Institute for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany
Author for correspondence (e-mail: noegel{at}uni-koeln.de )
Accepted 1 May 2002
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Summary |
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Key words: CH domain, Spectrin repeats, Klarsicht-like domain, Nuclear envelope
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Introduction |
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The identification of the Drosophila protein kakapo (also known as
groovin) (Gregory and Brown,
1998; Prokop et al.,
1998
; Strumpf and Volk,
1998
) and its mammalian homologue MACF (also described under the
names trabeculin-
and macrophin)
(Leung et al., 1999
;
Okuda et al., 1999
;
Sun et al., 1999
), as well as
BPAG1-a and BPAG1-b forms of the BPAG/dystonin locus
(Leung et al., 2001b
), brought
confusion to this classification, since these proteins, which are larger than
the other known plakin and spectrin proteins, harbor both domains found in
plakin and spectrin and may represent a fusion product of two precursor genes.
An alternative hypothesis proposes that the ancestral gene resembled the
shortstop/kakapo genes of Drosophila and
Caenorhabditis (Leung et al.,
2001a
). Owing to their multiple binding sites for microtubules,
intermediate filaments and microfilaments, these giant proteins are recognized
as cytolinkers, integrating the cytoplasmic cytoskeleton with membranes and
submembrane complexes (Fuchs and Yang,
1999
; Karakesisoglou et al.,
2000
).
The recently described protein calmin, which also possesses a N-terminal
actin-binding domain (ABD) of the -actinin type, does not fall into any
of the above categories, as it does not harbor either a coiled-coil rod domain
or any other common motifs (Ishisaki et
al., 2001
). Instead it has a C-terminal transmembrane domain (TMD)
that targets cytoplasmic reticular structures and therefore represents the
first integral membrane
-actinin-related protein known so far. However,
some transcripts of calmin are differentially spliced and lack the TMD. The
distribution pattern of the calmin isoforms did not overlap with that of the
actin cytoskeleton. Here we report the molecular characterization and cellular
localization of NUANCE, the largest known protein of the
-actinin
superfamily. The 796 kDa protein harbors multiple dystrophinlike spectrin
repeats but lacks domains characteristic for plakins. Hence, it is related to
the spectrin family of the
-actinin superfamily. Unlike other related
proteins, NUANCE is associated with the nuclear envelope (NE) via the
predicted TMD. This enormously large protein may harbor numerous binding sites
and serve as a scaffold for the assembly of various protein complexes.
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Materials and Methods |
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Production of the recombinant proteins in E. coli and
generation of the monoclonal antibodies
DNA fragments encoding the first 285 (6xHis-ABD) and 459 amino acid
residues (6xHis-ABD-1) were obtained by PCR using fragment 2 as a
template and inserted into pQE-30 (Qiagen). Recombinant proteins were induced
by IPTG in E. coli M15[pREP4] and purified using a Ni-NTA agarose
column according to the manufacturer (Qiagen). mAb K20-478 was produced by
immunizing mice with the purified 6xHis-ABD-1 protein with ImmunEasy
mouse adjuvant (Qiagen) as described earlier
(Olski et al., 2001).
Actin-binding assay
The F-actin co-sedimentation assay and the quantification of
6xHis-ABD bound to F-actin was performed as described previously
(Olski et al., 2001). For
high- and low-speed centrifugation assays, samples were pelleted at 125,000
g and at 20,000 g, respectively. Actin polymerization was
measured by recording the changes in fluorescence intensity of pyrene-labeled
-actin monomers as described previously
(Korenbaum et al., 1998
). The
fluorescence measurements were made using a Fluoroskan Ascent FL plate reader
(Labsystems). Polymerization of 8 µM actin alone or in the presence of
various amounts of the ABD was initiated by addition of 2 mM MgCl2
and 100 mM KCl.
Plasmid construction and transient transfections
The constructs GFP-ABD (residues 1-296) and GFP-ABD-S (residues 1-262 with
additional amino acids AYKN from exon 8a) were amplified from fragment 2 and
2a, which code for spliced variants, using primers with extensions for
BamHI and KpnI sites and cloned into
BglII/KpnI-cut pEGFP-C1 (Clontech). For the GFP-ABDsr1-2
construct (residues 1-531), fragment 2 was digested with BamHI and
EcoRI then ligated into BglII/EcoRI-cut pEGFP-C1
vector. For the GFP-sr15-21 (residues 5727-6596), the fragment 13a was excised
utilizing the EcoRI sites of pGEM T-Easy (Promega) and ligated into
EcoRI site of pEGFP-C1. For the GFP-Cterm1 construct (residues
6571-6885), the cDNA was amplified from clone 13 and ligated into
EcoRI-cut pEGFP-C2 vector. For the GFP-Cterm2 constructs, clone 13b
was digested with SacI and EcoRI and inserted into
corresponding sites of pEGFP-C1. The GFP-Cterm2tm (residues 6642-6834
with additional 14 amino acids from the extended exon 111) lacking the
putative TMD was generated from the GFP-Cterm2 by PstI digestion and
religation. To generate GFP-NUA
460-6643, the GFP-ABDsr1-2
was cut with KpnI and BamHI and ligated with the insert of
the GFP-Cterm1 (residues 6643-6885), which was amplified using the primers
with added-on KpnI and BglII sites. COS7 cells were
transfected by electroporation. The expression of the fusion proteins was
controlled by western blotting using a GFP-specific mAb.
