1 Department of Genetics, The Institute of Life Sciences, The Hebrew University
of Jerusalem, Jerusalem 91904, Israel
2 Department of Cell Biology, The Johns Hopkins University School of Medicine,
725 N. Wolfe Street, Baltimore, MD 21205, USA
3 Department of Molecular Biology and Genetics, 423 Biotechnology Building,
Cornell University, Ithaca, NY 14853, USA
Author for correspondence (e-mail:
klwilson{at}jhmi.edu
)
Accepted 15 November 2001
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Summary |
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Key words: Lamin, Lap2, LEM-domain, Nuclear envelope, Emery-Dreifuss muscular dystrophy
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Introduction |
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The nuclear envelope encloses and attaches to the chromosomes. The envelope
consists of the outer and inner membranes and their enclosed lumenal space,
nuclear pore complexes, and an underlying lamina
(Gant and Wilson, 1997). The
shape and structure of the nucleus depend on the nuclear lamina. The lamina
consists of a network of polymerized lamins (type V intermediate filament
proteins) (Moir et al., 2000
;
Stuurman et al., 1998
) plus a
growing number of laminbinding proteins
(Dechat et al., 2000
). Humans
express three B-type lamins (encoded by two genes) and four A-type lamins,
which arise by alternative splicing of LMNA
(Stuurman et al., 1998
;
Erber et al., 1999
). The
expression of A-type lamins is generally correlated with cell differentiation,
since all cells express at least one B-type lamin, but not all cells express
A-type lamins (Riemer et al.,
1995
).
Emerin belongs to the LEM family of nuclear proteins, most of which are
integral membrane proteins. This family includes emerin, found in C.
elegans to humans; LAP2, found only in vertebrates
(Foisner and Gerace, 1993);
MAN1, found in C. elegans to humans
(Lin et al., 2000
); otefin,
found in Drosophila (Goldberg et
al., 1998
); and Lem-3, found in C. elegans to mammals
(Lee et al., 2000
) (see IMAGE
clone number 1243400 for mouse Lem-3 ortholog). LEM proteins all share a
40-residue motif known as the LEM-domain
(Lin et al., 2000
). Emerin and
the ß-isoform of LAP2 are also similar outside the LEM-domain, suggesting
that their functions might be related. In biochemical experiments, both LAP2
and emerin bind directly to lamins; emerin in particular can interact with
both A- and B-type lamins (Fairley et al.,
1999
; Clements et al.,
2000
). In addition to binding lamins, both LAP2
(Furukawa, 1999
;
Shumaker et al., 2001
) and
emerin (Lee et al., 2001
)
interact with a small DNA-bridging protein named BAF, whose in vivo function
is unknown (barrier-to-autointegration factor)
(Lee and Craigie, 1998
;
Chen and Engelman, 1998
;
Zheng et al., 2000
). Emerin
localization at the nuclear envelope is proposed to be essential for its
function, since mutations that mislocalize emerin to the ER cause EDMD
(Ellis et al., 1998
). A-type
lamins contribute to localizing emerin at the nuclear inner membrane, since
emerin becomes localized to both the nuclear envelope and endoplasmic
reticulum (ER) network in LMNA-null mice
(Sullivan et al., 1999
).
However, the continued localization of emerin at the nuclear envelope in
LMNA-null mice suggested that other proteins, such as B-type lamins,
BAF, or other nuclear membrane proteins, might also help retain emerin at the
nuclear envelope.
Many models have been proposed to explain the symptoms of EDMD, ranging
from defects in mechanical instability or muscle cell regeneration, to defects
in gene expression, lamina structure or nuclear signaling
(Morris and Manilal, 1999;
Östlund et
al., 1999
; Gruenbaum et al.,
2000
; Wilson,
2000
). Testing such models will require a model organism with
powerful genetics. This model organism must also have nuclear envelope
proteins and dynamics that parallel the human nucleus. These criteria rule out
the use of single-celled eukaryotes such as S. cerevisiae, which do
not express lamins, LEM-domain proteins or BAF
(Cohen et al., 2000
). We have
chosen C. elegans as a possible model system to study the function of
emerin and the molecular mechanisms underlying EDMD. C. elegans is a
genetically tractable nematode with differentiated cells and tissues including
muscle (Wood, 1988
;
Culetto and Sattelle, 2000
).
The C. elegans nuclear envelope is a sophisticated yet simple version
of the human nuclear envelope. Sophisticated, because the C. elegans
nucleus shares many conserved nuclear envelope proteins with vertebrates, and
also breaks down completely during mitosis
(Lee et al., 2000
), with
nuclear structural dynamics similar to human nuclei. However, C.
elegans is comparatively simple because it encodes only a single B-type
lamin gene (lmn-1) (Riemer et
al., 1993
) that is essential for viability
(Liu et al., 2000
). By
contrast, humans have two B-type lamin genes and one A-type gene
(Stuurman et al., 1998
). There
are only three genes encoding LEM proteins in C. elegans: emr-1,
lem-2 and lem-3, encoding Ce-emerin, Ce-MAN1 and Ce-Lem3,
respectively (Lee et al.,
2000
), all of which are conserved in mammals
(Lin et al., 2000
;
Lee et al., 2000
) (IMAGE clone
1243400 for mouse Lem-3). However, humans have additional proteins at the
nuclear inner membrane that are unique to vertebrates, including a group of
alternatively spliced LEM-domain proteins named lamin associated polypeptide 2
(LAP2) (Foisner and Gerace,
1993
; Berger et al.,
1996
; Dechat et al.,
2000
). The conservation and relatively small number of LEM-domain
proteins in C. elegans means that it will ultimately be feasible to
investigate the function(s) of an entire family of LEM proteins in this single
organism.
Here we have investigated the localization, nuclear envelope interactions and loss-of-function phenotype for Ce-emerin. We show that Ce-emerin co-localizes at the nuclear envelope with Ce-lamin in C. elegans embryos, co-immunoprecipitates with Ce-lamin from embryonic lysates, and requires Ce-lamin for its localization at the envelope in vivo. However, no other nuclear envelope proteins tested depend on emerin for their envelope localization, including Ce-lamin. Our results also show that Ce-emerin is widely expressed, like human emerin, and similarly dispensible during embryonic development, suggesting that C. elegans is an appropriate genetic model for emerin function.
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Materials and Methods |
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Embryo lysate preparation and immunoprecipitation
Gravid wild-type N2 hermaphrodites were recovered from four 10 cm plates by
washing with 15 ml of M9 buffer (Lewis and
Fleming, 1995). The collected worms were pelleted by
centrifugation at 450 g for 1 minute, resuspended in 15 ml of
bleach solution (0.5% hypochlorite, 1N NaOH), and incubated at 22-24°C for
3 minutes with occasional shaking to dissolve the adult nematodes. The
released embryos were then pelleted at 450 g for 1 minute,
resuspended in 15 ml of bleach solution, repelleted, and washed twice with M9
buffer. The final pellet of isolated embryos was resuspended in 250 µl
homogenization buffer (HB; 15 mM Hepes pH 7.6, 10 mM KCl, 1.5 mM
MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 44 mM sucrose) containing 100
µg/µl protease inhibitors (aprotinin, leupeptin and PMSF) and 1 mM DTT.
The embryos were then crushed using about 25 strokes of a dounce homogenizer
(pestle A). After recovering the crushed embryos, the homogenizer was rinsed
with 250 µl of HB, and both this and the crushed embryos (total volume
500 µl) were combined in an eppendorf tube and centrifuged for 20
minutes at 14,000 g. The supernatant (embryo lysate) was
recovered, supplemented with 50 µl of cold buffer (15 mM Hepes, pH 7.6, 1 M
NaCl), and used as described below for immunoprecipitation reactions.
Co-immunoprecipitation was done as follows (modified from Lee and Schedl, 2002). Isolated N2 embryonic extracts (500 µl) were precleared by incubating for 30 minutes at 22-24°C with 100 µl uncoupled Protein A Sepharose (Amersham Pharmacia Biotech AB, Uppsala Sweden), followed by centrifugation at 4000 g for 2 minutes. The cleared extract supernatant was then incubated for 1 hour at 4°C (constant mixing) with 5 µl of either immune or preimmune rabbit antiserum against an N-terminal (serum 3930) or C-terminal (serum 3932) peptide of Ce-lamin. Samples were then supplemented with Protein A Sepharose beads (50 µl), incubated for 4 hours at 4°C, and centrifuged at 4000 g for 2 minutes. The beads were washed four times with HBS (HB containing 250 mM NaCl and protease inhibitors), resuspended in 25 µl of 2 x SDS gel-sample buffer and boiled for 5 minutes. Samples were loaded onto a 4-12% SDS-PAGE gradient gel, electrophoresed for 45 minutes at 200 volts, and blotted to nitrocellulose membrane. Membranes were then blocked for 2 hours in TBS-T containing 5% (weight/volume) nonfat dry milk. Blots were probed with rat anti-Ce-emerin serum 3598 (1:500 dilution) for 2 hours at 22-24°C, washed four times (5 minutes each) in TBS-T, and incubated for 1 hour with HRP-conjugated goat antirat secondary antibody (1:10,000 dilution; Jackson Immunoresearch, West Grove, PA). Blots were then washed four times (5 minutes each) in TBS-T, incubated with ECL reagents (Amersham) and exposed to film.
cDNA clones for Ce-emerin and Ce-lamin
The full-length cDNA encoding Ce-lamin has been described
(Liu et al., 2000). An EST
containing the entire open reading frame of Ce-emerin was obtained from Y.
Kohara (National Institute of Genetics, Japan; yk258d11) and confirmed by DNA
sequence analysis (not shown).
RNA-mediated interference (RNAi) experiments
Double-stranded RNA (dsRNA) corresponding to Ce-emerin was synthesized from
plasmid yk258d11 (Bluescript vector) using the Ambion Megascript T7 and T3
kits to synthesize single stranded RNAs. These RNAs were combined to form
dsRNA as previously described (Liu et al.,
2000). The dsRNA for Ce-lamin was synthesized as described
(Liu et al., 2000
). For
injection experiments, dsRNA (0.1-1 µg/µl) was injected into both gonads
of at least 10 adult hermaphrodites per construct as described
(Fire et al., 1998
;
Montgomery et al., 1998
). From
12 to 60 hours after injection, the adults and embryos were either examined
for viability as described (Liu et al.,
2000
), or fixed and stained by indirect immunofluorescence as
described above. For the lmn-1(RNAi) feeding experiments, pJKL483.1,
which contains the full length lamin cDNA subcloned into feeding vector L4440,
was used to transform E. coli HT115(DE3) cells. The transformed
bacteria were used to feed N2 nematodes as described
(Timmons et al., 2001
).
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Results |
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The expression pattern of endogenous Ce-emerin protein was determined by
indirect immunofluorescence in embryos, larvae and adult cells. To control for
antibody penetration into nematode tissues, we double-labeled with antibodies
against Ce-lamin, which is expressed in all C. elegans cells except
sperm (Liu et al., 2000).
Ce-emerin was detected at the nuclear envelope in all embryonic cells
(Fig. 2A), and was detected in
all cells examined throughout larval development, as shown for L1 and L3
larvae (Fig. 2B) and in adult
N2 hermaphrodites, including the gonad
(Fig. 2C), with one possible
exception. Ce-emerin was not detected in cells undergoing spermiogenesis that
had reached the stage of having condensed chromatin (data not shown). However,
we could not definitively conclude that emerin was absent from sperm, because
sperm lack Ce-lamin (Liu et al.,
2000
) and we therefore had no positive control for antibody access
into these cells during staining. Nevertheless, the nearly ubiquitous positive
staining seen for Ce-emerin throughout development and in adult animals was
similar to that seen for human emerin, which is found in most cell types
examined (Manilal et al.,
1999
).
|
Nuclear localization of other nuclear envelope proteins does not
depend on Ce-emerin
To determine whether lamin or other nuclear envelope proteins depend on
Ce-emerin for their localization, we created embryos deficient in Ce-emerin.
