1 Biochemistry Group, North East Wales Institute, Wrexham LL11 2AW, UK
2 Departments of Medicine and of Anatomy and Cell Biology, College of Physicians
and Surgeons, Columbia University, New York, NY 10032, USA
3 Laboratory of Cancer and Developmental Biology, NCI-FCRDC, PO Box B,
Frederick, MD 21702-1201, USA
* Author for correspondence (e-mail: morrisge{at}newi.ac.uk)
Accepted 3 April 2003
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Summary |
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Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of emerin in these foci suggests that the interaction of lamin A with emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect emerin binding by disrupting the structure of the putative emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (<2%) but, when they did so, emerin was absent, consistent with a direct effect of the mutations on emerin binding. The lipodystrophy mutation R482Q, which causes a different phenotype and is believed to act through an emerin-independent mechanism, was indistinguishable from wild-type in its localization and its ability to trap emerin at the nuclear rim.
The novel hypothesis suggested by the data is that EDMD/CMD1A mutations in the tail domain of lamin A/C work by direct impairment of emerin interaction, whereas mutations in the rod region cause defective lamina assembly that might or might not impair emerin capture at the nuclear rim. Subtle effects on the function of the lamina-emerin complex in EDMD/CMD1A patients might be responsible for the skeletal and/or cardiac muscle phenotype.
Key words: Nuclear lamina, Mouse knockout, Nuclear envelope, Emery-Dreifuss muscular dystrophy, Lipodystrophy
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Introduction |
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The A-type lamins, A and C, are produced from alternatively spliced mRNA
products of the gene on chromosome 1q21.3, lamin C being a shorter form of
lamin A (Lin and Worman,
1993). The emerin gene on chromosome Xq28
(Bione et al., 1994
) encodes a
254 amino acid, type II integral membrane protein that is anchored to the
inner nuclear membrane by its hydrophobic C-terminal tail
(Manilal et al., 1996
;
Nagano et al., 1996
) and
interacts directly with lamin A/C (Clements
et al., 2000
). This interaction involves the globular tail region
common to lamins A and C (Vaughan et al.,
2001
) and a central region (amino acids 70-164) of the emerin
molecule (Lee et al., 2001
). A
similar region of emerin (amino acids 107-175) is required for its
localization to the nuclear rim (Tsuchiya
et al., 1999
; Östlund et
al., 1999
), suggesting involvement of lamin A/C, and this was
confirmed by the location of emerin in the ER in the lamin A/C knockout mouse
(Sullivan et al., 1999
).
However, partial localization of emerin at the nuclear rim was still observed
in some knockout mouse tissues, suggesting that factors other than lamin A/C
can be involved (Sullivan et al.,
1999
).
The emerin-lamin A/C interaction is of particular interest because
mutations in both proteins cause Emery-Dreifuss muscular dystrophy (EDMD)
(Bione et al., 1994;
Bonne et al., 1999
). In
X-linked EDMD, most emerin mutations result in complete absence of emerin
protein (Manilal et al., 1998), although reduced emerin levels caused by
altered RNA splicing might also result in EDMD
(Holt et al., 2001b
). A few
missense mutations are known and these produce emerins that are more easily
extracted from the nucleus (Fairley et al.,
1999
). In autosomal dominant EDMD, most mutations are mis-sense
and evenly spread throughout the helical rod and globular tail domains common
to lamins A and C. It seems likely that these mutations exert a
dominant-negative effect on the function of the multisubunit lamin filaments
(Morris, 2001
). Both forms of
EDMD share the same clinical features of early joint contractures, selective
muscle wasting and cardiac conduction defects, and they are both extremely
variable in severity, suggesting that some specific function of the
emerin-lamin A/C complex is affected in both cases
(Morris, 2001
). Mutations in
lamin A/C are also responsible for one form of dilated cardiomyopathy (CMD1A)
(Fatkin et al., 1999
) and for a
limb-girdle muscular dystrophy with conduction defects (type1B)
(Muchir et al., 2000
). Because
these two diseases are closely related to EDMD phenotypically, their molecular
pathogenesis is likely to be similar, with variability caused by genetic
background (Morris, 2001
). In
three other diseases, the molecular effects of lamin A/C mutations might be
different, because their phenotypes are quite distinct from EDMD. These are
Dunnigan-type familial partial lipodystrophy (FPLD)
(Cao and Hegele, 2000
;
Shackleton et al., 2000
),
autosomal recessive Charcot-Marie-Tooth disorder type 2 (De Sandre-Giovannoli
et al., 2002) and inherited mandibuloacral dysplasia
(Novelli et al., 2002
). In
these cases, the mutations might cause gain or loss of specific lamin A/C
functions that do not involve the emerin interaction. In FPLD, for example,
binding to lamin A/C of a specific transcription factor in adipose tissue
might be affected (Lloyd et al.,
2002
).
