Changes in cytoskeletal organization during myotube formation in vitro

Abstract

The cytoskeletal changes accompanying CO25 myoblast differentia- tion, and transformation have been investigated using various techni- ques. A very significant change in microtubule organization was found to occur upon inducing differentiation by transferring the cells into a medium containing horse serum. Elongation of the cells was accompa- nied by a change in microtubule distribution such that all the micro- tubules ran parallel to the long axis of the cells and the radial organization from centrosomes was lost. The transient formation of primary cilia in the elongating cells was proceeded by an aggregation and fusion of all the centrioles wit- hin the cells. There seemed to be an apparent shift in microtubule nucleation centres from the centrosomes in myoblasts to a perinuclear distribution in myotubes. The capacity of these structures to nucleate microtubules was studied by allowing repolymerization after nocodazole treatment. The possible significance of these rather string changes in centriole organization, and in MTOC capacity, and the possible role of the primary cilia in myogenesis are discussed. During the course of the research the antibody MPM-13, supposed to be a centrosomal marker, was used. A very variable distribution of peri-centrosomally located granules was identified in myoblasts with MPM-13. Double immunostaining experiments demonstrated that MPM- 13 stained elements of the Golgi rather than a supposed centrosomal component. Study of the distribution of actin cables showed that stress fib- res were very rich in myoblasts, but decreased markedly in number as the myoblasts elongated. After fusion, an increase of F-actin cables occurred in newly formed myotubes, F-actin finally became highly or- ganised in a periodic distribution along the myofibrils in vitro. In transformed CO25 myoblasts, the distribution of F-actin was very variable. A few actin cables were seen in some cells which were attached to the substratum, but stress fibres were lacking in most cells, which were rounded cells in foci and not attached to the sub- stratum. A possible relationship of these changes to the focal con- tacts is considered.