Fluxes of Water through Aquaporin 9 Weaken Membrane-Cytoskeleton Anchorage and Promote Formation of Membrane Protrusions
Figure 7
Water fluxes across AQP9 induce blebs.
(A) A confocal time-lapse montage of HEK-293 cells stably overexpressing GFP-AQP9. The images are linearly adjusted and pseudo-colored in fire scale to visualize fluctuations in fluorescence intensity. The arrow is pointing towards GFP-AQP9 accumulation in the membrane Scalebar 10 µm. The red box represents the area of measurement for MFI in (B). A smoothing filter was applied to this image to reduce background. (B) MFI-measurement of the blebbing membrane throughout the time-lapse. The area of measurement is presented in (A). (C) Zoom in panel of the blebbing membrane presented in (A). (D) Representative confocal time-lapse montage of a blebbing HEK-293 cell expressing both GFP-AQP9 and tagRFP-LifeAct. Scalebar 5 µm. (E) The same montage as presented in (D) showing a longer acquisition time to illustrate the complete lifetime of the same bleb. (F) Ratio measurements obtained from the sequence presented in (E). The ratio is measured as AQP9 MFI in the bleb membrane divided by submembraneous actin MFI showing a rapid initial increase in ratio due to the absence of actin during bleb formation. The ratio subsequently decreases due to increasing actin fluorescence in the bleb. The insert is a representative image showing the area of measurement for AQP9 and actin.