Microtubule-associated protein 4 (MAP4) transcripts vary in different mouse tissues, with striated muscle (skeletal and cardiac) expressing 8and 9-kb transcripts preferentially to the more widely distributed 5.5and 6.5-kb transcripts (West, R. W., Tenbarge, K. M. and Olmsted, J. B. (1991). J. Biol. Chem. 266, 21886-21896). Cloning of the sequence unique to the muscle transcripts demonstrated that these mRNAs vary from the more ubiquitous ones by a single 3.2-kb coding region insertion within the projection domain of MAP4. During differentiation of the myogenic cell line, C2C12, muscle-specific MAP4 transcripts appear within 24 hours of growth in differentiation medium, and a larger MAP4 isotype (350×103Mr) accumulates to high levels by 48 hours of differentiation. In situ hybridization analyses of transcript distribution in mouse embryos demonstrated that muscle-specific transcripts appear early in myogenesis. To block the expression of the muscle-specific MAP4, stable lines of C2C12 cells were generated bearing an antisense construct with the musclespecific MAP4 sequence. Myoblast growth was unaffected whereas myotube formation was severely perturbed. Fusion occurred in the absence of the muscle MAP4 isotype, but the multinucleate syncytia were short and apolar, microtubules were disorganized and normal anisotropic myofibrils were absent. The patterns of expression of the muscle-specific transcripts and the antisense experiments indicate that this unique structural form of MAP4 plays a critical role in the formation and maintenance of muscle.

Microtubule-associated proteins (MAPs) are a diverse group of proteins that can bind to the surface of microtubules. MAPs are separated into two major classes. Motor MAPs, such as dynein and kinesin, play important roles in intracellular movements (Skoufias and Scholey, 1993). Structural MAPs, however, are postulated to have roles in the establishment and maintenance of the cytoarchitecture of cells (Olmsted, 1991; Wiche et al., 1991). Some structural MAPs may have general roles in cells whereas others may be involved in generating cell type or tissue-specific morphological characteristics (Matus, 1991; Chen et al., 1992). For example, the neural-specific MAPs, MAP2 and tau, are capable of causing non-neuronal cells to generate neuronal-like processes (reviewed in Kosik and McConlogue, 1994). Further, antisense experiments demonstrate the requirement of MAP2 (Caceres et al., 1992), tau (Caceres and Kosik, 1990) and MAP1B (Brugg et al., 1993) for neurite formation and maintenance. However, much less is known about the roles of ubiquitous MAPs, MAP1A and B, and MAP4, in non-neural tissues. The research described here examines MAP4 expression during myogenesis, a process that involves massive reorganization of the cytoarchitecture.

MAP4 is a thermostable protein of approx. 200×103Mr that is present in a variety of cell types and tissues (Bulinski and Borisy, 1980; Parysek et al., 1984; Murofushi et al., 1986). Like other structural MAPs, MAP4 has a domain that binds to microtubule surfaces, and a projection domain that presumably extends into the cytoplasm. MAP4 is encoded by a single gene (West et al., 1991) and studies of transcripts (Aizawa et al., 1990; West et al., 1991; Chapin et al., 1995) and proteins (Parysek et al., 1984) have identified multiple isotypes of MAP4, some of which are tissue-specific. RNA blot analyses demonstrated that most tissues contain major MAP4 transcripts of 5.5 and 6.5 kb, whereas additional unique transcripts of 10 kb exist in testis and transcripts of 8 and 9 kb in cardiac and skeletal muscle (West et al., 1991). Our previous work showed that the difference between the upper and lower band in each doublet was due to alternative polyadenylation (Code and Olmsted, 1992). We describe here the cloning of the sequence unique to the form of MAP4 found in striated muscle.

Differentiation of mammalian myoblasts into mature myotubes is accompanied by the production of muscle-specific gene products (e.g. Li et al., 1994; Gunning et al., 1987; Lin et al., 1994; Buckingham et al., 1992) and extensive changes in cell morphology. Several studies have characterized the distribution of microtubules in muscle (Warren, 1974; Goldstein and Cartwright, 1982), and both tubulin isotype composition (Lewis and Cowan, 1988) and posttranslational modification (Gundersen et al., 1989) change during myogenesis in cultured cell lines. The importance of microtubules in the normal ontogeny of muscle has been inferred from studies in which myotube formation was aberrant when microtubules were either disrupted in vivo (Warren, 1968) or disrupted or stabilized in cultured muscle cells (Holtzer et al., 1985; Saitoh et al., 1988). Here we report that during myogenesis, ubiquitous MAP4 is replaced by an isotype that is restricted to striated muscle. We describe the sequence of this muscle-specific form of MAP4 (hereafter called mMAP4), document the expression of mMAP4 transcripts and protein during the differentiation of a myogenic mouse cell line, and describe patterns of transcript expression during mouse embryogenesis. We also show that disruption of the expression of mMAP4 causes severe defects in the formation of myotubes in vitro.

