Properties of Lamin a Mutants Found in Emery-Dreifuss Muscular Dystrophy, Cardiomyopathy and Dunnigan- Type Partial Lipodystrophy

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RESEARCH ARTICLE 4435 Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan- type partial lipodystrophy

Cecilia Östlund1, Gisèle Bonne2, Ketty Schwartz2 and Howard J. Worman1,* 1Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA 2INSERM UR523, Institut de Myologie, GH Pitié-Salpétrière, 75651 Paris, France *Author for correspondence (e-mail: [email protected])

Accepted 6 September 2001 Journal of Cell Science 114, 4435-4445 (2001) © The Company of Biologists Ltd

SUMMARY

Autosomal dominant Emery-Dreifuss muscular dystrophy A/C, lamin B1 and lamin B2 were also altered in cells is caused by mutations in the LMNA gene, which encodes expressing these four mutants and three of them caused a lamin A and lamin C. Mutations in this gene also give rise loss of emerin from the nuclear envelope. In the yeast two- to limb girdle muscular dystrophy type 1B, dilated hybrid assay, the 15 lamin A mutants studied interacted cardiomyopathy with atrioventricular conduction defect with themselves and with wild-type lamin A and lamin B1. and Dunnigan-type partial lipodystrophy. The properties Pulse-chase experiments showed no decrease in the stability of the mutant lamins that cause muscular dystrophy, of several representative lamin A mutants compared with lipodystrophy and dilated cardiomyopathy are not known. wild-type. These results indicate that some lamin A We transfected C2C12 myoblasts with cDNA encoding mutants causing disease can be aberrantly localized, wild-type lamin A and 15 mutant forms found in patients partially disrupt the endogenous lamina and alter emerin affected by these diseases. Immunoﬂuorescence microscopy localization, whereas others localize normally in showed that four mutants, N195K, E358K, M371K and transfected cells. R386K, could have a dramatically aberrant localization, with decreased nuclear rim staining and formation of Key words: Nuclear envelope, Lamins, Intermediate ﬁlaments, intranuclear foci. The distributions of endogenous lamin Muscular dystrophy, Lipodystrophy, Cardiomyopathy