Cell culture
COS7 cells were grown in DME medium supplemented with 10% FBS (Sigma), 2 mM
glutamine, penicillin and streptomycin. For the propagation of human embryonic
kidney cells (293) pyruvate was added to the medium. Human T lymphocytes
(Jurkat), Burkitt's lymphoma cells BL-60 and B-JAB were maintained in RPMI
1640 containing 10% FBS, 2 mM L-glutamine, penicillin and streptomycin. For
Latrunculin A (Biomol) treatment, 70%-confluent COS-7 cells were fixed 15, 30
and 60 minutes after exposure to 1 µM LatA. The recovery was evaluated by
culturing the cells in LatA-free medium for another 30, 60 and 90 minutes. For
microtubule depolymerization the cells were incubated in medium containing 1
µM vincristine (Sigma) or 5 µg/ml colchicine (Fluka) for 4 hours before
processing for immunofluorescence. To promote the formation of lamellipodia,
confluent serum-starved monolayers of COS7 cells were wounded by scraping away
cells with a P1000 pipet tip. The coverslips were then incubated in complete
media for 6 hours prior to fixation.
Immunofluorescence
Adherent cells were allowed to attach onto glass coverslips for 1-16 hours,
rinsed with PBS, fixed in 3% paraformaldehyde and permeabilized with 0.5%
Triton X-100 in PBS for 5 minutes. Alternatively, cells were fixed with
methanol at -20°C for 10 minutes. Generally, no difference was observed
between the two fixation protocols except for the cells transfected with the
GFP-Cterm2 construct. All figures display cells fixed with paraformaldehyde
unless stated otherwise. For permeabilizing with digitonin, fixed cells
(3%-paraformaldehyde) were washed in ice-cold PBS and treated with 40 µg/ml
digitonin (Sigma) in PBS for 5 minutes on ice. For poorly adherent cells,
cover slips were pretreated with poly-L-lysine (1 mg/ml), and cells were
allowed to attach for at least 3 hours. Cells were incubated with mAbs against
vinculin (Sigma), annexin A7 (Selbert et
al., 1995), with the maD mAb, which is specific for ß-COP
(Pepperkok et al., 1993
), with
JOL2, which is specific for lamin A/C, with LN43, which is specific for
anti-lamin B2 (kind gift from Frans Ramaekers and Jos Broers) or with
polyclonal antibodies against Nup358 (kind gift from Elias Coutavas and
Günter Blobel) and against NO38/B23 (kind gift from M. Schmidt-Zachmann).
Cells washed with PBS were incubated with the appropriate secondary antibodies
conjugated to Cy3 (Sigma), Alexa488 or Alexa568 (Molecular Probes) and mounted
in Gelvatol/DABCO (Sigma). Factin was detected with TRITC-labeled phalloidin;
for nuclear staining the DNA-specific dye DAPI (Sigma) was used. Samples were
analyzed by wide-field fluorescence microscopy (DMR, Leica) or confocal laser
scanning microscopy (TCS-SP, Leica).
Cell fractionation and immunoblots
COS7 cells were harvested and washed in PBS, homogenized in lysis buffer
(1% SDS, 1 mM sodium vanadate and 100 mM Tris-HCl, pH 7.4), incubated at
95°C for 5 minutes and mixed with the protein sample buffer
(Laemmli, 1970) and heated
again for 5 minutes to 95°C. Proteins were separated on 3-15% gradient
SDS-PAGE then transferred onto PDVF membrane (Millipore). The membranes were
treated with mAb K20-478 followed by enhanced chemiluminescence.
For nuclei preparation, COS7 cells were sonicated in hypotonic buffer (10 mM HEPES, pH 7.5, 1.5 mM MgCl2, 1.5 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) supplemented with Protease Inhibitor Cocktail CompleteTM, Mini (Boehringer Mannheim). Nuclei were sedimented at 1,000 g at 4°C for 15 minutes. For further fractionation the supernatant was centrifuged at 100,000 g for 30 minutes at 4°C. Both pellets were resuspended in hypotonic buffer and analyzed on immunoblots with anti-NUANCE, anti-lamin B2 (LN43) and anti-annexin A7 mAbs.
Computer programs
For alignment of cDNA sequences and database mining, the GCG software
package and the BLAST (NCBI) program were used. Protein sequences were aligned
using the programs ClustalW and TreeView. Motif predictions and pattern
searches were performed with the ExPaSY (SIB) software package.
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Results |
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Genomic DNA organization
To analyse the intron-exon organization of NUANCE and to identify the
alternatively spliced isoforms, we searched the genomic database and found
that the NUANCE gene matched the publicly available working draft
sequences of human clones with the accession numbers AL355100.2, AL162832.3,
AL359235.2, AL355094.2, AF215937.1, AL352983.2 and AL161756.3 of chromosome 14
in contig NT_025892 mapped to 14q22.1-q22.3
(Fig. 1B). The human
NUANCE gene spans over 373 kb. About 6 kb downstream the
NUANCE gene is the estrogen receptor 2 gene (ESR2).
The NUANCE gene is split into 115 recognizable exons
(Fig. 1B,
Table 1). All exon-intron
boundaries are consistent with the consensus sequence for splice junctions
5'-GT...AG-3' (Breathnach and
Chambon, 1981). The intronic sequences comprise 94% of the total
gene length. The 5'UTR is interrupted by the longest 55,954 bp intron
located 42 bp upstream of the start codon, therefore translation starts in the
second exon. An alternatively spliced form with the exon 1a positioned 228 bp
upstream of exon 1 was found in the EST BI026470. The exon lengths vary from
50 nt in exon 114 to 2101 nt in the exon 48 coding for central coiled-coil
sequences.
Exons 3 to 9 encode the ABD region of NUANCE. One of the ABD fragments amplified by RACE-PCR (clone 2a) contained an additional exon 8a (Fig. 1C) inserted between exons 8 and 9. This leads to premature termination of the ORF and generation of an isoform comprising a truncated ABD, named ABD-S. Although the physiological relevance of ABD-S is not yet clear, the effect of ABD-S overexpression was analyzed in transfection experiments (see below; Fig. 9B,E). Several differentially spliced cDNAs corresponding to the 3' end of the NUANCE cDNA were found in the non-redundant NCBI database. The cDNAs with accession numbers AK001876, AL080133, AB023228, which correspond to a protein KIAA1011 (Syne-2), may represent a partial sequence of NUANCE, since their ORF is not interrupted by any Stop codons upstream of the methionine proposed as a translation initiation in AL080133. The cDNA with accession number AL117404 appears to contain a short isoform with a distinct 5' terminus (Fig. 1D). However, it can also result from amplification of unprocessed mRNA.
