RESEARCH ARTICLE
4435
Properties of lamin A mutants found in Emery-Dreifuss
muscular dystrophy, cardiomyopathy and Dunnigantype partial lipodystrophy
Cecilia Östlund1, Gisèle Bonne2, Ketty Schwartz2 and Howard J. Worman1,*
1Departments of Medicine and of Anatomy
2INSERM UR523, Institut de Myologie, GH
and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
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
is caused by mutations in the LMNA gene, which encodes
lamin A and lamin C. Mutations in this gene also give rise
to limb girdle muscular dystrophy type 1B, dilated
cardiomyopathy with atrioventricular conduction defect
and Dunnigan-type partial lipodystrophy. The properties
of the mutant lamins that cause muscular dystrophy,
lipodystrophy and dilated cardiomyopathy are not known.
We transfected C2C12 myoblasts with cDNA encoding
wild-type lamin A and 15 mutant forms found in patients
affected by these diseases. Immunofluorescence microscopy
showed that four mutants, N195K, E358K, M371K and
R386K, could have a dramatically aberrant localization,
with decreased nuclear rim staining and formation of
intranuclear foci. The distributions of endogenous lamin
INTRODUCTION
Emery-Dreifuss muscular dystrophy (EDMD) is characterized
by contractures of the elbows, Achilles’ tendons and posterior
neck, slow progressive muscle wasting and cardiomyopathy
with atrioventricular conduction block (Emery and Dreifuss,
1966; Rowland et al., 1979; Emery, 1989). Emery-Dreifuss
muscular dystrophy is inherited in both an autosomal dominant
(AD-EDMD, OMIM 181350) and an X-linked, recessive
(OMIM 310300) manner. In 1994, mutations in emerin, an
integral protein of the nuclear envelope inner membrane, were
shown to be responsible for X-linked EDMD (Bione et al.,
1994; Manilal et al., 1996; Nagano et al., 1996). The
connection between muscle disease and mutations in a nuclear
envelope protein was unexpected, but was further established
when Bonne et al. (Bonne et al., 1999) showed that ADEDMD, a phenotypically indistinguishable syndrome, is
caused by mutations in the LMNA gene, which encodes the
nuclear envelope proteins lamins A and C. Mutations in LMNA
have also been shown to cause limb girdle muscular dystrophy
1B (LGMD1B, OMIM 159001), a disease that differs from
AD-EDMD by displaying a different distribution of affected
skeletal muscle, a later occurrence of cardiac problems and an
absence of early contractures (Muchir et al., 2000), and dilated
cardiomyopathy without skeletal muscle pathology (OMIM
115200) (Fatkin et al., 1999; Bécane et al., 2000). LMNA
A/C, lamin B1 and lamin B2 were also altered in cells
expressing these four mutants and three of them caused a
loss of emerin from the nuclear envelope. In the yeast twohybrid assay, the 15 lamin A mutants studied interacted
with themselves and with wild-type lamin A and lamin B1.
Pulse-chase experiments showed no decrease in the stability
of several representative lamin A mutants compared with
wild-type. These results indicate that some lamin A
mutants causing disease can be aberrantly localized,
partially disrupt the endogenous lamina and alter emerin
localization, whereas others localize normally in
transfected cells.
Key words: Nuclear envelope, Lamins, Intermediate filaments,
Muscular dystrophy, Lipodystrophy, Cardiomyopathy
mutations that cause cardiac and skeletal muscle disease are
found throughout the gene, most being point mutations
resulting in amino acid substitutions. There appears to be no
clear correlation between the site of mutation and the severity
of disease (Bonne et al., 2000). By contrast, mutations in
LMNA clustering only in exons 8 and 11 have been shown to
cause Dunnigan-type familial partial lipodystrophy (FPLD,
OMIM 151660) (Cao and Hegele, 2000; Shackleton et al.,
2000; Speckman et al., 2000; Vigouroux et al., 2000). FPLD
is a rare, autosomal dominant disease characterized by the loss
of subcutaneous adipose tissue from the extremities and trunk
after the onset of puberty (Köbberling and Dunnigan, 1986).
