1 European Molecular Biology Laboratory, Cell Biology Division, Heidelberg,
Germany
2 Institut Jacques Monod, Paris, France
3 Max-Planck-Institut für molekulare Physiologie, Department of Physical
Biochemistry, 44202 Dortmund, Germany
4 Potsdam University, Department of Cell Biology, Potsdam, Germany
5 King's College London, Muscle Cell Biology, The Randall Centre, New Hunt's
House, London SE1 1UL, UK
* Author for correspondence (e-mail: mathias.gautel{at}kcl.ac.uk)
Accepted 24 August 2002
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Summary |
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Key words: Myosin, Microtubules, Titin, MURF2, Myofibril assembly, Connectin
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Introduction |
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The intermediates of myofibrillogenesis are hard to study and still not
fully characterised (Sanger et al.,
2000). This is partly due to the increasing number of recently
characterised components (Sanger and
Sanger, 2001
; Faulkner et al.,
2000
) and partly to difficulties in visualising the reorganization
of these components with sufficient resolution. In cultured cells,
stress-fibre like structures (SFLS), which contain some sarcomeric proteins
but lack the regular periodicity of mature sarcomeres, assemble first and then
undergo dramatic structural rearrangements, resulting finally in mature
sarcomeres (Dlugosz et al.,
1984
; Rhee et al.,
1994
). In vivo, this process appears to be similar but in the
heart, it seems to occur without some of the intermediates observed in culture
(Ehler et al., 1999
). During
the early stages of myofibrillogenesis in cultured cells, titin colocalises
with
-actinin in the Z-bodies of nascent myofibrils, and is thought to
be responsible for the anchorage of
-actinin
(Schultheiss et al., 1990
;
Sorimachi et al., 1997
;
Tokuyasu and Maher, 1987
;
Turnacioglu et al., 1996
;
Young et al., 1998
). Titin,
-actinin and actin sequentially form a regularly arranged scaffold on
SFLS, which progress on to form non-striated myofibrils or premyofibrils
(NSMF) and nascent striated myofibrils (naSMF). As late components, myomesin,
myosin-binding protein-C and finally myosin are incorporated. Nascent
myofibrils align and fuse with adjacent nascent myofibrils to form the Z-discs
of mature myofibrils (Dabiri et al.,
1997
). During these transitions, the spacing between
-actinin-containing Z-bodies increases from less than 1 µm in SFLS
and nascent myofibrils/premyofibrils to more than 2 µm in mature
myofibrils. Titin epitopes that initially colocalise at Z-bodies separate
during this process (Mayans et al.,
1998
; Van der Loop et al.,
1996
), indicating that stretching of the titin molecule, and
possibly exposure of binding sites for other myofibrillar proteins, is an
essential process for the assembly of sarcomeres. Clearly, coordination and a
series of regulatory processes are needed to organise the successive assembly
states over time and space.
The recent discovery that a family of microtubule-binding proteins, the
MURF proteins [first identified as putative interactors of the serum response
factor by Spencer et al. (Spencer et al.,
2000)], play a crucial role in myofibril assembly, points again to
microtubules as important elements of myofibril morphogenesis. The MURFs are
transcribed from three genes, (chromosomes 1p31.1-p33, 8q12-13, 2q16-21). They
contain an N- terminal RING, followed by a B-box zinc-finger domain and a
coiled-coil sequence, and thus belong to the RBCC subfamily of RING finger
proteins (Centner et al., 2001
;
Spencer et al., 2000
). MURFs
can multimerise via the coiled-coil domain, resulting in homo- or
heteromultimeres (Centner et al.,
2001
; Spencer et al.,
2000
). Intriguingly, the known cellular localisations of MURF1 and
MURF3 are variable and include microtubules and Z-disks (MURF3), or both
Z-disk and M-band (MURF1) (Centner et al.,
2001
; McElhinny et al.,
2002
; Spencer et al.,
2000
). Their exact functions are unclear, although roles in
cytoskeletal dynamics, transcription regulation and cell signalling are
conceivable. For MURF3, a stabilising effect on microtubules is established
(Spencer et al., 2000
). In
addition, MURF3 seems to be required for the initiation of myogenesis in
vitro: ablation of MURF3 in cell culture inhibits the expression of the early
myogenic regulatory factors, MyoD and myogenin. Reduced MURF3 expression also
diminishes myosin heavy chain (MHC) expression and concomitant myotube
formation (Spencer et al.,
2000
). Interestingly, a knock-out mouse model of MURF1 however
shows no primary defect in myofibrillogenesis
(Bodine et al., 2001
). The
temporal dynamics of MURFs in myofibril assembly, crucial for the
understanding of the mechanistic role in this process, is so far unknown.
