Barbed end–capping factors inhibit actin disassembly by cofilin, coronin, and Aip1. (A) Mean filament length over time under the following conditions: 2 μM cofilin, 1 μM coronin, 5 μM of actin monomer, and 2 mM ATP, as well as 200 nM Aip1 (red), no Aip1 (green), 5 μM CapZ (yellow), 200 nM Aip1 + 1 μM CytoD (black), or 200 nM Aip1 + 5 μM CapZ (blue). Fast monomer-insensitive disassembly required Aip1 (red vs. green, yellow). CytoD and CapZ inhibited disassembly in the full system (black, blue vs. red). (B) Bar graph showing the percentage of filaments that underwent endwise bursting or severing. The number of filaments analyzed is given below the bars. The percentages of filaments that either underwent endwise bursting or severing increased significantly in the presence of Aip1 (red vs. green, yellow) and decreased significantly when the barbed-end cappers CytoD and CapZ were added to the full system (black, blue vs. red; severing frequency of −CapZ vs. +CapZ, χ² = 4.9, df = 1, P < 0.05; all other pairwise comparisons, χ² > 40, df = 1, P < 0.001). (C) Polymer mass decay for bundles with either exposed barbed ends or pointed ends in 3 μM CytoD (brown and purple) or 10 μM CapZ (blue and green), as well as polymer mass decay for all filament bundles in the absence of barbed-end cappers (red). Disassembly of bundles with either exposed barbed ends or pointed ends were inhibited by CytoD/CapZ with equal efficacy. (D) Fraction of polymer mass disassembled as a function of CytoD concentration (red circles). Best fit of the data to a hyperbola (red curve) shows an IC₅₀ of 90 nM for inhibition of disassembly. Length of filament bundles polymerized for a fixed period of time in the presence of varying concentrations of CytoD is also shown (blue squares). Hyperbolic best fit (blue curve) shows an IC₅₀ of 30 nM for inhibition of polymerization. (E) Fraction of polymer disassembled as a function of CapZ concentration (red squares). The titration curve did not reach saturation; the red curve denotes best fit of the data to a straight line. The length of filament actin bundle polymerized in the presence of varying concentrations of CapZ is also shown (blue circles). Polymerization conditions were identical to those in D. Hyperbolic best fit (blue curve) gave an IC₅₀ of 30 nM for inhibition of polymerization. Error bars indicate SD. (F) CytoD does not inhibit cofilin-mediated severing. The bar graph shows the percentage of filaments that severed during a 400 s video either in the absence of CytoD (left) or in the presence of 1 μM CytoD (right). Conditions: 8 μM cofilin and 5 μM of actin monomer in assay buffer.