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 A∆10, 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 α-helical segments, which is
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 taildomains, 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.
MATERIALS AND METHODS
Plasmid construction
All cloning procedures were performed according to standard
methods (Sambrook et al., 1989). For expression in mammalian cells,
constructs that expressed FLAG-tagged polypeptides were made in
pSVK3 (Amersham Pharmacia Biotech, Inc., Piscataway, NJ), which
contains a multiple cloning site downstream from the SV-40 early
promoter. cDNA encoding prelamin A, cloned into the BamHI and
SalI restriction sites of plasmid pGBT9 (Ye and Worman, 1995), was
excised by restriction endonuclease digestion with SmaI and SalI and
ligated into pBFT4, digested with the same restriction enzymes.
pBFT4 is a pBluescript II KS- (Stratagene, La Jolla, CA) based
plasmid containing a Kozak sequence, ATG and the FLAG-tag coding
sequence 5′ to the multiple cloning site. The resulting plasmid was
digested with SpeI and XhoI to excise a cDNA encoding ATG-FLAGprelamin A that was ligated into pSVK3, digested with XbaI (an
isoschizomer of SpeI) and XhoI.
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 manufacturer’s
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 Dulbecco’s 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 manufacturer’s 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 antiLAP2 (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 568conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR)
and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse
IgG (Jackson ImmunoResearch Laboratories, Inc.). For DNAstaining, 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 β-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 manufacturer’s 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
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 SDSsample 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 wildFig. 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
are found in dilated cardiomyopathy, mutations in italics in FPLD and others in ADfinal 18 amino acids (Weber et al., 1989;
EDMD. Mutations shown in red are proteins that form aberrant intranuclear foci
Sinensky et al., 1994). For detection of expressed
when expressed in C2C12 cells (Fig. 1B; Table 1). (B) Cellular localization of
proteins, FLAG-epitopes were fused to the NFLAG-tagged wild-type and mutant forms of lamin A. The panels show laser
termini of the constructs. The mutations studied
scanning confocal immunofluorescence microscopy images of C2C12 cells
were: R60G, L85R, N195K and E203G, found in
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
FITC (green) and AlexaTM (red) channels where areas of colocalization appear
K486N, found in patients with FPLD (Cao and
yellow. Bar, 10 µm.
Hegele, 2000; Shackleton et al., 2000) (Fig. 1A).
In transfected cells, FLAG-tagged wild-type
M371K and R386K, showed a dramatically aberrant
lamin A localized to the nuclear periphery, showing
localization in many cells, accumulating in large intranuclear
colocalization with lamin B1, a marker for the lamina and
foci (Fig. 1B). These four mutants often showed a decrease in
nuclear envelope (Fig. 1B). A majority of the FLAG-tagged
nuclear rim localization, giving a more diffuse, nucleoplasmic
lamin A mutants showed no gross abnormalities in cellular
staining, which was also seen in cells lacking intranuclear foci
localization; however, four of the mutants, N195K, E358K,
4438
JOURNAL OF CELL SCIENCE 114 (24)
Table 1. Statistical analysis of nuclear foci in cells
expressing lamin A mutants
Cells without foci
Cells with foci
Cells with
foci (%)
WT
R60G
L85R
N195K*
199.33±0.33
199.33±0.66
199.00±0.58
164.33±6.44
0.67±0.33
0.67±0.66
1.00±0.58
35.67±6.44
0.3
0.3
0.5
17.8
E203G
E358K*
M371K*
R386K*
198.67±0.33
177.00±3.05
135.33±8.29
155.00±5.83
1.33±0.33
23.00±3.05
64.67±8.29
45.00±5.83
0.7
11.5
32.3
22.5
R453W
R482Q
R482W
K486N
196.67±1.45
199.00±0.58
199.00±0.58
198.33±0.88
3.33±1.45
1.00±0.58
1.00±0.58
1.67±0.88
1.7
0.5
0.5
0.8
W520S
R527P
T528K
L530P
197.67±0.88
195.33±1.45
196.67±0.66
198.33±0.88
2.33±0.88
4.67±1.45
3.33±0.66
1.67±0.88
1.2
2.3
1.7
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
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).
