Rho kinase activity promotes peripheral fascin-containing protrusions via a myosin-independent process. (A) Confocal images of C2C12 cells after 1 hour of adhesion to 50 nmol/l fibronectin (FN), either untreated or pretreated with specified inhibitors, fixed and stained for fascin-1. Arrowheads indicate examples of peripheral fascin-actin bundles in Y27632-treated cells. Scale bars, 10 μm. (B) Confocal images of C2C12 cells transiently expressing green fluorescent protein (GFP)-caldesmon or an inactive GFP-caldesmon-445 mutant after 1 hour of adhesion to 50 nmol/l FN. Cells were fixed and stained either for F-actin (left panels) or fascin-1 (right panels). In the anti-fascin-1 stained samples, arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Quantification of peripheral fascin-1 bundles/cell under the conditions shown in (A) and (B). Data are from 75 to 125 cells/condition and 3 independent experiments. *P < 0.001 versus control. (D) Measurement of the interaction of mRFP-fascin-1S39A with GFP-lifeact in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, Intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (E) Percentage FRET efficiency under each experimental condition. Each column represents the mean from fourteen cells per condition and three independent experiments; bars indicate SEM.