Force-dependent conformational switch of α-catenin controls vinculin binding

ARTICLE Received 4 Feb 2014 | Accepted 25 Jun 2014 | Published 31 Jul 2014 DOI: 10.1038/ncomms5525 Force-dependent conformational switch of a-catenin controls vinculin binding Mingxi Yao1,*, Wu Qiu2,3,*, Ruchuan Liu2,3, Artem K. Efremov1, Peiwen Cong1,4, Rima Seddiki5, Manon Payre5, Chwee Teck Lim1,6, Benoit Ladoux1,5, René-Marc Mège5 & Jie Yan1,3,6,7 Force sensing at cadherin-mediated adhesions is critical for their proper function. a-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 a-catenin adopts a stable auto-inhibitory conformation that makes the vinculin-binding site inaccessible. Here, by stretching single acatenin molecules using magnetic tweezers, we show that the subdomains MI vinculinbinding domain (VBD) to MIII unfold in three characteristic steps: a reversible step at B5 pN and two non-equilibrium steps at 10–15 pN. 5 pN unfolding forces trigger vinculin binding to the MI domain in a 1:1 ratio with nanomolar affinity, preventing MI domain refolding after force is released. Our findings demonstrate that physiologically relevant forces reversibly unfurl acatenin, activating vinculin binding, which then stabilizes a-catenin in its open conformation, transforming force into a sustainable biochemical signal. 1 Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore. 2 Department of Physics, National University of Singapore, Singapore 117542, Singapore. 3 College of Physics, Chongqing University, No. 55 Daxuecheng South Road, Chongqing 401331, China. 4 Singapore-MIT Alliance for Research and Technology, National University of Singapore, Singapore 117543, Singapore. 5 Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Paris 75013, France. 6 Department of Bioengineering, National University of Singapore, Singapore 117542, Singapore. 7 Centre for Bioimaging Sciences, National University of Singapore, Singapore 117546, Singapore. * These are joint first authors. Correspondence and requests for materials should be addressed to R.-M.M. (email: ) or to J.Y. (email: ). NATURE COMMUNICATIONS | 5:4525 | DOI: 10.1038/ncomms5525 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE C NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5525 ell–matrix and cell–cell adhesions are required in morphogenesis during embryogenesis, tissue development during fetal life, as well as tissue maintenance during adulthood1. In addition to mere cell membrane adhesion, finetuning of transmission of mechanical load from cell to extracellular matrix (ECM) and cell to cell is also essential to these processes2,3. The molecular mechanisms underlying cell– ECM mechanosensing processes have been partly unraveled. Although cell–ECM mechanotransduction may rely on more global adaptation of the actomyosin viscoelastic networks4 and activation of mechanosensitive channels5, pioneering works have demonstrated the existence of integrin-associated cytoplasmic proteins with buried sites of phosphorylation such as p130Cas6, and of protein–protein interactions such as talin7,8 that are unmasked upon myosin II-dependent stretching. The tensiondependent conformation switch of these proteins may thus initiate the force-dependent building of adaptor complexes linking cell–matrix adhesions to the tension-generating actomyosin network. By analogy, cadherin-associated adhesion complexes might have an essential role in transducing forces at cell–cell junctions9,10. These complexes are tension adaptive, actincytoskeleton-associated structures, responsive to both external load and tensile force produced by intracellular myosin motors11,12. The mechanism of mechanosensing at cell–cell contacts has only been very recently investigated13,14, and acatenin appears as a central component of the force transmission pathway. The aE isoform of a-catenin is expressed ubiquitously in early embryonic cells, and then restricted to epithelia. Its deletion is associated with impaired cadherin-mediated adhesion15,16, tissue growth, and homeostasis17–19. It has been recently hypothesized that aE-catenin may act as a mechanotransducer in the pathway that converts mechanical strain on cadherin adhesions into a cue for junction strengthening11. Because vinculin accumulates at mature cell–cell junctions upon actomyosin generated tension11,20–22 and binds aE-catenin23–25, it has been proposed that a-catenin functions in concert with vinculin. Further analysis of cadherin adhesion strengthening by cell doublet force separation measurement indicates that a-catenin, vinculin and their direct interaction are required for tension-dependent intercellular junction strengthening26. These proteins appear as key candidates for mechanotransduction at cell–cell junctions. Vinculin is a cytoplasmic actin-binding protein enriched in both focal adhesions and adherens junctions, essential for embryonic development27. At focal adhesions, vinculin has a critical function in linking integrins to F-actin. Vinculin is a compact globular protein composed of successive four a-helix bundles. Five of these a-helix bundles constitute the vinculin head binding to various partners such as talin, whereas the C-terminal constitutes the vinculin tail binding to F-actin. In the cytosol, vinculin is under an inactive head to tail conformation presenting only week affinity for actin. In contrast, vinculin captured at focal adhesions by force-dependent activated talin is stabilized under an open conformation characterized by head to tail dissociation, stabilized by binding of the head to talin and high affinity binding of the tail to F-actin28. a-Catenin is a complex protein with strong homology with the vinculin head domain, sharing a l-shape arrangement of a-helix bundles29. At cell–cell junctions, b-catenin directly binds to the N-terminus of a-catenin30,31 and to the intracellular tail of cadherins32,33, forming the cadherin/b/a-catenin complex. a-Catenin possesses a domain of homodimerization and dimerizes in solution (Fig. 1a: DD domain); however, this domain overlaps with a N-terminal b-catenin-binding domain, and homodimerization of a-catenin is inhibited by b-catenin 2 82 259 DD 396 VBD 631 M 393 277 αE-catenin FABD 671 841 αCM (275–735) Biot- His MI(VBD) MII MIII Force Streptavidin magnetic bead αCM VD1 Biot His Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Coverslip Figure 1 | Domain map and experimental setup. (a) Domain mapping of full-length and sub-domains of mouse aE-catenin. aE-catenin consists of an N-terminal dimerization domain (DD), followed by vinculin-binding domain (VBD; also referred to the MI domain). Interaction between VBD and two other modulation domains (MII-MIII) is suggested to inhibit vinculin binding. aE-catenin (...truncated)