Vimentin dynamics are regulated by aPKC during cell polarization. (A) Merged images of vimentin (green), tubulin (magenta), γ-tubulin (γ-tub; yellow), and DNA (blue) of astrocytes 4 d after transfection with indicated siRNAs (also see Fig. S3 C). Dashed lines indicate wound orientation. Bars, 20 µm. (B, left) Still fluorescence (Fluo.) images 3 min after photobleaching of vimentin-EGFP–expressing astrocytes 1 h 30 min after wounding. Cells were nucleofected with the indicated siRNAs (see Video 9 for small interfering aPKC [si-aPKC]). (Right) Line profiles showing the FRAP. (C and E) Percentages of cells displaying a symmetric profile of FRAP 1 h 30 min after wounding. Cells were nucleofected with the indicated siRNA or constructs and then were left untreated or treated with DMSO, PKCζ pseudosubstrate (PS; inhibitor of PKCζ; 10 µM was added 1 h before wounding), and wiskostatin (inhibitor of N-WASp; 2 µM was added 1 h after wounding). Statistics include 36–74 cells per condition from a minimum of three independent experiments. (D, left) Still fluorescence images acquired 3 min after photobleaching of Cdc42-depleted astrocytes expressing vimentin-RFP and either EGFP or PKCζ-EGFP (Video 10) 1 h 30 min after wounding. (Right) Line profiles showing the FRAP. 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). Bars: (main images) 10 µm; (insets) 2 µm. a.u., arbitrary units. (F) Schematic diagram showing the “front” polarity signaling triggering the polarized rearrangements of IFs and microtubules during astrocyte polarization. MT, microtubule.