Immunohistochemical analysis of wild-type mouse embryonic fibroblasts
(MEFs) showed emerin to be concentrated within the nuclear envelope, whereas
MEFs from lamin-A-knockout (lmna-/-) mice showed a
decrease in nuclear-envelope-associated emerin and a more general distribution
of emerin in the peripheral ER. Transfection of lmna-/-
MEFs with human lamin A cDNA corrected the localization of emerin to
the nuclear envelope. These results suggested that A-type lamin expression is
required for the correct localization of emerin
(Sullivan et al., 1999). In a
more recent study, lmna-/- MEFs were transfected with
three lamin A cDNAs containing pathogenic mis-sense mutations
(Raharjo et al., 2001
).
Wild-type lamin A and lamin A with a lipodystrophy mutation were able to
restore the nuclear envelope localization of emerin. However, two lamin A/C
mutants causing CMD1A and one causing EDMD were defective in relocating emerin
from the peripheral ER to the nuclear rim
(Raharjo et al., 2001
). A
transfection study with a wider range of lamin A/C mutants
(Östlund et al., 2001
)
showed that some EDMD and cardiomyopathy mutants caused the formation of
nuclear foci, or aggregates, and disrupted the assembly of endogenous lamins
in HeLa and mouse myoblast cell lines.
In the present study, we have advanced these earlier studies by transfecting lmna-/- MEFs with a much wider range of lamin A/C mutants, paying particular attention to the effects on the localization of emerin. As a result, we can now rationalize, for the first time, the biological effects of lamin A/C mutations with the known functions of different domains in the lamin A/C molecule. In the new model, EDMD mutations in the globular tail domain affect emerin interaction directly. Rod mutations affect assembly of the A-type lamina and we hypothesize that any effects on emerin localization at the nuclear rim result indirectly from this.
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Materials and Methods |
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Eukaryotic cells for transfection were grown in DMEM (Gibco) with 20%
decomplemented horse serum (Gibco), 2 mM L-glutamine and antibiotics. Cells
used were mouse embryonic fibroblasts from the lamin A null mouse (MEFs
lmna-/-) (Sullivan et
al., 1999), HeLa (human epithelial carcinoma cell line) and COS-7
(African green monkey kidney fibroblast cell line).
Transfection by electroporation was modified from the method of Espinos et
al. (Espinos et al., 2001).
Briefly, eukaryotic cells were trypsinized and centrifuged, and the cells
resuspended to approximately 2x106 cells ml-1 in
F10-Ham medium (Gibco). 200 µl cell suspension was mixed with 24 µg
plasmid (or 24 µg of each plasmid for co-transfections) and placed in an
electroporation cuvette with 2 mm gap (BioRad). Cells and plasmid were
electroporated at 1.5 kV [resistance = 200
; capacitance = 25 µF;
time constant (r)
0.8] using a BioRad Gene Pulser. Following
electroporation, the contents of the cuvette were mixed with 1.8 ml of
skeletal muscle cell growth medium (PromoCell, Heidelberg) supplemented with
10% decomplemented foetal bovine serum and plated on to four sterile glass
coverslips. Cell culture was continued for 48 hours to allow for adherence of
cells to the coverslips and expression of transfected plasmid. After the
incubation period, cells on coverslips were fixed in 50:50 acetone-methanol
for 5 minutes and stored at -80°C.
Immunohistochemistry
Coverslips were brought to room temperature and the cells washed four times
with casein buffer (0.1% casein in 154 mM NaCl, 10 mM Tris, pH 7.6). Double
labelling of cells was performed by first incubating cells on a coverslip with
50 µl primary polyclonal antisera for 1 hour at 37°C, washing four
times with casein buffer and then incubating with 50 µl of primary
monoclonal antibody (mAb) for 1 hour at 37°C. The anti-emerin antiserum
was prepared by immunization of a rabbit with a recombinant human emerin
fragment [amino acids 1-188 (Manilal et
al., 1996)], which is identical to that used by Raharjo et al.
