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Journal of Cell Science 112, 989-1001 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS9866
Dynamic distribution and formation of a para-sarcomeric banding pattern of
prosomes during myogenic differentiation of satellite cells in vitro
J. Foucrier1,*, M. C. Grand1, F. De Conto2,3, Y. Bassaglia1, G. Géraud2, K. Scherrer2 and I. Martelly1
1CRRET, UPRESA-CNRS 7053, Université Paris 12, Av. du Général de Gaulle, 94010 Créteil
2Institut Jacques Monod, Université Paris 7, 2, place Jussieu, 75251 Paris Cedex 05, France
3Istituto di Microbiologia, Università degli Studi, Viale A. Gramsci 14, 43100 Parma, Italy
Cedex, France
*Author for correspondence
Accepted 13 January; published on WWW 10 March 1999
SUMMARY
Myogenesis proceeds by fusion of proliferating myoblasts
into myotubes under the control of various transcription
factors. In adult skeletal muscle, myogenic stem cells are
represented by the satellite cells which can be cultured and
differentiate in vitro. This system was used to investigate the
subcellular distribution of a particular type of prosomes at
different steps of the myogenic process. Prosomes constitute
the MCP core of the 26S proteasomes but were first
observed as subcomplexes of the untranslated mRNPs;
recently, their RNase activity was discovered. A monoclonal
antibody raised against the p27K subunit showed that the
p27K subunit-specific prosomes move transiently into the
nucleus prior to the onset of myoblast fusion into myotubes;
this represents possibly one of the first signs of myoblast
switching into the differentiation pathway. Prior to fusion,
the prosomes containing the p27K subunit return to the
cytoplasm, where they align with the gradually formed
lengthwise-running desmin-type intermediate filaments and
the microfilaments, co-localizing finally with the actin
bundles. The prosomes progressively form discontinuous
punctate structures which eventually develop a pseudosarcomeric banding pattern. In myotubes just formed in
vitro, the formation of this pattern seems to preceed that
produced by the muscle-specific sarcomeric α-actin.
Interestingly, this pattern of prosomes of myotubes in
terminal in vitro differentiation was very similar to that of
prosomes observed in vivo in foetal and adult muscle. These
observations are discussed in relation to molecular
myogenesis and prosome/proteasome function.
INTRODUCTION
precursor cells into the syncitial myotubes, the cytoskeletal
framework is dramatically modified in relation to the creation of
a functional sarcomeric organization.
During the course of myogenic differentiation, numerous
factors may exert specific control at different key steps. Among
the factors acting in myoblast maturation, prosomes appear to
be important in view of their multiple biological roles. These
particles constitute the proteolytic core of the 26S proteasomes
(therefore, also called ‘multicatalytic proteinase’ (MCP) or
‘20S proteasomes’) and also have RNase activities. The 26S
proteasomes (‘MCP-complex’ or ‘MCP-C’) were originally
found as components of the untranslated mRNA complexes in
the cytoplasm and, more recently, as components of premRNPs and the nuclear matrix. They may thus be considered
as cellular factors playing an important role in the homeostasis
of cellular proteins by protein biosynthesis and breakdown (for
review see Coux et al., 1996; Scherrer and Bey, 1994).
The prosome particles, first found associated with untranslated
mRNPs, may be considered trans-acting factors acting at
transcriptional and post-transcriptional levels of gene expression.
Prosomes were found on interphase chromosomes, the nuclear
matrix and pre-mRNP (Pal et al., 1988; E. Pilotti et al.,
unpublished), and cytoplasmic mRNP but not in polyribosomes
Satellite cells, also called adult myoblasts or muscle precursor
cells, were first described by Mauro (1961). These mononucleate
cells are located between the basement membrane and the plasma
membrane of the muscle fiber. A large body of literature has
indicated that satellite cells provide myofiber nuclei in the
growing muscles. These cells are also activated in injured
muscles, where their progeny differentiate and fuse to reconstitute
the wounded muscle fibers (for review see Hartley and YablonkaReuveni, 1992). In contrast to some commonly used myogenic
cell lines such as C2.7, culture of primary satellite cells offers the
opportunity to observe complete myogenic differentiation very
similar to that occuring in vivo. Myoblasts proliferate, become
postmitotic, and differentiate into large multinucleate myotubes
which spontaneously contract about 11-12 days after plating
(Campion, 1983; Le Moigne et al., 1990).
The exit from cell cycling and the differentiation process most
likely involve significant alterations of the intracellular protein
pool mediated by reprogramming and cytoplasmic site-specific
targeting of mRNA expression, and by proteolytic activity.
Furthermore, myogenesis involves profound reorganization of the
cell structure. Thus, during the fusion of the individual myogenic
Key words: Prosome, Proteasome, Cytoskeleton, Satellite cell,
Myogenesis, Sarcomere
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J. Foucrier and others
(Schmid et al., 1984; Spohr et al., 1970); they inhibit mRNA
translation in vitro (Horsch et al., 1989). A minor fraction of
prosomes contains small RNAs of which the major component
was identified as tRNAlys3 (Nothwang et al., 1992). More
recently, an RNase activity of prosomes was discovered in two
of the α-type subunits (Petit et al., 1997; Pouch et al., 1995).
Moreover, as mentioned, the 20S particles have a multicatalytical proteinase (MCP) activity which is exerted in vivo
mainly if not exclusively within the context of a higher order
complex, the 26S proteasome. In yeast and higher eukaryotes the
particles have a closed molecular configuration that must be
opened to allow proteolysis (Groll et al., 1997). In the 26S
complex, the basic prosome particle is associated with an
extensive and variable combination of ATPases, proteases,
activators, and inhibitors, which confer very selective and
variable substrate specificities to the structure (Hilt and Wolf,
1996; Orino et al., 1991). However, it was shown most recently
that the proteasome activity can be fully replaced by another
multicatalytic proteinase system, most likely the ‘Tricorn’
proteases (Glas et al., 1998; Tamura et al., 1996).
Many different types of prosomes can exist since they form
variable mosaics of proteins within their 28 subunit structure.