INTRODUCTION mutations that cause cardiac and skeletal muscle disease are found throughout the gene, most being point mutations Emery-Dreifuss muscular dystrophy (EDMD) is characterized resulting in amino acid substitutions. There appears to be no by contractures of the elbows, Achilles’ tendons and posterior clear correlation between the site of mutation and the severity neck, slow progressive muscle wasting and cardiomyopathy of disease (Bonne et al., 2000). By contrast, mutations in with atrioventricular conduction block (Emery and Dreifuss, LMNA clustering only in exons 8 and 11 have been shown to 1966; Rowland et al., 1979; Emery, 1989). Emery-Dreifuss cause Dunnigan-type familial partial lipodystrophy (FPLD, muscular dystrophy is inherited in both an autosomal dominant OMIM 151660) (Cao and Hegele, 2000; Shackleton et al., (AD-EDMD, OMIM 181350) and an X-linked, recessive 2000; Speckman et al., 2000; Vigouroux et al., 2000). FPLD (OMIM 310300) manner. In 1994, mutations in emerin, an is a rare, autosomal dominant disease characterized by the loss integral protein of the nuclear envelope inner membrane, were of subcutaneous adipose tissue from the extremities and trunk shown to be responsible for X-linked EDMD (Bione et al., after the onset of puberty (Köbberling and Dunnigan, 1986). 1994; Manilal et al., 1996; Nagano et al., 1996). The Lamins are intermediate filament proteins that form the connection between muscle disease and mutations in a nuclear nuclear lamina, a meshwork on the nucleoplasmic side of the envelope protein was unexpected, but was further established inner nuclear membrane (Aebi et al., 1986; Fisher et al., 1986; when Bonne et al. (Bonne et al., 1999) showed that AD- McKeon et al., 1986; Stuurman et al., 1998; Worman and EDMD, a phenotypically indistinguishable syndrome, is Courvalin, 2000). Two general types of lamins have been caused by mutations in the LMNA gene, which encodes the identified in somatic cells, A-type and B-type. The somatic cell nuclear envelope proteins lamins A and C. Mutations in LMNA A-type lamins, lamin A, lamin C and lamin A∆10, are have also been shown to cause limb girdle muscular dystrophy alternative splice isoforms encoded by the LMNA gene (Lin 1B (LGMD1B, OMIM 159001), a disease that differs from and Worman, 1993; Machiels et al., 1996). Two different genes AD-EDMD by displaying a different distribution of affected encode the somatic cell B-type lamins, lamin B1 and lamin B2 skeletal muscle, a later occurrence of cardiac problems and an (Biamonti et al., 1992; Lin and Worman, 1995). Like all absence of early contractures (Muchir et al., 2000), and dilated intermediate filament proteins, lamins have a conserved central cardiomyopathy without skeletal muscle pathology (OMIM rod-domain containing three α-helical segments, which is 115200) (Fatkin et al., 1999; Bécane et al., 2000). LMNA responsible for the formation of coiled-coil dimers. The rod- 4436 JOURNAL OF CELL SCIENCE 114 (24) domain is flanked by N-terminal head and C-terminal tail- (D-MEM) containing 10% fetal bovine serum (Life Technologies, domains, which vary significantly between different proteins Gaithersburg, MD) at 37°C and 10% CO2. Cells were transfected in (Steinert and Roop, 1988). The lamins bind to some of the chamber slides using Lipofectamine PLUSTM (Life Technologies), integral membrane proteins of the inner nuclear envelope following the manufacturer’s instructions. The cells were overlaid (Worman and Courvalin, 2000). Lamin A interacts with emerin with the lipid-DNA complexes for approximately 23 hours, the first and lamina associated polypeptide 1 (LAP1) in vitro (Foisner five of which were in serum-free medium. The cells were then allowed to grow in fresh medium for 24 hours post-transfection before and Gerace, 1993; Clements et al., 2000; Sakaki et al., 2001). preparation for immunofluorescence microscopy. Because the functions of lamins A and C are poorly Fixation and labeling of cells for immunofluorescence microscopy understood, the mechanisms by which mutations cause were performed as described previously (Östlund et al., 1999). The different inherited diseases are not clear (Worman and monoclonal primary antibodies used were anti-FLAG M5 (Sigma, St Courvalin, 2000; Hutchison et al., 2001). Currently, it is not Louis, MO) diluted 1:200, anti-PCNA (proliferating cell nuclear known if any of the disease-causing mutations of lamin A alter antigen) P10 (Roche Molecular Biochemicals, Indianapolis, IN) protein localization, lamina structure, protein-protein diluted 1:20, anti-nuclear pore complex MAb414 (a gift from Tarik interactions or protein stability. We therefore investigated these Soliman, Laboratory of Cell Biology, Rockefeller University, New properties of mutant forms of lamin A found in patients with York, NY) diluted 1:5,000 and anti-lamin B2 X223 (a gift from Georg inherited diseases. Krohne, University of Würzburg, Würzburg, Germany) diluted 1:400. Polyclonal primary antibodies used were anti-lamin B1 (Cance et al., 1992) at a dilution of 1:2,000, anti-lamin A/C (Cance et al., 1992) at a dilution of 1:1,000, anti-emerin (a gift from Glenn Morris, North MATERIALS AND METHODS East Wales Institute, Wrexham, UK) at a dilution of 1:3,000 and