We therefore investigated the spatio-temporal relocalisation of the least well-characterised member of the MURF protein family, MURF2, during myogenesis in vitro. Since primary myofibril assembly can not be unambiguously studied in neonatal cardiomyocytes due to their pre-existing myofibrils, we investigate differentiating skeletal myoblasts. We show that MURF2 is a microtubule-binding protein involved in early myofibrillogenesis as it interacts with both microtubules and M-bands in forming myofibrils. Moreover, we find MURF2 in the nucleus under certain conditions, suggesting that it could also play a role in the transcription programme during differentiation of myocytes.
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Materials and Methods |
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Two-hybrid analysis
Ig domains from the titin C-terminal region, or MURFs were cloned into a
modified pLexA vector described previously
(Young et al., 1998) and
interactions with MURFs or titin in pGAD10 or MURFs in pLexA were monitored in
L40 cells (Vojtek et al.,
1993
) as described (Young et
al., 1998
). Titin domain nomenclature followed the human cardiac
titin sequence [EMBL X90568 (Labeit and
Kolmerer, 1995
)].
Antibodies
The polyclonal antibody -HPC is directed against the unique,
constitutive C-terminus of the 50, 60 and 27 kDa isoforms of MURF2. It was
raised by immunisation of New Zealand rabbits with the keyhole-limpet
hemocyanin-coupled peptide HP-C: DSEPARHIFSFSWLNSLNE following established
procedures. The antibody was affinity-purified on matrix-coupled HP-C using
standard methods.
The -tubulin-specific rat monoclonal antibody YL 1/2, specific for
the C-terminal EEY-epitope of tyrosinated tubulin
(Wehland et al., 1984
), was
used to stain tyrosinated microtubules. The
-tubulin-specific
monoclonal antibody ID5 (Rüdiger et
al., 1999
) was used to detect polyglutaminated tubulin and was a
kind gift of J. Wehland. Antibodies against titin recognised the Z-disk [T12
(Fürst et al., 1988a
)],
I-band [N2A (Gautel et al.,
1996
)] and A-band [T31
(Fürst et al., 1989a
)].
Monoclonal antibodies against tubulin (DM1A), skeletal myosin (MY-32) and
-skeletal and
-cardiac sarcomeric actins (5C5) were purchased
from Sigma-Aldrich.
Cell culture and immunofluorescence
The C57 myogenic cell line was established by Pinset and Montarras as
described previously (Pinset and
Montarras, 1998). Cells were grown in a mixture (1/1 volume) of
Dulbecco's modified Eagle's and MCDB202 medium (Cryo Bio System) supplemented
with 100 µg/ml penicillin, 100 units/ml streptomycin, 2 mM glutamine, 20%
fetal calf serum (FCS) and 2% UltroserSF (Gibco BRL Life Science). For
differentiation, cells were maintained in the previously described medium
containing 2% FCS and 10 µg/ml insulin. In all conditions, cells were grown
on gelatin-coated dishes or coverslips at 37°C in a 10% CO2
atmosphere. Double immunofluorescence experiments were performed as follow:
cells briefly rinsed with warm (37°C) BRB80 (8 mM K-Pipes pH 6.8, 1 mM
EGTA and 1 mM MgCl2), permeabilised with 0.1% Triton X-100 in BRB80 for 45
seconds and then fixed in methanol at -20°C for 7 minutes. Mouse
monoclonal anti
-tubulin (DM1A from Sigma), Titin or myosin antibodies
and affinity purified rabbit anti-MURF2 antibodies, diluted in PBS containing
0.1% saponin and 5% FCS, were applied for 1 hour at room temperature.
Following three washes with 0.1% saponin in PBS, primary antibodies were
visualized by fluorescent conjugated secondary antibodies (Molecular Probes)
for 30 minutes. Coverslips were mounted in AF1 solution (Citifluor) and
observed with an LSM 510 confocal microscope (Zeiss).
