Authors’ correction E. A. L. Fairley, J. Kendrick-Jones and J. A. Ellis (1999). The Emery-Dreifuss muscular dystrophy phenotype arises from aberrant targeting and binding of emerin at the inner nuclear membrane. J. Cell Science 112 (15), 2571-2582. All references to S54P or Ser54Pro are incorrect and should read as S54F or Ser54Phe, respectively.

Authors’ correction D. Gabriel, U. Hacker, J. Köhler, A. Müller-Taubenberger, J.-M. Schwartz, M. Westphal and G. Gerisch (1999). The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker . J. Cell Science 112 (22), 3995-4005. In Fig. 7 of this paper the numbers indicating seconds should be exchanged between panels C and E, as shown below.

Fig. 7

Authors’ correction K. P. Williams, P. Rayhorn, G. Chi-Rosso, E. A. Garber, K. L. Strauch, G. S. B. Horan, J. O. Reilly, D. P. Baker, F. R. Taylor, V. Koteliansky and R. B. Pepinsky (1999). Functional antagonists of sonic hedgehog reveal the importance of the N terminus for activity. J. Cell Science 112 (23), 4405-4414. In the discussion on page 4412, paragraph 3 line 21, the digit duplication assay was incorrectly quoted as mouse. The correct assay is chick embryo digit duplication. In addition, line 23 should state (S. Pagan, D. P. Baker, K. P. Williams and C. J. Tabin, unpublished data). Journal of Cell Science 112, 2571-2582 (1999) 2571 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0436

The Emery-Dreifuss muscular dystrophy phenotype arises from aberrant targeting and binding of emerin at the inner nuclear membrane

Elizabeth A. L. Fairley1,2, John Kendrick-Jones1 and Juliet A. Ellis2,* 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 2Department of Medical Genetics, Cambridge Institute of Medical Research, Wellcome Trust/MRC building, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY, UK *Author for correspondence (e-mail: [email protected])

Accepted 20 May; published on WWW 7 July 1999

SUMMARY

The product of the X-linked Emery-Dreifuss muscular multiple non-overlapping nuclear-membrane-targeting dystrophy is a single-membrane-spanning protein determinants. called emerin, which is localized to the inner nuclear Analysis of material immunoisolated using emerin membrane of all tissues studied. To examine whether a antibodies, from either undifferentiated C2C12 myoblasts number of the mutant forms of emerin expressed in or purified hepatocyte nuclei, demonstrated that both A- patients are mislocalized, we transfected GFP-emerin and B-type lamins and nuclear actin interact with emerin. cDNA constructs reflecting these mutations into This is the first report of interacting with emerin. undifferentiated C2C12 myoblasts and showed that The EDMD phenotype can thus arise by either the absence both wild type and all the mutant emerins are targeted or a reduction in emerin at the , and both to the nuclear membrane, but the mutants to a lesser of these disrupt its interactions with that of structural extent. Mutant Del236-241 (deletion in transmembrane components of the nucleus. We propose that an emerin- region) was mainly expressed as cytoplasmic nuclear protein complex exists at the nuclear envelope and aggregates, with only trace amounts at the nuclear that one of its primary roles is to stabilize the nuclear envelope. Complete removal of the transmembrane membrane against the mechanical stresses that are region and C-terminal tail relocated emerin to the generated in muscle cells during contraction. nucleoplasm. Mutations in emerin’s N-terminal domain had a less severe effect on disrupting nuclear envelope Key words: emerin, nuclear targeting, lamin binding, Emery-Dreifuss targeting. This data suggests that emerin contains muscular dystrophy.

INTRODUCTION nucleoplasm (Bonne et al., 1999). The unexpected finding that the EDMD arises due to defects in nuclear proteins suggests There are three major forms of muscular dystrophy, referred to that the pathophysiology of EDMD may be very different from as Duchenne, Becker and Emery-Dreifuss types, all the other types of muscular dystrophy. distinguishable by progressive wasting and Human emerin mRNA and protein show ubiquitous tissue cardiac abnormalities to varying degrees (reviewed by Emery distribution, with the highest expression in skeletal and cardiac 1989, 1996). The first two types are due to genetic defects in muscle (Bione et al., 1994; Manilal et al., 1996; Nagano et al., the cytoskeletal/plasma membrane-associated protein, 1996). Human emerin is a serine-rich protein of 254 amino dystrophin, which is part of the glycoprotein complex linking acids (Bione et al., 1994) with an Mr of 28,993. Structural actin to the extracellular matrix, whereas the third form arises analysis predicts emerin to be a type II membrane protein, with from genetic defects in nuclear proteins (Manilal et al., 1996; a transmembrane region 11 amino acids from the carboxyl Nagano et al., 1996; Bione et al., 1994, Bonne et al., 1999). X- terminus and a large hydrophilic N-terminal amino domain linked EDMD is due to the absence of or defects in emerin, orientated towards the nucleoplasm, containing 22 potential which is localized to the inner nuclear membrane (Yorifuji et phosphorylation sites for a range of kinases. Mutations occur al., 1997) in all tissues examined. It has been reported to be throughout the gene encoding emerin and there are no additionally present in the intercalated discs of heart (Cartegni mutational ‘hot spots’. The majority of the mutations so far et al., 1997), although this may be due to an antibody artifact studied produce no detectable emerin either by (Manilal et al., 1999). Recently, the gene product of the immunoblotting or immunohistochemistry in any tissue autosomal dominant form of EDMD has been identified as examined (Manilal et al., 1996, 1997, 1998a; Nagano et al., nuclear lamin A, an intermediate filament protein of the 1996; Mora et al., 1997; Ellis et al., 1998, 1999; Yates et al., 2572 E. A. L. Fairley and others