anti- LAP2 (a gift from Katherine Wilson, Johns Hopkins University, Plasmid construction Baltimore, MD) at a dilution of 1:500. Secondary antibodies used All cloning procedures were performed according to standard were Rhodamine RedTM-X-conjugated goat anti-rabbit IgG (Jackson methods (Sambrook et al., 1989). For expression in mammalian cells, ImmunoResearch Laboratories, Inc., West Grove, PA), AlexaTM 568- constructs that expressed FLAG-tagged polypeptides were made in conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) pSVK3 (Amersham Pharmacia Biotech, Inc., Piscataway, NJ), which and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse contains a multiple cloning site downstream from the SV-40 early IgG (Jackson ImmunoResearch Laboratories, Inc.). For DNA- promoter. cDNA encoding prelamin A, cloned into the BamHI and staining, 20 µg/ml propidium iodide (Sigma) was added to the SalI restriction sites of plasmid pGBT9 (Ye and Worman, 1995), was secondary antibody incubation. Labeled and washed slides were excised by restriction endonuclease digestion with SmaI and SalI and dipped in methanol, air-dried, and coverslips were mounted using ligated into pBFT4, digested with the same restriction enzymes. ProLong Antifade Kit (Molecular Probes, Inc.) or SlowFade Light pBFT4 is a pBluescript II KS- (Stratagene, La Jolla, CA) based Antifade Kit (Molecular Probes, Inc.). Immunofluorescence plasmid containing a Kozak sequence, ATG and the FLAG-tag coding microscopy was performed on a Zeiss LSM 410 confocal laser sequence 5′ to the multiple cloning site. The resulting plasmid was scanning system attached to a Zeiss Axiovert 100TV inverted digested with SpeI and XhoI to excise a cDNA encoding ATG-FLAG- microscope (Carl Zeiss, Inc., Thornwood, NY). Images were prelamin A that was ligated into pSVK3, digested with XbaI (an processed using Photoshop software (Adobe Systems, Inc., San Jose, isoschizomer of SpeI) and XhoI. CA) on a Macintosh G3 computer (Apple Computer, Inc., Cupertino, cDNAs encoding mutant forms of prelamin A, except the ∆NLA CA). mutant, which lacked amino acids 1-33, were made using the TransformerTM Site-Directed Mutagenesis Kit (CLONTECH Yeast two-hybrid assay Laboratories, Inc., Palo Alto, CA), following the manufacturer’s Saccharomyces cerevisiae strain Y187 (Clontech Laboratories, Inc.) instructions. ∆NLA was generated by polymerase chain reaction (Saiki was transformed with plasmids encoding lamins fused to the DNA et al., 1987), using the Gene Amp PCR System 2400 (Applied binding domain of the S. cerevisiae GAL4 protein (pGBT9) or Biosystems, Foster City, CA), with a SmaI restriction site engineered to the GAL4 transcriptional activation domain (pGAD424). at the 5′ end of the sense primer and an antisense primer spanning the Transformations and β-galactosidase assays were done according to ApaI site found at nucleotide 746 of prelamin A cDNA. Reaction the Matchmaker Two-Hybrid System manual (Clontech Laboratories, products were digested with SmaI and ApaI and ligated into prelamin Inc.). A-pBGT9 digested with the same enzymes. The resulting plasmid was digested with SmaI and SalI and ligated into the SmaI and SalI Pulse-chase experiment restriction sites of pSVF, a vector constructed by insertion of the COS-7 cells were grown in D-MEM containing 10% fetal bovine FLAG-epitope between the EcoRI and KpnI restriction sites of pSVK3. serum (Life Technologies) at 37°C and 5% CO2. The cells were For studies with the yeast two-hybrid system, wild-type prelamin transfected using Lipofectamine PLUSTM (Life Technologies), A cDNA in vectors pGAD424 and pGBT9 and lamin B1 cDNA in following the manufacturer’s instructions. The cells were overlaid vector pGBT9 were those previously described (Ye and Worman, with the lipid-DNA complexes for approximately 23 hours, the first 1995). cDNAs obtained by site-directed mutagenesis were excised five of which were in serum-free medium, and were then grown in from vector pSVK3 using the restriction endonucleases SmaI and SalI fresh medium for 24 hours before being subcultured at a 1:5 dilution and ligated into pGBT9 and pGAD424 digested with the same into 60 mm tissue culture dishes. After another 24 hours, cells were enzymes. ∆NLA was inserted into pGBT9 as described above and washed with D-MEM without methionine and then overlaid with 1 ml then digested with SmaI and SalI and ligated into the SmaI and SalI D-MEM without methionine, containing 28 µCi Pro-mix L-[35S] in sites of pGAD424. All cDNAs were sequenced using an ABI Prism vitro cell labeling mix (Amersham Pharmacia Biotech, Inc.). After 1 377 automated sequencer (Applied Biosystems). hour, cells were washed twice with phosphate-buffered saline (PBS) and either incubated in non-radioactive D-MEM with 10% fetal Cell culture, transfection and immunofluorescence bovine serum for an additional 3 or 25 hours or lysed immediately. microscopy For cell lysis, cells were washed three times in PBS and incubated for C2C12 cells (a gift from Hal Skopicki, Columbia University, New 40 minutes at 4°C in 440 µl lysis buffer (50 mM Tris-HCl, pH 8, 5 York, NY) were grown in Dulbecco’s modified Eagle medium mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate Lamin A mutations in disease 4437

