Activation of p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 leads to its interaction with fascin-1 and affects formation of filopodia. (A) Measurement of the interaction of wild-type or mutant forms of green fluorescent protein (GFP)-LIMK1 with monomeric red fluorescent protein (mRFP)-fascin-1 in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). 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. (B) Percentage FRET efficiency under each experimental condition. Each column represents the mean from fourteen to seventeen cells per condition and three independent experiments; bars indicate SEM. *P = 0.001 versus wildtype. (C) Role of LIMK1 activity in organization of filopodia. Live SW480 cells transiently transfected with GFP alone or GFP-LIMK1 (wild-type (WT), D460A or T508A) and mRFP-fascin-1, and protein localizations and cell edges were imaged using confocal microscopy. Arrowheads indicate points where GFP-LIMK1 and mRFP-fascin-1 colocalize in filopodia. Scale bars, 10 μm. (D) The number/cell and (E) length of filopodia were counted from images obtained as in (C), from 12 to 20 cells per condition and 4 independent experiments. *P < 0.05 versus GFP control. See Additional files 9 and 10 (movies 6 and 7) for the effects of LIMK1 mutants on filopodia.