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

Tissue fusion over non-adhering surfaces

Tissue fusion eliminates physical voids in a tissue to form a continuous structure and is central to many processes in development and repair. Fusion events in vivo, particularly in embryonic development, often involve the purse-string contraction of a pluricellular actomyosin cable at the free edge. However in vitro, adhesion of the cells to their substrate favors a closure mechanism mediated by lamellipodial protrusions, which has prevented a systematic study of the purse-string mechanism. Here, we show that monolayers can cover well-controlled mesoscopic non-adherent areas much larger than a cell size by purse-string closure and that active epithelial fluctuations are required for this process. We have formulated a simple stochastic model that includes purse-string contractility, tissue fluctuations and effective friction to qualitatively and quantitatively account for the dynamics of closure. Our data suggest that, in vivo, tissue fusion adapts to the local environment by coordinating lamellipodial protrusions and purse-string contractions

Bouncing or sticky droplets: impalement transitions on superhydrophobic micropatterned surfaces

When a liquid drops impinges a hydrophobic rough surface it can either bounce off the surface (fakir droplets) or be impaled and strongly stuck on it (Wenzel droplets). The analysis of drop impact and quasi static ''loading'' experiments on model microfabricated surfaces allows to clearly identify the forces hindering the impalement transitions. A simple semi-quantitative model is proposed to account for the observed relation between the surface topography and the robustness of fakir non-wetting states. Motivated by potential applications in microfluidics and in the fabrication of self cleaning surfaces, we finally propose some guidelines to design robust superhydrophobic surfaces.Comment: 7 pages, 5 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

Non-Amontons behavior of friction in single contacts

We report on the frictional properties of a single contact between a glassy polymer lens and a flat silica substrate covered either by a disordered or by a self-assembled alkylsilane monolayer. We find that, in contrast to common belief, the Amontons proportionality between frictional and normal stresses does not hold. Besides, we observe that the velocity dependence of the sliding stress is strongly sensitive to the structure of the silane layer. Analysis of the frictional rheology observed on both disordered and self-assembled monolayers suggests that dissipation is controlled by the plasticity of a glass-like interfacial layer in the former case, and by pinning of polymer chains on the substrate in the latter one.Comment: submitted to Eur. Phys. J.

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

Chiral edge current in nematic cell monolayers

Collectively migrating cells in living organisms are often guided by their local environment, including physical barriers and internal interfaces. Well-controlled in vitro experiments have shown that, when confined in adhesive stripes, monolayers of moderately active spindle-shaped cells self-organize at well-defined angle to the stripes' longitudinal direction and spontaneously give rise to a simple shear flow, where the average cellular orientation smoothly varies across the system. However, the impact of physical boundaries on highly active, chaotic, multicellular systems is currently unknown, despite its potential relevance. In this work, we show that human fibrosarcoma cells (HT1080) close to an interface exhibit a spontaneous edge current with broken left-right symmetry, while in the bulk the cell flow remains chaotic. These localized edge currents result from an interplay between nematic order, microscopic chirality, and topological defects. Using a combination of in vitro experiments, numerical simulations, and theoretical work, we demonstrate the presence of a self-organized layer of thorn 1/2 defects anchored at the boundary and oriented at a well-defined angle close to, but smaller than, 90 degrees with respect to the boundary direction. These self-organized defects act as local sources of chiral active stress generating the directed edge flows. Our work therefore highlights the impact of topology on the emergence of collective cell flows at boundaries. It also demonstrates the role of chirality in the emergence of edge flows. Since chirality and boundaries are common properties of multicellular systems, this work suggests a new possible mechanism for collective cellular flows.Theoretical Physic

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