1
Department of Biological Sciences, The University of Durham, South Road,
Durham, DH1 3LE, UK
2
Department of Biology and Biochemistry, The University of Brunel, Middlesex,
UB8 3PH, UK
3
Department of Molecular Cell Biology and Genetics, University Maastricht, PO
Box 616, Maastricht, 6200 MD, The Netherlands
4
Ernst-Moritz-Arndt-University, Institute of Human Genetics, Fleischmannstrasse
42-44, 17487 Greifswald, Germany
5
MRIC, North East Wales Institute, Plas Coch, Mold Road, Wrexham, LL11 2AW,
Wales
6
Department of Biological Sciences, The University of Dundee, Dundee, DD1 4HN,
Scotland
*
Author for correspondence (e-mail:
c.j.hutchison{at}durham.ac.uk
)
Accepted April 14, 2001
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SUMMARY |
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Key words: Lamins, Emerin, Nuclear envelope, Nuclear lamina, Emery-Dreifuss muscular dystrophy
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INTRODUCTION |
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The composition of the lamina varies according to cell type and stage of
differentiation. Two broad classes of lamins are expressed in vertebrates and
these are referred to as A-type and B-type. These lamins vary in primary
sequence and biochemical properties as well as in their expression patterns.
B-type lamins are expressed in all germ cells and somatic cells although
different B-type lamins are expressed in each (reviewed by Vaughan et al.,
2000). Lamins B1
and B2 are the major B-type lamins expressed in mammalian somatic
cells and these proteins are the products of separate genes (reviewed by Gant
and Wilson, 1997
). Lamins A,
C, C2 and A
10 comprise the A-type lamins and all are alternatively
spliced products of a single gene. Lamin A and C are the most abundant A-type
lamins and differ in that lamin C lacks a 90 amino acid C-terminal extension
possessed by lamin A but has five unique amino acids at its C-terminus
(reviewed by Quinlan et al.,
1995
). Both lamin A and lamin
C are expressed only in differentiated cells and during mouse development
appear at the time of organogenesis (Rober et al.,
1989
); however, they are
dispensable for development since a lamin A/C knockout mouse survives to
adulthood (Sullivan et al.,
1999
).
Recently, a number of different autosomal dominant diseases have been shown
to be caused by mutations in the gene encoding lamins A and C (reviewed by
Flier, 2000). These diseases include an autosomal dominant form of
Emery-Dreifuss muscular dystrophy (AD EDMD) (Bonne et al.,
1999; Raffaele et al.,
2000
), dilated cardiomyopathy
with conduction system disease (Fatkin et al.,
1999
) and a Dunnigan-type
familial partial lipodystrophy (Shackleton et al.,
2000
; Cao and Hegele,
2000
). It is currently unclear
why these very different diseases arise through mutations in the same
proteins. However, since a majority of disease phenotypes are caused by
missense mutations that occur in different parts of the proteins (Flier, 2000;
Raffaele et al., 2000
), one
possibility is that these lamins interact with a number of different nuclear
proteins and that different mutations affect different lamin interactions.
Emerin was first identified by positional cloning of a gene on chromosome
Xq28 that is mutated in individuals with X-EDMD. The emerin gene encodes a 254
amino acid type II integral membrane protein (Bione et al.,
1994). Structural analysis
predicts that emerin contains a transmembrane region at the C-terminus and a
large hydrophilic N-terminal domain with multiple putative phosphorylation
sites (Bione et al., 1994
). In
addition, emerin contains the LEM domain signature common to a number of
integral membrane proteins of the inner nuclear membrane (reviewed by
Hutchison et al., 2001
).
Emerin is a serine rich protein that migrates as a 34 kDa band on SDS-PAGE. It
is principally located at the INM in almost every tissue (Nagano et al.,
1996
: Manilal et al.,
1996
). In cardiac muscle,
emerin has also been located at intercalated disks (Cartegni et al.,
1997
), although this finding
was not substantiated by later studies (Manilal et al.,
1999
). In skeletal muscle
cells grown in culture, a fraction of emerin is located in the endoplasmic
reticulum (ER) (Fairley et al.,
1999
). The majority of lesions
in the emerin gene that cause EDMD are null mutations (Nagano et al.,
1996
, Manilal et al.,
1997
; Manilal et al.,
1998
; Mora et al.,
1997
; Ellis et al.,
1998
; Yates et al.,
1999
). However, some mutations
result in the production of modified forms of emerin (Manilal et al.,
1997
; Mora et al.,
1997
; Ellis et al.,
1998
; Wulff et al.,
1997
; Yates et al.,
1999
). These mutations occur
throughout the protein with no obvious hot-spots. Interestingly, some
mutations in the N-terminal `nucleoplasmic' domain cause mis-localisation of
emerin either to cytoplasmic membranes or to the nucleoplasm (Fairley et al.,
1999
; Ellis et al.,
1998
). This finding is
consistent with the observation that sequences within the N-terminal
nucleoplasmic domain are necessary and sufficient to target emerin to the INM
(Östlund et al.,
1999
).