BHK-21 cells were cultured and differentiated as described before
(Van der Ven and Fürst,
1998). Transfection with MURF plasmids was carried out using
Escort-III (Sigma) following the supplier's instructions. Neonatal rat
cardiomyocytes were isolated from day 2-4 Wistar pups essentially as described
(Auerbach et al., 1999
;
Young et al., 2001
) and
cultured in collagen-coated culture dishes at 37°C, 5% CO2
either in M199, 10% fetal calf serum, 10 µM phenylephrine, 10 µM
cytosine-arabinoside and penicilline/streptomycine or in low low-serum minimal
medium consisting of M199, 1% Nutridoma-HU (Roche biochemicals), 10 µM
cytosine-arabinoside and penicilline/streptomycine. Fixation followed the
protocols developed for plakophilin
(Mertens et al., 1996
) by
inclusion of MgCl2 to the fixation medium.
In vitro assembly of microtubules
C57 myoblasts and myotubes differentiated for 2 and 5 days were scraped
from dishes with a rubber policeman and centrifuged at 900 g for 5
minutes. Cell pellets resuspended with an equal volume of cold 2x
extraction buffer (2% Triton X100, 50 mM Tris/HCl pH 7.5, 2 mM EGTA, 2 mM
EDTA, 1% Nonidet P40, 200 mM NaCl, 2 mM sodium ortho-vanadate, 0.4 mM PMSF and
protease inhibitors) were then incubated on a rotary device at 4°C for 90
minutes, before being centrifuged as previously described. The supernatants
were then clarified by centrifugation at 50,000 g at 4°C for 15
minutes and protein quantification was performed by the Pierce Micro BCA assay
according to the supplier's protocol. 400 µg of total protein, supplemented
with 2 µg/ml of cytochalasine D or latrunculin A, were used for
microtubules polymerisation or depolymerisation experiments. Microtubule
polymerisation was performed by incubating the extracts with 2 mM MgGTP, 2 mM
AMP-PNP and 20 µM taxol at 37°C for 30 minutes. Microtubule
depolymerisation was obtained by incubating the extracts with 20 µM
nocodazole on ice for 30 minutes. After centrifugation through a 40% sucrose
cushion in BRB80 containing taxol or nocodazole (150,000 g, 30
minutes, 25°C), pellets were resuspended with 100 µl of 1% SDS in PBS.
In order to compare microtubule-associated proteins, western blot analyses
were performed with 20 µg of proteins solubilized from the taxol pellet and
identical volume of their corresponding pellet in presence of nocodazole.
Samples were separated on a 10% SDS gel, transferred to nitrocellulose
membranes, blocked and subsequently incubated with affinity purified
anti-MURF2 and anti-tubulin antibodies following standard procedures.
Myosin and titin binding assays
MURF2 was transfected in HeLa cells and soluble cell supernatants collected
as above. Myosin cosedimentation assays were carried out essentially as
described (Gruen and Gautel,
1999) using 40 µg of rabbit skeletal muscle myosin [prepared by
repeated high- and low-salt steps as described
(Gruen and Gautel, 1999
)] per
assay, and 40 µg of total soluble HeLa proteins from MURF2 transfected
cells. After incubation on ice for 20 minutes, myosin was pelleted (100,000
g, 30 minutes, 25°C) and the pellets collected. Pellets were run
on 12% SDS gels, blotted and bound MURF2 detected with the HPC antibody.
Titin binding was assayed in a column-based binding assay essentially as
described for titin--actinin (Young
et al., 1998
) using GST-fused titin domains A164-169 and GST alone
as control. Soluble proteins (40 µg) from MURF2 transfected HeLa cells were
passed over 20 µl of glutathione beads loaded with 20 µg of GST or
GST-titin fusion protein, washed in binding buffer (100 mM NaCl, 20 mM HEPES
pH 7, 1 mM EDTA, 1 mM DTT, 0.05% Tween-20) and bound proteins were eluted with
10 mM glutathione in binding buffer. Eluted fractions were run on 12% SDS gels
and MURF2 detected by western blot as above.
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Results |
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Endogenous MURF2 interacts with microtubules from skeletal muscle
cell
MURF3 has been reported to be associated both with microtubules as well as
with the mature Z-disk (Spencer et al.,
2000). Given the very high amino acid sequence homology of MURF1,
2 and MURF3 (Centner et al.,
2001
), we tested the microtubule binding ability of MURF2.
Microtubule sedimentation assays were performed using protein extracts from
the mouse C57 skeletal muscle cell line
(Pinset and Montarras, 1998
),
prepared at various times of differentiation, where MURF2p60 is
expressed in a differentiation-dependent way
(Fig. 2A). Cytoskeletal
fractions were prepared in the presence of either taxol or nocodazole in order
to stabilize, or to inhibit microtubule polymerization. However, as previously
described, some microtubules remain resistant to cold and nocodazole treatment
(Gundersen et al., 1989
).
