Humphries, J. D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043-1057

Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, England, UK.
The Journal of Cell Biology (Impact Factor: 9.83). 01/2008; 179(5):1043-57. DOI: 10.1083/jcb.200703036
Source: PubMed


Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

Download full-text


Available from: Christoph Ballestrem, Oct 04, 2015
29 Reads
    • "constitutively open conformation (Marg et al., 2010). Constitutively active T12 mutants also displayed slower exchange dynamics in focal adhesions but a similarly increased immobile fraction compared to phospho-mimicking vinculin (Cohen et al., 2006; Humphries et al., 2007). Interestingly, the force transmission in T12 mutants, in phospho-mimicking vinculin mutants and in rescue cells was similar, suggesting that the vinculin present in the focal adhesions of wild-type cells is mostly in an open, active conformation, which is in agreement with data from FRET-based experiments (Chen et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The focal adhesion protein vinculin connects the actin cytoskeleton via talin and integrin with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP, and actin-binding assays. Compared to cells expressing wildtype or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with non-phosphorylatable phenylalanine. Replacing tyrosines with phospho-mimicking glutamic acid restored cellular tractions, stiffness, adhesion strength as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 08/2015; DOI:10.1242/jcs.172031 · 5.43 Impact Factor
  • Source
    • "Moreover, experimental evidence has been presented in support of talin binding to the FAT domain of FAK via the talin-innate FERM domain, independently of FAK-paxillin binding (Lawson et al. 2012). Vinculin is essential for focal adhesions, since, on the one hand, vinculin interconnection with α-actinin yields the bonding of the β1- integrin with the filamentous-actin cytoskeleton and, on the other hand, the vinculin-talin interaction leads to membrane accumulation of activated β1-integrins concomitant with emerging focal adhesion growth (Humphries et al. 2007; Ziegler et al. 2008). Observations from mouse embryonic fibroblasts derived from vinculin knock-out animals reveal smaller focal adhesions, diminished adhesion on various ECM proteins, but faster migration as knock-out consequences (Xu et al. 1998), thereby suggesting that vinculin is important for the reinforcement of environmental cell attachment (Carisey and Ballestrem 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.
    Cell and Tissue Research 07/2014; 357(3). DOI:10.1007/s00441-014-1945-2 · 3.57 Impact Factor
  • Source
    • "More than 50 proteins have been found in focal adhesions, and these include scaffolding molecules, adapter proteins, small GTPases, kinases, phosphatases, and proteases (Zamir and Geiger, 2001). The adaptor protein vinculin, which undergoes a conformational changes when localizing to FA (Chen et al., 2005), is a pivotal regulator of focal adhesions through its interaction with other adhesion proteins (Humphries et al., 2007). Vinculin is comprised of three seven-helical bundle domains (D1, D2, and D3) and a four-helical bundle (D4) domain that is connected to a five-helical bundle (D5 or vinculin tail) domain via a proline-rich hinge region, and the D1–D4 domains form the vinculin head (Vh) domain (Bakolitsa et al., 2004; Borgon et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: C-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.
    Journal of Structural Biology 05/2014; 187(2):194-205. DOI:10.1016/j.jsb.2014.05.009 · 3.23 Impact Factor
Show more