Figure 1.
Schematic presentation of lamin A/C and the HBcAg-Ig-fold fusion protein.
(A) Tripartite structure of Lamin A and Lamin C consisting of an N-terminal head domain, a long central alpha-helical rod domain, and a globular C-terminal Ig-fold domain. Lamin A contains a CaaX signal motif for post-translational processing at its C-terminus. The two asterisks highlight the two mutations within the Ig-fold domain, R453W and R482W, respectively, which were chosen as antigens. The amino acid sequences comprising the respective mutant amino acids are highlighted. (B) Structure of the lamin A/C Ig-fold. The NMR structure obtained from Krimm et al. [11] was modified with the PyMOL Molecular Viewer program. The position and the side chains of the amino acids Arg453 and Arg482 are highlighted in red, and the first and the last amino acid of the Ig-fold (Ser428 and Val549, respectively) are indicated. (C) Structure of HBcAg. The crystal structure obtained from Wynne et al. [50] was modified with the PyMOL Molecular Viewer program. The immunodominant B-cell epitope c/e1 is highlighted in purple. (D) Recombinant HBcAg-Ig-fold protein. The Ig-fold, either in the wild-type sequence or with the R453W or R482W point mutation (indicated by an asterisk) was cloned into the c/e1 epitope of the His-HBcAg protein (aa 1–149), substituting amino acids Pro79 and Ala80. The Ig-fold is flanked by flexible glycine-rich linkers to allow proper folding of the inserted Ig-fold as well as of the HBcAg protein.
Table 1.
Oligonucleotides.
Figure 2.
R453W antibody is specific for the EDMD associated lamin A/C R453W point-mutant.
(A) HeLa, HeLa Flag-Lamin A wt, HeLa Flag-Lamin A R453W, and HeLa Flag-Lamin A R482W cell lysates were separated by 7.5% SDS-PAGE and analyzed by Western blotting with the mouse monoclonal antibodies anti R453W (panel I), anti Flag M2 (panel II), anti Lamin A/C 4C11 (panel III), and β-actin (panel IV). (B) Peptide competition of R453W antibody. Binding of R453W antibody to Flag-Lamin A R453W was competed with increasing amounts (0.44, 2.2, 11, and 55pmol) of mutant “R453W Peptide” (NH2-449GKFVWLRNK457-COOH) or wild-type “wt Peptide” (NH2-449GKFVRLRNK457-COOH).Competition efficiency was examined by 7.5% SDS-PAGE/Western blot analysis of HeLa Flag-Lamin A R453W cell lysates. Control antibodies were: anti R453W not subjected to competition, anti Flag M2, anti Lamin A/C 4C11. (C) Immunofluorescence of a mixed population of HeLa and HeLa Flag-Lamin A R453W cells, seeded at a ratio of 3∶1. Cell staining was performed with anti R453W (top panel), anti Flag M2 (middle panel), and anti Lamin A/C 4C11 (bottom panel). DNA was counterstained with Hoechst 33342. Bar scale, 20 µm.
Figure 3.
R482W antibody is specific for the FPLD-associated lamin A/C R482W point-mutant.
(A) 7.5% SDS-PAGE of HeLa, HeLa Flag-Lamin A wt, HeLa Flag-Lamin A R453W, and HeLa Flag-Lamin A R482W cell lysates followed by Western blot analysis with the mouse monoclonal antibodies anti R482W (panel I), anti Flag M2 (panel II), anti Lamin A/C 4C11 (panel III), and β-actin (panel IV). (B) Ig-fold competition of R482W antibody. Binding of anti R482W to Flag-Lamin A R482W was competed with recombinant HBcAg protein containing either the wild-type Ig-fold, “Ig-wt”, or the mutant Ig-fold, “Ig-R482W”, at increasing molar ratios. Competition efficiency was examined by 7.5% SDS-PAGE/Western blot analysis of HeLa Flag-Lamin A R482W cell lysates. Control antibodies were: anti R482W not subjected to competition, anti Flag M2, anti Lamin A/C 4C11. (C) Immunofluorescence of a mixed population of HeLa and HeLa Flag-Lamin A R482W cells, seeded at a ratio of 3∶1. Cells were stained with anti R482W (top panel), anti Flag M2 (middle panel), and anti Lamin A/C 4C11 (bottom panel). DNA was counterstained with Hoechst 33342. Bar scale, 20 µm.
