Fig. 7
Plectin deficiency leads to local hypo-osmotic shock-induced changes in cell volume. A Calcein AM loaded Plec+/+p53−/− and Plec−/−p53−/− astrocytes recorded in iso-osmotic (300 mOsm) (Ai,iii) and in hypo-osmotic (200 mOsm) conditions. The pseudocolored scale of 0–255 corresponds to fluorescence intensity, with cooler colors (blue) representing lower intensity and hotter colors (red) indicating higher intensity (and concentration) of calcein. (Aii,iv) Hypo-osmotic conditions triggered an increase in calcein fluorescence. Scale bar: 20 µm. t0, time point of induction of hypo-osmotic conditions; t0 + 10 s, time point 10 s after induction of hypo-osmotic conditions. B Recording of the fluorescence intensity of calcein over time in single astrocytes, reflecting the parameters of cell volume changes, i.e., Fmax (maximal increase in the volume), F0 (normalized baseline volume), ts,max (swelling duration), the (maximal) swelling rate, and tRVD,50% (half-time of RVD). C Representative recordings of the relative fluorescence changes (F/F0 × 100%) in Plec+/+p53−/− (Ci) and Plec−/−p53−/− (Cii) astrocytes. Dark gray denotes the swelling phase (SP); RVD is shaded in light gray. D Changes in the whole-cell volume. Note that the maximum relative fluorescence increase (Fmax/F0 × 100%) (Di), ts,max (Dii), the swelling rate (Diii), and tRVD,50% (Div) were similar between Plec+/+p53−/− and Plec−/−p53−/− astrocytes. E Time-lapse sequences of local peripheral regions (LPRs). Two LPRs from (A) are shown (marked with arrowheads) displaying changes in the fluorescence intensity of calcein (in pseudocolors) in Plec+/+p53−/− and Plec−/−p53−/− astrocytes. Scale bars: 10 µm. F Representative recordings of relative cell fluorescence changes (Fmax/F0 × 100%) measured in an LPR of a single Plec+/+p53−/− (Fi) and Plec−/−p53−/− (Fii) astrocyte (swelling phase (SP) in dark gray, RVD in light gray). G Note that the duration of swelling (tS,max) was increased (Gii) and the swelling rate (Giii) of LPR was reduced in Plec−/−p53−/− compared with Plec+/+p53−/− astrocytes (*P < 0.05, Mann–Whitney U test); the maximum relative fluorescence increase (Gi) and tRVD,50% (Giv) were similar. Numbers above the boxplots denote the number of cells (D) and LPRs (G) analyzed