Fig. 5: Twinfilin’s direct interaction with actin filament is essential for its effects on formin-CP decision complexes.
From: Multicomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein
a Domain diagram of wild-type and mutant mTwf1 constructs used here20. “x” denotes the location of mutations. b Schematic representation of the experimental strategy. Actin filaments were nucleated from coverslip-anchored formins by introducing a flow containing 1 µM G-actin (15% Alexa-488 labeled) and 0.5 µM profilin. The filaments were then exposed to a flow containing 1 µM unlabeled G-actin, 4 µM profilin, and 500 nM CP for about 10 s to convert formin-bound barbed ends (BF) to formin-CP-bound barbed ends or decision complexes (BF + C → BFC). These BFC complexes were then exposed to a flow containing PA only or supplemented with wildtype or mutant mTwf1. c Survival fraction of formin-CP-bound filaments (BFC complexes) as a function of time in the presence of PA only (black symbols, 92 filaments), or supplemented with 1 µM wild-type mTwf1 (black, 36 filaments), mTwf1 ADF mutant (green, 77 filaments), or mTwf1 tail mutant (blue symbols, 22 filaments). Experimental data (symbols) were fitted to a single-exponential function (lines) to determine BFC dissociation rate k-BFC. Error bars indicate 65% confidence intervals based on fits (see methods). d BFC dissociation rate for wild-type and mutant mTwf1, determined from data in (c). Error bars in (d) indicate 65% confidence intervals based on fits (see Methods). Source data are provided as a Source Data file.
