Regulated trafficking and targeting of membrane proteins to specific subcellular domains is an essential aspect of the organization of very large cells 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. During muscle differentiation and maturation, the Golgi complex undergoes striking changes. 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 allowed measurements of FRAP (fluorescence recovery after photobleaching) on live cells. We have demonstrated that the Golgi complex of myotubes is made of independent elements, which are localized at the endoplasmic reticulum (ER) exit sites. We have now shown that there is constant recycling of the proteins of the Golgi complex through the ER and, more importantly, that this retrograde cycling is necessary for the changes in Golgi complex during differentiation to occur. These results are important because they show that events as different as mitosis and differentiation affect the Golgi complex by similar pathways. We have been helped in this work by the finding that we could synchronize the differentiation of the C2 cultures by releasing them from a differentiation block induced by inhibitors of the MAP kinase p38. We will pursue this work.Single muscle fibers prepared from 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 and of the microtubules is fiber type dependent. To determine whether nerve-derived trophic factors or electrical activity are responsible for this effect, we have examined the distribution of the Golgi complex, of the ER exit sites, centrosomal proteins and microtubules in rat muscles that have been denervated and chronically stimulated for 2 weeks. We found that stimulation of a fast mucle with a fast stimulation pattern preserved the original distribution of all the markers, as did stimulation of a slow muscle with a slow stimulation pattern. However, cross-stimulation of a fast muscle with a slow stimulation frequency or of a slow muscle with a fast frequency led to changes in the distribution of the markers to different degrees. These results demonstrate that patterned electrical activity is responsible for the organization of the Golgi complex and that it remains plastic in the adult animal. In the future we will attempt to understand the molecular pathways responsible for this plasticity.