Although some tubulin was present in the nocodazole pellets
(Fig. 2B), higher amounts of
tubulin were observed in the taxol pellets indicating efficient microtubule
polymerisation and stabilization. MURF2p60 was not detected in
proliferating myoblasts, demonstrating the specific expression in
differentiating myogenic cells. Upon differentiation, MURF2p60 was
specifically detected in cytoskeletal fractions obtained from taxol treated
extracts (Fig. 2B).
Interestingly, the highest amount of cytoskeleton-associated MURF2 was
detected after 48 hours of differentiation, with a subsequent decline,
suggesting that the physical association between MURF2 and microtubules could
be a transient event occurring during the differentiation programme. To
confirm the cellular localisation of MURF2, double immunofluorescence
experiments were performed on differentiating C57 cells. Using specific
affinity-purified anti-MURF2 and anti tubulin antibodies, we observed that at
the beginning of differentiation, MURF2 colocalised with microtubules
(Fig. 2C). Later, although most
microtubules remained decorated with the anti-MURF2 antibody, some of the
MURF2 staining appeared as small rod and dot structures scattered in a
longitudinal and transverse orientation within the cytoplasm
(Fig. 2D, arrowhead). In fully
differentiated skeletal myofibrils, MURF2 labelling was not detected in mature
sarcomeres (Fig. 6). In
agreement with previous data (Spencer et
al., 2000
), overexpression of MURF1, 2 and 3 in C57 skeletal
myoblasts was lethal. We found that the cytotoxic effect was least pronounced
in myogenic BHK-21 cells (Van der Ven and
Fürst, 1998
). In these cells, MURF2 displayed a specific
differential localisation on post-translationally modified microtubules. Using
the antibody YL1/2 (Wehland et al.,
1984
) against tyrosinated tubulin, MURF2 was found to be
specifically excluded from tyrosinated microtubules
(Fig. 3A) but to localise with
polyglutaminated MTs (Fig. 3B)
labelled by the monoclonal antibody ID5
(Rüdiger et al., 1999
).
Altogether, these data demonstrate the association of MURF2 with microtubules
at early stages of skeletal muscle differentiation, and suggest that this
association is regulated by developmental post-translational tubulin
modification.
|
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|
MURF2 interaction with sarcomeric myosin and titin
Since endogenous MURF2 in muscle cells is largely associated with the
cytoskeleton, and several MURFs are co-expressed, we expressed MURF2
selectively in non-myogenic HeLa cells to assay for a possible interaction
with sarcomeric myosin and A-band titin. We observed that conventional
preparations of sarcomeric myosin contain copurified MURF2 protein, and that
exogenous MURF2 cosediments with myosin
(Fig. 2E). This suggests that
MURF2 can directly associate with myosin. Because of the high sequence
homology between MURF1 and MURF2, we also investigated the possibility that
MURF2 could interact with the MURF1-binding A-band titin region. Again, we
used HeLa-expressed MURF2 free of MURF1 or MURF3. In a pulldown assay using
GST-tagged titin A164-169, we observed binding of MURF2 to titin, but not to
GST (Fig. 2E) or other protein
constructs. These combined data suggest that MURF2 can interact with
sarcomeric myosin and that muscle myosin preparations actually contain traces
of MURF2, and furthermore, that MURF2 contains intrinsic titin-binding
properties previously undiscovered by yeast two-hybrid analysis.
Dynamics of MURF2 during skeletal muscle differentiation
MURF2 and sarcomeric actin
The actin cytoskeleton undergoes major remodelling during myocyte
differentiation, resulting ultimately in the highly ordered arrays of
sarcomeric, parallel actin filaments cross-linked with the antiparallel
filaments from the opposite sarcomere half at the Z-disk
(Luther et al., 2002). The
temporal and spatial distribution patterns of MURF2 were analysed by double
immunofluorescence experiments with the anti-MURF2 antibody and
anti-sarcomeric actin. In C57 cells, endogenous MURF2 and skeletal actin
remain mostly segregated during myofibrillogenesis
(Fig. 4). Although
MURF2-decorated microtubules aligned parallel to actin filaments (arrowheads
in 4B), strict colocalisation was rare and the bulk of both proteins were
found in separate compartments at the beginning of differentiation
(Fig. 4A). Later, in
premyofibrils, regions displaying cross-striated actin filaments showed no
MURF2 labelling (Fig. 4B). This
suggests that MURF2 does not follow the integrative route of most classical
sarcomeric proteins, which are found to colocalise with actin and
-actinin in premyofibrils or stress-fibre like structures.