A possible link between X-EDMD and AD EDMD is that A-type lamins form structural associations with emerin at the INM. To test this hypothesis we have investigated laminemerin interactions in vivo and in vitro. Our data suggest that the organisation of both lamin C in the lamina and of emerin at the INM is dependent upon lamin A.
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MATERIALS AND METHODS |
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Expression and immunoprecipitation of recombinant lamins, NUP-153 and
emerin
Plasmid pWW1 hLamin B1 was constructed by subcloning an Nco1-BamH1
fragment of human lamin B1 cDNA (Pollard et al.,
1990) into Nco1-BamH1 cut T7
expression vector pWW1 (Whitfield et al.,
1990
). Cloning of cDNA
encoding the first 188 amino acids of emerin from total human skeletal muscle
cDNA into the expression plasmid pMW172, and full length emerin cDNA into
plasmid pET17xb have already been described (Manilal et al.,
1996
; Manilal et al.,
1999
). Plasmids pET-1 hLamin A
and pET-1 hLamin C are described elsewhere (Moir et al.,
1990
; Moir et al.,
1991
). Nup-153 cDNA (gift from
Brian Burke, University of Calgary, Canada) was cloned into pRSET-A using
XhoI and PvuII. Recombinant lamins A, C, B1,
Nup-153, and the N-terminal 188 amino acids of emerin were expressed using the
TNTr Quick Coupled Transcription/Translation System (Promega) under
recommended conditions. Reticulolysates containing expressed lamins and emerin
were pooled and incubated at 4°C overnight. Pefablocr,
leupeptin, pepstatin and aprotinin (Boehringer Mannheim) were added to a final
concentration of 1 mM each. Reaction mixtures were pre-cleared using
paramagnetic Dynabeadsr M-280 (DYNAL). Lamins A and C were
recovered using mAbs Jol2, JoL4 or Jol5 (or the lamin A-specific Jol4)
conjugated to Dynabeadsr according to a method previously described
(Jenkins et al., 1993
).
Likewise, emerin was immunoprecipitated using mAbs MANEM3 and -5 (Manilal et
al., 1996
), and NUP-153 using
mAb 414.
Cell culture and preparation of SW13/20
Human cervix carcinoma (HeLa) and Human adrenal cortex carcinoma (SW13)
cells (gift from H. Herrmann, Heidelberg) were routinely maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum (FCS, v/v) and 1% penicillin/streptomycin (v/v) at 37°C in a
humidified atmosphere containing 5% CO2. Lymphoblastoid, lymphoma
and lung carcinoma cells were maintained in RPMI 1640 (GIBCO BRL) containing
non-essential amino acids, 15% FCS (v/v) and 1% penicillin/streptomycin. SW13
cells were transfected with pCDNA-EGFP-HLA, a plasmid containing human lamin A
fused to EGFP (gift from L. Karnitz, Mayo Clinic), using LipofectinTM
(GIBCO BRL), 15 µg of DNA per 2.5x105 cells and conditions
recommended for Lipofectin transfections. Stable clones were selected
using 300 µg/ml G418 (Calbiochem).
Cell fractionation
Cells were scraped from a 75 cm2 flask using a rubber policeman
then washed twice with phosphate-buffered saline (PBS) at 4°C. Cell lysis
occurred after incubation in cytoskeletal buffer (10 mM PIPES, pH 6.8, 300 mM
sucrose, 100 mM NaCl, 3 mM MgCl2 and 1 mM EGTA) containing 0.5%
Triton X-100 at 4°C for 5 minutes. Chromatin was removed by digestion with
200 units/ml RNase-free DNase in digestion buffer (10 mM PIPES, pH 6.8, 300 mM
sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 units/ml RNase
inhibitor (Boehringer Mannheim) containing 0.5% Triton X-100 at 30°C for
30 minutes. Nuclei were washed with extraction buffer (10 mM PIPES, pH 6.8,
250 mM ammonium sulphate, 300 mM sucrose, 3 mM MgCl2, and 1 mM
EGTA) twice for 5 minutes each time at 4°C. Nuclear matrix fractions were
solubilised in 8 M urea. Nuclei were pelleted after each step by
centrifugation at 1000 rpm for 5 minutes within a pre-chilled microcentrifuge.