(Raharjo et al., 2001
).
Primary antibodies, diluted in casein buffer, were as follows. Polyclonals:
1:100 rabbit anti-emerin; 5 µg ml-1 rabbit anti-FLAG (Sigma,
F7425); 1:100 rabbit anti-lamin-B1 (gift of L. Gerace)
(Schirmer et al., 2001
); 4
µg ml-1 goat anti-lamin-B (Santa Cruz Biotechnology, sc-6216).
Monoclonals: 1 µg ml-1 anti-LAP2 (Transduction Laboratories,
L74520); 20 µg ml-1 anti-FLAG M2 (Sigma, F3165); 1 µg
ml-1 anti-Xpress (Invitrogen); 1:3 monoclonal antibodies MANEM1 and
MANEM8 against emerin (Manilal et al.,
1996
). Following incubation with primary antibodies, cells were
washed four times with casein buffer. 50 µl per coverslip of secondary
antibody pairs (diluted in PBS containing 1% horse serum, 1% foetal bovine
serum and 0.1% bovine serum albumin) were then added. The secondary antibody
pairs were either 5 µg ml-1 goat anti-mouse ALEXA 546 and 5
µg ml-1 goat anti-rabbit ALEXA 488 (Molecular Probes, Eugene,
Oregon) or 20 µg ml-1 horse anti-mouse FITC (Vector Labs,
Burlingame, CA) and 50 µg ml-1 rabbit anti-goat TRITC (Sigma,
T6028). Secondary antibodies were incubated for 1 hour at 37°C. DAPI
(diamidino phenylindole; Sigma), which labels DNA, was added at 200 ng
ml-1 in PBS for the final 10 minutes of the incubation to
counterstain the nuclei. Cells were then washed four times in casein buffer
and mounted in Hydromount (BDH Merck). Cells were examined with a Nikon
Eclipse E600 epifluorescence microscope (Nikon UK, Kingston, Surrey, UK) with
a 60x objective (numerical aperture 1.40) and a BioRad MicroRadiance
2000 confocal scanning system attachment (BioRad, Hemel Hempstead, UK).
Sequential confocal scans were performed with an Argon 488 nm blue excitation
laser for green fluorescence from ALEXA 488 and FITC and with a helium/neon
543 nm green excitation laser for red fluorescence from ALEXA 546 and
TRITC.
The ability of transfected lamin A to relocate endogenous emerin to the nucleus of MEFs lmna-/- was used as an indicator of the degree of direct or indirect interaction of lamin A with emerin. Emerin interaction scoring was performed by two operators. The first operator selected a microscope field that contained between one and five strongly transfected cells and at least 20 non-transfected cells. The filter set was then changed and a second operator attempted to identify which of the cells in the field showed altered distribution of emerin. Altered distribution included a reduction in emerin from the peripheral ER or increased emerin in the nuclear membrane and nucleoplasm, or both. Cells were classed as either `correctly identified' or `incorrectly identified'. An alternative approach, in which the operator knew which cell was transfected and had to state whether emerin localization to the nucleus was increased or unaltered, was found to be too subjective, in our hands, to be reliable (see Results).
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Results |
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In lmna-/- MEFs, nearly all emerin is in the ER but
lamin A transfection resulted in relocalization to the nucleus
(Sullivan et al., 1999).
Raharjo et al. (Raharjo et al.,
2001
) showed that three EDMD/CMD mutations (L85R, N195K and L530P)
resulted in loss of ability to relocalize emerin to the nucleus, while the
lipodystrophy mutation (R482W) relocalized emerin as well as wild-type lamin
A. The main aim of the present study was to extend this work to a wider range
of lamin A mutants. Transfected MEFs (lmna-/-) were double
labelled with a rabbit polyclonal anti-emerin serum and an anti-tag mAb
specific for transfected lamin A. Fig.
1 shows that the antiserum is specific for emerin in MEF extracts.