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The ABD of NUANCE shares a high homology with the ABDs of the recently
identified proteins enaptin (S. Braune, MD Thesis, University of Cologne,
2001) and calmin (Ishisaki et al.,
2001). The ABDs of these three proteins differ from the
conventional ones as they have 30 amino-acids long linkers between two CH
domains (Fig. 3B). Across its
255 amino acid ABD region, NUANCE shares about 49% identity with enaptin, 45%
with calmin and 43% with ß-spectrin, plectin, dystonin and MACF. The
phylogenetic analysis of the ABDs suggested that NUANCE, enaptin and calmin
form a distinct family within the
-actinin superfamily
(Fig. 3C). BLAST searches with
human NUANCE cDNA identified several highly homologous mouse EST clones. On
the basis of the sequence of EST clones AI747790 and AA498987, we have
designed primers and amplified a partial mouse NUANCE cDNA. The amino-acid
sequence of the mouse and human NUANCE ABD is well conserved with an identity
of 97%.
A central rod domain contains coiled coils interrupted by several fragments
of random coils. Furthermore, four nuclear localization signals (amino acids
1188-1205, 1464-1467, 3629-3645 and 6115-6132) and two leucine zippers (amino
acids 2127-2148 and 5008-5029) were also predicted by computer analysis within
the rod domain (Fig. 3A).
Multiple repeated units weakly homologous to the triple-helical spectrin-like
repeats of dystrophin and utrophin can be recognized in the regions predicted
for two- and three-stranded coiled coils
(Fig. 3E). The repeats found in
NUANCE are less regular than the ones of dystrophin and utrophin
(Winder et al., 1995). The
alignment of the 22 best-defined NUANCE repeats are shown in
Fig. 3E. The helix A is
characterized by a highly conserved tryptophan followed by a hydrophobic
residue. The first helix (helix A) and the last one (helix C), which are
continuous in the following repeats, are relatively well defined, whereas the
middle helix B is less ordered. Random coils in the regions 4082-4232,
4332-4532 and 6347-6547 (Fig.
3A) may represent hinges providing additional flexibility to the
molecule.
In addition, the C-terminal region of NUANCE contains a 62 amino-acid
region similar to the Syne-1 and Drosophila Klarsicht proteins, which
are involved in nuclear migration and nuclear positioning
(Apel et al., 2000;
Mosley-Bishop et al., 1999
).
The highly conserved hydrophobic stretch of 22 amino acids (residues 6848 to
6872) in the Klarsicht-like domain was identified as a transmembrane region
(Fig. 3D). It is flanked by the
N-terminally positioned neck region and the C-terminal tail.
Tissue distribution of NUANCE mRNA
In a Human Tissue Multiple Expression array containing mRNA from a variety
of human tissues and cell lines we found that most of the tissues showed
detectable levels of NUANCE mRNA. The highest expression was detected in the
kidney, both adult and fetal, liver, stomach and placenta, and the lowest
levels were in skeletal muscle and brain
(Fig. 4A,
Table 2). The corpus callosum
and pituitary gland displayed a relatively strong signal in contrast to the
generally low levels of expression in other parts of the brain. Expression of
NUANCE in lymphatic organs was not uniform either. High levels of mRNA were
detected in spleen and lymphatic nodes; however they were relatively low in
the thymus and hardly detectable in bone marrow. Taken together with the
strong signal seen in peripheral blood lymphocytes, this suggests that NUANCE
is characteristic for mature lymphocytes. In the digestive system only
stomach, duodenum and salivary gland showed an elevated level of NUANCE mRNA
relative to the other parts of the gastrointestinal tract. In addition,
significant amounts of mRNA were also noted in the trachea, prostate, gonads,
thyroid and adrenal glands. In RNA from cancer-derived cell lines, NUANCE was
expressed at hardly traceable amounts with the exception of Daudi Burkitt's
lymphoma cells.
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NUANCE associates with nuclei
mAb K20-478 generated against the ABD of NUANCE recognized a protein of
about 800 kDa that cofractionated with nuclei and membranes of COS7 cells
(Fig. 4B,C). The mAbs against
lamin B2 as nuclear protein and annexin A7 as mainly cytosolic protein were
included as a control. Weaker bands below may represent degradation products
of NUANCE, although they could also be short isoforms or crossreactive
proteins. For size estimation, we included the A chain of EHS-laminin with a
molecular mass of about 400,000, which migrated significantly faster than
NUANCE (data not shown).
In immunofluorescence studies the mAb yielded a spotted rim-enriched
nuclear labeling in Burkitt's lymphoma cells BL-60, human embryonic kidney
cells 293 and COS7 cells (Fig.
5). In addition, a dotted pattern was detected in the cytoplasm,
which might result from association with vesicular structures or ER
(Fig. 5A,C). A similar pattern
was observed in C3H/10T1/2 mouse fibroblasts (data not shown). NUANCE is also
seen associated with bridges that occasionally connected nuclei in COS7 cells
(Fig. 5C,D). The punctate
NUANCE staining of the nuclear surface was reminiscent of that of
nucleoporins. A comparison of NUANCE and Nup358 distribution
(Wu et al., 1995) however
showed that both proteins are only partly colocalized
(Fig. 6A-C), which implies that
NUANCE is targeted to the NE without being localized to nuclear pores.