1999). However, a small number of mutations have been et al., 1998a). Phosphorylation of emerin may be involved in reported to produce modified forms of emerin (Mora et al., controlling these events. 1997; Manilal et al., 1998a; Ellis et al., 1998, 1999; Yates et In the present paper, we show that mutant forms of emerin al., 1999). Interestingly, despite the different mutations in expressed in a number of EDMD patients are targeted to the EDMD, producing varying effects on emerin expression, the inner nuclear membrane, but in a less efficient manner clinical phenotype of all these patients is similar. compared to wild type. These results suggest that emerin The functions of the integral membrane proteins in the inner contains multiple nuclear membrane localization signals, some nuclear membrane are not completely known, although the of which are involved directly in nuclear targeting and others available evidence suggests that they may have a structural role in retention at the nuclear membrane. We demonstrate that in maintaining nuclear architecture (Gerace and Foisner, 1994). wild-type emerin binds to both A- and B-type lamins and to Interactions between nuclear membrane components, nuclear nuclear actin, suggesting that interactions between and chromatin are crucially important for maintaining components are essential for skeletal and the structure of the nuclear membrane-chromatin organization function and that loss of integrity of the nuclear membrane may during interphase and for the disassembly and reformation of directly produce the muscular dystrophy phenotype. the nuclear membrane during mitosis. Emerin possesses two short regions of homology to rat lamina-associated polypeptide 2 (LAP2; Furukawa et al., 1995; Harris et al., 1995), another MATERIALS AND METHODS inner nuclear membrane protein, suggesting that emerin is a member of the nuclear lamina-associated protein family. This Cell lines and cell culture family includes lamina-associated polypeptide 1 (LAP1; COS-7, green monkey fibroblasts and C2C12 cells, a subclone of the Martin et al., 1995) and the lamin-B receptor (LBR; Soullam C2 mouse myoblast cell line (Yaffe and Saxel, 1977), were obtained from the European Collection of Cell Lines (ECACC) and cultured in and Worman, 1993). LAP2 has been shown to interact in a cell- Dulbecco’s Minimal Essential Medium (DMEM; Gibco BRL) cycle dependent manner with both lamin B1 and chromatin and supplemented with 10% fetal bovine serum (FBS; Sigma) and 2 mM the binding sites for both have been identified. At the onset of glutamine. To avoid spontaneous differentiation of the C2C12 cells, mitosis, LAP 2 is phosphorylated, causing it to dissociate from they were passaged at 75% confluence. the lamina network and chromatin and disperse throughout the ER (Yang et al., 1997a). Both the nuclear lamins and LAP2 Antibodies reassociate with the chromatin at late anaphase. This suggests The following antibodies against emerin were used: affinity-purified that reassembly of the nuclear envelope at the end of mitosis rabbit polyclonal antibodies AP2, AP5, AP8 and AP9 raised against involves sorting of integral membrane proteins to human emerin, as shown in Fig. 1 and as described previously (Ellis surfaces by binding interactions with lamins and chromatin et al., 1998). An affinity-purified rabbit polyclonal antibody, AP1, was raised against a bacterial fusion protein expressing residues 114-183 (Yang et al., 1997a). The cell cycle-dependent binding of LAP2 of rat recombinant emerin. This antibody is to the region of least to lamin B1 also controls the increase in nuclear volume seen homology between rat and human emerin, and recognizes rat emerin during interphase in cycling cells (Yang et al., 1997b), which with a greater affinity than the AP8 antibody. These affinity-purified allows them to enter S phase. antibodies were used at 1:3000 dilution on immunoblots and 1:100 in Recent data suggests that emerin has a role in cell cycle- both immunoprecipitation and immunofluorescence experiments. dependent events, since it is also localized at intranuclear sites, Antibodies used to label intracellular compartments in the emerin where it colocalizes with the nuclear lamins (Squarzoni et al., localization experiments included: a rat polyclonal antibody 1998; Manilal et al., 1998b) and binds tightly to unidentified MAC256, which recognizes resident endoplasmic reticulum (ER) insoluble matrix components (Ellis et al., 1998; Squarzoni et proteins (specific for KDEL motif) obtained from Dr Moreman al., 1998). In addition, emerin can occur in four differently (University of Georgia, USA) and used at 1:100; a rat monoclonal antibody against the lysosomal membrane glycoprotein, LAMP-1 phosphorylated forms, three of which appear to be associated (Developmental Studies Hybridoma Bank, University of Iowa, USA) with the cell cycle (Ellis et al., 1998). During mitosis emerin used at 1:400; a mouse monoclonal antibody β-actin (clone AC-74; becomes dispersed throughout the cell, no longer colocalizing Sigma No. A 5316) used at 1:50; a mouse monoclonal antibody lamin with the lamins, and then participates in the reconstitution of A (clone 133A2; McKeon et al., 1986) used at 1:300 and a rabbit membranes around the daughter nuclei at telophase (Manilal polyclonal antibody to lamin B (Moir et al., 1994) used at 1:50.

Fig. 1. Secondary structural features of emerin, LAP2 regions used to raise polyclonal antisera and the 221 254 position of mutations used in our study. The boxed 244 135 46 70140 220 254 section represents the full-length emerin protein. NLS Polyclonal antisera were raised against the regions shown by horizontal arrows labelled with the TM numbers given to the resulting affinity-purified LAP2 antisera (AP2, AP5, AP8, AP9). The positions of 644 S54P Del 95-99 P183T (Y15) Del 236-241 the mutations found in EDMD patients used in this (CM1) study are shown by the short vertical arrows (Y1) P183H (WG) (Y3) labelled S54P, Del 95-99, P183T/H, Del236-241. AP8 AP5 Other numbers show the positions of amino acids in AP2 the human emerin sequence. TM, transmembrane region; NLS, bipartite nuclear localization signal; AP9 LAP2, regions in emerin 41% identical to LAP2. Emerin and Emery-Dreifuss muscular dystrophy 2573

Table 1. Modified forms of emerin expressed in EDMD patients used in our study Nucleotide Nucleotide Protein expression Patient change position2 Mutation type Effect and size References Y1 (Del95-99) Del. 15 bp1 878 In-frame deletion Del. YEESY Reduced levels Ellis et al. (1998); Yates et al. (1999) (95-99) 31 kDa Y3 (Del236-241) Del. 18 bp 1763 In-frame deletion Del. VIVLFF Very reduced levels Yates et al. (1999); Ellis et al. (1999) (236-241) 34 kDa Y15 (P183T) C→A 1605 Missense Pro 183→Thr Normal levels Ellis et al. (1999); Yates et al. (1999) 34 kDa WG (P183H) C→A 1606 Missense Pro 183→His Normal levels Ellis et al. (1999) 34 kDa CM1 (S54P) C→T 394 Missense Ser 54→Phe Normal levels C. Muller, UP 34 kDa

1Deletion. 2Genomic numbering. UP, unpublished data. All mutations are listed in the EDMD database (Yates and Wehnert, 1999).

