Fig. 5. Effects of the N- and C-terminal domains of human CAP1 on actin dynamics. (A) Effect of the CAP1 domains on the turnover of F-actin. ATP-actin was polymerized as in Fig. 3A. After a steady state was reached, unlabeled ATP was added and the decline in fluorescence was recorded. 2.4 CT and 2.4 NT represent 2.4 µM CAP1-CT and 2.4 µM CAP1-NT, respectively. (B) Effect of the CAP1 domains on the rate of actin depolymerization. Gelsolincapped actin filaments (10%-pyrene labeled) were diluted in a solution containing the CAP1 domains with or without 0.1 µM of cofilin. When both CAP1 domains were simultaneously used, CAP1-NT was added at a final concentration of 1 µM (triangles, plots c and e). The gradual depolymerization was monitored as in Fig. 3B. The apparent rate constant (k_app) of each curve was calculated and plotted as a function of the amount of the respective CAP1-domains. 6 µM vitamin-D-binding protein (DBP) was used as a control actin-sequestering protein, which does not affect the depolymerization rate constant at the pointed end (Weber et al., 1994). (C,D) Effect of CAP1 domains on the rate of nucleotide exchange on G-actin. Mg-ADP-actin was mixed with cofilin or its solvent for 3 minutes. Then, ATP and CAP1 domains were simultaneously added and the exchange of actin-bound ADP to ATP was monitored as fluorescence increased. Final concentrations of actin, cofilin, CAP1-NT and ATP were 1 µM, 1.5 µM, 2 µM and 50 µM, respectively. The exponential curve was fitted for each trace and drawn in the graph C. The apparent rate constants (k_app) of exchange reactions were calculated and plotted as a function of the amount of CAP1 or CAP1-CT (D). (E) Subunit exchange assay using F-actin seeds with free barbed ends. Unlabeled F-actin was mixed with cofilin and/or the CAP1 domains, then a small amount of pyrene-labeled Mg-G-actin was immediately added. The incorporation of pyrene-actin into unlabeled F-actin was monitored as in Fig. 3C. The maximal rate (near initial rate) of each fluorescence trace was derived, normalized and plotted as a function of the amount of CAP1 or CAP1-CT. The results with the use of 1 µM cofilin were plotted by filled symbols, and those with the use of 1 µM CAP1-NT were shown by triangles.

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