Fig. 7. Plot of fluorescence intensities after photobleach. (A) Raw images of actin cones in a line bleach experiment. The data were used in the plot (B). Numbers in the upper-left corner indicate time after line bleach. Distance A (between green arrowheads) indicates distance from the front of actin cone to the front of bleached line. Distance B (between yellow arrowheads) is the distance of actin cone movement during the time indicated. Short yellow lines indicate the position of front of actin cone in each image. (B) Distances A and B of cone 1 and 2 in A are plotted. Distance A in cone 1 (black circle) and cone 2 (black square) did not change through the recording time suggesting no movement of bleached line relative to the front of the actin cone. However, distance B of cone 1 (white circle) and cone 2 (white square) increased linearly, suggesting constant movement of both actin cones. (C) Representative image of moving actin cones. Fluorescence intensity in the region indicated by rectangles (red:front and yellow:back) were measured and plotted in D. (D) Relative fluorescence intensities of GFP-actin in moving actin cones after photobleaching are plotted. A whole actin cone was photobleached and fluorescence recovery in front (black square) and back (black circle) were plotted separately. Relative intensity was calculated as fluorescence intensity at each time point/fluorescence intensity before photobleach in the region of interest. Six actin cones were examined for each plot. Bulk fluorescence of the actin cone recovers in about 12 minutes in both parts of the cone; however, the front of the cone recovers slightly more quickly than the back. (E) Representative plot of FRAP in actin cones before onset of movement. The fluorescence recovery plataued at 43.6 minutes after photobleach.

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