Isolation of mMAP4 cDNA clones from myogenic cells

To isolate muscle-specific transcripts identified by RNA blots (West et al., 1991), a λZAP II phage cDNA library constructed from C2C12 myotubes (gift from E. Olson, M. D. Anderson Cancer Center, University of Texas) was screened with a mixture of mouse MAP4 probes (M31, M4, M7; West et al., 1991) spanning the length of the ubiquitous MAP4 (uMAP4) sequence. Plaque-purified positive clones were then spot-blotted onto nitrocellulose, and hybridized separately with the individual probes used in the original screening mixture. One region was not represented in the clones isolated from the single screen of the cDNA library. This sequence was generated by RT-PCR (reverse transcriptase-polymerase chain reaction) using first-strand cDNA made from total mouse thigh muscle RNA, with one primer being anchored within the muscle-specific sequence and the other complementary to the sequence in uMAP4. This clone (mmPCR1521) was subcloned using the TA cloning method (Invitrogen; San Diego, CA). Sequences were obtained using the dideoxy sequencing method (Sanger et al., 1977) and the Sequenase kit (USB; Cleveland, OH). Sequences were confirmed by complete sequencing of both strands of overlapping clones. The complete sequence was analyzed using the UWGCG programs (Genetics Computer Group Program Manual, Version 7) and by searches (Altschul et al., 1990) through the NCBI databases using the BLAST network service.

Cell culture

C2C12 myoblast cells (CRL 1772; Blau et al., 1983) were obtained from ATCC (Rockville, MD), and maintained in DMEM (Gibco; Grand Island, NY) and 10% fetal calf serum (Hyclone; Logan, UT). Confluent cultures of myoblasts were transferred into DMEM with 10% horse serum to induce differentiation into myotubes. All cultures received fresh medium every 2–3 days.

Indirect immunofluorescence and microscopy

Myoblasts or myotubes cultured as described above were grown on chamber slides (Nunc; Naperville, IL), fixed in cold methanol and probed with rat monoclonal antibodies raised to mouse MAP4 (1F5 and 1C6) or α-tubulin antibody (Amersham; Arlington Heights, IL), followed by secondary antibodies conjugated with Texas Red (goat anti-mouse; Jackson ImmunoResearch, West Grove, PA) or fluorescein (goat anti-rat; Cappel, Durham, NC). Confocal images of the preparations were obtained using a Bio-Rad MRC-1000 confocal system and Nikon Diaphot microscope and were assembled using a Macintosh Centris 650 with Adobe Photoshop.

Protein preparation and analysis

We found mMAP4 was highly susceptible to proteolysis, even in the presence of protease inhibitors. Protein samples for analysis on gels were therefore prepared from normal and antisense C2C12 cultures by lysing whole cells directly in boiling Laemmli SDS sample buffer (Laemmli, 1970). Preparations from striated muscle (thigh) were made by mincing tissue on ice and then extracting immediately in boiling sample buffer. Proteins in lysates were separated by SDSPAGE (5% or 7.5% gels) using standard procedures, and then transferred to nitrocellulose or Immobilon-P (Millipore Corp., Bedford, MA) for immunoblot analysis. Immunoblots were prepared following standard procedures (Towbin et al., 1979), with immunoreactive species being detected with n-chloronapthol after blots had been incubated successively with rat monoclonal antibodies raised to mouse uMAP4 (1F5, 1C6), secondary antibodies coupled to biotin, and streptavidin peroxidase.

RNA preparation and analysis

Total RNA from myoblasts or differentiating myotubes was isolated according to standard protocols (Sambrook et al., 1989). Total cellular RNA from mouse tissues was isolated using the method described previously (West et al., 1991). For RNA blot analysis, 10 μg of total RNA were electrophoresed through a 0.9% agarose gel containing 3.7% formaldehyde. Gels were examined for loading consistency by ethidium bromide staining. Following capillary blotting of RNA to GeneScreen (DuPont-NEN; Boston, MA) or Hybond-N (Amersham; Arlington Heights, IL), hybridization with various MAP4 or myogenin cDNAs was performed as described previously (West et al., 1991), or with the Rapid-Hyb protocol (Amersham; Arlington Heights, IL) using randomly primed [32P]DNA probes (BRL Random Primers DNA labeling system). Blots were hybridized at 55°C at high stringency (50% formamide), and washed with 0.1× SSC at room temperature and 60°C. A myogenin cDNA clone was generously donated by Drs J. H. Morris and W. H. Klein (M. D. Anderson Cancer Center, University of Texas); the 1 kb EcoRI fragment of this clone within the coding region was used as a hybridization probe.

In situ hybridization

Sections of mouse embryos (days 8-16; Novagen; Madison, WI) were hybridized with 35S-uridine-triphosphate-labeled antisense or sense riboprobes using standard procedures (Angerer and Angerer, 1992). Probes were transcribed from 2 kb of previously described MAP4 sequence (M7; West et al., 1991), or from a 2 kb region of the mm1 clone that contained muscle-specific sequence. Probes were used at 30,000 cpm/μl (specific activity 15.7 μCi/ml) and slides were exposed for 10 days at 4°C. Structures were identified using an atlas of mouse development (Kaufman, 1992).

Antisense experiments with stably transfected cell lines

A plasmid bearing mMAP4 nucleotides 216-1214 in the reverse orientation was generated in the pcDNA3 vector (Invitrogen, San Diego, CA). C2C12 myoblasts were transfected with this construct using Lipofectamine reagent (GIBCO/BRL, Gaithersburg, MD) and stable lines were selected by growth in the presence of G418. For analysis of phenotype, myoblasts were grown in normal medium or switched to differentiation medium for up to 4 days. Cells were assayed for the presence of antisense transcripts using RT-PCR and for MAP4 protein by immunoblotting.

Expression of MAP4 in C2C12 cells

We initially examined the distribution and expression of MAP4 in differentiating C2C12 cells using reagents reacting with the ubiquitous form of MAP4 (uMAP4) characterized previously. C2C12 cells have been widely studied as a model for myogenesis (Blau et al., 1983; Li et al., 1994; Miller, 1990). Under standard tissue culture conditions, the cells proliferate as single cell myoblasts. When confluent cultures are transferred from fetal calf serum to horse serum, the myoblasts begin to elongate and fuse within 24 hours to form multinucleate myotubes, which become wider and longer over the next few days as additional myoblasts fuse.