(SDS), 1% Triton X-100) with 2% bovine serum albumin (BSA), 0.5 mM phenylmethylsulfonyl fluoride and 4.4 µl protease cocktail inhibitor (Sigma). The cells were then scraped off with a rubber policeman and sheared six times through a 21-gauge needle. After centrifugation for 10 minutes at 10,000 g, the supernatant was incubated overnight at 4°C with 30 µl anti-FLAG M2-agarose affinity gel (Sigma). The affinity gel was collected by centrifugation for 2 minutes at 325 g, washed three times in lysis buffer with 2% BSA, once with lysis buffer without BSA, once with 100 mM Tris-HCl (pH 6.8) and 0.5 M NaCl, and once with 100 mM Tris-HCl (pH 6.8). For the first, fourth and fifth washes, the gel was incubated for 5 minutes on a rotating wheel at 4°C. Twenty µl SDS- sample buffer (Laemmli, 1970) was added to the affinity gel and the samples were incubated for 15 minutes at 70°C and centrifuged for 2 minutes at 325 g. The proteins in the supernatants were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Gels were fixed for 30 minutes in 35% methanol, 10% acetic acid, incubated 30 minutes with Amplify (Amersham Pharmacia Biotech, Inc.), dried and exposed to Hyperfilm ECL (Amersham Pharmacia Biotech, Inc.) at –70°C. Other chemicals Unless otherwise indicated, routine chemicals were obtained from either Fisher Scientific Co. (Pittsburgh, PA) or Sigma. Enzymes for DNA cloning were obtained from either Fisher Scientific Co. or New England Biolabs (Beverly, MA).

