Focal adhesions are hotspots of keratin filament precursor formation
Windoffer R, Kölsch A, Wöll S, Leube RE,, 2006
Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia.
Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.
Tableau of time-lapse recordings (4 frames/min; display rate 10 frames/s) of phase contrast images (upper right) and fluorescence micrographs of a mammary epithelium-derived EpH4 cell that was transfected with cDNAs encoding HK18-YFP and actin-RFP (merged fluorescence images at lower right). Note the extension of an actin-rich lamellipodium and the subsequent formation of a new KF network in this region.
Tableau of phase contrast and epifluorescence recordings of an EpH4 cell producing fluorescent chimeras HK18-YFP and actin-RFP. Images were acquired every 15 s and are displayed at 10 frames/s. Note the continuous centripetal movement of KFPs along actin stress fibers in the lamellipodium.
Time-lapse fluorescence microscopy of HK18-YFP in an EpH4 cell that was treated with the actin polymerization inhibitor latrunculin B (10 µM). Images were taken every 20 s (display rate 10 frames/s). Note that addition of the drug prevents continuous inward-directed transport of KFPs but that new KFPs continue to appear, elongate and fuse.
Tableau of phase contrast and fluorescence micrographs depicting the distribution of HK18-YFP and paxillin-DsRed2 in a cDNA-transfected EpH4 cell (merged fluorescence images at upper right). Pictures were recorded every 15 s (display rate 10 frames/s). Note the successive appearance of paxillin-labeled FAs and KFPs in the emerging lamellipodium.
4D representation of fluorescence time-lapse recording of another region of the SK8/18-2 cell shown in Video 6 depicting HK18-YFP-positive KFs and RFP-zyxin-labeled FAs (correponding Fig. 3 B; recording rate 1 image/15s; display rate 25 frames/s). The red and green axes represent the image plane while the yellow axis corresponds to the time axis. The fluorescence of RFP-zyxin is shown in voxel representation resulting in orange rods that change only little over time as a consequence of the static nature of the FAs. Four KFPs are color-coded to highlight their dynamic behavior in relation to FAs.
High magnification images showing fluorescence emitted by RFP-zyxin and HK18-YFP keratin-YFP in a SK8/18-2 cell (corresponding Fig. 3 C; recording rate
1 image/15s; display rate 25 frames/s). Note the appearance, growth and mobility of KFPs in relation to FAs. The cell margin is at top.