|
MURF2 and the titin Z-disk and I-band regions
Titin follows a defined path of sarcomeric integration, starting from
dot-like aggregates on SFLS, which localise to Z-bodies. Initially, the Z-disk
portion becomes organised, followed by stretching-out of the molecule and the
integration of the C-terminal M-band portion. The association of MURF2 with
the A-band region of titin revealed by protein interaction analysis raises the
question of the time course and the distribution pattern of both proteins. We
therefore investigated the distribution of MURF2 in relation to the titin
Z-disk, I- and A-band using the anti-MURF2 antibody in combination with the
monoclonal anti-titin I-band N2A antibody
(Gautel et al., 1996) or with
the T12 anti-titin antibody [Z-band
(Fürst et al., 1988b
)].
At the beginning of differentiation, (Fig.
5A), stress fibre-like structures, stained with the titin N2A
antibody, are completely excluded from the labelling revealed with the MURF2
antibody. Titin T12 Z-disk epitopes were identified as dots and patches
aligned parallel to MURF2 positive filaments
(Fig. 5C). In young myotubes,
higher magnification showed that titin T12-positive spots can occasionally
colocalise with long MURF2-decorated microtubules
(Fig. 5D, arrowhead).
Occasional colocalisation of both proteins was also observed even with Z-disk
titin dots of a periodicity <1 µm
(Fig. 5D). The absence of
strict colocalisation at these early stages resembles the pattern of
sarcomeric actin and reflects the early association of titin and actin. When
sarcomeres start to form, MURF2 appears in small punctuate structures mostly
arranged in a linear pattern (Fig.
5E, arrowhead). These structures seemed to be in register with
more mature sarcomeric Z-disk titin displaying a 2 µm periodicity
(Fig. 5E, arrow). During
differentiation, colocalisation of MURF2 and titin I-band N2A could be
observed in cell regions where I band cross striation starts to be organised
(Fig. 5B). MURF2 and titin N2A
antibodies decorated regions where non-striated myofibrils differentiate into
striated myofibrils (Fig. 5B,
arrowhead). It is noteworthy that MURF2 was barely detectable in mature
sarcomeres (Fig. 5B, arrow).
All these results indicate that even though MURF2 can align with the Titin I
and Titin Z regions, these associations take place only transiently and in
different intracellular domains of differentiating cells.
|
MURF2 and the titin A-band region
A more complex picture was observed for the Titin A-band region
(Fig. 6). C57 cells were double
stained with the T31 antibody directed against a central A-band epitope of
titin (Fürst et al.,
1989a) in combination with the anti MURF2 antibody at different
developmental stages. Early during differentiation
(Fig. 6A), the MURF2 antibody
stained MURF2-decorated microtubules whereas titin was visible as fluorescent
spots dispersed longitudinally, apparently along SFLS and without obvious
colocalisation with MURF2. This pattern is similar to that observed for the
titin Z- and I-band epitopes. Titin A-band patches then accumulated and
assembled along MURF2 positive structures
(Fig. 6B). As shown by their
overlap, MURF2 and A-band titin particularly colocalised in maturing
non-striated myofibrils. In nascent striated myofibrils, MURF2 was seen mainly
as dotted, rather than continuous, structures arranged in a linear fashion,
sometimes on both sides of titin A-band labelled structures
(Fig. 6C, arrowhead). When
clear A-band doublets begun to form in nascent striated myofibrils, MURF2
showed lateral colocalisation with non-striated A-band titin on both sides of
the transversal titin bands (Fig.
6C, arrow). This pattern suggests that initially, MURF2 and A-band
titin were segregated, but transiently co-aligned during the progression of
the non-striated myofibril to the striated myofibril. These results also
confirm that MURF2 can associate with A-band titin, and suggest that this
interaction occurs transiently in non-striated myofibrils.
MURF2 and the assembly of sarcomeric myosin filaments
Sarcomeric myosin is integrated into the nascent sarcomere at a late stage
of myofibril formation, after the assembly of an initial titin/actin/M-band
scaffold (Ehler et al., 1999;
Van der Ven et al., 1999
).
