From the Department of Human Anatomy and Genetics,
University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom
and the § Medical Research Council Functional Genetics Unit,
Department of Human Anatomy and Genetics, University of Oxford, South
Parks Road, Oxford OX1 3QX, United Kingdom
Received for publication, September 11, 2000, and in revised form, October 24, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dystrophin coordinates the assembly of a complex
of structural and signaling proteins that are required for normal
muscle function. A key component of the dystrophin protein complex is The muscular dystrophies are a group of clinically and genetically
heterogeneous diseases characterized by progressive muscle wasting.
While mutations in many muscle genes are known to be responsible for
various forms of muscular dystrophy, the most common form is caused by
mutations in the Duchenne muscular dystrophy gene that encodes
dystrophin (1-3). Dystrophin is a large, actin-binding protein that is
pivotal in assembling a complex of membrane-associated and cytoplasmic
proteins, which make up the dystrophin protein complex
(DPC)1 (4, 5). The DPC can be
divided into three subcomplexes: the transmembrane dystroglycan
complex, the membrane-spanning sarcoglycan complex, and the
To further dissect these roles of Library Construction and Yeast Two-hybrid Screening--
The H2K
myoblast cell line was used to generate a cDNA library for yeast
two-hybrid screening (24, 25). Briefly, H2K myoblasts were
differentiated for 6 days into myotubes prior to harvest and generation
of poly(A)+ RNA using the Fast Track 2.0 system
(Invitrogen). cDNA was generated using methods described in Ref.
15. BstXI/EcoRI adaptors were ligated onto the
cDNA ends and subsequently cloned into the BstXI sites
of the yeast expression vector pYESTrp2 (Invitrogen). This fuses the
cDNAs with the B42 activation domain. The cDNA library was then
transformed into chemically competent Escherichia coli XL-1Blue, and purified plasmid DNA was extracted directly from the
plated library. The entire cDNA Library Screening--
The H2K cDNA library was
plated onto Hybond-N membranes (1 × 106 colonies) and
screened by hybridization with the 32P-labeled 5'
EcoRI/BamHI fragment of the SNIP4 cDNA clone
(see below). Hybridizing clones were isolated, purified, and sequenced using standard methods.
Northern Blotting--
Mouse multiple tissue Northern blots
(CLONTECH) were probed with the entire
32P-labeled SNIP4 sequence, generated by an
EcoRI digest. The membrane was hybridized overnight and then
washed to a stringency of 0.25× SSC (37.5 mM NaCl, 3.75 mM sodium citrate), 0.1% (w/v) SDS at 65 °C and exposed
overnight to film.
Antibodies--
The entire open reading frame of syncoilin was
produced as a thioredoxin fusion protein by subcloning the 1.8-kb
EcoRI insert from the SNIP4 cDNA clone into the
EcoRI site of pET32c (Novagen). This construct was
transformed into E. coli BL21 (DE3) Gold (Stratagene), and
SYNC-trx fusion protein synthesis was induced by overnight growth.
SYNC-trx was purified under denaturing conditions using the Talon resin
(CLONTECH) as per the manufacturer's instructions. Purified protein was used to immunize New Zealand White rabbits using
standard protocols. Affinity purification of the SYNC-FP antiserum was
performed by coupling the SYNC-trx fusion protein to SulfoLink coupling
gel (Pierce) according to the manufacturer's instructions. Eluted
SYNC-FP antibodies were further preabsorbed against purified
thioredoxin coupled to SulfoLink. Affinity-purified and preabsorbed
SYNC-FP antiserum was used in all subsequent applications. The
specificity of this antibody was determined by preabsorption of SYNC-FP
antiserum diluted 1:200 in phosphate-buffered saline (PBS) with a 1 mM concentration of the immunizing fusion protein SYNC-trx
for 1 h at room temperature. The preabsorbed serum was used to
label skeletal muscle quadricep sections as described below. The
following polyclonal antibodies are described elsewhere: dystrobrevin
antibodies Generation and Transfection of Expression Constructs--
The
syncoilin expression construct sync:pCIneo was made by cloning the
entire SNIP4 cDNA as an EcoRI fragment into the
EcoRI site of pCI-neo (Promega). The Myc-tagged
Immunoprecipitation Analysis--
Transfected COS-7 cells, H2K
myotubes, and flash-frozen mouse skeletal muscle were homogenized in
solubilization buffer (150 mM NaCl, 1% (v/v) Nonidet P-40,
0.05% (w/v) SDS, 50 mM Tris, pH 7.4, plus protease
inhibitors (Sigma)). Homogenates were incubated on ice for 30 min and
centrifuged at 14,000 rpm in a microcentrifuge for 30 min at 4 °C to
remove insoluble material. All subsequent steps were carried out at
4 °C. For immunoprecipitations, 1-2 mg of soluble protein in a
total volume of 1 ml was precleared with 100 µl of anti-rabbit
Magnabind beads (Pierce) on a blood mill for 2 h. After removal of
the beads using a magnetic particle concentrator (Dynal), the following
antibodies were added and incubated overnight: SYNC-FP (5 µl),
Protein Preparation and Western Blotting--
Skeletal muscle,
heart, kidney, liver, lung, and brain were dissected from a normal
adult C57 BL/10 mouse, flash-frozen in liquid nitrogen, and
subsequently homogenized separately in 8 ml of SDS/urea buffer. In
addition, skeletal muscle extracts were prepared from five different
strains of mice: a C57 normal mouse, a dystrophin-deficient
mdx mouse, a utrophin-deficient (utrn Immunofluorescence Microscopy--
8-µm cryosections of adult
skeletal muscle from normal C57 BL/10 and mdx mice were
prepared as described previously (31). Sections and transfected COS-7
cells were blocked in 10% (v/v) fetal calf serum PBS for 30 min at
room temperature prior to incubation with the primary antibodies. The
following antibodies were diluted in PBS: SYNC-FP (1:200), Radiation Hybrid Mapping--
The T31 Mouse-Hamster Radiation
Hybrid Mapping Panel (Research Genetics) (34) was screened to determine
mouse chromosomal localization of the syncoilin gene. The forward
primer (5'-ATGAGAAGCCAGCACTGACA-3') and the reverse primer
(5'-TCGGTGTTGGGAATCGGTTGA-3') were designed against the SNIP4 cDNA
sequence and resulted in a PCR product of 816 base pairs. Ninety-three
cell lines were analyzed and scored based on the presence or absence of
a PCR product. Data were submitted to the Jackson Laboratory Mapping
Panel (available on the World Wide Web) for multipoint analysis.
The human chromosomal localization of the syncoilin gene was determined
using the G3 Human-Hamster Radiation Hybrid Panel (Research Genetics).
Eighty-three cell lines were screened by PCR using the forward primer
(5'-ATCCATGAGCTTGTATTGCTC-3') and the reverse primer
(5'-ACTGGGCAGAGAGTCGC-3'). These primers were designed against the
corresponding human sequence (GenBankTM accession number
AL138800) and gave a PCR product of 393 base pairs. Data were submitted
to the Stanford Human Genome Center RH Data base (available on the
World Wide Web) for analysis.
Identification of a Novel Syncoilin Encodes a Novel Member of the Intermediate Filament
Superfamily--
The complete nucleotide sequence of the cDNA
clone SNIP4 predicts an uninterrupted open reading frame from its
5'-end with a stop codon at position 1422. The fourth predicted amino
acid encodes a methionine residue with an accompanying Kozak consensus sequence. To generate additional 5' sequence of the syncoilin transcript and to confirm that this residue is indeed the first methionine of the syncoilin protein, the H2K cDNA library was rescreened with the 5' EcoRI/BamHI fragment of
SNIP4. This screen identified a single clone that contained an
additional 43 nucleotides at its 5'-end compared with the original
SNIP4 cDNA clone. Within this additional sequence are two in-frame
stop codons, confirming that the start of translation does indeed occur
at the methionine present in the SNIP4 sequence. The nucleotide
sequence and complete open reading frame of syncoilin is shown in Fig.
2A (GenBankTM
accession number AJ251641). This gives a protein with a predicted molecular mass of 53.6 kDa and pI of 4.5.
To determine whether the syncoilin sequence is similar to other known
proteins, a gapped BLAST search (36) of the nonredundant protein
sequence data base was performed at the National Center for
Biotechnology Information (available on the World Wide Web) using the SEG filter option. This search showed that syncoilin is a
novel sequence. Using the COILS prediction program (available on the
World Wide Web), syncoilin was found to have a region containing coiled-coils. These coiled-coils were significantly similar to those
found in intermediate filament proteins. The top 107 highest scoring
proteins in the BLAST search (E < 4 × 10-6) were all members of the intermediate filament family.
