Figure 2. Cofilin fragments... | Rockefeller University Press

Figure 2.

$Cofilin fragments fascin-induced bundles slower than single filaments. (A) Schematics of the sequential steps to investigate fascin-induced two-filament bundle fragmentation by cofilin. Inside a microfluidic chamber, actin filaments were grown from randomly positioned surface-anchored seeds and aligned by the flow (step 1). Once elongated, filaments were aged and allowed to form two-filament bundles in the presence of fascin and actin for 15 min (step 2). They are then exposed to cofilin, actin, and fascin (step 3). Note that isolated filaments did not bundle and were used as reference single filaments for side-by-side comparison. (B) Result from a typical experiment showing the fragmentation, over time, of single actin filaments (n = 53) and two-filament bundles (n = 47) upon exposure to 200 nM cofilin, 0.15 µM actin, and 200 nM fascin. 95% confidence intervals are shown as shaded surfaces. There is an approximately ninefold difference in the rate of decay of those two populations, as obtained by single exponential fits (lines). (C) Fragmentation rates of single actin filaments and two-filament bundles when exposed to 200 nM cofilin. Rates are obtained from exponential fits as shown in B. Dashed lines indicate paired data points from single filament and bundle populations acquired simultaneously in the same microfluidics chamber (N = 3 independent experiments; n = 53, 21, 29 single filaments; n = 47, 21, 30 two-filament bundles). Rates and error bars are obtained from exponential fits as shown in B. (D) Actin filaments were polymerized from spectrin–actin seeds adsorbed onto micron-sized glass beads to create larger bundles in an experiment otherwise similar to the one shown in A. Note that non-productive spectrin–actin seeds on beads are targeted by cofilin (blue), as revealed by the cofilin fluorescence appearing on the surface of the bead. (E) Survival fractions of intact 5-µm long segments of single actin filaments (n = 12) or filament bundles (n = 30, average size 9.3 (±3.2) filaments per bundle), upon exposure to 200 nM cofilin and 200 nM fascin, as a function of time. 95% confidence intervals are shown as shaded surfaces. There is a ∼40-fold difference in the rates at which these two populations decrease, as obtained by single exponential fits (lines). (F) Schematics of the reactions that lead to cofilin-induced severing. The fragmentation of single filaments (left) results from the severing of one cofilin cluster, whereas the fragmentation of two-filament bundles (right) requires the severing of two “co-localized” cofilin clusters, one on each filament.$

Cofilin fragments fascin-induced bundles slower than single filaments. (A) Schematics of the sequential steps to investigate fascin-induced two-filament bundle fragmentation by cofilin. Inside a microfluidic chamber, actin filaments were grown from randomly positioned surface-anchored seeds and aligned by the flow (step 1). Once elongated, filaments were aged and allowed to form two-filament bundles in the presence of fascin and actin for 15 min (step 2). They are then exposed to cofilin, actin, and fascin (step 3). Note that isolated filaments did not bundle and were used as reference single filaments for side-by-side comparison. (B) Result from a typical experiment showing the fragmentation, over time, of single actin filaments (n = 53) and two-filament bundles (n = 47) upon exposure to 200 nM cofilin, 0.15 µM actin, and 200 nM fascin. 95% confidence intervals are shown as shaded surfaces. There is an approximately ninefold difference in the rate of decay of those two populations, as obtained by single exponential fits (lines). (C) Fragmentation rates of single actin filaments and two-filament bundles when exposed to 200 nM cofilin. Rates are obtained from exponential fits as shown in B. Dashed lines indicate paired data points from single filament and bundle populations acquired simultaneously in the same microfluidics chamber (N = 3 independent experiments; n = 53, 21, 29 single filaments; n = 47, 21, 30 two-filament bundles). Rates and error bars are obtained from exponential fits as shown in B. (D) Actin filaments were polymerized from spectrin–actin seeds adsorbed onto micron-sized glass beads to create larger bundles in an experiment otherwise similar to the one shown in A. Note that non-productive spectrin–actin seeds on beads are targeted by cofilin (blue), as revealed by the cofilin fluorescence appearing on the surface of the bead. (E) Survival fractions of intact 5-µm long segments of single actin filaments (n = 12) or filament bundles (n = 30, average size 9.3 (±3.2) filaments per bundle), upon exposure to 200 nM cofilin and 200 nM fascin, as a function of time. 95% confidence intervals are shown as shaded surfaces. There is a ∼40-fold difference in the rates at which these two populations decrease, as obtained by single exponential fits (lines). (F) Schematics of the reactions that lead to cofilin-induced severing. The fragmentation of single filaments (left) results from the severing of one cofilin cluster, whereas the fragmentation of two-filament bundles (right) requires the severing of two “co-localized” cofilin clusters, one on each filament.

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