Titin has been proposed to act as a molecular ruler that directs the assembly
of thick filaments in the sarcomere
(Trinick, 1992
). An intact
microtubule network dynamics is also required for the correct assembly of
sarcomeres (Antin et al., 1981
;
Holtzer et al., 1985
;
Saitoh et al., 1988
). Due to
the interaction of MURF2 with microtubules and its transient association with
A-band titin, we investigated the localisation of MURF2 in relation to
sarcomeric myosin during differentiation. Double immunofluorescence was
performed with anti-MURF2 and anti-myosin heavy chain antibodies. Myofibril
assembly progresses from microscopically less ordered structures to the highly
ordered contractile apparatus; as long as new sarcomeres and myofibrils
develop, these distinct steps are observed simultaneously in a single cell
(Fig. 7B).
|
Of all sarcomeric markers, myosin showed the earliest colocalisation with MURF2. Arrays of MURF2 labelled microtubules largely colocalised with parallel bundles of sarcomeric myosin in non-striated myofibrils (Fig. 7A). As differentiation proceeded, non-striated myofibrils co-aligned with more mature stages, nascent striated myofibrils and mature myofibrils. Clear colocalisation of sarcomeric myosin and MURF2 could then be observed in long filamentous structures (Fig. 7B). In these structures, MURF2 was found in strictest localisation with myosin, at the lateral boundaries of the nascent striated myofibrils where myosin lacked the exact alignment in-register found in fully mature sarcomeres (Fig. 7C, arrowhead). Interestingly, when myosin was organised in a typical periodic A-band pattern, MURF2 disappeared from the sarcomere (Fig. 7C, arrow). Altogether, these results reveal that MURF2 associates closely with sarcomeric myosin all along myogenic differentiation until mature sarcomeres are organized.
Nuclear translocation of MURF2 in cardiomyocytes
MURF1 and -3 were reported to be localised at the sarcomeric Z- and M-bands
of cardiomyocytes, with a surprising dual localisation of MURF1 at both sites
in cardiomyocytes (Centner et al.,
2001; Spencer et al.,
2000
). For the understanding of MURF functions, it is important to
establish whether, and where MURF2, whose cellular localisation in both
skeletal and cardiac myocytes was unknown to date, was present in the
sarcomeres of cardiomyocytes. In neonatal rat cardiomyocytes cultivated for 72
hours under high-serum conditions and
-adrenergic stimulation, we
detected MURF2 at the sarcomeric M-band as well as in a weak diffuse cytosolic
pattern (Fig. 8A). In these
cells, no nuclear localisation of MURF2 could be observed in agreement with
(Spencer et al., 2000
). In
neonatal rat cardiomyocytes that had been serumstarved for 36 hours, MURF2
(green) is largely diffusely distributed in the cytosol but notably found in a
speckled pattern in the nucleus (Fig.
8B, arrow). This pattern persists in cells re-exposed to serum for
3-12 hours. There is also significant accumulation of MURF2 at the nuclear
envelope (Fig. 8B, arrowhead),
the site of other proteins involved in SUMO-regulated nuclear transport like
RanGAP. The diffuse cytosolic staining suggests a mobile pool of protein.
These combined data identify MURF2 as a protein with triple cellular
localisation: on microtubules, at M-bands, and in the nucleus. The latter
localisation seems to correlate with the differentiation state of the
cardiomyocyte and is induced by serum starvation.
|
![]() |
Discussion |
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MURF2 belongs to the MURF family of muscle-specific RING/B-box zinc-finger
proteins (Centner et al., 2001)
first identified by Spencer et al.
(Spencer et al., 2000
) in a
search for ligands of the serum response factor (SRF). A second MURF member,
MURF1, was identified as a ligand of titin close to the M-band, and about 12
nm N-terminal to the kinase domain. It was hence speculated that MURFs might
be regulators of the titin kinase domain
(Centner et al., 2001
). In
adult cardiac muscle, MURF1 was found both at the Z-disk and the M-band; MURF3
was assigned to the Z-disk. MURF1 was recently proposed to play a role in
thick filament assembly (McElhinny et al.,
2002
). However, a MURF1 knockout mouse shows no defects in primary
myofibrillogenesis (Bodine et al.,
2001
), but rather a resistance to atrophy in agreement with the
up-regulation of MURF1 under these conditions. These observations suggest that
the functions of MURF1 in myofibril assembly are partly redundant or
dispensable. To understand MURF functions, a detailed analysis of the role in
myofibril assembly is clearly required. The previous studies on MURF1 and
MURF3 have focussed on cardiac myocytes, where primary myofibril assembly
cannot be studied due to the preformed myofibrils. In this study, we therefore
present the first detailed temporal analysis of a MURF-protein, MURF2, during
myogenic differentiation in skeletal muscle.