Immunofluorescence analysis of cells expressing
foci-forming lamin A mutants
To investigate further the composition of the abnormal
nuclear foci formed by four of the lamin A mutants and their
downstream effects on the nuclear envelope, we used several
different markers to perform a detailed immunofluorescence
microscopy analysis. Mislocalization of the lamin A mutants
was often accompanied by disturbances in the localization of
endogenous lamin B1. In many cells, lamin B1 was found in
the interior of the intranuclear foci, with the mutant lamin A
being more concentrated at the edges (Fig. 2). This phenotype
was seen to a variable degree, being more frequent with
mutants E358K and R386K (approximately 90% of cells with
Fig. 2. Aberrant cellular localization of lamin A mutants is often
accompanied by a change in lamin B1 localization. The panels show
laser scanning confocal immunofluorescence microscopy images of
C2C12 cells expressing either wild-type lamin A (WT) or lamin A
with missense mutations as indicated, fused to a FLAG-epitope. The
bottom panels show the ∆NLA mutant, which lacks the N-terminal
head-domain, fused to a FLAG-epitope. Arrows show lamin B1
localized to the nuclear envelope. Antibodies used were monoclonal
antibodies against FLAG recognized by FITC-conjugated secondary
antibodies (left) and polyclonal antibodies against lamin B1
recognized by AlexaTM-conjugated secondary antibodies (middle).
The pictures to the right show an overlay of the FITC (green) and
AlexaTM (red) channels. Bars, 10 µm.
foci), where the foci often were large, than with mutants
N195K (20-50%) and M371K (10-50%). The mislocalization
of lamin B1 was, however, not complete, and in virtually
all cells, some lamin B1 could still be detected at the
nuclear periphery (Fig. 2, arrows). In occasional cells, lamin
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 headdomain (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
A mutants. The panels show laser scanning confocal immunofluorescence
et al., 2001).
microscopy images of C2C12 cells transfected with the prelamin A E358K or
To obtain additional information about the effects
∆NLA mutants fused to a FLAG-epitope. (A) Staining with monoclonal
of foci-forming lamin A mutants on the nuclear
antibodies against lamin B2 (left panel) and polyclonal antibodies against
lamina, we performed double labeling with antilamin A/C (middle panel). Arrows indicate concentration of lamin B2 by
FLAG antibodies, recognizing the exogenous lamin
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
rhodamine (red) channels. Bars, 10 µm.
lamin B1 (Fig. 5). As with lamin B1, however, some
lamin A/C still remained at the nuclear periphery in
the majority of the cells. In two separate experiments
seen in a subset of all transfected cells, also some cells
with 100 transfected cells counted, peripheral lamin A/C was
transfected with wild-type lamin A. Similar results have been
present in 85-100% of cells expressing mutants N195K,
reported in transfected HeLa cells by Raharjo et al. (Raharjo
E358K and M371K, and 70-90% of cells expressing mutants
et al., 2001). All foci-forming mutants except E358K led to
R386K and ∆NLA (Fig. 5, arrows).
a loss of emerin from the nuclear envelope in a significantly
Lamin A has been shown to bind to emerin in vitro
greater number of cells than wild-type lamin A (Fig. 6B). We
(Clements et al., 2000; Sakaki et al., 2001) and, in cells from
also investigated loss of emerin from the nuclear envelope in
mice lacking A-type lamins, emerin is partly mislocalized to
cells expressing three mutants that do not form foci (R60G,
the cytoplasm (Sullivan et al., 1999). To investigate whether
E203G and K486N). None of these mutants led to an
mutant forms of lamin A caused a mislocalization of emerin
increased loss of emerin from the nuclear envelope, compared
in the presence of wild-type lamin A, the situation in
to wild-type (data not shown). Emerin, similar to nuclear pore
heterozygous patients, we stained C2C12 cells expressing the
complex proteins and LAP2, did not localize to the
foci-forming lamin A mutants with antibodies against emerin
intranuclear lamin foci, further indicating that the foci are not
(Fig. 6A). A loss of emerin from the nuclear envelope was
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 FITCconjugated 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
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
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.
possible using this assay to determine whether the proteinprotein interactions formed are the coil-coiled ones in the
normal lamina, these results suggest that the primary laminlamin dimerization is not disturbed in these mutants.