RESULTS

Some lamin A mutants have a dramatically abnormal intranuclear localization To determine if the intracellular localizations of mutant forms of lamin A differ from that of the wild-type, we transiently transfected C2C12 mouse myoblasts with plasmids expressing wild- Fig. 1. (A) Lamin A and the localization of mutations. The N-terminus is on the left. type and mutated forms of prelamin A. Prelamin Domains of lamin A are represented by: white, globular head and tail regions and A is a precursor form of mature lamin A, which linker regions in the rod-domain; stripes, α-helical rod-domain regions 1a, 1b and 2; is processed by endoproteolytic cleavage of the black, the most highly conserved regions of the rod-domain. Underlined mutations final 18 amino acids (Weber et al., 1989; are found in dilated cardiomyopathy, mutations in italics in FPLD and others in AD- Sinensky et al., 1994). For detection of expressed EDMD. Mutations shown in red are proteins that form aberrant intranuclear foci proteins, FLAG-epitopes were fused to the N- when expressed in C2C12 cells (Fig. 1B; Table 1). (B) Cellular localization of termini of the constructs. The mutations studied FLAG-tagged wild-type and mutant forms of lamin A. The panels show laser were: R60G, L85R, N195K and E203G, found in scanning confocal immunofluorescence microscopy images of C2C12 cells transfected with wild-type prelamin A (WT) or prelamin A with missense mutations patients with dilated cardiomyopathy (Fatkin et as indicated. Arrows show cells with nuclear foci, insets show nucleoplasmic al., 1999); E358K, M371K, R386K, R453W, staining in cells expressing mutant protein but lacking foci. Antibodies used were W520S, R527P, T528K and L530P, identified in mouse monoclonal antibodies against FLAG recognized by FITC-conjugated patients with AD-EDMD (Bonne et al., 1999; secondary antibodies and rabbit polyclonal antibodies against lamin B1 recognized Bonne et al., 2000); and R482Q, R482W and by AlexaTM-conjugated secondary antibodies. The pictures show an overlay of the K486N, found in patients with FPLD (Cao and FITC (green) and AlexaTM (red) channels where areas of colocalization appear Hegele, 2000; Shackleton et al., 2000) (Fig. 1A). yellow. Bar, 10 µm. In transfected cells, FLAG-tagged wild-type lamin A localized to the nuclear periphery, showing M371K and R386K, showed a dramatically aberrant colocalization with lamin B1, a marker for the lamina and localization in many cells, accumulating in large intranuclear nuclear envelope (Fig. 1B). A majority of the FLAG-tagged foci (Fig. 1B). These four mutants often showed a decrease in lamin A mutants showed no gross abnormalities in cellular nuclear rim localization, giving a more diffuse, nucleoplasmic localization; however, four of the mutants, N195K, E358K, staining, which was also seen in cells lacking intranuclear foci 4438 JOURNAL OF CELL SCIENCE 114 (24)

Table 1. Statistical analysis of nuclear foci in cells expressing lamin A mutants Cells with Cells without foci Cells with foci foci (%) WT 199.33±0.33 0.67±0.33 0.3 R60G 199.33±0.66 0.67±0.66 0.3 L85R 199.00±0.58 1.00±0.58 0.5 N195K* 164.33±6.44 35.67±6.44 17.8 E203G 198.67±0.33 1.33±0.33 0.7 E358K* 177.00±3.05 23.00±3.05 11.5 M371K* 135.33±8.29 64.67±8.29 32.3 R386K* 155.00±5.83 45.00±5.83 22.5 R453W 196.67±1.45 3.33±1.45 1.7 R482Q 199.00±0.58 1.00±0.58 0.5 R482W 199.00±0.58 1.00±0.58 0.5 K486N 198.33±0.88 1.67±0.88 0.8 W520S 197.67±0.88 2.33±0.88 1.2 R527P 195.33±1.45 4.67±1.45 2.3 T528K 196.67±0.66 3.33±0.66 1.7 L530P 198.33±0.88 1.67±0.88 0.8 ∆NLA* 113.00±2.64 87.00±2.64 43.5

Numbers shown are means from three experiments±s.e. unless otherwise stated. 200 transfected cells of each type were studied in each experiment. Nuclear foci smaller than 0.7 µm were not counted, as lamin foci smaller than this size frequently can be seen in all cells, including untransfected cells. Asterisks (*) denote statistically signiﬁcant differences between mutant and wild-type (WT) cells in the number of cells with nuclear foci as determined by χ2-test (P<0.001). In all other cases, there were no signiﬁcant differences (P>0.05).

