The vinculin C-terminal hairpin mediates F-actin bundle formation, focal adhesion, and cell mechanical properties

Abstract

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.

FIGURE 1.

Vt NT deletions within the strap do not affect actin binding and bundling. A, binding of WT Vt to F-actin at actin concentrations ranging from 0 to 60 μm. The solid line provides visual guidance; binding to F-actin reaches a plateau at actin concentrations of ∼20 μm or higher; WT Vt concentration, 10 μm; B, compared with WT Vt, both the ΔN5 and Δstrap Vt variants show slightly enhanced actin binding. Vt variant concentration was 10 μm; actin concentration was 0–30 μm. C, Vt variants with partial (ΔN5) and complete deletion of the strap (Δstrap) show comparable actin bundling to that of WT Vt. Vt variant concentration was 10 μm; actin concentration was, 20 μm.

FIGURE 2.

Vinculin CT hairpin is critical for bundling F-actin, but not for binding F-actin. A, compared with WT Vt, Vt variants containing deletions of residues within the CT hairpin does not alter binding to F-actin. Vt variant concentration used was 10 μm; actin concentrations ranged from 0 to 30 μm; B, deletion of C-terminal residues 1062 to 1066 (Vt ΔC5) impairs Vt-induced F-actin bundling. Vt concentration was 10 μm; actin concentration was 20 μm. Vt protein was not present in the control runs; C, deletion within the Vt C-terminal hairpin reduces the amount of Vt associated with F-actin bundles. Vt concentration was 10 μm; actin concentration was 20 μm. In the absence of Vt, a protein band at ∼23 kDa was not observed in the control run.

FIGURE 3.

Visualization of actin bundles in the presence and absence of WT Vt and Vt ΔC5 using fluorescence microscopy. Removal of the CT hairpin significantly impairs F-actin bundling. A, in the absence of Vt, only F-actin fragments are observed; B, when incubated with WT Vt, F-actin forms thick and stable actin bundles; C, upon incubation with Vt ΔC5, F-actin forms randomly oriented thin fragments, similar to those observed in A. Samples were prepared using the procedure described for the actin bundling assay. The nominal actin and Vt concentrations during image acquisition are 50 and 25 nm, respectively. Scale bar, 25 μm.

FIGURE 4.

Removal of residues within the Vt C-terminal hairpin does not alter Vt conformation. A, two-dimensional ¹H-¹⁵N HSQC spectral overlay of WT Vt (black) and Vt ΔC2 (red). Note that Thr-1062 and Trp-1064 resonances show significant chemical shift changes, whereas Lys-915, Met-930, and Lys-1035 show slight chemical perturbations (within a line width). SC, side chain. The weak resonance associated with Gln-1066 represents its minor conformation. B, CD spectra overlay of WT Vt (black) and Vt ΔC2 (red). Left panel, far UV; right panel, near UV.

FIGURE 5.

Vt forms a distinct dimer (∼45 kDa) in the presence of F-actin. Cross-linking experiments were conducted at room temperature for 40 min. Final Vt concentration was 2.5 μm; BS3 concentration was 25 μm; Western blots of WT Vt and Vt ΔC5 in the presence of increasing concentrations of actin probed against Vt (A and B) or actin (C). The relative intensity ratio of the lower native dimer band to the upper (actin-induced) dimer band for WT Vt gradually increases when the A/V ratio is raised from 0 to 6. Cross-linking samples were run on SDS-PAGE gels (B) to observe the Vt, monomer band (black arrow) or Tris acetate 7% gradient gels (A and C) to observe dimer and trimer species. Although Vt ΔC5 is impaired in actin bundling, a distinct dimer is formed in the presence of actin (lower band, ∼40 kDa). The smaller dimer band is distinct in molecular weight and likely differs from the actin-induced dimer that is formed by WT Vt (C). Gray arrows in both panels indicate the positions of Vt dimer bands (gray solid arrow for the actin-induced dimer and the gray dashed arrow for the native dimer or a different dimer species in the case of Vt ΔC5). The black dashed arrow indicates a Vt trimer species.

FIGURE 6.

Vt CT hairpin deletion affects cell adhesion. A, Vin^−/− MEFs were transfected with WT or ΔC5 vinculin and plated on FN. After fixation, F-actin and FAs were stained using phalloidin and GFP-tagged vinculin variants, respectively. Scale bar is 25 μm. B–D, adhesion number per cell (B), cell area (C), and adhesion size (D) were analyzed (n = 19; *, p < 0.01). Box plots indicate median values and capture 50% of data in boxes and 80% in the lines.

FIGURE 7.

The Vt CT hairpin is necessary for the mechanical response to force on integrins. a, within measurement errors, WT vinculin and ΔC5 vinculin have comparable basal stiffness. Spring constant was calculated for the first pulse of force applied to FN-coated beads bound to Vin^−/− MEFs transfected with WT or ΔC5 vinculin (n = 15). b, upon applying pulses of a constant force, relative stiffness of Vin^−/− MEFs transfected with WT vinculin increases, whereas the stiffness of Vin^−/− MEFs transfected with ΔC5 vinculin does not change within errors. For the relative stiffness measurements, two force pulses were applied to FN-coated beads bound to Vin^−/− MEFs transfected with either WT or ΔC5 vinculin (n = 15).

FIGURE 8.

Model for actin-induced vinculin tail oligomerization and bundling of F-actin. According to our data (see Fig. 1A and supplemental Fig. S2) and the dimer model (31, 32), optimal bundling of actin by Vt is expected to occur at an A/V ratio of 2:1, assuming Vt binds to two sites on an F-actin unit and upon binding to actin, Vt dimerizes which in turn promotes bundling of actin. When actin (red) is in excess and Vt (green) is saturated, the bundling efficiency decreases as shown by our cross-linking data and F-actin bundling saturation curves. We hypothesize that the lack of reinforcement by Vt to bundle F-actin when actin is in excess, results in reduced bundling efficiency as well as formation of actin-induced dimer species.