Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions

Abstract

In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and there are contradictory models with respect to the determinants of adhesion site localization. Here, we report turnover rates and protein-protein interactions in a range of talin rod domain constructs varying in helix bundle structure. We conclude that several bundles of the C terminus cooperate to regulate targeting and concomitantly tailor high affinity interactions of the talin rod in cell adhesions. Intrinsic control of ligand binding activities is essential for the coordination of adhesion site function of talin.

FIGURE 1.

FA localization of talin domain constructs. A, ligand binding sites for F-actin (ABS), β-integrins (IBS), vinculin (VBS), and the dimerization site (DS) of talin are shown. Constructs are indicated (bars), numbers refer to amino acid residues of the N/C terminus, and the relative strength of FA targeting is summarized (strong, weak, or none). Constructs used by others (19, 28), which we have re-analyzed for the purposes of comparison, are indicated (light gray bars). B, representative fluorescence images of GFP-tagged talin variants co-stained for vinculin to visualize FAs (red). Construct information is given as α-helices number (H) of the talin rod. Insets showing enlarged views of vinculin (v, top) or talin variants (t, middle) in FAs and the merged image (m, bottom) refer to selected regions (frames). Size bar, 20 μm (inset, 8 μm).

FIGURE 2.

Structure and FA localization of talinC domain constructs. A, top view of talinC and ribbon model of helix bundle structures for the ABS-3/DS domain (18). VBS helices H50 and H58 are highlighted in blue. B, scheme and localization observed for talinC domain constructs (see Fig. 1A); asterisks indicate an altered FA morphology, and ag indicates the observation of protein aggregates in cells. For talin H47-H56, some strongly stained FAs were observed. Size bar, 20 μm (inset, 8 μm). C, representative fluorescence images of GFP-tagged talinC constructs co-stained for vinculin (see Fig. 1B).

FIGURE 3.

Accessibility of VBS helices in talinC. In a pulldown assay, the interaction of recombinant Vh with T7-tagged talin domain constructs was determined using αT7 antibody to immobilize purified talin fragments on magnetic beads. The Ala-50 to Ile mutant of vinculin head (Vh A50I) was used as specificity control. Note the increased stability of a four-helix bundle in the Vh A50I mutant limits interactions to binding partners that expose very high affinity VBS helices. 1, H47-62; 2, H47-62:V2087A, L2091A; 3, H57-62; 4, H57-62:V2360A, I2352A; 5, H60-62; 6, H52-56; 7, H50-56; 8, H50-56:V2087A, L2091A; 9, H52-5; 10, H52-58:V2360A, I2352A; 11, H47-56; 12, H47-58; 13, H47-62 (amino acids 2524); 14, H47-51; 15, H47-51:V2087A, L2091A; 16, H47-50; 17, H47-50:V2087A, L2091A; blank, no partner added.

FIGURE 4.

FRAP analysis of GFP-tagged talinC in FAs. A, for the regions indicated, fluorescence intensities were monitored over time, and mean gray values were determined and plotted. Size bar, 10 μm. B, intensity values corrected for background and acquisition photobleaching were normalized (see the formula). Averaged curves (>10 adhesions, several cells) provided the percentage of mobile (MF) and immobile (IF) fraction of talin molecules in FAs. C, regression analysis comparing mono- and biexponential fit of fluorescence recovery revealed two kinetic pools for talinC in FAs, which represent tethered (half-life times, t_½(I) = 2.8 ± 0.5 s) and bound protein (t_½(II) = 33.5 ± 3.6 s), respectively. a.u., arbitrary units.

FIGURE 5.

FRAP analysis of talinC variants in adhesion sites. In C₂C₁₂ cells, residency times and mobility distribution of talinC and domain constructs thereof were determined using biexponential fit of normalized FRAP recovery curves. For each construct, half-life times (± S.D.) for tethered t_½(I) (gray) and bound t_½(II) (black) protein fractions are indicated together with the distribution between kinetic pools of tethered (I, light color), bound (II, dark color) and immobile protein (IF, white) in FAs. A, talinC kinetics depend on both β-integrin binding to IBS-2A/B and F-actin binding to ABS-3/DS. B, integrin binding to IBS-2A and IBS-2A/B is inhibited. C, high affinity interactions of talinC require co-stimulation of several binding partners (see “Results” for further information). a.u., arbitrary units.

FIGURE 6.

Activation model of ligand binding in the talin C terminus. FA targeting of talinC (1) is induced by low affinity binding of IBS-2A/B and ABS-3 to their respective ligands (black arrows). Activation of high affinity states in talinC (2) is reversible (bold arrows), depends on additional factors such as mechanical stress and vinculin (blue lines, blue arrow), and is abolished if low affinity ligand binding to either domain is inhibited (red).