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To clarify whether NUANCE is associated with the inner (INM) or outer
nuclear membrane (ONM) we compared the immunostaining of the digitonin- and
Triton X-100-permeabilized cells. Digitonin disrupts the plasmalemma leaving
the intracellular membranes, including the NE, intact
(Adam et al., 1990). As a
result, the mAb can access ONM-associated antigens but not the proteins facing
the nucleoplasm. The anti-NUANCE mAb stained only the nuclear periphery of the
digitonin-permeabilized cells, which was similar to the staining for
cytoplasm-facing nucleoporin 358 (Fig.
6J-L). The anti-lamin B2 mAb taken for a control did not yield any
staining in digitonin-treated cells in interphasel; instead it labeled mitotic
cells on the same cover slip (data not shown). This suggests that NUANCE
associates with the ONM with its N-terminus facing the cytoplasm. However, it
does not exclude its further association with the inner membrane of the
envelope. In the Triton X-100-treated cells we also observed a labeling in the
nucleus (Fig. 6D-I). It was
especially enriched at nucleoli, which were identified using antiserum against
the nucleolus protein NO38/B23 (G-I). Furthermore we always observed a diffuse
cytoplasmic NUANCE staining that accumulated at the nuclear invagination
harboring the Golgi apparatus (Fig.
6J), as visualized with a ß-COP-specific mAb (data not
shown).
In mitotic cells NUANCE remains associated with the NE during its breakdown, which marks the end of prophase and beginning of prometaphase (Fig. 5A,B, arrows). This indicates a stable association of NUANCE with the NE. In prometaphase, NUANCE is accumulated at condensed chromosomes (Fig. 5A,B, large arrowheads) and is diffusely present throughout the cytoplasm at later stages (Fig. 5A, small arrowhead).
Sensitivity to agents affecting the cytoskeleton
The distribution pattern of NUANCE did not overlap with that of the actin
cytoskeleton in spite of the presence of the predicted conserved ABD
(Fig. 7A-C). To test whether
disruption of the actin cytoskeleton affects NUANCE localization, we have
treated COS7 cells with Latrunculin A (LatA), a drug depolymerizing actin
filaments owing to its G-actin-sequestering activity
(Yarmola et al., 2000).
Already after 15 minutes exposure to 1 µM LatA, the actin filament meshwork
was partially disassembled, and actin-rich foci were formed (data not shown).
By 30 minutes, actin accumulated in the central area of the cell in which the
NUANCE-positive `cloud' was often seen at the invagination of the nuclei
(Fig. 7D-F). Along with further
disassembly of the actin cytoskeleton, the nuclei became less flattened and
acquired an irregular shape with wrinkled invaginations from the side where a
diffuse cytoplasmic pool of NUANCE and actin aggregates accumulated
(Fig. 7G-I). This effect on
nuclei was fully reversible. After 3 hours washout of LatA their shape was
completely restored (not shown). This observation implies that the nuclear
shape is controlled by the actin cytoskeleton and that NUANCE may have a
structural role in maintaining the nuclear architecture.
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In wound-healing assays performed on confluent monolayers of COS7 cells, NUANCE was detected at the leading edge of migrating cells colocalized with F-actin (Fig. 7J-L); this indicates partial redistribution of NUANCE in polarized cells.
We have also examined the pattern of NUANCE distribution upon treatment with vincristine and colchicine, drugs that disrupt selectively microtubules in the cell. However, no changes in nuclear morphology or in NUANCE distribution were observed (data not shown).
Domain analysis
The N-terminal actin-binding domain binds to F-actin in vitro and in
vivo
As our immunofluorescence analysis revealed that association of NUANCE with
the actin cytoskeleton is limited to the leading edge in migrating cells and
to the perinuclear patches in LatA-treated cells, we examined the
functionality and the subcellular localization of the ABD. For biochemical
characterization of NUANCE we have used the recombinantly expressed
ABD-containing construct 6xHis-ABD. The 6xHis-ABD associates with
F-actin filaments in a high-speed cosedimentation assay
(Fig. 8A). Some 6xHis-ABD
protein was present in the pellet without added actin; however it was always
enriched in the pellets in the presence of actin-containing samples. The assay
was performed with different concentrations of 6xHis-ABD in order to
quantify binding to F-actin. The Kd value was determined to be
3.8±1 µM, and saturation was achieved at a 1:1 molar ratio. The
presence of 6xHis-ABD also had an effect on the polymerization kinetics
of pyrene-labeled actin (Fig.
8C). It shortened the elongation time and increased the rate of
actin polymerization in a concentration-dependent manner. Moreover, in
low-speed F-actin co-sedimentation assays most of the actin was detected in
the pellet fraction, suggesting that NUANCE ABD acts to bundle F-actin
(Fig. 8B).
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We expressed GFP-fused ABD in COS7 cells and examined its subcellular
distribution in vivo. In contrast to the endogenous protein, the GFP-ABD
containing the 285 N-terminal amino acids of NUANCE was associated with all
microfilament structures detectable by TRITC-phalloidin, stress fibers,
lamellar meshwork and cortical actin (Fig.
9A,D). The GFP-ABD-S fusion protein, which corresponds to a short
alternatively spliced isoform where insertion of an exon 8a introduced a stop
codon in the second CH domain, showed a slightly different pattern. The
GFP-ABD-S weakly associated with the middle parts of stress fibers but was
enriched at their ends, which colocalized with the vinculin-labeled focal
adhesions (Fig. 9B,E).
Interestingly, the small focal complexes at the extreme edge of lamellipodia,
which were not associated with stress fibers yet
(Nobes and Hall, 1995), did
not recruit the GFP-ABD-S (Fig.
9B,E). The GFP-ABD-S association with cortical actin was mainly
confined to the bundles in retracting concave parts but not to the protruding
lamellas. We also noted the formation of multiple spikes and filopodia in the
cells strongly overexpressing GFP-ABD or GFP-ABD-S (data not shown). The
filopodia-rich phenotype was presumably caused by the increased formation of
actin bundles, which is in agreement with our in vitro observations. These
proteins might therefore exhibit a dominant-negative effect. Moderate GFP-ABD
expression did not seem to affect the organization of the actin cytoskeleton
in COS7 cells.
