Vimentin filament dynamics during astrocytoma random migration. (A) Phase contrast (left) and fluorescence (right) images of a motile astrocytoma cell (U373 cell line) expressing vimentin-EGFP (Video 1; total time, 120 min). (B) Still images of the vimentin network acquired 0 and 5 s after photobleaching of a vimentin-GFP– and tubulin-mCherry–expressing astrocytoma cell (Video 2; total time, 2 min; see also Fig. S1, B–D). mCherry-tubulin images are shown in Fig. S1 C. (C) Green and red fluorescence images of a vimentin-Dendra2–expressing astrocytoma cell just after (t = 0) and 40 s after photoconversion by a 405-nm flash illumination (purple) in the yellow rectangle (Video 3; total time, 2 min). Kymographs are boxed in yellow and show the fluorescence intensity profile along the yellow arrow in the corresponding image over time. The colored lines highlight the nucleus and protrusion forward movements (green), the retrograde flow (blue), and the anterograde (red) and retrograde (orange) movements of IFs on kymographs. The higher-magnification images of regions indicated by a cyan or purple dashed box are shown in the corresponding dotted line boxes. (D) Merged images of an astrocytoma coexpressing GFAP-EGFP and vimentin-RFP just before and 50 s after photobleaching with a zoom on moving filaments. (E) Merged images of an astrocytoma coexpressing nestin-EGFP and vimentin-RFP just before and 70 s after photobleaching with a zoom on filaments. Bars: (main images) 10 µm; (insets) 2 µm.