Differential immunoreactivity for Z-DNA in rat myoblast nuclei during their terminal differentiation.

L6 myoblasts in vitro accomplish the process of terminal differentiation from dividing mononucleated cells to quiescent plurinucleated myotubes, synthesizing muscle-specific proteins. They have been tested, using paraformaldehyde and acetic acid fixations and immunocytochemical techniques, for the presence of Z-DNA at different stages: namely after 3, 5 and 8-9 days of culture. The nuclei of the actively dividing 3-day myoblasts were strongly Z-DNA and B-DNA-positive. The inhibition of replication by araC did not diminish the reaction. In the myotubes, the nuclei became Z-DNA-negative but were still B-DNA-positive. In contrast, the nuclei of a non-fusing alpha-amanitin-resistant mutant (Ama102) stayed Z-DNA-positive. These results tend to show that during the process of terminal differentiation Z-DNA either becomes less accessible or is present in undetectable amounts. In circular DNAs, it has been shown that the presence of Z-DNA depends on their negative supercoiling. In addition, the presence of closed superhelical loops of nuclear DNA has been demonstrated in several mammalian cell types; moreover, the density of DNA topological turns in these loops varies during cellular differentiation and malignant transformation. The relationship between these results and ours is discussed.

Metabolic changes in the extracellular matrix during differentiation of myoblasts of the L6 line and of a Myo- non-fusing mutant.

In the present study we have characterized by biochemical and immunochemical methods the changes which take place in collagen, laminin and fibronectin biosynthesis during the differentiation of clonal skeletal myoblasts of the L6 line. Time-course experiments showed that the relative rate of synthesis of collagen increased significantly during the cell-cell contact step of myogenesis and decreased later on. The major collagens synthesized were types I and III, found mainly as soluble precursors in the culture medium. Types IV and V collagens were detected exclusively in the cell layer. The relative amounts of types I and III collagens remained unchanged during myogenesis, while types IV and V collagens increased as the cells of the L6 line fused. In a non-fusing alpha-amanitin-resistant mutant of the L6 line (Ama 102), the rate of collagen synthesis was largely depressed and its rate of degradation was increased as compared with the fusing wild type. The synthesis of laminin was very low in cells of the fusing wild type, but abundant and associated with the cell layer of the Myo- mutant. The appearance of a muscle-specific extracellular matrix is a complex process involving changes in the organization, the biosynthesis and remodelling of its macromolecules of the extracellular matrix.

Modulation of differentiation in vitro. II. Influence of cell spreading and surface events on myogenesis.

We examined the influence of attachment and spreading on myogenesis by adding polylysine-covered beads at different times after plating the cells on a plastic substratum. We show that polylysine per se acting on the cell surface can modulate myogenesis independently of cell spreading. Thus cell shape would not be the limiting factor for the division and differentiation of L6 myoblasts. Multinucleation of the cells was found to be first enhanced by the addition of polylysine-covered beads to replicating myoblasts, although the final percentage of fusion attained by these cultures was lower than in the controls. A similar phenomenon was observed concerning myosin synthesis. No such effect could be observed when the beads were added to a nonfusing mutant or to fibroblasts. Our results show that this phenomenon is specific. We postulate that some of the surface molecules necessary for this process appear on myoblasts shortly before they fuse.

Alterations in glycosylation of plasma membrane proteins during myogenesis.

Highly purified plasma membranes were obtained from cells of the L6 line at three characteristic stages of myogenesis: Actively proliferating cells; post-mitotic, confluent myoblasts which have already aligned; and fused myotubes. Differential glycosylation of the plasma membrane proteins of these cells was detected by staining polyacrylamide gels of the separated components with three lectins of different specificity: Concanavalin A (conA), wheat germ agglutinin (WGA) and phytohemagglutinin (PHA) Els. Four kinds of developmentally regulated changes could be identified. 1. Those which took place only at confluency (160, 150, 90, 85, 60, 43 and 40 kD for conA binding, 190 kD for WGA binding, 190 and 110 kD for PHA Els binding. 2. Those which took place only at fusion (135, 51.5 and 38 kD for conA, 160 and 150 kD for WGA and 150 kD for PHA Els binding). 3. Those where the phenomena initiated at confluency continue during fusion (66.5 and 32 kD for conA and 120 kD for PHA binding). 4. Those where opposite changes take place at confluency and at fusion (48 kD for conA, 180, 98 and 85 kD for PHA binding). These results suggest that most developmentally regulated changes in glycosylation take place during the first cell-cell contact step of myogenesis. Metabolic labelling experiments showed that, on the contrary, only few alterations in the accumulation of plasma membrane proteins take place prior to the main burst of fusion.

Modulation of differentiation in vitro. I. Influence of the attachment surface on myogenesis.

Myogenic cells of the L6 line proliferate and fuse in culture to form myotubes that actively synthesize muscle-specific proteins such as myosin. We show that the expression of the differentiated phenotype can be influenced by the electrical charges of the substratum on which the cells were grown. Negatively charged surfaces did not influence the developmental program of the cells although positively charged ones interfered with myogenesis. The interaction operates primarily by interfering with the mitotic cycle, which is slowed down, with fusion which is blocked, and with myosin synthesis, which is reduced. Our results show that growth of the cells on positively charged surfaces prevents the switching of a large fraction of the population from a proliferative state to a differentiating program. We postulate that this interference might operate through the slowdown in DNA replication. The cell culture method described represents a good model for studying the different steps involved in the differentiation of L6 cells.

Differentiation in vivo and in vitro of pyruvate kinase isozymes in rat muscle.

The M type isozymes of Pyruvate-kinase have been studied by isoelectrofocusing in thin layer acrylamide ampholine gel, during the ontogeny of rat muscle in vivo and during the differentiation in vitro of myoblasts of a line established by Yaffe. In both cases, multiple subbands have been seen; the most acid (pHi 5.2) was the predominant band in myoblasts and in fetal muscle at the 15th day. Several more cathodic bands appear sequentially in vitro as in vivo, one of them corresponding to the "M2" or "K" band (predominant in kidney). The most cathodic band M1 (pHi 7.3), characteristic of the adult muscle, appears at the 9th day of culture in vitro in multinucleated myotubes and at the 20th day of fetal life in vivo. Kinetic results confirm these electrofocusing results, showing in fetal muscle and in myoblasts a sigmoid saturation curve of pyruvate kinase activity with phosphoenolpyruvate as substrate. This allosteric kinetic is progressively replaced by a Michaëlian kinetics in vitro as in vivo. Consequently, the studies in vitro may serve as a model for myogenesis in vivo, and may contribute to the understanding of the significance of the multiple forms of pyruvate-kinase during this myogenesis.

Gene expression during the differentiation of myogenic cells of the L6 line.

The problem of the mechanistic relationship among the different phenotypic expressions in an established myogenic line was approached by blocking cell fusion at different developmental stages, by addition of cytochalasin B. The addition of the drug to cultures at the time when the first two myotubes appeared on the dish, blocked fusion, but did not affect DNA synthesis, expression of myosin, phosphorylase, phosphocreatine kinase, phosphorylase kinase or glycogen synthetase, nor the organization of the elements of the hexagonal lattice. It is concluded that cell fusion is not a prerequisite for the expression of the differentiated phenotype.