Cofilin at low concentration quickly binds and severs oxidized actin filament.Figure 1A. Three steps of a typical experiment (see also Materials and Methods). Filaments are polymerized with 0.6-1 µM ATP-G-actin and aged for 15 minutes with ATP-G-actin at critical concentration (0.1 µM) to maintain the filament length. This solution is supplemented with MICAL1 and NADPH to oxidize filaments. Tpm can also be added at this step to fully decorate filaments B. Fraction (1/17th) of a typical field of view. In a microfluidic chamber, actin filaments (yellow) are anchored by their pointed ends and align with the flow. C. Time-lapse images showing the assembly of cofilin-1 domains (blue) and subsequent disassembly of actin filaments (yellow). The time-lapse of an oxidized filament shows filament severing at 6s; the first cofilin domain nucleation at 2s; growth of 4 individual domains as their fluorescence intensity increases; and severing (lightning symbol) at the top domain, 2s after its nucleation. D. Global measurement of the severing of filaments exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 20 (from 1 representative experiment) and 50 (1 experiment) for non-oxidized and oxidized actin filaments, respectively. P-value = 8.10-8 (log-rank test). Data information: Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.  E. Nucleation of the first cofilin domain onto 5 µm-long actin segments. Filaments are exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 60 filaments, from 1 experiment for each condition. P-value = 4.10-15 (log-rank test). Data information: Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces. F. Growth rate of single cofilin domains, normalized by the cofilin concentration. N = 50 (from 3 independent experiments) and 20 (1 experiment) domains on non-oxidized and oxidized filaments, respectively. Measurements were obtained using 20 and 100 nM cofilin (non-oxidized actin) and 10 nM cofilin (oxidized actin). Note that this normalized growth rate does not depend on the cofilin concentration (Appendix Fig S4A). Bars: mean and S.D. P-value = 7.10-8 (t-test). G. Filament severing rate at single cofilin domains. Time t=0 is defined for every domain as the frame on which they nucleate. N = 163 (from 2 independent experiments) and 203 (3 experiments) cofilin domains on non-oxidized and oxidized actin respectively. Measurements were performed at 100 nM cofilin (non-oxidized actin) and 10, 20 or 30 nM cofilin (oxidized actin). Since the severing rate does not depend on the cofilin concentration data for 10, 20 and 30 nM cofilin on oxidized filaments were pooled. Measurements on non-oxidized filaments were done at 100 nM mCherry-cofilin-1. P-value = 9.10-41 (log-rank test). Data information: Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.