Figure 5
Actin dynamics after PA-Rac1 activation and deactivation.
(a) Live-cell images showing actin assembly during PA-Rac1 activation. RAW 264 cells were co-transfected with pTriEx/mCherry-PA-Rac1 and pYFP-β-actin. The left top panel shows a merged image of the phase-contrast, mCherry and YFP signals before the photoactivation of PA-Rac1, confirming the expression of mCherry-PA-Rac1 and YFP-β-actin. The laser-irradiated area is indicated with a blue rectangle. The other panels show selected time-lapse phase-contrast (upper) and YFP-β-actin images (lower; green) captured at the indicated times. Time 0 indicates the initiation of photoactivation. The bar indicates 5 μm. (b) Disassembly of actin from the same cellular region as shown in Fig. 5a after ceasing the 445 nm laser irradiation. The left top panel shows a merged image of the phase-contrast, mCherry and YFP signals obtained when the irradiation was stopped (time 0 is equivalent to time 11.7 min in Fig. 5a). The rectangle with broken lines indicates the previously irradiated area in Fig. 5a. The other panels show selected time-lapse phase-contrast (upper) and YFP-β-actin images (lower; green) captured at the indicated times. The elapsed times after ceasing irradiation are shown at the top of each panel. The bar indicates 5 μm. (c) Quantification of the YFP-β-actin level in the photoactivated area. The YFP-β-actin signal intensity ratio was calculated and is shown as a % of the control area. The graph shows the accumulation of YFP-β-actin in the photoactivated area during PA-Rac1 photoactivation; and the disappearance of YFP-β-actin from the photoactivated area after ceasing PA-Rac1 photoactivation. Data presented are the mean ± standard error of six independent experiments (n = 7).Time 0 corresponds to initiation of PA-Rac1 photoactivation. Arrow indicates the time point at which irradiation was stopped (12 min after photoactivation).