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 FITCconjugated 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
lamin A
To investigate whether representative mutant lamin A proteins
were less stable than wild-type lamin A, we performed pulsechase 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
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 FLAGepitopes, 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
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.
DISCUSSION
We have investigated 15 lamin A mutants in patients with
dilated cardiomyopathy, AD-EDMD and FPLD. These
mutations are dominant and patients carry one mutant and one
wild-type copy of the LMNA gene. There are many possible
mechanisms for how these mutations may cause disease, such
as changes in the stability, localization and interactions of the
mutant proteins.
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 wildtype 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, α-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
Lamin A mutations in disease
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 wildtype 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 musclespecific 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 laminlamin 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
4443
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 tissuespecific nature of the diseases caused by mutations in the Atype 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 δ 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.
4444
JOURNAL OF CELL SCIENCE 114 (24)
This work was supported by grants from the Muscular Dystrophy
Association (MDA) to H.J.W. and the Human Frontiers Science
Program to G.B. and H.J.W. Also, C.Ö. received support from the
Sweden-America Foundation/Thanks to Scandinavia, Inc. and
Karolina Widerström’s Foundation, and G.B. and K.S. received
support from Association Française contre les Myopathies (AFM)
grant 7434. H.J.W. is also an Irma T. Hirschl Scholar. The confocal
microscopy facility used for part of this project was established by
National Institutes of Health grant 1S10-RR10506 and is supported
by National Institutes of Health Grant 5 P30-CA13696 as part of the
Herbert Irving Cancer Center at Columbia University. We thank
Sudhindra Swamy and Theresa Swayne (Columbia University) for
help with the confocal microscopy; Jean-Claude Courvalin and
Brigitte Buendia (Institut Jacques Monod) and Brian Burke
(University of Calgary) for helpful discussions; Glenn Morris (North
East Wales Institute), Tarik Soliman (Rockefeller University), Georg
Krohne (University of Würzburg) and Katherine Wilson (Johns
Hopkins University) for antibodies; and Hal Skopicki (Columbia
University) for C2C12 cells.
REFERENCES
Aebi, U., Cohn, J., Buhle, L. and Gerace, L. (1986). The nuclear lamina is
a meshwork of intermediate-type filaments. Nature 323, 560-564.
Bécane, H.-M., Bonne, G., Varnous, S., Muchir, A., Ortega, V.,
Hammouda, E. H., Urtizberea, J.-A., Lavergne, T., Fardeau, M.,
Eymard, B. et al. (2000). High incidence of sudden death with conduction
system and myocardial disease due to lamins A and C gene mutation. Pacing
Clin. Electrophysiol. 23, 1661-1666.
Biamonti, G., Giacca, M., Perini, G., Contreas, G., Zentilin, L., Weighardt,
F., Guerra, M., Della Valle, G., Saccone, S., Riva, S. et al. (1992). The
gene for a novel human lamin maps at a highly transcribed locus of
chromosome 19 which replicates at the onset of S-phase. Mol. Cell. Biol.
12, 3499-3506.
Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G. and
Toniolo, D. (1994). Identification of a novel X-linked gene responsible for
Emery-Dreifuss muscular dystrophy. Nat. Genet. 8, 323-327.
Bonne, G., Raffaele di Barletta, M., Varnous, S., Bécane, H.-M.,
Hammouda, E.-H., Merlini, L., Muntoni, F., Greenberg, C. R., Gary, F.,
Urtizberea, J.-A. et al. (1999). Mutations in the gene encoding lamin A/C
cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat. Genet.
