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Figures
- Fig. 1.
KFPs are predominantly formed at the leading edge of migrating cells. (A-A‴) A wound was induced by scratching a confluent monolayer of mammary-epithelium-derived EK18-1 cells that produce fluorescent HK18-YFP with a microinjection needle. Fluorescence images were then recorded until the wound had closed. Selected images are shown in A-A‴, demonstrating the continuous and collective convergence of both edges. Multiple KFPs were observed in lamellipodial protrusions of the approaching cells, which can be best appreciated in accompanying supplementary material Movie 1, and are marked by arrows in selected cells. (B-C″) Fluorescence images (inverse representation) are shown of a migrating EK18-1 cell with a large lamellipodium containing multiple KFPs that are depicted at higher resolution in C-C″. Corresponding phase-contrast images are shown in D-D″. The images are taken from a 15 minute time-lapse recording of fluorescence and phase contrast (see supplementary material Movie 2). (E) The kymogram was prepared from supplementary material Movie 2, corresponding to the boxed green area in B. It shows the appearance of multiple KFPs in the lamellipodium in proximity to the plasma membrane (yellow line). The blue line depicts the position of first KFP detection. The subsequent inward-directed movement of KFPs until integration into the peripheral KF network (red line) occurs at similar speed in each instance (slope of line). Scale bars: 10 μm.
- Fig. 2.
KFP formation persists in the presence of protein biosynthesis inhibitors. (A-D‴) Hepatocellular-carcinoma-derived PLC cells of clone PK18-5 stably producing keratin chimera HK18-YFP were treated at 20 minutes with the translation inhibitors cycloheximide (17 μM; A-A‴; high magnifications of boxed region in B-B‴) or puromycin (1 μg/ml; C-C‴; high magnifications of boxed region in D-D‴). The images were taken from supplementary material Movies 3 and 4. Note that KFP formation and KFP integration into the KF network are not prevented in either instance. Arrows in B-B‴ and D-D‴ depict newly appearing KFPs, as determined from the corresponding movies. (E,E′) Kymograms were prepared from the fluorescence recorded along the dashed lines in B and D, respectively, which further demonstrates ongoing KFP formation (marked by arrows) and inward-directed movement after drug application (addition of drugs at 20 minutes). N, nucleus. Scale bars: 10 μm.
- Fig. 3.
The KF network translocates continuously towards the nucleus. (A,B) Fluorescence micrographs showing a section of a PK18-5 cell (projected images of 22 focal planes) at time points 0 and 68 minutes (cell periphery at left, nucleus at right) observed by time-lapse imaging (60 second intervals; see corresponding supplementary material Movie 5). (C) Vector visualization of the direction and speed of KFs in this cell segment. Z-axis projections of all volume were superimposed on grids of overlapping ROIs at 10-pixel spacing. Between consecutive frames, each ROI was rigidly registered by the SSD criterion using a weighted window to emphasize central parts. Assuming long-term local stability of keratin movement, medians of the translocation vectors were computed for each grid point over time. (D) Velocities from C depicted in pseudo-colour representation.
- Fig. 4.
Inward-moving KFs disassemble into non-filamentous subunits. (A-G) Images taken from a 250 minute time-lapse recording of HK18 fluorescence in a PK18-5 cell (overview shown in G, with the boxed area delineating a region shown at high magnification in A-F). The confocal 4D data sets were recorded at 12 bit (recording interval 5 minutes; 11 planes each) for 250 minutes (supplementary material Movie 6). The coloured lines in A-F represent borders of ROIs that were defined in the outer part of the KF network between characteristic KF bundles. The borders were traced manually in the projection images of each time point of the recording to ensure proper delineation. The individual ROIs are depicted in (Ha-d). Note that all ROIs moved from the cell periphery towards the nucleus (N), that is, from left to right, during the recording. The size of each ROI decreased continuously over time (H). In addition, the relative fluorescence intensity in each ROI (or AOI) decreased over time, with a fast component in the beginning and a slow component later on (I; same colour code as in H). The black line in I shows the fluorescence intensity of the entire cell over time. Scale bars: 2 μm (A), 10 μm (G). (J-L) Projected fluorescence images (19 planes) and diagram from a time-lapse series of a PK18-5 cell in which a single fluorescent KF fragment was ‘isolated’ by bleaching the surrounding KFs. The nucleus (outside the depicted area) is to the left, the cell periphery to the right. The isolated KF fragment is shown at higher magnification at different time points in K. The red line depicts the border of the manually assigned ROIs, the intensities of which are plotted in L over time, revealing continuous fluorescence loss. Scale bars: 2 μm (J), 0.5 μm (K). (M-N) Projected images (25 planes) at time point 0 minutes and a diagram derived from a subsequent time-lapse series (supplementary material Movie 7). The PK18-5 cell (N, nucleus) was bleached in such a way that fluorescent filament ‘fragments’ remained in two sectors. This allowed measurement of fluorescence in these singled out KFs. ROIs that are shown in green in M′ were manually adjusted at each time point. The fluorescence of these mobile ROIs was measured over time and compared to the fluorescence in fixed ROIs (red in M′) within unbleached parts of the cell. The results for the three mobile and fixed ROIs are summarized in the graph in N. Note the fluorescence decrease in the green ROIs due to filament disassembly, whereas it stays constant in the red ROIs, where fluorescence loss from filament disassembly is compensated by incoming new filaments. Scale bar: 10 μm (M).
- Fig. 5.
KFs disassemble to support KF assembly in the peripheral cytoplasm. (A-B‴) Two representative FRAP experiments are depicted that were performed in PK18-5 cells producing HK18-YFP. (A) The peripheral network was bleached throughout the entire circumference in a zone extending ~5 μm from the plasma membrane to the cell interior (area between red and yellow lines). The shape of the bleached and non-bleached areas was adjusted by hand to account for cellular shape shifts. Within 15 minutes, considerable fluorescence recovery was observed in the periphery (compare A′ and A″). The histograms below summarize the results of fluorescence quantification in this and three similarly treated cells. The red bars correspond to the average fluorescence recovery in the cell periphery, whereas the yellow bars correspond to the average fluorescence measured in the central cytoplasm. (B) Half of a PK18-5 cell was bleached. Fluorescence recovery measured in the boxed ROI (red), which encompasses mainly central filaments, was ~60% within 60 minutes (red bars in histograms below). By contrast, filaments in the corresponding unbleached ROI (yellow box) decreased within the same time period (yellow bars). For comparison, an ROI in a neighbouring cell that was not bleached at all (blue box) shows slight increase in fluorescence, possibly due to de novo synthesis (blue bars). Corresponding supplementary material Movie 8 presents an image series of 3D reconstructions of the altering fluorescence patterns. (C-F) Photoactivatable GFP fused to keratin 8 (HK8-paGFP) was expressed in PLC cells. HK8-paGFP fluorescence was activated with UV light (405 nm) in different cell regions. Subsequently, fluorescence was monitored at 488 nm by time-lapse fluorescence microscopy. Note the appearance of fluorescence in the non-activated parts of the cells (C′,E′). In addition, fluorescence concentrated in the perinuclear domain, whereas peripheral fluorescence was generated from new filament formation. Quantification of fluorescence (in percent of total fluorescence) shows gradual depletion of photoactivated regions (yellow) and simultaneous fluorescence increase in non-activated areas (red). D, n=3 and F (derived from E,E′). N, nucleus. Scale bars: 10 μm.
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