Pefablocr (2 mM), leupeptin (10 µM), pepstatin (1 µM), and
aprotinin (0.5 µM) were included throughout.
Construction and expression of GST/GFP fusion proteins
The construction of plasmid pGEX-XLaminB12+ is described elsewhere
(Ellis et al., 1997
). This
construct expresses amino acid residues 34-420 from Xenopus lamin
B1 as a GST fusion protein. GST-XLaminB1
2+ fusion protein
was expressed then purified from Escherichia coli BL21 cells using a
method described previously (Ellis et al.,
1997
). The
pEGFP-XLaminB1
2+ plasmid was constructed by subcloning the
SalI-NotI insert of pGEX-XLaminB1
2+ into pEGFP-C1
(Clontech) cut with SalI and Bsp120L. Plasmid pEGFP-emerin was
constructed by sub-cloning full length emerin from pET17xb into pEGFP-C1
(Clontech) cut with BamHI and HindIII. Plasmids pS65T-lamC
and pS65T-lamA have been described previously (Broers et al.,
1997
). HeLa and SW13 cells
grown on coverslips to 20% confluence were transfected with relevant plasmids
(1-2 µg/coverslip) using the calcium-phosphate method (Graham and van der
Eb, 1973
). The cells were
grown overnight and media replaced, with expression allowed to proceed for a
further 36-48 hours.
Immunofluorescence microscopy
Lymphoblast/lymphoma cells were centrifuged onto coverslips at 300 rpm for
3 minutes prior to fixation. Cells grown on coverslips were washed twice in
PBS and fixed in ice-cold 1:1 (v/v) methanol-acetone for 10 minutes. Nuclear
matrices were prepared as described elsewhere (Dyer et al.,
1997). Coverslips were washed
3 times, with 0.5% newborn calf serum (NCS, v/v) in PBS. Hybridoma cell
culture supernatants (undiluted) containing mAbs Jol2, Jol4 and LN43 (gift
from Birgit Lane, Dundee) were used to stain nuclear lamins A/C, A and
B2 respectively. Likewise, supernatants of mAbs MANEM3 and -5 were
used to immunodetect emerin, and LAP17 (gift from Roland Foisner, Vienna) to
detect LAP 2ß. Goat anti-lamin B (Santa Cruz Biotechnology) and rabbit
anti-ER (gift from Daniel Louvard, Paris) and anti-calreticulin (Calbiochem)
were used at recommended dilutions to stain lamin B1 and ER,
respectively. Affinity purified rabbit anti-lamin C was used at a dilution of
1/50, as described previously (Venables et al.,
2000
). Primary antibodies were
added for 1 hour at room temperature. Coverslips were washed three times with
PBS, then incubated with appropriate Rhodamine (TRITC)-conjugated affinipure
secondary antibodies (donkey anti-goat, goat anti-mouse, and goat anti-rabbit;
Jackson Immunoresearch) for a further hour at room temperature. After several
washes in PBS, coverslips were mounted face down in Mowiol (Calbiochem)
containing 1 µg/ml DAPI. Immunostained samples were viewed using a Zeiss
axiovert 10 microscope with a plan-APOCHROMAT 63x/1.40-oil immersion
lens and equipped with a Digital Pixel Instruments 12-bit CCD camera. Images
were captured using IP Lab Scientific Imaging Software (Scanalytics).
Additionally, a Zeiss LSM 410 confocal laser scanning microscope was used
(63x/1.40 oil immersion lens) for imaging of emerin and ER within SW13
cells.
Gel electrophoresis and immunoblotting
Recombinant proteins and nuclear matrix fractions were resolved on 10%
SDS-PAGE and transferred to nitrocellulose according to established protocols
(Jenkins et al., 1993).