Although emerin has homology with LAP2 and MAN1 proteins in their shared LEM
domain (reviewed in Morris,
2001
), previous studies have also shown that the antiserum does
not cross-react with LAP2 (Raharjo et al.,
2001
). A wide range of changes in the distribution of emerin was
observed after transfection with wild-type or mutant lamin A, ranging from
emerin remaining in the cytoplasmic ER
(Fig. 2B; cf. untransfected
Fig. 2A) to emerin being
completely relocalized with the transfected lamin A to the nucleus
(Fig. 2E). In between, however,
were cells with increased nuclear emerin but little apparent decrease in the
ER (Fig. 2C,D) and, conversely,
cells in which cytoplasmic emerin was decreased with little apparent nuclear
change (data not shown). In many cases, it was difficult to decide whether
transfection with lamin A mutants had affected the endogenous emerin or not.
Further study might show whether this variability is due to cell cycle
differences, mixed cell lineages in MEFs or lamin A expression level.
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|
Effect of pathogenic lamin A mutations on relocalization of emerin to
the nuclear rim and on formation of nuclear foci
To quantify the effects of mutations more objectively, we adopted a blind
method of assessment, the observer being shown fields of view containing one
or several transfected cells (strongly positive for tagged lamin A) plus a
four- to 20-fold excess of untransfected cells. The observer was told how many
transfected cells were in the field but could not see the lamin A tag
fluorescence. Cells correctly identified as transfectants from emerin
redistribution (into nucleus, out of cytoplasm or both) were scored as `hits'
(the random hit rate by chance alone would be less than 20%).
Cells transfected with wild-type lamin A in pcDNA4 plasmid were easy to
identify from the dramatic changes in emerin distribution and the hit rate of
95% reflects this (Table 1).
This hit rate was hardly affected by the R482Q lipodystrophy mutation,
confirming earlier results with the R482W mutation
(Raharjo et al., 2001). When
pSVK3 plasmids were used, it was more difficult to see changes in emerin
distribution and the hit rate for wild-type lamin A was only 72%. This might
be because the SV40 promoter in pSVK3 gives lower expression levels than the
CMV promoter in pcDNA4. The effects of the EDMD and CMD mutations, which were
all expressed in the pSVK3 plasmid, are also shown in
Table 1. The hit rate was
significantly reduced from 72% to 41-50% for six mutants, the L85R and N195K
studied previously (Raharjo et al.,
2001
) and four new mutants, R386K, R453W, W520S and R527P. These
mutations seriously impaired the ability to relocalize emerin to the nuclear
rim but did not appear to abolish it completely. A novel observation from this
study is the identification of two rod mutations, E358K and M371K, that cause
no loss of ability to relocate emerin
(Table 1). Both mutations are
near the C-terminal end of the rod domain. All five rod mutants therefore
cause visible defects either in lamin A assembly (E358K and M371K) or in
emerin capture (L85R) or in both (N195K and R386K). Because L85R specifically
affects lamin C assembly (Raharjo et al.,
2001
), we could say that all five rod mutations cause defective
assembly of the nuclear lamina.
Because most or all of these rod mutations cause lamin A to accumulate in intranuclear foci in about 10% of cells, we determined whether emerin colocalized in these foci. MEFs (lmna-/-) were transfected with each mutant lamin A construct and examined by indirect immunofluorescence using antibody against emerin (Fig. 3). The foci formed by four of the lamin A rod mutants were positive for emerin, N195K (Fig. 3A), E358K (Fig. 3B), M371K and R386K (data not shown). The fifth mutant, L85R, rarely produces lamin A foci, but these rare foci are also positive for emerin (Fig. 3C). Emerin staining was usually more intense in the larger foci (>1.5 µm) but was also present in smaller foci.
|
Three mutations in the globular tail domain (R453W, W520S and R527P)
produced abnormal foci rarely or not at all
(Table 1), but they did impair
emerin relocalization (Table
1). The three-dimensional structure of this domain suggests that
these mutations might disturb the overall structure of the domain and thus
affect emerin binding (Dhe-Paganon et al.,
2002; Krimm et al.,
2002
). The lipodystrophy R482Q mutation, by contrast, is on an
outlying surface and might not disrupt the domain folding
(Dhe-Paganon et al., 2002
;
Krimm et al., 2002
),
consistent with normal emerin relocalization after transfection
(Table 1). Large nucleoplasmic
foci were occasionally seen with R453W and R527P but emerin did not accumulate
at these foci (Fig. 3D,E). This
important difference from the rod mutants is consistent with the hypothesis
that tail mutations, unlike rod mutations, act primarily by directly blocking
emerin interaction.
