectives. Coefficients at the macroscopic level are derived from considerations at the cellular scale. For instance the stiffness of the signal integration process turns out to have a strong effect on collective motion. Furthermore the bottom-up scaling allows to perform preliminary mathematical analysis and write efficient numerical schemes. This model is intended as a predictive tool for the investigation of bacterial collective motion

Modeling E. coli Tumbles by Rotational Diffusion. Implications for Chemotaxis

The bacterium Escherichia coli in suspension in a liquid medium swims by a succession of runs and tumbles, effectively describing a random walk. The tumbles randomize incompletely, i.e. with a directional persistence, the orientation taken by the bacterium. Here, we model these tumbles by an active rotational diffusion process characterized by a diffusion coefficient and a diffusion time. In homogeneous media, this description accounts well for the experimental reorientations. In shallow gradients of nutrients, tumbles are still described by a unique rotational diffusion coefficient. Together with an increase in the run length, these tumbles significantly contribute to the net chemotactic drift via a modulation of their duration as a function of the direction of the preceding run. Finally, we discuss the limits of this model in propagating concentration waves characterized by steep gradients. In that case, the effective rotational diffusion coefficient itself varies with the direction of the preceding run. We propose that this effect is related to the number of flagella involved in the reorientation process

Etalement de polymeres liquides: une etude experimentale sur surfaces solides de haute energie et sur surfaces chimiquement modifiees

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78328 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Collective migration of epithelial cells

Non UBCUnreviewedAuthor affiliation: Institut CurieFacult

Croissance et densification d'un épithélium en géométrie confinée

Un épithélium est un tissu formé de cellules étroitement juxtaposées dont la fonction est d'isoler des organes entre eux ou vis-à-vis du milieu extérieur. Nous étudions la croissance d un épithélium en géométrie confinée. En utilisant des techniques de microfabrication, nous avons développé un protocole de traitement de surface permettant de confiner un tissu dans une zone adhésive pendant plusieurs semaines. La résolution spatiale de cette technique est micrométrique, et nous autorise la conception de motifs adhésifs de diverses géométries. Dans notre étude, leurs tailles sont telles que les cellules s'y comportent collectivement. Nous analysons la croissance d un épithélium de cellules Madine Derby Canine (MDCK) dans des domaines adhésifs circulaires. La migration et la densification du tissu sont étudiées par PIV (Particle image velocimetry) et d autres techniques d analyse d image. Nous caractérisons les champs de vitesse et observons des oscillations de grande amplitude de la vitesse dont la période correspond à l hypothèse d une onde de contraintes se propageant dans l épithélium. Nous caractérisons également l apparition d un bourrelet tridimensionnel de cellules à la périphérie de l épithélium, rappelant les premières étapes de la tubulogénèse. Dans deux autres expériences, en utilisant une géométrie inverse, nous étudions le recouvrement d'un épithélium sur une région anti-adhésive. Nous montrons que ce recouvrement nécessite un câble d actomyosine supracellulaire, et que la tension de ce câble s'opposant à une tension de surface définit une taille critique au-delà duquel le motif anti-adhésif ne peut pas être recouvert par l épithéliumEpithelium consists of closely packed cells that make up the inside or outside lining of body s surfaces. We study the growth of an epithelium in a confined geometry. Using microfabrication techniques, we developed a surface treatment protocol allowing tissue confinement inside an adhesive area over a few weeks. The technique achieves a micrometer resolution and any geometry of the adhesive area is feasible. In our study, the size is such that cells behave collectively. We analyse the growth of epithelium with Madine Derby Canine (MDCK) cells in circular adhesive regions. Migration and densification of the tissue are studied with PIV (Particle Image Velocimetry) and others image analysis techniques. We characterize velocity field and observe large amplitude oscillations of the velocity, whose period match the hypothesis of stress wave propagating through the epithelium. We also characterize the appearance of a tridimensionnal rim of cells at the periphery of the epithelium, similar to the first step of tubulogenesis. In two other experiments, using opposite geometry, we study how the epithelium can cover an anti-adhesive region. We show the covering requires a supracellular actomyosine cable, and the cable tension balanced by a surface tension defines a critical size beyond which the anti-adhesive region cannot be covered by the epitheliumPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF