Regulated trafficking and targeting of membrane proteins to specific cellular domains is especially crucial for the very large excitable cells of the nervous system, such as neurons and muscle fibers. The goal of this project is to understand how subcellular domains are organized in these cells during differentiation, and how they are subsequently shaped by cellular activity. We believe that the formation of such domains depends on changes in the organization of the Golgi complex, the strategic cellular center for membrane protein sorting and targeting. In mitotic cells, its organization is dictated by the needs of cell division. In post-mitotic differentiated cells, in contrast, its organization changes according to cell morphology and needs. The changes in the organization of the Golgi complex during muscle differentiation and maturation are striking. Neither their mechanism nor their regulation is understood. The mouse muscle cell line C2 is our model to study differentiation. During differentiation, the Golgi complex appears to fragment into small stacks of cisternae which are positioned along the outer nuclear membrane of the myotube nuclei and in rows in the cytoplasm. Permanently transfected cell lines expressing the Golgi complex enzyme alpha-mannosidase II tagged with the fluorescent protein GFP have been cloned and characterized. Measurements of FRAP (fluorescence recovery after photobleaching) on myoblasts and myotubes have demonstrated that the Golgi complex of myotubes is made of independent elements, which are not in rapid dynamic exchange with the ER, as has been suggested in mitotic cells. It has also shown that these elements are relatively immobile and are localized at the ER exit sites. These results suggest a model in which the changes that occur during differentiation of muscle cells resemble the events taking place in other mammalian cells when microtubules are severed. In the near future, we will continue to investigate this model.Single muscle fibers prepared from several rat muscles are used as a model to study the changes in the Golgi complex during muscle maturation in vivo. In mature muscle fibers, small stacks of cisternae are found throughout the fibers, both near the surface and in the myofibrillar core, ensuring that protein trafficking can be locally controlled in all areas of the large fibers. We have observed that the distribution of the Golgi complex is fiber type dependent. A quantitative study by confocal light microscopy has shown that about 75% of all Golgi elements are found in a thin layer of cytoplasm at the surface of the slow fibers, whereas Golgi elements are more uniformly distributed throughout fast fibers. At the neuromuscular junction, however, the organization is independent of fiber type. The differential distribution of the Golgi complex is explained by a fiber type dependent distribution of microtubules. This differential distribution may have important physiological consequences for muscle fiber metabolism. Future work will examine whether muscle activity or nerve-derived factors are responsible for the fiber type differences. - skeletal muscle, differentiation,subcellular organelles, Golgi complex, C2C12, rat soleus, fiber type, confocal microscopy.
