Journal Staff

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.Funding Agencies|Swedish Research Council for Medicine and Health|2007-34832009-66492010-3045|</p

Characterization of AQP9-induced filopodia.

<p>(A) Representative confocal images of HEK-293 cells transfected with tagRFP-AQP9 or empty vector together with GFP-Mem to label the membrane. Intensities have been adjusted linearly to visualize the relative expression and localization of both fluorophores. Scalebar 10 µm. (B) Quantification of peripheral filopodia in HEK-293 cells transfected with GFP-AQP9 or GFP-Mem. The data is presented as mean number of filopodia/µm perimeter(±SEM; n = 34–43 cells/group). (C) Representative confocal images of HEK-293 cells transfected with tagRFP-AQP9 and GFP-Mem. Images are pseudo-colored in fire scale to visualize the differences between the two vectors in the filopodia. The intensities have been adjusted linearly to visualize the relative distribution of both fluorophores. The lower panel represents enlargement of the green box. Scalebar 10 µm. (D) Ratiometric measurements of mean fluorescence intensity (MFI) in the filopodial membrane divided by MFI in the cell body membrane in HEK-293 cells transfected with both tagRFP-AQP9 and GFP-Mem. Measurement areas are illustrated in the schematic image. The data is presented as mean (± SEM, n = 51 filopodia/group). (E) Montage of a representative confocal time-lapse of a HEK-293 cell overexpressing GFP-AQP9 pseudo-colored in fire scale to visualize AQP9 localization in growing filopodia. The linear intensity has been adjusted to visualize differences in fluorescent intensity. Scalebar 2 µm. (F, left panel) An enlarged image from (E) showing the points of measurements for the profile plots presented in the right panel. (F, right panel) Intensity profile plots of filopodia during growth to visualize AQP9 accumulation in filopodial tips.</p

Disruption of actin dynamics inhibits the formation of new filopodia.

<p>(A) Representative confocal images of HEK-293 cells stably overexpressing GFP-AQP9 before and 15 min after treatment with 1 µM Cyt D or 500 nM Jasplakinolide. The red arrows point towards distended filopodia. Scalebar 10 µm. (B) Quantification of peripheral filopodia before, and 10–15 min after treatment with 1 µM Cyt D. The data is presented as mean (±SEM, n = 5–43 cells/group). (C, left panel) Quantification of the relative filopodial tip area of GFP-AQP9 expressing cells before (Ctrl), and 15 min after the addition of 1 µM Cytochalsin D. The filopodial tips are defined by the fluorescent area occupied in a 2×2 µm ROI of the filopodial tips. The data is presented as mean (±SEM) of fold change compared to untreated cells (Ctrl; n = 13–22 filopodia/group).(C, right panel) Representative examples of a filopodia before and after treatment with Cyt D. The red box illustrates the area of measurement for the data presented in the left panel. (D) A confocal time-lapse montage of GFP-AQP9 fluorescence, pseudo-colored in fire scale, in HEK-293 cells 10 min after treatment with 10 µM Cytochalsin D. The images illustrate a bleb-like protrusion that recoils back towards the cell body after treatment with actin dynamics inhibitors. The linear intensity is adjusted to visualize differences in fluorescence intensity. Scalebar 1 µm.</p

Model for AQP9-induced membrane protrusion.

<p>(A) A migrating cell with lamellipodia, filopodia, and blebs where an increased influx of water corresponds to a darker blue tone. (B1) Local accumulation of AQP9 by vesicle transport and/or lateral membrane diffusion enables a localized increased influx of water across the cell membrane. The influx is driven by an osmotic gradient, likely created by the transmembrane ion distribution (not shown). (B2) The rapid influx of water creates a localized hydrostatic pressure between the membrane and the cytoskeleton pushing the membrane outwards, thus initiating a membrane protrusion. (B3) The influx of water increases the hydrostatic pressure locally. In parallel, actin polymerization is promoted by the exposure of previously membrane-anchored barbed ends and the rapid diffusion of actin monomers in the now diluted, less viscous cytoplasm leading to an elongating filopodium. (B4) Then the rapid water-induced elongation reaches a critical distance from the actin, resulting in termination of the filopodial elongation likely due to equilibration of the water along the filopodium and loss of counter-pressure obtained from the actin cytoskeleton. (B5) The rate of the actin polymerization catches up with the water-induced protrusion and thereby stabilizes the structure. Based on the rate of water flux and equilibration, the filopodium can either protrude once more, or remain at its present length.</p

Water fluxes across AQP9 induce blebs.

<p>(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.</p

Addition of H₂O to the medium causes filopodial bleb-like protrusions.

<p>(A) Confocal time-lapse montage of a HEK-293 cell stably overexpressing GFP-AQP9. During acquisition, 20 µl of H₂O was added to the medium (2 ml) with a pipette directed towards the cell, yielding a rapid but transient reduction in local osmolarity. The images are pseudo-colored in fire scale to visualize variations in fluorescence intensity. White arrows are pointing towards a representative bleb-like protrusions formed during image acquisition. Scale bar 10 µm. (B, upper panel) An enlarged image of a single filopodium during acquisition, before and after the addition of H₂O. The white arrow shows the direction and length of measurement presented in the lower panel. The images are linearly adjusted and pseudo-colored in fire scale to visualize variations in fluorescent intensity. (B, lower panel) Intensity profile plots measured along the filopodia as shown by the white arrow. The red arrows are pointing towards peaks in fluorescent intensity before and after the addition of H₂O. (C) Quantification of the percentage of filopodia that developed filopodial bleb-like protrusions subsequent to the addition of 20 µl of H₂O after pre-treatment with AQP9-inhibitors. HEK-293 cells overexpressing GFP-AQP9 were pretreated with 1, 5 and 10 µM Hg²⁺ or with 25 µM of HTS13286. Control cells represents untreated HEK-293 cell overexpressing GFP-AQP9. Data is presented as mean (±SEM, n = 4–7 experiements/group). (D, left panel) Phase contrast images of primary human macrophages. The cell in the lower panel is treated with 25 µM of the novel AQP9 inhibitor HTS13286. (D, right panel) Cropped and inverted time lapse montage of the cells displayed in the left panel. During image acquisition 20 µl of H₂O was added to the medium (2 ml). Magenta arrows are pointing towards filopodial bleb-like protrusions. Scale bar 10 µm.</p