Figure 5.
Interfilament cooperativity leads to bundle fragmentation. (A) Schematic representation of the two routes leading to the fragmentation of a two-filament actin bundle, after the first cofilin cluster has appeared, at a rate kinit,1.2Lfil, where Lfil is the segment length of each filament of the bundle. For route 1, the initial cofilin cluster severs at its two boundaries (see main text), at a rate ksev,1, before another cofilin cluster is initiated on the other filament of the bundle, in the region facing the first cofilin cluster. Subsequently, the initiation of a cluster on the remaining filament, followed by its severing, leads to bundle fragmentation, but these two final steps were never observed experimentally (shaded steps). For route 2, a cofilin cluster forms on the second filament in the region facing the first cofilin cluster before the latter severs, leading to the presence of two clusters in the same region. The sequential severing of the two clusters fragments the bundle. (B) An example illustrating the route 1 type of events: a single cofilin cluster (blue) severs a region of one filament in a two-filament bundle, while no other cofilin cluster has formed on the adjacent filament. The graph shows the intensity of the cofilin cluster in the dashed rectangle, as a function of time. (C) Fraction of bundles for which the first cofilin cluster severs, leaving behind no detectable cofilin, as a function of time. The fit of the experimental curve (see Materials and methods) yields ksev,1 = 3 × 10−3 s−1 and kinit,2 = 1 × 10−4 sub−1.s−1 (n = 71 events). The dashed line shows the best fit, using a chi-square minimization procedure (see Materials and methods). Three additional experiment repeats are shown in Fig. S10. In total, N = 4 independent experiments, with n = 101, 71, 17, 28 events, yield the following average values of ksev,1 = 1.5 × 10−3 s−1 and kinit,2 = 4.7 × 10−5 sub−1.s−1. (D) Average fluorescence intensity of cofilin clusters over a 1 µm wide segment on single filaments (light blue, n = 10) and on two-filament bundles (dark blue, n = 29), as a function of time, in the presence of 200 nM fascin and 200 nM cofilin, normalized by the maximum fluorescence intensity on single filaments. (E) Examples of cofilin intensity on a single filament (light blue) and a two-filament bundle (dark blue) as a function of time. The drop in intensity for the single filament is accompanied by the fragmentation of the single filament. For the two-filament bundle, a drop in the intensity to a lower value reveals the existence of two “co-localized” cofilin clusters. The second drop in intensity is accompanied by the complete fragmentation of the bundle. (F) Fraction of colocalized cluster regions that have not yet had a severing event, versus time (n = 31 events). The dashed line shows the best fit by a computed curve (see Materials and methods), using a chi-square minimization procedure. Three experimental repeats, with n = 31, 33, 39 events each, yield an average ksev,2 = 1.06 (±0.2).10−3 s−1 (± SD). (G) Fraction of cofilin clusters remaining after the first severing event and that have not yet had the second severing event, versus time (n = 53 events, pooled from four independent experiments). Fit of the experimental curve by a single exponential yields ksev,final = 2.25 (±0.6).10−3 s−1 (±95% confidence interval).

Interfilament cooperativity leads to bundle fragmentation. (A) Schematic representation of the two routes leading to the fragmentation of a two-filament actin bundle, after the first cofilin cluster has appeared, at a rate kinit,1.2Lfil, where Lfil is the segment length of each filament of the bundle. For route 1, the initial cofilin cluster severs at its two boundaries (see main text), at a rate ksev,1, before another cofilin cluster is initiated on the other filament of the bundle, in the region facing the first cofilin cluster. Subsequently, the initiation of a cluster on the remaining filament, followed by its severing, leads to bundle fragmentation, but these two final steps were never observed experimentally (shaded steps). For route 2, a cofilin cluster forms on the second filament in the region facing the first cofilin cluster before the latter severs, leading to the presence of two clusters in the same region. The sequential severing of the two clusters fragments the bundle. (B) An example illustrating the route 1 type of events: a single cofilin cluster (blue) severs a region of one filament in a two-filament bundle, while no other cofilin cluster has formed on the adjacent filament. The graph shows the intensity of the cofilin cluster in the dashed rectangle, as a function of time. (C) Fraction of bundles for which the first cofilin cluster severs, leaving behind no detectable cofilin, as a function of time. The fit of the experimental curve (see Materials and methods) yields ksev,1 = 3 × 10−3 s−1 and kinit,2 = 1 × 10−4 sub−1.s−1 (n = 71 events). The dashed line shows the best fit, using a chi-square minimization procedure (see Materials and methods). Three additional experiment repeats are shown in Fig. S10. In total, N = 4 independent experiments, with n = 101, 71, 17, 28 events, yield the following average values of ksev,1 = 1.5 × 10−3 s−1 and kinit,2 = 4.7 × 10−5 sub−1.s−1. (D) Average fluorescence intensity of cofilin clusters over a 1 µm wide segment on single filaments (light blue, n = 10) and on two-filament bundles (dark blue, n = 29), as a function of time, in the presence of 200 nM fascin and 200 nM cofilin, normalized by the maximum fluorescence intensity on single filaments. (E) Examples of cofilin intensity on a single filament (light blue) and a two-filament bundle (dark blue) as a function of time. The drop in intensity for the single filament is accompanied by the fragmentation of the single filament. For the two-filament bundle, a drop in the intensity to a lower value reveals the existence of two “co-localized” cofilin clusters. The second drop in intensity is accompanied by the complete fragmentation of the bundle. (F) Fraction of colocalized cluster regions that have not yet had a severing event, versus time (n = 31 events). The dashed line shows the best fit by a computed curve (see Materials and methods), using a chi-square minimization procedure. Three experimental repeats, with n = 31, 33, 39 events each, yield an average ksev,2 = 1.06 (±0.2).10−3 s−1 (± SD). (G) Fraction of cofilin clusters remaining after the first severing event and that have not yet had the second severing event, versus time (n = 53 events, pooled from four independent experiments). Fit of the experimental curve by a single exponential yields ksev,final = 2.25 (±0.6).10−3 s−1 (±95% confidence interval).

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