Effect of Cyt. D on the Toca-1/N-WASP phenotypes.
(A). Cells were transfected with Toca-1 and N-WASP cDNA as described in the Material and methods sections. After 36 hr cells were chosen for either tubulation or membrane vesicle formation. Using time-lapse microscopy the change in phenotype was followed after addition of Cyt. D (4 µM) for 60 min. Panels show images at zero time (a, a′) and at 60 min (b, b′) of a representative cell. (a′b′) are enlargements of areas in panels a–b, respectively, showing (a′b′, Cyt. D) vesicle to tubule transitions. Bar = 10 µm. (B). Cells were then scored for presence of vesicles (vesicle index), tubules (tubule index) and vesicle motility as described in the Material and methods section. (C) Shows AP-FRET analysis of Toca-1/N-WASP in tubules or vesicles after Cyt. D treatment. (a) Images show typical cells used and the ROI. Green/red tracings show changes in intensity of the ROI before and after photobleaching. (b) A statistical analysis of FRET data with controls and FRET pairs. Bar = 10 µm. (D) FRAP analysis of the protein dynamics within the tubules/vesicles, with and without Cyt. D. Images show typical cells used and the graphs below show the bleach followed by the recovery profile. Time in sec. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.