Nuclear foci formed by lamin A rod mutants contain emerin
The fine detail of the emerin and lamin distribution in large foci was
examined by collecting z-series of 0.25 µm sections through the
nuclei. Fig. 4A shows the red
lamin A signal and the green emerin signal in one optical section of a MEF
(lmna-/-) transfected with N195K. The nuclear foci consist
of transfected lamin A at the periphery with emerin trapped inside. Because
the foci are very intensely stained by antibody, care was taken to avoid
saturation of the digital image and loss of resolution by using the `SetCol
LUT' facility of the confocal microscope, as recommended by the manufacturers.
In an xz section through one of the foci and the emerin is clearly
inside a ring of transfected lamin A (Fig.
4B). Similar observations were made with the large nuclear foci
formed by the other lamin A rod mutants. The large foci appear to occupy the
entire thickness of the nucleus (1.5-3.5 µm) and might be contiguous with
the upper and lower nuclear surfaces, although it is difficult to establish
this because the vertical resolution of the microscope is only 0.5-0.6 µm,
at best. Fig. 4C,D shows
controls showing a MEF (lmna-/-) transfected with N195K
lamin A but with pre-immune rabbit serum in place of the anti-emerin serum, to
confirm that the green fluorescence in Fig.
4A,B is authentic emerin. Two highly specific mAbs against emerin
(Manilal et al., 1996) were
also used to confirm the presence of emerin in nuclear foci of human HeLa
cells transfected with the N195K mutant lamin A
(Fig. 5). The mAb labelling
experiment could not be performed on MEFs because the mAbs do not recognize
mouse emerin. The emerin in the nuclear foci produced by lamin A rod mutants
was not reported in earlier studies
(Östlund et al., 2001
;
Raharjo et al., 2001
),
although the presence of endogenous A-type and B-type lamins was observed.
Part of the explanation for this might be that, in smaller foci that do not
traverse the whole nucleus (Fig.
4E-G), emerin tends to concentrate on the membrane side of the
foci (asterisks in Fig. 4E, and
compare the other two foci in Fig.
4E with Fig. 4F).
Fig. 4E-G shows three confocal
sections (top to bottom) of a nucleus in which one of the foci is associated
with the lower part of the nucleus (arrow in
Fig. 4G), whereas three other
foci (lower right of Fig. 4E)
are associated with the upper part of the nucleus. If this nucleus represents
an early stage in the development of foci (and this is purely hypothetical,
requiring time-lapse confirmation), we speculate that foci might grow from the
upper or lower lamina and attract emerin, causing it to invaginate into the
foci, possibly with its associated membrane.
Fig. 4 also shows that the foci
contain lamin B but very little LAP2, confirming previous observations
(Östlund et al., 2001
;
Raharjo et al., 2001
).
|
|
Transfected lamin A also colocalizes with abnormally distributed
lamin B at the nuclear rim
Cells with a `burst end' or `cage' appearance at one pole of their nucleus
occur frequently in lmna-/- MEFs stained for B-type lamins
(Sullivan et al., 1999).
Transfected lamin A colocalizes with the lamin B in these abnormal nuclei and
does not correct the unusual lamin B distribution
(Fig. 6). The colocalization
was independent of either the lamin A expression level or the mutation (data
not shown). This result is consistent with assembly of an A-type lamina upon a
preexisting B-type lamina, in which case the mutations in lamin A do not
appear to disrupt its assembly onto the B-type lamina.
|
Co-expression of wild-type lamin A reduces mislocalization of mutant
lamin A into nuclear foci
In autosomal dominant laminopathies, mutant lamin A from one allele is
invariably co-expressed with normal lamin A from the other allele in all cells
of affected patients. To mimic this heterozygous condition in disease, cells
[MEFs (lmna-/-) or COS-7] were co-transfected with both
wild-type (in pcDNA4 vector) and N195K mutant lamin A (in pSVK3 vector). The
transfected protein products were detected by their different fusion tags
(mouse anti-Xpress mAb and rabbit anti-FLAG polyclonal). High levels of
co-transfection (>50%) were obtained by optimizing the electroporation
conditions. In cotransfected cells, wild-type and mutant lamin A usually
co-localized at the nuclear rim and in the nuclear interior
(Fig. 7A). Of 400
co-transfected MEFs and COS cells examined, only two COS cells had large
nuclear foci and these contained both wild-type and mutant lamins
(Fig. 7B). Co-expression of the
wild-type lamin A clearly reduced mislocalization of N195K lamin A into
nuclear foci.