Spectrin repeats 1-2 and 15-21 of NUANCE seem to mediate membrane
targeting
To explore the role of the spectrin repeats in actin association, we
constructed a fusion protein GFP-ABDsr1-2 harboring a 531 amino acid fragment
containing the ABD and the first two repeats
(Fig. 9C,F). Costaining the
GFP-ABDsr1-2-expressing cells with TRITC-phalloidin showed overall
colocalization of the fusion protein with filamentous actin (data not shown).
However, the GFP-ABDsr1-2 was strongly attracted to the subplasmalemmal
regions, especially in protruding lamellas
(Fig. 9C,F). This implies a
role for the two spectrin repeats in membrane binding. The targeting of the
stress fibers and focal contacts was reduced in comparison with the GFP-ABD
and GFP-ABD-S proteins. The anti-NUANCE mAb recognized the GFP-ABDsr1-2
protein (Fig. 9F) as well as
the other two ABD-containing chimeras (data not shown) and showed an identical
pattern.
GFP-sr15-21 harboring spectrin repeats from 15 to 20 followed by the random coil stretch and a part of repeat 21 were used for the examination of the further spectrin repeats. The distribution pattern of the GFP-sr15-21 suggests its association with the intracellular membranes and with vesicular structures colocalizing with ß-COP, a component of the COP coat (Fig. 9G,J). No association with the NE was detected.
The C-terminal transmembrane domain associates with the nuclear
envelope
The NUANCE staining pattern appeared to be similar to the subcellular
distribution of Syne-1, a protein that is associated with the NE in skeletal,
cardiac and smooth muscle cells (Apel et
al., 2000). As Syne-1 is highly homologous to the C-terminal part
of NUANCE, the targeting of NUANCE to the NE could result from the
Klarsicht-like domain at the C-terminus. To test our assumption we have
prepared two C-terminal constructs, GFP-Cterm1 and GFP-Cterm2, comprising the
Klarsicht-like domain and the preceding spectrin repeats. The GFP-Cterm1
protein possesses spectrin repeats 21 and 22, whereas the GFP-Cterm2 harbors
only one complete repeat 22 but contains the additional 14 amino acids gained
from the alternatively spliced exon 111. Both fusion proteins showed clear
nuclear rim staining along with diffuse cytoplasmic staining in the Golgi area
(Fig. 9H, shown for
GFP-Cterm1). Moreover, both constructs seemed to displace endogenous NUANCE
from the NE but did not interfere with the nucleoplasmic staining
(Fig. 9K). In heavily
overexpressing cells both GFP-Cterm1 and GFP-Cterm2 localized in patches at
the NE and also showed a reticular or vesicular pattern in the cytoplasm (data
not shown). To inspect whether the overexpression of the GFP-Cterm1 affects
the nuclear pore distribution, we stained the transfected cells with the
anti-Nup358 antibody. Confocal analysis reveals a mutually exclusive pattern
of GFP patches and anti-Nup358 staining
(Fig. 10A-C), suggesting the
displacement of nuclear pores from the regions of GFP-Cterm1 accumulation. The
appearance of lamins A/C and B, detected by the mAbs JOL2 and LN43, in
contrast, was not affected in GFP-Cterm1- and GFP-Cterm2-expressing cells
(data not shown). The overexpression of the C-terminal constructs did not
hamper mitotic progression in COS7 cells. GFP-Cterm2, like the endogenous
protein, remained associated with the NE during prophase, whereas the
anti-Nup358 staining was largely diffuse
(Fig. 10D-F), indicating at
least partial disassembly of nuclear pores.
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GFP-Cterm2tm, which is identical to GFP-Cterm2, but lacks the
hydrophobic membrane-spanning sequence and the C-terminal tail, confirmed the
importance of the TMD for the nuclear association. This protein was found in
all cellular compartments when overexpressed in COS7 cells. However, in cells
fixed with methanol, only vesicular staining in the cytoplasm remained
(Fig. 9I). No nuclear staining
was detected, and the localization of the endogenous NUANCE was not affected
by the overexpression of the GFP-Cterm2
tm
(Fig. 9L). Thus, the TMD is
necessary for recruitment to the NE. These results suggest that NUANCE is an
integral protein of the NE that shares the TMD with the other proteins
exhibiting a similar subcellular localization.
Having identified the intracellular locations for N-terminal and C-terminal
parts of NUANCE we generated a construct GFP-NUA460-6643
where the first 459 amino acids were directly fused to the C-terminal 315
amino acids that correspond to the Cterm1 construct. In transfected COS7
cells, the GFP-NUA
460-6643 was arranged in various structures
from fine filaments to large aggregates
(Fig. 10G-L). In most of the
cells, the GFP-NUA
460-6643 was observed at the NE envelope,
enriched at one side of the nucleus (Fig.
10J-L). Actin was often attracted to the
GFP-NUA
460-6643 fibers and aggregates; however,
GFP-NUA
460-6643 was never seen in association with the
dynamic submembranous actin cytoskeleton.
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Discussion |
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NUANCE appears to be recruited to the NE through the Klarsicht-like
C-terminal domain harboring a TMD. Drosophila Klarsicht protein is
required for migration of nuclei in developing retinal photoreceptors
(Mosley-Bishop et al., 1999).
