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
*Author for correspondence (e-mail: hjw14{at}columbia.edu)
Accepted September 6, 2001
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SUMMARY |
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Key words: Nuclear envelope, Lamins, Intermediate filaments, Muscular dystrophy, Lipodystrophy, Cardiomyopathy
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INTRODUCTION |
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Lamins are intermediate filament proteins that form the nuclear lamina, a meshwork on the nucleoplasmic side of the inner nuclear membrane (Aebi et al., 1986; Fisher et al., 1986; McKeon et al., 1986; Stuurman et al., 1998; Worman and Courvalin, 2000). Two general types of lamins have been identified in somatic cells, A-type and B-type. The somatic cell A-type lamins, lamin A, lamin C and lamin A10, are alternative splice isoforms encoded by the LMNA gene (Lin and Worman, 1993; Machiels et al., 1996). Two different genes encode the somatic cell B-type lamins, lamin B1 and lamin B2 (Biamonti et al., 1992; Lin and Worman, 1995). Like all intermediate filament proteins, lamins have a conserved central rod-domain containing three
-helical segments, which is responsible for the formation of coiled-coil dimers. The rod-domain is flanked by N-terminal head and C-terminal tail-domains, which vary significantly between different proteins (Steinert and Roop, 1988). The lamins bind to some of the integral membrane proteins of the inner nuclear envelope (Worman and Courvalin, 2000). Lamin A interacts with emerin and lamina associated polypeptide 1 (LAP1) in vitro (Foisner and Gerace, 1993; Clements et al., 2000; Sakaki et al., 2001).
Because the functions of lamins A and C are poorly understood, the mechanisms by which mutations cause different inherited diseases are not clear (Worman and Courvalin, 2000; Hutchison et al., 2001). Currently, it is not known if any of the disease-causing mutations of lamin A alter protein localization, lamina structure, protein-protein interactions or protein stability. We therefore investigated these properties of mutant forms of lamin A found in patients with inherited diseases.
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MATERIALS AND METHODS |
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cDNAs encoding mutant forms of prelamin A, except the NLA mutant, which lacked amino acids 1-33, were made using the TransformerTM Site-Directed Mutagenesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA), following the manufacturers instructions.
NLA was generated by polymerase chain reaction (Saiki et al., 1987), using the Gene Amp PCR System 2400 (Applied Biosystems, Foster City, CA), with a SmaI restriction site engineered at the 5' end of the sense primer and an antisense primer spanning the ApaI site found at nucleotide 746 of prelamin A cDNA. Reaction products were digested with SmaI and ApaI and ligated into prelamin A-pBGT9 digested with the same enzymes. The resulting plasmid was digested with SmaI and SalI and ligated into the SmaI and SalI restriction sites of pSVF, a vector constructed by insertion of the FLAG-epitope between the EcoRI and KpnI restriction sites of pSVK3.
For studies with the yeast two-hybrid system, wild-type prelamin A cDNA in vectors pGAD424 and pGBT9 and lamin B1 cDNA in vector pGBT9 were those previously described (Ye and Worman, 1995). cDNAs obtained by site-directed mutagenesis were excised from vector pSVK3 using the restriction endonucleases SmaI and SalI and ligated into pGBT9 and pGAD424 digested with the same enzymes. NLA was inserted into pGBT9 as described above and then digested with SmaI and SalI and ligated into the SmaI and SalI sites of pGAD424. All cDNAs were sequenced using an ABI Prism 377 automated sequencer (Applied Biosystems).
Cell culture, transfection and immunofluorescence microscopy
C2C12 cells (a gift from Hal Skopicki, Columbia University, New York, NY) were grown in Dulbeccos modified Eagle medium (D-MEM) containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD) at 37°C and 10% CO2. Cells were transfected in chamber slides using Lipofectamine PLUSTM (Life Technologies), following the manufacturers instructions. The cells were overlaid with the lipid-DNA complexes for approximately 23 hours, the first five of which were in serum-free medium. The cells were then allowed to grow in fresh medium for 24 hours post-transfection before preparation for immunofluorescence microscopy.
