Figure 6
Persistent PA-Rac1 activation perturbs the recruitment of the Citrine-FYVE domain, a specific probe for PI(3)P, to macropinosomes.
(a) RAW 264 cells were co-transfected with pTriEx/mCherry-PA-Rac1 and pCitrine-FYVE. Photoactivation of PA-Rac1 was performed by repeated blue laser irradiation for more than 20 min. The first panel shows a merged image of the phase-contrast, mCherry (red) and Citrine (green) signals obtained before the photoactivation of PA-Rac1, confirming the expression of mCherry-PA-Rac1 and Citrine-FYVE. The 445 nm laser-irradiated area is indicated with a blue rectangle. The other panels show time-lapse merged images of the phase-contrast and Citrine-FYVE fluorescence (green), captured at the indicated times. Time 0 indicates the initiation of photoactivation. The bar indicates 5 μm. (b) After a 5-min photoactivation period, the laser irradiation was stopped. Note that the 5-min pulse photoactivation efficiently induced the recruitment of PI(3)P probes to newly formed macropinosomes. The bar indicates 5 μm.