It was found that these mosaics change with the cell type and
physiological state, for example when the cells are treated by
interferon γ (Ortiz-Navarrete et al., 1991), or when myoblasts
fuse into myotubes (De Conto et al., 1997).
Since the original observation (Grossi de Sa et al., 1988)
showing colocalization of prosomes with intermediary
filaments (IFs), the preferential association of some prosomal
subpopulations within the cytoskeleton has been widely
investigated in various types of epithelial cells, in fibroblasts
and muscle cells (Arcangeletti et al., 1992; De Conto et al.,
1997; Olink-Coux et al., 1992, 1994). Preliminary observations
had given evidence for association of prosomes not only with
the IFs but also with the actin-containing filaments (Foucrier
et al., 1994; Grand et al., 1994); this observation was recently
fully confirmed by Arcangeletti et al. (1997b). The association
of prosomes with the cytoskeleton was more recently shown to
undergo changes during the fusion of C2.7 myoblasts (De
Conto et al., 1997). These data may be of particular interest
since the cytoskeleton, and more specifically the microfilament
system, are known to be directly implicated in translation by
‘carrying’ the polyribosomes and possibly providing ‘tracks’
for mRNA translocation leading to the anchoring of transcripts
in specific cellular regions (Grossi de Sa et al., 1988; Hesketh,
1991; Hesketh and Pryme, 1991; Hovland et al., 1996; Scherrer
and Bey, 1994; Singer, 1992; Wilhelm and Vale, 1993).
Taken together, all these observations have led to the
hypothesis that the prosomes associated with the mRNAs might
serve in targeting gene expression to regions of the cell where
their cognate proteins are used. Recently, a basic assumption
within this working hypothesis was confirmed by the
demonstration of Fulton and co-workers (Cripe et al., 1993;
Fulton and Alftine, 1997) that specific muscular and
cytoskeletal mRNAs are intercalated into the sarcomeric
structure of skeletal muscle of the chicken forming a regular
banding pattern similar to that of the prosomes. In this context,
it seemed of particular interest to investigate the behaviour of
prosomes during the course of myogenesis when important
morpho-functional changes occur in relation to the progressive
sarcomeric compartmentation of the functional myotubes.
In view of the profound reprogramming of the translation
machinery accompanying the reorganization of the cytoskeletal
systems during myotube formation, we investigated the
relationship between prosomes and the cytoskeleton and in
particular the intermediate filament and actin systems in the
satellite cells prior to and during fusion, with the aim of better
understanding the possible metabolic functions of the
prosomes in myogenesis.
To study whether changes in prosome cytodistribution and
preferential location of these particles occur during the different
steps of the myogenic process, we used several antibodies raised
against prosomes (p-mAbs). Here we show that according to the
stages of differentiation and/or maturation of the cells, the
cytoplasmic prosomes containing the p27K subunit move first
to the nucleus and then back. Thereafter, when cells assume a
fusiform shape, their preferential localization is on longitudinal
fibrillar structures, in a progressively ‘pearls on a string’ manner
and gradually organizing into a pseudo-sarcomeric pattern
similar to that observed in vivo in the adult striated muscle.
MATERIALS AND METHODS
Satellite cell cultures
Limb muscles of 2 month old rats (Wistar strain) were aseptically
removed. After elimination of tendons, connective tissue and blood
vessels, muscles were minced in Ca2+/Mg2+-free PBS buffer. The
fragments were rinsed three times in the same buffer, then incubated
with gentle stirring for 2 hours at 37°C in 0.15% pronase in Ham’s F12
medium, 10 mM Hepes/NaOH (pH 7.3) supplemented with 10% FCS
(Gibco Laboratories, Grand Island, NY/USA). After decanting, the
supernatant was diluted with Dulbecco’s medium (1:1) and centrifuged
20 minutes at 300 g. After re-suspension of the pellet, centrifugation
was repeated under the same conditions. After final rinsing in
Dulbecco’s medium supplemented with 10% FCS and 10% horse serum
(Gibco), the cells were plated on gelatinated four-chamber glass slides
(2000 cells/cm2). Cultures were maintained at 37°C in a humidified
atmosphere with 5% CO2. The medium was replaced every 4 days.
Preparation of biological samples
Adult male rats (Wistar strain) were sacrificed and Soleus muscles
were removed maintaining their state of contraction. The limbs of
foetuses on day 19 of gestation were also used. After immediate
immersion in chilled isopentane, both types of biological samples
were frozen in liquid nitrogen and stored at −80°C.
Prosomal antibodies
Six monoclonal antibodies (p-mAbs) raised against prosomal proteins
from duck erythroblasts and HeLa cells and known to cross-react with
mammalian cells were used (Olink-Coux et al., 1994; Grossi de Sa et
al., 1988); these mAbs are available from ICN Biomedicals (Orsay
Cedex, France). The prosomal subunits tested were: p27K (hybridoma
clone IB5); p29K (clone GD6); p31K (clone AA4); p23K (clone
35A); p25K (clone 7A11); p30/33K (clone 62A32). The prosome
subunits named according to our terminology (Grossi de Sa et al.,
1988) correspond to the human subunit names published by Hendil,
Tanaka and colleagues (Kristensen et al., 1994) in the following
manner: p23K, subunit HC7-I; p25K, HC3; p27K, Pros-27 (or iota);
p29K, HC9; p31K, HC8; p30-33K, HC2.
Cell fractionation
After 11 days of primary satellite cell culture in Petri dishes (60 mm
diameter), the cells were scraped off and washed twice in PBS.
Hypotonic buffer (10 mM triethanolamine, pH 7.4, 10 mM KCl, 10
mM MgCl2, 10 mM MnCl2, 5 mM 2-mercaptoethanol) was added to
the suspension (dilution 1:9) and cells were disrupted at 4°C using a
Prosome behaviour in myogenic satellite cells
Dounce homogenizer. Lysis was monitored by phase contrast
microscopy. Sucrose (1 M) was added to restore the isotonicity of the
suspension (0.25 M final). Nuclei and mitochondria were removed by
centrifugation (4000 g for 10 minutes and 16000 g for 15 minutes,
respectively) as described by Goodridge et al. (1979). The
postmitochondrial supernatant was removed carefully, so as not to stir
up the lysosomes from the pellet surface, and stored at −80°C until use.
