Fig. 3: Direct visualization of formin, CP, and mTwf1 at barbed ends.

a Representative time-lapse images of a multicolor single-molecule TIRF experiment (see Supplementary Movie 4). Actin filaments were initiated by incubation of 0.5 µM G-actin (15% Alexa-488 labeled, 0.5% biotin-labeled), 1 µM profilin, and 50 pM 649-mDia1 (magenta). 649-mDia1 bound filaments were then exposed to PA and 10 nM 549-CP (yellow) with or without mTwf1. The white box around each filament end indicates the location of the barbed end. Insets show individual and merged channels localized at the barbed end. Scale bar, 2 µm. b Fluorescence images of a 13  ×  13-pixel box around the barbed end of the filament from (a) show the formation and dissociation of a mDia1–CP complex at the barbed end. The Formin channel is magenta and the CP channel is yellow. c Fluorescence intensity and length records of the filament in (a) with formation and resolution of the decision complex in the absence of mTwf1. d Same as (c) but in the presence of 20 nM mTwf1. Gray shaded boxes indicate the period when both 549-CP and 649-mDia1 were simultaneously present at the barbed end, i.e., the BFC complex. e Distribution of lifetimes of 649-mDia1:549-CP decision complexes at barbed ends in presence of 0 nM mTwf1 (left, n = 110 BFC complexes), 5 nM mTwf1 (center, n = 60 BFC complexes), and 20 nM mTwf1 (right, n = 57 BFC complexes). f Mean lifetimes (±sem) of 649-mDia1:549-CP decision complexes at barbed ends as a function of mTwf1 concentration, determined from data in (e). g Fluorescence intensity and length records of a filament with formation and resolution of the decision complex formed in the presence of 100 pM 649-mDia1, 10 nM unlabeled CP, and 40 nM 549-mTwf1. The gray shaded box indicates the time duration when both 549-mTwf1, 649-mDia1, and unlabeled CP were simultaneously present at the barbed end (BFCT). h Cropped fluorescence images of a 13  ×  13-pixel box around the barbed end of the filament in (g) show the formation and dissociation of a 649-mDia1–unlabeled CP complex in the presence of 549-mTwf1. The formin channel is magenta and twinfilin channel is yellow. Source data are provided as a Source Data file.