Immunoblots from the emerin immunoprecipitation experiments were on Qiagen columns according to the QIAprep Spin Miniprep kit probed with rabbit polyclonal antibody to actin (against C11 peptide; protocol). A time course of emerin expression was conducted to Sigma No. A 2066) used at 1:1000; rat polyclonal antibody to lamin identify the optimum time for the correct localization of wild-type A/C (Moir et al., 1994) and the aforementioned rabbit polyclonal emerin. Cells were then fixed and immunolabelled as described below. antibody to lamin B, were both used at 1:1000. Expression levels of the GFP-emerin constructs were examined by immunoblotting the transfected cells for emerin expression with Mutant human emerin cDNA constructs affinity-purified antibody AP8. A full-length human emerin cDNA clone in plasmid pBluescript KS− (Ellis et al., 1998) was used as the starting template to make the Immunofluorescence microscopy mutant cDNA plasmid constructs. All the constructs chosen were For microscopic visualization, transfected cells were washed 3 times known to occur in EDMD patients and to express modified forms of with phosphate buffered saline (PBS; 145 mM NaCl, 7.5 mM emerin and are shown in Table 1. The position of the mutations in the Na2HPO4, 2.5 mM NaH2PO4, pH 7.4), prior to being fixed in either emerin gene are shown in Fig. 1. Site-directed mutagenesis was 4% (w/v) paraformaldehyde for 20 minutes (for Rhodamine- performed on the wild-type cDNA emerin clone by PCR, Phalloidin and endogenous emerin staining), methanol for 10 minutes incorporating restriction sites HindIII and NdeI at the 5′ end and (for immunolabelling for lysosomal marker, LAMP 1) or 2% (w/v) BamHI at the 3′ end, using the primers listed in Table 2. paraformaldehyde/0.1% (w/v) glutaraldehyde mix for 25 minutes (for PCR was performed in a total reaction volume of 50 µl with 0.4 ng immunolabelling for lamins A and B and the ER marker MAC256). wild-type cDNA plasmid DNA as template. Final reagent concentrations in the PCR reaction mixture were 20 mM Tris-HCl, µ µ pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 400 M dNTPs and 50 pmol/ l Table 2. Primers used to generate modified forms of of each primer (Table 2); 5 units Pfu polymerase (Clontech). The emerin samples were denatured at 95¡C for 3 minutes and then for 1 minute at 94¡C, 1 minute at 43¡C and 1.5 minutes at 74¡C for 5 cycles. All Wild-type emerin 5′-TTTTA AGC TTA CAT ATG GAC AAC TAC DNA samples were annealed and amplified for 25 cycles (1 minute (1-254) GCA GAT- at 94¡C, 1 minute at 55¡C, 1 minute at 74¡C), except for the primers 3′-TTTTGGA TCC TGG CTC CCT CTA GAA that were used to synthesize the P183H mutant, which required an GGG GTT- annealing temperature of 50¡C. The application was finished with a Del236-241 5′-TTTTA AGC TTA CAT ATG GAC AAC TAC final extension at 74¡C for 7 minutes. GCA GAT- The PCR fragments generated were cloned into the HindIII and 3′-TTTTGGA TCC TGG CTC CCT CTA GAA BamHI sites of mammalian expression vector pEGFP-C2 (Clontech), GGG GTT GCC TTC TTC AGC CTG CAT which contains the Green Fluorescent Protein (GFP). Another emerin GAA GTG GTA AAT AAA GAC CAG GAA cDNA clone was constructed in pEGFP-C1 by subcloning residues 1- AAG CAG CAG CTG- 220 from the fusion protein FP9 construct in vector pET-29b (see Del95-99 5′-AGA AAG GGC TAC AAT GAC GAC TAC below) to provide a mutant form of emerin, which lacks the TTC ACC ACC AGG ACT TAT- transmembrane and C-terminal tail region. 3′-ATA AGT CCT GGT GGT GAA GTA GTC All constructs made were sequenced in both directions using a GTC ATT GTA GCC CTT GCT- Sequence Version 2.0 DNA Sequencing Kit (Amersham Life Science) Serine 54 Proline 5′-CCC AGC TCG TTC GCC GCC TCC- and Thermo Sequence dye terminator cycle sequencing pre-mix kit 3′-GGA GGC GGC GAA CGA GCT GGG- (Amersham Life Science). Proline 183 Histidine 5′-TCC TAT TAT CAT ACT TCC TCC- Transient transfections 3′-GGA GGA AGT ATG ATA ATA GGAÐ Undifferentiated C2C12 myoblasts were transfected according to Proline 183 Threonine 5′-TCC TAT TAT ACT ACT TCC TCC- the SuperFectª Transfection Protocol of Qiagenª, using a 3′-GGAGGA AGT AGT ATA ATA GGA- Superfect:DNA ratio of 1:6. For immunofluorescence experiments, subconfluent cells were plated on coverslips (22 mm2) in 6-well Restriction enzyme sites are underlined. plates, 1 day prior to being transfected with 2 µg of DNA (purified Mutated DNA bases are shown in bold. 2574 E. A. L. Fairley and others

Fig. 2. Immunofluorescence labelling of endogenous emerin and nuclear lamin A by confocal immunofluorescence microscopy. (A) Comparison of endogenous emerin levels in COS-7 cells and undifferentiated C2C12 myoblasts. Localization of emerin is shown in red and β-actin in green. Affinity-purified antiserum AP8 was used for COS-7 cells (1:100) and affinity-purified antiserum AP1 was used on the C2C12 myoblasts (1:100). (B) Colocalization of emerin and lamin A in undifferentiated C2C12 myoblasts. Emerin is shown in red (i) and lamin A in green (ii) with an overlay of the two signals shown in (iii). Immunolabelling was performed as described in Materials and methods. Bars, 10 µm.

