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First published online 14 November 2006
doi: 10.1242/jcs.03255

Journal of Cell Science 119, 4974-4985 (2006)
Published by The Company of Biologists 2006
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New insights into the molecular basis of desmoplakinand desmin-related cardiomyopathies

Karine Lapouge1,*,{ddagger}, Lionel Fontao1,*, Marie-France Champliaud1, Fabienne Jaunin1, Miguel A. Frias2, Bertrand Favre1,§, Denise Paulin3, Kathleen J. Green4 and Luca Borradori1,¶

1 Clinic of Dermatology, University Hospital, Geneva, Rue Micheli-du-Crest 14, 1211-Geneva 14, Switzerland
2 Division of Endocrinology, Diabetology and Nutrition, University Hospital, Geneva, Rue Micheli-du-Crest 14, 1211-Geneva 14, Switzerland
3 Biologie Moléculaire de la Différenciation, Université Paris-7, 2 Place Jussieu, 75005 Paris, France
4 Departments of Pathology and Dermatology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611, USA


Figure 1
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  		Fig. 1. Human wild type desmin, vimentin and the C-terminal half of DP showing the predicted subdomains with the corresponding amino acid positions. The location of a potential protein kinase A consensus site within the C-terminal extremity of DP is indicated in bold, while the position of Ser2849 is underlined.

 

Figure 2
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  		Fig. 2. Expression and localization of DP and desmin in monkey cardiomyocytes was detected by immunofluorescence microscopy (A-C). Desmin was stained with a rabbit antiserum (A) and DP with a monoclonal mouse antibody (B). The two proteins codistribute at intercalated discs, whereas desmin is also found along the cardiac muscle striations (C). Bar, 5 µm.

 

Figure 3
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  		Fig. 3. Ability of DP to become codistributed with both the desmin network and keratin intermediate filaments in a transfected human keratinocyte cell line, that stably expresses desmin (A-F) as well as with the desmin network in transfected primary rat cardiomyocytes (G-I). The cells were transiently transfected with cDNA encoding DP BCS2849G, GFP-tagged at its N-terminus (A-I). Transfected cells were double-stained with an anti-GFP antiserum (A,D) and either a monoclonal antibody directed against desmin (B) or a monoclonal anti-keratin 14 antibody (E), and subsequently examined by laser confocal microscopy. Overlays are shown in C and F. Primary rat cardiomyocytes were transiently transfected with cDNA encoding GFP-tagged DP-BCS2849G (G-I). The GFP is localized in the fluorescein channel. Cells were also stained with a monoclonal anti-desmin antibody (overlays in G and F) or with both the rabbit NW161 anti-DP antiserum and a monoclonal anti-desmin antibody (overlays in I). DP-BCS2849G is found colocalized with desmin filaments that are either still longitudinal (overlay in G) or already rearranged into a cross-striated pattern (overlay in H). At intercalated discs, DP-BCS2849G colocalizes with endogenous DP and desmin (white color in I, arrows). Notably, in cells in which the GFP-DP-BCS2849G construct strongly stained the region of intercalated discs, the staining pattern of endogenous desmin extending from the junctional region into the cytoplasm appeared disorganized (arrowhead) and reduced (I) suggesting that the DP-tail acted in a dominant negative fashion by competing with the endogenous DP for sites of IF attachment at intercalated discs. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Codistribution potential of the DP tail with the intermediate filament desmin network. Representatives of double immunofluorescence analysis of IF-free SW13 cells transiently transfected with cDNAs encoding desmin (A,C,E,G) tagged at the C-terminus with an HA epitope and deletion mutants DP-BCS2849G (B), DP-BC (D), DP-BL (F), DP-CS2849G (H) and tagged at their N-terminus with a Myc epitope. When co-expressed, both DP BCS2849G and DP-BC colocalized with desmin. In some transfected cells, disruption and collapse of the desmin network was observed (see e.g. D and E). The DP mutant containing the B subdomain and the linker region, DP-BL2194-2566, also codistributed with desmin, while the DP mutant protein containing the C subdomain and the C-terminal extremity, DP-CS2849G, did not colocalize with desmin but was present as cytoplasmic aggregates in transfected SW13 cells. Bars, 15 µm.

