Figure 4.

Vinculin modulates E-cadherin mechanosensing. (A, left) The relative number of wt, ko, and vinculin-reconstituted F9 cells that adhered to E-cadherin–COMP-coated wells after 45 min is shown. (right) Phase-contrast images of ko and reconstituted cells on E-cadherin–COMP. (B) The force-independent stiffening of E-cadherin junctions as a function of the bead–cell contact time. After increasing periods of bead–cell contact, the oscillating field (0.3 Hz at 10 Gauss) was switched on for 10 s to quantify the elastic shear modulus (Pa/nm). (C) Junctional stiffness in wt versus ko cells in response to increasing applied shear stress. After 20 min of bead–cell contact, the field strength was increased stepwise in 10-s intervals with no pause between successive changes in the magnetic field. Each data point represents the mean. (D) The force-induced stiffening of Fc–E-cadherin–coated beads bound for 20 min to wt, ko, and vinculin-reconstituted F9 cells was measured using a modulated 0.3-Hz field (20 Gauss) for 50 s. (E, left) The mean intensity profile of vinculin IF plotted against the distance from the bead center for ∼80 unforced and forced beads. (right) Total vinculin intensity above baseline at unforced and forced beads measured in a 1-µm-wide area around the maximum of fluorescence intensity (gray). Error bars represent SD (A, triplicates; B–D, n > 300 [approximately one bead/cell]; E, n = ∼80).