Figure 1.
Figure 1. Tension in actin filament prevents or delays severing by cofilin. (A) A schematic drawing of the experimental setup. One end of a rhodamine-labeled actin filament was tethered to a NEM-myosin–coated bead (10 µm in diameter; left) fixed on a coverslip, and the other end of the filament was tethered to a small NEM-myosin–coated bead (3 µm in diameter; right) trapped by optical tweezers. (B) A fluorescence image of an actin filament attached to a bead before application of cofilin. The filament was suspended in the flow by perfusion (from left to right). (C–E) The filament was severed at 16 s (shown by an arrow) from the onset of cofilin application (see also Video 2). In contrast, when the actin filament (D) was stretched (E) by moving the microscope stage (a bead at the bottom middle of the panel was on the trapping point), severing of the filament by cofilin was prevented (E; see alsoVideo 1). (F) The distribution of the delay between cofilin application and the severing of the nontensed actin filaments (n = 27 from 24 independent experiments). (G) The duration of time observing actin filaments without being severed by cofilin (solid bars) was significantly prolonged (P < 0.01; n = 15 from 15 independent experiments) when the filament was tensed with optical tweezers (∼30 pN). Actin filaments were not severed (for >50 s) when control F-buffer solution was perfused (hatched bars in F for nontensed filaments and in G for tensed filaments). (H) A schematic drawing of the experimental setup used to apply magnetic force to actin filament. Actin filaments are pulled toward the electric magnet (indicated by a red arrow). (I) The plot shows the number of magnetic beads in a 1-mm2 area pulled toward the electromagnet during 2 min of observation after 250 nM cofilin application. The data (mean ± SEM; n = 7) were plotted with red squares against the force (from 3.4 to 0.17 pN). A similar plot in gelsolin (25 nM; mean ± SEM; n = 12) is shown with black circles. Vertical bars denote SEM. *, P < 0.05; **, P < 0.01, using the Mann-Whitney test.

Tension in actin filament prevents or delays severing by cofilin. (A) A schematic drawing of the experimental setup. One end of a rhodamine-labeled actin filament was tethered to a NEM-myosin–coated bead (10 µm in diameter; left) fixed on a coverslip, and the other end of the filament was tethered to a small NEM-myosin–coated bead (3 µm in diameter; right) trapped by optical tweezers. (B) A fluorescence image of an actin filament attached to a bead before application of cofilin. The filament was suspended in the flow by perfusion (from left to right). (C–E) The filament was severed at 16 s (shown by an arrow) from the onset of cofilin application (see also Video 2). In contrast, when the actin filament (D) was stretched (E) by moving the microscope stage (a bead at the bottom middle of the panel was on the trapping point), severing of the filament by cofilin was prevented (E; see alsoVideo 1). (F) The distribution of the delay between cofilin application and the severing of the nontensed actin filaments (n = 27 from 24 independent experiments). (G) The duration of time observing actin filaments without being severed by cofilin (solid bars) was significantly prolonged (P < 0.01; n = 15 from 15 independent experiments) when the filament was tensed with optical tweezers (∼30 pN). Actin filaments were not severed (for >50 s) when control F-buffer solution was perfused (hatched bars in F for nontensed filaments and in G for tensed filaments). (H) A schematic drawing of the experimental setup used to apply magnetic force to actin filament. Actin filaments are pulled toward the electric magnet (indicated by a red arrow). (I) The plot shows the number of magnetic beads in a 1-mm2 area pulled toward the electromagnet during 2 min of observation after 250 nM cofilin application. The data (mean ± SEM; n = 7) were plotted with red squares against the force (from 3.4 to 0.17 pN). A similar plot in gelsolin (25 nM; mean ± SEM; n = 12) is shown with black circles. Vertical bars denote SEM. *, P < 0.05; **, P < 0.01, using the Mann-Whitney test.

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