Nitrocellulose membranes were washed with blocking buffer (5% milk powder
(w/v), 0.1% Tween-20 in PBS) for 1 hour at room temperature. Undiluted cell
culture supernatants containing mAbs Jol2, Jol4 and LN43 were used to detect
lamins A/C, A and B2, respectively. Goat anti-lamin B1
(Santa Cruz Biotechnology) was used at 1/200. Affinity purified rabbit
anti-lamin C was used at a dilution of 1/100. All primary antibodies were
incubated with membranes for 30 minutes at room temperature. Membranes were
rinsed with blocking buffer several times then incubated with appropriate
HRP-conjugated secondary antibodies (rabbit anti-mouse, DAKO; goat
anti-rabbit, BIO-RAD; donkey anti-goat, Jackson Immunoresearch) for 30 minutes
at room temperature. ECL reagents (Amersham Life Science) were used for the
immunological detection of proteins after membranes were rinsed in PBS.
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RESULTS |
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Abnormal targeting of emerin in a cell line SW13 with altered
expression and organisation of lamins A and C
To investigate lamin-emerin interactions in vivo we compared the
distribution of emerin and the lamin B binding protein LAP2ß in two cell
lines with very different lamin complements. The expression of the different
lamins in HeLa cells was investigated by immunoblotting with specific
antibodies against lamins A/C, A, C, B1 and B2. All four
lamins were expressed at high levels (Fig.
2A). A similar investigation was performed on the adrenal cortex
carcinoma cell line SW13 (Paulin-Levasseur et al.,
1989). In this cell line lamin
A was expressed at greatly reduced levels (compared with HeLa) and was
undetectable with some antibodies (JoL4). By contrast, lamins B1
and B2 were expressed at similar levels in SW13 and HeLa, whereas
lamin C was expressed at reduced but readily detectable levels
(Fig. 2B). Next, the
distribution of lamins A and C were compared in HeLa and SW13 cells by
immunofluorescence. Using anti-lamin-C-specific anti-sera, lamin C was
detected predominantly in the nuclear rim in HeLa cells
(Fig. 2C). In SW13 cells lamin
C was detected at low levels in the nuclear rim but at high levels in the
nucleolus (Fig. 2D). (Note that
the nucleolar distribution of lamin C was confirmed by co-staining with Ki67
antibody and in all subsequent experiments co-staining with Ki67 was used to
confirm nucleolar localisation of lamin C in the absence of lamin A; data not
shown.) Using the lamin-A-specific mAb JoL4, lamin A was detected in nuclear
speckles and at the nuclear rim in HeLa cells but was undetectable in SW13
cells (Fig. 2C,D). Lamins
B1 and B2 were both localised exclusively at the nuclear
rim in HeLa and SW13 cells (data not shown). These data suggested that, in
SW13 cells, lamin A was expressed at very low levels (compared with HeLa),
whereas a significant fraction of lamin C was mis-localised to the
nucleolus.
|
Next we compared the distribution of LAP2ß and emerin in HeLa and SW13
cells. In each cell line, LAP2ß was located at the NE
(Fig. 2E,F). In HeLa, emerin
was detected as a distinct nuclear rim stain indicating its location at the NE
(Fig. 2E). By contrast, in SW13
cells, emerin was located both at the NE and within the cytoplasm. To
investigate the site of emerin localisation within the cytoplasm, SW13 cells
were co-stained with MANEM3 and antibodies against calreticulin. As a control,
SW13 cells were co-stained with MANEM3 and the mitochondrial marker p32
(Matthews and Russell, 1998).
Confocal microscopy revealed that the cytoplasmic fraction of emerin
co-localised exclusively with calreticulin
(Fig. 3) but did not
co-localise with p32 (data not shown). These data suggest that in SW13 cells a
significant fraction of emerin resides in the ER.
|
To further investigate the behaviour of emerin in HeLa and SW13 cells, cDNA encoding full-length human emerin was sub-cloned into pEGFP (pEGFP-emerin) and expressed in each cell line following transient transfection. As expected, in HeLa cells GFP-emerin was localised exclusively in the NE (Fig. 4E). When the same construct was transfected into SW13 cells the majority of GFP-emerin was localised in the cytoplasm, where it accumulated in large granular structures (Fig. 4A-D). Co-staining with calreticulin revealed that although the granular structures were distinct from the majority of the ER they did contain ER proteins (Fig. 4D). Transfected cells were also co-stained with specific antibodies against lamins C, B1 or B2. Surprisingly, a readily detectable fraction of lamin C (Fig. 4A), but no lamin B1 (Fig. 4B) or B2 (Fig. 4C), relocated from the nucleus to co-distribute with GFP-emerin in some of the cytoplasmic granules.
|
A number of conclusions can be drawn from these data. First, the absence of lamin A from the NE correlates with a significant fraction of lamin C being mis-localised to the nucleolus and a fraction of emerin residing in the ER. Second, when GFP-emerin is overexpressed in SW13, it forms cytoplasmic aggregates, probably within an ER sub-domain that traps some lamin C but no lamins B1 or B2. Taken together, these data suggest that emerin interacts with lamins A and/or C in vivo.