Western blotting
After SDS-PAGE according to the method of Laemmli (1970),
proteins were electrophoretically transferred onto nitrocellulose
membranes (0.45 mm) according to the method of Towbin et al.
(1979) in the presence of 0.1% SDS in the transfer buffer. The protein
blot was satured for 2 hours at 20°C with 5% skimmed milk in PBS.
Incubation of the nitrocellulose membranes with each different pmAb (1:300 in the saturating buffer) was for 90 minutes at room
temperature, followed by three washings with PBS. A further
incubation was for 60 minutes with the alkaline phophataseconjugated second antibody (goat anti-mouse IgG AP conjugate;
Promega) diluted in the saturating buffer (1:5000). The membranes
were washed three times in PBS, and developed for 10 minutes using
an extratemporaneous preparation containing NBT (nitro blue
tetrazolium) substrate and BCIP (5-bromo-4-chloro-3-indolylphosphate) substrate (Promega) diluted in alkaline phosphatase buffer
(100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2). The
reaction was arrested by washing with PBS containing 20 mM EDTA.
Immunofluorescence microscopy
At given times, the cultures were washed with PBS, dried, and fixed in
acetone for 10 minutes at room temperature and kept at −20°C until use.
For immunolabelling, the indirect immunofluorescence technique
was used. All incubations were at room temperature in a humid
chamber. Antibodies were diluted in PBS (pH 7.4) containing 1% Triton
X-100, 1% bovine serum albumin (BSA) and 0.02% natrium azide.
Preparations were pre-incubated for 15 minutes with normal goat
serum (Eurobio, Paris, France), then incubated for 1 hour with the pmAb raised against the p27K subunit (dilution 1:20). After three
washes for 10 minutes in PBS, the second incubation was carried out
for 1 hour in darkness. The mAbs were revealed by sheep anti-mouse
IgG antibodies coupled to fluorescein isothiocyanate (FITC)
(Amersham, Aylesbury, UK; diluted 1:30).
For double immunofluorescence, the first incubation was performed
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using a mixture containing the anti-p27K mAb (final dilution 1:20)
and a rabbit polyclonal anti-desmin antibody (Euro-diagnostica,
Dardilly, France; final dilution 1:20). The primary antibodies were
secondarily detected with a mixture containing FITC-conjugated
sheep anti-mouse IgG and Texas Red-conjugated donkey anti-rabbit
IgG (Amersham, Aylesbury, UK) used at 1:25 final dilution. To detect
both p27K and actin the first incubation was with Texas Red-labelled
phalloidin diluted 1:1000 (Sigma, St Louis, USA) mixed with the antip27K antibody. In all cases, the preparations were washed three times
after the second incubation and mounted in Mowiol (Calbiochem, La
Jolla, USA). Negative controls were realized on specimens incubated
either with nonimmune serum or with the incubation buffer alone,
followed by the second conjugated antibody.
Longitudinal sections (5 to 8 µm thickness) of adult or fœtal frozen
muscles were made with a cryotome and air dried prior to fixation and
then fixed in acetone for 10 minutes at room temperature. The
p27K prosomal antigen was detected using the same indirect
immunofluorescence technique as described above concerning the
satellite cells cultivated in vitro.
The immunofluorescence preparations were observed with a
Polyvar microscope (Reichert-Leica) in epifluorescence or by
confocal microscopy.
Confocal laser scanning microscopy
Confocal laser scanning microscopy was performed using a MRC-600
(Bio-Rad, UK), mounted on an Optiphot II Nikon microscope equipped
with a ×60 objective (Plan Apo; NA 1.4). Detection of fluorescein and
Texas Red was performed using an argon ion laser adjusted at 488 nm,
and an helium-neon ion laser adjusted at 543 nm, respectively. For each
optical section, double fluorescence images were acquired in sequential
mode, fluorescein first, Texas Red second. The emitted signal was
treated by Kalman filter (average of 8 images) in order to increase the
signal to noise ratio. The pinhole of the confocal system was closed to
a minimum to yield the thinnest possible optical section and a step of
500 nm between consecutive optical sections was chosen.
Photographs were printed on sublimation laser printer (Colorease
Kodak) with Photoshop software (Adobe Systems, Inc.).
RESULTS
Satellite cells grown in vitro proliferate, align laterally at a
Fig. 1. Histology of in vitro myogenesis. (A) 4-day adult rat satellite cells prior to cell fusion. (B) 12-day culture showing a network of
plurinucleated myotubes. (A-B) May-Grunwald-Giemsa staining. (C) Phase contrast of a mature contracting myotube with cross-striations
related to the functional sarcomeric organization. Bars, 20 µm.
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J. Foucrier and others
kDa
Fig. 2. Presence of prosomal subunits in the post-mitochondrial
supernatant of satellite cells. Proteins precipitated from the
postmitochondrial supernatant fraction were separated by gel
electrophoresis, transferred to a nitrocellulose membrane and then
separately immunoreacted with a panel of different p-mAbs (antip23K, anti-p25K, anti-p27K, anti-p29K, anti-p31 and anti-p30/33K,
lanes 2 to 7, respectively). On the left side (lane 1), molecular mass
markers were: phosphorylase b (97.4 kDa), BSA (66.2 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin
inhibitor (21.5 kDa) and lactalbumin (14.4 kDa).
specific
post-mitotic
stage,
undergo
spontaneous
differentiation, and finally fuse into multinucleated myotubes.
In our experimental conditions, myogenic differentiation was
not synchronous since the same medium was used during the
entire time of culture (2 weeks). The first fusions were observed
about 6 days after plating. In older cultures, large myotubes
were seen with hundreds of nuclei. They eventually contracted
spontaneously from days 11 to 12 onwards (Fig. 1A-C).
