FIGURE 4:
Effect of Capu mutations on elongation and processivity. (A–D) TIRF microscopy observation of actin filament (gray) elongation from immobilized F-actin seeds (green) in the presence of wild-type CapuCT or designated mutants. White arrowheads denote the barbed ends of fast-growing dim filaments (CapuCT bound); yellow arrowheads denote the ends of slow-growing bright filaments (no CapuCT bound). Scale bars, 10 μm. The same conditions were used for all TIRF experiments: 0.6 μM actin (20% Oregon green labeled), 1 nM CapuCT, and 3 μM Chic (Drosophila profilin). (E) Quantification of elongation rates for CapuCT and CapuCT mutants. At least 10 filaments (represented by dots) were analyzed from at least two independent experiments to obtain the mean elongation rate (represented by a line). The p values were calculated using the Mann–Whitney U test and are shown for samples that are significantly different from wild-type CapuCT. (F) Quantification of processivity shown as a decay plot. Solid lines represent the fraction of total filaments that have CapuCT bound at the barbed end. The total number of filaments analyzed for each mutant is shown in parentheses. Dashed lines represent the exponential curve fits of the data used to determine koff.