The cells fixed in 4% (w/v) paraformaldehyde were washed 3×10 analysis was performed using the GraphPad Prism version 2.0a minutes in PBS, permeabilised in 0.2% (w/v) PBS/Triton X-100 for software. 5 minutes, washed 3×10 minutes in PBS and blocked with PBS/0.2% (w/v) fish gelatin (Sigma) 4×10 minutes, prior to being incubated with Expression and purification of fusion proteins the primary antibody for 1 hour in a humidified chamber. Those Four bacterial fusion proteins expressing fragments of human cells fixed with methanol were washed 3×10 minutes in PBS, then recombinant emerin spanning residues 1-70 (FP8), 70-140 (FP2), 1- blocked with PBS/0.2% (w/v) fish gelatin 4×10 minutes prior to 174 (FP3) and 1-220 (FP9) were expressed and purified in the primary antibody incubation. Those cells fixed with 2% (w/v) expression plasmid pET-29b (C-terminal his6 tag; Novagen, Madison, paraformaldehyde/0.1% (w/v) glutaraldehyde were washed 3×10 WI) and a fifth fusion protein (residues 244-254) was expressed and minutes in PBS, permeabilised with PBS/Triton X-100 for 5 minutes, purified as a glutathione S-transferase (GST) fusion in pGEX-4T-3 washed 3×10 minutes in PBS, quenched in 1 mg/ml NaBH4 in PBS (Pharmacia Biotech Inc.). Fusion proteins were expressed and purified for 10 minutes, washed 3×10 minutes in PBS, blocked with PBS/0.2% as described previously in Ellis et al. (1998). (w/v) fish gelatin 4×10 minutes prior to primary antibody incubation. After antibody incubation all cells were washed thoroughly 4×10 Overlay binding assay of emerin-fusion proteins onto minutes with PBS/fish gelatin for 5 minutes and incubated with the undifferentiated C2C12 cell lysates and purified rat appropriate secondary antibody for 30 minutes in a humidified hepatocyte nuclei chamber. The secondary antibodies included a goat anti-rabbit IgG Undifferentiated C2C12 myoblasts were harvested by scraping them conjugated with Texas Red (Amersham Life Sciences) at 1:400, and off tissue culture flasks, washed in PBS, resuspended in PBS, a sheep anti-mouse IgG conjugated with FITC (Amersham Life sonicated briefly, loaded into sample buffer and proteins separated by Sciences) at 1:150. Thereafter the coverslips were washed thoroughly 10% SDS-PAGE according to Laemmli (1970) before transferring 3×10 minutes in PBS/fish gelatin and 3×PBS for 5 minutes, rinsed in onto Hybond ECL nitrocellulose membrane (Amersham Life Science) deionised water and placed on slides washed in 70% methanol and air according to Burnette (1981). The membranes were washed and dried. 20 µl of Mowiol mounting medium (87% (v/v) glycerol, 0.2M blocked in PBS/6% (w/v) dried skimmed milk/0.1% (v/v) Tween-20 Tris-HCl, pH 8.5, 12% (w/v) Mowiol) were added. The samples were (blocking buffer) 4× for 15 minutes at room temperature. Blots were stored in the dark at room temperature prior to examination. overlaid with fusion proteins, at final concentrations of 0.25-12.5 Immunofluorescence microscopy was performed on an MRC-1024 µg/ml, for 1 hour at room temperature, with rocking, in blocking Laser Scanning Confocal Imaging System (Bio-Rad). Scanning was buffer. Blots were then washed in blocking buffer 4× for 15 minutes. done with a Nikon PlanApo 60× lens having a numerical aperture of The blots were then processed by immunoblotting with the panel of 1.4 using Texas Red emission filter 605 DF32 and FITC emission rabbit polyclonal affinity-purified antibodies raised against the human filter 522 DF32. Computerized images were processed by mrc2M emerin-fusion proteins (AP8, AP2, AP9, AP5; see Fig. 1 and Ellis et (Michio Ono; e-mail [email protected]:), Adobe Photoshop al., 1998). Immunoblotted bands were visualized by enhanced 4.0 and Adobe illustrator 7.0 software on a Power Macintosh chemiluminescence (ECL; Amersham Life Sciences) in conjunction 4400/200. The Lasersharp Processing Screen software was used with autoradiography. to quantify the fluorescence intensity of GFP-emerin present at To determine the specificity of any protein-protein interaction the nuclear envelope. These fluorescence intensities were measured identified, the salt concentration in both the blocking, binding and per unit area (100 pixels) of nuclear envelope, to allow washing stages of the overlay procedure was varied between 145 mM comparisons between the transfectants expressing different forms of and 500 mM, by supplementing the PBS with NaCl. We also GFP-emerin. The average fluorescence intensity was quantified from examined whether any fusion protein binding observed could be a 25-cell unbiased selection, for each construct expressed. Statistical blocked, by incubating the fusion protein with the appropriate affinity- Emerin and Emery-Dreifuss muscular dystrophy 2575