Syncoilin was most similar in sequence to rat
The human orthologue of syncoilin was identified by a BLAST search of
the human expressed sequence tag data base and the human high
throughput genomic sequence data base at the National Center for
Biotechnology. The sequence of human syncoilin was assembled from a
human genomic sequence (chromosome 1 clone RP5-887B19, map
p34.1-34.3, GenBankTM Accession Number AL138800) that
overlaps with three expressed expressed sequence tags
(GenBankTM accession numbers AI281998, W24127, and
AI074110), and the conceptual translation is shown in Fig.
2B. Comparison of the human and mouse sequences reveals an
overall identity of 76%.
Syncoilin Is Expressed in Mammalian Skeletal Muscle and
Heart--
To determine the expression pattern of syncoilin, we
hybridized a mouse multiple tissue Northern blot with the entire SNIP4 cDNA clone. As Fig. 3A
illustrates, syncoilin transcripts were found to be highly expressed in
skeletal muscle and heart, two tissues known to be rich in
To investigate the protein products of these transcripts, a
syncoilin-specific polyclonal antibody called SYNC-FP was generated. The entire open reading frame of syncoilin contained within the SNIP4
cDNA clone was fused in frame to thioredoxin and expressed in
E. coli, and the purified fusion protein was used for
immunizing rabbits. The resulting SYNC-FP antiserum was
affinity-purified against the thioredoxin-syncoilin fusion protein and
subsequently preabsorbed against purified thioredoxin. Fig.
3B shows that probing a mouse multiple tissue Western blot
with SYNC-FP led to the detection of two protein products in the heart
of ~55 and 64 kDa. The presence of two bands suggests that syncoilin
may undergo alternative splicing. Alternatively, syncoilin may be
subject to proteolytic processing, resulting in the production of a
smaller protein species in the heart. The 64-kDa protein was also
detected strongly in skeletal muscle, while a faint 55-kDa band was
detected in the lung. To determine which of these protein products
corresponds to the syncoilin sequence encoded by SNIP4, the SNIP4
cDNA was cloned into pCI-neo to generate the expression construct
sync:pCIneo, which was then transfected into COS-7 cells. Western blots
of transfected COS-7 cell extracts probed with SYNC-FP revealed that
the relative mobility of sync:pCIneo is identical to the 64-kDa protein
detected in skeletal muscle and heart (Fig. 3B,
final lane). This result suggests that the larger
protein product detected on the multiple tissue Western blot
corresponds to the protein identified in the yeast two-hybrid screen.
In Vitro Binding of
Cotransfection of COS-7 cells with sync:pCIneo and m32Myc:pCIneo
resulted in the apparent colocalization of syncoilin and
To demonstrate a direct interaction between syncoilin and
Colocalization of In Vivo Association of
Immunoprecipitation experiments performed using skeletal muscle
extracts and the SYNC-FP antibody resulted in the precipitation of a
small amount of Syncoilin Expression Is Increased in Dystrophic Muscle--
To
investigate the localization of syncoilin in dystrophin-deficient
muscle, mdx and normal C57 quadricep sections were
immunostained with the SYNC-FP antibody. As Fig.
8A shows, syncoilin
immunoreactivity in mdx muscle was found to be maintained at
NMJs but was increased at both the sarcolemma and within muscle fibers.
This increase in muscle fiber staining appeared to correlate with
regenerating muscle fibers, as identified by the presence of centrally
located nuclei (data not shown). This observation contrasts markedly
with the localization of
To determine whether this increase in syncoilin immunoreactivity in
mdx muscle is reflected in an increase in levels of the protein, Western blots of total protein from skeletal muscle of normal
and mdx muscle were probed with SYNC-FP. In addition, muscle extracts from a utrophin-deficient mouse (utrn Syncoilin Is Located on Mouse Chromosome 4 and Human
Chromosome 1--
To determine the mouse and human chromosomal
localizations of syncoilin, radiation hybrid panels were screened by
PCR using primers specific to the syncoilin gene. The mouse syncoilin
gene is located on chromosome 4 at position 59.9-60
centimorgans, between markers D4Mit203 (LOD 18.4) and D4Mit72
(LOD 14.0). The human syntenic region is 1p32-36, and mapping to
chromosome 1 was confirmed by the G3 Human-Hamster Hybrid Panel. The
syncoilin gene was located between markers SHGC-30801 (LOD 12.98) and
SHGC-57292 (LOD 12.32), which correspond to D1S2569 and D1S2676,
respectively. D1S2676 has been mapped by fluorescence in
situ hybridization to chromosome 1p33-34.3 (Sanger Center
Cytogenetics), and a BLAST data base search with the mouse cDNA
matched a human chromosome 1 sequence in the interval p34.1-34.3
(GenBankTM accession number AL138800). The localization of
syncoilin to human chromosome 1p makes it a candidate gene for
muscle-eye-brain disease, which has been mapped to 1p32-34 by linkage
analysis (39).
Here we describe the interaction between The intermediate filaments, along with actin-containing microfilaments
and tubulin-containing microtubules, make up the three major classes of
cytoskeletal filaments in multicellular animals (37, 38). There are
over 50 members of the intermediate filament protein superfamily, and
these have been divided into six classes based on sequences comparisons
throughout their rod domains. The rod domain promotes coiled-coil
interactions between two individual intermediate filament proteins to
initiate the formation of 10-nm diameter filaments. Syncoilin is most
similar to class III and class IV intermediate filaments including
Overexpression of the syncoilin cDNA in COS-7 cells did not result
in the formation of intracellular filaments, indicating that syncoilin
cannot self-assemble into 10-nm structures under these conditions. One
explanation for this may be that syncoilin is a component of
heterotypic polymers, in which case it would require a second
intermediate filament family member to form functional filaments. A
number of classes of intermediate filament proteins, including the
class I and II keratins and the class IV neurofilament proteins, are
known to form obligate heteropolymers in vivo, resulting in
intermediate filaments that consist of at least two different proteins
(38). Furthermore, recent evidence has shown that desmin, the major
intermediate filament protein in mature muscle, forms heteropolymeric
filaments with a newly described member of the intermediate filament
superfamily called synemin (40). Since both desmin and the large
intermediate filament protein nestin are localized to the NMJ of
normal skeletal muscle, these are candidates for associating with
syncoilin to form heterofilaments (41-44). A second explanation for
syncoilin's lack of self-assembly is that this protein may not
participate in filament formation per se but instead
associates with intermediate filaments to perform an accessory role.
Synemin, for example, was initially proposed to play a role in
cross-linking intermediate filaments, prior to its identification as a
bona fide intermediate filament protein (45).
Syncoilin is highly expressed in skeletal and cardiac muscle, tissues
both rich in Circumstantial evidence supports the idea that the
syncoilin/ Most DPC proteins, including The involvement of intermediate filament proteins in muscle disease has
been described. Mice that lack desmin develop skeletal and cardiac
myopathy (53), and recent studies have shown that mutations in the
human desmin gene cause a distinct "desmin myopathy," a disease
that is often associated with cardiomyopathy (54). In a number of the
cases examined, a mutant desmin protein was produced and interfered
with the normal assembly of intermediate filaments, resulting in
fragility of the myofibrils (54). Another example of the role of
intermediate filaments in muscle disease is illustrated by Emery
Dreifuss muscular dystrophy. The autosomal dominant form of Emery
Dreifuss muscular dystrophy is caused by mutations in the lamin A/C
gene (55). Lamin A and C are class V intermediate filament proteins of
the nuclear lamina, a lattice structure localized between chromatin and
the inner nuclear membrane (38). While it is not understood how
mutations in the lamin A/C gene result in muscular dystrophy, it has
been suggested that lamins A and C are involved in protecting the
nuclear membrane from the mechano-physical stress encountered during
muscle contraction and relaxation (56). Finally, loss of the
intermediate filament-associated protein plectin also results in
muscular dystrophy and epidermal bullosa in humans and mice (57,
58).
Mapping of the syncoilin gene to human chromosome 1p34.1-34.3 makes it
a candidate gene for muscle-eye-brain disease (39). This disease is
characterized by congenital muscular dystrophy with consistent ocular
and central nervous system involvement that commonly manifests itself
in myopia and severe mental retardation (59). While syncoilin was not
detected in adult mouse brain by Northern or Western analysis, there is
a possibility that low levels of this gene are expressed in the brain
and eye. Alternatively, syncoilin may be developmentally expressed. The
association of syncoilin with In summary, we have shown that -dystrobrevin, a dystrophin-associated protein whose absence results
in neuromuscular junction defects and muscular dystrophy. To gain
further insights into the role of
-dystrobrevin in skeletal muscle,
we used the yeast two-hybrid system to identify a novel
-dystrobrevin-binding partner called syncoilin. Syncoilin is a new
member of the intermediate filament superfamily and is highly expressed
in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is
concentrated at the neuromuscular junction, where it colocalizes and
coimmunoprecipitates with
-dystrobrevin-1. Expression studies in
mammalian cells demonstrate that, while
-dystrobrevin and syncoilin
associate directly, overexpression of syncoilin does not result in the
self-assembly of intermediate filaments. Finally, unlike many
components of the dystrophin protein complex, we show that syncoilin
expression is up-regulated in dystrophin-deficient muscle. These data
suggest that
-dystrobrevin provides a link between the dystrophin
protein complex and the intermediate filament network at the
neuromuscular junction, which may be important for the maintenance and
maturation of the synapse.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystrobrevin-syntrophin cytoplasmic complex (5, 6). All of these
subcomplexes are thought to act in concert to provide a link between
the extracellular matrix and the actin cytoskeleton (7) and thereby
provide mechanical support to the muscle membrane (8, 9). Loss of the
DPC from the sarcolemma through the absence of dystrophin is thought to disrupt membrane integrity, ultimately leading to loss of muscle function (10).