To define the distribution of MAP4 in the cells of these differentiating cultures, double-label immunofluorescence analyses were performed. As shown in Fig. 1A, interphase and mitotic C2C12 myoblasts have distributions of microtubules resembling those of other cultured cells, and distribution of MAP4 in myoblasts (Fig. 1B) parallels that of tubulin. Tubulin and MAP4 also colocalize in multinucleate myotubes (compare Fig. 1C,D), but microtubules are now arranged in long, parallel arrays that extend the length of the multinucleate syncytium. These data indicate that MAP4 distribution parallels microtubules during myogene-sis, even though the cytoskeleton undergoes dramatic rearrangements.

Fig. 1.

Indirect immunofluorescence of myoblast and myotube cultures, demonstrating the distribution of MAP4 and tubulin in C2C12 cells. Confocal images of myoblast (A,B) or myotube (C,D) cultures (grown for 4 days in differentiation medium), fixed with methanol and labeled for double immunofluorescence with tubulin (A,C) and MAP4 (B,D) antibodies. The MAP4 and tubulin distributions are coincident in both types of culture, but the microtubules reorganize upon myotube formation into arrays running parallel to the long axis of the syncytium. Single label controls showed no overlap of signals.

Fig. 1.

Indirect immunofluorescence of myoblast and myotube cultures, demonstrating the distribution of MAP4 and tubulin in C2C12 cells. Confocal images of myoblast (A,B) or myotube (C,D) cultures (grown for 4 days in differentiation medium), fixed with methanol and labeled for double immunofluorescence with tubulin (A,C) and MAP4 (B,D) antibodies. The MAP4 and tubulin distributions are coincident in both types of culture, but the microtubules reorganize upon myotube formation into arrays running parallel to the long axis of the syncytium. Single label controls showed no overlap of signals.

To determine if C2C12 cells contain transcripts characteristic of the unique MAP4 species in adult skeletal muscle, total RNA was isolated from C2C12 myoblasts and myotubes, and the transcripts were compared with those in skeletal muscle by northern analysis (Fig. 2). Lane 1 illustrates the mRNAs detected in adult skeletal muscle using a probe (M4) spanning the initial third of the coding sequence (West et al., 1991). Prominent transcripts of 8 and 9 kb, and less abundant species of 5.5 and 6.5 kb, are evident. In contrast, myoblasts (lane 2) contain only the doublet of smaller transcripts characteristic of most cultured cells and tissues. RNA prepared from C2C12 cells cultured for 8 days in differentiation medium, however, contains the transcripts typical of adult muscle (lane 3). The relative abundance of the upper doublet of transcripts in the C2C12 myotubes is comparable to that in RNA from adult muscle preparations.

Fig. 2.

Differentiation is accompanied by accumulation of tissue-specific MAP4 mRNAs. Total RNA from adult mouse thigh muscle (lane 1), C2C12 myoblasts (lane 2) or C2C12 myotubes grown in differentiation medium for 8 days (lane 3) were analyzed by northern blotting. The blot was probed with a subclone (M4; West et al., 1991) of mouse MAP4 coding sequence that does not contain the muscle-specific sequence. Positions of markers are indicated at right. The smear of hybridization extending below the 5.5kb species in lane 1 is due to degradation in this sample, and was not seen in other muscle RNA preparations (see Fig. 6A).

Fig. 2.

Differentiation is accompanied by accumulation of tissue-specific MAP4 mRNAs. Total RNA from adult mouse thigh muscle (lane 1), C2C12 myoblasts (lane 2) or C2C12 myotubes grown in differentiation medium for 8 days (lane 3) were analyzed by northern blotting. The blot was probed with a subclone (M4; West et al., 1991) of mouse MAP4 coding sequence that does not contain the muscle-specific sequence. Positions of markers are indicated at right. The smear of hybridization extending below the 5.5kb species in lane 1 is due to degradation in this sample, and was not seen in other muscle RNA preparations (see Fig. 6A).

Identification of muscle-specific MAP4 sequence

In order to characterize the sequences specific for mMAP4 transcripts, a cDNA library constructed from C2C12 myotube RNA was screened. A single screen using probes spanning the length of uMAP4 sequence identified 60 clones. Clones were classified according to size of insert and by patterns of hybridization to probes representing different regions of uMAP4 sequence. Inserts from each class were then sequenced to identify novel sequences contiguous with those determined previously. A schematic illustration of the location of the new sequence with respect to uMAP4 sequence is shown in Fig. 3A, and the positions of the clones from which the mMAP4 sequence was derived (solid lines) are illustrated in Fig. 3B. One region of mMAP4 was not represented in the recovered clones; this section (dashed line) was obtained by PCR amplification of single-stranded cDNA generated from mouse thigh muscle total RNA. Amplification between an anchoring primer in the new sequence and a primer in uMAP4 sequence yielded a 1300-base pair product. The fact that the amplimer could be generated with these primers using cDNA from skeletal muscle indicated that the new sequence was continuous with uMAP4 transcripts derived from this tissue.

Fig. 3.

