Gundersen G G, Khawaja S, Bulinski J C
Department of Biology, University of California, Los Angeles 90024.
J Cell Biol. 1989 Nov;109(5):2275-88. doi: 10.1083/jcb.109.5.2275.
Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.
微管（MTs）被认为在肌生成过程中细胞形状和细胞内组织的变化中发挥作用。然而，微管参与这些形态发生事件的机制尚不清楚。作为阐明微管在肌生成中作用的第一步，我们使用针对酪氨酸化（Tyr）、去酪氨酸化（Glu）和乙酰化（Ac）微管蛋白的特异性抗体，研究了分化的大鼠L6肌肉细胞中翻译后修饰形式的微管蛋白的积累和亚细胞分布。Glu微管蛋白和Ac微管蛋白都是稳定微管的组成成分，而Tyr微管蛋白是动态微管的主要成分。在增殖的L6成肌细胞中，与其他类型的增殖细胞一样，与Tyr微管蛋白水平相比，Glu微管蛋白水平非常低。然而，当我们将增殖的L6细胞转移到分化培养基中时，我们观察到Glu微管蛋白在细胞微管中迅速积累。通过免疫荧光检测，Glu微管蛋白的增加首先在融合前成肌细胞的微管中被检测到，并且特异性定位于与细胞伸长部分相关的微管。在分化后期观察到的多核肌管中的微管维持了在融合前成肌细胞中观察到的Glu微管蛋白的升高水平。当对分化早期（更换培养基后不到1天）的细胞进行Glu微管蛋白和肌肉特异性标记物肌肉肌球蛋白的免疫染色时，我们发现Glu微管蛋白的增加先于肌肉肌球蛋白的积累。因此，Glu微管的形成是肌生成中非常早期的事件之一。Ac微管蛋白在L6肌生成过程中也增加；然而，乙酰化的增加发生在肌生成后期，即在融合已经发生之后。由于去酪氨酸化与肌生成的早期事件在时间上相关，我们研究了融合前成肌细胞微管中Glu微管蛋白积累的机制。我们发现，在分化开始时L6细胞微管的稳定性增加，这表明我们观察到的早期去酪氨酸化增加是伸长的成肌细胞中微管动态性降低的一种表现。我们得出结论，建立一个稳定性增强且翻译后修饰亚基水平升高的定向微管阵列可能参与了肌生成过程中发生的亚细胞重塑。