-Dystrobrevin is a member of the dystrophin family of proteins and a
key component of the DPC that binds directly to dystrophin and
syntrophin (11-16). In mammalian muscle, there are at least three
isoforms,
-dystrobrevin-1 (94 kDa),
-dystrobrevin-2 (62 kDa), and
-dystrobrevin-3 (42 kDa), which are generated by alternative splicing of the single
-dystrobrevin gene (15, 17, 18). While
-dystrobrevin-1 and -2 contain dystrophin and syntrophin binding
sites,
-dystrobrevin-3 lacks both of these sites (19). The exact
function of the individual isoforms remains unclear, but the recent
characterization of the
-dystrobrevin knock-out mouse has shed new
light on the combined functions of these proteins in skeletal muscle.
-Dystrobrevin-deficient mice have neuromuscular junction (NMJ)
abnormalities (20). While NMJs form normally, the postsynaptic
apparatus matures abnormally, resulting in destabilized acetylcholine
receptor (AChR) clusters and altered synaptic morphology.
-Dystrobrevin-deficient mice also develop mild muscular dystrophy, similar to that of the dystrophin-deficient mdx mouse (21). Unlike the mdx mouse, however, where the loss of dystrophin
results in the loss of other components of the DPC at the sarcolemma
(22), loss of
-dystrobrevin results in neither the perturbation of the DPC nor the disruption of the structural integrity of the muscle
membrane (21). This result implies that other mechanisms, including
aberrant cell signal transduction, are likely to contribute to the
pathogenesis of muscular dystrophy (21, 23). Thus, it has been proposed
that the
-dystrobrevins have both a structural role at the NMJ and a
signaling role in muscle.
-dystrobrevin in skeletal muscle,
we have screened for new interacting proteins using the yeast
two-hybrid system. Here we describe the interaction between
-dystrobrevin and a novel member of the intermediate filament superfamily, which we have named syncoilin. Syncoilin is concentrated at the NMJ of normal skeletal muscle, where it colocalizes and coimmunoprecipitates with
-dystrobrevin-1. This interaction is likely to be important in maintaining the integrity of the NMJ in
normal muscle and provides a link between the DPC and the intermediate filament cytoskeletal network.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystrobrevin-2 coding sequence including
the muscle-expressed variable region-3 (vr3) sequence was PCR-amplified
using the following primers: forward,
5'-GTGAATTCAGAATGATTGAAGATAGTGGA-3'; reverse,
5'-CACTCGAGTGTTAGTTAAGACCTGCAGTA-3'. The amplified PCR product was
subcloned into the EcoRI/XhoI sites of the yeast
bait vector pHybLex/Zeo to generate
DB2:pHybLex. This placed the
-dystrobrevin-2 sequence in frame with the DNA binding domain of the
LexA transcriptional activator. A bait strain was created by
transforming
DB2:pHybLex into Saccharomyces cerevisiae
strain L40 as described in the manufacturer's instructions (Hybrid
Hunter; Invitrogen). The bait strain was cotransformed with the H2K
cDNA library, and transformants were plated onto minimal yeast
medium lacking histidine, tryptophan, uracil and lysine,
containing 300 µg/ml Zeocin (Invitrogen) and 5 mM
3-aminotriazole. Plates were incubated at 30 °C for 5 days, and
yeast colonies that grew on histidine-deficient medium were restreaked onto fresh selective plates and assayed for
-galactosidase activity as per the manufacturer's instructions.
Prey plasmids were isolated from yeast and electroporated into E. coli XL-1Blue electrocompetent cells (Stratagene). The 5'-end of
each clone was sequenced using a vector primer. To confirm the
interaction in yeast, purified prey plasmids were retransformed with
the
DB2:pHybLex bait and with the empty bait vector pHybLex/Zeo and
tested for growth on selective plates and
-galactosidase activity.
Other control yeast strains used were the positive control strain
containing pHybLex/Zeo-FOS2 and pYESTrp-Jun plasmids (Invitrogen) and
the negative control containing the empty bait and prey vectors
pHybLex/Zeo and pYESTrp2.
1CT-FP (26),
CT-FP (26),
2-PEP (27), and
-PAN
(27); the anti-dystrophin antibody 2166 (28); the anti-utrophin
antibody URD40 (28); and the
2-syntrophin antibody 2045 (29). The
9E10 monoclonal anti-Myc antibody was purchased from Covance (Berkeley,
CA). The MANDRA1 anti-dystrophin monoclonal antibody was kindly
supplied by Prof. Glenn Morris (30). For immunofluorescence studies,
rhodamine Red-X-conjugated donkey anti-rabbit IgG (Jackson
Immunoresearch), Alexa 488-conjugated goat anti-mouse IgG (Molecular
Probes, Inc., Eugene, OR), and Alexa 488
-bungarotoxin (Molecular
Probes) were used. For Western blot analysis, horseradish
peroxidase-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch)
was used.
-dystrobrevin-1 and -2 expression constructs containing the
muscle-specific vr3 sequence were generated by digesting the cDNA
clones m24 (murine
-dystrobrevin-1 (15)) and m32 (murine
-dystrobrevin-2 (15)) with BglII and inserting a
double-stranded DNA insert encoding the Myc epitope (EQKLISEEDL),
generated by annealing a pair of phosphorylated oligonucleotides
(5'-GATCTGGAGCAAAAGCTCATTTCTGAAGAGGACTTG-3', 5'-GATCCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCA-3'). The vr3 sequence was
generated by PCR on reverse-transcribed first strand cDNAs from
skeletal muscle and inserted using the restriction enzymes Bsu361 and ClaI into the appropriate region of
m24 and m32, replacing the Bsu361/ClaI fragment
without the vr3 sequence with the vr3-containing sequence. The modified
cDNA clones were excised by SpeI-NotI
digestion and inserted in the sense orientation into pCI-neo cut with
NheI and NotI. The constructs pDp116 (encoding
rat Dp116) and
2syn:pCIneo (encoding
2-syntrophin) are described
elsewhere.2 All expression
constructs were prepared in E. coli XL-1Blue and purified
using the Qiagen Plasmid Maxikit ready for transfection. 24 h
prior to transfection, COS-7 cells were seeded onto coverslips in
six-well plates at a density of 1.5 × 105 cells/well
or 2 × 106 cells/15-cm dish. Cells were transfected
with 1 µg of plasmid DNA/well or 15 µg of DNA/dish using Fugene-6
(Roche Molecular Biochemicals) following the manufacturer's
instructions. After 24 h, cells on coverslips were washed twice in
PBS, fixed in ice-cold 4% (w/v) paraformaldehyde in PBS for 15 min,
and permeabilized in 0.1% (v/v) Triton X-100 in PBS for 10 min prior
to being processed for immunofluorescence microscopy as described
below. Cells plated on dishes were washed three times in PBS prior to
being processed for immunoprecipitation analysis.
1CT-FP (4 µl),
CT-FP (8 µl), 2166 (5 µl), 2045 (8 µl),
and URD40 (10 µl). Control experiments without antibody were set up
in parallel. Immune complexes were captured by the addition of
anti-rabbit Magnabind beads overnight. Beads were washed four times
with 1 ml of solubilization buffer and eluted in 60 µl of SDS/urea
buffer (75 mM Tris, pH 6.8, 3.8% (w/v) SDS, 4 M urea, 5% (v/v)
-mercaptoethanol, 20% (v/v) glycerol) and analyzed by Western blotting.