Detection of specific prosomal subunits by western
blotting
In order to determine the presence and nature of the particular
types of prosomes present in satellite cells, one-dimensional
gel electrophoresis and western blotting were carried out on
the precipitated proteins of the post-mitochondrial supernatant
of cells collected on day 11 after plating. The immunoreactions
shown in Fig. 2 indicate that only three (anti-p27K, anti-p31K
and anti-p30/33K) of the six different p-mAbs tested, clearly
cross-reacted with prosomal proteins in this subcellular
fraction, analyzed after denaturation of proteins by SDS. The
p27K-specific p-mAb gave the strongest staining reaction,
showing a large protein band; a second minor band was
observed corresponding to a protein of about 38 kDa which
corresponds to an alternate minor product encoded by the same
gene (Bey et al., 1993; Schmid et al., 1984). Compared to the
anti-p27K p-mAb, the anti-p30/33K p-mAb gave a minor
reaction and recognized a protein of 30 kDa. Very faint positive
reactions were produced by the p25K- and p31K-, and no or
insignificant reactions were seen with the p23K- and p29Kspecific p-mAbs. It seems that in fusing myoblasts only some
of the commonly found prosomal subunits are easily detected.
In view of this analysis, we choose the p27K antigen for
subsequent investigation.
Intra-sarcomeric banding of the p27K antigen in
foetal and adult muscles
To appreciate the indirect immunofluorescence data shown
below, it should be kept in mind that in all cells tested thus far,
prosomal antigens were never found as free subunits outside of
the particles (discussed by Scherrer and Bey, 1994), with some
notable exceptions (Martins de Sa et al., 1989; Hendil et al.,
Fig. 3. Immunolocalization of the p27K prosomal antigen in skeletal muscle frozen sections. (A) adult myofibers and (B) foetal myotubes at
day 19 of gestation using the monoclonal anti-p27K antibody. In both tissue sections, the p27K-specific p-mAb showed a typical doublet
banding pattern in a cross-striated fashion. Bar, 10 µm.
Prosome behaviour in myogenic satellite cells
1995). The immunofluorescence labelling patterns shown here
accordingly represent prosome particles and not free antigens.
In histological preparations of adult and foetal rat muscles, a
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very intense and reproducible labelling pattern was observed by
immunofluorescence using the p27K p-mAb. The obvious
regular transverse striated pattern shows single or typically
Fig. 4. Reorganization of the p27K prosomal antigen during myogenic cell differentiation in vitro. The p27K prosomal antigen was detected by
indirect immunofluorescence labelling after fixation of satellite cells with acetone at successive stages of differentiation. (A,B) Proliferating
cells on days 3 and 4 of culture, respectively. (C) Post-mitotic myoblasts on days 6-7. (D,E) First stages of fusion on days 8-11. (F,G) Days 1113. (H) Days 13-14. Note the appearance of the typical pattern of striated p27K doublets in the mature myotube (arrows). Bar, 30 µm.
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J. Foucrier and others
double banding in both adult muscle fibers and foetal mature
myotubes (Fig. 3A,B). In the foetal muscle, this banding pattern
seems to be gradually generated from filaments running
lengthwise. These observations have been the object of separate
detailed investigation to be published elsewhere (Y. Bassaglia
et al., unpublished); here we report the in vitro generation of
this prosome pattern from the individual satellite cells.
Co-distribution of prosomes with the proteins of the
cytoskeletal networks
Data presented in Fig. 4D indicate that, in the course of cellular
fusion, the p27K labelling appeared along fibrillar longitudinal
Dynamics of the p27K specific prosome
cytodistribution in myoblasts fusing into myotubes
in vitro
Immunofluorescence of the p27K antigen in satellite cells
entering myogenic differentiation revealed different patterns of
localization depending on the time of culture. In proliferating
satellite cells at day 3 after plating, labelling was mainly
detected in the cytoplasm, with a more pronounced perinuclear
cone-like localization. The labelling was heterogeneous and in
patches (Fig. 4A). Interestingly, after 4 to 5 days in culture, the
labelling was strongest in the nucleus where it appeared first
with a granular and later a reticular pattern, while faint
labelling was still seen in the cytoplasm (Fig. 4B). By 6-7 days
in culture, corresponding to the first post-mitotic stage when
mononucleated cells start to show spindle-like morphology and
align with each other, the labelling shifted from the nucleus
back to the cytoplasm (Fig. 4C). This labelling appeared as a
very intense and granular fluorescence preferentially in the
perinuclear area of these still pre-fusion myoblasts.
During cellular fusion at day 8-11 the labelling appeared
along fibrillar longitudinal structures in the young myotubes
containing few nuclei. In such cells, the nuclei are no longer
stained (Fig. 4D). Spots of labelling appeared progressively in
a ‘pearls on a string’ manner which, at first, seemed randomly
distributed along the fibrils (Fig. 4E). Subsequently, as fusion
proceeded, the cytodistribution of the p27K antigen
progressively changed to a transverse striated pattern starting
from the periphery of the myotubes. This was seen extensively
from day 11 to 13 (Fig. 4F,G). In mature myotubes (days 1314), the prosome distribution forms a clear sarcomere-like
striation, with double transverse bands (Fig. 4H). Interestingly,
this pattern is very similar to the distribution of the p27K
antigen, which can be observed in rat foetal myogenesis at later
stages (17-19 days) of gestation (see above Fig. 3B) and evokes
the cross-striations seen in adult muscle (Fig. 3A).
The transient nuclear localization of prosomes in
proliferating cells
The obvious shift of the p27K-type prosomes to the nucleus and
back was quite surprising. To study this in detail the movement
of specific prosomes containing the p27K subunit at between 3
and 7 days of culture, were observed by confocal microscopy
(Fig. 5). An analysis of subsequent focal planes on cells early
in culture (day 3), produced serial optical images showing a
predominantly cytoplasmic localization, the nuclei (arrow)
being largely negative (Fig. 5A). In older cultures (days 4-5),
Z-series allowed us to clearly locate the p27K antigen inside the
nuclei of still mononuclear myoblasts (Fig. 5B); labelling in the
nuclei was granular, as already observed (Fig. 4B). Thereafter,
the nuclei progressively lose their staining; in post-mitotic
spindle-shaped cells, prior to their fusion into myotubes (days
6-7), the labelling was concentrated in cytoplasmic patches
mainly in the perinuclear area (Fig. 5C).