Schematics of the muscle-specific sequence relative to ubiquitous mouse MAP4 sequence. (A) The position of the muscle-specific MAP4 insertion (▴) is indicated relative to the MAP4 domain structure determined previously (West et al., 1991). Shaded boxes represent various domains, and numbers indicate the nucleotide positions. Lines represent 5′ and 3′ untranslated regions. Alternative polyadenylation signals (Code and Olmsted, 1992) are indicated (AAUAAA). (B) The clones used to identify the muscle-specific sequence are indicated as lines below the box representing the domain of the muscle variant. The dashed line represents a PCR product amplified from mouse muscle cDNA that provided a sequence not represented in the isolated library clones.

Fig. 3.

Schematics of the muscle-specific sequence relative to ubiquitous mouse MAP4 sequence. (A) The position of the muscle-specific MAP4 insertion (▴) is indicated relative to the MAP4 domain structure determined previously (West et al., 1991). Shaded boxes represent various domains, and numbers indicate the nucleotide positions. Lines represent 5′ and 3′ untranslated regions. Alternative polyadenylation signals (Code and Olmsted, 1992) are indicated (AAUAAA). (B) The clones used to identify the muscle-specific sequence are indicated as lines below the box representing the domain of the muscle variant. The dashed line represents a PCR product amplified from mouse muscle cDNA that provided a sequence not represented in the isolated library clones.

The new sequence comprised 3183 bases, which corresponds to the estimated size difference between the musclespecific transcripts and the 5.5/6.5 kb uMAP4 transcripts characterized previously. The mMAP4 sequence, shown in Fig. 4, represents an in-frame insertion between bases 2402 and 2403 of uMAP4 sequence. The uMAP4 sequence has been found in all cell types and adult mouse tissues examined (West et al., 1991). In contrast, analysis of RNA from various adult tissues with probes unique to mMAP4 demonstrated this sequence is only in skeletal and cardiac muscle. Notably, it was not detectable in uterus, which contains smooth muscle, or in testis, which has an uncharacterized MAP4 mRNA of approx. 10 kb (Fig. 5). The new sequence was used to search the GenBank database for related sequences, the most similar of which is an expressed sequence tag isolated from a human skeletal muscle cDNA library. This nucleic acid sequence (accession no. Z25124) was 74.5% similar/72.7% identical to mouse mMAP4 over the 340 nucleotides reported, and is likely to represent the human mMAP4 sequence.

Fig. 4.

The nucleotide and amino acid sequences of the muscle variant region of mouse MAP4. The unique sequence of 3183 nucleotides, coding for 1061 amino acids, is shown. The sequence was confirmed by sequencing both strands. The bordering nucleotides and amino acid present in the uMAP4 sequence are highlighted in bold. Nucleotide 1 corresponds to position 2402 of the previously described sequence, and nucleotide 2 begins the new sequence. These sequence data were submitted on 12/23/93 and have been available from GenBank since 6/15/94 under accession number UO8819.

Fig. 4.

The nucleotide and amino acid sequences of the muscle variant region of mouse MAP4. The unique sequence of 3183 nucleotides, coding for 1061 amino acids, is shown. The sequence was confirmed by sequencing both strands. The bordering nucleotides and amino acid present in the uMAP4 sequence are highlighted in bold. Nucleotide 1 corresponds to position 2402 of the previously described sequence, and nucleotide 2 begins the new sequence. These sequence data were submitted on 12/23/93 and have been available from GenBank since 6/15/94 under accession number UO8819.

Fig. 5.

The novel mMAP4 sequence is present only in striated muscle cells. A portion of the mMAP4 sequence was used to probe total RNA samples from a variety of adult mouse tissues. The novel sequence is detected only in differentiating C2C12 cultures, skeletal muscle from thigh, and heart.

Fig. 5.

The novel mMAP4 sequence is present only in striated muscle cells. A portion of the mMAP4 sequence was used to probe total RNA samples from a variety of adult mouse tissues. The novel sequence is detected only in differentiating C2C12 cultures, skeletal muscle from thigh, and heart.

The derived ORF for the new sequence comprises 1061 amino acids, and is continuous within the previously described mouse uMAP4 sequence (West et al., 1991). The new coding region lies within the projection domain immediately aminoterminal to the highly conserved proline-rich (P) region. The muscle-specific sequence is relatively neutral in charge, with no preponderant clustering of acidic or basic residues. Thus, it generates a novel charge domain within the molecule that is intermediate (pI=7.6) between the relatively acidic projection domain (pI=4.2) and the basic microtubule binding domain (pI=11.4) defined previously. Like the KDM repeats in uMAP4, this region of the protein contains multiple potential phosphorylation sites for casein kinase II (24 sites) and pkC (19 sites). Whether these sites affect the flexibility or function of mMAP4 remains to be determined.

Accumulation of MAP4 transcripts and protein during muscle differentiation

The preceding data suggested that we had identified a new sequence corresponding to the muscle-specific transcript of MAP4, and we tested this by examining RNA expression in differentiating C2C12 cells. Total RNA was isolated from nonconfluent cultures of myoblasts (day 0) and from cells that had been in differentiation medium for 1 to 7 days. RNA from adult thigh skeletal muscle was included as a control. As shown in Fig. 6A, a mMAP4 probe detects only the 8and 9-kb transcripts in RNA samples from adult thigh muscle. In contrast, no signal is observed in C2C12 myoblasts (lane 0). However, mMAP4 transcripts accumulate concurrently with the onset of myoblast fusion (during day 1), and these transcripts are maintained in differentiated cultures. These data demonstrate that the new sequence identified is specific for the large transcripts of MAP4 found in muscle, and that these transcripts are induced during C2C12 cell differentiation. A similar analysis in which samples were harvested every 4 hours during the first 24 hours of differentiation demonstrated that the mMAP4 transcripts first appear between 12 and 16 hours after the switch to differentiation medium (data not shown).

