Figure 2
The deactivation following activation of PA-Rac1 is required for the closure of macropinosomes.
(a) We added FDx, a fluid-phase probe, to the extracellular medium while photoactivating and incubate for 5 min followed by washing with cold PBS. Then, phase-contrast and FDx images were immediately acquired. Photoactivation was not turned off until washing out FDx. The left panel is a merged image of FDx and phase-contrast before washing out FDx. The middle (FDx, Phase merged) and right (FDx only) images were acquired immediately after washing out. Of note, FDx was largely released from the bubble-like structures by washing out. (b) FDx was added and incubated for 5 min as the same as Fig. 2a. Photoactivation was turned off 2 min before washing out FDx with PBS. A significant amount of FDx was observed in bubble-like structures after washing out. (c) After confirming bubble-like structures by the PA-Rac1 activation, FM4-64 lipophilic dye was added to the extracellular medium at different time points of 0 min (PA-Rac on), 0.5 min and 5 min after turning off the photoactivation. The phase-contrast (upper) and FM4-64 fluorescence (lower) images were acquired within 1 min after the FM 4-64 addition. The membrane of unclosed pre-macropinosomes can be labelled with FM4-64 (open arrowheads at 0 min and 0.5 min), but closed macropinosomes cannot be labelled with FM4-64 (filled arrowheads at 0.5 min and 5 min). The bar indicates 10 μm. (d) Quantitation of macropinosome closure after PA-Rac1 deactivation. By repeating the experiments shown as Fig. 2c, the percentage of FM4-64-negative closed macropinosomes in the total of phase-bright macropinocytic structures was calculated. The data presented are the mean ± standard error of four independent experiments. *p < 0.05, Student's t-test.