Fig. 3. Intracellular localization of wild type and modified forms of GFP-emerin constructs (as indicated) expressed by transient transfection into undifferentiated C2C12 myoblasts. GFP-emerin cDNA constructs were transfected into C2C12 cells, as described in Materials and methods. Cells were fixed with 4% (w/v) paraformaldehyde. The intracellular location of emerin was monitored by GFP fluorescence (green). Cells were counterstained with Rhodamine- Phalloidin (red) to visualize F- actin. GFP-emerin fluorescence is shown separately to clarify GFP- emerins trafficking through the ER. Bars, 10 µm. purified emerin antibody, prior to using the fusion protein in the with 1 µg of non-immune rabbit IgG for 1 hour at 4¡C with rotation, overlay procedure. Overlays onto an E. coli cell lysate were performed and captured with 20 µl of Protein A-Sepharose (Sigma: 50 mg/ml as a control against non-specific binding. slurry in PBS) for 1 hour with rotation at 4¡C. The Protein A- Sepharose was removed by centrifuging at 11,600 g for 5 minutes at Immunoprecipitation 4¡C and the supernatants transferred to fresh tubes. Samples were Immunoprecipitation experiments were performed on both either incubated with affinity-purified rat emerin polyclonal antibody undifferentiated C2C12 myoblasts and purified rat hepatocyte nuclei AP1 (final dilution of 1:100) or with the corresponding pre-immune (a kind gift from H. Kent, MRC, Cambridge, UK) using the same sera (1:100), with rotation, overnight at 4¡C. The samples were then protocol. C2C12 cells were used at 60% confluence (108 cells/sample) centrifuged at 11,600 g for 20 minutes at 4¡C to remove any insoluble and the purified nuclei at 5×107/sample. For immunoprecipitating immune complexes. Supernatants were transferred to fresh tubes and from the tissue culture cells, medium was removed, the cell monolayer 50 µl of Protein A-Sepharose was added per sample followed by washed in PBS, and the flasks placed on ice for 30 minutes. 0.5 ml rotation for 2 hours at 4¡C. The beads were collected by centrifuging of extraction buffer (1% (v/v) Triton X-100, 50 mM Tris-HCl, pH 7.4, at 11,600 g for 5 minutes at 4¡C, and washed five times in wash buffer 2 2 mM MgCl2, and 100 mM - 1 M NaCl) was added to each 75cm (1% (v/v) Triton X-100, 50 mM Tris-HCl, 2 mM MgCl2, 150 mM- flask used, which were left on ice for a further 30 minutes with 1.5 M NaCl). A range of salt concentrations was tried both in the intermittent agitation. The lysate was collected and sonicated 4× for extraction and wash stages to optimize immunoprecipitation 15 second bursts (set at 40%) on a sonicator ultrasonic processor XL conditions and to investigate the strength of any emerin-protein (Misonix Incorporated, NY, USA) and then centrifuged at 11,600 g interactions identified. Residual detergent was removed from the for 10 minutes at 4¡C. The supernatant was collected and preincubated beads by washing them twice in 50 mM Tris-HCl, pH 7.4. Samples 2576 E. A. L. Fairley and others were rotated for 10 minutes between each wash. Samples were microscope (Fig. 3). Transfection efficiency was between 5% prepared for SDS-PAGE by heating to 95¡C for 5 minutes in sample and 10%. After 22 hours of expression, wild-type GFP-emerin buffer (Laemmli, 1970) to release the immunocomplexes from the was correctly localized to the nuclear rim and in the ER beads. Proteins were separated by 10% SDS-PAGE (Laemmli, 1970) outlining the nuclear rim (Fig. 3wt), as reported previously and either silver stained (Ansorge, 1985) or immunoblotted (Burnette, (Cartegni et al., 1997). Mutant GFP-emerin constructs were 1981) for emerin, actin and nuclear lamins. transfected and examined in the same manner. In all the transfected cells overexpressed emerin was additionally present in large randomly sized cytosolic aggregates to RESULTS varying degrees, although more were present in the transfectants expressing the mutant emerins. These aggregates Localization of endogenous emerin in COS-7 cells probably result from precipitation or aggregation of and undifferentiated C2C12 myoblasts overexpressed transfected proteins. Similar levels of GFP- The localization of immunofluorescent endogenous emerin in emerin expression were detected between the different both COS-7 cells and undifferentiated C2C12 myoblasts is transfectants when the cells were harvested and shown in Fig. 2A. The rabbit polyclonal affinity-purified immunoblotted for GFP-emerin (Fig. 4), except with Del236- antibodies AP1 (rat emerin) and AP8 (human emerin) gave the 241, which showed consistently lower levels of expression. same immunofluorescence pattern for emerin in both cell lines, The fluorescence intensity of each GFP-emerin construct but each antibody was more sensitive for the species of emerin expressed at the nuclear envelope was determined using the it was raised against (data not shown). In both cell types, confocal lasersharp processing software (Fig. 5). For each emerin is localized to the nuclear rim, but internal nuclear foci transfectant, we measured the fluorescence intensity of the and ER staining close to the nuclear rim was also observed. nuclear envelope from 25 cells. Fluorescence intensities were Interestingly, we observed more cytoplasmic emerin in quantified per unit area (100 pixels) of the nuclear envelope undifferentiated C2C12 myoblasts than in COS-7 cells. We and to allow comparisons between the wild type and mutants to others (Cartegni et al., 1997) have observed this phenomenon be made. The mean fluorescence intensity of wild-type GFP- in other adherent cell lines. Quantitatively there appears to be emerin at the nuclear envelope was 39.94±4.18 per 100 pixels the same amount of endogenous emerin in both cell types. (mean ± s.e.m.). All the mutant forms exhibited less GFP Emerin was shown to colocalize with lamins at the nuclear rim fluorescence at the nuclear envelope than wild type (Fig. 5). and in the intranuclei foci at interphase (Fig. 2B), as previously The mean fluorescence intensities were 9.48±1.63 per 100 reported by Manilal et al. (1998b). No plasma membrane pixels (mean ± s.e.m.; P<0.001) for Del95-99; 20.05±3.84 staining was seen, nor any colocalization with cytoskeletal (mean ± s.e.m.; P<0.0001) for S54P; 13.53±2.99 (mean ± actin (Fig. 2A). s.e.m.; P<0.0001) for P183H and 20.23±4.51 (mean ± s.e.m.; P<0.0024) for P183T. The targeting of the three missense Emerin targeting in transfected undifferentiated mutations was less severely affected compared to the deletion C2C12 myoblasts mutations, and similarly affected with respect to one another. The majority of EDMD patients exhibit the null phenotype; The most severely mistargeted mutant, Del236-241, was however, in the small number of patients who express mutant present in only trace amounts at the nuclear envelope, forms of emerin, it is not known whether these are mistargeted therefore no fluorescence intensities were measured for this or, if correctly localized, they are dysfunctional. To answer mutant. this question, GFP-constructs, mimicking the mutant forms We also transiently expressed in undifferentiated C2C12 of emerin identified in patients, were introduced into myoblasts, a mutant form of emerin representing a prematurely undifferentiated C2C12 myoblasts by transfection, and their truncated form (residues 1-220), which lacks both the localization monitored from the onset of expression using transmembrane region and C-terminal tail. This construct was fluorescence imaging with a confocal laser scanning localized exclusively to the nucleoplasm, with no nuclear rim

Fig. 4. Immunoblot showing the expression levels of the GFP-emerin constructs transfected into undifferentiated C2C12 myoblasts. Transfected cells were subjected to 15% SDS-PAGE and immunoblotted for actin (1:1000), emerin and GFP- emerin with affinity-purified antibody AP8 (1:3000), and the blot developed by ECL. The level of endogenous actin in each lane acted as the control for protein loading. Lane 1, untransfected C2C12 cells; lane 2, pEGFP vector alone; lane 3, pEGFP- emerin wild type; lane 4, pEGFP-Del236-241; lane 5, pEGFP-Del95-99; lane 6, pEGFP-S54P; lane 7, pEGFP-P183T; lane 8, pEGFP-P183H. The positions of molecular mass standards (Mwt. kDa) are shown. Emerin and Emery-Dreifuss muscular dystrophy 2577