Fig. 6.

Muscle-specific MAP4 mRNA transcripts and protein accumulate during differentiation of C2C12 cells. Total RNA was isolated from cultures of myoblasts (lane 0), cultures in differentiation medium for 1 to 7 days (lanes 1-7) or from adult mouse thigh muscle (adult). (A) Blot probed with a subclone of the mm1 clone (see Fig. 3) that contains only the muscle-variant sequence. Transcripts accumulate within 24 hours of the switch to differentiation medium. (B) The same samples, probed with a portion of a myogenin cDNA clone. (C) Western blot analysis of total protein isolated from C2C12 myoblasts (lane 0), cultures in differentiation medium for 1 to 7 days (lanes 1-7) or from adult mouse thigh muscle (adult), probed with MAP4 monoclonal antibodies that recognize sequences common to uMAP4 and mMAP4. Previously characterized uMAP4 protein migrates at approx. 220×103Mr (arrowhead). A higher molecular mass protein of approx. 350×103Mr is also present (arrow) and accumulates to high levels by 48 hours in differentiating C2C12 cultures; this represents the mMAP4 isotype. The day-3 sample contains slightly less total protein than the other lanes. Degradation of mMAP4 protein is evident in older cultures; this varies with the preparation.

Fig. 6.

Muscle-specific MAP4 mRNA transcripts and protein accumulate during differentiation of C2C12 cells. Total RNA was isolated from cultures of myoblasts (lane 0), cultures in differentiation medium for 1 to 7 days (lanes 1-7) or from adult mouse thigh muscle (adult). (A) Blot probed with a subclone of the mm1 clone (see Fig. 3) that contains only the muscle-variant sequence. Transcripts accumulate within 24 hours of the switch to differentiation medium. (B) The same samples, probed with a portion of a myogenin cDNA clone. (C) Western blot analysis of total protein isolated from C2C12 myoblasts (lane 0), cultures in differentiation medium for 1 to 7 days (lanes 1-7) or from adult mouse thigh muscle (adult), probed with MAP4 monoclonal antibodies that recognize sequences common to uMAP4 and mMAP4. Previously characterized uMAP4 protein migrates at approx. 220×103Mr (arrowhead). A higher molecular mass protein of approx. 350×103Mr is also present (arrow) and accumulates to high levels by 48 hours in differentiating C2C12 cultures; this represents the mMAP4 isotype. The day-3 sample contains slightly less total protein than the other lanes. Degradation of mMAP4 protein is evident in older cultures; this varies with the preparation.

To compare the accumulation of the mMAP4 transcripts with those of another gene that has been shown to be activated during differentiation in C2C12 cultures (Wright et al., 1989; Edmondson and Olson, 1989), the same samples were probed with a myogenin cDNA clone. As shown in Fig. 6B, myogenin is present at very low levels in myoblast cultures, and is induced within 24 hours to high levels. Consistent with other findings (Wright et al., 1989), myogenin mRNA was barely detectable in adult muscle tissue. This difference in myogenin transcript levels between differentiated C2C12 cells and adult tissue suggests that the differentiated state of C2C12 cells is not equivalent to that of adult muscle (see Discussion).

To determine if a larger protein isotype of MAP4 was induced during the course of myogenesis in culture, proteins were prepared for immunoblot analysis with monoclonal antibodies that recognize sequences common to uMAP4 and mMAP4 (Fig. 6C). Proliferating confluent C2C12 myoblasts have the characteristic MAP4 complex at approx. 220×103Mr (day 0) (arrowhead). In this preparation, a less prevalent isotype of approx. 350×103Mr is also detected in day-0 cultures (arrow); this low level is seen in myoblast cultures that are confluent, but not in those that are dividing rapidly. However, this larger MAP4 isoform accumulates to high levels after cultures have been in differentiation medium for 48 hours (day 2) and remains abundant thereafter. The increasing amounts of signal below the uMAP4 band as differentiation proceeds is consistent with our finding that mMAP4 protein is highly susceptible to degradation. The large isoform is also present in adult mouse thigh, and two degradation products that are consistently observed are also detected. Because the appearance of the larger MAP4 protein coincides with the appearance of the mMAP4 transcripts during differentiation, we conclude that this is mMAP4 protein. Like other structural MAPs (Noble et al., 1989; Lee et al., 1988; Irminger-Finger et al., 1990), including uMAP4 (West et al., 1991), the mMAP4 protein migrates at a higher molecular mass (approx. 350×103Mr) than the mass predicted from the sequence (232×103Mr).

In situ hybridization detects muscle MAP4 transcripts early in myogenesis

To examine whether expression of mMAP4 transcripts occurred early during muscle development in vivo, the distribution of MAP4 transcripts in mouse embryos was analyzed by in situ hybridization. Myogenic regulatory factors can first be detected by embryonic day 8 –8.5, and transcripts for structural proteins such as α-cardiac and α-skeletal actin are first detected between 8.5 and 9.5 days post-coitum (reviewed in Buckingham et al., 1992). Therefore, we examined embryos from days 8 to 16 of gestation to determine where mMAP4 mRNA is expressed. Patterns obtained with the sequence unique to mMAP4 were compared to those obtained with probes common to all MAP4 sequences, and hybridizations with sense strand probes served as controls.