Fig. 5. Nucleo-cytoplasmic displacements of the p27K antigen prior to
myoblast fusion observed by confocal microscopy. Observations were
performed after simple labelling with the p27K p-mAb of satellite cells
at 3 to 6-7 days of culture. Consecutive confocal sections (shown in A
and C, from top left to bottom right, and in B from left to right) give
successive profiles of the p27K antigen in the nucleus and the
cytoplasm. Note in A and B, 3 days and 5 days of culture, respectively,
the translocation of the antigen from the cytoplasm to the nucleus, and
back between 5 days and 6-7 days of culture (B-C), in the myoblasts
prior to fusion. In the postmitotic stages, nuclear labelling was no
longer found. Arrows: nuclei. Bars: 15 µm (A); 10 µm (B,C).
Prosome behaviour in myogenic satellite cells
structures resembling some of those constituting the
cytoskeleton. Additional experiments using double-label
immunofluorescence were carried out to identify the type of
cytoskeletal filaments with which the p27K-type prosomes
might be associated. Since desmin, an intermediate filament
protein, was identified as a specific marker of muscular cells
(Lazarides, 1980), its distribution in correlation with the p27K
subunit was examined first. Although the p27K labelling showed
regular fine spots along straight filamentous structures in very
young myotubes (Fig. 6B) desmin, despite aligning in an
analogous pattern, presented a more felt-like aspect (Fig. 6A).
When testing for the actin/prosome association, similar
lengthwise-running filaments were observed which also
seemed to be occupied by p27K prosomes (Fig. 6C,D). In these
slightly more mature myotubes the beginning of a striated
pattern can be observed in the prosome- as well as actinpatterns.
Fig. 6. Immunodetection of the p27K antigen and two
different cytoskeletal elements in young myotubes.
Double labelling of myogenic cells during the fusion
stage was performed with the p27K p-mAb (B,D) and
either an anti-desmin polyclonal antibody (A) or
Texas Red-conjugated phalloidin (C).
(A,B) Distribution of desmin vs p27K antigen. At the
opposite to the diffuse desmin labelling pattern (A), a
rough pseudo-sarcomeric organization of p27K is
shown in association with the fibrillar structures (B).
(C,D) Distribution of actin vs p27K antigen. In both
types of labelling, a tiny transverse striation is
observed. The more intensely labelled actin structures
(C) are also observed in (D), corresponding to the
p27K labelling, but with a very weak intensity. Bars,
25 µm.
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In order to strengthen these initial observations, we
performed confocal microscopy. Single optical sections were
tested for colocalisation of prosomes with desmin and/or actin.
Fig. 7 shows some of the data obtained by this investigation.
In early stages of cell fusion, on day 8 of culture (Fig. 7AC), partial overlap between the labelling of the desmin (red)
and the prosomal antigen (green) can be seen (Fig. 7C)
particularly in filamentous bundles near some of the nuclei. In
contrast to the desmin pattern, in these zones a very discrete
discontinous organization of the p27K labelling seems to begin
(Fig. 7B). This difference increases progressively during the
course of fusion. Thus, only a part of the p27K type of
prosomes colocalizes, in the peri-nuclear zone with desmin
fibrils present as a fine reticular network throughout the
cytoplasm. At day 13 of culture, prosomes seem to be
distributed over a large part of the young myotubes, but are
regularly located in a spotted pattern along fibrillar structures
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J. Foucrier and others
in the thin cellular sections corresponding to individual
confocal planes (Fig. 7D-F). In more mature myotubes, a strict
superposition of desmin and p27K was not observed except in
some rare rough patches. In Fig. 7G-I, a repetitive distribution
of triplet bands can be noticed aligned at a 90° angle along the
fibrillar axis, comprising a desmin band flanked on each side
Fig. 7. Confocal microscopy
showing localization of desmin
and p27K antigen at different
stages of the myogenic
differentiation. (A,D,G: desmin;
B,E,H: p27K; C,F,I:
superposition of the labelling
with desmin in red and p27K in
green). Double-label indirect
immunolocalization was
performed using for the first
incubation, a mixture of antip27K mAbs and a polyclonal
anti-desmin antibody.
(A,B,C) In the course of the cell
fusion at day 8 of culture,
desmin (red) is present under
straight filaments. p27K (green)
shows a cytoplasmic
distribution with sometimes a
perinuclear concentration and
also a discrete segregation in
aligned spots. (D,E,F) In young
myotubes at day 13 of culture,
desmin forms a faint labelled
network meanwhile p27K in the
narrow cellular parts is largely
distributed along fibrillar
structures in a discrete and
regular pattern of spots (arrow).
(G,H,I) Mature myotube
showing typical transverse
labelling associated with the
setting up of the sarcomeric
organization. Bar, 25 µm.
by the p27K antigen. Such triplets were regularly separated
from each other by less densely labelled spaces.
In contrast to this weak decoration of desmin filaments by
the p27K specific p-mAb, more extensive codistribution of
microfilaments and the p27K type prosomes was found in
young myotubes (Fig. 6C,D; Fig. 8). In fact, the fibrillar
Prosome behaviour in myogenic satellite cells
structures which were the most extensively phalloidin-labelled
filaments were also most strongly decorated with the anti-p27K
antibody (Figs 6C,D, 8D,E).
A peripheral but noteworthy observation was that prosome
997
and phalloidin labelling seemed to interfere with each other
(data not shown). In the case of consecutive deposition of the
conjugated phalloidin and the p27K specific p-mAb, according
to the sequence of deposition and the nature of the marker, the
A
B
C
D
E
F
G
H
I
Fig. 8. Observation in
confocal microscopy of
localization of actin and p27K
antigen at different stages of
the myogenic differentiation.
(A,D,G: p27K; B,E,H: actin;
C,F,I: superposition of p27K
and actin label). Detection of
actin was made using Texas
Red-labelled phalloidin.
