Figure 6

FRAP experiments reveal widely different actin recovery half-times in transduced Ra2 cells. Ra2 cell populations as indicated were superinfected with lentivectors expressing β-actin-YFP and used for FRAP. A) Panels (top, LIMK1-WT80 cells; two lower panels, LIMK1-DN80 cells) show time-lapse series of live Ra2 cells where the ROI bleached by the laser beam is indicated by arrows (the frame before and after bleach). In the top and middle row (LIMK1-WT80 and LIMK1-DN80 cells, respectively) the ROI covers podosomal structures, while the lower panel (LIMK1-DN80 cells) shows a ROI covering the opposing plasma membranes of two cells. B) Mean pixel intensity of β-actin-YFP fluorescence is depicted over time before and after bleach (bleach is at time point three) for LIMK1-WT80 cells, together with whole cell and background intensity (white symbols), and for LIMK1-DN80 cells (black). C) Mean pixel intensity of β-actin-YFP fluorescence was fitted to a curve for FRAP recovery to obtain the mean half-time of recovery for either plasma membrane or podosome ROI's for the indicated cell lines or drug-treated cells. The ordinate shows the recovery time in seconds and mean and SEM is based on FRAP curves obtained from at least ten different cell profiles.