Fixation and labeling of cells for immunofluorescence microscopy were performed as described previously (Östlund et al., 1999). The monoclonal primary antibodies used were anti-FLAG M5 (Sigma, St Louis, MO) diluted 1:200, anti-PCNA (proliferating cell nuclear antigen) P10 (Roche Molecular Biochemicals, Indianapolis, IN) diluted 1:20, anti-nuclear pore complex MAb414 (a gift from Tarik Soliman, Laboratory of Cell Biology, Rockefeller University, New York, NY) diluted 1:5,000 and anti-lamin B2 X223 (a gift from Georg 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 East Wales Institute, Wrexham, UK) at a dilution of 1:3,000 and anti-LAP2 (a gift from Katherine Wilson, Johns Hopkins University, Baltimore, MD) at a dilution of 1:500. Secondary antibodies used were Rhodamine RedTM-X-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), AlexaTM 568-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). For DNA-staining, 20 µg/ml propidium iodide (Sigma) was added to the secondary antibody incubation. Labeled and washed slides were dipped in methanol, air-dried, and coverslips were mounted using ProLong Antifade Kit (Molecular Probes, Inc.) or SlowFade Light Antifade Kit (Molecular Probes, Inc.). Immunofluorescence microscopy was performed on a Zeiss LSM 410 confocal laser scanning system attached to a Zeiss Axiovert 100TV inverted microscope (Carl Zeiss, Inc., Thornwood, NY). Images were processed using Photoshop software (Adobe Systems, Inc., San Jose, CA) on a Macintosh G3 computer (Apple Computer, Inc., Cupertino, CA).
Yeast two-hybrid assay
Saccharomyces cerevisiae strain Y187 (Clontech Laboratories, Inc.) was transformed with plasmids encoding lamins fused to the DNA binding domain of the S. cerevisiae GAL4 protein (pGBT9) or to the GAL4 transcriptional activation domain (pGAD424). Transformations and ß-galactosidase assays were done according to the Matchmaker Two-Hybrid System manual (Clontech Laboratories, Inc.).
Pulse-chase experiment
COS-7 cells were grown in D-MEM containing 10% fetal bovine serum (Life Technologies) at 37°C and 5% CO2. The cells were transfected using Lipofectamine PLUSTM (Life Technologies), following the manufacturers instructions. The cells were overlaid with the lipid-DNA complexes for approximately 23 hours, the first five of which were in serum-free medium, and were then grown in fresh medium for 24 hours before being subcultured at a 1:5 dilution into 60 mm tissue culture dishes. After another 24 hours, cells were washed with D-MEM without methionine and then overlaid with 1 ml D-MEM without methionine, containing 28 µCi Pro-mix L-[35S] in vitro cell labeling mix (Amersham Pharmacia Biotech, Inc.). After 1 hour, cells were washed twice with phosphate-buffered saline (PBS) and either incubated in non-radioactive D-MEM with 10% fetal bovine serum for an additional 3 or 25 hours or lysed immediately. For cell lysis, cells were washed three times in PBS and incubated for 40 minutes at 4°C in 440 µl lysis buffer (50 mM Tris-HCl, pH 8, 5 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate (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).
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RESULTS |
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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 0.7 µm were counted, as many cells, including untransfected controls, frequently contained smaller foci of lamins in their nuclei. A localization of wild-type lamin A in small intranuclear foci has been reported previously (Moir et al., 1994; Broers et al., 1999).
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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 -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 ß-galactosidase, which can be measured using a colorimetric assay (Fields and Song, 1989). We expressed the 15 mutant forms of prelamin A described above as fusion proteins with the GAL4 activation domain, or 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 possible using this assay to determine whether the protein-protein interactions formed are the coil-coiled ones in the normal lamina, these results suggest that the primary lamin-lamin dimerization is not disturbed in these mutants.
Several lamin A mutants are as stable as wild-type lamin A
To investigate whether representative mutant lamin A proteins were less stable than wild-type lamin A, we performed pulse-chase analyses. COS-7 cells were transfected with cDNAs encoding wild-type or mutant prelamin A with FLAG-epitopes fused to their N-termini. The cells were pulsed with [35S]-methionine for 1 hour and then harvested or grown in nonradioactive media for 3 or 25 hours. Cell lysates were incubated with anti-FLAG M2-agarose affinity gel and the immunoprecipitates were separated by SDS-PAGE and exposed to Hyperfilm ECL. Representative results are shown in Fig. 7. At time-point zero (no chase), both prelamin A and the slightly smaller mature lamin A could be seen in all samples from transfected cells. After 3 hours of growth in nonradioactive media, no prelamin A could be seen and the total levels of radiolabeled FLAG-tagged lamin A had decreased; it could, however, still be detected in all cases. After 25 hours of incubation, very small amounts of radiolabeled lamin A could still be detected in all samples. These results showed that the representative lamin A mutants N195K, M371K, R386K, R453W, R482W and K486N were as stable as wild-type lamin A. Mutant R386K seemed slightly more stable than the others after 3 hours of chase. Wild-type lamin A and these lamin A mutants were also processed normally from prelamin A to the mature protein.