(A,B,C) Co-expression of
actin and the p27K antigen at
a precise stage of the cell
fusion. Notice the perinuclear
distribution of actin
aggregates. (D,E,F) At a later
stage, dotted line structures
decorated by p27K mAb
coincide with actin stress
fiber-like structures. The first
actin striated organized
structures tightly colocalized
with the transverse prosomal
distribution. (G,H,I) Codistribution in a mature
myotube. Alternation or
partial overlapping of the
labellings can be observed in
I. Bars, 10 µm.
998
J. Foucrier and others
intensity of the labelling of the second marker appeared always
attenuated when compared to the first one. This suggests a
competitive interaction between the labelled phalloidin and the
p-mAbs in the decoration of the actin cytoskeletal structure and
the prosomal particles, respectively.
Confocal analysis indicated that, in the early stages of fusion,
the p27K-type prosomes were present in a diffuse cytoplasmic
distribution while actin was detected either in a rough granular
form around the nuclei or as fibrillar elements sometimes
decorated by the p27K-specific mAb (Fig. 8A-C). At a later stage,
a typical longitudinal fibrillar organization is revealed by both
phalloidin and the p27K p-mAb (Fig. 8D-F). Interestingly, these
fibrillar stuctures showing transverse striations after labelling by
the p27K p-mAb also appear striated by phalloidin, although to
a lesser degree. Moreover, some regions where no tranverse actin
striations can be observed already show an obvious pseudosarcomeric distribution of the p27K prosomal antigen.
In mature contracting myotubes, a typical sarcomeric
structure is usually observed, showing partial superposition of
the actin and prosomal p27K labelling (Fig. 8G-I).
These results show that when the post-mitotic myoblasts
fuse, after transient and partial colocalization with desmin and
gradually pronounced coincidence with still lengthwise-running
cytoskeletal actin filaments, an early distribution of prosomes
occurs according to a pseudo-sarcomeric organization along
these actin structures (Fig. 8D-F). At the same time the
initiation of a typical cross-striated distribution pattern of actin
appears weakly prior to that of desmin which arises later in
older myotubes close to maturation when the p27K-specific
prosome banding is fully developed (see Fig. 4H).
DISCUSSION
This study was performed with the aim of investigating the
localization of prosomes (20S-proteasomes) and the possible
association of these particles with cytoskeletal structures
during the growth and differentiation of rat satellite cells in
primary culture. These cells offer the advantage of developing
a more complete myogenic differentiation pattern than
observed with myoblastic cell lines, and form in vitro
contracting myofibrils.
The main results reported in the present paper relate to the
fact that prosomes containing the p27K subunit undergo a
sequence of dynamic distribution patterns in the course of in
vitro myogenic differentiation. In early stages of culture,
prosomes were mainly localized in the cytoplasm of myoblasts.
Then, surprisingly, they move transiently to the nucleus of prefusion myoblasts and then return, to be redistributed in the
cytoplasm, in association with cytoskeletal elements. A gradual
shift to the cytoskeletal actin system takes place after a transitory
localization in a perinuclear zone occupied by intermediate
filaments of desmin type. Then, remarkably, a pseudosarcomeric banding pattern was formed by the prosomes,
superposed onto the actin filaments. Most interestingly, in the
fusing satellite cells which form myotubes, the prosomes seem
to assume this pseudo-sarcomeric banding pattern shortly before
the formation of the mature sarcomeric structure, as usually
detected by α-actin banding. The same striking banding pattern
of prosomes containing the p27K subunit, intercalated into the
sarcomeric structure, was also found in foetal and adult skeletal
muscle, as shown here and detailed elsewhere (Y. Bassaglia et
al., unpublished). Preliminary notice of this observation was
given previously (Foucrier et al., 1994; Grand et al., 1994) and
a parallel investigation on fusing C2.7 myoblasts was published
recently (De Conto et al., 1997).
The sequence of events observed here on prosome
cytodistribution recalls closely that reported by several authors
concerning the expression and structural organization of
different intermediate filament proteins or sarcomeric
constituents during differentiation of myogenic cells in vitro
(Gard and Lazarides, 1988; van der Ven et al., 1992, 1993).
Most important in the context of our investigation are also
the observations by Fulton and collaborators showing a
gradual building-up of a highly specific para-sarcomeric
cytolocalization, similar to that of prosomes, of mRNA of
muscle-specific proteins and cytoskeletal constituents (Cripe et
al., 1993; Fulton and Alftine, 1997; Morris and Fulton, 1994).
Prior biochemical investigations, which are the basis of this
study in which mainly indirect immunofluorescence was used,
have shown that various types of prosomes occur, differing by the
‘mosaic’ structure of their subunit composition which can be
detected specifically by the different monoclonal and polyclonal
antibodies in our possession (De Conto et al., 1997; discussed by
Scherrer and Bey, 1994). Prosomes isolated from satellite cells,
as shown here, produce the regular electrophoretic pattern of
prosomal proteins which migrate with molecular masses of 20 to
36 kDa. It is of interest that not all of the antibodies used reacted
with the prosomes of satellite cells and, in particular, there was a
predominance of prosomes reacting with the p27K specific pmAbs. The p27K prosome subunit (also called ‘iota’; see
Materials and Methods) is one of the most ubiquitous prosome
proteins, present in many tissues and cell types, although
restricted in early embryogenesis to the mesoderm layer (Pal et
al., 1988). It was extensively studied in earlier investigations; we
have determined its cDNA sequence (Bey et al., 1993) and are
studying its genomic organization. The Pros-27 gene is of the αsubunit type, is one of the carriers of the RNase activity of
prosomes, and includes a consensus site for RNA binding
proteins (Bey et al., 1993; Petit et al., 1997; Pouch et al., 1995).
Therefore, the p27K-mAb was chosen for this investigation
although other antibodies were tested as well. The prosomes
containing the p27K subunit form the most clearcut banding
pattern, in fusing satellite cells as well as in adult skeletal muscle.
