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Supplementary Material

JCS068080 Supplementary Material

Files in this Data Supplement:

  • Movie 1 -

    Movie 1. KF-network reorganization after scratch wounding (see corresponding Fig. 1A). Time-lapse recording (3-minute intervals; display rate 30 frames/second) of HK18-YFP fluorescence in a confluent monolayer of EK18-1 cells after scratch wounding.

  • Movie 2 -

    Movie 2. KFP appearance at the leading edge of migrating cells (see corresponding Fig. 1B-D). Tableau of time-lapse recordings of HK18-YFP fluorescence (left) and corresponding phase contrast (right) of a migrating EK18-1 cell displaying multiple emerging KFPs in the proceeding lamellipodium. The bottom panel shows high-power views of part of the lamellipodium. In this instance, cellular movement was compensated by an image-intensity-based method. The images were acquired every 30 seconds and are displayed at 30 frames/second.

  • Movie 3 -

    Movie 3. Persistence of KF-network formation in the presence of the protein biosynthesis inhibitor cycloheximide (see corresponding Fig. 2A-B,E). Time-lapse fluorescence microscopy of a PK18-5 cell treated with the protein biosynthesis inhibitor cycloheximide (17 µM). Images were acquired every 20 second and are displayed at 30 frames/second. The inhibitor was added after recording 50 pictures. Note the continuous formation of new precursors.

  • Movie 4 -

    Movie 4. Persistence of KF-network formation in the presence of the protein biosynthesis inhibitor puromycin (see corresponding Fig. 2C-E′). Time-lapse fluorescence recording of a PK18-5 cell treated with the protein biosynthesis inhibitor puromycin (1 µg/ml). Images were taken every 20 seconds and are displayed at 30 frames/second. The inhibitor was added after recording 50 pictures. Note the continuous formation of new KFPs in the cell periphery.

  • Movie 5 -

    Movie 5. Detection of continuous inward-directed KF-network motility (see corresponding Fig. 3). Time-lapse fluorescence recording of HK18-YFP in a section of a PK18-5 cell (projected images of 22 focal planes). Recording interval was 60 seconds (display rate 25 frames/second). The frames are aligned to the first frame to compensate for cell movement. Note the continuous inward movement of KFs within the network.

  • Movie 6 -

    Movie 6. Detection of inward-directed KF-network motility and loss of KFs by ROI tracking (see corresponding Fig. 4A-F). Time-lapse fluorescence recording of HK18-YFP in a segment of a PK18-5 cell (periphery at left, nucleus at right). Projection views of 11 focal planes are shown for each time point (recording intervals 60 seconds; 30 frames/second). ROIs demarcated in the lower panel were defined manually and the respective margins are shown by coloured lines. Note the continuous inward-directed flow and loss of fluorescent filaments during inward translocation of each ROI.

  • Movie 7 -

    Movie 7. Detection of inward-directed KF-network motility and loss of KFs by ROI tracking (see corresponding Fig. 4M-M′). Time-lapse fluorescence recording of a HK18-YFP-producing PK18-5 cell after bleaching of three centripetal segments. Shown are projections of 25 focal planes, recording intervals were 2 minutes (display rate 50 frames/second). Note the inward movement of unbleached KF bundles and continuous loss of fluorescence without fragmentation, which are best seen at higher magnification at right.

  • Movie 8 -

    Movie 8. Detection of a continuous KF-network turnover cycle by FRAP (see corresponding Fig. 5B). Time-lapse fluorescence recordings were prepared from two PK18-5 cells producing HK18-YFP after bleaching half of one of the cells. The movie presents a series of projected images (10 focal planes) after registration at a display rate 25 frames/second. Note the peripheral recovery of fluorescence and the continuous centripetal motility of the keratin system. Image stacks were recorded every 5 minutes.