Figure 3. Cofilin cluster initiation is... | Rockefeller University Press

Figure 3.

$Cofilin cluster initiation is slowed down by fascin-induced filament bundling. (A) Time-lapse images of a two-filament bundle (yellow) showing the initiation of cofilin clusters (blue). (B) The fraction of 5-µm segments of single filaments (light blue, n = 63 segments) or 2.5-µm segments of two-filament bundles (dark blue, n = 47 segments) with at least one cofilin cluster, over time. 95% confidence intervals are shown as shaded surfaces. Black lines are single exponential fits. (C) The rate of initiation of cofilin clusters (log-scale), per cofilin binding site along actin filaments, measured from six independent experiments (n > 30 segments for each population), in the presence of 200 nM cofilin, 200 nM fascin, and 0.15 µM actin. For each condition, the black dot represents the average, and the error bars represent the SD of the distribution. Rates were obtained from exponential fits as shown in B. Dashed lines indicate paired data points from populations acquired simultaneously in the same microchamber. The paired data shows consistently a sixfold difference in the cofilin cluster initiation rate. The P value is from a paired t test. (D) Impact of fascin concentration on the initiation rate of cofilin clusters on two-filament bundles, normalized by the rate on single filaments (n = 4, 9, and 4 independent experiments at 100, 200, and 500 nM fascin respectively, with >20 segments analyzed for each experiment). Rates were derived as shown in B. All conditions with 200 nM cofilin and 0.15 µM actin. For each fascin concentration, the error bar represents the SD of the distribution. (E) Timelapse images showing the appearance of cofilin clusters (blue) on either a single actin filament or a seven-filament bundle (yellow) grown from a micrometer-size glass bead, in conditions similar to A. (F) Impact of bundle size on the initiation rate of cofilin clusters (per cofilin binding site), normalized by the rate on single filaments, when exposed to 200 nM cofilin, 200 nM fascin and 0.15 µM actin (N = 1 experiment, with 22, 10, 20, 40, and 25 segments analyzed for single filaments, and bundles of size 2, 3, 4–5, and 5–8 filaments, respectively). Horizontal error bars are SD for bundle size, and vertical error bars are the SD of the normalized cofilin initiation rates.$

Cofilin cluster initiation is slowed down by fascin-induced filament bundling. (A) Time-lapse images of a two-filament bundle (yellow) showing the initiation of cofilin clusters (blue). (B) The fraction of 5-µm segments of single filaments (light blue, n = 63 segments) or 2.5-µm segments of two-filament bundles (dark blue, n = 47 segments) with at least one cofilin cluster, over time. 95% confidence intervals are shown as shaded surfaces. Black lines are single exponential fits. (C) The rate of initiation of cofilin clusters (log-scale), per cofilin binding site along actin filaments, measured from six independent experiments (n > 30 segments for each population), in the presence of 200 nM cofilin, 200 nM fascin, and 0.15 µM actin. For each condition, the black dot represents the average, and the error bars represent the SD of the distribution. Rates were obtained from exponential fits as shown in B. Dashed lines indicate paired data points from populations acquired simultaneously in the same microchamber. The paired data shows consistently a sixfold difference in the cofilin cluster initiation rate. The P value is from a paired t test. (D) Impact of fascin concentration on the initiation rate of cofilin clusters on two-filament bundles, normalized by the rate on single filaments (n = 4, 9, and 4 independent experiments at 100, 200, and 500 nM fascin respectively, with >20 segments analyzed for each experiment). Rates were derived as shown in B. All conditions with 200 nM cofilin and 0.15 µM actin. For each fascin concentration, the error bar represents the SD of the distribution. (E) Timelapse images showing the appearance of cofilin clusters (blue) on either a single actin filament or a seven-filament bundle (yellow) grown from a micrometer-size glass bead, in conditions similar to A. (F) Impact of bundle size on the initiation rate of cofilin clusters (per cofilin binding site), normalized by the rate on single filaments, when exposed to 200 nM cofilin, 200 nM fascin and 0.15 µM actin (N = 1 experiment, with 22, 10, 20, 40, and 25 segments analyzed for single filaments, and bundles of size 2, 3, 4–5, and 5–8 filaments, respectively). Horizontal error bars are SD for bundle size, and vertical error bars are the SD of the normalized cofilin initiation rates.