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First published online 13 June 2006
doi: 10.1242/jcs.03009

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Expression of disease-causing lamin A mutants impairs the formation of DNA repair foci

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

View larger version (59K): [in a new window]   Fig. 1. Assembly properties of GFP-tagged lamin A constructs. (A) Domain structure of the lamin A protein indicating disease-causing mutations that have been analysed in this study. (B) Western blot analysis of HeLa cell lysates expressing GFP-tagged lamin constructs, probed with anti-GFP and anti-lamin A antibodies. Unprocessed and processed forms of lamins are indicated by arrowheads, lamin A del50 is marked by asterisks, and A and C indicate lamin A and lamin C. Molecular mass markers indicated on the left are: phosphorylase b, 94 kDa; albumin, 67 kDa; and ovalbumin, 43 kDa. (C) Immunofluorescence analysis of GFP-tagged lamin A constructs transiently transfected into HeLa cells and counterstained with DAPI. Ut, untransfected cells; WT, wild-type GFP-lamin A; del50, lamin A del50; numbers represent positions of altered amino acid residues indicated in A. Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 2. Localisation of emerin. (A) HeLa cells transfected with the indicated lamin A constructs were fixed and stained with antibody to emerin, and counterstained with DAPI. Arrowheads indicate transfected cells showing mislocalisation of emerin. (B) Quantitative analysis of transfected cells showing normal emerin staining. Bar, 10 µm.

View larger version (81K): [in a new window]   Fig. 3. Optimisation of treatment with DNA-damaging agents. (A) Western blot analysis of untransfected HeLa cells and cells transfected with wild-type GFP-lamin A, treated with cisplatin (CP) for 0-24 hours, or UV irradiation with a recovery period of 0.5 or 24 hours, and probed with antibodies to PARP-1 and lamin A/C. Arrowheads indicate cleavage products as a result of apoptosis. Wild-type GFP-lamin A is indicated by an asterisk. Molecular mass markers indicated on the left are: phosphorylase b, 94 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; and carbonic anhydrase, 30 kDa. (B) HeLa cells treated with cisplatin for 4 hours or UV irradiation with a recovery time of 0.5 hours were stained with antibody to lamin A/C (LA-2B3). (C) HeLa cells treated with cisplatin for 0-24 hours were stained with antibody to -H2AX, and counterstained with DAPI. Arrowheads indicate fragmented nuclei. Percentages of apoptotic cells after cisplatin treatment were <3% (4h), 5% (8h), 15% (16h) and 40% (24h); and after UV irradiation, <3% (0.5h) and 50% (24h). Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 4. Formation of -H2AX foci after DNA damage. HeLa cells transfected with the indicated GFP-tagged lamin A constructs (green) were (A) treated with cisplatin for 4 hours or (B) irradiated with UV and allowed to recover for 0.5 hours, fixed and stained with antibody to -H2AX (red) and counterstained with DAPI. Bar, 10 µm. (C,D) Quantitative analysis of -H2AX staining with cisplatin-treated and UV-treated HeLa cells, respectively. Percentage of untransfected (Ut) or transfected cells positive (solid bars) or negative (open bars) for -H2AX foci are plotted.

View larger version (77K): [in a new window]   Fig. 5. Localisation of 53BP1 after DNA damage. (A) Untreated HeLa cells or cells treated with cisplatin for 4 hours or UV irradiation with a recovery time of 0.5 hours were stained with antibody to 53BP1 and counterstained with DAPI. (B) HeLa cells transfected with the indicated GFP-tagged lamin A constructs (green) were treated with cisplatin for 4 hours, fixed and stained with antibody to 53BP1 (red) and counterstained with DAPI. Arrows indicate transfected cells showing diffuse 53BP1 staining. Bar, 10 µm. (C) Quantitative analysis of cells showing 53BP1 staining in nuclear foci after cisplatin treatment. Ut, untransfected.

View larger version (86K): [in a new window]   Fig. 6. Localisation of ATR and ATM kinases in untreated cells. (A) HeLa cells transfected with a FLAG-ATR construct (ATR) or cotransfected with the indicated GFP-tagged lamin A constructs (green) were fixed and stained with anti-FLAG antibody (red), and counterstained with DAPI. Arrows indicate cells showing exclusive cytoplasmic staining of ATR. (B) As in A, except that a FLAG-ATM construct (ATM) was used. Bar, 10 µm. (C) Quantitative analysis of transfected cells expressing ATR, including nuclear and/or cytoplasmic staining (open bars), or ATM (solid bars), both normalised to wild type as 100%.

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