Examination of embryos at selected stages showed increasing accumulation of the muscle-specific transcripts in muscle structures as development progressed. Day-11 embryos showed expression of mMAP4 transcripts in the pre-thoracic muscles and somites (data not shown). Examples from day-13 embryos, in which expression of the mMAP4 mRNA is very prominent, are shown in Fig. 7. All skeletal muscles (head and neck muscles, tongue, intercostal muscle and diaphragm) accumulate mMAP transcripts (Fig. 7B,D,F). In contrast, uMAP4 is expressed at relatively uniform levels in most tissues (Fig. 7A,C,E). mMAP4 transcripts accumulate in the axial musculature that has given rise to dorsal muscles (dm), intercostal muscles (ic) and pre-thoracic muscles (pt) (Fig. 7B). More caudally, intercostal muscles (ic) show high levels of muscle transcript (Fig. 7D). Transcripts are also evident in the intrinsic muscle of the tongue (Fig. 7F). In older embryos (days 14-16), overall patterns for mMAP4 transcripts are the same as those seen for day 13 (data not shown). These data demonstrate expression of the mMAP4 transcripts also occurs early in myogenesis in vivo, and that it parallels the patterns seen for other structural proteins that comprise the skeletal muscle cytoskeleton.

Fig. 7.

In situ hybridization analysis of mMAP4 and uMAP4 expression in day-13 mouse embryos. Sections of day-13 mouse embryos were probed with antisense RNA probes specific either to uMAP4 (A,C,E) or to mMAP4 transcripts (B,D,F). Shown are regions including dorsal muscle (dm), intercostal muscle (ic), pre-thoracic muscle (pt; A,B); more caudal intercostal (ic) muscles, diaphragm (d; C,D) and tongue (E,F). See text for further description. Bar, 0.1 mm.

Fig. 7.

In situ hybridization analysis of mMAP4 and uMAP4 expression in day-13 mouse embryos. Sections of day-13 mouse embryos were probed with antisense RNA probes specific either to uMAP4 (A,C,E) or to mMAP4 transcripts (B,D,F). Shown are regions including dorsal muscle (dm), intercostal muscle (ic), pre-thoracic muscle (pt; A,B); more caudal intercostal (ic) muscles, diaphragm (d; C,D) and tongue (E,F). See text for further description. Bar, 0.1 mm.

Requirement for muscle MAP4 expression during in vitro myogenesis

To examine how mMAP4 might function in myogenesis, experiments were designed to perturb the expression of the musclespecific transcripts. Initial observations were made by treating differentiating cultures with antisense oligonucleotides that bracketed the MAP4 start site; controls were either sense or mismatched oligonucleotides. A plasmid expressing an antisense construct specific to mMAP4 sequence was also injected into myotubes. In both cases, aberrant, swollen myotubes were seen in the cells treated with antisense reagents. Creatine kinase levels, however, were comparable to those seen in normal cultures. To develop a better system in which to study this effect, we generated stable cell lines bearing the mMAP4 antisense construct. The prediction was that myoblasts would grow normally because they naturally lack the mMAP4, but that differentiative events dependent on the expression of these sequences would be altered. Expression of the antisense transcript in stably transfected myoblasts was confirmed by RT-PCR (data not shown). As shown in Fig. 8A, myoblasts derived from a stable cell line containing the antisense plasmid appear identical to those lacking the plasmid (compare to Fig. 1). In contrast, the morphology of cells containing the antisense plasmid that had been in differentiation medium for 4 days is severely altered relative to the control (Fig. 8B,C). Most striking is the presence of multinucleate syncytia, which lack a polarized morphology. The formation of the multinucleate syncytia indicates that fusion can occur in differentiating cultures bearing the antisense construct, but the aberrant shapes indicate that elaboration of the elongate myotube is perturbed. The apolar shapes coincided with the coalescence of the nuclei in the center of the syncytia, and the arrangement of microtubules in radial or disorganized arrays rather than the alignment parallel to an elongate axis typical of normal myotubes.

Fig. 8.

C2C12 cells expressing sequence antisense to mMAP4 mRNA are normal as myoblasts, but have an aberrant phenotype upon differentiation. Confocal images of myoblast (A) or myotube (B,C) cultures (grown for 4 days in differentiation medium), fixed with methanol and labeled with tubulin antibodies to visualize the microtubule array. (A) Stably transfected proliferating myoblasts expressing the antisense transcript have interphase and mitotic microtubule arrays indistinguishable from those of normal C2C12 myoblasts (compare with Fig. 1A,C). (B) Differentiating cultures of antisense C2C12 cells demonstrate aberrant phenotypes. Shown at low magnification are three multinucleate syncytia (arrows) with variable numbers of nuclei. The microtubule arrays in these cells lack the parallel organization observed in normal myotubes.(C) Higher magnification of a typical antisense-expressing myotube.

Fig. 8.

C2C12 cells expressing sequence antisense to mMAP4 mRNA are normal as myoblasts, but have an aberrant phenotype upon differentiation. Confocal images of myoblast (A) or myotube (B,C) cultures (grown for 4 days in differentiation medium), fixed with methanol and labeled with tubulin antibodies to visualize the microtubule array. (A) Stably transfected proliferating myoblasts expressing the antisense transcript have interphase and mitotic microtubule arrays indistinguishable from those of normal C2C12 myoblasts (compare with Fig. 1A,C). (B) Differentiating cultures of antisense C2C12 cells demonstrate aberrant phenotypes. Shown at low magnification are three multinucleate syncytia (arrows) with variable numbers of nuclei. The microtubule arrays in these cells lack the parallel organization observed in normal myotubes.(C) Higher magnification of a typical antisense-expressing myotube.

