The rate of initiation of cofilin clusters decreases with bundle size. (A) Size distribution of fascin-induced bundles formed from actin filaments polymerized from individual beads in the presence of 200 nM fascin. Average size = 9.7 (±4.8) filaments per bundle, n = 179 bundles. Size is determined by the actin fluorescent intensity relative to the intensity of single actin filaments (n = 10 filaments). (B) Fraction of 5-µm segment filament bundles harboring at least one cofilin cluster, over time, as a function of bundle size (single filaments n = 22, 2-fil. n = 10, 3-fil. n = 20, 4–5-fil. n = 40 and 5–8-fil. bundles n = 25). Bundle sizes were determined based on the relative fluorescence intensity of actin compared to single filaments. (C) Impact of bundle size on the rate of appearance of cofilin clusters, normalized by the rate on single filaments and by the number of filaments in bundles. Values are obtained from exponential fits of curves shown in A. Error bars are SD.