(Fig. 1B, insets). The diffuse, nucleoplasmic staining was particularly common for mutant R386K, with approximately 95% of transfected cells with or without large intranuclear foci showing this pattern. Mutants N195K, E358K and M371K showed a mostly diffuse, nucleoplasmic localization in 50-70% of transfected cells. Not all cells expressing the four foci-forming lamin A mutants contained large intranuclear foci. We counted 200 transfected cells expressing wild-type lamin A and each of the 15 mutants for nuclear foci. The results of these studies showed that there were significant numbers of cells with large intranuclear foci only in cells expressing the N195K, E358K, M371K and R386K mutants (Table 1). Only foci larger than µ Fig. 2. Aberrant cellular localization of lamin A mutants is often 0.7 m were counted, as many cells, including untransfected accompanied by a change in lamin B1 localization. The panels show controls, frequently contained smaller foci of lamins in their laser scanning confocal immunofluorescence microscopy images of nuclei. A localization of wild-type lamin A in small C2C12 cells expressing either wild-type lamin A (WT) or lamin A intranuclear foci has been reported previously (Moir et al., with missense mutations as indicated, fused to a FLAG-epitope. The 1994; Broers et al., 1999). bottom panels show the ∆NLA mutant, which lacks the N-terminal head-domain, fused to a FLAG-epitope. Arrows show lamin B1 Immunofluorescence analysis of cells expressing localized to the nuclear envelope. Antibodies used were monoclonal foci-forming lamin A mutants antibodies against FLAG recognized by FITC-conjugated secondary To investigate further the composition of the abnormal antibodies (left) and polyclonal antibodies against lamin B1 recognized by AlexaTM-conjugated secondary antibodies (middle). nuclear foci formed by four of the lamin A mutants and their The pictures to the right show an overlay of the FITC (green) and downstream effects on the nuclear envelope, we used several AlexaTM (red) channels. Bars, 10 µm. different markers to perform a detailed immunofluorescence microscopy analysis. Mislocalization of the lamin A mutants was often accompanied by disturbances in the localization of foci), where the foci often were large, than with mutants endogenous lamin B1. In many cells, lamin B1 was found in N195K (20-50%) and M371K (10-50%). The mislocalization the interior of the intranuclear foci, with the mutant lamin A of lamin B1 was, however, not complete, and in virtually being more concentrated at the edges (Fig. 2). This phenotype all cells, some lamin B1 could still be detected at the was seen to a variable degree, being more frequent with nuclear periphery (Fig. 2, arrows). In occasional cells, lamin mutants E358K and R386K (approximately 90% of cells with B1 was missing from one pole of the nucleus (Fig. 2, mutant Lamin A mutations in disease 4439 N195K), or from buds protruding out from the nucleus. This phenotype has been described in cells from mice lacking A-type lamins (Sullivan et al., 1999) and fibroblasts from patients with FPLD (Vigouroux et al., 2001). A similar pattern of nuclear foci was observed previously when a mutant form of lamin A (∆NLA), lacking the nonhelical head- domain (amino acids 1-33), was microinjected into mammalian cells or added to Xenopus laevis interphase egg extracts (Spann et al., 1997). When FLAG-tagged ∆NLA was expressed in our system, the same pattern was seen in many cells (Fig. 2) (Table 1). Lamin B2 was also partly mislocalized in some cells with mutant lamin A-containing nuclear foci. Representative results for the lamin A E358K mutant and ∆NLA are shown in Fig. 3A. Although apparently slightly less affected than lamin B1, lamin B2 was sometimes missing from one pole of the nucleus or concentrated near the nuclear foci (Fig. 3A, arrows). However, the peripheral distribution of the integral inner nuclear membrane protein LAP2β appeared normal in all cells (Fig. 3B). We also examined the distribution of nuclear pore complexes in cells containing aberrant lamin foci. Nuclear pore complexes showed a normal localization, even in cells with large intranuclear lamin foci (Fig. 4). They were not present in the foci, even when the foci were situated near the nuclear periphery (Fig. 4, N195K). As for lamin B1, there were occasional cells where the nuclear pore complexes were excluded from one pole of the cell (Fig. 4, M371K), as has also been reported in mouse cells lacking A-type lamins and fibroblasts from patients with FPLD (Sullivan et al., 1999; Vigouroux Fig. 3. Analysis of lamin B2 and LAP2 localization in cells expressing lamin et al., 2001). A mutants. The panels show laser scanning confocal immunofluorescence To obtain additional information about the effects microscopy images of C2C12 cells transfected with the prelamin A E358K or of foci-forming lamin A mutants on the nuclear ∆NLA mutants fused to a FLAG-epitope. (A) Staining with monoclonal lamina, we performed double labeling with anti- antibodies against lamin B2 (left panel) and polyclonal antibodies against FLAG antibodies, recognizing the exogenous lamin 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. A, and anti-lamin A/C antibodies, recognizing the (B) Staining with monoclonal antibodies against FLAG (left panel) and exogenous protein as well as endogenous lamins A polyclonal antibodies against LAP2 (middle panel). The monoclonal and C. This analysis showed an accumulation of antibodies were recognized by FITC-conjugated secondary antibodies and the endogenous lamins A and C inside the foci, similar to polyclonal antibodies were recognized by rhodamine-conjugated secondary the localization frequently seen with endogenous antibodies. The panels to the right show an overlay of the FITC (green) and lamin B1 (Fig. 5). As with lamin B1, however, some rhodamine (red) channels. Bars, 10 µm. lamin A/C still remained at the nuclear periphery in the majority of the cells. In two separate experiments with 100 transfected cells counted, peripheral lamin A/C was seen in a subset of all transfected cells, also some cells present in 85-100% of cells expressing mutants N195K, transfected with wild-type lamin A. Similar results have been E358K and M371K, and 70-90% of cells expressing mutants reported in transfected HeLa cells by Raharjo et al. (Raharjo R386K and ∆NLA (Fig. 5, arrows). et al., 2001). All foci-forming mutants except E358K led to Lamin A has been shown to bind to emerin in vitro a loss of emerin from the nuclear envelope in a significantly (Clements et al., 2000; Sakaki et al., 2001) and, in cells from greater number of cells than wild-type lamin A (Fig. 6B). We mice lacking A-type lamins, emerin is partly mislocalized to also investigated loss of emerin from the nuclear envelope in the cytoplasm (Sullivan et al., 1999). To investigate whether cells expressing three mutants that do not form foci (R60G, mutant forms of lamin A caused a mislocalization of emerin E203G and K486N). None of these mutants led to an in the presence of wild-type lamin A, the situation in increased loss of emerin from the nuclear envelope, compared heterozygous patients, we stained C2C12 cells expressing the to wild-type (data not shown). Emerin, similar to nuclear pore foci-forming lamin A mutants with antibodies against emerin complex proteins and LAP2, did not localize to the (Fig. 6A). A loss of emerin from the nuclear envelope was intranuclear lamin foci, further indicating that the foci are not 4440 JOURNAL OF CELL SCIENCE 114 (24)

