Effect of Fhod3 on in vitro actin assembly. A, effect of Fhod3 on spontaneous assembly of actin filaments. 10% pyrene-labeled actin (2 μm) was incubated in the presence of Fhod3-ΔN-(wt), Fhod3-ΔN-(I1127A), or mDia1-FH1FH2 at a concentration of 25 nm. Actin polymerization was monitored by fluorescence enhancement of pyrene-labeled actin. B, effect of profilin on Fhod3-mediated actin assembly. Actin (2 μm) was incubated with 25 nm Fhod3-ΔN-(wt), Fhod3-ΔN-(I1127A), or mDia1-FH1FH2 in the presence of profilin (2 μm). C, effect of Fhod3 on the barbed end assembly. 20% pyrene-labeled actin (0.5 μm) was incubated with 25 nm Fhod3-ΔN-(wt) or Fhod3-ΔN-(I1127A) in the presence of spectrin-actin seeds (5 μg/ml).