Similarly, a role in the migration of micronuclei in myotubes and/or their
anchoring at the postsynaptic apparatus was ascribed to Syne-1
(Apel et al., 2000
), a protein
that is highly homologous to the C-terminal part of NUANCE. Syne-1 is
selectively associated with the nuclei that lie beneath the postsynaptic
membrane at the neuromuscular junctions in skeletal, cardiac and smooth muscle
cells. Syne-1 is strongly expressed in the muscle and brain, tissues with the
lowest level of NUANCE expression. Since the N-terminus of the longest Syne-1
isoform has not yet been identified, it is plausible that the full-length
Syne-1 protein also possesses an N-terminal ABD and therefore may represent a
muscle/brain counterpart of NUANCE. Nuclear positioning and migration are
essential for the movement of pronuclei during fertilization, normal mitotic
and meiotic cell division and various morphogenetic processes during metazoan
development. Nuclear migration has been directly linked to a human disease,
the brain developmental disorder lissencephaly
(Morris, 2000
). Migration of
neurons involves nucleokinesis, a process whereby cells acquire an elongated
shape or extend a frontal protrusion, and the nucleus migrates to its new
position within the cytoplasm. The nuclear translocation within the cell can
also be associated with cell motility during tumor cell invasion. In moving
fibroblasts, the classical model for cell motility studies, the nucleus
remains centrally anchored within the cell despite drastic morphological
changes. However, the human lung adenocarcinoma cells, which generally display
a fibroblast-like motility, can be switched to the characteristic pattern of
cell translocation with nucleokinesis as a distinct step in response to an
autocrine motility factor (Klominek et
al., 1991
). The motility steps of the invasion process have been
proposed to comprise protrusion of the tumor cell pseudopodia through
surrounding tissues, followed by nucleokinesis and, finally, retraction of the
pseudopodia behind the nucleus.
On the basis of the data accumulated on NUANCE, we can envision several
roles for it. Rapid shrinkage of the nuclei accompanied by the accumulation of
cytoplasmic NUANCE and actin at the side of the nucleus in the LatA-treated
cells suggests that the actin cytoskeleton is implicated in controlling
nuclear shape and that this process could be mediated by NUANCE. In addition
to a role in nuclear positioning and/or migration, NUANCE may also contribute
to the mechanical connection between cell surface receptors, cytoskeletal
filaments and nuclear scaffolds. Such a coordinated response to mechanical
stress was demonstrated by micromanipulation of integrins, which resulted in
changes in nuclear shape that may influence gene expression
(Chicurel et al., 1998).
Maintenance of the nuclear shape and polarity could be essential for many cell
types undergoing mechanical stress in vivo, as for instance, for lymphocytes
when transgressing the blood vessels.
Eight integral proteins of the NE have so far been identified. Six of them
are located at the INM. These are lamina-associated polypepetide (LAP)1
(Senior and Gerace, 1988),
LAP2/thymopoietin (Foisner and Gerace,
1993
), p58/lamin B receptor (LBR)
(Worman et al., 1988
), emerin
(Nagano et al., 1996
), nurim
(Rolls et al., 1999
) and MAN1
(Lin et al., 2000
). Two
integral proteins, POM121 and gp210, have been found to be specific for the
nuclear pore membrane (Gerace et al.,
1982
; Soderqvist and Hallberg,
1994
). All of them are arranged with their N-termini facing the
nucleoplasm. The discovery of NUANCE as the first example of a protein, which
seems to associate specifically with the ONM, implies that the ONM represents
a membrane subdomain that is biochemically and functionally distinct from the
peripheral ER. Recent studies demonstrated that the dynein-dynactin complex is
recruited to the cytoplasmic face of the NE during late G2 and early prophase
prior to the NE breakdown and plays a role in tearing the NE at the beginning
of mitosis (Beaudouin et al.,
2002
; Salina et al.,
2002
). Our data suggest that the N-terminal part of NUANCE faces
the cytoplasm thereby allowing the protein to represent a platform for
anchoring the dynein-dynactin complexes at the ONM. Interestingly, the NE
wrinkles observed at the sites of invaginations of the `kidney bean' - shaped
nuclei in the LatA-treated cells resemble the microtubules containing
finger-like projections of the prophase and prometaphase cells. Furthermore,
accumulation of F-actin and the cytoplasmic NUANCE at the sites of nuclear
invaginations upon LatA treatment imply a link between NUANCE and
pericentrosomal astral complex. Although at present we have no evidence of a
functional relationship between NUANCE and NE breakdown, it is possible that
NUANCE is involved in the spatial organization of microtubule-dependent
machinery linking it to the NE.
Our results with digitonin-treated cells do not exclude an association of
NUANCE with the INM where it might face the nucleoplasm with its ABD and
interact with nuclear actin whose role is still elusive
(Rando et al., 2000). NUANCE
staining was also observed throughout the nucleoplasm and in nucleoli. These
forms might well represent differentially spliced variants lacking the TMD.
Such isoforms may build a fibrous meshwork in the nucleoplasm by
oligomerizating via leucine zippers of the coiled coil. Although we have not
identified TMD-lacking transcripts in our screen yet, their existence is quite
plausible since many membrane proteins including calmin are differentially
spliced and give rise to soluble isoforms
(Ishisaki et al., 2001
). It
can be speculated that NUANCE plays a role in the organization of the
intranuclear space. It was shown that actin is associated with the
nucleoplasmic filaments of nuclear pore complexes and is involved in nuclear
export (Hofmann et al., 2001
).
A role of NUANCE in nuclear export would explain its partial colocalization
with nuclear pores. Actin may also be important for packaging the RNA into a
Balbiani ring particles and their translocation through the nuclear pores to
the cytoplasm (Percipalle et al.,
2001
). In a complex with actin, NUANCE may play a role in location
and transportation of proteins and RNA within the nucleoplasm. The observation
of NUANCE in nucleoli is particularly intriguing. The nucleoli are structures
responsible mainly for ribosomal biogenesis; however they also participate in
processing or export of some mRNAs and tRNAs
(Schneiter et al., 1995
;
Bertrand et al., 1998
) and are
involved in sequestering cell cycle regulating proteins
(Visintin and Amon, 2000
).
