Vimentin transport directionality is triggered by cell polarization. (A) Merged images of vimentin (green), tubulin (magenta), and DNA (blue) of nonmigrating astrocytes plated on either 120-µm (polarized cells) or 50-µm disks (nonpolarized cells). Cells were nucleofected with the indicated siRNAs. Bars, 10 µm. (B) Ratio between peripheral and perinuclear vimentin intensity in each condition. Each experiment was repeated three times with ∼50 cells per condition and per repeat. Error bars display the SEM for the three repeats. (C and D, left) Still fluorescence (Fluo.) images (6 min after photobleaching) of vimentin-EGFP–expressing astrocytes: five to eight cells were plated on 120-µm diameter micropatterns (C), and single cells were plated on a 50-µm diameter circular micropatterns (D). Bars: (main images) 10 µm; (insets) 2 µm. (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). a.u., arbitrary units. (E) Percentages of cells displaying a symmetric fluorescence recovery (32 polarized nonmigrating cells and 15 nonpolarized nonmigrating cells out of three and five independent experiments, respectively). (F) Quantification of the vimentin retrograde flow. Error bars display SEM for the three repeats with 32 and 15 cells per condition, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G) Schematics of IF dynamics during cell polarization and migration. Arrows correspond with colored labels on the right.