Emerin is localised in the ER in a range of cell lines that display
abnormal levels of expression and distributions of lamins A and C
Birkitt's lymphoma cell lines
A comparison of emerin and lamin C localisation in HeLa and SW13 cell lines
indicated that lamin A might organise both proteins at the NE. To investigate
the generality of this phenomenon we compared the distribution of emerin and
LAP2ß in other human cell lines that are deficient for lamin A/C
expression or which display altered distributions of lamin C. A Burkitt's
lymphoma cell line (Ramos) did not express lamin A and expressed reduced
levels of lamin C. Moreover, in immunofluorescence experiments lamin C was
localised exclusively in the nucleolus. The levels of expression and
distribution of lamins B1 and B2 appeared normal
(Table 2). LAP2ß was
localised exclusively at the NE in Ramos cells. By contrast, emerin was
distributed exclusively in the ER (Table
2).
|
Small cell lung carcinomas
Previous investigations have shown that small cell lung carcinomas express
greatly reduced levels of lamins A and C compared with non-small-cell lung
carcinomas (Kaufman et al.,
1991). Therefore, we compared
the distribution of LAP2ß and emerin in non-small-cell and small cell
lung carcinoma cell lines. The non-small-cell lung carcinoma line used in this
investigation (NCl-H125) expressed high levels of lamins A, C, B1
and B2 that were distributed mainly at the NE
(Table 2). In this cell line,
LAP2ß and emerin were both located exclusively at the NE. In common with
most examples (Broers et al.,
1993
), the small cell lung
carcinoma cell line used in the study (NL-SCSC2) did not express lamin A or
lamin C but expressed high levels of lamins B1 and B2.
LAP2ß was localised at the NE in the small cell lung carcinoma, whereas
emerin was localised in the ER (Table
2).
EDMD cell lines
Finally, we compared emerin localisation in EBV-transformed lymphoblastoid
cell lines (LCL) obtained from a control donor and from a patient with
autosomal dominant EDMD (AD EDMD). The control LCL expressed relatively low
levels of lamins A and C but both were located in the nuclear rim
(Table 2;
Fig. 5A). Lamin B1
and B2 expression and distribution
(Table 2) in this cell line
appeared normal. Although some emerin was located in the ER (presumably as a
consequence of the relatively low-level expression of lamins A/C), the
majority was located at the NE (Fig.
5A). The AD EDMD cell line was obtained from a patient having a
missense mutation in the lamin A/C tail (T528K). In this patient expression of
lamins A and C was variable, with some cells expressing apparently normal
levels of the proteins at the NE, whereas in other cells both proteins were
absent (Table 2;
Fig. 5B). Again, lamins
B1 and B2 appeared normal both in terms of level of
expression and distribution (Table
2). The variable level of A-type lamin expression in the AD EDMD
patient permitted a side-by-side comparison of emerin distribution in those
cells expressing lamins A/C and in those that do not. Cells in which lamins A
and C were expressed and localised at the NE (lamin C is shown in
Fig. 5B) also contained
significant quantities of emerin at the NE. By contrast, in adjacent cells
that did not express lamins A and C, emerin was located exclusively in the
ER/NE (Fig. 5B; the ER
localisation was confirmed by costaining with anti-calreticulin (not shown)).
Therefore, in a range of cell lines, localisation of emerin at the NE
correlates with expression and localisation of lamins A and C at the NE. In
addition, lamin C localisation at the NE may also depend upon lamin A.
|
Stable and transient expression of GFP-lamin A in SW13 cells causes
the relocalisation of lamin C and emerin to the NE
Our immunofluorescence data have revealed a strong correlation between
abnormal expression and distribution of lamins A and C and localisation of
emerin in the ER. To investigate whether this represented a causal
relationship we carried out transient and stable transfection experiments on
SW13 with GFP-lamins. Initially, we selected a number of cell lines that had
been stably transfected with GFP-lamin A. One such cell line (SW13/20) is
shown here, since it is representative. Stable transfection with GFP-lamin A
in SW13 resulted in levels of expression of the fusion protein that were
approximately fourfold higher than levels of expression of lamin C (not
shown). Importantly, all A-type lamins (including endogenous lamin C) were
located predominantly in the NE rather than in the nucleolus
(Fig. 6A). In SW13/20 cells
both LAP2ß (Fig. 6C) and
emerin (Fig. 6B) were localised
exclusively at the NE. Next we performed transient transfection experiments
with GFP-lamins A, B1 and C. Typically in these experiments 15% of
cells expressed the GFP-fusion protein. When SW13 was transfected with
GFP-lamin A, the GFP-fusion protein was localised at the NE
(Fig. 7A,B). Importantly, the
endogenous lamin C became predominantly co-localised with the GFP rather than
in the nucleolus (Fig. 7B).
