Fig. 3. Effects of human CAP1 on actin dynamics. (A) His₆-CAP1 promotes the turnover of F-actin in the presence of cofilin. 6 µM 1,N⁶-etheno ATP (ATP)-actin was polymerized with or without cofilin and/or His₆-CAP1. The intensity of ATP fluorescence is higher when it is bound to actin (Wang and Taylor, 1981). After a steady state was reached, excess unlabeled ATP was added to chase actin-bound ATP, and the decline in fluorescence was recorded. Although binding of cofilin to ATP-F-actin was observed to increase the basal intensity of fluorescence, it was possible to semi-quantitatively evaluate CAP1 activity. The legends of each graph state the protein concentration in µM. `Cof' designates bacterially produced porcine cofilin without a His-tag. (B) Effect of His₆-CAP1 on the rate of actin depolymerization. Gelsolin-capped actin filaments (10%-pyrene labeled) were diluted 20-fold in the presence or absence of His₆-CAP1 and/or cofilin. The fluorescence intensity was recorded to monitor gradual depolymerization. Pyrene-labeled actin is more fluorescent when it is in F-actin than in its unpolymerized state (Kouyama and Mihashi, 1981). (C) Subunit exchange assay using F-actin seeds with free barbed ends. Unlabeled F-actin was mixed with cofilin and/or His₆-CAP1, then a small amount of pyrene-labeled Mg-G-actin was immediately added. The incorporation of pyrene-actin into unlabeled F-actin was monitored by the change in fluorescence. Final concentrations of pyrene-actin and unlabeled actin were 0.2 µM and 2.0 µM, respectively.

Return to article