Figure 5. The rate of actin delivery depends on the distance between profilin and the barbed end.

The experimental conditions were as follows: 0.75 μM actin (33% Oregon Green-labeled) in microscopy buffer. The data were collected by TIRF microscopy. (A) FH1-tethered profilin can bind the barbed end either via FH1-mediated actin delivery (upper reaction scheme) or via a direct binding event (lower reaction scheme). (B) Time series of TIRF micrographs of actin filament elongation in the absence and presence of PRF₃₈FH2, PRF₅₆FH2 and PRF₉₇FH2. White and red arrowheads indicate the barbed ends of free and formin-bound actin filaments, respectively. (C) Dependence of the total (black data), FH1-mediated (purple data) and FH2-mediated (orange data) barbed end elongation rates mediated by the PRF_xxFH2 constructs on the number of residues separating the profilin and Bni1p’s FH2 domain. Error bars are standard errors of the mean elongation rates measured for at least 15 formin-bound filaments. (D) Dependence of the fluorescence intensities of actin filaments polymerized by the Bni1p PRF_xxFH2 constructs on the number of residues separating the profilin and Bni1p’s FH2 domain. Fluorescence intensities of formin-bound filaments were normalized to the intensities of filaments that are not formin-bound. Error bars are standard errors of the mean intensities of at least 15 filaments.