 

Figure 5
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  		Fig. 5. Dot-blot overlay assays of purified recombinant polymerized intermediate filament vimentin (B) and desmin (C) with radiolabeled fragments of the DP tail. 35S-radiolabeled c-myc-tagged recombinant forms of DP, DP-BCS2849G, DP-BC{Delta}51, DP-BL, DP-CS2849G, DP-C{Delta}51 and DP-L were generated by coupled in vitro transcription/translation and analyzed by SDS-PAGE and autoradiography (A). The occasional detection of additional radiolabeled protein bands most likely reflect the presence of partially transcribed and translated products (A). Different amounts of polymerized vimentin (B) and desmin (C) were immobilized on a nitrocellulose membrane by dot blotting and incubated with radiolabeled DP-BCS2849G, DP-BC{Delta}51, DP-BL, DP-CS2849G, DP-C{Delta}51, DP-L. Protein loading was verified by amino-black staining of the spotted proteins (P). The binding efficiency of DP-BCS2849G to both desmin and vimentin varied according to the amount of immobilized filaments. While binding of DP to desmin abruptly decreased when the amount of desmin was below 0.5'g, the association of DP with vimentin was maintained for up to 0.05'g immobilized vimentin. Furthermore, DP-BC{Delta}51 and DP-BL exhibited greatly reduced binding for both desmin and vimentin compared with DP-BCS2849G. Finally, DP-L, DP-CS2849G, and DP-C{Delta}51 did not detectably interact with either desmin or vimentin.

 

Figure 6
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  		Fig. 6. Yeast two-hybrid analysis of interactions between various subdomains of the C-terminal tail of DP fused to GAL4-BD and vimentin or desmin proteins fused to GAL4-AD. + and -, indicate growth or no growth respectively on selective media, tested as described under Materials and Methods. # indicates no growth on medium lacking adenine. * indicates that the interaction was tested between DP fused to GAL4-AD and desmin mutants fused to GAL4-BD.

 

Figure 7
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  		Fig. 7. Yeast two-hybrid analysis of interactions between various constructions of desmin and vimentin proteins fused to GAL4-AD and the DP-BCS2849G fused to GAL4-BD. + and -, indicate growth or no growth, respectively, on selective media, tested as described under Materials and Methods. # indicates no growth on medium lacking adenine.

 

Figure 8
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  		Fig. 8. Interaction of DP with wild-type desmin and tailless desmin in GST pull-down experiments. (A) 35S-radiolabeled recombinant forms of wild-type desmin and tailless desmin were generated by coupled in vitro transcription/translation and analyzed by SDS-PAGE and autoradiography. Their Mr was 54K and 48K, respectively, as predicted on the basis of their cDNA. Ladder: molecular mass markers, Mr from top to bottom: 175K, 83K, 62K, 47.5K and 32.5K. (B) GST-fusion proteins of DP-BCS2849G and of BP230-BC were immobilized on glutathione agarose beads and incubated with either 35S-labeled wild-type desmin or tailless desmin. After washing, samples were resolved by SDS-PAGE and 35S-labeled bound proteins were visualized by autoradiography (C). Note that wild-type and tailless desmin showed a tendency to proteolytic degradation under the conditions of the GST pull-down experiments. (B) Coomassie blue staining of the gel depicted in panel B to verify equimolar loading of GST fusion proteins. (D) Different amounts of polymerized wild-type and tailless desmin were immobilized on a nitrocellulose membrane by dot blotting and incubated with radiolabeled DP-BC. Protein loading was verified by amino-black staining of the spotted proteins. The binding efficiency of DP-BC to both wild-type and tailless desmin was not significantly different and abruptly decreased when the amount of desmin was below 0.5'g. Note that binding of radiolabeled DP-BCS2849G to wild-type desmin was comparable with that of DP-BC.

 

Figure 9
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  		Fig. 9. The potential of the DP tail to become codistributed with the desmin network is affected by sequences contained within the rod and tail domains of desmin. Intermediate-filament-free SW13 cells were transiently co-transfected with the cDNAs encoding DP BCS2849G, Myc-tagged at the N-terminus (B,D,F) and tailless desmin (Desmin{Delta}T) (A,C), or desmin I451M (E). Although in most transfected cells, DP and tailless desmin were found together in spot-like and large cytoplasmic aggregates (C,D), in ~5% of the transfected cells the recombinant DP-BCS2849G was found diffusely distributed in the cytoplasm without obvious colocalization with tailless desmin (A,B). In a few cells expressing both desminI451M and DP-BCS2849G, DP-BCS2849G did not become coaligned with desminI451M, but retained a cytoplasmic distribution (E,F). Bar, 20 µm.

 

Figure 10
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  		Fig. 10. Binding sites for various IF proteins on the tail of DP. The linker region between the B and C plakin-repeats and the C-terminal extremity contain sequences critical for IF binding and ensure binding specificity. The B and C plakin-repeat domains participate in the association by providing additional binding sites and/or affecting the proper folding of the linker region and C-terminal extremity. While the linker region and the B plakin repeat of DP contain the minimal sequences required for its association with desmin, the C plakin repeat and C-terminal extremity contribute to efficient binding.

 

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© The Company of Biologists Ltd 2006