NUANCE would make an ideal candidate for organizing the nucleoli matrix and
ensuring its compartmentalization and integrity.
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Adam, S. A., Marr, R. S. and Gerace, L. (1990). Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111,807 -816.[Abstract]
Ahn, A. H. and Kunkel, L. M. (1993). The structural and functional diversity of dystrophin. Nat. Genet. 3,283 -291.[Medline]
Apel, E. D., Lewis, R. M., Grady, R. M. and Sanes, J. R.
(2000). Syne-1, a dystrophin- and Klarsicht-related protein
associated with synaptic nuclei at the neuromuscular junction. J.
Biol. Chem. 275,31986
-31995.
Beaudouin, J., Gerlich, D., Daigle, N., Eils, R. and Ellenberg, J. (2002). Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108, 83-96.[Medline]
Bertrand, E., Houser-Scott, F., Kendall, A., Singer, R. H. and
Engelke, D. R. (1998). Nucleolar localization of early tRNA
processing. Genes Dev.
12,2463
-2468.
Breathnach, R. and Chambon, P. (1981). Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50,349 -383.[Medline]
Brown, A., Bernier, G., Mathieu, M., Rossant, J. and Kothary, R. (1995). The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1. Nat. Genet. 10,301 -306.[Medline]
Chicurel, M. E., Singer, R. H., Meyer, C. J. and Ingber, D. E. (1998). Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions. Nature 392,730 -733.[Medline]
Foisner, R. and Gerace, L. (1993). Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. Cell 73,1267 -1279.[Medline]
Fuchs, E. and Yang, Y. (1999). Crossroads on cytoskeletal highways. Cell 98,547 -550.[Medline]
Gerace, L., Ottaviano, Y. and Kondor-Koch, C. (1982). Identification of a major polypeptide of the nuclear pore complex. J. Cell Biol. 95,826 -837.[Abstract]
Gregory, S. L. and Brown, N. H. (1998). Kakapo,
a gene required for adhesion between and within cell layers in
Drosophila, encodes a large cytoskeletal linker protein related to
plectin and dystrophin. J. Cell Biol.
143,1271
-1282.
Hofmann, W., Reichart, B., Ewald, A., Muller, E., Schmitt, I.,
Stauber, R. H., Lottspeich, F., Jockusch, B. M., Scheer, U., Hauber, J. and
Dabauvalle, M. C. (2001). Cofactor requirements for nuclear
export of Rev response element (RRE)- and constitutive transport element
(CTE)- containing retroviral RNAs. An unexpected role for actin. J.
Cell Biol. 152,895
-910.
Ishisaki, Z., Takaishi, M., Furuta, I. and Huh, N. (2001). Calmin, a protein with calponin homology and transmembrane domains expressed in maturing spermatogenic cells. Genomics 74,172 -179.[Medline]
Karakesisoglou, I., Yang, Y. and Fuchs, E.
(2000). An epidermal plakin that integrates actin and microtubule
networks at cellular junctions. J. Cell Biol.
149,195
-208.
Klominek, J., Sundqvist, K. G. and Robert, K. H. (1991). Nucleokinesis: distinct pattern of cell translocation in response to an autocrine motility factor-like substance or fibronectin. Proc. Natl. Acad. Sci. USA 88,3902 -3906.[Abstract]
Korenbaum, E., Nordberg, P., Bjorkegren-Sjogren, C., Schutt, C. E., Lindberg, U. and Karlsson, R. (1998). The role of profilin in actin polymerization and nucleotide exchange. Biochemistry 37,9274 -9283.[Medline]
Kozak, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15,8125 -8148.[Abstract]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Leung, C. L., Sun, D., Zheng, M., Knowles, D. R. and Liem, R.
K. (1999). Microtubule actin cross-linking factor (MACF): a
hybrid of dystonin and dystrophin that can interact with the actin and
microtubule cytoskeletons. J. Cell Biol.
147,1275
-1286.
Leung, C. L., Liem, R. K., Parry, D. A. and Green, K. J.
(2001a). The plakin family. J. Cell Sci.
114,3409
-3410.
Leung, C. L., Zheng, M., Prater, S. M. and Liem, R. K.
(2001b). The BPAG1 locus: Alternative splicing produces multiple
isoforms with distinct cytoskeletal linker domains, including predominant
isoforms in neurons and muscles. J. Cell Biol.
154,691
-697.
Lin, F., Blake, D. L., Callebaut, I., Skerjanc, I. S., Holmer,
L., McBurney, M. W., Paulin-Levasseur, M. and Worman, H. J.
(2000). MAN1, an inner nuclear membrane protein that shares the
LEM domain with lamina-associated polypeptide 2 and emerin. J.
Biol. Chem. 275,4840
-4847.
Matsudaira, P. (1994). Actin crosslinking proteins at the leading edge. Semin. Cell Biol. 5, 165-174.[Medline]
Morris, N. R. (2000). Nuclear migration. From
fungi to the mammalian brain. J. Cell Biol.
148,1097
-1101.
Mosley-Bishop, K. L., Li, Q., Patterson, L. and Fischer, J. A. (1999). Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye. Curr. Biol. 9,1211 -1220.[Medline]
Nagano, A., Koga, R., Ogawa, M., Kurano, Y., Kawada, J., Okada, R., Hayashi, Y. K., Tsukahara, T. and Arahata, K. (1996). Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy. Nat. Genet. 12,254 -259.[Medline]
Nobes, C. D. and Hall, A. (1995). Rho, rac and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibres, lamellipodia and filopodia. Cell 81,53 -62.[Medline]
Okuda, T., Matsuda, S., Nakatsugawa, S., Ichigotani, Y., Iwahashi, N., Takahashi, M., Ishigaki, T. and Hamaguchi, M. (1999). Molecular cloning of macrophin, a human homologue of Drosophila kakapo with a close structural similarity to plectin and dystrophin. Biochem. Biophys. Res. Commun. 264,568 -574.[Medline]
Olski, T. M., Noegel, A. A. and Korenbaum, E.