Further analyses showed additional defects in the antisense population (Fig. 9). Polarized light microscopy illustrates that normal C2C12 differentiated cells form anisotropic myofibrils (Fig. 9A), whereas such structures are undetectable in antisense-expressing cultures (Fig. 9B). The antisense syncytia (Fig. 9D) are also markedly shorter than their normal counterparts (Fig. 9C). Quantification of this difference (Fig. 9E) indicates antisense syncytia are 2–3 times shorter than control myotubes: the average length of the myotubes in normal cultures was 498±225 μm (n=70; range 130–1060 μm), whereas that in the antisense cultures is 192±104 μm (n=153; range 70–670 μm).

Fig. 9.

Differentiating C2C12 cells expressing sequence antisense to mMAP4 mRNA lack organized myofibrils, are shorter than their normal counterparts and lack mMAP4 protein. (A,B) Polarized light images of normal (A) and antisense (B) C2C12 cells grown in differentiation medium for 4 days. Myofibrils are apparent in the normal cultures (arrowheads) but are absent in the antisense-expressing cultures. Bar, 50 μm. (C,D) Phase contrast images of normal (C) and antisense (D) C2C12 cultures grown for 4 days in differentiation medium. Bar, 50 μm. (E) Histogram of the lengths of normal (black bars) and antisense (hatched bars) multinucleate cells present in cultures grown for 4 days in differentiation medium. Lengths were determined from low-power photographs of fields of cells, and the same number of fields was examined for each cell type. (F) Immunoblots, probed as described in Fig. 5C, of protein from normal (N) and antisense (A) myogenic cultures that are either undifferentiated (undiff.) or differentiated for 4 days (diff.). All undifferentiated and differentiated cultures contain approx. 220×103Mr MAP4 (short arrow), whereas the 350×103Mr isotype (long arrow) typical of normal differentiated myotubes is absent in the differentiated antisense cultures.

Fig. 9.

Differentiating C2C12 cells expressing sequence antisense to mMAP4 mRNA lack organized myofibrils, are shorter than their normal counterparts and lack mMAP4 protein. (A,B) Polarized light images of normal (A) and antisense (B) C2C12 cells grown in differentiation medium for 4 days. Myofibrils are apparent in the normal cultures (arrowheads) but are absent in the antisense-expressing cultures. Bar, 50 μm. (C,D) Phase contrast images of normal (C) and antisense (D) C2C12 cultures grown for 4 days in differentiation medium. Bar, 50 μm. (E) Histogram of the lengths of normal (black bars) and antisense (hatched bars) multinucleate cells present in cultures grown for 4 days in differentiation medium. Lengths were determined from low-power photographs of fields of cells, and the same number of fields was examined for each cell type. (F) Immunoblots, probed as described in Fig. 5C, of protein from normal (N) and antisense (A) myogenic cultures that are either undifferentiated (undiff.) or differentiated for 4 days (diff.). All undifferentiated and differentiated cultures contain approx. 220×103Mr MAP4 (short arrow), whereas the 350×103Mr isotype (long arrow) typical of normal differentiated myotubes is absent in the differentiated antisense cultures.

The correlation of the phenotype in the differentiated cultures with the expression of mMAP4 protein was analyzed by immunoblotting using antibodies that recognize sequences common to uMAP4 and mMAP4. As shown in Fig. 9F, the amount of uMAP4 protein (short arrow) is comparable in normal myoblasts that were never transfected (N; undiff) or that were stably transfected with the muscle MAP4 antisense construct (A; undiff). Control cultures that have been differentiated for 4 days have both the uMAP4 and the mMAP4 (long arrow) proteins (N; diff). However, the mMAP4 species is absent from the antisense cultures that have been maintained in differentiation medium for the equivalent time period (A; diff). These data show that the mMAP4 protein is specifically repressed in cells bearing the mMAP4 antisense construct. Immunoblotting shows that the levels of tubulin are the same in control and antisense cultures (data not shown). Collectively, these data suggest that suppression of muscle MAP4 results in the aberrant microtubule organization and shape of the multinucleate myotubes.

We describe here the sequence and expression characteristics of a tissue-specific form of the ubiquitous microtubule associated protein, MAP4. This alternatively spliced variant has a long neutrally charged insertion between the acidic projection domain and basic microtubule binding domain described previously (West et al., 1991). RNase protection and PCR analyses of the complete coding region of RNAs from muscle tissue indicate that this constitutes the major, and probably the only, muscle-specific variation in MAP4 (unpublished data). Variants in the conserved PGGG repeats of the microtubule binding domain of MAP 2 (Kindler et al., 1990), tau (Lee, 1990) and MAP4 (West et al., 1991; Chapin et al., 1995) have been described, and RNase protection analyses indicate that the 4-repeat form of the PGGG domain is used most extensively in muscle (Chapin et al., 1995, and our unpublished observations). Variations in the projection domains of both MAP 2 (Papandrikopoulou et al., 1989; Garner and Matus, 1988) and tau (Goedert et al., 1992; Couchie et al., 1992) are suggested to influence spacing between microtubules during neuronal morphogenesis (Chen et al., 1992). By analogy, it seems likely that this novel muscle-specific variation in the projection domain determines a unique role for mMAP4 in the structuring of microtubules in muscle that is distinct from the more general roles ascribed to uMAP4 in mitosis and/or microtubule dynamics in interphase cells (Olson et al., 1995; Ookata et al., 1995).