Emerin was also localised at the NE in cells expressing GFP-lamin A but not in
surrounding untransfected cells (Fig.
7A, arrowhead; the arrow identifies a mitotic cell lying adjacent
to the cell expressing GFP-lamin A; the cytoplasmic emerin observed next to
the transfected cell is within the mitotic cell). GFP-lamin B1 was
also localised to the NE in transfected SW13 cells but failed to cause the
relocalisation of endogenous lamin C or emerin to the NE (data not shown).
Finally, when SW13 was transfected with GFP-lamin C, the fusion protein
accumulated in nucleoplasmic granules (Fig.
7C,D; note that when GFP-lamin C is transiently expressed in HeLa
cells it localises exclusively to the NE (data not shown)). These granules did
not influence the distribution of endogenous lamins (endogenous lamin C
remained in the nucleolus; Fig.
7C) and emerin remained in the cytoplasm
(Fig. 7D). As with previous
experiments the nucleolar distribution of lamin C and the ER localisation of
emerin was confirmed by co-localisation with Ki67 and calreticulin,
respectively (data not shown). These data strongly support the view that
localisation of emerin to the NE depends upon the presence of lamins A and C
within the lamina. Furthermore, the data also suggest that the presence of
lamin A within the lamina is necessary (but possibly not sufficient) for lamin
C localisation to the NE.
|
|
Dominant negative mutants of lamin B1 selectively
eliminate lamins A and C from the lamina and cause emerin to accumulate in
cytoplasmic granules
To further investigate the relationship between emerin localisation at the
NE and the presence of lamins A and C in the lamina, we used dominant negative
mutants of lamin B1 to specifically disrupt A-type lamins. We have
previously described the creation of a dominant negative mutant of lamin
B1 that is capable of disrupting the lamina of sperm pronuclei
assembled in vitro (Ellis et al.,
1997). This mutant protein
(delta 2+) was fused to GFP and expressed in transient transfection assays in
HeLa cells. Following transfection, GFP-delta 2+ accumulated as small
nucleoplasmic granules that formed over 48 hours
(Fig. 8A-D). Both lamin A
(Fig. 8A) and lamin C
(Fig. 8B) relocated from the
nuclear lamina to the nucleoplasmic granules over the same period of time. By
contrast, lamins B1 (Fig.
8C) and B2 (Fig.
8D) remained in the nuclear lamina. To confirm that lamins
B1 and B2 remained in the nuclear lamina, transfected
cells were extracted in situ with detergents, nucleases and ammonium sulphate.
Following this procedure both B-type lamins were retained in the insoluble
lamina, demonstrating that their solubility properties were unaffected by the
presence of the mutant protein (data not shown). Thus in these transfection
experiments, GFP-delta 2+ exerted a dominant effect over A-type lamins,
causing their redistribution from the lamina to nucleoplasmic granules, but
had seemingly no effect on B-type lamins. Next we investigated the effects of
the dominant negative mutants on emerin and LAP2ß distribution. When HeLa
cells were transfected with GFP-delta 2+ and then stained with anti-emerin
antibodies, the majority of emerin was located in cytoplasmic granules rather
than the NE (Fig. 9A). By
contrast, LAP2ß remained in the NE
(Fig. 9B).
|
|
The cytoplasmic granules observed in transfected HeLa were unlike the ER distribution of emerin observed in lamin A/C-deficient cell lines. Therefore, to investigate the location of the cytoplasmic emerin, transfected cells were stained with anti-calreticulin antibodies (TRITC) and anti-emerin antibodies (Cy5). A typical result is shown (Fig. 10) and reveals that emerin did not co-localise with the majority of calreticulin in transfected cells. Instead. emerin was mainly located in granules lying close to the NE. However, calreticulin did accumulate within these granules (Fig. 10, arrowheads) suggesting that the granules were within the ER.