21, 285-288.
Bonne, G., Mercuri, E., Muchir, A., Urtizberea, A., Bécane, H.-M., Recan,
D., Merlini, L., Wehnert, M., Boor, R., Reuner, U. et al. (2000). Clinical
and molecular genetic spectrum of autosomal dominant Emery-Dreifuss
muscular dystrophy due to mutations of the lamin A/C gene. Ann. Neurol.
48, 170-180.
Bravo, R. and Macdonald-Bravo, H. (1987). Existence of two populations
of cyclin/proliferating cell nuclear antigen during the cell cycle: association
with DNA replication sites. J. Cell Biol. 105, 1549-1554.
Broers, J. L. V., Machiels, B. M., van Eys, G. J. J. M., Kuijpers, H. J. H.,
Manders, E. M. M., van Driel, R. and Ramaekers, F. C. S. (1999).
Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins.
J. Cell Sci. 112, 3463-3475.
Cance, W. G., Chaudhary, N., Worman, H. J., Blobel, G. and CordonCardo, C. (1992). Expression of the nuclear lamins in normal and neoplastic
human tissue. J. Exp. Clin. Cancer Res. 11, 233-246.
Cao, H. and Hegele, R. A. (2000). Nuclear lamin A/C R482Q mutation in
Canadian kindreds with Dunnigan-type familial partial lipodystrophy. Hum.
Mol. Genet. 9, 109-112.
Clements, L., Manilal, S., Love, D. R. and Morris, G. E. (2000). Direct
interaction between emerin and lamin A. Biochem. Biophys. Res. Commun.
267, 709-714.
Eissenberg, J. C., James, T. C., Foster-Hartnett, D. M., Hartnett, T., Ngan,
V. and Elgin, S. C. (1990). Mutation in a heterochromatin-specific
chromosomal protein is associated with suppression of position-effect
variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 87,
9923-9927.
Emery, A. E. H. (1989). Emery-Dreifuss syndrome. J. Med. Genet. 26, 637641.
Emery, A. E. H. and Dreifuss, F. E. (1966). Unusual type of benign X-linked
muscular dystrophy. J. Neurol. Neurosurg. Psychiatry 29, 338-342.
Fatkin, D., MacRae, C., Sasaki, T., Wolff, M. R., Porcu, M., Frenneaux,
M., Atherton, J., Vidaillet, H. J., Jr, Spudich, S., De Girolami, U. et al.
(1999). Missense mutations in the rod domain of the lamin A/C gene as
causes of dilated cardiomyopathy and conduction-system disease. New.
Engl. J. Med. 341, 1715-1724.
Fields, S. and Song, O. (1989). A novel genetic system to detect proteinprotein interactions. Nature 340, 245-246.
Fisher, D. Z., Chaudhary, N. and Blobel, G. (1986). cDNA sequencing of
nuclear lamins A and C reveals primary and secondary structural homology
to intermediate filament proteins. Proc. Natl. Acad. Sci. USA 83, 64506454.
Foisner, R. and Gerace, L. (1993). Integral membrane proteins of the nuclear
envelope interact with lamins and chromosomes, and binding is modulated
by mitotic phosphorylation. Cell 73, 1267-1279.
Holt, I., Clements, L., Manilal, S., Brown, S. C. and Morris, G. E. (2001).
The R482Q lamin A/C mutation that causes lipodystrophy does not prevent
nuclear targeting of lamin A in adipocytes or its interaction with emerin.
Eur. J. Hum. Genet. 9, 204-208.
Hutchison, C. J., Alvarez-Reyes, M. and Vaughan, O. A. (2001). Lamins in
disease: why do ubiquitously expressed nuclear envelope proteins give rise
to tissue-specific disease phenotypes? J. Cell Sci. 114, 9-19.
Izumi, M., Vaughan, O. A., Hutchison, C. J. and Gilbert, D. M. (2000).
Head and/or CaaX domain deletions of lamin proteins disrupt preformed
lamin A and C but not lamin B structure in mammalian cells. Mol. Biol. Cell
11, 4323-4337.
