Fig. 7

SMIFH2 enhances the detachment of actin filaments from mDia1 formin in vitro. a The constitutively active mDia1 formin construct (FH1FH2DAD) was anchored to the glass surface of a microfluidic chamber by one of their FH2 domains using an anti-His antibody (as in ref. ³⁸, see Methods). Actin filaments were grown first in the presence of Alexa488-labeled actin to form fluorescent segments at their tips. Once the formin had nucleated a filament, it was exposed, from time zero onward, to 1 µM unlabeled actin and 4 µM profilin, in the absence or presence of 100 µM SMIFH2. Images show frames corresponding to the beginning (time zero, left column) and 280 s (right column) following addition of unlabeled actin into the flow chamber in the absence (top row) and presence (bottom row) of SMIFH2 inhibitor in solution. The representative fragments of epifluorescent image field of view of the labeled segments of actin filaments are seen. The number of filaments decreased with time due to their detachment from immobilized formin (see Supplementary Movies 23 and 24). Scale bar, 10 μm. b The time at which each filament detached was recorded and the survival fraction of the filaments at each time point was calculated. See Methods for more details. Graphs of the survival fraction curves with 95% confidence interval shaded area around the actual data are presented. To estimate the significance of difference between the two survival fraction curves, the logrank test³⁹ was used. Calculated p value (Python software) was equal to 0.0009 (***)