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DISCUSSION |
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Mutant lamins are as stable as wild-type lamins
Disease phenotypes may be caused by haploinsufficiency of lamins A and C due to low levels of expression or rapid degradation of the mutant lamin protein. One mutation causing AD-EDMD is a nonsense mutation at the codon for amino acid six (Bonne et al., 1999; Bécane et al., 2000); in this case a reduction of lamins A and C most likely caused the disease. In patients with this mutation, both lamin A/C and emerin were correctly localized to the nuclear envelope as shown by immunolabeling of a cardiac biopsy (Bonne et al., 1999), although there was a decrease in the amount of lamin A/C in cardiac tissue, as shown by western blot (Bécane et al., 2000). Our studies indicate no decrease in the stability of several mutant lamins, including those in AD-EDMD, cardiomyopathy and FPLD. Therefore, a simple haploinsufficiency of lamins A can not be the only pathophysiological mechanism. Studies of mutant lamins A and C in fibroblasts from patients carrying the LMNA mutations causing R482Q and R482W substitutions showed these proteins to be present at similar levels to the wild-type proteins in control cells (Vigouroux et al., 2001). Further studies of the levels of mutant lamins A and C in cells from patients carrying other LMNA mutations would provide important confirmation of our results on the stability of the mutant proteins.
Mutations in the lamin A rod-domain can cause an abnormal intranuclear localization of the protein
Mutant proteins may be unable to carry out their normal functions, or disrupt the functions of associated proteins, because of mislocalization in cells. We examined the intracellular localization of 15 mutant lamin A proteins by immunofluorescence microscopy. The most striking feature in these studies was the formation of intranuclear foci by four of the lamin A mutants (N195K, E358K, M371K and R386K), which was often accompanied by a mislocalization of some of the endogenous lamins. These cells also had a decrease in the localization of the mutant lamins to the nuclear periphery. A nucleoplasmic localization and formation of intranuclear foci have also been reported when the N195K lamin A mutant was expressed in HeLa cells (Raharjo et al., 2001). The four lamin proteins that formed intranuclear foci all had mutations introducing a lysine into the rod-domain. Three out of the four proteins had mutations at the end of the central, -helical rod-domain, which is evolutionary well conserved between all intermediate filaments (Stuurman et al., 1998). A lamin A mutation (R377H) causing LGMD1B has also recently been mapped to this region (Muchir et al., 2000). The conserved segments of the rod-domain have been shown to play crucial roles in the assembly of intermediate filament dimers into higher order oligomers (Stuurman et al., 1998). Mutations in the corresponding regions of keratin K5 and K14 are responsible for the majority of skin diseases caused by keratin mutations, for example the Dowling-Meara type of epidermolysis bullosa simplex (McLean and Lane, 1995). This disease and others caused by keratin mutations are blistering skin disorders resulting from epithelial fragility. It is conceivable that mutations in lamins A and C cause nuclear fragility. Nuclei of embryonic fibroblasts from mice lacking A-type lamins and Xenopus nuclei assembled in egg extracts with the
NLA mutant have been shown to be more fragile than wild-type nuclei (Spann et al., 1997; Sullivan et al., 1999). One hypothesis is that an increased fragility of nuclei can be particularly harmful to muscle cells, where they are subjected to mechanical stress (Worman and Courvalin, 2000; Hutchison et al., 2001). This could explain the muscle-specific nature of AD-EDMD and cardiomyopathy but does not explain why the specialized cardiomyocytes involved in atrioventricular conduction may be more readily affected than contractile cardiomyocytes (Morris, 2000).
The nuclear stress-hypothesis as a pathophysiological mechanism of disease is less likely in the case of FPLD. The restriction of missense mutations causing FPLD to two regions in exon 8 and 11 implies that they encode a domain important for a specific lamin A function, for example interaction with a protein important for adipocyte survival. This is further suggested by the concentration of the majority of FPLD mutations to codon 482 (Cao and Hegele, 2000; Shackleton et al., 2000; Speckman et al., 2000; Vigouroux et al., 2000). In our studies, as well as in the studies by others (Holt et al., 2001; Raharjo et al., 2001), all using transiently transfected cells, there were no gross abnormalities in the localization or stability of lamin A-containing mutations found in FPLD. However, fibroblasts from FPLD patients with the R482Q and R482W lamin A mutations, and some transfected fibroblasts overexpressing the R482W mutant, show more subtle nuclear abnormalities (Vigouroux et al., 2001).