/
) mouse
(31), a dystrophin- and utrophin-deficient (dko) mouse (32), and an
mdx mouse transgenic for full-length utrophin (Fio) (33) and
the tissue treated as above. These mice were all ~10 weeks old. 25 µg of total protein or 10 µl of each immunoprecipitation experiment
was separated on 10% SDS-polyacrylamide gels and electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell). After blocking the membranes for 1 h in Tris-buffered saline
containing 0.1% (v/v) Tween 20 (TBS-T) and 5% (w/v) nonfat dry milk,
blots were incubated for 1 h with the following primary
antibodies: SYNC-FP (1:500),
1CT-FP (1:1000),
CT-FP (1:250),
URD40 (1:250), and 2166 (1:500). Membranes were washed twice in TBS-T
and once in blocking buffer prior to incubation for 1 h with
horseradish peroxidase-conjugated donkey anti-rabbit IgG diluted 1:3000
in blocking buffer. Membranes were washed extensively in TBS-T and were
developed using the BM chemiluminescence substrate system according to
the manufacturer's instructions (Roche Molecular Biochemicals). The
Seescan Optical Density System was used to measure the optical density
of bands obtained on exposed film. The mean optical density was
measured for each band by drawing a standardized box around the band.
This value was normalized by subtracting the mean background optical
density measured immediately above and below the band.
1CT-FP
(1:500),
2-PEP (1:50),
-PAN (1:100), 9E10 (1:200), MANDRA1
(1:200), 2045 (1:200). They were then incubated for 1 h at room
temperature. Following two 5-min PBS washes, the secondary antibodies
(1:200) were applied for 1 h at room temperature. Secondary
antibodies applied to muscle sections also included
-bungarotoxin
(1:500). Sections and cells were washed twice in PBS, mounted with
Vectashield (Vector Laboratories), and viewed under a Leica DMRE
microscope. Photographs were taken with a Leica DMLD camera.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Dystrobrevin-binding Protein Using
Yeast Two-hybrid Analysis--
Previously, we have described three
-dystrobrevin isoforms,
-dystrobrevin-1, -2, and -3, in murine
skeletal muscle, and these are shown schematically in Fig.
1A (15, 17, 19). To identify
proteins that interact with
-dystrobrevin in skeletal muscle, we
performed a yeast two-hybrid screen using the bait construct
DB2:pHybLex, encoding the entire
-dystrobrevin-2 open reading
frame including the muscle-expressed vr3 sequence (15). Attempts
to produce a bait containing the full-length
-dystrobrevin-1 sequence resulted in the nonspecific activation of the yeast
HIS3 and lacZ reporter genes. The
DB2:pHybLex
bait plasmid was screened against a cDNA library generated from
mouse H2K myotubes. 1.2 × 106 yeast colonies were
screened, and a total of 28 positive clones were identified that
activated the lacZ and HIS3 reporter genes. Among
the prey plasmids isolated were dystrophin, utrophin, and syntrophin
cDNAs, demonstrating the success of this screen in identifying
known binding partners for
-dystrobrevin (12, 14, 16, 35).
Importantly, no known components of signaling cascades were identified.
Rather, we identified three identical cDNA clones, SNIP4, SNIP12,
and SNIP88, that showed strong interaction with
DB2:pHybLex in this
system. These 2.1-kb clones were found to encode the entire open
reading frame of a novel protein that we have called syncoilin. To
confirm the syncoilin/
-dystrobrevin-2 interaction in the yeast
system, the SNIP4 cDNA was retransformed into the yeast strain L40
together with the bait plasmid
DB2:pHybLex. The resulting
cotransformants grew vigorously on medium lacking histidine and also
demonstrated
-galactosidase activity (Fig. 1B).
Cotransformation of SNIP4 with the empty bait vector pHybLex/Zeo confirmed that syncoilin did not nonspecifically activate the yeast
reporter genes (Fig. 1B). Control experiments for the yeast two-hybrid system utilized the transcription factors Fos and Jun as a
positive interaction and the empty bait and prey vectors that failed to
transactivate the yeast reporter genes (Fig. 1B).
View larger version (29K):
[in a new window]
Fig. 1.
-Dystrobrevin-2 interacts with
syncoilin in the yeast two-hybrid system. A, schematic
representation of the
-dystrobrevin isoforms in skeletal muscle. All
isoforms are generated by alternative splicing of the
-dystrobrevin
gene. The identifiable domains are boxed. EF,
EF-hand region, predicted calcium binding domain; ZZ, ZZ
domain, putative zinc binding domain (60); vr3, variable
region 3; cc, coiled-coil domain (13); Y, unique
tyrosine kinase substrate domain of
-dystrobrevin-1. The dystrophin
binding site (DBS) (13, 16) and syntrophin binding sites
(SBS 1 and 2) (61) are also shown.
B,
-galactosidase filter assays and growth assays on
yeast cotransformed with the bait and prey constructs indicated. Yeast
containing interacting clones demonstrated
-galactosidase activity
with the production of the blue coloration and also grew
vigorously on histidine-deficient medium
(
HIS).
View larger version (126K):
[in a new window]
Fig. 2.
Sequence of mouse syncoilin.
A, the cDNA sequence and complete conceptual translation
are shown. The predicted molecular mass of syncoilin is 53.6 kDa with a
pI of 4.5. This sequence was deposited in the EMBL data base under EMBL
accession number AJ251641. B, multiple sequence alignment of
mouse and human syncoilin with class III and IV intermediate filament
proteins, -internexin, vimentin, peripherin, glial fibrillary acidic
protein (GFAP), neurofilament triplet proteins NF-L and
NF-H, and chick synemin. The top part shows the
alignment of mouse and human syncoilin sequences throughout their
N-terminal head domains. The rest of B shows the alignment
between mouse and human syncoilin rod domain sequences and those found
in other intermediate filament proteins. The alignment has been colored
using CHROMA (available on the World Wide Web) according to a
75% consensus: negatively charged (
) residues (D and
E) in red, positively charged (+) residues
(H, K, and R) in dark
blue, aliphatic (l) residues (I,
L, and V) in light gray
on yellow, tiny (t) residues
(A, G, and S) in light
green, aromatic (a) residues (F,
H, W, and Y) in blue
on yellow, charged (c) residues
(D, E, H, K, and
R) in pink, small (s) residues
(A, C, D, G, N,
P, S, T, and V) in
dark green, polar (p) residues
(C, D, E, H, K,
N, Q, R, S, and
T) in light blue, big (b)
residues (E, F, H, I,
K, L, M, Q, R,
W, and Y) in dark gray
on yellow, and hydrophobic (h)
residues (A, C, F, G,
H, I, L, M, T,
V, W, and Y) in black
on yellow. The subdivisions of the intermediate
filament helical rod into helical "domains" (38) are shown. The
last residues shown for mouse and human syncoilins represent their most
C-terminal amino acids. Hs, Homo sapiens;
Mm, Mus musculus; Gg, Gallus
gallus. The human syncoilin sequence represents conceptual
translations of a human genomic sequence (chromosome 1 clone
RP5-887B19, map p34.1-34.3, GenBankTM accession number
AL138800) that overlap with three expressed sequence tags
(GenBankTM accession numbers AI281998, W24127, and
AI074110).
-internexin
(E = 5 × 10-16), a class IV
intermediate filament protein (37). Similarity to class III or IV
intermediate filaments such as NF-L (E = 5 × 10-14), peripherin (E = 9 × 10-11), glial fibrillary acidic protein (E = 4 × 10-10), vimentin (E = 1 × 10-9), and synemin (E = 3 × 10-9) was also apparent (Fig. 2B). Although
coiled-coils are present in several different nonhomologous proteins,
such as myosins, kinesins, and desmosomal proteins, the greatest
similarity of syncoilin to these 107 intermediate filament sequences
strongly suggests that it is a novel member of the intermediate
filament protein family. All intermediate filament proteins possess a
central
-helical coiled-coil region containing ~300 amino acid
residues, with nonhelical and divergent N- and C-terminal regions (38). It should be noted that the length of the coiled-coil region in syncoilin (296 amino acids) and the alignment, without insertions or
deletions, of its sequence against the intermediate filament-diagnostic helices 1A, 1B, 2A, and 2B (38) are entirely consistent with its
assignment as an intermediate filament homologue (Fig. 2B). Syncoilin has a N-terminal head domain of 156 residues and a short C-terminal tail domain of 18 amino acids. Both head and tail regions do
not show any sequence similarity to sequences in the data base.
-dystrobrevin transcripts (15). Two syncoilin transcripts were
detected in skeletal muscle and heart, one of ~3.0 kb and a larger
transcript of ~4.5 kb. On longer exposure, weak signals of 3.0 and
4.5 kb were also detected in lung and testes. The same Northern blot
was also hybridized with a
-actin cDNA control probe to confirm
that each lane contained a similar amount of mRNA (Fig.
3A, lower panel).
View larger version (43K):
[in a new window]
Fig. 3.
