Nanoscale architecture of integrin-based cell adhesions

Abstract

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.

Figure 1. iPALM imaging of a plasma membrane marker, integrin α_v and actin

a–c, Plasma membrane marker CAAX–tdEos. a, Top view; b, histogram and Gaussian fits for the z positions (white box in a) of PA-FP molecules (red) and nonspecific fluorescence adsorbed to substrate (blue); c, side view (red box in a). d, e, Integrin α_v–tdEos. d, Top view; e, side view (right), histogram and fits (left). f, g, Actin–mEos2. f, Top view; g, side view (right), histograms and fits (left). The vertical distribution of actin is non-Gaussian, so the focal adhesion peak fit is not shown. Colours in a, c–g indicate the vertical (z) coordinate relative to the substrate (z=0nm, red). Scale bars: 500 nm (c, e, g).

Figure 2. Protein stratification of the focal adhesion core

Top view and side view iPALM images of focal adhesions (white boxes, top-view panels) and corresponding z histograms and fits. a, b, FAK–tdEos; c, d, paxillin–tdEos; e, f, vinculin–tdEos; g, h, zyxin–mEos2; i, j, VASP–mEos2; k, l, α-actinin–mEos2. The vertical distribution of α-actinin is non-Gaussian, so the focal adhesion peak fit is not shown. Paxillin and α-actinin shown are C-terminal PA-FP-tagged (N-terminal fusions in Supplementary Figs 21 and 22). Colours: vertical (z) coordinate relative to the substrate (z=0nm, red). Scale bars: 5 μm (a, c, e, g, i, k) and 500 nm (b, d, f, h, j, l).

Figure 3. Talin orientation in focal adhesions

a, Schematic diagram, with important domains and binding sites indicated for Talin PA-FP fusions (FERM, protein 4.1, ezrin, radixin, moesin domain; VBS, vinculin binding sequence; IBS, integrin binding site). Talin-N, N-terminal fusion; Talin-C, C-terminal fusion (Supplementary Table 3). b–f, Top view and side view iPALM images of focal adhesions (white boxes, top-view panels) and corresponding z histograms and fits for talin-N–tdEos (b, c) and talin-C–tdEos (d–f). Colours: vertical (z) coordinate relative to the substrate (z=0nm, red). Scale bars: 5 μm (b, d) and 500 nm (c, e, f).

Figure 4. Nanoscale architecture of focal adhesions

a, b, Peak position (z_centre)(a) and width parameter (σ_vert)(b) of PA-FP fusions in focal adhesions. Notched boxes, 1st and 3rd quartiles, median and confidence interval; whiskers, 5th and 95th percentiles; +, means, outliers also shown. (See also Supplementary Table 1.) c, z_centre protein positions (nm) versus focal adhesion area (μm²) or aspect ratio for both N- (red) or C- (blue) terminal fusions of talin, paxillin and α-actinin. Each point corresponds to individual focal adhesion measurements. d, Schematic model of focal adhesion molecular architecture, depicting experimentally determined protein positions. Note that the model does not depict protein stoichiometry.