Effects of lamin mutations on protein-protein interactions
To examine the ability of mutant lamins to self-interact and to interact with wild-type lamins, we used the yeast two-hybrid assay. Our results showed no indication of impaired lamin-lamin interactions at this level. However, the two-hybrid assay does not show whether the complexes formed are the usual coiled-coil dimers. Neither would it detect disturbances of higher order lamin interactions, such as filament formation. Subtle defects of lamin filament formation are at this time difficult to detect because lamins, as opposed to cytoplasmic intermediate filaments, do not form stable filaments in vitro.
Lamin A binds to LAP1 and emerin (Foisner and Gerace, 1993; Clements et al., 2000; Sakaki et al., 2001). The interaction between lamin A and emerin is especially intriguing because mutations in these two proteins both cause the EDMD phenotype. In cells from mice lacking A-type lamins, emerin is partially mislocalized to the cytoplasm (Sullivan et al., 1999). These mice show typical signs of EDMD. Heterozygous mice, having only one wild-type copy of the LMNA gene, have a slight mislocalization of emerin but are healthy (Sullivan et al., 1999). We have studied the localization of emerin in C2C12 cells expressing mutant forms of lamin A. Our studies showed an increased loss of endogenous emerin from the nuclear envelope when C2C12 cells, which have wild-type lamin A in addition to the mutant forms, were transfected with mutants N195K, M371K, R386K or NLA. However, as cells transfected with wild-type lamin A also exhibited a loss of emerin from the nuclear envelope, although to a significantly lower extent, it is not clear whether the emerin loss was due to specific disturbances in the interactions between emerin and mutant lamins or to a loss of peripheral lamins caused by the mutants. Alternatively, other effects of lamin A overexpression in cells could lead to emerin mislocalization, but whatever these effects may be, certain lamin A mutants enhance them. An increased loss of emerin from the nuclear envelope has also been reported in HeLa cells expressing the lamin A mutants L85R, N195K and L530P, compared with wild-type (Raharjo et al., 2001).
Do lamins have a role in gene expression and DNA replication?
Alternative hypotheses, which could explain the tissue-specific nature of the diseases caused by mutations in the A-type lamins, are that these proteins are involved in specific gene expression or DNA replication events, or both. A-type lamins have been shown to bind proteins involved in transcriptional regulation such as the retinoblastoma protein (Mancini et al., 1994), and ectopic expression of lamin A has also been shown to induce muscle-specific genes in undifferentiated cells (Lourim and Lin, 1992). A role for the inner nuclear membrane in the regulation of gene expression has previously been suggested by the interaction between the lamin B receptor, an integral protein of the inner nuclear membrane, and human orthologues of Drosophila HP1 (Ye and Worman, 1996; Ye et al., 1997). In Drosophila, HP1 suppresses the expression of normally active euchromatic genes translocated near heterochromatin (Eissenberg et al., 1990). Several other nuclear proteins involved in neuromuscular disease are involved in gene expression, either at the level of transcription, splicing or mRNA transport (Morris, 2000). The nuclear lamins are also required for DNA replication during the S-phase and the lamin foci formed in Xenopus egg extracts containing the NLA mutant were previously shown to contain the DNA polymerase
cofactor PCNA (Spann et al., 1997). PCNA, however, did not accumulate in the foci formed by lamin mutants in C2C12 cells in our studies. The reason for this discrepancy is not clear; one possibility is that PCNA localization is dependent on cell-cycle phase. Although in vitro assembled Xenopus nuclei are in S-phase, it is not clear whether cells containing nuclear foci can enter S-phase. It may also be due to different fixation techniques; our cells were fixed with methanol, which yields a staining pattern where only PCNA bound to specific nuclear structures is recognized by the antibody (Bravo and Macdonald-Bravo, 1987). There may also be differences in lamina structure between mammalian cells versus Xenopus egg extracts, which has been suggested by Izumi et al. (Izumi et al., 2000); they showed that PCNA is not colocalized with
NLA foci in CHO cells.
The recent results of others (Raharjo et al., 2001; Vigouroux et al., 2001) and those reported in this study provide a starting point towards understanding the properties of disease-causing lamin A/C mutants. They also exclude some possible pathogenetic mechanisms, such as haploinsufficiency of lamin A, absence of prelamin A processing or cytoplasmic sequestration, as the cause of disease in most instances. Further work is necessary to connect the behavior of mutant nuclear lamins and their resulting effects on nuclear envelope structure to muscular dystrophy, cardiomyopathy and lipodystrophy.
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ACKNOWLEDGMENTS |
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