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. membrane invaginations of the nuclear envelope but separate structures inside the nucleus. Finally, we examined the association of PCNA and DNA with the aberrant lamin foci. PCNA, a cofactor of DNA polymerase δ, which was shown previously to localize inside intranuclear foci containing ∆NLA in Xenopus egg extract experiments (Spann et al., 1997), very rarely accumulated inside the lamin A foci in our experiments (data not shown). DNA appeared to be mostly excluded from the foci (data not shown). Lamin A mutants interact with other lamins in the yeast two-hybrid assay Lamins, like other intermediate ﬁlaments, form parallel dimers through coiled-coil interactions between their α-helical rod-domains (Stuurman et al., 1998). To investigate if the ability of mutant lamin A molecules to interact was impaired, we performed a two-hybrid assay. Lamin A, prelamin A, lamin B1 and lamin C have previously been shown to interact with themselves and with each other in the yeast two-hybrid system (Ye and Worman, 1995). In this system, an interaction between a protein fused to the GAL4 activation domain and a protein fused to the GAL4 DNA-binding domain causes the expression of β-galactosidase, which can be possible using this assay to determine whether the protein- measured using a colorimetric assay (Fields and Song, 1989). protein interactions formed are the coil-coiled ones in the We expressed the 15 mutant forms of prelamin A described normal lamina, these results suggest that the primary lamin- above as fusion proteins with the GAL4 activation domain, or lamin dimerization is not disturbed in these mutants. the GAL4 DNA-binding domain, in S. cerevisiae. All of the mutant proteins interacted with themselves, wild-type prelamin A and wild-type lamin B1 (data not shown). No differences were seen between the mutant and wild-type lamins in the two-hybrid interaction assay. Although it is not

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. Lamin A mutations in disease 4441

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 χ2-test. NS denotes no significant difference.

Several lamin A mutants are as stable as wild-type exposed to Hyperﬁlm ECL. Representative results are shown lamin A in Fig. 7. At time-point zero (no chase), both prelamin A and To investigate whether representative mutant lamin A proteins the slightly smaller mature lamin A could be seen in all were less stable than wild-type lamin A, we performed pulse- samples from transfected cells. After 3 hours of growth in chase analyses. COS-7 cells were transfected with cDNAs nonradioactive media, no prelamin A could be seen and the encoding wild-type or mutant prelamin A with FLAG-epitopes total levels of radiolabeled FLAG-tagged lamin A had fused to their N-termini. The cells were pulsed with [35S]- decreased; it could, however, still be detected in all cases. After methionine for 1 hour and then harvested or grown in 25 hours of incubation, very small amounts of radiolabeled nonradioactive media for 3 or 25 hours. Cell lysates were lamin A could still be detected in all samples. These results incubated with anti-FLAG M2-agarose affinity gel and the showed that the representative lamin A mutants N195K, immunoprecipitates were separated by SDS-PAGE and M371K, R386K, R453W, R482W and K486N were as stable 4442 JOURNAL OF CELL SCIENCE 114 (24)

