Figure 6.
Figure 6. Vimentin dynamics is regulated by Cdc42 during cell polarization. (A) Merged images of vimentin (green), tubulin (magenta), and DNA (blue) of astrocytes before or 1 h 30 min after wounding. Cells were nucleofected with indicated siRNAs (also see Fig. S3 B). The orientation of the wound is shown with dashed lines. Bars, 20 µm. (B, left) Still fluorescence (Fluo.) images 3 min after photobleaching from a FRAP experiment performed with vimentin-EGFP–expressing astrocytes 1 h 30 min after wounding. Wounding was done 4 d after cell nucleofection with the indicated siRNAs (see Video 8 for small interfering Cdc42). The contrast was increased in the zoom of Cdc42-depleted cell to better show the emergence of vimentin filaments in the photobleached region. (Right) fluorescence intensity profiles showing the fluorescence recovery at different time points after photobleaching. (C) Percentages of cells displaying a symmetric FRAP 1 h 30 min after wounding. Cells were nucleofected with the indicated siRNA or treated with DMSO, ML141 (inhibitor of Cdc42; 10 µM added 1 h before wounding), and NSC23761 (inhibitor of Rac1; 50 µM added 1 h before wounding; 44–66 cells per condition with a minimum of three independent experiments). (D, left) Still fluorescence images acquired 3 min after photobleaching of vimentin-EGFP–expressing astrocytes 10 h after wounding. Control or Cdc42-depleted cells were left treated with DMSO or with bradykinin (10 µM) 1 h before the FRAP experiment. (Right) Fluorescence intensity profiles along the corresponding arrows showing the fluorescence recovery at different time points after photobleaching. Filled arrows point to the slopes observed in the fluorescence recovery profile, indicating a symmetric or asymmetric recovery of fluorescence. Note that in the case of asymmetry, the fluorescence recovery occurs only from the rear side of the photobleached area (shown by a single filled arrow). (B and D) Bars: (main images) 10 µm; (insets) 2 µm. a.u., arbitrary units. (E) Percentages of cells displaying a symmetric profile of FRAP after 10 h of migration. Control or Cdc42-depleted cells were left treated with DMSO or bradykinin (10 µM; 1 h; 37–60 cells per condition with a minimum of three independent experiments). (F) Quantification of the vimentin retrograde flow in the experiments described in E. Error bars display SEM for the three repeats. *, P < 0.05; ***, P < 0.001. (G) Schematics representing the mechanisms involved in IF dynamics after Cdc42 activation by Bradykinin treatment. Arrows correspond with colored labels on the right.

Vimentin dynamics is regulated by Cdc42 during cell polarization. (A) Merged images of vimentin (green), tubulin (magenta), and DNA (blue) of astrocytes before or 1 h 30 min after wounding. Cells were nucleofected with indicated siRNAs (also see Fig. S3 B). The orientation of the wound is shown with dashed lines. Bars, 20 µm. (B, left) Still fluorescence (Fluo.) images 3 min after photobleaching from a FRAP experiment performed with vimentin-EGFP–expressing astrocytes 1 h 30 min after wounding. Wounding was done 4 d after cell nucleofection with the indicated siRNAs (see Video 8 for small interfering Cdc42). The contrast was increased in the zoom of Cdc42-depleted cell to better show the emergence of vimentin filaments in the photobleached region. (Right) fluorescence intensity profiles showing the fluorescence recovery at different time points after photobleaching. (C) Percentages of cells displaying a symmetric FRAP 1 h 30 min after wounding. Cells were nucleofected with the indicated siRNA or treated with DMSO, ML141 (inhibitor of Cdc42; 10 µM added 1 h before wounding), and NSC23761 (inhibitor of Rac1; 50 µM added 1 h before wounding; 44–66 cells per condition with a minimum of three independent experiments). (D, left) Still fluorescence images acquired 3 min after photobleaching of vimentin-EGFP–expressing astrocytes 10 h after wounding. Control or Cdc42-depleted cells were left treated with DMSO or with bradykinin (10 µM) 1 h before the FRAP experiment. (Right) Fluorescence intensity profiles along the corresponding arrows showing the fluorescence recovery at different time points after photobleaching. Filled arrows point to the slopes observed in the fluorescence recovery profile, indicating a symmetric or asymmetric recovery of fluorescence. Note that in the case of asymmetry, the fluorescence recovery occurs only from the rear side of the photobleached area (shown by a single filled arrow). (B and D) Bars: (main images) 10 µm; (insets) 2 µm. a.u., arbitrary units. (E) Percentages of cells displaying a symmetric profile of FRAP after 10 h of migration. Control or Cdc42-depleted cells were left treated with DMSO or bradykinin (10 µM; 1 h; 37–60 cells per condition with a minimum of three independent experiments). (F) Quantification of the vimentin retrograde flow in the experiments described in E. Error bars display SEM for the three repeats. *, P < 0.05; ***, P < 0.001. (G) Schematics representing the mechanisms involved in IF dynamics after Cdc42 activation by Bradykinin treatment. Arrows correspond with colored labels on the right.

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