MURF2 was isolated on affinity beads of an A-band titin fragment
(Iakovenko and Gautel, 2000)
containing the binding site for MURF1, possibly due to its ability to form
heterodimers with MURF1 and MURF3 (Centner
et al., 2001
); Spencer et al.,
2000
). However, we demonstrate here for the first time that MURF2
also displays intrinsic titin-binding properties which may contribute to the
cellular localisation near the M-band of mature cardiac sarcomeres. Whereas
MURF1 appears to remain expressed in all striated muscles at all stages of
differentiation, we find that MURF2 is down-regulated in mature skeletal
myotubes and is excluded from mature skeletal sarcomeres. These results are in
good agreement with northern blot analysis which suggested that MURF2 is
expressed at best weakly in adult cardiac and skeletal tissues
(Centner et al., 2001
), and
that MURF1 is strongly expressed in fetal heart muscle
(Dai and Liew, 2001
). Our data
together with those of Spencer et al.
(Spencer et al., 2000
) show
that both MURF3 and MURF2 proteins are microtubule associated proteins but
that expression and localisation remain distinct, indicating that MURF2 and
MURF3 could fulfil different tasks during differentiation. Whereas MURF3 was
detected in proliferating myoblasts and in adult skeletal and cardiac muscle
(Spencer et al., 2000
), MURF2
shows temporal dynamics in its expression in skeletal muscle cells with
highest levels early after the onset of differentiation
(Fig. 2A,B).
During differentiation, MURF2 distribution follows the morphological
reorganisation undergone by the microtubule cytoskeleton
(Fischman, 1970;
Gundersen et al., 1989
;
Okazaki and Holtzer, 1965
). At
early stages of differentiation, the MURF2 antibody stains distinct
microtubules all along the length of the nascent myotube, while at later
stages, segmented rod-like and dotty structures were observed. These various
morphologies reflect the reorganization of the microtubule network
(Cartwright and Goldstein,
1982
; Gundersen et al.,
1989
; Warren,
1974
), and/or specific intracellular relocalisation of MURF2. This
association is also evident in cells treated with nocodazole where MURF2
adopts a diffuse distribution whereas titin on SFLS is not impaired (data not
shown).
The closest association between MURF2 and sarcomeric proteins is observed
for sarcomeric myosin, well before the integration of myosin into nascent
myofibrils. Although sarcomeric myosin appears as one of the first
myofibrillar proteins, the striated A-band arrangement is observed only at a
very late stage (Person et al.,
2000; Rudy et al.,
2001
; Van der Ven et al.,
1999
). MURF2 is the first MURF protein known to show morphological
myosin association. In agreement with this, we also detected MURF2 in
preparations of sarcomeric myosin (Fig.
2E). According to our immunofluorescence data, MURF2 and myosin
association take place at the beginning of differentiation, when the
microtubule network is still preserved on large scale. Interestingly, myosin
filament assembly is independent of actin filaments
(Guo et al., 1986
;
Holtzer et al., 1997
;
LoRusso et al., 1997
;
Rhee et al., 1994
;
Sanger et al., 1986
;
Schultheiss et al., 1990
;
Wang and Wright, 1988
), but
requires the microtubule cytoskeleton for the formation and the organization
of the thick filaments in A-bands (Antin et
al., 1981
; Toyama et al.,
1982
).
In contrast, actin and Z-disk titin co-assemble early on and initiate the
formation of a Z-disk-titin-M-band scaffold into which myosin is finally
integrated. Our observations suggest that the Z-disk/titin/M-band scaffold
only transiently co-aligns with MURF2-containing microtubules during sarcomere
formation. However, both myosin and A-band titin colocalise with MURF2 in
non-striated and nascent striated myofibrils, parallel and closely apposed to
striated myofibrils. Many observations showed that the organisation of the
N-terminal titin domains precede those of the C-terminal regions, revealing
the sequential and structural order of titin molecule unravelling
(Ehler et al., 1999;
Fürst et al., 1989b
;
Komiyama et al., 1993
;
Mayans et al., 1998
;
Schultheiss et al., 1990
;
Soeno et al., 1999
;
Van der Loop et al., 1996
;
Van der Ven et al., 1999
). The
temporal order of the spatial relationship of MURF2 with respect to Z-disk,
I-band and A-band titin are in good agreement with an involvement in the
straightening and stretching of the giant titin molecule during sarcomere
formation. Co-alignment of MURF2 and Z-disk titin takes place at the beginning
of differentiation, as indicated by the integrity of the microtubule network
staining revealed with MURF2 antibody (Fig.