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DISCUSSION |
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Evidence for a hierarchy of lamina associations mediated by lamin
A
We investigated emerin localisation in two different human cell lines that
were deficient for synthesis of lamins A and C and two further cell lines that
were deficient for lamin A synthesis and in which lamin C was mis-localised to
the nucleolus. In each cell line either all or a majority of emerin was
mis-localised to the ER. In addition, we used a dominant negative mutant of
lamin B1 that selectively eliminates lamins A and C but not lamins
B1 and B2 from the NE of HeLa cells. A consequence of
eliminating lamins A and C from the NE was that emerin relocated from the NE
to the ER, where it formed insoluble inclusions.
Our data suggest that lamin A has a central role in tethering both emerin and lamin C to the NE. The following evidence supports this model. Association of lamin C with the NE in two of the cell lines reported here is dependent upon the presence of lamin A, and in its absence lamin C organisation is disrupted such that most (SW 13) or all (Ramos) is mis-localised to the nucleolus. In SW 13 cells the failure of lamin C to be incorporated into the lamina is a direct consequence of the absence of lamin A since transient or stable expression GFP-lamin A causes significant re-localisation of endogenous lamin C to the NE. The failure of lamin C to be incorporated into the lamina in the absence of lamin A may result from weak associations between lamin C and lamin B filaments or because lamin C is not isoprenylated and carboxy methylated and cannot accumulate at the NE on its own. Thus lamin A may be required to carry lamin C to the NE or to mediate its association with lamina filaments or both. It is unclear why, in the absence of lamin A, lamin C becomes mis-localised to the nucleolus. However, it is possible that the nucleolus is a default location for some proteins that might otherwise form damaging structures (e.g. aggregates) when they are unable to accumulate at their normal sites of assembly.
Evidence that lamin C incorporation into the lamina is dependent on the
presence of lamin A has been reported previously. When fluorescently labelled
lamin C was microinjected into Swiss 3T3 cells it forms small aggregates in
the nucleoplasm which persist for several hours (an analogous situation was
observed in this study when GFP-lamin C was transiently expressed in SW 13
cells). When lamin A and lamin C were injected together into Swiss 3T3 cells
they are both incorporated into the NE rapidly (Pugh et al.,
1997). In agreement with these
findings, when lamin C was transfected into cells arrested in S-phase it
remained in the nucleoplasm, whereas lamin A was incorporated into the NE
under similar conditions. However, if the transfected cells were released from
S-phase and permitted to divide, presumably allowing transfected lamin C to
interact with soluble lamin A, the lamin C became incorporated into the NE
during the following G1 phase (Horton et al.,
1992
).
We observed that emerin is mis-localised to the ER in four different human
cell lines that display abnormal expression or localisation of lamins A and C.
Moreover, expression of GFP-lamin A in one of these lines resulted in
relocation of emerin from the ER to the NE. In all cell lines employed in the
study, lamins B1 and B2 were expressed and localised
normally and LAP2ß was localised at the NE. Thus, even though emerin can
bind to lamin B1 in vitro, the presence of this protein in the
lamina is not sufficient to anchor emerin at the INM. Instead these data
suggest that the presence of lamins A and C at the NE is necessary for emerin
localisation at the INM. The data reported here is entirely consistent with
the recent description of the lamin A/C-knockout mouse. In lamin
A/C-/- mice, emerin is located mainly in the ER in most tissues
(Sullivan et al., 1999).
Consistent with this data, when we used dominant negative mutants of lamin
B1 to selectively eliminate lamins A and C from the NE in HeLa
cells, emerin relocated from the INM to inclusions within the ER.
Our data can be explained by a hierarchical series of associations between
lamin A, lamin C and emerin. In this hierarchy, lamin A may mediate the
association of lamin C with the lamina and, once present, lamin C may
stabilise the association of emerin with the INM. In previous studies
associations between lamins A/C and emerin have been reported but the
possibility that lamin A and lamin C perform different functions at the INM
were not considered. For example, a clear temporal correlation between emerin
and lamin A/C association with the reforming NE at telophase has been reported
(Manilal et al., 1999).
