Stretching single talin rod molecules activates vinculin binding

Abstract

The molecular mechanism by which a mechanical stimulus is translated into a chemical response in biological systems is still unclear. We show that mechanical stretching of single cytoplasmic proteins can activate binding of other molecules. We used magnetic tweezers, total internal reflection fluorescence, and atomic force microscopy to investigate the effect of force on the interaction between talin, a protein that links liganded membrane integrins to the cytoskeleton, and vinculin, a focal adhesion protein that is activated by talin binding, leading to reorganization of the cytoskeleton. Application of physiologically relevant forces caused stretching of single talin rods that exposed cryptic binding sites for vinculin. Thus in the talin-vinculin system, molecular mechanotransduction can occur by protein binding after exposure of buried binding sites in the talin-vinculin system. Such protein stretching may be a more general mechanism for force transduction.

Fig. 1.

(A) Structure of the 12 helices that form the TR 482 to 889 [Protein Data Bank (PDB) 1xwx]. Color coding: blue, N terminus; green, middle; and red, C terminus. VBS helices are numbered and represented with cartoon models, and the rest of the protein is shown with tube models. (B) Under the application of a force in the direction indicated by the black arrows in (A), TR starts to unfold. Once helix 12 exposed its VBS, Vh (PDB 1u6h), represented in yellow, reorganizes to bind to it. (C) X-ray structure of the complex Vh-VBS-helix 12, PDB 1u6h. Images were generated by using the VMD (Visual Molecular Dynamics) program (www.ks.uiuc.edu/research/vmd/).

Fig. 2.

Representation of the device used to measure the binding events. The Ni-NTA (labeled N) grafted slides containing the TR fixed through its 6×His N terminus (labeled H) to the glass and with the avidinated magnetic bead bound to its biotinylated C terminus (labeled B) was placed over the objective. Alexa 488–Vh was added to the slides for the period of the incubation. The TR and Vh structures are represented in green and yellow, respectively. The arrow shows the direction of the movement of the beads when they are pulled using the magnetic tweezers.

Fig. 3.

Diagram of photobleaching events of Alexa 488–Vh bound to (A) TR, (B) dimeric tandem TR, and (C) α-actinin. Histograms show the number of beads per photobleaching event. In all cases, blue, gray, and green colors correspond with no force, 2-pN force, and 12-pN force applied, respectively. The TR, talin dimeric tandem (positive control), and α-actinin (negative control) showed maximally 1 and 3, 2 and 6, and 1 and 1 photobleaching events (black arrows) when no force and 12 pN force, respectively, was applied.

Fig. 4.

AFM experiments. (A) Diagram of the polyprotein designed for AFM, I27₂–TR–I27₂. (B) Force extension trace for I27₂-TR-I27₂. (C) Histogram of the contour length increments (ΔL_c) for the unfolding peaks of the TR (120 traces). (D) Force-clamp trace obtained by stretching I27₂-TR-I27₂ at 20 and 150 pN of force. The red and black lines represent extension and force, respectively. (E) Probability of unfolding versus time for TR. Average recordings of TR unfolding at 20 pN (15 traces), 30 pN (14 traces), 40 pN (17 traces), and 50 pN (22 traces). A single exponential (colored smooth lines) is fitted to each average trace (in black). (F) The rate constant of unfolding as a function of force. Data are represented as mean ± SD; error bars were calculated by using the bootstrapping method (26).