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Catenin Controls Vinculin Binding

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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,Rene´-Marc Me`ge5 & 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 a- catenin molecules using magnetic tweezers, we show that the subdomains MI vinculin- binding domain (VBD) to MIII unfold in three characteristic steps: a reversible step at B5pN 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 a- catenin, 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, Universite´ 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: [email protected]) or to J.Y. (email: [email protected]). NATURE COMMUNICATIONS | 5:4525 | DOI: 10.1038/ncomms5525 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5525 ell–matrix and cell–cell adhesions are required in morphogenesis during embryogenesis, tissue development 82 259 396 631 during fetal life, as well as tissue maintenance during C α adulthood1. In addition to mere cell membrane adhesion, fine- DD VBD M FABD E-catenin tuning of transmission of mechanical load from cell to 277 393 671 841 extracellular matrix (ECM) and cell to cell is also essential to 2,3 αC (275–735) these processes . The molecular mechanisms underlying cell– M ECM mechanosensing processes have been partly unraveled. Although cell–ECM mechanotransduction may rely on more global adaptation of the actomyosin viscoelastic networks4 and Biot- His 5 activation of mechanosensitive channels , pioneering works have MI(VBD) M M demonstrated the existence of integrin-associated cytoplasmic II III proteins with buried sites of phosphorylation such as p130Cas6, 7,8 and of protein–protein interactions such as talin that are Force unmasked upon myosin II-dependent stretching. The tension- dependent 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 Streptavidin have an essential role in transducing forces at cell–cell magnetic bead V junctions9,10. These complexes are tension adaptive, actin- D1 cytoskeleton-associated structures, responsive to both external load and tensile force produced by intracellular myosin α CM Biot motors11,12. The mechanism of mechanosensing at cell–cell contacts has only been very recently investigated13,14, and a- catenin 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 His 15,16 associated with impaired cadherin-mediated adhesion , tissue Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ Cu2+ growth, and homeostasis17–19. It has been recently hypothesized that aE-catenin may act as a mechanotransducer in the pathway Coverslip that converts mechanical strain on cadherin adhesions into a cue Figure 1 | Domain map and experimental setup. (a) Domain mapping of for junction strengthening11. Because vinculin accumulates at full-length and sub-domains of mouse aE-catenin. aE-catenin consists of an mature cell–cell junctions upon actomyosin generated N-terminal dimerization domain (DD), followed by vinculin-binding domain tension11,20–22 and binds aE-catenin23–25, it has been proposed (VBD; also referred to the M domain). Interaction between VBD and two that a-catenin functions in concert with vinculin. Further analysis I other modulation domains (M -M ) is suggested to inhibit vinculin binding. of cadherin adhesion strengthening by cell doublet force II III aE-catenin contains a C-terminal F-actin-binding domain (FABD) that binds separation measurement indicates that a-catenin, vinculin and to F-actin and transmits mechanical forces generated by actomyosin their direct interaction are required for tension-dependent contraction. The aC construct (residues 275–735) is enclosed in the top intercellular junction strengthening26. These proteins appear as M bracket. (b) Schematics of experimental setup. A single aC molecule is key candidates for mechanotransduction at cell–cell junctions. M tethered between a cover glass surface and a paramagnetic bead through Vinculin is a cytoplasmic actin-binding protein enriched in NTA-His tag and streptavidin-biotin linkages, respectively. The modulatory both focal adhesions and adherens junctions, essential for M (VBD) domain is shown in green, M and M domains in yellow. The embryonic development27. At focal adhesions, vinculin has a I II III vinculin-binding helix is marked in red. The V molecule, which is also a critical function in linking integrins to F-actin. Vinculin is a D1 helix bundle, is shown in silver. Force was applied to the paramagnetic bead compact globular protein composed of successive four a-helix using a pair of permanent magnets. aC constructs were stretched in the bundles. Five of these a-helix bundles constitute the vinculin head M absence or in the presence of V of various concentrations. The model of binding to various partners such as talin, whereas the C-terminal D1 aC is adopted from PDB structure 4IGG. constitutes the vinculin tail binding to F-actin. In the cytosol, M 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 binding29,34. The C-terminus of a-catenin contains an F-actin- an open conformation characterized by head to tail dissociation, binding site25,35, which associates the tertiary cadherin/b-catenin/ stabilized by binding of the head to talin and high affinity binding a-catenin complex to the actin filaments36. Although direct of the tail to F-actin28. binding has not been observed between the purified components a-Catenin is a complex protein with strong homology with the of this complex in solution37, it is still acknowledged that vinculin head domain, sharing a l-shape arrangement of a-helix a-catenin dynamically links the complex to F-actin directly, bundles29. At cell–cell junctions, b-catenin directly binds to the indirectly or both, allowing force transduction and strengthening N-terminus of a-catenin30,31 and to the intracellular tail of of adhesions13,26,38. a-Catenin binds to other actin-binding cadherins32,33, forming the cadherin/b/a-catenin complex. proteins, such as vinculin18,24,25,39, ZO-1 (refs 25,40), afadin41 a-Catenin possesses a domain of homodimerization and and formin-1 (ref. 42), through sites distributed in the central dimerizes in solution (Fig. 1a: DD domain); however, this part of the molecule. The actin-binding domain of a-catenin domain overlaps with a N-terminal b-catenin-binding domain, located at the C-terminus of the molecule (Fig. 1a: FABD and homodimerization of a-catenin is inhibited by b-catenin domain) appears to bind to the side of actin filaments, inducing 2 NATURE COMMUNICATIONS | 5:4525 | DOI: 10.1038/ncomms5525 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5525 ARTICLE conformational changes of individual filaments and preventing Thus, we provide here a molecular mechanism by which forces the binding of the branching complex Arp2/3 and the severing are locally transformed into biochemical signal at cadherin- protein cofilin43. Thus, a-catenin binding to actin may favour mediated adhesions, which has significant implications for assembly of unbranched filaments that are more protected from adherens junction assembly and regulation. severing than dynamic, branched filament arrays44. The a-catenin central domain (aCM, Fig. 1a: VBD þ M domains) is an adhesion modulation domain25,45, composed of Results 23,46 a vinculin-binding domain MI or VBD , followed by the
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