Cytoskeletal organization and synthesis in substrate‐independent and ‐dependent myogenesis in chick embryos

Abstract

Chick embryo myoblasts were fused in suspension culture to form myoballs by modification of previous procedures that excluded the use of divalent ion chelators and antimitotic drugs and included the continuous presence of serum in order to analyze the organized appearance and synthesis of major cytoskeletal proteins during cell attachment and spreading. The organization of the major cytoskeletal proteins actin, tubulin, and vimentin was assessed by fluorescence microscopy under these conditions as well as under conditions in which the myoballs were allowed to attach and spread on a collagen-coated substrate. Actin, detected by fluorescence microscopy, stained myoballs diffusely and was reorganized to form stress fibers in the attached and spreading myoball. Nuclei were segregated to a centrally located lattice of microtubules. The microtubule-specific drugs nocodazole and taxol prevented myoball spreading and the establishment of myotube polarity, respectively. Vimentin appeared as wavy ribbons in a perinuclear position around attached and spreading myoballs. In parallel studies, the synthesis of these cytoskeletal proteins was analyzed by radioisotopic labeling and polyacrylamide gel electrophoresis. These studies showed that myoballs possess altered ratios of actin and tubulin isoforms and of phosphorylated and nonphosphorylated vimentin compared to myotubes. These ratios rapidly change to the myotube pattern when myoballs are allowed to attach to solid substrata. Thus, although both myoballs and myotubes undergo muscle-specific differentiation, their cytoskeletal proteins are morphologically and biochemically distinct.

Differential splicing-in of a proline-rich exon converts alphaNAC into a muscle-specific transcription factor.

“…Overexpression of meltrin-a, a transmembrane protein impli cated in myoblast fusion, has also been shown to induce myosac formation (Yagami-Hiromasa et al 1995). Simi larly, inhibition of protein kinase C, as well as inhibition of substrate attachment, leads to an identical phenotype (Lin et al 1987;Daniels and Sandra 1990). Thus, it is conceivable that interactions of myoblasts with the ex tracellular matrix via transmembrane receptors trigger a protein kinase C-dependent signaling cascade leading to myotube fusion and that skNAC may be implicated in this transduction pathway.…”

“…Several adhesion macromolecules have been implicated in myo blast fusion, including neural cell adhesion molecule (N-CAM) (Dickson et al 1990;Knudsen et al 1990a), N-cadherin (Knudsen et al 1990b), very late activation antigen 4 (VLA-4) (Rosen et al 1992), vascular cell adhesion mol ecule 1 (VCAM-1) (Rosen et al 1992), and meltrin-a (Yagami-Hiromasa et al 1995). Among others, manipula tions that inhibit protein kinase C (Lin et al 1987) or attachment to extracellular substratum (Daniels and Sandra 1990) affect myoblast fusion. Moreover, fusion is inhibited in myoblasts overexpressing the c-myc protooncogene (Crescenzi et al 1994), a site-specific DNAbinding transcriptional activator (Liischer and Eisenman 1990).…”

Cells under pressure – the relationship between hydrostatic pressure and mesenchymal stem cell chondrogenesis

“…The authors investigated the changes in cytoskeletal elements under pressure, as alterations in these structures relate to cell morphology and shape that are influenced by the surrounding environment and stimuli. Indeed, previous studies have indicated that prevention of F-actin polymerisation induces chondrogenesis through development of a rounded cell shape (Daniels and Solursh, 1991;Daniels and Sandra, 1990;Woods et al, 2005). Steward et al (2013) demonstrated that within stiff hydrogels, more actin development is observed in unloaded controls, leading to a reduced chondrogenic response as compared to that found for cells in softer gels.…”