Fig. 4: Visualization and characterization of twinfilin’s interactions with CP-bound barbed ends.

a Schematic of three-color single-molecule experiments with labeled CP and labeled mTwf1. Actin filaments were assembled from of 1 µM G-actin (15% Alexa-488 labeled, 1.4% biotin-labeled) and then capped by 10 nM 649-CP. These filaments were then exposed to 20 nM 549-mTwf1. The green arrow denotes polymerization. b Binding of labeled twinfilin on CP-bound filament barbed ends recorded at 1 s time resolution. Kymographs show Alexa-488 actin (top), 649-CP (second from top), 549-mTwf1 (third from top), and merge (bottom). Magenta bars denote episodes in which a 549-mTwf1 molecule was present at the 649-CP-bound barbed end of the filament. c Schematic of the two-color single-molecule experiments with unlabeled CP and labeled mTwf1. Actin filaments were polymerized from 1 µM G-actin (15% Alexa-488 labeled, 1.4% biotin-labeled) and 0.5 µM profiling, then capped by unlabeled CP. The capped filaments were then exposed to 15 nM 549-mTwf1. Arrival and departure of 549-mTwf1 molecules at the barbed end were recorded until the filament started depolymerizing, i.e., CP dissociated. The green arrow denotes polymerization, and the red arrow denotes depolymerization. d Distribution of residence times of 549-mTwf1 on unlabeled CP-bound filament barbed ends (n = 510 binding events across 16 filaments). Mean dwell time = 1.9 ± 0.1 s (±sem). The histogram represents 99% of all binding events (remaining 1% outliers are not shown). 100% of binding events were included in calculations of mean dwell time. e Top: time records of 549-mTwf1 fluorescence intensity at the unlabeled CP-bound barbed end of an actin filament. Intensity is integrated over a 5 × 5-pixel square centered around the barbed end of the filament. Bottom: presence (1) or absence (0) of 549-mTwf1 molecules at the CP-649-bound filament barbed end shown above. Inset: cropped fluorescence images of a 10  ×  10-pixel box around the barbed end of the filament showing the arrival, presence, and departure of a single 549-mTwf1 molecule at the CP-bound barbed end. Source data are provided as a Source Data file.