Köbberling, J. and Dunnigan, M. (1986). Familial partial lipodystrophy: two
types of an X-linked dominant syndrome, lethal in the hemizygous state. J.
Med. Genet. 23, 120-127.
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of
the head of bacteriophage T4. Nature 227, 680-685.
Lin, F. and Worman, H. J. (1993). Structural organization of the human gene
encoding nuclear lamin A and nuclear lamin C. J. Biol. Chem. 268, 1632116326.
Lin, F. and Worman, H. J. (1995). Structural organization of the human gene
(LMNB1) encoding nuclear lamin B1. Genomics 27, 230-236.
Lourim, D. and Lin, J. J. (1992). Expression of wild-type and nuclear
localization-deficient human lamin A in chick myogenic cells. J. Cell Sci.
103, 863-874.
Machiels, B. M., Zorenc, A. H. G., Endert, J. M., Kuijpers, H. J. H., van
Eys, G. J. J. M., Ramaekers, F. C. S. and Broers, J. L. V. (1996). An
alternative splicing product of the lamin A/C gene lacks exon 10. J. Biol.
Chem. 271, 9249-9253.
Mancini, M. A., Shan, B., Nickerson, J. A., Penman, S. and Lee, W.-H.
(1994). The retinoblastoma gene product is a cell cycle-dependent, nuclear
matrix-associated protein. Proc. Natl. Acad. Sci. USA 91, 418-422.
Manilal, S., Nguyen T. M., Sewry, C. A. and Morris, G. E. (1996). The
Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane
protein. Hum. Mol. Genet. 5, 801-808.
McKeon, F. D., Kirschner, M. W. and Caput, D. (1986). Homologies in both
primary and secondary structure between nuclear envelope and intermediate
filament proteins. Nature 319, 463-468.
McLean, W. H. I. and Lane, E. B. (1995). Intermediate filaments in disease.
Curr. Opin. Cell Biol. 7, 118-125.
Moir, R. D., Montag-Lowy, M. and Goldman, R. D. (1994). Dynamic
properties of nuclear lamins: lamin B is associated with sites of DNA
replication. J. Cell Biol. 125, 1201-1212.
Morris, G. E. (2000). Nuclear proteins and cell death in inherited
neuromuscular disease. Neuromusc. Disord. 10, 217-227.
Muchir, A., Bonne, G., van der Kooi, A. J., van Meegen, M., Baas, F.,
Bolhuis, P. A., de Visser, M. and Schwartz, K. (2000). Identification of
mutations in the gene encoding lamins A/C in autosomal dominant limb
girdle muscular dystrophy with atrioventricular conduction disturbances
(LGMD1B). Hum. Mol. Genet. 9, 1453-1459.
Nagano, A., Koga, R., Ogawa, M., Kurano, Y., Kawada, J., Okada, R.,
Hayashi, Y. K., Tsukahara, T. and Arahata, K. (1996). Emerin deficiency
at the nuclear membrane in patients with Emery-Dreifuss muscular
dystrophy. Nat. Genet. 12, 254-259.
Östlund, C., Ellenberg, J., Hallberg, E., Lippincott-Schwartz, J. and
Worman, H. J. (1999). Intracellular trafficking of emerin, the EmeryDreifuss muscular dystrophy protein. J. Cell Sci. 112, 1709-1719.
Raharjo, W. H., Enarson, P., Sullivan, T., Stewart, C. and Burke, B. (2001).
Nuclear envelope defects associated with LMNA mutations causing dilated
Lamin A mutations in disease
cardiomyopathy and Emery-Dreifuss muscular dystrophy. J. Cell Sci. 114,
4447-4457.
Rowland, L. P., Fetell, M., Olarte, M., Hays, A., Singh, N. and Wanat, F.
E. (1979). Emery-Dreifuss muscular dystrophy. Ann. Neurol. 5, 111-117.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn,
G. T., Mullis, K. B. and Erlich, H. (1987). Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science 239,
487-491.
