Drosophila fascin S289A can rescue fascin function in nurse cells without interacting with actin. (A) Expression of wild-type (wt) fascin in sn28 mutant flies rescued the formation of actin bundles and fertility. Note that wild-type fascin (green) colocalizes with actin bundles (purple). The S289D mutation failed to rescue fertility, bundle assembly, or sterility. In contrast, fascin S289A rescued fertility but did not colocalize with actin bundles. Note that in the high magnification image (bottom), fascin S289A localized to the barbed ends of actin filaments at the cell membrane (arrows). Boxes represent the regions magnified at the bottom. (B) FRET/FLIM analysis of GFP-fascin transgenes with LifeAct-TagRFP in nurse cells. Lifetime of the GFP is represented by a color code from no interaction (blue) to high interaction (red). (C) Quantification of FRET efficiency between Drosophila fascin transgenes and F-actin. Both S289A/D significantly disrupted the ability of fascin to interact with F-actin. n = 6; ***, P < 0.001. Error bars show means ± SD. Bars, 5 µm.
