Rho kinase activity promotes the interaction of fascin-1 with actin. (A) Percentage FRET efficiency of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in SW480 cells on laminin (LN) under control conditions or after inhibition of Rho kinases by Y27632. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. *P < 0.05 versus control. (B) Confocal images of the same non-fixed SW480 cell transiently expressing GFP-lifeact, before and after treatment with Y27632 (see Additional file 4, movie 1). The boxed 25 × 25 μm regions in the lefthand panels are enlarged in the zoomed right panels. Scale bars, 10 μm. (C,E) Fascin-1 and F-actin dynamics in SW480 cells transiently expressing GFP-fascin-1 and mRFP-lifeact, (C) without or (E) with Y27632 treatment (see Additional files 5 and 6, movies 2 and 3). (C,E) Left panels show representative cells from four independent confocal time-lapse movies. Scale bars, 10 μm. Right panels show zoomed images from the boxed 10 × 15 μm regions in the lefthand panels. (D,F) Fluorescence line-scan analysis of GFP-fascin-1 and mRFP-lifeact in a single filopodium from (D) control, or (E) Y27632-treated cells at three timepoints (i to iii). (C,E) Yellow arrows indicate the filopodia analyzed; (E) arrowheads indicate another example of an unstable filopodium. Scale bars, 10 μm.
Jayo et al. BMC Biology 2012 10:72 doi:10.1186/1741-7007-10-72