50 Wild type P183T 40 P183H S54P Del95-99 30 *** **

Fig. 5. Bar chart showing the nuclear envelope fluorescence 20 *** intensities of the GFP-emerin constructs expressed in *** undifferentiated C2C12 cells. Undifferentiated C2C12 10 myoblasts were transiently transfected with either wild type or mutant GFP-emerin constructs as described in Materials fluorescence intensity/100 pixels and methods. The nuclear envelope fluorescence intensities 0 from 25 cells were quantified and statistically analyzed for Del95-99 S54P P183H P183T Wild type each transfectant, as described in Materials and methods. Values are means ± s.e.m. **P<0.01; ***P<0.001. GFP-emerin constructs staining (Fig. 6), suggesting that the major determinant for the actin (Fig. 7B, lane 7), and this was stable at 500 mM NaCl. nuclear membrane localization of emerin is within the region This could be blocked either by allowing 2.5 µg F-actin to bind spanning residues 221-254. to 1.25 µg FP9 for 2 hours at room temperature, prior to overlaying onto actin (Fig. 7B, lane 8) or by allowing 1.25 µg Overlay of bacterial emerin-fusion proteins onto cell FP9 to bind to 10 µg C2C12 cell lysate for 2 hours at room lysates temperature, prior to overlaying onto actin (Fig. 7B, lane 9). In To identify interacting proteins and the region in emerin addition, if we incubated 1.25 µg FP9 with 15 µg F-actin for involved in these potential protein-protein interactions, C2C12 2 hours at room temperature prior to overlaying onto C2C12 cell lysate proteins were separated electrophoretically, cell lysates, and immunoblotted with emerin antibody AP9, no transferred to nitrocellulose membranes and overlaid with the bands at all were visualized (data not shown). bacterially expressed emerin-fusion proteins. Only the fusion These results suggest that emerin is able to interact with protein FP9 (residues 1-220) bound to C2C12 cell lysates using actin, and that residues 174-220 are involved in binding to actin this overlay procedure. Since no binding was seen with the and also to the 64 and 74 kDa bands. In addition, the interaction fusion protein expressing residues 1-174, it suggests that between emerin and all three proteins can be blocked by prior residues 174-220 contain the sites that interact with other incubation of fusion protein FP9 with an excess of actin, proteins. Three bands of approx. 42, 64 and 74 kDa were suggesting that the actin, 64 kDa and 74 kDa binding sites on identified as binding to emerin in the C2C12 cell lysates (Fig. emerin are very close to one another or overlap. 7A). Similar results were obtained on purified rat hepatocyte nuclei, human skeletal muscle, HeLa cells and lymphoblastoid Coimmunoprecipitation of proteins interacting with cells (data not shown). The affinity-purified rabbit polyclonal emerin antisera AP2, AP8 and AP9 gave the same results and could To identify proteins interacting with emerin, mild detergent be used interchangeably, but AP5 (which recognizes residues solubilisation is required to ensure that emerin is isolated under 244-254) did not. These interacting bands did not appear in a non-denaturing conditions in order to retain protein-protein gel overlay onto an E. coli cell lysate (Fig. 7A, lane 1), and interactions. We adapted extraction conditions reported for the could be blocked by incubating affinity-purified antibody with LBR (Simos and Georgatos, 1992) and LAP1 proteins (Maison 1.25 µg of fusion protein FP9 (Fig. 7A, lane 2), prior to the gel et al., 1997) for extracting emerin, using a basic extraction overlay. The 42 kDa band predominated and no real increase buffer of 1% (v/v) Triton X-100, 50 mM Tris-HCl, pH 7.4, 2 in binding to this component was seen after 1.25 µg/ml fusion mM MgCl2, with varying amounts of NaCl (100 mM to 1 M). protein was added (Fig. 7B, lane 5), whereas increased binding The optimal extraction conditions chosen were those where we was seen for the 64 and 74 kDa bands as the fusion protein could demonstrate by immunoblotting of the cell lysate that we concentration was increased (Fig. 7A, lanes 3-7). The had maximum extraction of emerin and nuclear lamins, but interaction with the 42 kDa protein was stable at 500 mM which still retained protein-protein interactions in the NaCl, but was severely reduced with the 64 and 74 kDa subsequent immunoprecipitation (data not shown). The final proteins. Alongside the gel overlay, we immunoblotted the extraction buffer contained 150 mM NaCl, and the captured C2C12 cell lysates for actin (Fig. 7A, lane 8) and on the basis immunocomplexes were washed at 300 mM NaCl. of size we speculated that the 42 kDa is actin. A direct interaction between emerin and a nuclear To see if the 42 kDa band was actin, the emerin bacterial component has not been previously reported. We therefore fusion proteins FP2, FP3, FP8 and FP9 were overlaid onto examined our immunoprecipitates for lamin A/C, lamin B and rabbit skeletal muscle α-actin (prepared as described in Pardee actin. The size of the bands obtained were compared with an and Spudich, 1982). 2.5 µg F-actin was loaded per gel track immunoblot of the cell lysate/extracted rat nuclei run alongside and each track overlaid with 1.25 µg/ml of each bacterial (Fig. 8A,C). Emerin antibody AP1 immunoprecipitated emerin fusion protein (Fig. 7B). Binding to actin by the emerin-fusion from both C2C12 (Fig. 8A, lane 7) and purified rat hepatocyte proteins was analyzed by immunoblotting with the affinity- nuclei (Fig. 8C, lane 13). The pre-immune sera did not purified emerin antibodies. Only fusion protein FP9 bound to immunoprecipitate emerin (Fig. 8A,C, lanes 5 and 10). 2578 E. A. L. Fairley and others

purified rat nuclei (Fig. 8C, lane 11), even on lowering the washing stringency to 200 mM, which increased the amount of emerin and lamin B being immunoprecipitated (Fig. 8C, lane 13). In this track we blocked the signal from the heavy IgG chain (by adding excess rabbit IgG-alkaline phosphatase antibody first) to increase the sensitivity of detecting a minor band. The immunocomplexes isolated with the pre-immune sera were also immunoblotted for nuclear lamins and actin, and were negative (Fig. 8A,C, lanes 5 and 10). To examine whether immunoprecipitating with the nuclear lamin antibodies would isolate emerin, we performed the reciprocal experiment. The lamin A/C antibody did not immunoprecipitate any immunocomplexes, but the lamin B antibody immunoprecipitated itself and emerin but not actin (Fig. 8C, lane 14), suggesting that the actin and lamin B binding sites in emerin are the same or at least in close proximity to one another. Silver staining SDS-PAGE gels of the immunoisolates, did not reveal any other protein bands that were specifically immunoprecipitated with any of the antibodies. To examine the strength of any ionic interactions between emerin, actin and the lamins, the stringency of the washing conditions of the immunocomplexes isolated were altered. When we raised the salt concentration to 500 mM in the washing buffer, we severely reduced the amounts of both lamin A/C and emerin immunoprecipitated equally, but not the amount of actin (Fig. 8B). This somewhat surprising result may be explained if the stoichiometry of binding of emerin to actin is only a few emerin molecules per actin filament (which may contain many actin monomers), in which case a decrease in the amount of actin immunoprecipitated compared to emerin would be difficult to detect. Alternatively, the actin-emerin interaction may be through covalent bonds or disulphide bridges. The strength of the emerin-protein interactions were not affected by whether the immunoprecipitation was performed under reducing or non-reducing conditions. We were unable to detect lamin B when we washed at 500 mM salt (data not shown), suggesting the interaction between emerin and lamin B is substantially weaker than between lamin A and emerin.

