Figure 3. In vitro activities of the FH2 domains of TgFRM1 and TgFRM2 in actin assembly.

(A) Spontaneous assembly time course of actin at 2.5 µM in the absence (control) and in the presence of 19 nM WT or mutated forms of F1 and F2 as indicated. (B) F1 concentration dependence of the nucleating activity. Actin was polymerized at 2.5 µM in the presence of F1-WT as indicated. (C) Compared nucleating efficiencies of F1, F1-R/A, F2, at different concentrations of formins. (D) The IR/AA double mutation abolishes the nucleating activity of F1. Actin was polymerized in the absence and in the presence of F1-WT or F1-IR/AA as indicated. (E) Effect of R/A and IR/AA mutations on the nucleating activity of F2. Actin was polymerized in the absence and in the presence of F2-WT, F2-R/A or F2-IR/AA as indicated. (F) F2-IR/AA blocks barbed end growth with high affinity. Barbed end growth of actin (2.5 µM) was initiated by spectrin-actin seeds in the absence and in the presence of F2-IR/AA at the indicated concentrations. (G) The initial rate of barbed end growth (from panel F and additional data) was plotted versus the concentration of F2-IR/AA. Inset: double reciprocal plot of the data, indicating that F2-IR/AA blocks barbed ends with a Kd of 6.5 nM. (H) Effect of F1 and F2 on depolymerization of filaments at barbed ends. Depolymerization of filaments was measured by diluting 40-fold a 2.5 µM F-actin solution (70% Pyrenyl-labeled) in F buffer in the absence and presence of F1-WT, F2-WT and F1-R/A as indicated.