This was done by the RNA interference (RNAi) method, in which mRNA production
from a specific gene is disrupted by injecting adult hermaphrodites with
double-stranded RNA (dsRNA) corresponding to the targeted gene
(Fire et al., 1998;
Montgomery et al., 1998
). We
used a 500 base pair dsRNA, corresponding to the entire open reading frame of
the Ce-emerin gene, emr-1 (Lee et
al., 2000
). Emerin protein was not detected by indirect
immunofluorescence in emr-1(RNAi) embryos
(Fig. 3A), relative to
uninjected controls (Fig. 3A,
`WT'). In these experiments, embryos were double-stained for endogenous
Ce-lamin as a positive control for antibody access, and to rule out the
possibility that loss of Ce-emerin might affect Ce-lamin localization
(Fig. 3, right panels).
Ce-lamin localized normally in the absence of Ce-emerin, demonstrating that
emerin is not structurally required for lamin assembly in C. elegans.
We also localized two other inner membrane proteins: fellow LEM protein
Ce-MAN1 (Lee et al., 2000
),
and a conserved non-LEM protein named UNC-84
(Malone et al., 1999
;
Dreger et al., 2001
) (K.K.L.,
D. Starr, M.C. et al., unpublished). Both Ce-MAN1 and UNC-84 were localized at
the nuclear envelope in emr-1(RNAi) embryos
(Fig. 3B), as were nuclear pore
complexes detected using monoclonal antibody mAb414
(Fig. 3B). Thus, of the nuclear
proteins tested (Ce-lamin, Ce-MAN1, UNC-84 and nucleoporins), none depended on
Ce-emerin. The RNAi method was quite effective at removing Ce-emerin. Thus,
even though we cannot rule out the possible activity of undetectable residual
Ce-emerin, we concluded that emerin is not structurally (stoichiometrically)
required for the localization or retention of the B-type lamin in C.
elegans, fellow LEM protein Ce-MAN1, or unrelated nuclear envelope
protein, UNC-84.
|
Ce-emerin is not required for viability in embryos or adults
We then examined animals depleted of Ce-emerin for any phenotype during
development. We found that the emr-1(RNAi) embryos with no detectable
Ce-emerin developed at normal rates into fertile adult nematodes. Because the
emr-1 (RNAi) embryos remained emerin-depleted through adulthood, we
also examined the Ce-emerin depletion phenotype in adult nematodes. Gonad
cells, which usually gave the brightest staining for Ce-emerin in adults, had
undetectable Ce-emerin as shown by double-staining for Ce-lamin
(Fig. 4, green) and Ce-emerin
(Fig. 4, red; note the
nonspecific red staining of the cuticle). These emerin-depleted adults had no
detectable phenotype: they displayed normal movement and feeding behavior, and
produced viable fertile offspring: emr-1 (RNAi) animals and control
N2 animals had similar brood sizes (averaging 206 for emr-1 (RNAi)
and 208 for N2; n=5), and aged at similar rates (50-58% not moving
well and 35% dead by day 23, and all dead by day 26 at 20°C; n=20
each). This result was expected, since emerin loss in humans has no detectable
effect until childhood, and then selectively affects a few specific tissues
(Emery, 1989). We concluded
that Ce-emerin is not essential in C. elegans.
