Bud6(S) functions as an NPF for Bnr1 but does not affect the elongation rate of Bnr1-capped filaments. (A) Actin filament densities observed by TIRF-M (450 s after the start of the reaction) for reactions containing 0.5 µM actin + 5.0 µM yeast profilin with no further proteins (left), 0.2 nM C-Bnr1 (middle), or 0.2 nM C-Bnr1 + 100 nM Bud6(S) (right). The data shown are one representative example of three independent experiments. (B) Quantification of filament densities for reactions in A. Each bar represents a mean of nine fields of view from three independent experiments; error bars show SEM. (C) Time-lapse TIRF-M performed as in A. Yellow arrowheads indicate observed filaments. The data shown are one representative example of three independent experiments. (D) Plot of filament length versus time for individual filaments observed in C. Filaments were measured every 60 s and were normalized to a length of 0 µm at 0 s. The data shown are one representative example of three independent experiments. (E) Mean elongation rates for filaments observed as in C. Rates were determined by measuring the mean slopes of filament growth as in D. Data shown are from three independent experiments, in which for each condition in each experiment, >15 filaments were measured; error bars show SEM. (F) Bulk actin assembly assays performed as in Fig. 3 C using 2 µM actin, 5 µM yeast profilin, 2 nM C-Bnr1, and where indicated, 200 nM Bud6(S), Bud6-35(S), or Bud6-8(S). The data shown are one representative example of two independent experiments. AU, arbitrary unit. Bars, 20 µm.