Figure 4.
R482W antibody recognizes a discontinuous epitope in the lamin A/C Ig-fold.
(A) Equal amounts of peptide pR482 (472QNGDDPLLTYRFPPKFTLKAG492), pW482 (472QNGDDPLLTYWFPPKFTLKAG492), pR453 (449GKFVRLRNK457), pW453 (449GKFVWLRNK457) and equal amounts of recombinant HBcAg Ig-fold wt, HBcAg Ig-fold R453W, and HBcAg Ig-fold R482W were spotted onto PVDF membranes. Blots were incubated either with anti R482W antibody (upper left panels) or with anti R453W antibody (upper right panels). Anti lamin A/C (middle panels) and Ponceau S staining (bottom panels) confirmed equal spotting of recombinant HBcAg Ig-fold proteins or peptides, respectively. (B) 12.5% SDS-PAGE of bacterial lysates expressing His-tagged HBcAg Ig-fold R482W fragments of increasing size followed by Western blot analysis with R482W antibody (left panel). Equal amounts of recombinant proteins were confirmed by incubation with anti-His antibody (right panel). Numbers correspond to amino acids of lamin A/C.
Figure 5.
R453W and R482W antibodies recognize the respective disease causing mutant A-type lamins in primary patient fibroblasts.
(A) Western blot analysis of wild type, EDMD (R453W), and FPLD (R482W) primary human fibroblast lysates with anti R453W (panel I), anti R482W (panel II), anti Lamin A/C 4C11 (panel III), and anti β-actin (panel IV). (B) Immunofluorescence of wild-type and EDMD primary human fibroblasts. Cells were stained with anti R453W (top panels) and anti Lamin A/C 4C11 (bottom panels). The DNA was counterstained with Hoechst 33342. Bar scale, 20 µm. (C) Immunofluorescence of wild-type and FPLD primary human fibroblasts. Cells were stained with anti R482W (top panels) and anti Lamin A/C 4C11 (bottom panels). The DNA was counterstained with Hoechst 33342. Bar scale, 20 µm. (D) Immunoprecipitation of wild-type and mutant lamin A/C from wild-type, EDMD, and FPLD cell lysates. The precipitated proteins were separated by 7.5% SDS-PAGE and analyzed by Western blotting with either the anti R453W or anti R482W antibody (top panels), or with anti Lamin A/C 4C11 (bottom panels).
Figure 6.
EDMD patient cells contain an increased number of transnuclear tubules.
(A) Immunofluorescence of wild-type (top panel), EDMD (middle panel), and FPLD (bottom panel) primary human fibroblasts. Cells were co-stained with anti Lamin A/C N-18 and anti Lamin A/C 4C11, anti R453W, or anti R482W, respectively. Mid plane sections, xz-projections (above), and yz-projections (right) of the nuclei are shown. Arrowheads indicate transnuclear tubules through which the respective projection planes cut. Bar scale, 10 µm. (B) Number of transnuclear tubules per cell for wild-type, EDMD, and FPLD fibroblasts. n = 44 (wild-type), n = 46 (EDMD), n = 50 (FPLD). ** p<0.02; * p = 0.13. (C) Merged image of a mid plane section, xz-projection (above), and yz-projection (right) of an EDMD nucleus. Bar scale, 10 µm.
Figure 7.
Wild-type and mutant R453W lamin A/C are present in distinct, non co-localizing foci in EDMD nuclei.
(A) Immunofluorescence of two EDMD nuclei. Cells were co-stained with anti Lamin A/C N-18 and anti R453W. Fluorescence intensity of the lamin A/C staining by the two antibodies along the dashed lines in the magnified images of the nucleoplasm (4th image in each row) and of the nuclear rim (last image in each row) are shown above or below the respective images. Areas of magnification (3.5-fold) are indicated by white squares in the merged images of each nucleus. Bar scale, 10 µm. (B) Cellular fractionation of wild-type and EDMD cells. Whole cell lysate (WC), cytoplasmic (C), whole nuclear (N), low-salt soluble nuclear (LS), low-salt insoluble nuclear (LP), high-salt soluble nuclear (HS), and high-salt insoluble nuclear (HP) fractions were separated by 7.5% SDS-PAGE and analyzed by Western blotting with the indicated antibodies.