Cofilin and fascin compete to bind filaments in bundles. (A) Time-lapse images showing the growth of a cofilin cluster (blue) on a two-filament bundle (yellow). (B) Cofilin cluster growth rates on single filaments and two-filament bundles, in the presence of 200 nM cofilin, 200 nM fascin, and 0.15 µM Alexa488(10%)-G-actin (N = 4 repeats, each shown in a different color, with at least 10 cofilin clusters observed in each condition). Small data points are individual measurements (one per cluster) and the large points are the averages per repeat. Dashed lines indicate paired averages from populations acquired simultaneously in the same microchamber. The P value is from a paired t test. (C) Cofilin cluster growth rates as a function of bundle size (n = 11, 4, 6, 7, 6, and 9 cofilin clusters analyzed on single filaments and bundles of an average size of 2, 3, 4, 7, and 11 filaments, respectively). Error bars are SD. (D) Time-lapse images showing a two-filament bundle with Alexa568-fascin (green) and exposed to eGFP-cofilin1 (blue). (E) Fluorescence intensity of fascin and cofilin, integrated over the region shown in D (dashed rectangle), as a function of time. (F) Schematics of the irreversible departure of fascin, caused by a cofilin cluster growing on a two-filament bundle.