Adult skeletal muscle is built by successive waves of developmental changes. Myoblasts, undifferentiated cells committed to a muscle fate, first differentiate. They exit the mitotic cycle, start expressing proteins specific to skeletal muscle, elongate, and fuse with each other to form long multinucleated myotubes. Myotubes undergo further morphological changes that turn them into muscle fibers which, upon innervation, adopt one of several possible types. Very little is understood of the regulation and of the mechanism of the morphological changes that muscle cells undergo. Differentiation affects each component of the muscle cytoskeleton and of the muscle subcellular organelles. The most dramatic reorganization of a subcellular organelle is that of the Golgi complex, which is essential for the processing of transmembrane and secreted proteins. It was shown 20 years ago that during myoblast differentiation, the Golgi complex is reorganized and appears to follow the proteins that nucleate microtubules. Our work is focused on this reorganization: how are microtubules reorganized during muscle differentiation and maturation and what role do microtubules play in the reorganization of the Golgi complex? Working with skeletal muscle cultures and with rodent muscle fibers, we have established, a few years ago, that the Golgi complex is fragmented into hundreds of small elements during muscle differentiation. In myotubes, Golgi elements are found along the nuclear membrane and between nuclei. In muscle fibers, this organization is further differentiated between slow-twitch and fast-twitch fibers. In all, Golgi elements are not distributed randomly; instead they are found next to the sites through which proteins are exported from the endoplasmic reticulum to the Golgi complex. These sites are known as ER exit (or export) sites. The proximity of Golgi elements and of ER exit sites suggests that the mechanism of fragmentation of the Golgi complex during differentiation involves retrograde cycling through the endoplasmic reticulum. This mechanism accounts fragmentation of the Golgi complex following depolymerization of microtubules in all undifferentiated mammalian cells. We therefore proposed a model in which Golgi complex reorganization during muscle differentiation results directly from the reorientation of the microtubules. Things are however more complicated since the ER exit sites themselves are reorganized during differentiation. We have also shown that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is fiber type-dependent and is controlled by the pattern of contractile activity. In the past year, we have progressed in several respects. i) We have asked whether the reorganization of the ER exit sites during differentiation (aggregation along the nuclear membrane, for example) could be due to differences in the organization of the ER itself. We have expressed fluorescently tagged proteins targeted to the ER in myoblasts and myotubes of the muscle cell line C2. We have used the confocal microscope to estimate differences in the coefficient of diffusion of the tagged proteins in the outer nuclear membrane of myoblasts and myotubes. We found that ER proteins were diffusing as fast in the nuclear membrane of myotubes as that of myoblasts, suggesting that the intrinsic motility of proteins in the outer nuclear membrane does not explain the reorganization of ER exit sites. It is then likely that ER exit site changes take place by interaction with a muscle-specific protein expressed during differentiation. ii) We have also progressed in our attempts to identify the hierarchy of changes and the signaling pathways involved in the reorganization of the microtubules and Golgi complex during differentiation. Using drugs that either destabilize microtubules or stabilize them massively, we have observed that the redistribution of microtubule-organizing sites is resistant to alterations in the microtubule network. We have also observed that redistribution of microtubule-organizing sites is a prerequisite to the reorganization of the Golgi complex, whereas redistribution of ER exit sites is not. Finally, we observed that reorganization of the Golgi complex is initiated but only rarely completed in the absence of a normal microtubule network. iii) At the molecular level, we have continued to examine the role of GSK3-beta in the reorganization of the microtubules and Golgi complex during differentiation. Treatment of muscle cultures with a ruthenium complex specific inhibitor of the kinase reduced microtubule dynamics and partially inhibited the reorganization of the Golgi complex. Myoblasts also failed to align and fuse. In cell migration signaling pathways which are sensitive to microtubule stabilization by inhibitors of GSK3-beta, the microtubule-plus-end tracking protein EB1 has shown to play an important role. Without EB1, tubulin could not be glutamylated, which means that they were not stabilized. In contrast, in new experiments using siRNAs, we have obtained results suggesting that EB1 is not necessary for the glutamylation of muscle microtubules or for the normal reorganization of the Golgi complex. In contrast, EB1 depletion seems to prevent the normal elongation and fusion of myoblasts. We therefore are now able to uncouple some of the changes of the microtubule network and associated organelles taking place during muscle differentiation and reorganization. We also are progressing in the identification of pathways involved in these changes. Microtubules and Golgi complex are essential components of every mammalian cell. Defects in microtubules and in the secretory pathway are now known to be involved in several cardiac and skeletal muscle pathologies. Yet, microtubule cytoskeleton and associated organelles have mostly been studied in proliferating cells. We understand very little of their organization in muscle. These studies should help us in better understanding these basic cellular processes.
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