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First published online 19 February 2008
doi: 10.1242/jcs.013755

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Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption

Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria

View larger version (51K): [in this window] [in a new window]   Fig. 1. Epiplakin binds to keratins via multiple sites. (A) Double immunofluorescence microscopy of primary keratinocytes and primary hepatocytes using antibodies as indicated. Inserts show twofold magnifications of boxed areas; localization of epiplakin dot-like structures along a keratin filament is indicated by arrows. Scale bar: 10 µm. (B) Domain structure of mouse epiplakin (Spazierer et al., 2003) showing 16 repeat domains, linkers (white bars) and modules (grey ellipses). Modules that are virtually identical are shown in light gray, those with lower homology in dark gray. (C,D) Blot overlay of keratins (isolated from mouse keratinocytes) with 10 µg/ml of the protein indicated. Positions of K5 and K14 (arrows) and of size markers (bars) are indicated. PRD, plakin-repeat domain; Li, linker; M, module. Note that M8 and M9 have identical amino acid sequence. (E) Co-sedimentation of K5 and K14 with epiplakin-GST fusion proteins. Proteins bound to epiplakin-GST-Sepharose beads were analyzed by immunoblotting (IB) using antibodies to K5 and K14; GST coupled to Sepharose beads was used as negative control. For IB, keratinocyte protein lysates were used as positive controls (lysate).

View larger version (95K): [in this window] [in a new window]   Fig. 2. Ca2+-mediated differentiation of primary mouse keratinocytes is independent of epiplakin. (A) Immunoblotting of protein extracts from keratinocytes grown in the presence of 1.3 mM Ca2+ for 1, 3 or 5 days, using antibodies as indicated. Note the upregulation of epiplakin and involucrin (differentiation marker) compared with tubulin (loading control). (B) Primary keratinocytes isolated from wild-type (+/+) and epiplakin-deficient (–/–) mice were grown in the presence of 1.3 mM Ca2+ for the times indicated and then subjected to immunofluorescence microscopy using antibodies as indicated. Scale bar: 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Colocalization and subcellular co-distribution of epiplakin with keratin aggregates after OA-induced filament disruption in wild-type (+/+) and epiplakin-deficient (–/–) keratinocytes. (A) Primary mouse keratinocytes, treated with OA for 2, 4 and 6 hours were immunolabeled using antibodies as indicated. Merged images show complete colocalization of epiplakin with keratin. Compare with untreated cells (negative control) shown in Fig. 1A. Scale bar: 10 µm. (B) Immunoblotting (upper four panels) of soluble and insoluble cell fractions obtained from 2, 4 and 6 hour OA-treated keratinocytes using antibodies to epiplakin, tubulin, and phospho (p)-K5. Coomassie Blue staining of high salt-extracted keratins is shown in bottom panel.

View larger version (106K): [in this window] [in a new window]   Fig. 4. Recruitment of epiplakin to keratin filaments and aggregates after application of various types of stress. Primary mouse keratinocytes isolated from wild-type (+/+) or epiplakin-deficient (–/–) mice were treated with orthovanadate (OV) for 2 minutes and 10 minutes, or were exposed to 150 mM urea or UV (400 J/m2) prior to immunolabeling using antibodies as indicated. Merged images of wild-type cells show colocalization of epiplakin and keratin. Scale bar: 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Phosphatase-inhibitor-induced keratin filament disruption in differentiated keratinocytes proceeds faster in the absence of epiplakin. (A) Primary wild-type (+/+) and epiplakin-deficient (–/–) keratinocytes differentiated in medium containing 1.3 mM Ca2+ were treated with OA for 2, 4 and 6 hours and immunolabeled with antibodies to pan-keratin. Arrows indicate examples of cells with partially disrupted keratin filament networks. Arrowheads indicate examples of cells with totally disrupted keratin networks. Scale bar: 10 µm. (B) Decrease of cells with intact filaments during the first 2 hours after onset of OA-induced filament breakdown (left panel) and increase of cells with totally disrupted filament networks over a 2 hour period, starting when the first cells of this type appeared (right panel). Mean values ± s.d. of four independent experiments are based on >100 cells evaluated per time point. (C) Statistical analysis of keratinocytes with complete, partial or no filament breakdown upon okadaic acid incubation. Note that the trend of faster keratin network collapse in epiplakin–/– compared with wild type (+/+) cells was observed in four independent experimental series, regardless of the genetic background of the mice used as cell donors. One representative series is shown. Relative proportions of intact (filamentous), partially disrupted, and disrupted keratin filament networks are based on >100 cells evaluated per time point. (D) Increase of cells without intact filament networks upon incubation with orthovanadate over a 9 minute period. Mean values ± s.d. of cells without intact filaments per time-point are based on >100 cells evaluated per time point.

View larger version (107K): [in this window] [in a new window]   Fig. 6. Forced expression of epiplakin fragments in immortalized wild-type mouse keratinocytes. Cells were transfected with indicated epiplakin cDNA constructs, and subjected to immunofluorescence microscopy either directly (left column) or after incubation with OA (+OA) for 5 hours. Epiplakin-GFP-fusion proteins expressed were visualized using antibodies as indicated. Merged images show colocalization of overexpressed fragments after OA incubation only in the cases of module 9 and PRD9. Scale bar: 10 µm.