(2001). Parvin, a 42 kDa focal adhesion protein, related to the
alpha-actinin superfamily. J. Cell Sci.
114,525
-538.
Pascual, J., Castresana, J. and Saraste, M. (1997). Evolution of the spectrin repeat. Bioessays 19,811 -817.[Medline]
Pepperkok, R., Scheel, J., Horstmann, H., Hauri, H. P., Griffiths, G. and Kreis, T. E. (1993). Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell 74, 71-82.[Medline]
Percipalle, P., Zhao, J., Pope, B., Weeds, A., Lindberg, U. and
Daneholt, B. (2001). Actin bound to the heterogeneous nuclear
ribonucleoprotein hrp36 is associated with Balbiani ring mRNA from the gene to
polysomes. J. Cell Biol.
153,229
-236.
Prokop, A., Uhler, J., Roote, J. and Bate, M.
(1998). The kakapo mutation affects terminal arborization and
central dendritic sprouting of Drosophila motorneurons. J.
Cell Biol. 143,1283
-1294.
Puius, Y. A., Mahoney, N. M. and Almo, S. C. (1998). The modular structure of actin-regulatory proteins. Curr. Opin. Cell Biol. 10, 23-34.[Medline]
Rando, O. J., Zhao, K. and Crabtree, G. R. (2000). Searching for a function for nuclear actin. Trends Cell Biol. 10,92 -97.[Medline]
Rivero, F., Kuspa, A., Brokamp, R., Matzner, M. and Noegel, A.
A. (1998). Interaptin, an actin-binding protein of the
alpha-actinin superfamily in Dictyostelium discoideum, is developmentally and
cAMP-regulated and associates with intracellular membane compartments.
J. Cell Biol. 142,735
-750.
Rolls, M. M., Stein, P. A., Taylor, S. S., Ha, E., McKeon, F.
and Rapoport, T. A. (1999). A visual screen of a GFP-fusion
library identifies a new type of nuclear envelope membrane protein.
J. Cell Biol. 146,29
-44.
Ruhrberg, C. and Watt, F. M. (1997). The plakin family: versatile organizers of cytoskeletal architecture. Curr. Opin. Genet. Dev. 7,392 -397.[Medline]
Salina, D., Bodoor, K., Eckley, D. M., Schroer, T. A., Rattner, J. B. and Burke, B. (2002). Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell 108,97 -107.[Medline]
Schneiter, R., Kadowaki, T. and Tartakoff, A. M. (1995). mRNA transport in yeast: time to reinvestigate the functions of the nucleolus. Mol. Biol. Cell 6, 357-370.[Abstract]
Selbert, S., Fischer, P., Pongratz, D., Stewart, M. and Noegel,
A. A. (1995). Expression and localization of annexin VII
(synexin) in muscle cells. J. Cell Sci.
108, 85-95.
Senior, A. and Gerace, L. (1988). Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina. J. Cell Biol. 107,2029 -2036.[Abstract]
Soderqvist, H. and Hallberg, E. (1994). The large C-terminal region of the intergral pore membrane protein, POM121, is facing the nuclear pore complex. Eur. J. Cell Biol. 64,186 -191.[Medline]
Strumpf, D. and Volk, T. (1998). Kakapo, a
novel cytoskeletal-associated protein is essential for the restricted
localization of the neuregulin-like factor, vein, at the muscle-tendon
junction site. J. Cell Biol.
143,1259
-1270.
Sun, Y., Zhang, J., Kraeft, S. K., Auclair, D., Chang, M. S.,
Liu, Y., Sutherland, R., Salgia, R., Griffin, J. D., Ferland, L. H. et al.
(1999). Molecular cloning and characterization of human
trabeculin-alpha, a giant protein defining a new family of actin-binding
proteins. J. Biol. Chem.
274,33522
-33530.
Tinsley, J. M., Blake, D. J., Roche, A., Fairbrother, U., Riss, J., Byth, B. C., Knight, A. E., Kendrick-Jones, J., Suthers, G. K., Love, D. R. et al. (1992). Primary structure of dystrophin-related protein. Nature 360,591 -593.[Medline]
Visintin, R. and Amon, A. (2000). The nucleolus: the magician's hat for cell cycle tricks. Curr. Opin. Cell Biol. 12,372 -377.[Medline]
Wiche, G. (1998). Domain structure and
transcript diversity of plectin. Biol. Bull.
194,381
-383.
Wiche, G., Becker, B., Luber, K., Weitzer, G., Castanon, M. J., Hauptmann, R., Stratowa, C. and Stewart, M. (1991). Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil. J. Cell Biol. 114,83 -99.[Abstract]
Winder, S. J., Gibson, T. J. and Kendrick-Jones, J. (1995). Dystrophin and utrophin: the missing links! FEBS Lett. 369,27 -33.[Medline]
Wolf, E., Kim, P. S. and Berger, B. (1997).
MultiCoil: a program for predicting two- and three-stranded coiled coils.
Protein Sci. 6,1179
-1189.
Worman, H. J., Yuan, J., Blobel, G. and Georgatos, S. D. (1988). A lamin B receptor in the nuclear envelope. Proc. Natl. Acad. Sci. USA 85,8531 -8534.[Abstract]
Wu, J., Matunis, M. J., Kraemer, D., Blobel, G. and Coutavas,
E. (1995). Nup358, a cytoplasmically exposed nucleoporin with
peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A
homologous domain, and a leucine-rich region. J. Biol.
Chem. 270,14209
-14213.
Yarmola, E. G., Somasundaram, T., Boring, T. A., Spector, I. and
Bubb, M. R. (2000). Actin-latrunculin A structure and
function. Differential modulation of actin-binding protein function by
latrunculin A. J. Biol. Chem.
275,28120
-28127.
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