Syncoilin expression in mouse skeletal and
cardiac muscle. A, Northern blot probed with the SNIP4
cDNA clone demonstrating that two transcripts of ~3.0 and 4.5 kb
were detected in skeletal muscle and heart. To demonstrate that
equivalent levels of RNA were present in each sample, the blot was
stripped and rehybridized with a -actin cDNA control probe
(lower panel). B, Western blot
analysis of syncoilin. A multiple tissue Western blot was probed with
the antibody SYNC-FP, which detected a protein of 64 kDa in skeletal
muscle. In heart, two proteins of 64 and 55 kDa were detected, and a
faint band of ~55 kDa was detected in lung. Recombinant syncoilin
expressed in COS-7 cells is shown on the end
panel and demonstrates that the 64-kDa protein detected in
skeletal muscle and heart corresponds to the syncoilin protein isolated
from the yeast two-hybrid screen. The molecular mass markers are shown
on the left in kDa.
-Dystrobrevin and Syncoilin in Transfected
COS-7 Cells--
To confirm the interaction between
-dystrobrevin-2
and syncoilin, both proteins were expressed in COS-7 cells. First,
sync:pCIneo and the Myc-tagged
-dystrobrevin expression constructs
m32Myc:pCIneo (encoding full-length
-dystrobrevin-2 and including
the vr3 sequence) and m24Myc:pCIneo (encoding full-length
-dystrobrevin-1 and including the vr3 sequence) were separately
transfected into COS-7 cells. Expression of syncoilin was detected
using the SYNC-FP polyclonal antibody, and the expression of
dystrobrevin was detected using the anti-Myc monoclonal antibody 9E10.
As shown in Fig. 4, the transfection of
syncoilin (Fig. 4A) and
-dystrobrevin-2 (Fig. 4B) and
-dystrobrevin-1 (Fig. 4C) into COS-7
cells each resulted in a very similar pattern of protein expression:
strong cytoplasmic labeling particularly concentrated around the
nucleus, coupled with intense staining of the cell periphery.
-Dystrobrevin-1 and -2 immunoreactivity was also detected strongly
in the nucleus of transfected cells (Fig. 4, B and
C). It should also be noted that overexpression of syncoilin
in COS-7 cells did not result in the formation of intracellular
filaments. This implies that syncoilin does not self-assemble into
10-nm intermediate filaments under these conditions. Instead, a second
intermediate filament protein may be required for heteropolymeric
filament formation.
View larger version (63K):
[in a new window]
Fig. 4.
Syncoilin colocalizes with
-dystrobrevin-1 and -2 in COS-7 cells. The
localization of syncoilin,
-dystrobrevin-2, and
-dystrobrevin-1
in transfected COS-7 cells was determined by transfecting cells with
the expression constructs sync:pCIneo (A), m32Myc:pCIneo
(B), and m24Myc:pCIneo (C), respectively.
Expression of syncoilin was detected using SYNC-FP, whereas
dystrobrevin was labeled using the anti-Myc monoclonal antibody 9E10.
The subcellular distribution of all three proteins was very similar
with strong cytoplasmic immunolabeling concentrated in particular round
the nucleus coupled with intense staining of the cell periphery (see
arrow in A). Cotransfection of syncoilin with
either
-dystrobrevin-2 (D, E, and
F) or
-dystrobrevin-1 (G, H, and
I) resulted in the clear colocalization of dystrobrevin and
syncoilin immunoreactivity. D and G, syncoilin
immunolabeling; E and H, dystrobrevin
immunolabeling. F and I, overlay of syncoilin and
dystrobrevin immunoreactivity. In contrast, cotransfection of syncoilin
with the dystrophin isoform Dp116 (pDp116) did not result in the
colocalization of the two proteins (J, K, and
L). J, syncoilin immunolabeling; K,
Dp116 immunolabeling detected with the anti-dystrophin monoclonal
MANDRA1; L, overlay of syncoilin and Dp116 immunoreactivity.
*, a cell singly transfected with sync:pCIneo but not pDp116. As a
control for these experiments,
-dystrobrevin-1 (m24Myc:pCIneo) and
2-syntrophin (
2syn:pCIneo) expression constructs were
cotransfected into COS-7 cells (M-O). There is clear
colocalization of
2-syntrophin immunolabeling (M), as
detected with the anti-
2-syntrophin polyclonal antibody 2045 and
-dystrobrevin-1 staining (N). O, overlay of
2-syntrophin and
-dystrobrevin-1 immunoreactivity.
Scale bar, 50 µm.
-dystrobrevin-2 immunoreactivity as detected by double
immunofluorescent labeling of transfected cells (Fig. 4,
D-F). Identical observations were made when sync:pCIneo and
m24Myc:pCIneo were cotransfected into COS-7 cells (Fig. 4,
G-I), indicating that syncoilin colocalizes with both
-dystrobrevin-1 and -2 in this system. To investigate whether
syncoilin colocalized with Dp116, a C-terminal dystrophin isoform
containing related sequences to
-dystrobrevin, pDp116 was
cotransfected with sync:pCIneo. The expression of Dp116 in transfected
cells was detected with the anti-dystrophin monoclonal antibody
MANDRA1. In this case, syncoilin and Dp116 immunoreactivity did not
colocalize in cotransfected cells (Fig. 4, J-L). In control experiments, COS-7 cells were cotransfected with m24Myc:pCIneo and a
known interactor,
2-syntrophin which was also expressed in pCI-neo.
2-Syntrophin was detected with the polyclonal antibody 2045. Clearly,
2-syntrophin expression colocalizes with
-dystrobrevin-1 expression in transfected COS-7 cells (Fig. 4, M-O).
However, these data do not demonstrate a true interaction between
syncoilin and
-dystrobrevin, since both proteins are shown to be
targeted to the same intracellular compartments (Fig. 4,
A-C).
-dystrobrevin in cotransfected COS-7 cells, cell extracts were prepared and subject to immunoprecipitation analysis (Fig.
5, A-F). Using the antibody
1CT-FP, which specifically detects
-dystrobrevin-1, syncoilin was
immunoprecipitated strongly from cell lysates transfected with
m24Myc:pCIneo and sync:pCIneo (Fig. 5A). Likewise,
immunoprecipitation with SYNC-FP coimmunoprecipitated
-dystrobrevin-1 (Fig. 5B). Similar results were obtained
with
-dystrobrevin-2 and syncoilin. Using the antibody
CT-FP as
the precipitating antibody (an antibody that detects both
-dystrobrevin-1 and -2), syncoilin was detected in the resulting
immune complexes (Fig. 5C).
-Dystrobrevin-2 was partially
obscured by the IgG heavy chains in reciprocal immunoprecipitations but
can be detected in the COS-7 cell lysate (Fig. 5D).
Syncoilin was not, however, immunoprecipitated with Dp116 (Fig.
5E), whereas
2-syntrophin strongly immunoprecipitated
with
-dystrobrevin-1 (Fig. 5F). Taken together, these
results confirm the colocalization observations and demonstrate that
in vitro, syncoilin interacts with both
-dystrobrevin-1
and -2.
View larger version (46K):
[in a new window]
Fig. 5.
In vitro interaction between
syncoilin and -dystrobrevin-1 and -2 in
transfected COS-7 cells. Protein extracts prepared from cells
cotransfected with expression constructs as indicated were
immunoprecipitated with SYNC-FP,
1CT-FP, or
CT-FP. Immune
complexes were detected with either SYNC-FP (A and
C) or
CT-FP (B and D). Syncoilin
was strongly precipitated by
1CT-FP (A), and in
reciprocal immunoprecipitations,
-dystrobrevin-1 was precipitated
with the SYNC-FP antibody (B). Syncoilin was also
precipitated with
CT-FP, the antibody detecting
-dystrobrevin-2
(C). In reciprocal immunoprecipitation,
-dystrobrevin-2
was obscured by IgG heavy chains (D) but can be detected in
the COS-7 cell lysate (lys). In contrast, syncoilin was not
immunoprecipitated by the anti-dystrophin antibody 2166 in cells
cotransfected with pDp116 and sync:pCIneo (E). As a positive
control,
-dystrobrevin-1 was strongly immunoprecipitated with the
2-syntrophin antibody 2045 (F). lys, COS-7
cell lysate; sync, syncoilin;
DB1,
-dystrobrevin-1;
DB2,
-dystrobrevin-2. *, rabbit
IgG heavy chain.
-Dystrobrevin and Syncoilin in Skeletal
Muscle--
To determine the localization of syncoilin in skeletal
muscle, normal C57 mouse quadricep cryosections were labeled with the antibody SYNC-FP. Fig. 6, A,
E, and I, illustrates that syncoilin was
concentrated at NMJs of normal muscle, as identified by double labeling
the sections with
-bungarotoxin (Fig. 6, B, F,
and J). On longitudinal sections, syncoilin appears to be
localized to the contractile apparatus in a stripelike pattern,
consistent with the labeling of Z-lines (Fig. 6,
I and J). Some weak sarcolemmal labeling was also
observed, predominantly on type I fibers as determined by NADH staining
(data not shown). This syncoilin immunostaining was completely
abolished by incubating the antibody with the immunizing fusion protein
(Fig. 6, M and N). The labeling pattern of
syncoilin most closely resembles that of
-dystrobrevin-1, which was
concentrated at the NMJs of normal muscle and also weakly detected at
the sarcolemma (Fig. 6, C, D, G,
H, K, and L). Fig. 6, E and
G, show serial muscle sections labeled with SYNC-FP and
1CT-FP, respectively. The staining patterns demonstrate that
-dystrobrevin-1 and syncoilin immunoreactivity colocalize in
skeletal muscle. Using the antibody
2-PEP,
-dystrobrevin-2 immunoreactivity was also shown to be enriched at NMJs, although strong
sarcolemmal labeling was also observed (Fig. 6, O and
P). Thus, it is possible that syncoilin is also associated
with
-dystrobrevin-2 at NMJs.
