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Hemidesmosome-Related Keratin Filament Bundling and Nucleation
Moch M and Leube RE

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin interme-diate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassem-bled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin fila-ments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Treatment of HaCaT keratinocytes with latrunculin B disrupts actin dynamics within a few minutes in a dosedependent manner. The video presents timelapse fluorescence microscopy of HaCaT cells producing mAppleActin before and after addition of either 2 μM or 1 μM latrunculin B. The cells treated with the higher concentration show a stronger decrease in actindynamics and a pronounced disassembly of actin structures. Note that there are adjacent non or weaklytransfected cells. The recordings show only bottom focal planes. The original resolution was downscaled in the video with a bicubic filter to preserve detail.

 

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Treatment of HaCaT keratinocytes with 20 μM nocodazole stops microtubule dynamics instantaneously. The image series shows fluorescence recordings of EB3GFP in HaCaT cells before and after addition of 20 μM nocodazole. The drug was washed out after 10 min incubation, resulting in slow reassembly of EB3GFP positive microtubules in the cell periphery. The recording shows maximum intensity projections of lower focal planes (z = 0 to 1.05 μm; 4 planes), and the signal was processed as annotated in the video.

 

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Simultaneous disruption of actin filaments (3 μM latrunculin) and microtubules (20 μM nocodazole) induces partial keratin network retraction, keratin filament bundling, and formation of new keratin filament bundles in the cell periphery near the cell bottom (compare recordings at z = 0 nm with those at z = 700 nm). The fluorescence images were recorded in HaCaT B10 cells producing fluorescent keratin 5YFP.

 

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Treatment  of  HaCaT  keratinocytes  with  latrunculin  B  disassembles  focal  adhesions, whereas nocodazole does not affect focal adhesions. The video shows timelapse fluorescence recordings of paxillinGFP and keratin 5mCherry in HaCaT cells before and after addition of 3 μM latrunculin B (top panels) or 20 μM nocodazole (lower panels). The recordings show maximum intensity projections of lower focal planes (z = 0 to 1.05 μm; 4 planes).  The  original  resolution  was  downscaled  in  the  video  with  a  bicubic  filter  to  preserve detail. In addition see corresponding Figure A4.

 

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Keratin network reorganization is independent of focal adhesions in the absence of actin filaments and microtubules. The image series shows the fluorescence of keratin 5mCherry  and  talinGFP  in  a  HaCaT  cell  of  a  monolayer  that  was  treated  with  3 μM latrunculin B and 20 μM nocodazole. The recording shows maximum intensity projections of lower focal planes (z = 0 to 1.05 μm; 4 planes) at original resolution. See corresponding Figure 5 for more details.

 

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Growing keratin filaments appear at nascent hemidesmosomes after actin and microtubule disruption. The image series shows the fluorescence of keratin 5mCherry and integrin β4GFP in a HaCaT cell of a monolayer that was treated with 3 μM latrunculin B and 20 μM nocodazole. See annotations in the video and corresponding Figure 6 for more details.

 

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Keratin  filaments  are  assembled  at  and  interconnect  hemidesmosomes.  The  image  series  shows  the  fluorescence  of  keratin  14mCerulean  and  integrin β4YFP  in  a migrating HaCaT cell within a monolayer a few minutes after a scratch was induced. The recording shows the bottom confocal plane at original resolution with enlarged panels that were upscaled by bilinear filtering. See corresponding Figure 7 for more details.

 

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