Figure 5.

Reduction of Ca²⁺ results in loss of order concurrent with loss of adhesion. (a) HaCaT cells were transfected with Dsg3-ΔEA-GFP (cyan) and DP-mCherry (magenta). Cells were imaged before (0 min) and after an exchange of normal Ca²⁺ media. Shown are intensity and order factor images for time = 0 and 30 min. Scale bars, 5 μm. (b) Shown here is ROI time lapse of Dsg3-ΔEA-GFP, DP-mCherry, and order factor. Scale bars, 1 μm. (c) Given here is a plot of Dsg3-ΔEA-GFP order factor (red; mean ± SD) and the projected order factor (gray; mean ± SD) as a function of time. (d) Shown here is a pixel-by-pixel order factor plotted as a function of S/B for the ROI over the time course (time progresses from dark to light blue). (e) HaCaT cells were transfected with Dsg3-ΔEA-GFP (cyan) and DP-mCherry (magenta). Intensity and order factor images are shown for time = 0 and 30 min after switch from normal (∼3 mM) to low (∼0.03 mM) Ca²⁺ media. Scale bars, 5 μm. (f) Shown here is the time lapse of cell border ROI of Dsg3-ΔEA-GFP, DP-mCherry, and order factor showing dynamics after calcium switch. Scale bars, 1 μm. (g) Plot of Dsg3-ΔEA-GFP order factor (yellow; mean ± SD) and the projected order factor (gray; mean ± SD) from the ROI are shown as a function of time. Order factor was fit to an exponential decay (solid line) with the equation $y=0.47e^{5.5t^{-1}}$ (R² = 0.94). (h) Pixel-by-pixel order factor was plotted as a function of S/B for the ROI over the time course (time progresses from dark to light blue). (i) Population average and projected order factor were plotted as a function of time. The average Dsg3-ΔEA-GFP switch to normal calcium (red; n = 6 cells) and average Dsg3-link-GFP order factor after switch to low calcium (blue; n = 4 cells) were fit by linear regression (solid lines). The average Dsg3-ΔEA-GFP order factor switch to low Ca²⁺ (n = 8 cells) was fit with an exponential decay $y=0.38e^{{4.5}^{-t}}$ (R² = 0.97). To see this figure in color, go online.