View larger version (47K):
[in a new window]
Fig. 6.
Syncoilin and -dystrobrevin-1
colocalize in normal skeletal muscle. C57 mouse quadricep
cryosections were double-labeled with SYNC-FP (A,
E, and I) and
-bungarotoxin (B,
F, and J). Syncoilin immunoreactivity was
concentrated at the NMJ, with much weaker labeling detected at the
sarcolemma of some muscle fibers. On longitudinal muscle sections,
syncoilin immunoreactivity was detected in a stripelike pattern within
the muscle fibers, presumably due to labeling of the contractile
apparatus (arrow). Preincubation of SYNC-FP with the
immunizing fusion protein abolished all labeling (M and
N).
-Dystrobrevin-1 was also concentrated at NMJs, as
shown by immunolabeling with
1CT-FP (C, G, and
K) and
-bungarotoxin (D, H, and
L). Colocalization of syncoilin and
-dystrobrevin-1
immunoreactivity was demonstrated by labeling serial sections with
SYNC-FP (E) and
1CT-FP (G). Note the
colocalization of immunolabeling at the NMJs and also at the sarcolemma
of some muscle fibers. *, identical muscle fibers in serial sections.
-Dystrobrevin-2 was also enriched at NMJ but was also detected at
the sarcolemma, as illustrated using the antibody
2-PEP
(O) and
-bungarotoxin (P). Scale bars, 50 µm.
-Dystrobrevin-1 and Syncoilin--
To
demonstrate an in vivo association between syncoilin and
-dystrobrevin, a series of immunoprecipitation experiments were performed using extracts from cultured myotubes and mouse skeletal muscle. Cultured H2K myotubes were extracted in solubilization buffer,
and immunoprecipitations were performed using the antibodies SYNC-FP
and
1CT-FP (Fig. 7, A and
B). Using SYNC-FP as the precipitating antibody, a small
amount of
-dystrobrevin-1 was detected in the resulting immune
complexes (Fig. 7A). Reciprocal experiments using
1CT-FP
for immunoprecipitation resulted in the detection of a faint syncoilin
band (Fig. 7B). These precipitated complexes were also
tested for the presence of utrophin and dystrophin, which are known
binding partners for
-dystrobrevin in skeletal muscle. Utrophin was
found to be strongly immunoprecipitated with
1CT-FP, but only a very
weak signal was detected in the SYNC-FP precipitates (Fig.
7C). Meanwhile, dystrophin was only weakly detected in these myotube extracts and was not detected at all in SYNC-FP
immunoprecipitates (Fig. 7D). A faint dystrophin band was
observed in
1CT-FP immune complexes, demonstrating an interaction
between
-dystrobrevin-1 and dystrophin.
View larger version (43K):
[in a new window]
Fig. 7.
In vivo association of syncoilin
and -dystrobrevin-1 in cultured myotubes and
skeletal muscle. Solubilized proteins extracted from H2K myotubes
(A-D) and skeletal muscle (E) were subject to
immunoprecipitation analysis using antibodies SYNC-FP and
1CT-FP.
A, immunoblot probed with
1CT-FP showing that
-dystrobrevin-1 was precipitated using SYNC-FP as the
immunoprecipitating antibody in H2K myotubes. B, in the
reciprocal immunoprecipitation probed with SYNC-FP, syncoilin was
precipitated using
1CT-FP. C, immune complexes were also
probed with the anti-utrophin antibody URD40 and resulted in the
detection of a faint utrophin band in SYNC-FP immunoprecipitates. A
strong utrophin band was detected in
1CT-FP immunoprecipitates.
D, immune complexes probed with the anti-dystrophin antibody
2166. A faint full-length dystrophin band was detected in
1CT-FP
immunoprecipitations, along with a substantial band of ~70 kDa. This
band possibly corresponds to the detection of the dystrophin isoform
Dp71 that is not expressed in normal adult muscle, although it is not
detected in the H2K protein lysate (H2K Lys). Alternatively,
this band may correspond to a dystrophin breakdown product.
E,
-dystrobrevin-1 was immunoprecipitated by SYNC-FP and
URD40 in mouse skeletal muscle. Immune complexes were probed with
1CT-FP to reveal
-dystrobrevin-1 complexed with syncoilin and
utrophin. It is notable that utrophin was associated with
-dystrobrevin-1 isoforms with and without the alternatively spliced
vr3 sequence, while syncoilin was only complexed with
-dystrobrevin-1 containing the vr3 sequence. SM Lys,
skeletal muscle protein lysate; sync, syncoilin;
DB1+vr3,
-dystrobrevin-1 containing the vr3
sequence;
DB1
vr3,
-dystrobrevin-1
without the vr3 sequence; Utr, utrophin; Dys,
dystrophin. *, rabbit IgG heavy chains.
-dystrobrevin-1 (Fig. 7E). A reciprocal immunoprecipitation using
1CT-FP did not result in the detection of
syncoilin (data not shown). The anti-utrophin antibody URD40 was
included in this set of immunoprecipitations as a control, and it can
be seen that this antibody results in the precipitation of ~7.5 times
as much
-dystrobrevin-1 as SYNC-FP. Thus, only a small proportion of
syncoilin is associated with
-dystrobrevin and vice
versa. It was also noticeable that the SYNC-FP antibody only
resulted in the precipitation of the larger
-dystrobrevin-1 isoform
containing the vr3 sequence in myotubes and skeletal muscle (Fig. 7,
A and E). This result suggested that syncoilin
might interact with
-dystrobrevin-1 via the vr3 sequence. We tested this possibility using the yeast two-hybrid system by generating a vr3
bait and cotransforming yeast with this new bait and the SNIP4 prey.
The vr3 bait did not interact with syncoilin in this system (data not
shown), implying that syncoilin does not bind to the alternatively
spliced region of
-dystrobrevin in skeletal muscle.
-dystrobrevin in mdx muscle,
which, like other components of the DPC, has greatly reduced
immunoreactivity at the sarcolemma (22). Fig. 8A illustrates
such immunolabeling of normal and dystrophin-deficient muscle sections
with the antibody
-PAN, which detects all three
-dystrobrevin
isoforms in skeletal muscle. Like syncoilin,
-dystrobrevin remains
enriched at NMJs in mdx muscle.
View larger version (27K):
[in a new window]
Fig. 8.
Syncoilin expression in dystrophic
muscle. A, localization of syncoilin and
-dystrobrevin in normal C57 and mdx quadricep
cryosections. In normal muscle, SYNC-FP immunoreactivity was
concentrated at NMJs, as identified by double labeling with
-bungarotoxin (
-BT). mdx muscle
demonstrated increased immunostaining at the sarcolemma and within
dystrophic muscle fibers (arrowhead). NMJs were still
labeled in mdx muscle but were harder to identify due to the
overall increase in immunostaining. Using the antibody
-PAN, which
detects all three
-dystrobrevin isoforms in skeletal muscle,
-dystrobrevin immunoreactivity was depleted from the sarcolemma of
mdx muscle fibers while maintained at NMJs. Scale
bar, 50 mm. B, Western blot of total protein
extracted from skeletal muscle of a normal C57 mouse, an mdx
mouse, a utrophin-deficient (utrn
/
) mouse, a dystrophin
and utrophin double knock-out (dko) mouse, and an mdx mouse
transgenic for full-length utrophin (Fio), probed with SYNC-FP.
Syncoilin levels were increased 6.4-fold in mdx muscle and
12-fold in dko muscle compared with C57 controls as assessed by optical
density measurements. In the Fio mouse, levels of syncoilin were
reduced to almost control levels. C, an identical Western
blot probed with
CT-FP, which detects
-dystrobrevin-1 and -2 in
skeletal muscle. While levels of
-dystrobrevin-1 were largely
unaffected in dystrophic muscle, levels of
-dystrobrevin-2 were
reduced substantially in mdx and dko muscle. Molecular mass
markers, in kDa, are shown.