Only the p31K prosomes show an analogous pattern, whereas
all other prosomal antibodies tested give diffuse or no staining
at all. It is of interest that, as shown in a parallel investigation
on C2.7 myoblast fusion, which remains abortive and does not
lead to genuine sarcomere formation, some prosome particles,
studied by individual subunit-specific p-mAbs (p23 and p31K),
follow different patterns of temporal and spatial change (De
Conto et al., 1997). Remarkably, in this same study, it was also
found by two-dimensional electrophoresis that prosomes change
their subunit composition during the myogenic process.
Two arguments allow us to confirm the specificity of the
observed labelling patterns using our fixation conditions. In the
first place, classical controls of immunocytological analysis
were always negative: all immunofluorescence was abolished
when the primary antibody was omitted. Furthermore, the fact
that when using the same p-mAb and the same fixation
procedure a different distribution of prosomes is seen at
specific stages of myotube formation, represents in itself an
internal control, certifying the validity of the observations.
Prosome behaviour in myogenic satellite cells
Prosomes are very stable, and to our knowledge, prosome
subunits were never found in a free form outside the particles (see
discussion by Scherrer and Bey, 1994) except under heat-shock
conditions (Martins de Sa et al., 1989) or, in the case of the αsubunit zeta found in HeLa cell extracts, using a particular
technique (Hendil et al., 1995). Indeed, de novo synthesized
prosomal proteins are immediately incorporated into preprosomes (Frentzel et al., 1994). Thus, the changes in distribution
of the p27K antigen reflect the evolution of prosomes containing
the p27K subunit and not free proteins outside the particles. It is
not known at the present time if the in vivo proteolytically active
26S proteasomes follow the cytodistribution of their prosomal
core, identified here by the anti-p27K mAb.
Among the large range of prosome/proteasome properties,
four may be considered to be possibly related to our
observations: (1) their proteolytic activity (Coux et al., 1996);
(2) their RNase activity (Petit et al., 1997; Pouch et al., 1995)
and, possibly related, RNA-binding properties concerning premRNPs as well as mRNPs (discussed by Scherrer and Bey
(1994); (3) their interaction with the cytoskeleton (Arcangeletti
et al., 1997b); and (4) their presence on chromatin and the
nuclear matrix (Pal et al., 1988; E. Pilotti et al., unpublished).
Taking into account these different properties which have been
assigned to prosomes and/or proteasomes, it is tempting to
assume that the cytodistribution of prosomes during the
myogenic differentiation process reflects a possible role in some
of the sequential physiological and molecular events of
myogenesis. In our system of rat satellite cell culture, it has been
previously shown that the maximal number of cells in S phase
occurs 4-5 days after plating, corresponding at that time to a
proliferative phase. Thereafter, the cells become post-mitotic and
fuse into myotubes (Lassale et al., 1989). At the molecular level,
this involves the sequential expression of a set of muscle specific
proteins such as creatine kinase, myosin (Lagord et al., 1996) or
phosphoglycerate mutase (Castella-Escola et al., 1990), under the
control of muscle-specific transcription factors of the bHLH
family. Among these factors, we demonstrated that MyoD and
myogenin are successively turned on, and reach a maximal level
of expression at days 5 and 8, respectively (Lagord et al., 1996).
In light of our observations on prosomes, the prefusion phase
of mononucleated myoblasts might be subdivided into 3 stages,
occurring sequentially between days 3 and 7 after plating: (1)
a clear proliferative stage of un-differentiated cells, followed
(2) by a phase when some types of prosomes move to the
nuclei, and a final stage (3) when prosomes re-enter the
cytoplasm. We do not know at present if the two latter stages
involve still dividing myoblasts. One might therefore assume
that phases (2) and (3), when the prosomal nucleo-cytoplasmic
forth and back movement takes place, are related to the
commitment of postmitotic mononucleated cells to the
terminal differentiation pathway. However, for the time being,
this suggestion remains hypothetical.
The movement of the 27K prosomes to the nucleus, which
takes place at the onset of myogenic fusion, could possibly relate
to either the selective degradation of regulatory proteins as
reported for the cell cycle-related cyclins, or the processing of
precursor proteins into active regulatory (transcription) factors
(Novak et al., 1991). Proteasomes are known to play an essential
role in cell cycle control (Ghislain et al., 1993; Gordon et al.,
1993) and their nuclear localization could be related in some
cases to a specific proteolytic role in the regulation of cell cycle
999
type proteins, such as cyclins, and other short lived nuclear
proteins as c-fos, jun, myc (Jariel-Encontre et al., 1997; Tsurumi
et al., 1996). Such a role might possibly be considered in
myogenic fusion since successive waves of cyclin proteins have
been described in a study of the L6 α1 myogenic cell line
(Borycki et al., 1995). However, in early cleavage stage embryos,
when cyclin cycling is most rapid and fully active, the prosomes
remain located in the cytoplasm, and move to the nucleus only
selectively in mid-blastula transition (Pal et al., 1988, 1994) at
the onset of zygotic transcription. Remodelling of the chromatin
structure including structural proteins and transcription factors
might therefore constitute an alternative hypothesis involving
proteolytic activities. To sort out these possibilities it will be
important to know if only the prosomes or also the 26S
proteasomes with their additional 19S components are present
on chromatin and the nuclear matrix. New recent observations
(E. Pilotti et al., unpublished) to be taken into account indicate
that, in C2.7 myoblasts, prosomes containing the p27K subunit
occupy the nuclear matrix predominantly localized in the
perinucleolar zone, and are present as well on chromatin and premRNA. One part of the prosomes seem to be linked to salt- and
detergent-labile structures (interestingly, prosomes are less
easily extracted than histones) whereas others remain with the
nuclear matrix, with about 50% in a RNase-labile form. The
prosomes which move to the nucleus might, therefore, function
at the level of chromatin and pre-mRNA; both are linked to the
nuclear matrix, the former to the chromatin scaffold via the
MARs (matrix attachment regions), and the latter to the dynamic
matrix on which processing and transport of (pre-)mRNA
occurs. Such a distribution can be observed directly in the case
of the lampbrush (interphase) chromosomes of P. waltl, which
have been studied by several p-mAbs (Pal et al., 1988).
