Article Vinculin Regulates the Recruitment and Release of Core Focal Adhesion Proteins in a Force-Dependent Manner Alex Carisey, 1 Ricky Tsang, 1,5 Alexandra M. Greiner, 2,5,6 Nadja Nijenhuis, 1,3 Nikki Heath, 1 Alicja Nazgiewicz, 1 Ralf Kemkemer, 2 Brian Derby, 3 Joachim Spatz, 2,4 and Christoph Ballestrem 1, * 1 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, England, UK 2 Department of New Materials and Biosystems, Max Planck Institute for Metals Research, Heisenbergstrasse 3, 70569 Stuttgart, Germany 3 School of Materials, University of Manchester, Oxford Road, Manchester M13 9PL, England, UK 4 Department of Biophysical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany Summary Background: Cells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory proteins that, all together, form focal adhesions (FAs). The molecular basis of how these sensing units are regulated, how they are implicated in transducing mechanical stimuli, and how this leads to a spatiotemporal coordination of FAs is unclear. Results: Here we show that vinculin, through its links to the talin-integrin complex and F-actin, regulates the transmission of mechanical signals from the extracellular matrix to the acto- myosin machinery. We demonstrate that the vinculin interac- tion with the talin-integrin complex drives the recruitment and release of core FA components. The activation state of vinculin is itself regulated by force, as underscored by our observation that vinculin localization to FAs is dependent on actomyosin contraction. Using a variety of vinculin mutants, we establish which components of the cell-matrix adhesion network are coordinated through direct and indirect associa- tions with vinculin. Moreover, using cyclic stretching, we demonstrate that vinculin plays a key role in the transmission of extracellular mechanical stimuli leading to the reorganiza- tion of cell polarity. Of particular importance is the actin- binding tail region of vinculin, without which the cell’s ability to repolarize in response to cyclic stretching is perturbed. Conclusions: Overall our data promote a model whereby vinculin controls the transmission of intracellular and extracel- lular mechanical cues that are important for the spatiotem- poral assembly, disassembly, and reorganization of FAs to coordinate polarized cell motility. Introduction The ability of cells to communicate with their environment is essential for all developmental and physiological processes. Cells sense the chemical and mechanical properties of their environment through cell-matrix adhesion sites known as FAs. In FAs, integrins, which are the main adhesion receptors binding to extracellular matrix proteins, are linked to the actin cytoskeleton by a large number of FA plaque proteins [1, 2]. The appearance of FAs is dependent on the tension exerted by the contractile actomyosin machinery [3, 4]. Inhibition of pathways that lead to myosin II activation results in the disas- sembly of adhesion clusters [5, 6], indicating that tensile forces contribute to the stability of FAs. However, the way that cells sense and transmit forces that lead to the reorganization of these structures is not clear. Vinculin is one of the core FA proteins appearing in the early stages of FA formation in small dot-like adhesion complexes at the cell periphery that mature into larger streak-like FAs [7]. The presence of talin is required for vinculin recruitment to FAs [8, 9], and paxillin may contribute to this [10]. Through interactions with the talin-integrin complex and the actin cyto- skeleton [11–13], vinculin is ideally positioned to coordinate force-induced signals. The hypothesis that vinculin is part of the force machinery regulating FAs derives from the observa- tions that its recruitment to FAs correlates with subcellular areas of increased tensile forces in cells [14] and that tensile forces act on vinculin itself [15]. However, the precise function of vinculin as a force-transducing protein remains unclear. Structurally, vinculin consists of a headpiece and a tail region separated by a flexible proline-rich neck region [12]. It can adopt either an inactive, globular conformation or an active, extended conformation. In the inactive state, a head- tail interaction masks binding sites [16]. The activated form of vinculin primarily localizes to FAs [17] where the binding sites for its many partners, including talin and a-actinin (which bind to the head domain), VASP, vinexins, ponsin, and Arp2/3 (which bind to the neck region), and paxillin, F-actin, and PIP 2 (which bind to the tail domain) are exposed [18]. The interac- tion with talin, the main regulator of integrin activation [19], is essential for the role of vinculin in FA stabilization [20], whereas binding of vinculin to F-actin contributes to the ability of the cell to exert tensile forces on the extracellular matrix (ECM). Binding of both actin and talin together is hypothesized to be necessary for the full activation of vinculin [21, 22]. In this study, we test the hypothesis that vinculin coordi- nates force-mediated signals. We used a combination of techniques, including atomic force spectroscopy and a variety of imaging methods, to show how vinculin coordinates core FA proteins that regulate polarized cell migration. In addition, we show that vinculin, via its actin binding tail, is involved in the transmission of mechanical stimuli, which is essential for cellular responses to cyclic stretching forces. Results Vinculin Activity Governs the Recruitment and Release of FA Components In previous experiments, we have shown that vin880, a vinculin form without its C-terminal actin-binding tail (Figure 1A), stabi- lizes FAs such that they become resistant to tension-releasing 5 These authors contributed equally to this work 6 Present address: Department of Cell Biology and Neurobiology, Institute of Zoology, Karlsruhe Institute of Technology, Haid-und-Neu-Strasse 9 (MRI), 76131 Karlsruhe, Germany *Correspondence: christoph.ballestrem@manchester.ac.uk Current Biology 23, 271–281, February 18, 2013 ª2013 Elsevier Ltd. Open access under CC BY license. http://dx.doi.org/10.1016/j.cub.2013.01.009