|
The nuclear localization of Ce-emerin is dependent on Ce-lamin
In mice that lack A-type lamins, emerin is found both in the nuclear
envelope and ER. To test definitively for dependence on lamins, we made
lamin-deficient lmn-1 (RNAi) embryos
(Liu et al., 2000; see
Materials and Methods). These lmn-1 (RNAi) embryos were then
triple-stained for DNA, Ce-lamin, and Ce-emerin
(Fig. 5). Ceemerin staining at
the nuclear envelope was not detectable in lamin-deficient embryos
(Fig. 5A, `injection'). This
result showed that either the expression or nuclear envelope localization of
Ce-emerin requires Ce-lamin. The background cytoplasmic staining for Ce-emerin
appeared to increase in lamin-deficient cells. However, this signal was too
weak to rigorously conclude that Ce-emerin dispersed into the ER in the
absence of Ce-lamin. Lamin-dependent nuclear localization of Ce-emerin was
also seen in animals fed with bacteria that expressed lmn-1 dsRNA;
with this method, individual embryonic nuclei became lamin-depleted at
different times (Liu et al.,
2000
). With the feeding method, Ce-emerin was detected only in
nuclear envelopes with high residual levels of Ce-lamin
(Fig. 5, `feeding', arrow), and
was reduced in nuclei with reduced or low staining for Ce-lamin
(Fig. 5, `feeding',
arrowheads). Together these results clearly showed that Ce-emerin depends on
lamins for stable localization at the nuclear inner membrane in C.
elegans embryos.
|
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Discussion |
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The physiological effects of emerin deficiency on tendon growth and cardiac
function in people cannot be directly studied in nematodes, since nematodes
lack tendons and heart. However, nematodes do have differentiated and
specialized muscle cells, and highly conserved muscle proteins
(Waterston, 1988). Our
RNAi-depletion results suggest that emerin is not required for C.
elegans development, and might also be non-essential in adults, with the
caveat that trace amounts of Ce-emerin in emr-1(RNAi) adults might
have covered a putative essential function. It is also possible that a
phenotype for Ce-emerin depletion will only appear when worms are physically
stressed by growth conditions found in nature. Although these RNAi results are
negative, they are consistent with the human null phenotype for emerin; human
emerin is not essential for development, and its loss causes no known
phenotype in most human tissues that express emerin, except for heart,
skeletal muscle and tendons. One potentially critical difference between
nematodes and humans is that the disease progresses very slowly (years) in
humans, whereas nematodes live less than 4 weeks. Thus, if the disease
mechanism is strictly linked to physical trauma or longterm mechanical
disruption of muscle cell function (Morris
and Manilal, 1999
; Fairley et
al., 1999
), which cannot currently be ruled out, then nematodes
will not provide a model for disease. Alternatively, disease may arise from
the disruption of specific emerin-dependent interactions required for gene
expression (Wilson, 2000
).
Supporting this model, the emerin-related protein LAP2ß can directly
repress the expression of a reporter gene in mammalian cells
(Nili et al., 2001
).
Our present work shows that the nuclear envelope localization of Ce-emerin
is lamin-dependent during development, and that emerin is expressed in nearly
all cell types in C. elegans, similar to human emerin. The only cells
we detected in C. elegans that might lack emerin are the amoeba-like
sperm cells, which also fail to stain for Ce-lamin
(Liu et al., 2000). Emerin
mRNA is present in human testis (Small et
al., 1997
), but it is not known whether emerin is present in human
sperm. In humans, the non-myocyte cells of the heart are among the few cell
types known to lack emerin (Manilal et
al., 1999
). Thus, the presence of emerin at the nuclear envelope
in a wide range of cells and tissues is a conserved feature of emerin from
humans to nematodes. Given the complexity of nuclear envelope structure and
function, the possible functional overlap between emerin and other LEM-domain
proteins, and the possibility that emerin might have multiple roles (e.g.
lamin-related, BAF-related and novel), C. elegans will be a useful
genetic system for dissecting the functional inter-relationships among LEM
proteins, lamins and other nuclear envelope proteins, as well as the
mechanisms of Emery-Dreifuss muscular dystrophy.
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Acknowledgments |
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Footnotes |
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References |
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