EMBO Reports
16
(2)
250-257
(2014)
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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
M S-Medecine Sciences
30
(8-9)
736-738
(2014)
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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
Journal of Cell Science
127
(8)
1660-1671
(2014)
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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.
Contact
Tissue Mechanobiology Division Prof. Benoît Ladoux Principal Investigator
Max-Planck-Zentrum für Physik und Medizin Kussmaulallee 2 91054 Erlangen, Germany
