Marc Y. Fiszman

Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis.

Primary isolates of chick leg muscle myoblasts cultured on hyaluronic acid substrates have been examined by transmission electron microscopy for evidence of myoblast fusion and subsequent differentiation. Even though these cells form close contacts, no evidence of multinucleated myotubes is found in these cultures. Two-dimensional SDS-polyacrylamide gel electrophoresis shows that the muscle macromolecular biosynthetic program is not initiated in these hyaluronic acid fusion-blocked cells. Further, these fusion-blocked myoblasts continue replicating while cultured on hyaluronic acid surfaces. The inhibition of both fusion and the myogenic expressional program is reversed by replating these myoblasts onto a denatured collagen (gelatin) substrate; both the synthesis of muscle-specific proteins and the formation of multinucleated myotubes are observed when these subcultured cells are introduced onto gelatin substrates. These observations indicate that the hyaluronic acid inhibition of fusion is not permanent and is manifested in a way different from other fusion blockers in that hyaluronic acid inhibits both fusion and the myogenic expressional program.

A skeletal muscle cell line isolated from a mouse teratocarcinoma undergoes apparently normal terminal differentiation in vitro

Abstract A muscle cell line was obtained as a clonal isolate from a contractile zone of cultured cells derived from a mouse teratocarcinoma. The cell line (C17-S1-D-T984) differentiates in vitro when the cells become confluent, with the formation of multinucleated myotubes. The muscle fibres contract spontaneously and electron microscopy demonstrates the presence of organized sarcomeric structures. In addition to the presence of the contractile proteins, there is also an accumulation of acetylcholine receptors and the appearance of the M form of creatine phosphokinase, all characteristic markers of differential skeletal muscle.

Expression of myogenic differentiation and myotube formation by chick embryo myoblasts in the presence of sodium butyrate

Abstract The presence of Na butyrate in cultures of chick embryo myoblasts interferes with normal differentiation of these cells. At a concentration of 3 mM it reversibly inhibits the formation of myotubes without affecting the normal program of biochemical differentiation. This effect is not related to the inhibition of cell growth produced by the same concentration of butyrate but may be due to modifications of the cell membranes by the drug.

In vivo splicing of the beta tropomyosin pre-mRNA: a role for branch point and donor site competition.

The chicken beta tropomyosin gene contains two sets of alternatively spliced, mutually exclusive exons whose utilization is developmentally regulated. Exons 6A and 6B are used in nonmuscle cells (or undifferentiated muscle cells) and skeletal muscle cells, respectively. A complex arrangement of cis-acting sequence elements is involved in alternative splicing regulation. We have performed an extensive mutational analysis on the sequence spanning the region from exon 6A to the constitutive exon 7. A large number of mutant minigenes have been tested in transfection assays of cultured myogenic cells, and the splicing products have been analyzed by cDNA polymerase chain reaction. We demonstrate that in undifferentiated myoblasts, exon 6B is skipped as a result of a negative control on its selection, while exon 6A is spliced as a default choice. We provide evidence that the focal point of such a regulation is localized in the intron upstream of exon 6B and probably involves the blockage of its associated branch point. In differentiated myotubes, in contrast, both exons are accessible to the splicing machinery. We show that the preferential choice of exon 6B in this splicing environment depends on the existence of a competition between the two exons for the flanking constitutive splice sites. We demonstrate that both the donors and the branch points of the two exons are involved in this competition.

Influence of the excision shock on the protein metabolism ofVicia faba L. meristematic root cells

UsingVicia faba root meristems we have shown that protein synthesis was dramatically changed after excision. The amino-acid incorporation dropped to 13% of the level in the unexcised control. This downshift was a direct consequence of the breakdown of polysomes which are converted into monosomes. In order to perform an analysis of the protein pattern by two-dimensional gel electrophoresis, endogenous proteolytic activity, which is high in broad bean root, had to be inhibited. Therefore, several protease inhibitors were tested and a very efficient inhibitor pool was obtained which could be used during the preparation of meristematic cell extracts. Protein-pattern analysis showed important differences between the unexcised control and excised apices. The number of proteins synthesized after excision droped from 250 in the control to 80, as a consequence of polysome breakdown. Futhermore, we present evidence that new and apparently specific proteins are synthesized in response to this excision shock.