DISCUSSION

We have previously shown, by immunoblotting lymphoblastoid cell lines derived from EDMD patients, that there are a small number of mutations in the emerin gene which allow mutant emerin proteins to be expressed (Ellis et al., 1998). In the present paper we show that when cDNA Fig. 6. Intracellular localization of the GFP-emerin 1-220 construct constructs reflecting these mutations are expressed in expressed by transient transfection into undifferentiated C2C12 undifferentiated C2C12 myoblasts, all are targeted to the myoblasts. The cells were transfected with GFP-emerin-1-220 (green) nuclear membrane, but less efficiently than wild type. It is and double-labelled (red) for MAC256 (ER marker), LAMP1 generally believed that all EDMD patients exhibit the same (lysosomal marker), or lamin A and lamin B (nuclear lamina), with clinical phenotype, therefore X-linked EDMD can arise the overlay showing the extent of protein colocalization. Bars, 10 µm. regardless of whether emerin is totally absent or expressed in a modified form. The emerin mutant with a deletion in the transmembrane region (Del236-241), was present in the least Coimmunoprecipitated with emerin were lamin B and lamin amount at the nuclear envelope, in agreement with protein A/C from both C2C12 (Fig. 8A, lanes 6 and 7, respectively) studies conducted on muscle biopsies and lymphoblastoid and purified nuclei extracts (Fig. 8C, lanes 9, lamin A/C, 11 cell lines derived from two unrelated EDMD families who and 13, lamin B). Actin was only coimmunoprecipitated with both possess this mutation (Manilal et al., 1998; Ellis et al., emerin in the C2C12 cell lysates (Fig. 8A, lane 7), and not from 1999). The muscle samples taken from patients expressing Emerin and Emery-Dreifuss muscular dystrophy 2579

Fig. 7. Gel overlay of bacterial fusion proteins onto cell lysates (A) and purified sarcomeric α-actin (B). Proteins were separated by 10% SDS-PAGE and immunoblotted onto nitrocellulose membranes for incubation with emerin- fusion proteins under a variety of conditions, as described in Materials and methods. (A) FP9 was overlaid onto a bacterial cell lysate (lane 1) or C2C12 cell lysates (lanes 2-7) in increasing amounts of FP9 (lane 3, zero; lane 4, 0.25 µg/ml; lane 5, 1.25 µg/ml; lane 6, 2.5 µg/ml; lane 7, 12.5 µg/ml). Preincubation of affinity-purified antibody AP9 (1:1000) with 1.25 µg of FP9 prior to overlaying is shown in lane 2. Lanes 1-7 were immunoblotted with AP8 at 1:3000 after fusion protein binding, and bands visualized by ECL and autoradiography. A C2C12 cell lysate was immunoblotted directly for actin (lane 8) for size comparison. (B) Gel overlay of emerin-fusion proteins onto pure skeletal muscle α- actin. Fusion proteins were overlaid onto a blot of actin (lane 4, FP8; lane 5, FP3; lane 6, FP2; lane 7, FP9). FP9 binding to actin could be blocked either by preincubating 1.25 µg FP9 with 2.5 µg actin prior to overlay (lane 8) or preincubating 1.25 µg FP9 with 10 µg C2C12 cell lysate (lane 9). Lanes 4-9 were immunoblotted with affinity-purified antibody AP8 subsequent to fusion protein overlay. Controls included immunoblotting directly for pure actin (lane 2) and actin in C2C12 cell lysates (lane 3) with actin antibody, and overlaying FP9 onto C2C12 cell lysates and immunoblotting with AP9 to show the position of the 42 kDa band in cell lysates with respect to actin (lane 1). this emerin mutation were shown to possess normal 220, with its molecular mass of 51 kDa, may therefore enter emerin mRNA controls. This suggests that a functional the nucleus by either of these mechanisms. transmembrane helix is required for emerin stability (Manilal Multiple regions have been identified in both the amino et al., 1998), and may explain why we see a lot of this domain of LAP2 and LBR (Furukawa et al., 1995, 1998; modified form of emerin aggregated in our transfectants. The Soullam and Worman 1993; 1995) and in the first lack of nuclear envelope localization of this construct, and of transmembrane segment of LBR (Smith and Blobel, 1993), the construct lacking the transmembrane and C-terminal tail, which promote localization to the nuclear rim. LAP2 has two suggest that the major nuclear envelope-targeting determinant non-overlapping regions in its N-terminal domain, which of emerin lies within residues 221-254. Similar results have independently promote nuclear rim localization. The first is in been reported by Cartegni et al. (1997), who demonstrated residues 1-296, which also includes the chromatin binding that a Del227-254 construct is localized to the nucleoplasm, region, and the second spans residues 298-409 and is involved and that a construct of GFP-227-254 targets to the nuclear in associating with nuclear lamins (Furukawa et al., 1995, envelope. Taken together these results would suggest that 1998; Furukawa and Kondo, 1998). The first nuclear targeting residues 236-241 of the transmembrane region contain the signal (residues 1-226) includes residues 114-152, which share major determinant for nuclear envelope targeting. Emerin 41% identity with residues 6-44 of emerin (Bione et al., 1994), also contains a consensus bipartite nuclear localization and contains a bipartite NLS. This suggests that this region of sequence (NLS; Fig.1; Dingwell and Laskey, 1991) in its N- identity functions as a common nuclear targeting region. The terminal domain (Ellis et al., 1998) also present in LAP2 transmembrane region in LAP2 is not required for nuclear rim (Furukawa et al., 1995). NLSs are non-operational in localization, but is required for efficient membrane integration membrane proteins, but once the restraint of the (Furukawa et al., 1995). The transmembrane region in LAP2 transmembrane region is removed, they have been shown to (residues 410-433) lies within the carboxyl-terminal region direct the remaining portion of the protein into the (residues 409-442), which exhibits 41% identity to residues nucleoplasm (Soullam and Worman, 1995). However, for 221-254 of emerin (Bione et al., 1994). We can thus assign a soluble proteins of >42-60 kDa, import into the nucleus can function of membrane insertion to this sequence. also occur by passive diffusion through the lateral channels Mutations in the N-terminal domain of emerin may also of the nuclear pore complex (Paine, 1975). Construct GFP-1- affect its ability to be retained at the nuclear membrane, by 2580 E. A. L. Fairley and others

