Regulation of kinesin-1– and dynein-1– dependent transport drives the polarization of the IF network. (A) Epifluorescence images of vimentin (green), tubulin (magenta), and DNA (blue) in migrating astrocytes (before wounding, 1 h 30 min, 4 h, and 10 h after wounding) 4 d after nucleofection with the indicated siRNAs (also see Fig. S3 A). Dashed lines indicate wound orientation. (B) Ratios between front and perinuclear vimentin fluorescence (Fluo) intensities before wounding (confluent cells) or at different times after wounding. Cells were nucleofected with the indicated siRNAs. The results are shown as means ± SEM of at least three independent experiments with ∼50 cells per time point, per condition, per repeat experiment; ∼1,800 cells were used in total. (C–E) Fluorescence images acquired 6 min after photobleaching during a FRAP experiments on vimentin-EGFP–expressing astrocytes in confluent cells (C) 1 h 30 min (D) and 8 h (E) after wounding (Video 7). Fluorescence intensity profiles along corresponding arrows show either a symmetric (C and E) or asymmetric (D) recovery of fluorescence. The higher-magnification images of regions indicated by a dotted line box are shown in corresponding dotted line boxes. Bars: (main images) 10 µm; (insets) 2 µm. 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). a.u., arbitrary units. (F) Percentages of cells displaying a symmetric fluorescence recovery in each condition (33–54 cells per condition in a minimum of three independent experiments). (G) Speed of the retrograde flow in each condition was extracted from the same data as in F. Means ± SEM of three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.