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. as wild-type lamin A. Mutant R386K seemed slightly more studies of the levels of mutant lamins A and C in cells from stable than the others after 3 hours of chase. Wild-type lamin patients carrying other LMNA mutations would provide A and these lamin A mutants were also processed normally important confirmation of our results on the stability of the from prelamin A to the mature protein. mutant proteins. Mutations in the lamin A rod-domain can cause an DISCUSSION abnormal intranuclear localization of the protein Mutant proteins may be unable to carry out their normal We have investigated 15 lamin A mutants in patients with functions, or disrupt the functions of associated proteins, dilated cardiomyopathy, AD-EDMD and FPLD. These because of mislocalization in cells. We examined the mutations are dominant and patients carry one mutant and one intracellular localization of 15 mutant lamin A proteins by wild-type copy of the LMNA gene. There are many possible immunofluorescence microscopy. The most striking feature mechanisms for how these mutations may cause disease, such in these studies was the formation of intranuclear foci by four as changes in the stability, localization and interactions of the of the lamin A mutants (N195K, E358K, M371K and mutant proteins. R386K), which was often accompanied by a mislocalization of some of the endogenous lamins. These cells also had a Mutant lamins are as stable as wild-type lamins decrease in the localization of the mutant lamins to the Disease phenotypes may be caused by haploinsufficiency of nuclear periphery. A nucleoplasmic localization and lamins A and C due to low levels of expression or rapid formation of intranuclear foci have also been reported when degradation of the mutant lamin protein. One mutation causing the N195K lamin A mutant was expressed in HeLa cells AD-EDMD is a nonsense mutation at the codon for amino acid (Raharjo et al., 2001). The four lamin proteins that formed six (Bonne et al., 1999; Bécane et al., 2000); in this case a intranuclear foci all had mutations introducing a lysine into reduction of lamins A and C most likely caused the disease. In the rod-domain. Three out of the four proteins had mutations patients with this mutation, both lamin A/C and emerin were at the end of the central, α-helical rod-domain, which is correctly localized to the nuclear envelope as shown by evolutionary well conserved between all intermediate immunolabeling of a cardiac biopsy (Bonne et al., 1999), filaments (Stuurman et al., 1998). A lamin A mutation although there was a decrease in the amount of lamin A/C in (R377H) causing LGMD1B has also recently been mapped cardiac tissue, as shown by western blot (Bécane et al., 2000). to this region (Muchir et al., 2000). The conserved segments Our studies indicate no decrease in the stability of several of the rod-domain have been shown to play crucial roles in mutant lamins, including those in AD-EDMD, cardiomyopathy the assembly of intermediate filament dimers into higher and FPLD. Therefore, a simple haploinsufficiency of lamins A order oligomers (Stuurman et al., 1998). Mutations in the can not be the only pathophysiological mechanism. Studies of corresponding regions of keratin K5 and K14 are responsible mutant lamins A and C in fibroblasts from patients carrying the for the majority of skin diseases caused by keratin mutations, LMNA mutations causing R482Q and R482W substitutions for example the Dowling-Meara type of epidermolysis showed these proteins to be present at similar levels to the wild- bullosa simplex (McLean and Lane, 1995). This disease and type proteins in control cells (Vigouroux et al., 2001). Further others caused by keratin mutations are blistering skin Lamin A mutations in disease 4443 disorders resulting from epithelial fragility. It is conceivable lower extent, it is not clear whether the emerin loss was due to that mutations in lamins A and C cause nuclear fragility. specific disturbances in the interactions between emerin and Nuclei of embryonic fibroblasts from mice lacking A-type mutant lamins or to a loss of peripheral lamins caused by the lamins and Xenopus nuclei assembled in egg extracts with the mutants. Alternatively, other effects of lamin A overexpression ∆NLA mutant have been shown to be more fragile than wild- in cells could lead to emerin mislocalization, but whatever type nuclei (Spann et al., 1997; Sullivan et al., 1999). One these effects may be, certain lamin A mutants enhance them. hypothesis is that an increased fragility of nuclei can be An increased loss of emerin from the nuclear envelope has also particularly harmful to muscle cells, where they are subjected been reported in HeLa cells expressing the lamin A mutants to mechanical stress (Worman and Courvalin, 2000; L85R, N195K and L530P, compared with wild-type (Raharjo Hutchison et al., 2001). This could explain the muscle- et al., 2001). specific nature of AD-EDMD and cardiomyopathy but does not explain why the specialized cardiomyocytes involved in Do lamins have a role in gene expression and DNA atrioventricular conduction may be more readily affected than replication? contractile cardiomyocytes (Morris, 2000). Alternative hypotheses, which could explain the tissue- The ‘nuclear stress-hypothesis’ as a pathophysiological specific nature of the diseases caused by mutations in the A- mechanism of disease is less likely in the case of FPLD. The type lamins, are that these proteins are involved in specific restriction of missense mutations causing FPLD to two regions gene expression or DNA replication events, or both. A-type in exon 8 and 11 implies that they encode a domain important lamins have been shown to bind proteins involved in for a specific lamin A function, for example interaction with a transcriptional regulation such as the retinoblastoma protein protein important for adipocyte survival. This is further (Mancini et al., 1994), and ectopic expression of lamin A has suggested by the concentration of the majority of FPLD also been shown to induce muscle-specific genes in mutations to codon 482 (Cao and Hegele, 2000; Shackleton et undifferentiated cells (Lourim and Lin, 1992). A role for the al., 2000; Speckman et al., 2000; Vigouroux et al., 2000). In inner nuclear membrane in the regulation of gene expression our studies, as well as in the studies by others (Holt et al., 2001; has previously been suggested by the interaction between the Raharjo et al., 2001), all using transiently transfected cells, lamin B receptor, an integral protein of the inner nuclear there were no gross abnormalities in the localization or stability membrane, and human orthologues of Drosophila HP1 (Ye of lamin A-containing mutations found in FPLD. However, and Worman, 1996; Ye et al., 1997). In Drosophila, HP1 fibroblasts from FPLD patients with the R482Q and R482W suppresses the expression of normally active euchromatic lamin A mutations, and some transfected fibroblasts genes translocated near heterochromatin (Eissenberg et al., overexpressing the R482W mutant, show more subtle nuclear 1990). Several other nuclear proteins involved in abnormalities (Vigouroux et al., 2001). neuromuscular disease are involved in gene expression, either at the level of transcription, splicing or mRNA transport Effects of lamin mutations on protein-protein (Morris, 2000). The nuclear lamins are also required for DNA interactions replication during the S-phase and the lamin foci formed in To examine the ability of mutant lamins to self-interact and to Xenopus egg extracts containing the ∆NLA mutant were interact with wild-type lamins, we used the yeast two-hybrid previously shown to contain the DNA polymerase δ cofactor assay. Our results showed no indication of impaired lamin- PCNA (Spann et al., 1997). PCNA, however, did not lamin interactions at this level. However, the two-hybrid assay accumulate in the foci formed by lamin mutants in C2C12 does not show whether the complexes formed are the usual cells in our studies. The reason for this discrepancy is not coiled-coil dimers. Neither would it detect disturbances of clear; one possibility is that PCNA localization is dependent higher order lamin interactions, such as filament formation. on cell-cycle phase. Although in vitro assembled Xenopus Subtle defects of lamin filament formation are at this time nuclei are in S-phase, it is not clear whether cells containing difficult to detect because lamins, as opposed to cytoplasmic nuclear foci can enter S-phase. It may also be due to different intermediate filaments, do not form stable filaments in vitro. fixation techniques; our cells were fixed with methanol, which Lamin A binds to LAP1 and emerin (Foisner and Gerace, yields a staining pattern where only PCNA bound to specific 1993; Clements et al., 2000; Sakaki et al., 2001). The nuclear structures is recognized by the antibody (Bravo and interaction between lamin A and emerin is especially intriguing Macdonald-Bravo, 1987). There may also be differences in because mutations in these two proteins both cause the EDMD lamina structure between mammalian cells versus Xenopus phenotype. In cells from mice lacking A-type lamins, emerin egg extracts, which has been suggested by Izumi et al. (Izumi is partially mislocalized to the cytoplasm (Sullivan et al., et al., 2000); they showed that PCNA is not colocalized with 1999). These mice show typical signs of EDMD. Heterozygous ∆NLA foci in CHO cells. mice, having only one wild-type copy of the LMNA gene, have The recent results of others (Raharjo et al., 2001; Vigouroux a slight mislocalization of emerin but are healthy (Sullivan et et al., 2001) and those reported in this study provide a starting al., 1999). We have studied the localization of emerin in C2C12 point towards understanding the properties of disease-causing cells expressing mutant forms of lamin A. Our studies showed lamin A/C mutants. They also exclude some possible an increased loss of endogenous emerin from the nuclear pathogenetic mechanisms, such as haploinsufficiency of lamin envelope when C2C12 cells, which have wild-type lamin A in A, absence of prelamin A processing or cytoplasmic addition to the mutant forms, were transfected with mutants sequestration, as the cause of disease in most instances. Further N195K, M371K, R386K or ∆NLA. However, as cells work is necessary to connect the behavior of mutant nuclear transfected with wild-type lamin A also exhibited a loss of lamins and their resulting effects on nuclear envelope structure emerin from the nuclear envelope, although to a significantly to muscular dystrophy, cardiomyopathy and lipodystrophy. 4444 JOURNAL OF CELL SCIENCE 114 (24)

This work was supported by grants from the Muscular Dystrophy Emery, A. E. H. and Dreifuss, F. E. (1966). Unusual type of benign X-linked Association (MDA) to H.J.W. and the Human Frontiers Science muscular dystrophy. J. Neurol. Neurosurg. Psychiatry 29, 338-342. Program to G.B. and H.J.W. Also, C.Ö. received support from the Fatkin, D., MacRae, C., Sasaki, T., Wolff, M. R., Porcu, M., Frenneaux, Sweden-America Foundation/Thanks to Scandinavia, Inc. and M., Atherton, J., Vidaillet, H. J., Jr, Spudich, S., De Girolami, U. et al. Karolina Widerström’s Foundation, and G.B. and K.S. received (1999). Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease. New. support from Association Française contre les Myopathies (AFM) Engl. J. Med. 341, 1715-1724. grant 7434. H.J.W. is also an Irma T. Hirschl Scholar. The confocal Fields, S. and Song, O. (1989). 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