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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    • "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.
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    • "Focal complexes are the earliest microscopically visible integrin containing structures, which appear as spots where the diameter of the spot is in the low nanometer range, that is from $ 100 nm down to $ 30 nm or less [4]. Differently, Focal adhesions are integrin-based multiprotein complexes, and thus they typically contain a finite, but generally large, number of focal complexes, and may be therefore several micrometres in diameter [5]. Focal adhesions mechanically link the extracellular matrix with the termini of actin bundles [6] and, for their nature, are sensitive to the biochemical characteristics, rigidity and spatial organization [7] [8] of the cell/ external environment interface. "
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