Oligosaccharyltransferase activity is markedly increased during differentiation of a nonfusing myoblast cell line

We have studied several aspects of glycoprotein synthesis in myoblast differentiation by using a nonfusing myoblast cell line, BC3H1. Previous studies showed that transfer of proliferating undifferentiated BC3H1 cells to mitogen-depleted medium results in the cells' withdrawal from the cell cycle and induction of a variety of muscle-specific gene products [E. N. Olson, L. Glaser, J. P. Merlie, R. Sebane, and J. Lindstrom (1983) J. Biol. Chem. 258, 13946-13953]. Because cell surface glycoproteins have been implicated in myoblast differentiation, in the present study we measured the amount of oligosaccharyltransferase in microsomes isolated from BC3H1 cells at various stages of differentiation. By using an acceptor peptide containing the sequence-Asn-Leu-Thr-, enzyme activity was measured by formation of [3H]glycopeptide. In addition, active enzyme protein was measured with a 125I-labeled photoreactive derivative of the acceptor tripeptide. Both of these independent assay methods revealed a marked increase in oligosaccharyltransferase when differentiation was induced by serum depletion. Moreover, mitogenic stimulation of differentiated cells resulted in a return of oligosaccharyltransferase to near basal levels. This reversible increase in this key enzyme in protein glycosylation occurred despite the fact that both total protein and glycoprotein synthesis were depressed during differentiation. These data indicate that during myogenesis the level of oligosaccharyltransferase is regulated in parallel with a number of muscle-specific gene products. These results are discussed in the context of regulation of the pathway of glycoprotein synthesis.     

Constant expression of mouse calpastatin isoforms during differentiation in myoblast cell line, C2C12

C2C12 is a myoblast cell line which is used to studydifferentiation into multinucleated cells in vitro. Addition of calpain inhibitors, calpeptin orE-64d, to the culture medium prevented the myoblasticfusion of C2C12 cells. Immunoblot studies usingaffinity-purified antibody, revealed that the expressedlevels of mouse calpastatin remained unaltered duringC2C12 cell fusion. The detected calpastatin migratedas a protein of 130 kDa on SDS-polyacrylamide gelelectrophoresis. The estimated molecular mass wassomewhat greater than that in mouse liver anderythrocytes, and much greater than that reported inrat myoblasts. The 130 kDa isoform may contain anadditional N-terminal region designated XL domainfound in bovine calpastatin.     

Sequence analysis of the rDNA spacer ofParacentrotus lividus and observations about pre-rRNA processing

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchinParacentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor which has the same 5 terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.Abbreviations bp base pair(s) - Kb Kilobase(s) or 1000 bp - nt nucleotide(s) - rDNA DNA encoding rRNA - rRNA ribosomal RNA - S sedimentation constant     

Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.     

Alterations in surface proteins during myogenesis of a rat myoblast cell line.

Lactoperoxidase-catalyzed iodination and SDS-polyacrylamide gradient gel electrophoresis were used to examine the surface proteins of cultures of an embryonic rat myoblast cell line during myogenesis. We observed several consistent alterations in the proteins iodinated during the periods of alignment and fusion. In addition, we examined the surface proteins of cultures where fusion was inhibited by phospholipase C (PLC), and of cultures of several nonfusing variants of our original line. Many of the proteins which appeared during “normal” myogenesis were not seen in PLC-treated cultures, while the appearance and loss of three low molecular weight proteins were accelerated. The nonfusing variants often accumulated large amounts of many of the proteins which appeared during alignment in normal cultures. This accumulation was demonstrated by Coomassie blue stain intensities as well as by the extent of surface iodination. The three low molecular weight proteins were heavily iodinated in one class of variant, but did not disappear as in normal cultures. One protein of apparent molecular weight 66,000 (66K) was iodinated during alignment but was inaccessible during fusion. Coomassie blue staining of the gels showed that the actual appearance and disappearance of the 66K protein band from the membrane were coincident with alignment and fusion. While this band was not seen in fluorograms from gels of PLC-treated cultures and some of the nonfusing variants, a 66K band was invariably stained by Coomassie blue, and in PLC-treated cultures appeared to accumulate with time. In the variants there appeared to be a correlation between the availability of the 66K protein for iodination and the appearance of the low molecular weight proteins.     

Proliferation, differentiation and maturation of a mouse epidermal keratinocyte cell line

The spontaneous development of a cell line of neonatal mouse C3H/He epidermal cells is described. The culture has been serially passaged at 29 °C over 18 months in the absence of any dermal support. The cell morphology of the 18th passage is reported. During early growth phase, the morphology of the cell layers was similar to that observed in the basal and differentiating strata of the epidermis: numerous tonofilament bundles and desmosome-filament complexes were observed. During late growth phase, maturation and vertical stratification occurred: demonstrated by the tonofilament accumulation, cell organelle degradation, nuclear pyknosis, presence of keratohyalin granules and horny cell layers with thickened membranes. Hemidesmosome-like structures were shown. No basal lamina or membrane coating granules were detectable. The 18th passage cultured cells did not induce tumors in nude mice. This keratinocyte cell line is not permanent, however: a malignant transformation occurred after 25 subcultures which resulted in an undifferentiated cell population.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Changes in plasma membrane glycoproteins during differentiation of an established myoblast cell line and a non-fusing α-amanitin-resistant mutant

The glycoproteins of purified plasma membranes from mononucleated myoblasts and from myotubes of the L₆ line were characterized according to their apparent molecular weight (MW) and to their ability to bind concanavalin A (conA). We identified 25 proteins in membranes from mononucleated myoblasts and fused myotubes which specifically bound the lectin. Comparison with the pattern of membrane glycoproteins of a non-fusing mutant allowed us to identify developmentally regulated changes in the accumulation of 8 proteins with an apparent MW of 160, 80, 60, 51.5, 43, 40, 38, and 27 Kilodalton (kD), and changes in the glycosylation of six others which migrate at 215, 150, 135, 90, 85, and 32 kD. Two of them (160 and 38 kD) appeared at fusion, whereas the 51.5 kD band could not be identified in plasma membrane from myotubes. As conA inhibits fusion of myoblasts, it is suggested that at least some of these proteins might be involved in this process.     

Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.