(A–C) Typical cases of filament severing (red arrowheads) within or near cofilin clusters (white arrowheads). The observation buffers were F-buffer containing 1 mM ADP (A and C) and 1 mM ATP (B). Concentration of cofilin was 40 nM (A and C) and 75 nM. The first break in (A) was inside a cluster, while the second was at or near the junction between a bare zone and a cluster. Red, blue, and green arrowheads show severing points within cofilin clusters, in bare zone close to cofilin clusters, and in bare zones more than half a helix away from a cofilin cluster, while white arrowheads show cofilin clusters. Bars: 25 nm; Z-scale: 0–12 nm. See Videos 6–8. (D) Classification of severing sites into four categories: (1) in ‘far’ bare zone (between a tall and a short black arrow or between two short black arrows, indicated by green bars in (E)); (2) in bare zone half helices immediately neighboring a cofilin cluster (between a tall black and a tall orange arrow, indicated by blue bars in (E)); (3) in ‘end’ cofilin cluster half helices immediately neighboring bare zones (between a tall and a short orange arrow, indicated by red bars in (E)); and (4) in ‘inner’ cofilin cluster half helices (between two short orange arrows, indicated by a red bar in (E)). Comparison of the last two categories demonstrates that severing within cofilin clusters occurs preferentially near the ends. Note, however, that this comparison does not necessarily show a quantitative difference in the susceptibility to severing between end and inner half helices, since the number of end and inner helices examined are not the same. (E) A schematic summary of the proposed distributions of bound cofilin molecules (red spheres), segments of normal (yellow) and shortened (orange) helical pitch, and normal (black arrows) and tall (orange arrows) crossover points. Free cofilin molecules tend to bind to the supertwisted bare zone on the pointed-end side of the cluster (gray arrows), driving the growth of the cluster in the pointed-end direction. This is most certainly an oversimplification, ignoring a number of complex issues, some of which are discussed in the main text.