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![]() |
Discussion |
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In AD-EDMD/CMD1A, nearly all lamin A/C mis-sense mutations are found
between amino acids 35 and 386 or between amino acids 442 and 541 (i.e. within
the rod domain or the globular tail). No mis-sense mutations between amino
acids 387 and 441 have been reported. The results of the present study
(Fig. 8) are consistent with
the simple hypothesis that mutations in the rod domain cause defective lamin
assembly, whereas tail-domain mutations impair the interaction of lamin A with
emerin. Four out of five rod mutants of lamin A frequently produced abnormal
nuclear foci in transfected cells (Fig.
8); the exception, L85R, rarely produced foci, but transfection
with lamin C containing the L85R mutation has been shown to produce frequent
foci (Raharjo et al., 2001).
Frequent formation of abnormal foci is a likely symptom of defective lamina
assembly. In all five cases, we found that the foci contained emerin,
indicating that direct interaction with emerin was not impaired. Two of the
three AD-EDMD tail mutants produced foci rarely, and these rare foci were
negative for emerin, consistent with direct impairment of emerin interaction.
The ability of lamin A to capture emerin at the nuclear rim when expressed in
lmna-/- MEFs clearly requires the ability to bind emerin
directly and, indeed, all three tail mutants showed impaired ability to
capture emerin at the nuclear rim. Two of the five rod mutants, E358K and
M371K, were able to capture emerin as well as the control. Three rod mutants,
however, showed reduced capture of emerin at the nuclear rim, although some
capture of emerin still occurred. This suggests that there might be additional
structural requirements for emerin capture by the lamina. Mutations causing
structural changes that prevent a close approach of the lamin A molecule to
the nuclear membrane, for example, might indirectly prevent emerin capture. A
three-dimensional structure of the rod region of lamin A might throw light on
the different emerin capture ability of rod mutations, in the same way that
the three-dimensional structure of the lamin A tail domain has elucidated the
special properties of the lipodystrophy mutations at R482
(Dhe-Paganon et al., 2002
;
Krimm et al., 2002
). Some rod
mutations cause changes in lamin C that are not evident in lamin A
(Raharjo et al., 2001
) so, if
we view the lamina as a complex of lamins A, B and C, there might be defects
in emerin capture by rod mutants in vivo that are not evident with transfected
lamin A alone. It might be possible to use quantitative in vitro studies of
emerin interaction with mutant lamins [e.g. BIAcore
(Holt et al., 2001a
) or
pull-downs (Lee et al., 2001
)]
to distinguish direct effects of lamin A mutations on emerin binding from
their indirect effects on emerin relocalization.
|
A crucial observation in the present study is that nuclear foci formed by
lamin A rod mutants contain emerin, whereas foci formed by tail mutants do
not. This differs somewhat from our earlier studies, which found no emerin in
foci in mouse C2C12 myoblasts using the same rabbit anti-emerin serum
(Östlund et al., 2001).
Our monoclonal antibodies do not recognize mouse emerin. However, by
transfecting HeLa cells, we could confirm the presence of endogenous human
emerin in foci formed by the N195K mutant using specific mAbs against two
different emerin epitopes (Fig.
5), although emerin staining was sometimes less intense than in
MEFs (Fig. 5A). We are
currently investigating differences between cell lines and
culture/transfection conditions used in this and our earlier study that might
affect emerin localization or alter the pathway of nuclear focus formation.
Endogenous lamin A/C in normal cells might reduce the amount of emerin in
nuclear foci compared with lmna-/- MEFs. Time is another
factor that might influence the number and composition of foci, all our
studies having been done 48 hours after transfection.
