Figure 7.

FRAP of the K255E mutant of α-actinin-4 in the mitotic cortex of HeLa cells. (a) Averaged long-term recovery of mutant α-actinin-4-GFP after photobleaching at the mitotic cortex: control (N = 19, blue curve), tension-reduced (N = 19, red curve, incubated with para-nitroblebbistatin at 10 μM). Fluorescence intensities were normalized. Recovery curves were averaged and fitted with an exponential recovery with a single timescale (black dashed lines). Red and blue shaded regions indicate the respective standard error of the mean for each condition. (b and c) Distribution of parameters from fitting individual recovery curves with an exponential function (same data as a): (b) distributions of fit recovery times τ (p-value: 0.6) and (c) distributions of recovering fractions. (d) Estimated fraction of cortical α-actinin-4 in control and tension-reduced conditions for an enlarged data set. Median values of fit recovery timescales are 0.6 in both conditions (control: N = 60, P.N.Bleb.: N = 59, p-value: 0.3). (e) Cortical tensions in control and blebbistatin-treated conditions corresponding to measurements presented in (a). (f) Averaged recovery of mutant α-actinin-4-GFP during 300 s after photobleaching (large data set). Depicted are normalized fluorescence intensities within the bleached cortex region ${\tilde{I}}_{bl}\left(t\right)$. Recovery curves were averaged and fitted with an exponential recovery with two timescales (black dashed lines; see Materials and Methods). Red and blue shaded regions indicate the respective standard error of the mean for each condition. (g) Decay of the bleached fraction $\left(1-{\tilde{I}}_{bl}\left(t\right)\right)$ reveals different timescales of relaxations of recovery dynamics (same data as f). Note the logarithmic y axis. (h–j) Distribution of parameters from fitting individual recovery curves with a double-exponential function including a fast and a slow recovery timescale corresponding to data in (f). (h) Distributions of fitted fast recovery times τ_f are not significantly different in control and tension-reduced conditions (p-value: 0.56). (i) Slow recovery times τ_s are significantly larger in tension-reduced conditions. (j) Distributions of the amplitude A_f of the slow recovering fraction are not significantly changed by cortical tension reduction (p-value: 0.55) (measurements are representative for at least two independent experiments. n.s., p > 0.05; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). To see this figure in color, go online.