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First published online 28 September 2004
doi: 10.1242/jcs.01407

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Mechanical stress induces profound remodelling of keratin filaments and cell junctions in epidermolysis bullosa simplex keratinocytes

¹ Cancer Research UK Cell Structure Research Group, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK
² Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK
³ Centre for High Resolution Imaging & Processing, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK

View larger version (125K): [in a new window]   Fig. 1. Mechanical stretch induces keratin fragmentation in DM-EBS keratinocytes. Wild-type cells, NEB-1 (A,C,E) and DM-EBS cells, KEB-7 (B,D,F) were subjected to mechanical stress using an oscillating stretch with frequency of 4 Hz and amplitude of 12% for varying times. Cells were stained for keratin 14 using monoclonal antibody LL001. (A,B) Before stretch; (C,D) after 30 minutes stretch; (E,F) after 120 minutes stretch. All cells show concentric compaction and wrinkling of keratin filaments but only the EBS cells show accumulation of peripheral aggregates of keratin after stretch. By 120 minutes of stretch, the EBS cells show severely disrupted keratin networks with any remaining filaments concentrated around the nucleus. Bars, 10 µm.

View larger version (168K): [in a new window]   Fig. 2. Keratin aggregate formation is delayed where desmosomes are numerous. DM-EBS cells (KEB-7) stretched for 10 minutes and stained for keratin 14 using monoclonal antibody LL001. Short periods of stretch reveal that desmosomes provide initial resistance to keratin fragmentation and highlight areas of the cell where the keratin network is least resistant to the force of stretch, i.e. at apices where three cells meet and along free edges. Inset shows the presence of two distinct types of keratin aggregate: solid keratin aggregates (arrow) and hollow ring structures (asterisk). Bars, 10 µm.

View larger version (74K): [in a new window]   Fig. 3. Mechanical stretch causes relocation of desmoplakin from desmosomes in DM-EBS keratinocytes. KEB-7 cells were stretched using a cyclic stretch with a frequency of 4 Hz and amplitude of 12% for varying times. (A) Control cells before stretch; (B) DM-EBS cells before stretch; (C) Control cells after 120 minutes of stretch; (D) DM-EBS cells after 120 minutes of stretch. Elongation of desmoplakin staining is seen in the majority of control cells after stretch (C, arrow). Desmoplakin staining remains localized to desmosomes where the keratin network is maintained on at least one cell edge (D, arrow). Cells were stained using a polyclonal antibody against keratin 5 (BL18) and a monoclonal antibody against desmoplakin (11-5F). Bars, 10 µm. (E) Desmoplakin staining elongates in response to stretch in wild-type keratinocytes. Forty regions of desmoplakin staining were measured before and after stretch. Results show that desmoplakin staining elongates approximately threefold in response to stretch, suggesting an inherent elasticity within desmosomes that involves desmoplakin.

View larger version (62K): [in a new window]   Fig. 4. Keratin aggregate formation begins in areas rich in hemidesmosome proteins. (A) Unstretched DM-EBS (KEB-7) keratinocytes stained with polyclonal antibody BL18 against keratin 5 (green) and monoclonal antibody 233 against BP180 (red). Hemidesmosome components are usually concentrated in regions lacking many desmosomes at the periphery. These regions (arrows) coincide with aggregate formation early in stretch as seen in Fig. 2. Bar, 10 µm. (B) Localisaton of keratin aggregates with hemidesmosomal and desmosomal components after 10 minutes stretch. Cells stretched for 10 minutes were scored for colocalisation of keratin aggregates with desmoplakin, BP180 or plectin. 300 cells were counted for each antibody and results expressed as the average number per 100 cells. This shows that after short periods of stretch keratin aggregates form more commonly in areas of the cell high in hemidesmosomal components.

