Figure 4.

Cell-squishing experiments: fluorescence of α-actinin-4 (Actn4-EGFP) increases at the cortex after transient mechanically induced increase in actin cortex tension. (a) A schematic of the experimental procedure. Cells were mechanically perturbed by a fast lowering of the AFM cantilever onto the cell (see Materials and Methods). Thereby, a mechanical stretch of cortical area was induced. Cells were in mitotic arrest and treated with the ROCK inhibitor Y27632 (5 μM). ROCK inhibition was necessary to avoid blebbing during mechanical cell squishing. (b) Exemplary force and cantilever height readout during the experiment. As the cantilever is lowered, cellular confinement force increases. Afterwards, the confinement force relaxes to a new steady-state force. (c) An exemplary time series of confocal micrographs of the equatorial plane of the cell recorded during a squishing experiment. Fluorescence changes are typically too small to be assessed by eye. Scale bar, 10 μm. (d) Averaged time evolution of cortical tension (gray curve) and normalized α-actinin-4 fluorescence at the cortex (red curve) during and after mechanical squishing of the cell. Averages were taken over 10 cells. Red and grey shaded regions indicate the respective standard errors of the mean of normalized fluorescence and tension. Mechanical tension peaks right after cantilever lowering has been stopped. Then, tension drops because of viscoelastic relaxation of deformation-induced cortical mechanical tension (12). Concomitantly, we observe a peak of α-actinin-4 fluorescence at the cortex at ∼30 s shortly after the peak in mechanical tension at the actin cortex. (e) Our model on protein dynamics provides an excellent fit (dashed black line) to the measured time evolution of cortical α-actinin-4 fluorescence (as depicted in d). Fitting parameters are given in Table 1, second row. According to our model, the transient peak in cortical intensity at t ≈ 30 s stems from the tension increase and the resulting increase in cross-linking lifetime k_off(σ)⁻¹. The elevated stationary-state value at long times can be accounted for by an increase of the surface/volume ratio due to increased cell confinement. To see this figure in color, go online.