Morphological and biochemical differentiation in RSV transformed chick embryo myoblasts.

Chick embryo myoblasts have been transformed with a temperature sensitive mutant of Rous Sarcoma virus (RSV ts68). At permissive temperature (36 degrees C) it is shown that transformed myoblasts have lost their ability to form myotubes as well as to express the biochemical markers of myogenic differentiation. Upon a shift to the non-permissive temperature (41 degrees C), the normal program of differentiation is restored; myotubes are formed which express muscle specific proteins.

Butyrate-treated chick embryo myoblasts synthesize new proteins.

Abstract Upon treatment with sodium butyrate, chick embryo myoblasts synthesize two new major proteins, BIP 1 and BIP 2 which have the same molecular weight but differ in charge. These proteins are found free in the cytoplasm. Their synthesis starts within a few hours following addition of butyrate, reaches a constant rate after 40 h in the presence of the drug, and stops upon its removal. At all times BIP 1 and BIP 2 (Butyrate-Induced Proteins) remain at a constant ratio of approx. 2. Their appearance is correlated with the appearance of new species of messenger RNA (mRNA). Only myoblasts seem to synthesize the BIPs upon butyrate treatment. At the present time their function is completely unknown. Evidence is presented that the BIPs are not involved in the inhibition of myotube formation, since a derivative of butyrate, the methylester of butyric acid, can prevent fusion of myoblasts without inducing the synthesis of the BIPs.

Regulated splicing of an alternative exon of beta-tropomyosin pre-mRNAs in myogenic cells depends on the strength of pyrimidine-rich intronic enhancer elements.

Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.

Fast and slow chicken skeletal muscles contain different α and β tropomyosins

Abstract Avian tropomyosin has been purified from fast skeletal muscles (breast muscle and posterior latissimus dorsi: PLD) and from a slow skeletal muscle (anterior latissimus dorsi: ALD) and the α and β subunits have been further separated using preparative gel electrophoresis. These subunits have been subjected to partial proteolysis using different proteolytic enzymes. In this communication we show that this procedure allows to distinguish not only between fast and slow α tropomyosin but also between fast and slow β tropomyosin. Furthermore we have raised an antiserum against the fast α tropomyosin and we present evidence to show that this antiserum does not cross-react with the slow α tropomyosin. These results are taken to indicate that all these tropomyosin subunits represent different gene products.

Changes in ganglioside metabolism during in vitro differentiation of quail embryo myoblasts

The metabolism of gangliosides was studied during the in vitro differentiation of both normal quail myoblasts and myoblasts which have been transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV). These transformed cells can be maintained undifferentiated if incubated at 35 degrees C, but they will differentiate when shifted to 41 degrees C. (D. Montarras and M. Y. Fiszman (1983) J. Biol. Chem. 258, 3882-3888). The analysis of [14C]Glucosamine-labeled gangliosides by two-dimensional thin-layer chromatography reveals variations in the metabolism of the gangliosides during the process of differentiation. During the formation of myotubes, it was observed that the accumulation of GD1a is reduced, while the accumulation of GD3 is increased. Therefore, this results in the variation of the ratio GD3/GD1a which increases from 1.8 to 25 in the case of clones of transformed myoblasts, and from 0.5 to 1.7 in the case of uninfected myoblasts. These variations which have been observed seem to be specific of the myogenic differentiation since they cannot be reproduced when differentiation is inhibited by BUdR treatment or when fibroblasts reach confluency and are blocked in the G1 phase of cell cycle. Furthermore, the transformed myoblasts in vitro are shown to be a good model system since their gangliosides composition is very similar to that of muscle cells in vivo.