Nanoscale architecture of integrin-based cell adhesions. Nature

National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nature (Impact Factor: 41.46). 11/2010; 468(7323):580-4. DOI: 10.1038/nature09621
Source: PubMed


Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ∼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

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Available from: Gleb Shtengel
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    • "Recent advances in microscopy have allowed us to gain a deeper insight into the precise location of vinculin within a FA. Elegant super-resolution microscopy experiments have placed vinculin within a 'force-transduction layer' where it links actin filaments to the extracellular matrix (ECM), through talin and integrin[10,35]. This imaging work supports functional molecular studies that show separate roles for the head domain of vinculin in regulating integrins (through its association with talin) and of the tail in regulating the link to the actomyosin machinery[30]. "
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    ABSTRACT: External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a variety of diseases. Cells contain several types of adhesions, linking them to their external environment. It is through these adhesions that forces are both sensed (from the outside inwards) and applied (from inside to out). Furthermore, several adhesion-based proteins are sensitive to changes in intracellular forces, utilising them for activation and regulation. Here, we outline how vinculin, a key component of integrin-mediated adhesions linking the actin cytoskeleton to the extracellular matrix (ECM), is regulated by force and acts as force transducing protein. We discuss the role of vinculin in vivo and its place in health and disease; summarise the proposed mechanisms by which vinculin is recruited to and activated at integrin-ECM adhesions; and discuss recent findings that place vinculin as the major force sensing and transmitting component of cell-matrix adhesion complexes. Finally, we discuss the role of vinculin in regulating the cellular responses to both the physical properties of the external environment and to externally applied physical stimuli.
    Full-text · Article · Nov 2015 · Experimental Cell Research
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    • "The initial connection between integrins and f-actin in nascent adhesions can mature to focal complexes (FC) or eventually FA. Therefore many components must be recruited which transform the structures into signalling hubs (Hu et al., 2007; Moser et al., 2009; Kanchanawong et al., 2010; Ciobanasu et al., 2013; Schiller and Fässler, 2013; Pinon et al., 2014). Their proper spatiotemporal organisation is critical for optimal migration (Gupton and Waterman-Storer, 2006) as they integrate biochemical and mechanical signals regulating the cell behaviour (Schwartz and Horwitz, 2006; Ciobanasu et al., 2013; Lieber et al., 2013). "
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    ABSTRACT: The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required.
    Full-text · Article · Oct 2015 · European Journal of Cell Biology
    • "Adherent cells are anchored through focal adhesions to the extracellular matrix (ECM), which is essential for force transduction, cell spreading and migration (Choquet et al., 1997; Beningo et al., 2001; Bershadsky et al., 2006). Focal adhesions consist of clusters of transmembrane adhesion proteins of the integrin family (Damsky et al., 1985; Gallant et al., 2005; Kanchanawong et al., 2010) and numerous intracellular proteins including α-actinin and talin (Harburger and Calderwood, 2009). "
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    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.
    No preview · Article · Aug 2015 · Journal of Cell Science
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