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Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy

¹ Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
² INSERM UR523, Institut de Myologie, GH Pitié-Salpétrière, 75651 Paris, France

View larger version (79K): [in a new window]   Fig. 1. (A) Lamin A and the localization of mutations. The N-terminus is on the left. Domains of lamin A are represented by: white, globular head and tail regions and linker regions in the rod-domain; stripes, -helical rod-domain regions 1a, 1b and 2; black, the most highly conserved regions of the rod-domain. Underlined mutations are found in dilated cardiomyopathy, mutations in italics in FPLD and others in AD-EDMD. Mutations shown in red are proteins that form aberrant intranuclear foci when expressed in C2C12 cells (Fig. 1B; Table 1). (B) Cellular localization of FLAG-tagged wild-type and mutant forms of lamin A. The panels show laser scanning confocal immunofluorescence microscopy images of C2C12 cells transfected with wild-type prelamin A (WT) or prelamin A with missense mutations as indicated. Arrows show cells with nuclear foci, insets show nucleoplasmic staining in cells expressing mutant protein but lacking foci. Antibodies used were mouse monoclonal antibodies against FLAG recognized by FITC-conjugated secondary antibodies and rabbit polyclonal antibodies against lamin B1 recognized by AlexaTM-conjugated secondary antibodies. The pictures show an overlay of the FITC (green) and AlexaTM (red) channels where areas of colocalization appear yellow. Bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 2. Aberrant cellular localization of lamin A mutants is often accompanied by a change in lamin B1 localization. The panels show laser scanning confocal immunofluorescence microscopy images of C2C12 cells expressing either wild-type lamin A (WT) or lamin A with missense mutations as indicated, fused to a FLAG-epitope. The bottom panels show the NLA mutant, which lacks the N-terminal head-domain, fused to a FLAG-epitope. Arrows show lamin B1 localized to the nuclear envelope. Antibodies used were monoclonal antibodies against FLAG recognized by FITC-conjugated secondary antibodies (left) and polyclonal antibodies against lamin B1 recognized by AlexaTM-conjugated secondary antibodies (middle). The pictures to the right show an overlay of the FITC (green) and AlexaTM (red) channels. Bars, 10 µm.

View larger version (81K): [in a new window]   Fig. 3. Analysis of lamin B2 and LAP2 localization in cells expressing lamin A mutants. The panels show laser scanning confocal immunofluorescence microscopy images of C2C12 cells transfected with the prelamin A E358K or NLA mutants fused to a FLAG-epitope. (A) Staining with monoclonal antibodies against lamin B2 (left panel) and polyclonal antibodies against lamin A/C (middle panel). Arrows indicate concentration of lamin B2 by nuclear foci and absence of lamin B2 from one pole of some nuclei. (B) Staining with monoclonal antibodies against FLAG (left panel) and polyclonal antibodies against LAP2 (middle panel). The monoclonal antibodies were recognized by FITC-conjugated secondary antibodies and the polyclonal antibodies were recognized by rhodamine-conjugated secondary antibodies. The panels to the right show an overlay of the FITC (green) and rhodamine (red) channels. Bars, 10 µm.

View larger version (63K): [in a new window]   Fig. 4. Nuclear pore complexes are not present in nuclear foci. The panels show laser scanning confocal immunofluorescence microscopy images of C2C12 cells transfected with prelamin A with missense mutations as indicated. Short arrows indicate lack of pore complexes in the nuclear foci and long arrows their absence from one pole of some nuclei. Antibodies used were monoclonal antibodies against nuclear pore complex protein p62 (NPC) (left panel) and polyclonal antibodies against lamin A/C (middle panel). The monoclonal antibodies were recognized by FITC-conjugated secondary antibodies and the polyclonal antibodies were recognized by rhodamine-conjugated secondary antibodies. The panels to the right show an overlay of the FITC (green) and rhodamine (red) channels. Bars, 10 µm.

View larger version (76K): [in a new window]   Fig. 5. Endogenous lamin A/C is localized both to the nuclear foci and to the nuclear rim. Staining with monoclonal antibodies against FLAG, recognizing exogenous protein, and polyclonal anti-lamin A/C antibodies, recognizing both exogenous and endogenous A-type lamins. The monoclonal antibodies were recognized by FITC-conjugated secondary antibodies and the polyclonal antibodies were recognized by rhodamine-conjugated secondary antibodies. The panels show an overlay of the FITC (green) and rhodamine (red) channels. Areas of colocalization appear yellow. Arrows show lamin A/C localized to the nuclear envelope. Bars, 10 µm.

View larger version (55K): [in a new window]   Fig. 6. Some lamin A mutations increase the loss of emerin from the nuclear envelope in transfected cells. (A) Laser scanning confocal immunofluorescence microscopy images of C2C12 cells transfected with wild-type (WT) or indicated mutated prelamin A, fused to a FLAG-epitope. In cells expressing mutants causing an increase in emerin loss from the nuclear envelope, examples are shown both of cells with correctly localized and mislocalized emerin. Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (left) and polyclonal antibodies against emerin recognized by rhodamine-conjugated secondary antibodies (middle). The panels to the right show an overlay of the FITC (green) and rhodamine (red) channels. Bars, 10 µm. (B) Percentage of cells with lack of emerin in the nuclear envelope in cells expressing wild-type (WT) or mutant lamin A. One hundred cells each in two separate experiments were studied. P values denote a statistically significant difference between the mutant and wild-type as determined by 2-test. NS denotes no significant difference.

View larger version (87K): [in a new window]   Fig. 7. Pulse-chase analysis of wild-type and mutant forms of lamin A. COS-7 cells were transiently transfected with wild-type (WT) or mutant forms of prelamin A containing FLAG-epitopes, as indicated above the panels. The cells were pulse-labeled with [35S]-methionine for 1 hour. After 0, 3 or 25 hours (indicated above the panels) of incubation in non-radioactive media (chase), cell lysates were prepared and immunoprecipitated using M2 anti-FLAG agarose. The panels show autoradiograms of precipitated proteins separated by SDS-PAGE. Migration of molecular mass markers is indicated on the left. Plus signs (+) show prelamin A; asterisks (*) show mature lamin A. All experiments were performed in triplicate; representative samples are shown. Owing to the large number of samples and the importance of rapid handling, not all samples could be prepared at once. The results from different experiments varied slightly in labeling efficiency and level of background. The first five panels and the last three panels are taken from two separate experiments. Immunoprecipitates from transfected cells also contained a protein with an apparent molecular mass of 100 kDa. The identity of this protein is not clear but it was absent in untransfected cells and recognized by anti-FLAG and anti-lamin A/C antibodies on western blots (data not shown), suggesting that it is an aberrantly expressed form of lamin A.