(A) Representative time-lapse images from TIRF microscopy experiments where Oregon green-labeled actin filaments were polymerized and tethered, and then 1 μM Cof1, Cof2, or ADF was flowed in. Severing events are indicated by yellow arrow heads. (B) Analysis of severing activities, in which each data point is the cumulative severing events per micron of filament at that time point, averaged from 3 independent experiments (20 filaments each). Data were fit to an exponential association curve. (C) Average time to half-maximal severing, calculated from exponential curve fits of data in (B). Error bars, SEM. Statistical significance was determined using a one-way ANOVA analysis and Bonferroni Multiple comparison test; * indicates p < 0.05. (D) Representative time-lapse images from TIRF microscopy experiments as in (A) except using 150 nM Cof1, Cof2, or ADF. (E) Analysis of severing activities as in (B), except that data were averaged from 4 independent experiments (20 filaments each). (F) Average time to half-maximal severing, calculated as in (C).* indicates p < 0.05; ** indicates p < 0.01.