Sakaki, M., Koike, H., Takahashi, N., Sasagawa, N., Tomioka, S., Arahata,
K. and Ishiura, S. (2001). Interaction between emerin and nuclear lamins.
J. Biochem. 129, 321-327.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press.
Shackleton, S., Lloyd, D. J., Jackson, S. N. J., Evans, R., Niermeijer, M.
F., Singh, B. M., Schmidt, H., Brabant, G., Kumar, S., Durrington, P.
N. et al. (2000). LMNA, encoding lamin A/C, is mutated in partial
lipodystrophy. Nat. Genet. 24, 153-156.
Sinensky, M., Fantle, K., Trujillo, M., McLain, T., Kupfer, A. and Dalton,
M. (1994). The processing pathway of prelamin A. J. Cell Sci. 107, 61-67.
Spann, T. P., Moir, R. D., Goldman, A. E., Stick, R. and Goldman, R. D.
(1997). Disruption of nuclear lamin organization alters the distribution of
replication factors and inhibits DNA synthesis. J. Cell Biol. 136, 12011212.
Speckman, R. A., Garg, A., Du, F., Bennet, L., Veile, R., Arioglu, E.,
Taylor, S. I., Lovett, M. and Bowcock, A. M. (2000). Mutational and
haplotype analyses of families with familial partial lipodystrophy (Dunnigan
variety) reveal recurrent missense mutations in the globular C-terminal
domain of lamin A/C. Am. J. Hum. Genet. 66, 1192-1198.
Steinert, P. M. and Roop, D. R. (1988). Molecular and cellular biology of
intermediate filaments. Annu. Rev. Biochem. 57, 593-625.
4445
Stuurman, N., Heins, S. and Aebi, U. (1998). Nuclear lamins: their structure,
assembly, and interactions. J. Struct. Biol. 122, 42-66.
Sullivan, T., Escalante-Alcalde, D., Bhatt, H., Anver, M., Bhat, N.,
Nagashima, K., Stewart, C. L. and Burke, B. (1999). Loss of A-type
lamin expression compromises nuclear envelope integrity leading to
muscular dystrophy. J. Cell Biol. 147, 913-919.
Vigouroux, C., Magré, J., Vantyghem, M.-C., Bourut, C., Lascols, O.,
Shackleton, S., Lloyd, D. J., Guerci, B., Padova, G., Valensi, P. et al.
(2000). Sex-determined expression of mutations in Dunnigan-type familial
partial lipodystrophy and absence of coding mutations in congenital and
acquired generalized lipoatrophy. Diabetes 49, 1958-1962.
Vigouroux, C., Auclair, M., Dubosclard, E., Pouchelet, M., Lepanse, S.,
Capeau, J., Courvalin, J.-C. and Buendia, B. (2001). Nuclear envelope
disorganization in fibroblasts from lipodystrophic patients with
heterozygous R482Q/W mutations in lamin A/C gene. J. Cell Sci. 114,
4459-4468.
Weber, K., Plessmann, U. and Traub, P. (1989). Maturation of nuclear lamin
A involves a specific carboxy-terminal trimming, which removes the
polyisoprenylation site from the precursor: implications for the structure of
the nuclear lamina. FEBS Lett. 257, 411-414.
Worman, H. J. and Courvalin, J.-C. (2000). The inner nuclear membrane.
J. Membr. Biol. 177, 1-11.
Ye, Q. and Worman, H. J. (1995). Protein-protein interactions between
human nuclear lamins expressed in yeast. Exp. Cell Res. 219, 292-298.
Ye, Q. and Worman, H. J. (1996). Interaction between an integral protein of
the nuclear envelope inner membrane and human chromodomain proteins
homologous to Drosophila HP1. J. Biol. Chem. 271, 14653-14656.
Ye., Q., Callebaut, I., Pezhman, A., Courvalin, J.-C. and Worman, H. J.
(1997). Domain-specific interactions of human HP1-type chromodomain
proteins and inner nuclear membrane protein LBR. J. Biol. Chem. 272,
14983-14989.