Similar temporal correlations were reported in live GFP-imaging studies
(Haraguchi et al., 2000
). In
addition, one study reported a striking spatial correlation in which lamins
A/C and emerin co-associate with the reforming NE in discrete foci at
telophase, whereas lamin B1 and LAP2ß re-associates throughout
the NE (Dabauvalle et al., 1999). This spatial correlation between lamin A/C
and emerin incorporation at the reforming NE is all the more striking because
A-type lamins do not return to the nucleus until after the formation of a
transport-competent envelope, whereas emerin-containing membranes associate
with chromatin much earlier. Although all three studies indicated a structural
complex involving emerin, lamin A and lamin C at the INM, none of the studies
was able to distinguish between different roles for lamin A and lamin C in the
complex. Five observations led us to present a new hypothesis to explain
lamin-emerin associations at the INM. (1) emerin's preferred in vitro binding
partner is lamin C. (2) Emerin is never present in the NE in the absence of
lamin C. (3) Lamin C is not incorporated into the lamina in the absence of
lamin A. (4) Lamin C can be sequestered into emerin aggregates in the ER when
GFP-emerin is overexpressed in lamin A-deficient cells. (5) We have
demonstrated previously that lamin A is incorporated into the lamina through
association with lamin B filaments (Dyer et al.,
1999
). Therefore, we propose
that the hierarchy of lamina associations involving emerin is as follows:
lamin A associates with lamin C as either dimers or tetramers in the
nucleoplasm. Lamin A/C then associates with B-type lamina filaments, this
association being mediated by lamin A. The association of lamin A with the
lamina allows incorporation of lamin C but in the absence of lamin A, lamin C
accumulates at default sites in the nucleus. Once incorporated into the
lamina, lamin C associates with emerin at the INM. The association between
emerin and lamin C may stabilise and tether both proteins at the lamina. Thus
in the absence of lamin C, emerin is not stably associated with the lamina and
we speculate that, in the absence of emerin, lamin C may not be stably
associated with the INM. This model provides an explanation for the
accumulation of lamin C in cytoplasmic aggregates when GFP-emerin was
overexpressed in SW 13 cells. Presumably, in this instance, emerin aggregates
were a preferred location compared with the default location in nucleoli.
Our data suggest that emerin can bind to lamin B1 in vitro.
However, we found no evidence to suggest that B-type lamins influences emerin
behaviour in vivo. B-type lamins have a number of specific binding partners at
the NE, including LAP2ß and LBR (reviewed by Vaughan et al.,
2000). It is possible that
potential emerin binding sites on B-type lamins, organised as lamina
filaments, are unavailable because they are occupied by other INM proteins
with higher binding affinities. Thus, although emerin can associate with
B-type lamins, it does not do so in living cells.
Disease causing mutations in the lamin A/C gene and their
relationship to lamina structure
Since the report early last year that mutations in the gene encoding lamins
A and C cause AD-EDMD, it has become clear that a large range of lamin A/C
mutations give rise to a number of diseases (Bonne et al.,
1999; Brodsky et al.,
2000
; Cao et al.,
2000
; Fatkin et al.,
1999
; Raffaele et al.,
2000
). The mutations so far
identified map to different regions of the lamin A/C protein depending upon
the disease. Missense mutations causing EDMD map either to highly conserved
residues in the lamin A/C tail (Bonne et al.,
1999
; Raffaele et al.,
2000
), or to equally highly
conserved residues throughout the coiled-coil domain (Raffaele et al.,
2000
). The spread of mutations
causing AD-EDMD suggest that lamin A/C makes a number of molecular
interactions at the INM and that different mutations disrupt different
interactions. Reported interactions are with emerin (present study; Fairley et
al., 1999
; Clements et al.,
2000
), lamin B (present study;
Dyer et al., 1999
), lamin A to
lamin C (present study; Pugh et al.,
1997
), the chromatin binding
protein LAP2
(Dechat et al., 2000), LAPIC (Powell and Burke,
1990
), the tumour suppressor
protein p110RB (Ozaki et al.,
1994
) and chromatin
(Höger et al.,
1991
; Glass et al.,
1993
). If the model presented
above is correct, mutations in lamin A/C and perhaps in emerin might give rise
to loss or weakened association of lamin C with the lamina in EDMD. This in
turn would lead to the possibility of abnormal lamin C complexes forming
elsewhere in the nucleus (e.g. the nucleolus). Indeed, in a preliminary
investigation of LCLs from 20 EDMD patients, abnormal nucleoplasmic
distributions of lamin C were observed in a majority of cases (E. Wang, M.W.
and C.J.H. unpublished). Given the range of associations reported for A-type
lamins, abnormal nuclear localisation of lamin C may result in significant and
deleterious gain of function. Therefore, abnormal nuclear distributions of
lamin C rather than an absence of emerin from the INM may promote EDMD and
other lamin diseases.
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ACKNOWLEDGMENTS |
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