/
) that
does not have muscular dystrophy (31), a dystrophin- and
utrophin-deficient double knock-out mouse (dko) that has severe
muscular dystrophy (32), and an mdx mouse expressing a
full-length utrophin transgene that largely rescues the dystrophic
phenotype (Fio) (33) were also included. Fig. 8B shows the
resulting Western blot and reveals a clear correlation between levels
of syncoilin protein and muscle damage; the more dystrophic the muscle,
the greater the levels of syncoilin. It is estimated using mean optical
density measurements of the resulting bands that levels of syncoilin in
mdx muscle are ~6.4-fold greater than in normal control
muscle and in dko muscle that levels are increased 12-fold. In Fio
muscle, levels of syncoilin are similar to the control, presumably
reflecting the rescue of the dystrophic phenotype by the utrophin
transgene. These results for syncoilin contrast with changes in the
levels of the
-dystrobrevins in dystrophic muscle. While levels of
-dystrobrevin-1 are largely unaffected in mdx and dko
muscle, levels of
-dystrobrevin-2 are significantly reduced (Fig.
8C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystrobrevin and
syncoilin, a novel member of the intermediate filament superfamily. Syncoilin is so named due to its localization at the neuromuscular synapse and the presence of predicted coiled-coils within its primary
sequence. Using a combination of yeast two-hybrid analysis, in
vitro expression assays, and in vivo colocalization and
coimmunoprecipitation experiments, we propose that
-dystrobrevin-1
and syncoilin form a complex at the NMJ, suggesting a link between the
intermediate filament network and the subsynaptic DPC.
-internexin (class IV) and vimentin and peripherin (class III).
Flanking this rod domain are the N-terminal head domain and C-terminal
tail domain that vary considerably in size and sequence among the
intermediate filament classes. The head domain of syncoilin lacks
significant sequence similarity with all known proteins.
-dystrobrevin. The loss of
-dystrobrevin through
targeted disruption of the
-dystrobrevin gene results in a number of
NMJ abnormalities including the irregular distribution of AChRs and a
50% reduction in the number of junctional folds (20). These results
imply that there must be a molecular link between
-dystrobrevin and
elements important for maintaining synaptic structure. Here we have
shown that in skeletal muscle, syncoilin immunoreactivity is
concentrated at the NMJ, where it colocalizes with
-dystrobrevin-1.
In addition, immunoprecipitation analysis showed that a proportion of
syncoilin and
-dystrobrevin-1 associate in vivo. This
association at the postsynaptic membrane may offer a route for
understanding the effects of
-dystrobrevin deficiency at the
NMJ.
-dystrobrevin-1 interaction may be required for NMJ
integrity and for the correct localization of AChRs. Intermediate
filament proteins have been implicated in synapse organization. For
example, desmin, a protein enriched at the NMJ and found near the
AChR-rich crests of the junctional folds (41, 43), together with actin, has been proposed to form a submembraneous support for AChRs at the NMJ
(46). This desmin-containing network is suggested to be important in
mediating the excitation of AChRs to the sarcomeric contraction system
(46). Similarly, in the central nervous system, the NMDA receptor
subunit NR1 interacts directly with the intermediate filament protein
neurofilament subunit NF-L. This interaction was also identified using
yeast-two hybrid screening and is thought to be important for anchoring
or localizing NMDA receptors in the neuronal plasma membrane (47).
Thus, there is a clear precedent for the association of intermediate
filaments with neurotransmitter receptors and receptor-associated proteins.
-dystrobrevin-1 and -2, are depleted
from the sarcolemma of dystrophin-deficient muscle (19, 35). Syncoilin
however, shows increased immunoreactivity within mdx muscle
fibers and at the sarcolemma. Increased syncoilin levels also appear to
correlate with the severity of the muscular dystrophy, implying that
syncoilin may be a marker for muscle fiber damage or regeneration.
Interestingly, the muscle intermediate filament proteins desmin and
vimentin, which are also up-regulated in regenerating muscle fibers,
are thought to be part of the regenerative process, rather than
representing an immediate response to muscle damage (48). Syncoilin is
not the first DPC-associated protein that has been found to be
up-regulated in dystrophin-deficient muscle. Others include utrophin
(49, 50), the sarcoglycan-binding protein filamin-2 (51), and biglycan,
which binds
-dystroglycan (52).
-dystrobrevin and the involvement of
different intermediate filament proteins in muscle disease makes
syncoilin a good functional as well positional candidate for this disorder.
-dystrobrevin-1 binds to a novel
member of the intermediate filament superfamily, syncoilin, that is
highly expressed in skeletal and cardiac muscle. It is possible that
this interaction is important for the maintenance of the mature NMJ and
in efficient synaptic transmission. The marked up-regulation of
syncoilin in dystrophic muscle suggests that this protein may also be
involved in the pathogenesis of muscular dystrophy. Further molecular
and cellular studies will be required to understand the interactions
and function of syncoilin within the intermediate filament network, the
importance of its association with the DPC through
-dystrobrevin,
and its possible involvement in muscle-eye-brain disease.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jenny Morgan for the H2K myoblast cell line and Colin Akerman for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was funded by the Wellcome Trust and the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ251641.
¶ A Wellcome Prize Student.
Present address: Dept. of Anatomy and Cellular Neurobiology,
University of Ulm, Albert Einstein Allee 11, 89069 Ulm, Germany.
** To whom correspondence should be addressed: MRC Functional Genetics Unit, Dept. of Human Anatomy and Genetics, University of Oxford, South Parks Rd., Oxford, OX1 3QX, United Kingdom. Tel.: 44-1865-272179; Fax: 44-1865-272-427; E-mail: kay.davies@anat.ox.ac.uk.
A Wellcome Trust Senior Fellow.
Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M008305200
2 M. A. Benson, S. E. Newey, E. Martin-Rendon, R. Hawkes, and D. J. Blake, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DPC, dystrophin protein complex; NMJ, neuromuscular junction; AChR, acetylcholine receptor; vr3, variable region-3 sequence; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; kb, kilobase pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hoffman, E. P., Brown, R. H., Jr., and Kunkel, L. M. (1987) Cell 51, 919-928[Medline] [Order article via Infotrieve] |
2. | Straub, V., and Campbell, K. P. (1997) Curr. Opin. Neurol. 10, 168-175[Medline] [Order article via Infotrieve] |
3. | Blake, D. J., and Davies, K. E. (1997) in Protein Dysfunction and Human Genetic Disease (Swallow, D. M. , and Edwards, Y. H., eds) , pp. 219-241, BIOS Scientific, Oxford |
4. | Yoshida, M., and Ozawa, E. (1990) J. Biochem. (Tokyo) 108, 748-752[Abstract] |
5. | Ervasti, J. M., and Campbell, K. P. (1991) Cell 66, 1121-1131[Medline] [Order article via Infotrieve] |
6. | Yoshida, M., Suzuki, A., Yamamoto, H., Noguchi, S., Mizuno, Y., and Ozawa, E. (1994) Eur. J. Biochem. 222, 1055-1061[Abstract] |
7. | Ervasti, J. M., and Campbell, K. P. (1993) J. Cell Biol. 122, 809-823[Abstract] |
8. | Menke, A., and Jockusch, H. (1991) Nature 349, 69-71[CrossRef][Medline] [Order article via Infotrieve] |
9. | Petrof, B. J., Shrager, J. B., Stedman, H. H., Kelly, A. M., and Sweeney, H. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3710-3714[Abstract] |
10. | Brown, S. C., and Lucy, J. A. (1993) Bioessays 15, 413-419[Medline] [Order article via Infotrieve] |
11. | Wagner, K. R., Cohen, J. B., and Huganir, R. L. (1993) Neuron 10, 511-522[Medline] [Order article via Infotrieve] |
12. | Ahn, A. H., and Kunkel, L. M. (1995) J. Cell Biol. 128, 363-371[Abstract] |
13. | Blake, D. J., Tinsley, J. M., Davies, K. E., Knight, A. E., Winder, S. J., and Kendrick-Jones, J. (1995) Trends Biochem. Sci. 20, 133-135[CrossRef][Medline] [Order article via Infotrieve] |
14. | Dwyer, T. M., and Froehner, S. C. (1995) FEBS Lett. 375, 91-94[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Blake, D. J.,
Nawrotzki, R.,
Peters, M. F.,
Froehner, S. C.,
and Davies, K. E.
(1996)
J. Biol. Chem.
271,
7802-7810 |
16. |
Sadoulet-Puccio, H. M.,
Rajala, M.,
and Kunkel, L. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12413-12418 |
17. | Ambrose, H. J., Blake, D. J., Nawrotzki, R. A., and Davies, K. E. (1997) Genomics 39, 359-369[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sadoulet-Puccio, H. M.,
Khurana, T. S.,
Cohen, J. B.,
and Kunkel, L. M.
(1996)
Hum. Mol. Genet.
5,
489-496 |
19. |
Nawrotzki, R.,
Loh, N. Y.,
Ruegg, M. A.,
Davies, K. E.,
and Blake, D. J.
