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First published online 21 October 2008
doi: 10.1242/jcs.030312

Journal of Cell Science 121, 3737-3746 (2008)
Published by The Company of Biologists 2008
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A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice

Roland Bornheim1,*,{ddagger}, Martin Müller1,*, Uschi Reuter1, Harald Herrmann2, Heinrich Büssow3 and Thomas M. Magin1,§

1 Institut für Biochemie and Molekularbiologie, Abteilung für Zellbiochemie und LIMES, Universität Bonn, Nussallee 11, 53115 Bonn, Germany
2 Department of Molecular Genetics, B065, German Cancer Research Center, 69120 Heidelberg, Germany
3 Anatomisches Institut der Universität Bonn, Nussallee 10, 53115 Bonn, Germany


Figure 1
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  		Fig. 1. Vimentin expression constructs and eye phenotype. (A) Schematic representation of the constructs used for expression of wildtype, R113C point-mutant (VimR113C) and C-terminal truncated (Vim{Delta}C2B) vimentin transgene. All constructs are based on the mouse vimentin gene with its own regulatory elements. (B) Note turbid lens in a 22-week-old animal expressing the VimR113C mutant (arrow).

 

Figure 2
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  		Fig. 2. Histology of eye of vimentin wildtype and VimR113C transgenic mice. (A,B) Haematoxylin and eosin (H&E) stained, paraffin-embedded lens sections of 45-week-old (A) VimR113C transgenic and (B) wt mice. (A',B') Higher magnifications of the insets is shown in A and B. Note that VimR113C lenses are much more fragile than wt lenses; they display a less regular organisation of fibre cells. Scale bars: 70 µm, (A,B), 60 µm (A',B').

 

Figure 3
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  		Fig. 3. Age-dependent aggregate and cataract formation. (A) Immunoblot analysis of endogenous and transgene vimentin in extracts of lenses from 4-month-old wt and VimR113C mice. Endogenous and transgenic vimentin was detected using a vimentin antibody. Owing to the Mycepitope present in the mutated vimentin, it migrates slower than endogenous vimentin. (B-E) Immunofluorescence analysis of lenses. Cryosections of newborn and 5-month-old animals were stained for total (green) and Myc-tagged vimentin (red). (B,D) Vimentin expression and localisation in the wt. (C,E) Vimentin expression in VimR113C mice. In newborn mice, the distribution of VimR113C protein is similar to wt vimentin but less homogenous. (E) Aggregate formation in fibre cells of adult animals. Note the high number and large size of vimentin aggregates in lenses from adult compared with those from new born animals (C',C",E',E"). Bar: 20 µm.

 

Figure 4
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  		Fig. 4. Ultrastructure of lens fibre cells in mutant mice. (A,B) EM of vimentin aggregates in lens fibre cells of 11-week-old VimR113C mice. Vimentin aggregates appear amorphous with sharp boundaries and are not surrounded by membranes. At this stage, the plasma membrane is not yet strongly affected. (C,D) Notice the highly pleated organisation of the plasma membrane in Vim{Delta}C2B mice. (D) Vimentin aggregates and membrane reorganisation in Vim{Delta}C2B mice at high resolution. Aggregates are dense, different in size and not attached to or enclosed by a membrane. Arrowheads mark irregular membrane invaginations. (E) Semi-thin section of a lens from a 22-week-old Vim{Delta}C2B transgenic mouse. An arrow marks the disrupted basal lamina of the lens capsule. Lens fibres break through basal lamina and protrude into the vitreous body. Scale bars: 2 µm (A) 1 µm (B, D), 3 µm (C), 20 µm (E).

