Figure 4. Twinfilin’s direct binding to Capping Protein accelerates the disassociation of V-1 to promote capping of filaments.

(A, B) Seeded elongation assays comparing the rates of actin assembly from spectrin-F-actin seeds (grey) in the presence of 0.5 µM actin (10% pyrene-labeled), 25 nM muscle Capping Protein (CapZ), 500 nM V-1, and variable concentrations of mTwf1 (A) or mTwf1-11 (B) as indicated. Data shown are representative curves from experiments performed three independent times. (C) Fluorescence anisotropy measurement of 100 nM HiLyte488-labeled mTwf1 tail peptide mixed with 1 μM mouse Capping Protein (CP) and variable concentrations of CBR fragment of CARMIL or V-1. Rates for each condition averaged from three independent experiments. (D) Stopped-flow fluorescence assays measuring the kinetics of dissociation of 50 nM TAMRA-V-1 from 1 μM CP upon addition at time zero of 2.5 μM unlabeled V-1 and variable concentrations of mTwf1 as indicated. Apparent dissociation rates are listed for each condition. (E) Apparent dissociation rates of TAMRA-V-1 for different concentrations of mTwf1 are from (D); and for 12 µM mTwf1−11 = 1.0 ± 0.003 s⁻¹. Anisotropy values for each condition were averaged from five independent experiments.