Related Experiment Videos
Possible involvement of a cell surface glycoprotein in the differentiation of skeletal myoblasts
Abstract:
From a highly myogenic permanent line of rat skel-myoblasts (L6), we have isolated two classes of single step concanavalin A-resistant mutants. The RI class is about 2-fold and RII about 5-fold more resistant than the parental cells to the lethal action of concanavalin A. In all of the mutants, both the morphological differentiation (i.e. fusion to form myotubes) and biochemical differentiation, measured by the appearance of creatine kinase and acetylcholine receptors, are absent. The biochemical lesion in the RI type of mutants is not known, but RII type of mutants is unable to catalyze transfer of mannose from GDP-mannose into a lipid-linked form. Concanavalin A binding to separated membrane proteins from RII type of mutants on polyacrylamide gels is reduced 80% compared to wild type cells. In the RI type of mutants, however, only one major band, approximately 46,000 daltons, does not bind concanavalin A to the same extent as the wild type cells. In somatic cell hybridizations, RI type of mutants complements the RII type. In the hybrids, fusion as well as creatine kinase and acetylcholine receptors reappear, although not to the same extent as in the wild type cells. The 46,000-dalton band also reappears in the complementing hybrids. Thus, this protein may play some crucial role in myogenesis.
Insights
Researchers identified concanavalin A-resistant rat skeletal muscle cell mutants (L6). These mutants lack differentiation, but a specific 46,000-dalton protein
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Myogenesis involves complex differentiation processes in skeletal muscle cells.
- Concanavalin A (ConA) is a lectin that binds to specific carbohydrate residues on cell surfaces.
- Mutant cell lines are valuable tools for dissecting cellular pathways.
Purpose of the Study:
- To isolate and characterize concanavalin A-resistant mutants of rat skeletal myoblasts (L6).
- To investigate the role of ConA binding proteins in myoblast differentiation.
- To identify potential molecular defects underlying resistance and differentiation defects.
Main Methods:
- Isolation of single-step concanavalin A-resistant mutants (RI and RII classes).
- Assessment of morphological (myotube fusion) and biochemical (creatine kinase, acetylcholine receptors) differentiation.
- Analysis of concanavalin A binding to membrane proteins via polyacrylamide gel electrophoresis.
- Somatic cell hybridization experiments.
Main Results:
- RI mutants showed 2-fold, RII mutants showed 5-fold ConA resistance.
- All mutants exhibited absent morphological and biochemical differentiation.
- RII mutants had impaired mannose transfer to lipid-linked forms and reduced ConA binding to membrane proteins.
- RI mutants showed reduced ConA binding to a specific 46,000-dalton protein.
- RI and RII mutants complemented each other in somatic cell hybrids, restoring differentiation and the 46,000-dalton protein.
Conclusions:
- A 46,000-dalton protein is implicated in myogenesis, potentially through its interaction with ConA.
- Defects in this protein or related pathways disrupt skeletal myoblast differentiation.
- Somatic cell hybridization is effective in identifying complementing genetic defects.