Figure 2

Rho activity promotes the interaction of fascin-1 with actin: detection by a novel fascin-1/lifeact fluorescence resonance energy transfer (FRET) system. (A,B) Measurement of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in (A) C2C12 cells on fibronectin (FN) or (B) SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) 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 FRET. In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). (B) Representative images of GFP and lifetime plot in the absence of an acceptor, or in presence of mRFP-fascin-1S39D, which does not bundle F-actin. In each panel, lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C),Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. *P < 0.001 versus control.