Fig. 2: Effect of twinfilin on capping protein (CP)—formin decision complex.
From: Multicomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein
a Experimental strategy schematic. Actin filaments were nucleated from coverslip-anchored formins by flowing 1 µM G-actin (15% Alexa-488 labeled) and 0.5 µM profilin. Filaments were then exposed to flow containing 1 µM unlabeled G-actin, 4 µM profilin, and 1 µM CP for ~10 s to convert formin-bound barbed ends (BF) to formin-CP-bound barbed ends (BF + C → BFC). BFC complexes were then exposed to flow containing PA only or with a range of mTwf1 concentrations. b Representative filament kymographs are transitioning to the BFC state upon exposure to 1 µM CP. Upon CP removal from solution and exposure to PA (with or without mTwf1), filaments resume elongation following CP dissociation from BFC (top, BFC → BF + C) or detach from formin (bottom, BFC → BC + F). White arrowheads denote BFC complex dissociation. c Dissociation fraction of formin-CP-bound filaments (BFC) as a function of time in the presence of PA with/without a range of mTwf1 concentrations. Experimental data (symbols) are fitted to a single-exponential function (lines) to determine the rate of BFC disassembly. Number of filaments analyzed per condition (0 to 1 µM mTwf1): 31, 51, 30, 44, 42, and 50. d BFC dissociation rate into BC or BF as a function of mTwf1 concentration, determined from (c). e Fraction of BFC (black symbols) filaments transitioning to BF (magenta symbols) or BC (yellow symbols). Experimental data (symbols) are fitted to exponential fits (lines), such that k-BFC = k’-F + k’-C, where k’-F is the formin dissociation rate from BFC (BFC → BC + F) and k’-C is CP dissociation rate from BFC (BFC → BF + C). Conditions – left (0 nM mTwf1, 50 filaments), center (50 nM mTwf1, 37 filaments), right (1 µM mTwf1, 30 filaments). See Supplementary Fig. 2 for the range of mTwf1 concentrations. f Dissociation rate of CP (k’-C) or Formin (k’-F) from BFC complexes g Percentages of BFC complexes in (e) transitioning to BC (yellow) or BF (magenta) at different mTwf1 concentrations. Error bars in (d) indicate 65% confidence intervals based on fits (see methods). Source data are provided as a Source Data file.