View larger version (59K): [in a new window]   Fig. 5. Fragmentation of the keratin network leads to relocation of desmoplakin and plectin, but not desmogleins, from desmosomes and hemidesmosomes. DM-EBS (KEB-7) keratinocytes were stretched for 120 minutes and stained for keratin 5 (green) using rabbit polyclonal antibody BL18 and mouse monoclonal antibodies against desmoplakin (11-5F) (A); desmoglein 2 (Dsg2) (B); HD1/plectin (HD121) (C) (all red). 3D maximum-intensity volumetric projections were generated by deconvolution microscopy. Desmoplakin is associated with small fragments of keratin in areas close to desmosomal junctions (A). In areas towards the nucleus there is a decrease in the number of small fragments and an increase in the number of ring structures, which have desmoplakin (A, arrow) and plectin (C) associated with them. Desmoglein 2, a major transmembrane component of desmosomes, does not relocalise in response to stretch (B). Bars, 5 µm.

View larger version (62K): [in a new window]   Fig. 6. Keratin rings are associated with components of hemidesmosome and desmosome junctions in cells with mutant keratin. Fragmentation of the mutant keratin network in response to stretch leads to small fragments of keratin associated with desmoplakin (A). Keratin fragmentation results in the formation of ring structures, which are intercalated with patches of desmoplakin (B). HD1/plectin associates with these ring structures and is particularly located at adhesion points between rings, associated with the formation of chains of rings (C). BP180 (D), plakoglobin (E), BP230 (F) and plakophilin (G) are also associated with keratin rings. The hemidesmosomal transmembrane protein, ß4 integrin, showed no specific localisation with keratin fragments or keratin rings (H and I). Single optical slices were obtained using deconvolution microscopy after 120 minutes of stretch. Cells were stained using polyclonal antibody BL18 against keratin 5 (green) and monoclonal antibodies against desmoplakin (11-5F), HD1/plectin (HD121), BP180 (233), plakoglobin (CBL175), BP230 (IE5), plakophilin (PP1-5C2) (all red) and ß4 integrin (CD104) (blue). Bars, 1 µm.

View larger version (159K): [in a new window]   Fig. 7. Keratin rings are composed of small fragments of keratin. DM-EBS keratinocytes were stretched for 120 minutes and prepared for transmission electron microscopy. Whorls and rings of keratin filament bundles were frequently observed in the cell periphery (A). Solid aggregates of keratin, reminiscent of the diagnostic hallmark of DM-EBS, were also seen (B,C). Keratin rings were observed and the irregular form of the rings suggests they may be formed of annealed stretches of filament bundles complexed with some associated proteins (D,E). In some cases these structures dipped in or out of the plane of section (E,). Bars, 500 nm.

View larger version (139K): [in a new window]   Fig. 8. DM-EBS keratinocytes recover from stretch by resorption of keratin rings and rebuilding of the keratin network. DM-EBS keratinocytes were stretched for 120 minutes followed by continued incubation at 37°C. Cells were fixed at various time points. Cells quickly begin to recover from stretch-induced malformations of the keratin network by formation of thickened bands of keratin close to the cell edge (arrows) (A,B and G). By 30 minutes after cessation of stretch, the majority of rings and aggregates have gone and the keratin network is being reformed (C). This is completed by 1 hour (D), and a degree of remodelling continues with time (E,F). We examined the keratin band, which often forms during early recovery (G, arrow), and found that keratin rings are resorbed in this zone (H). Cells were stained with monoclonal antibody LL001 against keratin 14 (A-G) and polyclonal antibody BL18 against keratin 5 and monoclonal antibody 11-5F against desmoplakin (red) (H). Images G and H were obtained from 3D maximum-intensity volumetric projections using deconvolution microscopy. Bars, 5 µm.

View larger version (18K): [in a new window]   Fig. 9. Model for the formation of ring structures in DM-EBS keratinocytes in response to mechanical stretch. Breakage of mutant keratin results in small fragments of keratin attached to hemidesmosomal and desmosomal junctions. The subsequent loss of tension within the keratin filaments leads to the progressive disassembly of these junctions. Cytoplasmic proteins (coloured squares) from these junctions relocalise to the cytoplasm where they interact with keratin filaments which form ring structures presumably as breakage results in `sticky-ends'. Transmembrane components (green) do not relocalise and remain associated with the membrane.