Cofilin-1 Binding Dynamics in Actin Filaments

The procedure is described in detail in Wioland et al. (2017 (link)). Briefly, single, fluorescently labeled, rabbit alpha-skeletal actin filaments are aged for 15 minutes and exposed to eGFP-cofilin-1 in a microfluidics chamber, in F-buffer (5 mM Tris HCl pH 7.4, 50 mM KCl, 1 mM MgCl2, 0.2 mM EGTA, 0.2 mM ATP, 10 mM DTT and 1 mM DABCO), at room temperature (RT). Acquisition is performed on a Nikon TiE inverted microscope, controlled by micromanager, using epifluorescence with a 120W Xcite lamp (Lumen dynamics) and images were acquired by an sCMOS Orca-Flash4.0 camera (Hamamatsu). All experiments were performed at least twice and at least one representative movie was analyzed as described in (Wioland et al. (2017 (link)). The Welch’s unequal variances t test was used to test for significant differences in the domain growth rates, using the ‘scipy’ python package.