ARTICLES Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells M. Reffay 1,2 , M. C. Parrini 3 , O. Cochet-Escartin 1 , B. Ladoux 2,4 , A. Buguin 1 , S. Coscoy 1 , F. Amblard 1 , J. Camonis 3,5 and P. Silberzan 1,5 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. When cells of an epithelium are presented in vitro with a free surface—created, for example, by a wound 1 or by the release of a physical barrier 2 —they migrate collectively while still maintaining strong cell–cell adhesions. This collective migration 3,4 , characterized by coordinated long-range displacements within the monolayer, often coexists with a strong fingering of the leading edge where the pluricellular migration fingers are preceded by leader cells 2,5,6 . Interestingly, similar structures are also observed in vivo in diverse situations such as morphogenesis 1 or cancer invasion 7,8 . At the start of migration, a cell initially similar to the others changes its phenotype into a ‘leader cell’. Its neighbours at the border do not undergo this transformation, and thus long migration fingers develop. The initiation of the leader cell is determined by mechanical cues resulting from, and acting on, intracellular biochemical activities 9,10 . Of particular interest in this context is the distribution of the mechanical traction forces within these fingers and at the leader cell, and the coordination of these forces and the local biochemical activity of GTPases such as RhoA and Rac1. In contrast to single- cell migration, the mechanics of collective migration has been investigated only in the past few years 11–14 . Similarly, the roles of RhoA and Rac1—whose importance in single-cell motility 15,16 and in cell mechanics 17 has long been recognized—have only recently been explored in more collective processes such as embryogenesis-related collective migration 18,19 , invasion from tumours 20 and wound-healing assays 9,21 . In this study, we directly investigated the correlation between GTPase activity and the mechanical forces in migration fingers by mapping in parallel the traction forces developed on the surface by the cells that constitute these structures and the local activity of RhoA and Rac1 in these cells. The experiments were conducted with epithelial Madin–Darby canine kidney (MDCK) cells using the ‘model wound assay’, in which cells are cultured in the apertures of micro-stencils. Peeling off these stencils triggers migration and finger formation without damaging the cells 2 (Supplementary Fig. 1A, B). Forces were then dynamically mapped using a dense array of soft micro-pillars as the underlying substrate, with each pillar acting as an independent force sensor 22 . The local activities of RhoA and Rac1 were measured at the basal plane of live cells transfected with the appropriate fluorescence resonance energy transfer (FRET)-based biosensor 23 . RESULTS Migration fingers are mechanical global entities As previously reported 2 , fingers began to form two hours after the start of the experiments. Traction forces were measured in well- formed, long, mature structures (length d 0 , 40 μm < d 0 < 100 μm, 1 Laboratoire Physico-chimie Curie—UMR 168, Institut Curie, Centre de Recherche, CNRS, UPMC, Paris, F-75248, France. 2 Laboratoire Matière et Systèmes Complexes—UMR 7057, Université Paris Diderot, CNRS, Paris, F-75251, France. 3 Analysis of Transduction Pathways Group—U830, Institut Curie, Centre de Recherche, Inserm, Paris, F-75248, France. 4 Present address: Institut Jacques Monod (IJM), CNRS UMR 7592 & Université Paris Diderot, Paris F75205, France and Mechanobiology Institute, National University of Singapore 117411, Singapore. 5 Correspondence should be addressed to J.C. or P.S. (e-mail: jacques.camonis@curie.fr or pascal.silberzan@curie.fr) Received 15 March 2013; accepted 10 January 2014; published online 23 February 2014; DOI: 10.1038/ncb2917; corrected online 26 February 2014 NATURE CELL BIOLOGY VOLUME 16 | NUMBER 3 | MARCH 2014 217 © 2014 Macmillan Publishers Limited. 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