Figure 3.
mDia1 mutants that activate SRF induce F-actin accumulation. (A) Active mDia1 mutants reorganize cellular F-actin structures. NIH3T3 cells were transfected with plasmids expressing active mDia1 derivatives (FH1/FH2, 0.1 μg; FH2, 1.0 μg) and C3 transferase (0.1 μg) as indicated. F-actin was visualized with FITC-phalloidin. The mDia proteins are shown schematically at the top right, with the essential FH2 sequences shown as boxes. Transfected cells were detected by mDia1 derivative epitope tag immunofluorescence. (B) Inactive mDia proteins do not reorganize cellular F-actin structures. NIH3T3 cells were transfected with plasmids expressing inactive mDia1 derivatives F1F2Δ1, F1F2Δ2, and F1F2ΔC2 (1.0 μg each) as indicated. F-actin was visualized with FITC-phalloidin. Transfected cells were detected by mDia1 derivative epitope tag immunofluorescence. (C) Active mDia1 proteins decrease G: F-actin ratio. NIH3T3 cells were transfected with plasmids expressing FH1/FH2 (0.1 μg), or FH2, F1F2Δ1, F1F2Δ2, and F1F2ΔC2 (1 μg) together with an expression plasmid encoding Flag-tagged wild-type β-actin (0.5 μg). Detergent-soluble and insoluble cell extract fractions were prepared as in MATERIALS AND METHODS, and the amount of Flag-actin in each evaluated by SDS-PAGE and immunoblotting with M2 anti-Flag antibodies. SwinholideA and jasplakinolide treated controls are shown in the bottom panel. A representative experiment is shown (N = 3). (D) SRF activation correlates with ability to promote F-actin assembly. NIH 3T3 cells were transfected with plasmids expressing the indicated mDia1 derivatives as above and the mean F-actin content of transfected cells determined, relative to untransfected cells in the same population, using the FACS. Data represent the mean ± SEM of three independent experiments.