Fig. 8. Coimmunoprecipitation of proteins interacting with emerin in C2C12 cell lysates (A,B) and in purified rat hepatocyte nuclei (C). The immunocomplexes captured by immunoprecipitation with affinity-purified antibody AP1 (lanes 6, 7, 9, 11, 13) or pre-immune sera (lanes 5 and 10) were subjected to SDS-PAGE and immunoblotted for emerin (lanes 5, 7, 10 and 13), lamin A/C (lanes 7 and 9), lamin B (lanes 6, 11 and 13) and actin (lanes 7, 11 and 13). The size of the bands obtained were compared with an immunoblot of the cell lysate/rat nuclei run alongside; emerin (lane 1); actin (lanes 2 and 12); lamin A/C (lanes 3 and 8) and lamin B (lanes 4 and 12). Purified rat nuclei were also immunoprecipitated with lamin B antibody and immunoblotted for lamin B and emerin (lane 14). (B) The strength of the interaction between emerin, actin and the nuclear lamins was investigated by washing the immunoprecipitates with AP1, at either 350 mM or 500 mM salt, and immunoblotting for lamin A/C, emerin and actin. Where necessary, heavy chain IgG was either immunoblotted separately or blocked (lane 13), so as to prevent reduction of the ECL signal. affecting its interaction with other nuclear components. The required to determine whether the N-terminal mutations overlay binding assay technique, in combination with studied here are mistargeted to the inner nuclear membrane coimmunoprecipitation experiments, identified lamin A/C, because of disruptions in lamin interactions affecting nuclear lamin B and actin as binding to residues 174-220 in emerin. membrane retention, or because of aberrant nuclear membrane The inner nuclear membrane proteins LAP1, LAP2 and LBR targeting signals or due to contributions from both effects. have all been shown to bind to lamin B (Maison et al., 1997; The observation that emerin antisera coimmunoprecipitate Furukawa et al., 1998; Ye and Worman, 1994) and LAP1 has cytoplasmic actin was an unexpected finding. Physiologically been shown in addition to bind to lamin A (Foisner and Gerace, it is most likely that emerin is binding to nuclear actin, but 1993). The lamin B binding region in LAP2 coincides with one under the conditions of cell lysis employed in our of its nuclear envelope targeting domains found in the amino- immunoprecipitation experiments emerin is exposed to the terminal domain (Furukawa et al., 1998; Furukawa and Kondo, total pool of cellular actin (approximately 10% of total cell 1998), suggesting that a major mechanism for localization of protein is actin), which probably accounts for the large amount integral membrane proteins at the inner nuclear membrane of actin being immunoprecipitated. Bundles of actin filaments involves binding to lamins, thus preventing diffusion through whose distribution changes with respect to changes in nuclear the continuous nuclear envelope/endoplasmic reticulum functional states and appear to maintain the linear integrity of membrane system. Two of our constructs with mutations at polytene have been reported (Parfenov et al., P183 are within the region identified in emerin as interacting 1995). In addition it has been reported that the carboxyl with lamins (residues 174-220) and they exhibited reduced terminus of lamin A interacts with nuclear actin (Sasseville and targeting and retention at the nuclear rim, but not as severely Langelier, 1998), suggesting that actin is also a structural as the Del95-99 and Del236-241 constructs. Further studies are component of the nucleus. An actin-based motor link to the Emerin and Emery-Dreifuss muscular dystrophy 2581 lamina and lamina-associated proteins could provide a 1996). At interphase this complex includes the LBR, LBR dynamic network which, by causing structural changes in kinase, nuclear lamins A and B, p18 (an 18 kDa polypeptide) chromatin, could be involved in the transduction of messages and p34/p32 (a 34 kDa protein). The LBR kinase regulates from the to various (Sasseville and LBRs interaction with p34/p32 at its N-terminal domain by Langelier, 1998). site-specific phosphorylation. The function of this complex is It is likely that the interaction of inner nuclear membrane not clear, although regulating its internal interactions may proteins with themselves and with other nuclear components control nuclear architecture or link the nuclear lamina to plays a major role in regulating nuclear architecture. However, regulatory factors involved in different aspects of gene it is far from obvious how a disruption in location/function of expression (Nikolakaki et al., 1996). Emerin has been shown a protein with a nuclear location could influence muscle cell to be phosphorylated both at interphase and in a cell cycle- integrity. Actin and lamin filaments have been reported to dependent manner (Ellis et al., 1998), and this modification mechanically connect the plasma membrane to the nuclear may control the interactions between the components of the envelope (Maniotis et al., 1997). A mechanical tug on the cell proposed emerin-nuclear protein complex. It may be that surface has been shown to change the molecular organization regulation of the interactions between these components of both the nucleus and the cytoplasm (Maniotis et al., 1997), produces changes in nuclear structure necessary for muscle but there is no evidence that a disruption of nuclear integrity function and any defects in these interactions and/or regulation affects plasma membrane stability. However, because fully may be the underlying cause of EDMD. differentiated cardiac and skeletal muscle cells are non- dividing and long-lived, the nuclear membrane in these cells is This work was funded by the Muscular Dystrophy Group of Great required to provide stability over a long time. Emerin may be Britain and Northern Ireland and the Medical Research Council (MRC involved in the molecular interactions necessary to maintain studentship to E.A.L.F.). We would like to thank Tony P. Hodge and this stability. Sabine M. Gonsior for their help with the transfections and confocal immunofluorescence imaging, to Sean Munro for immunolabelling The recent discovery that an autosomal dominant EDMD reagents, to Roy Quinlan for lamin A antibodies, Rebecca J. Carter (Bonne et al., 1999) arises due to defects in the gene encoding for help with the statistics and to J. 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