mMAP4 is expressed early in myogenesis, as assessed either by analysis of cultured cell lines or by examination of embryos. In C2C12 cells, the expression of mMAP4 transcripts starts within 24 hours following the onset of differentiation. This pattern of expression corresponds to a period characterized by the reorganization of myofibrils from a non-striated form to the striated arrangement described in other cell culture models (Lin et al., 1994). In mouse embryos, mMAP4 transcripts can be detected by day 11 of development. This corresponds to a period when myogenic cells begin to elongate, and some immature myofibrils first appear (Furst et al., 1989). By day 13, when fusion of myoblasts begins in vivo, mMAP4 is detected at high levels in all the developing skeletal muscles of the embryo. Thus, both in vivo and in vitro, mMAP4 is first expressed when the onset of establishment of myofibril architecture and cellular morphogenesis is most active.

Our data indicated that in differentiated C2C12 cultures, the mMAP4 transcripts do not completely supplant the ubiquitous MAP4 sequences. The persistence of uMAP4 transcripts in differentiated cultures probably reflects the presence of myoblasts and/or less differentiated cells than are found in adult muscle. This is supported by the finding that myogenin message, which is down-regulated in adult muscle (Wright et al., 1989), is still present in the most highly differentiated C2C12 cultures (data presented here; Wright et al., 1989), as well as by the observations of others that the ‘differentiation index’ for C2C12 cells grown under the conditions used here is only 30-50% (Miller, 1990). Despite an incomplete conversion in the cultured cell system, it is apparent that the regulation of the alternative splicing pathway for MAP4 is not limited to the myogenic process but is also maintained in the committed state, as the muscle variant transcripts are present in adult skeletal and cardiac muscle, even after such factors as myogenin are downregulated.

The results of our antisense experiments indicate that mMAP4 plays a significant role in the morphological differentiation of the myotube. The absence of the mMAP4 protein does not alter the fusion of myoblasts into multinucleate syncytia, but the generation of the polarized morphology characteristic of normal myotubes is affected. These data argue that formation of multinucleate syncytia alone is not sufficient to cause microtubule re-organization in myogenic cells. Interestingly, antisense inhibition of another structural protein expressed early in myogenesis causes a quite different phenotype. When the synthesis of desmin, a muscle-specific intermediate filament protein, was repressed, the myogenic pathway was blocked prior to fusion and no syncytia were formed (Li et al., 1994). Collectively, these data indicate that the timing of the requirement for different structural proteins varies during myotube formation, with mMAP4 appearing to have a key role in the post-fusion elongation of multinucleate syncytia. While a wealth of experiments have demonstrated that assembled myotubes can be disrupted with drugs (Antin et al., 1981; Toyama et al., 1982), or with antibodies or mutant forms of proteins (Schafer et al., 1995), our data represent an unusual example in which repression of a protein at the onset of differentiation results in the improper structuring of postfusion myotubes.

What sort of functions might mMAP4 play in myogenesis, a time during which massive remodeling of cytoplasmic structures occurs? At the primary level, mMAP4 could be involved in the re-organization of microtubules from the radial arrays present in myoblasts to the parallel arrays typical of mature myotubes. Both modification (Gundersen et al., 1989) and isotype composition (Lewis and Cowan, 1988) of tubulin change as microtubules undergo these rearrangements. Since we have found that the projection domain of uMAP4 strongly influences the way in which the binding domain interacts with microtubules (Olson et al., 1995), the muscle-specific insertion of a long peptide between these two domains may contribute to generating a novel microtubule array that has very different dynamics than that in myoblasts. Changes in the binding affinity of the mMAP for microtubules, as well as the presence of the extended projection domain in mMAP4, may both help to stabilize the long parallel arrays of microtubules in the myotube, in a manner analogous to the roles proposed for MAP 2 and tau in stabilizing neuritic microtubules. In turn, these arrays, either passively or actively, could be crucial in establishing the organization of the contractile apparatus. The fact that mMAP4 remains up-regulated in adult tissue suggests that it has an ongoing role in maintaining proper functioning in the mature myotube. In addition to the realignment of microtubules and the organization of the contractile elements, myotube formation involves the reorganization of a number of other cellular organelles: the nuclei in the myotubes are redistributed to the periphery of the syncytium, the endoplasmic reticulum re-aligns along the myotube axis, and the Golgi apparatus and pericentriolar material become distributed circumferentially around the nucleus rather than localized to one side (Tassin et al., 1985; Ralston, 1993). Potentially, the organization of the contractile system, the redistribution of organelles or the establishment of the new membranous systems of the sarcoplasmic reticulum and t-tubules, may be assisted by microtubules and microtubule-associated proteins. Our current investigations are focused on a further understanding of the role that the muscle-specific form of MAP4 may have in these dynamic events during myogenesis and in the function of adult muscle.

We are grateful to Hiram D. Lyon for assistance with the in situ hybridization protocols and manuscript preparation, Rob Code for RNase protection information on muscle and other tissue variants within mouse MAP4, Dr E. Olson for the C2C12 myotube cDNA library, Drs J. H. Morris and W. Klein for the myogenin cDNA clone, Dr Robert Summers for generous assistance with confocal microscopy, Dr Susan Zusman and Carol Roote for assistance with polarizing light microscopy and Dr Robert Angerer for critical reading of the manuscript. This work was supported by NIH grant GM 22214 to J.B.O. M. E. M. was partially supported by Interdepartmental Training Grant in Genetics and Regulation NIH 5 T32 GM07102.

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