The current view of lamina assembly is a sequential one, with B-type lamins
followed by lamin A and, finally, lamin C
(Raharjo et al., 2001;
Vaughan et al., 2001
). Our
studies are consistent with this view. Thus, nuclei with B-type lamins absent
from one pole, or abnormally distributed at one pole, occur in
lmna-/- MEFs (Sullivan
et al., 1999
) and transfected lamin A often followed a similar
pattern. Lamin A enters nuclei through nuclear pores, whereas emerin in the ER
enters around nuclear pores by diffusion and is usually captured at the
nuclear rim by lamin A/C and/or other proteins
(Östlund et al., 1999
).
Emerin and lamins, however, are also detected by antibodies in the nucleoplasm
of cultured cells, often in discrete structures that might be invaginations of
the nuclear membrane. The relationship between these small structures and the
much larger mutant foci is not clear but it is possible that the latter form
by a similar mechanism to the former or that the former act as seeds for the
growth of the latter. Invaginations of the nuclear membrane would be one way
to explain how emerin with its hydrophobic transmembrane sequence can be found
in the interior of the nuclear foci. In
Fig. 4E-G, there is a clear
indication that foci might begin to develop at the lamina, attracting emerin
initially at the nuclear rim and eventually as invaginations into the interior
of the foci. It is not clear whether such structures are really an early stage
in the development of foci, but it might be possible to resolve this question
by time-lapse studies of transfected GFP/lamin-A in living cells. More
detailed analysis of the lipid and protein composition of nuclear foci and
immunoelectron microscopy might also help us to understand how these
structures develop and how they relate to normal lamina assembly.
A simplified conclusion about AD-EDMD/CMD1A phenotypes suggested by this
study is that they might be caused by defective formation of the
emerin/lamin-A/C complex. Some function of the complex that has yet to be
identified but is of particular importance in skeletal and cardiac muscle, in
which the diseases are manifested, is presumably affected
(Morris, 2001). It must be
remembered also that lamin A mutations reduce, rather than completely prevent,
emerin relocalization and that abnormal nuclear foci are only formed in a
small proportion of transfected cells. Furthermore, transfection of
lmna-/- MEFs mimics the homozygous situation, whereas
autosomal dominant diseases are manifested in heterozygotes. Our
co-transfection experiment of N195K and wild-type lamin A attempted to mimic
the heterozygous state and abnormalities were rare. This is perfectly
consistent with the fact that most tissues are unaffected in EDMD and CMD1A
and that the characteristic cardiac abnormalities appear very late in human
development (at least 10-20 years after birth, in most cases)
(Morris, 2001
). Mandibuloacral
dysplasia is caused by a homozygous R527H mutation, quite similar to the R527P
EDMD mutation studied here. The clinical features of this disease are quite
unlike EDMD, affecting a wider range of tissues, although they often include
FPLD features (Novelli et al.,
2002
). The good correlation between defective emerin/lamin-A
complex formation and a range of EDMD/CMD1A mutations in lamin A/C strongly
suggests that the explanation of the cardiac and skeletal muscle defects in
EDMD/CMD1A lies in altered emerin-lamina interactions.
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References |
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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.[Medline]
Bonne, G., DiBarletta, M. R., Varnous, S., Becane, 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.[CrossRef][Medline]
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.[CrossRef][Medline]
Des Sandre-Giovannoli, A., Chaouch, M., Kozlov, S., Vallat, J. M., Tazir, M., Kassouri, N., Szepetowski, P., Hammadouche, T., Vandenberghe, A., Stewart, C. L. et al. (2002). Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse. Am. J. Hum. Genet. 70,726 -736.[CrossRef][Medline]
Dhe-Paganon, S., Werner, E. D., Chi, Y. I. and Shoelson, S.
E. (2002). Structure of the globular tail of nuclear lamin.
J. Biol. Chem. 277,17381
-17384.
Espinos, E., Liu, J. H., Bader, C. R. and Bernheim, L. (2001). Efficient nonviral DNA-mediated gene transfer to human primary myoblasts using electroporation. Neuromusc. Disord. 11,341 -349.[CrossRef][Medline]
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.
Fairley, E. A., Kendrick-Jones, J. and Ellis, J. A.
(1999). The Emery-Dreifuss muscular dystrophy phenotype arises
from aberrant targeting and binding of emerin at the inner nuclear membrane.
J. Cell Sci. 112,2571
-2582.