5D). Since overlap of MURF2 and I-band titin is only observed on
irregular cross-striated sarcomeres (Fig.
5B), MURF2 seems to align with I-band titin only briefly. As
expected due to the biochemical association with titin, MURF2 localises with
A-band titin. Since definite striations of A-band titin epitopes are formed at
a late stage of differentiation, one could hypothesise that MURF2 interacts
with the folded-up titin molecule when their A-band portion starts to stretch
out from the Z-disk portion.
The early and persistent association of MURF2 with both sarcomeric myosin
and microtubules, and the parallel alignment of microtubules and their
associated proteins with nascent striated myofibrils suggest that microtubules
are indeed involved in translocating myosin filaments to the sites of final
sarcomere assembly. MURFs appear to act as a transient adaptor between
sarcomeric proteins, especially myosin and titin, and the microtubule network.
At the beginning of differentiation, the MURF2/myosin complex interacts with
microtubules, allowing the dispersal of myosin throughout the myotube along
the microtubule network. During differentiation, MURF2 thus brings myosin
filaments at the vicinity of maturing titin filaments. MURF2 homo- or
hetero-multimeres could then coordinate binding sites on myosin and titin.
MURF1, which interacts most strongly with A-band titin
(McElhinny et al., 2002), may
provide the link to the titin filament, although we show here that titin
binding is not an exclusive property of MURF1. This may explain why a knockout
of MURF1 is without consequences for primary sarcomere formation
(Bodine et al., 2001
). This
proposed role of MURFs provides an explanation how A-band titin and myosin are
finally brought in tight register, summarised synoptically in the sketch in
Fig. 9. The processes of active
transport of myosin, and the stretching out and aligning of nascent
myofibrils, must require force and hence the activity of molecular motors. It
will be interesting to see whether these are microtubule-based and whether
such motors associate with MURFs.
|
We found that MURF2 is expressed in multiple isoforms, some of which are
tissue-specific. MURF2p60B is generated by alternative reading
frame use, the first description of this novel splice mechanism
(Klemke et al., 2001) in a
muscle protein. Apart from creating the potential for innumerable permutations
in complex formation during sarcomere assembly, the differentially expressed
MURFs may have other, muscle-type-specific functions. The ablation of MURF3 by
antisense RNA dramatically suppresses myogenic differentiation on the
transcriptional level (Spencer et al.,
2000
) apart from impairing myofibril formation. This may suggest a
role in the control muscle differentiation apart from that of a transient
structural adaptor during myofibril formation. A role for MURF3 in muscle gene
transcription could be inferred from the putative interaction with SRF
(Spencer et al., 2000
). MURF1,
also known as SMRZ (Dai and Liew,
2001
), was found to localise to the nucleus when transfected into
C2C12 myoblasts, as well as to translocate in cardiac myocytes
(McElhinny et al., 2002
).
MURF1 can also interact via the highly conserved RING domain with the
ubiquitin-like SUMO-2/SMT3b (Dai and Liew,
2001
), linking MURFs to potential roles in nuclear transport,
transcription regulation and signal transduction
(Müller et al., 2001
).
Our data provide the first evidence that endogenous MURF2 is translocated to
the nucleus and the nuclear lamina in response to stimulation of serum-starved
cardiac myocytes. MURF2 can thus shuttle between three cellular compartments:
microtubules, M-bands and the nucleus. However, MURF2 is detectable in the
nucleus of neonatal rat cardiomyocytes only briefly after shifting
serum-starved cells from low to high-serum conditions, indicating an
involvement in nuclear signalling in a narrow time window and related to the
stress of serum-withdrawal. Although we could not observe MURF2 in skeletal
myotube nuclei, we are currently investigating the localisation in skeletal
muscle tissues. Whether MURFs act as transcriptional co-activators or
co-repressors in the nucleus in addition to a more structural involvement in
sarcomere assembly will now need to be elucidated. Our observations suggest
that the sarcomere not only receives input from many signal transduction
pathways, but may also relay information to the transcriptional machinery and
could thus regulate muscle-specific gene expression as first proposed by
Iakovenko and Gautel (Iakovenko and
Gautel, 2000
). MURFs emerge as novel components of this crosstalk,
and the various signals resulting in stress-induced MURF translocation now
need to be identified.
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Acknowledgments |
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References |
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