Rôle des isoformes non musculaires de la Myosine-II dans la biogenèse des jonctions adhérentes et la migration collective

Adherens junction formation and remodeling is essential for many biological processes like embryo compaction, tissue morphogenesis and wound healing. It is now well described that non-muscle myosin II (NMII) acts as a mechanical support and force-generator for E-cadherin junctions during collective migration and morphogenesis. However, the contribution of NMII during early steps of junction formation remains obscure, probably because of the technical difficulty to catch such a transient event. In this work, we investigate the role of non-muscle myosin II isoforms (NMIIA and NMIIB) during adherens junction biogenesis in MDCK cells, using an in vitro reductionist approach. This system, based on chemically switchable micropatterns allows a spatio-temporal control of adherens junction formation. Our observations on MDCK cells show that the cells form irreversible E-cadherin based contacts, junction elongation is accompanied by the repolarization of actin cytoskeleton and nucleus-centrosome axis. Using isoform-specific ShRNA for NMIIA and IIB, we show that they have distinct contributions to junction formation and dynamics. 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.La formation et le remodelage des jonctions intercellulaires sont essentiels pour de nombreux processus biologiques tels que la compaction et la morphogenèse de l’embryon, la formation et la cicatrisation des tissus, le maintien de l’homéostasie tissulaire. Il est maintenant bien décrit que la myosine II non musculaire (NMII) agit comme un générateur de force et un support mécanique pour les jonctions adherens (E-cadhérine-dépendantes) lors de la migration collective et de la morphogenèse. Cependant, la contribution de NMII pendant les premières étapes de la formation de jonctions adherens reste mal connue, probablement en raison de la difficulté technique à capter un tel évènement transitoire mais complexe. Dans ce travail, nous avons étudié le rôle des isoformes non musculaires de la myosine II (NMIIA et NMIIB) au cours de la biogenèse des jonctions adherens dans les cellules MDCK, en utilisant une approche réductionniste in vitro. Cette approche, basée sur l’utilisation de substrats de culture micropatternés, chimiquement activables, mais permit un contrôle spatio-temporel de la formation des contacts intercellulaires. Mes travaux montrent que les cellules forment des contacts irréversibles base de E-cadhérine. L’élongation de ces contacts est accompagnée de la repolarisation du cytosquelette d’actine et de l’axe noyau-centrosome. En utilisant des shRNA spécifiques aux isoformes NMIIA et IIB, j’ai montré que ces deux isoformes ont contributions distinctes la formation et la dynamique des jonctions. NMIIA et NMIIB régulent différemment la biogenèse des jonctions par association avec des réseaux d'actine distincts. L'analyse de la dynamique des jonctions, de l'organisation de l'actine et des forces mécaniques a révélé que NMIIA fournit la force de traction mécanique nécessaire au renforcement et la maintenance des jonctions cellulaires. Le NMIIB est impliquée dans le clustering de la E-cadhérine, le maintien d'une couche d'actine branchée reliant les complexes de cadhérine et les fibres d'actine péri-jonctionnelles conduisant la création d'un stress mécanique anisotrope. Ces données révèlent des fonctions complémentaires imprévues de NMIIA et NMIIB dans la biogenèse et l'intégrité des jonctions adherens

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

International audienceN-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

Myosin II isoforms play distinct roles in adherens junction biogenesis

\u3cp\u3eAdherens 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.\u3c/p\u3

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

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Nature of active forces in tissues: how contractile cells can form extensile monolayers

Actomyosin machinery endows cells with contractility at a single cell level. However, at a tissue scale, cells can show either contractile or extensile behaviour based on the direction of pushing or pulling forces due to neighbour interactions or substrate interactions. Previous studies have shown that a monolayer of fibroblasts behaves as a contractile system 1 while a monolayer of epithelial cells 2,3 or neural crest cells behaves as an extensile system. 4 How these two contradictory sources of force generation can coexist has remained unexplained. Through a combination of experiments using MDCK (Madin Darby Canine Kidney) cells, and in-silico modeling, we uncover the mechanism behind this switch in behaviour of epithelial cell monolayers from extensile to contractile as the weakening of intercellular contacts. We find that this switch in active behaviour also promotes the buildup of tension at the cell-substrate interface through an increase in actin stress fibers and higher traction forces. This in turn triggers a mechanotransductive response in vinculin translocation to focal adhesion sites and YAP (Yes-associated protein) transcription factor activation. Our studies also show that differences in extensility and contractility act to sort cells, thus determining a general mechanism for mechanobiological pattern formation during cell competition, morphogenesis and cancer progression

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

International audienceThe directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis 1,2 . 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 3,4 . In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation 5–7 . Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations 5,6,8,9 can arise from single cell behavior through a sustained memory of cell polarity

Alginate Bead Based Hexagonal Close Packed 3D Implant for Bone Tissue Engineering

Success of bone tissue engineering (BTE) relies on the osteogenic microarchitecture of the biopolymeric scaffold and appropriate spatiotemporal distribution of therapeutic molecules (growth factors and drugs) inside it. However, the existing technologies have failed to address both the issues together. Keeping this perspective in mind, we have developed a novel three-dimensional (3D) implant prototype by stacking hexagonal close packed (HCP) layers of calcium alginate beads. The HCP arrangement of the beads lead to a patterned array of interconnected tetrahedral and octahedral pores of average diameter of 142.9 and 262.9 μm, respectively, inside the implant. The swelling pattern of the implants changed from isotropic to anisotropic in the <i>z</i>-direction in the absence of bivalent calcium ions (Ca²⁺) in the swelling buffer. Incubation of the implant in simulated body fluid (SBF) resulted in a 2.7-fold increase in the compressive modulus. The variation in the relaxation times as derived from the Weichert viscoelasticity model predicted a gradual increase in the interactions among the alginate molecules in the matrix. We demonstrated the tunability of the spatiotemporal drug release from the implant in a tissue mimicking porous semisolid matrix as well as in conventional drug release set up by changing the spatial coordinates of the “drug loaded depot layer” inside the implant. The therapeutic potential of the implant was confirmed against <i>Escherichia coli</i> using metronidazole as the model drug. Detailed analysis of cell viability, cell cycle progression, and cytoskeletal reorganization using osteoblast cells (MG-63) proved the osteoconductive nature of the implant. Expression of differentiation markers such as alkaline phosphatase, runx2, and collagen type 1 in human mesenchymal stem cell <i>in vitro</i> confirmed the osteogenic nature of the implant. When tested <i>in vivo</i>, VEGF loaded implant was found capable of inducing angiogenesis in a mice model. In conclusion, the bead based implant may find its utility in non-load-bearing BTE