Gruenbaum, Y., Wilson, K. L., Harel, A., Goldberg, M. and Cohen, M. (2000). Review: Nuclear laminsstructural proteins with fundamental functions. J. Struct. Biol. 129,313 -323.[CrossRef][Medline]
Holt, I., Clements, L., Manilal, S., Brown, S. C. and Morris, G. E. (2001a). 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.[CrossRef][Medline]
Holt, I., Clements, L., Manilal, S. and Morris, G. E. (2001b). How does a g993t mutation in the emerin gene cause Emery-Dreifuss muscular dystrophy? Biochem. Biophys. Res. Commun. 287,1129 -1133.[CrossRef][Medline]
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.
Krimm, I., Östlund, C., Gilquin, B., Couprie, J., Hossenlopp, P., Mornon, J. P., Bonne, G., Courvalin, J. C., Worman, H. J. and Zinn-Justin, S. (2002). The Ig-like structure of the C-terminal domain of lamin A/C, mutated in muscular dystrophies, cardiomyopathy and lipodystrophy. Structure 10,811 -823.[CrossRef][Medline]
Lee, K. K., Haraguchi, T., Lee, R. S., Koujin, T., Hiraoka, Y. and Wilson, K. L. (2001). Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF. J. Cell Sci. 114,4567 -4573.[Medline]
Lin, F. and Worman, H. J. (1993). Structural
organization of the human gene encoding nuclear lamin A and nuclear lamin C.
J. Biol. Chem. 268,16321
-16326.
Lloyd, D. J., Trembath, R. C. and Shackleton, S.
(2002). A novel interaction between lamin A and SREBP1:
implications for partial lipodystrophy and other laminopathies.
Hum. Mol. Genet. 11,769
-777.
Manilal, S., Nguyen, T. M., Sewry, C. and Morris, G. E.
(1996). The Emery-Dreifuss muscular dystrophy protein, emerin, is
a nuclear membrane protein. Hum. Mol. Genet.
5, 801-808.
Morris, G. E. (2001). The role of the nuclear envelope in Emery-Dreifuss muscular dystrophy. Trends Mol. Med. 7,572 -577.[CrossRef][Medline]
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., Tsukuhara, T. and Arahata, K. (1996). Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy. Nat. Genet. 12,254 -259.[Medline]
Novelli, G., Muchir, A., Sangiuolo, F., Helbling-Leclerc, A., D'Apice, M. R., Massart, C., Capon, F., Sbraccia, P., Federici, M., Lauro, R. et al. (2002). Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C. Am. J. Hum. Genet. 71,426 -431.[CrossRef][Medline]
Östlund, C., Ellenberg, J., Hallberg, E.,
Lippincott-Schwartz, J. and Worman, H. J. (1999).
Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy
protein. J. Cell Sci.
112,1709
-1719.
Östlund, C., Bonne, G., Schwartz, K. and Worman, H. J. (2001). Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy. J. Cell Sci. 114,4435 -4445.[Medline]
Raharjo, W. H., Enarson, P., Sullivan, T., Stewart, C. L. and Burke, B. (2001). Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy. J. Cell Sci. 114,4447 -4457.[Medline]
Schirmer, E. C., Guan, T. and Gerace, L.
(2001). Involvement of the lamin rod domain in heterotypic lamin
interactions important for nuclear organization. J. Cell
Biol. 153,479
-489.
Shackleton, S., Lloyd, D. J., Jackson, S. N., 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.[CrossRef][Medline]
Stuurman, N., Heins, S. and Aebi, U. (1998). Nuclear lamins: their structure, assembly and interactions. J. Struct. Biol. 122,42 -66.[CrossRef][Medline]
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.
Tsuchiya, Y., Hase, A., Ogawa, M., Yorifuji, H. and Arahata,
K. (1999). Distinct regions specify the nuclear membrane
targeting of emerin, the responsible protein for Emery-Dreifuss muscular
dystrophy. Eur. J. Biochem.
259,859
-865.
Vaughan, A., Alvarez-Reyes, M., Bridger, J. M., Broers, J. L., Ramaekers, F. C., Wehnert, M., Morris, G. E., Whitfield, W. G. F. and Hutchison, C. J. (2001). Both emerin and lamin C depend on lamin A for localization at the nuclear envelope. J. Cell Sci. 114,2577 -2590.[Medline]
Wilson, K. L., Zastrow, M. S. and Lee, K. K. (2001). Lamins and disease: insights into nuclear infrastructure. Cell 104,647 -650.[Medline]