During the subsequent steps of myogenesis, namely the
cellular fusion and the maturation of myotubes, prosomes were
first found transiently associated with desmin in the perinuclear
area, then they were lined up along longitudinally oriented
fibrillar actin structures where they formed progressively small
patches, and finally they showed the surprising sarcomeric
banding pattern.
Recent evidence on primary cell cultures of rat embryonic
myoblasts showed involvement of prosomes and 26S
proteasomes in the myogenic process and more specifically
during the fusion step. Thus, a significant increase in the level
of the p27K subunit, as seen for the p23K and p31K subunits
was observed in fusing C2.7 myoblasts (De Conto et al., 1997).
Moreover, dose-dependent inhibition of the fusion process takes
place using either inhibitors of the proteasome activities or
antisense oligonucleotides specifically targeted to the mRNA of
the p27K subunit (Gardrat et al., 1997). Globally speaking, the
dynamic pattern of cytoplasmic prosome distribution observed
during the later myogenic stages was quite similar to those of
myofibrillar constituents, reported in the myogenesis of several
types of muscle cells grown in vitro (van der Ven et al., 1993).
It also evokes closely the sequence of events and the eventual
positioning of specific mRNAs in the sarcomeric structure
demonstrated by Fulton and co-workers (Cripe et al., 1993;
Fulton and Alftine, 1997; Morris and Fulton, 1994). The
question arises of whether these closely synchronized events are
going on in a parallel but independent manner or are
interrelated? The fact that the pseudo-sarcomeric prosome
banding develops prior to the insertion of α-actin into the
1000 J. Foucrier and others
sarcomeric structure, apparently observed during satellite cell
fusion (this study) as well as in foetal myogenesis (Y. Bassaglia
et al., unpublished; Grand et al., 1994), relates the prosomes to
the formation rather than to the turnover of the myofibrils.
A discrepancy exists over the interpretation of the data on the
proteolytic role of proteasomes in muscle cells. If the ubiquitinproteasome system has recently been proposed to represent the
major pathway responsible for accelerated muscular proteolysis
related to pathological conditions, or for the turnover of
disorganized myofibrillar proteins (Mitch and Goldberg, 1996;
Solomon and Goldberg, 1996), muscle degradation seems
mainly related to calpain or cathepsin type proteases and, in
vitro, proteasomes act poorly in disorganization of the
sarcomeric structures (Taylor et al., 1995). Moreover, it is not
evident why a proteolytic activity, involving prior
ubiquitinylation of proteins or not, would necessitate the
creation of a specific permanent organization of proteasome
bands in the sarcomeric structure. In this context, it is of interest
that the 26S proteasomes involved in antigen presentation,
monitored by the LMP2/LMP7 subunits, seem to diffuse freely
in the cytoplasm (Reits et al., 1997), whereas in our previous
investigations, a majority of the prosomes was always found to
co-localize with cytoskeletal structures. Thus, any speculation
concerning the proteolytic activities of prosomes in myogenesis
and muscle function, although not impossible, seems
premature. In the same manner, extensive work will be
necessary in muscle cells to appreciate the possible RNase
activities of prosomes which have been mainly related to the
degradation of mRNA involving the (AUUUA)n signal (Petit et
al., 1997), which is particularly abundant in unstable mRNAs.
In opposition, the observations of Fulton and collaborators
on mRNA cyto-localization may be closer to our initial
hypothesis concerning the putative role of prosomes in the
control of protein biosynthesis, possibly related to the
cytoskeleton-bound phases (Singer, 1992). They have used in
situ hybridization to follow the gradual building-up of a parasarcomeric cytolocalization of specific mRNAs coding for
different muscle cell constituents, in parallel to that of the
cognate proteins observed by immunofluorescence (Cripe et al.,
1993; Fulton and Alftine, 1997; Morris and Fulton, 1994). This
represents strong evidence for co-translational assembly of the
sarcomeric structure and its constitutive molecular complexes.
Moreover, as observed here for the prosomes, this specific
mRNA banding occurs after a phase of nuclear and more diffuse
cytoplasmic distribution, with occasional peri-nuclear
concentration towards the cellular poles, and prior to alignment
with lengthwise running filamentous structures which gradually
organize into a regular but discontinuous pattern (Morris and
Fulton, 1994). The similarity of this sequence of events with the
reorganization of the cytoskeleton on the one hand, and with
that of prosome re-localization on the other is striking.
We have already reported and discussed the relationship
between prosomes to the cytoskeleton within our investigations
of in vitro myotube formation from fusing C2.7 myoblasts; the
hypothesis of a possible involvement of prosomes in
cytoskeleton-related mRNA transport and localization was
formulated there (De Conto et al., 1997). The exact biological
significance of the apparent cytoskeleton-binding properties of
prosomes observed here again remains to be elucidated.
However, our results corroborate other data showing that the
majority of the prosomes are linked to the cytoskeleton of
either IF (Olink-Coux et al., 1994), or IF as well as actin types
(Arcangeletti et al., 1997a; De Conto et al., 1997).
The use of satellite cells and possibly some other systems of
in vitro myogenesis might allow a better understanding of the
function of prosomes in myogenesis in relation to other
myogenic factors. The fact that the prosome banding pattern, in
the final stages of in vitro satellite cell fusion, closely resembles
that observed in foetal myogenesis as well as in adult skeletal
muscle seems to validate this model. More detailed studies (in
progress) may enable these questions to be answered and may
assign specific functions to the prosomes and the 26Sproteasomes in myogenesis, within their implied roles in gene
expression by both, protein biosynthesis and breakdown.
We thank Dr C. Arcangeletti and the late Dr D. Péchinot for helpful
comments and also M. Barre and G. Carpentier for advice with image
analysis, and Michel Ronemus and Fayçal Bey for help with the
manuscript. This work was supported by grants from the Centre
National de la Recherche scientifique (CNRS), and the Ministère de
l’Enseignement Supérieur et de la Recherche (MESR), the
Association pour la Recherche sur le Cancer (ARC), and the
Association Française contre les Myopathies (AFM).
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1002 J. Foucrier and others
Prosome behaviour in myogenic satellite cells 1003