Microtubule assembly in cultured myoblasts and myotubes following nocodazole induced microtubule depolymerisation

Abstract

When myoblasts fuse into myotubes, the organisation of the cytoskeleton changes dramatically. For example, microtubules emanate in a radial array form the centrosome in myoblasts, but form linear arrays not linked to a centrosome in myotubes. It is not clear how these linear arrays are formed and nucleated. They could arise in a number of ways: by nucleation and release from centrosomal like structures, cytoplasmic assembly, breakage/severing or nucleation from non-centrosomal sites. To test which of the above mechanisms or combination of mechanisms are responsible we investigated the re-formation of microtubules after depolymerisation by nocodazole, using antibodies against pericentrin, gamma-tubulin, EB1, and tyrosinated alpha-tubulin. In myoblasts, we found that when microtubules were allowed to recover after complete depolymerisation with nocodazole, microtubule recovery began within 1 min and was complete after 5 min. Microtubules grew out from the centrosome, which was positively stained for gamma-tubulin or pericentrin. In untreated myotubes, microtubules were arranged in linear arrays, with EB1 at their ends. The pericentriolar protein, pericentrin was arranged in a band around the nucleus as well as discrete spots in the cytoplasm. In contrast, the microtubule nucleating protein gamma-tubulin was not found in a band around the nucleus, but was found in several punctuate spots throughout the cytoplasm. Further, when microtubules were allowed to recover, after complete depolymerisation with nocodazole, recovery was not as rapid as that seen in myoblasts, and we found that regrowth began with the formation of short microtubule fragments throughout the cytoplasm. Gamma-tubulin was associated with these fragments. These results suggest that in myotubes, nucleation of microtubules can be non-centrosomal.

Fig. 1.

Triple immunostained images of myoblasts immunostained for α-tubulin (green), nuclei (blue) and either pericentrin (A, B and C), γ-tubulin (D, E and F) or EB1 (G, H and I) in red. The figure shows both untreated myoblasts and myoblasts after treatment with nocodazole to induce microtubule depolymerisation. A, D and G show control myoblasts, B, E and H show myoblasts immediately after nocodazole treatment, and C, F and I shows myoblasts after 5 min recovery following nocodazole treatment. Scale Bar: 20 μm.

Fig. 2.

Triple immunostained images of myotubes immunostained for α-tubulin (green), nuclei (blue) and either pericentrin (A, B and C), γ-tubulin (D, E and F) or EB1 (G, H and I) in red. The figure shows both untreated myotubes and myotubes after treatment with nocodazole to induce microtubule depolymerisation. A, D and G show control myotubes, B, E and H show myotubes 1 min after nocodazole treatment, and C, F and I show myotubes after 5 min recovery following nocodazole treatment. The arrows are pointing to undifferentiated cells lying close to the myotubes, in which microtubule asters can be seen, typical of re-growth in myoblasts. These aster formations are not seen in the myotubes. Scale Bar: 20 μl. The separate panels below, show single magnified images of re-growing microtubules in myotubes following 2 and 5 min recovery after nocodazole treatment. The staining for γ-tubulin and α-tubulin are shown separately, as well as the merged image. The arrows point to regrowing microtubules in which a spot of γtubulin appears to be associated with re-growing microtubules.

Fig. 3.

Stills from a time-lapse image of fusing myoblasts. The numbers in the bottom left corner indicate the time in minutes. At time = 0, two myoblasts can be seen interacting, at an angle of 45° (arrows 1 and 2). These two myoblasts subsequently fuse between 20 and 30 min later. The resulting myotube (mt1) then interacts with a second myotube (mt2) after 160 min, and these two myotubes fuse 40-80 min later (between t = 240 and 280). The white arrows point to the myoblasts and myotubes. After each fusion event the newly formed myotubes elongates. The phase contrast image has been inverted. Scale bar 20 μl.