(1998)
J. Cell Sci.
111,
2595-2605 |
20. | Grady, R. M., Zhou, H., Cunningham, J. M., Henry, M. D., Campbell, K. P., and Sanes, J. R. (2000) Neuron 25, 279-293[Medline] [Order article via Infotrieve] |
21. | Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ohlendieck, K., and Campbell, K. P. (1991) J. Cell Biol. 115, 1685-1694[Abstract] |
23. | Bredt, D. S. (1999) Nat. Cell Biol. 1, 89-91 |
24. | Jat, P. S., Noble, M. D., Ataliotis, P., Tanaka, Y., Yannoutsos, N., Larsen, L., and Kioussis, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5096-5100[Abstract] |
25. | Morgan, J. E., Beauchamp, J. R., Pagel, C. N., Peckham, M., Ataliotis, P., Jat, P. S., Noble, M. D., Farmer, K., and Partridge, T. A. (1994) Dev. Biol. 162, 486-498[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Blake, D. J.,
Nawrotzki, R.,
Loh, N. Y.,
Gorecki, D. C.,
and Davies, K. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
241-246 |
27. | Newey, S. E., Gramolini, A. O., Wu, J., Holzfeind, P., Jasmin, B. J., Davies, K. E., and Blake, D. J. (2001) Mol. Cell. Neurosci. 17, 127-140[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Blake, D. J.,
Hawkes, R.,
Benson, M. A.,
and Beesley, P. W.
(1999)
J. Cell Biol.
147,
645-658 |
29. |
Loh, N. Y.,
Newey, S. E.,
Davies, K. E.,
and Blake, D. J.
(2000)
J. Cell Sci.
113,
2715-2724 |
30. | Nguyen, T. M., Ginjaar, I. B., van Ommen, G. J., and Morris, G. E. (1992) Biochem. J. 288, 663-668[Medline] [Order article via Infotrieve] |
31. |
Deconinck, A. E.,
Potter, A. C.,
Tinsley, J. M.,
Wood, S. J.,
Vater, R.,
Young, C.,
Metzinger, L.,
Vincent, A.,
Slater, C. R.,
and Davies, K. E.
(1997)
J. Cell Biol.
136,
883-894 |
32. | Deconinck, A. E., Rafael, J. A., Skinner, J. A., Brown, S. C., Potter, A. C., Metzinger, L., Watt, D. J., Dickson, J. G., Tinsley, J. M., and Davies, K. E. (1997) Cell 90, 717-727[CrossRef][Medline] [Order article via Infotrieve] |
33. | Tinsley, J., Deconinck, N., Fisher, R., Kahn, D., Phelps, S., Gillis, J. M., and Davies, K. (1998) Nat. Med. 4, 1441-1444[CrossRef][Medline] [Order article via Infotrieve] |
34. | Flaherty, L., and Herron, B. (1998) Mamm. Genome 9, 417-418[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Peters, M. F.,
Sadoulet-Puccio, H. M.,
Grady, M. R.,
Kramarcy, N. R.,
Kunkel, L. M.,
Sanes, J. R.,
Sealock, R.,
and Froehner, S. C.
(1998)
J. Cell Biol.
142,
1269-1278 |
36. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
37. | Herrmann, H., and Aebi, U. (2000) Curr. Opin. Cell Biol. 12, 79-90[CrossRef][Medline] [Order article via Infotrieve] |
38. | Fuchs, E., and Weber, K. (1994) Annu. Rev. Biochem. 63, 345-382[CrossRef][Medline] [Order article via Infotrieve] |
39. | Cormand, B., Avela, K., Pihko, H., Santavuori, P., Talim, B., Topaloglu, H., de la Chapelle, A., and Lehesjoki, A. E. (1999) Am. J. Hum. Genet. 64, 126-135[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Bellin, R. M.,
Sernett, S. W.,
Becker, B.,
Ip, W.,
Huiatt, T. W.,
and Robson, R. M.
(1999)
J. Biol. Chem.
274,
29493-29499 |
41. | Sealock, R., Murnane, A. A., Paulin, D., and Froehner, S. C. (1989) Synapse 3, 315-324[Medline] [Order article via Infotrieve] |
42. | Askanas, V., Bornemann, A., and Engel, W. K. (1990) Neurology 40, 949-953[Abstract] |
43. | Bilak, M., Askanas, V., and Engel, W. K. (1994) Synapse 16, 280-283[Medline] [Order article via Infotrieve] |
44. |
Vaittinen, S.,
Lukka, R.,
Sahlgren, C.,
Rantanen, J.,
Hurme, T.,
Lendahl, U.,
Eriksson, J. E.,
and Kalimo, H.
(1999)
Am. J. Pathol.
154,
591-600 |
45. | Bilak, S. R., Sernett, S. W., Bilak, M. M., Bellin, R. M., Stromer, M. H., Huiatt, T. W., and Robson, R. M. (1998) Arch. Biochem. Biophys. 355, 63-76[CrossRef][Medline] [Order article via Infotrieve] |
46. | Mitsui, T., Kawajiri, M., Kunishige, M., Endo, T., Akaike, M., Aki, K., and Matsumoto, T. (2000) J. Cell. Biochem. 77, 584-595[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Ehlers, M. D.,
Fung, E. T.,
O'Brien, R. J.,
and Huganir, R. L.
(1998)
J. Neurosci.
18,
720-730 |
48. | Tews, D. S., Goebel, H. H., Schneider, I., Gunkel, A., Stennert, E., and Neiss, W. F. (1997) Exp. Neurol. 146, 125-134[CrossRef][Medline] [Order article via Infotrieve] |
49. | Helliwell, T. R., Man, N. T., Morris, G. E., and Davies, K. E. (1992) Neuromusc. Disord. 2, 177-184[CrossRef][Medline] [Order article via Infotrieve] |
50. | Mizuno, Y., Nonaka, I., Hirai, S., and Ozawa, E. (1993) J. Neurol. Sci. 119, 43-52[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Thompson, T. G.,
Chan, Y. M.,
Hack, A. A.,
Brosius, M.,
Rajala, M.,
Lidov, H. G.,
McNally, E. M.,
Watkins, S.,
and Kunkel, L. M.
(2000)
J. Cell Biol.
148,
115-126 |
52. |
Bowe, M. A.,
Mendis, D. B.,
and Fallon, J. R.
(2000)
J. Cell Biol.
148,
801-810 |
53. | Li, Z., Colucci-Guyon, E., Pincon-Raymond, M., Mericskay, M., Pournin, S., Paulin, D., and Babinet, C. (1996) Dev. Biol. 175, 362-366[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Dalakas, M. C.,
Park, K. Y.,
Semino-Mora, C.,
Lee, H. S.,
Sivakumar, K.,
and Goldfarb, L. G.
(2000)
N. Engl. J. Med.
342,
770-780 |
55. | Bonne, G., Di Barletta, M. R., Varnous, S., Becane, H. M., Hammouda, E. H., Merlini, L., Muntoni, F., Greenberg, C. R., Gary, F., Urtizberea, J. A., Duboc, D., Fardeau, M., Toniolo, D., and Schwartz, K. (1999) Nat. Genet. 21, 285-288[CrossRef][Medline] [Order article via Infotrieve] |
56. | Wilson, K. L. (2000) Trends Cell Biol. 10, 125-129[CrossRef][Medline] [Order article via Infotrieve] |
57. | Smith, F. J., Eady, R. A., Leigh, I. M., McMillan, J. R., Rugg, E. L., Kelsell, D. P., Bryant, S. P., Spurr, N. K., Geddes, J. F., Kirtschig, G., Milana, G., de Bono, A. G., Owaribe, K., Wiche, G., Pulkkinen, L., Uitto, J., McLean, W. H., and Lane, E. B. (1996) Nat. Genet. 13, 450-457[Medline] [Order article via Infotrieve] |
58. |
Andra, K.,
Lassmann, H.,
Bittner, R.,
Shorny, S.,
Fassler, R.,
Propst, F.,
and Wiche, G.
(1997)
Genes Dev.
11,
3143-3156 |
59. | Santavuori, P., Somer, H., Sainio, K., Rapola, J., Kruus, S., Nikitin, T., Ketonen, L., and Leisti, J. (1989) Brain Dev. 11, 147-153[Medline] [Order article via Infotrieve] |
60. | Ponting, C. P., Blake, D. J., Davies, K. E., Kendrick-Jones, J., and Winder, S. J. (1996) Trends Biochem. Sci. 21, 11-13[CrossRef][Medline] [Order article via Infotrieve] |
61. | Newey, S. E., Benson, M., Ponting, C. P., Davies, K. E., and Blake, D. J. (2000) Curr. Biol. 10, 1295-1298[CrossRef][Medline] [Order article via Infotrieve] |