 

Figure 5
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  		Fig. 5. Increased proteasome activity and reversibility of vimentin aggregates in vivo. (A,B) Immunofluorescence analysis of lenses from 4-month-old VimR113C transgenic and wt mice, stained with antibodies against total (green) and transgenic vimentin (red). Panels 1-3 show higher magnification of lens areas that represent different stages of fibre cell differentiation. Notice that towards the centre of the lens that contains the oldest fibre cells, vimentin aggregates become dissolved, suggesting that they are transient. (C) Fluorescence-based proteasome assay of extracts from lenses and from cells stably transfected with VimR113C. In both settings, a 30% increase in proteasome activity in the mutant, compared with the wt, was detected. (D) SDS-PAGE and immunoblotting for ubiquitin of VimR113C and wt lens extracts after immunoprecipitation with a vimentin antibody. Substantially increased ubiquitylation of vimentin was detected in the transgenic lenses (indicated by arrows). The position of the Ig heavy chain is indicated. (E) Immunoblot for Hsp70 in soluble and insoluble lens extracts of wt and VimR113C transgenic mice. In both fractions, Hsp70 expression was upregulated in VimR113C lenses. Scale Bars: 100 µm (A,B), 15 µm (panels 1-3).

 

Figure 6
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  		Fig. 6. Unaltered distribution and expression of beaded filaments. (A-C) Immunofluorescence analysis of lens cryosections of 5-month-old VimR113C and wt transgenic mice. Sections were stained using antibodies against filensin, CP49 (both green) and Myc-tagged transgene vimentin (red). Expression and localisation of filensin and mutant vimentin is shown in (A) wt and in (B) VimR113C mice; (C) shows CP49. Merged images document unaltered expression and localisation of major lens proteins in the presence of vimentin aggregates that show no colocalisation. (D,E) Immunoblot detection of filensin and CP49 in soluble (sol.) and insoluble (insol.) lens extracts of wt and VimR113C transgenic mice. (D',E') Similar loadings of the corresponding Coomassie-stained SDS-gels. No changes in the amounts and in the processing of major lens proteins were detected. `Fragment R' filensin marks a major processing product of filensin, in agreement with previous findings (Sandilands et al., 1995bGo). Scale bar: 20 µm.

 

Figure 7
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  		Fig. 7. Distribution of cytoskeletal and vimentin-associated proteins. (A,B) Immunofluorescence analysis of lens cryosections of (A) wt and (B) VimR113C mice. Sections were stained using phalloidin to visualise F-actin. In mutant lenses actin localisation was not altered. (C,D) Double labelling of lens sections showing plectin (red) and vimentin (green). In control sections, both proteins were distributed underneath the plasma membrane. In VimR113C lens sections, the submembraneous localisation of plectin was retained, whereas vimentin formed extensive aggregates. (E,F) Double immunofluorescence showing synemin (red) and vimentin (green). Synemin, which can heteropolymerise with vimentin, was relocalised to some but not all vimentin aggregates, suggesting that the interaction of vimentin and synemin is maintained, whereas their interaction to membrane attachment complexes is severed. Scale bars: 10 µm (A,B), 20 µm (C-F).

 

Figure 8
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  		Fig. 8. Normal focal adhesions in fibroblasts expressing VimR113C. Double immunofluorescence of focal adhesion proteins in wt and VimR113C 3T3L1 fibroblasts. (A,C) Interaction of vimentin with talin and vinculin at the focal adhesions (arrow in inset). (B,D) Unaltered localisation of talin and vinculin in the presence of cytosolic vimentin aggregates (arrows). (E,G) Colocalisation of vinculin and talin with β1-integrin at focal adhesions. (F,H) Localisation of talin and vinculin at focal adhesions, marked by β1-integrin is unaltered in VimR113C-expressing fibroblasts. These data suggest that VimR113C-containing IFs are unable to interact stably with focal adhesions. Scale bar: 20 µm.

 

Figure 9
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  		Fig. 9. Model of interaction between vimentin and focal adhesion proteins. (A) In fibroblasts, the vimentin-IF network is connected to integrins through interactions with synemin and vinculin or through plectin. (B) Expression of mutant vimentin in fibroblasts causes aggregate formation and severed connection of IF to focal adhesions. The presence of synemin in vimentin aggregates and the unaltered localisation of vinculin and plectin at focal adhesions suggest a stronger association with vimentin compared with other proteins of the focal adhesion.

 

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© The Company of Biologists Ltd 2008