To study the events taking place during differentiation we use the C2 mouse muscle cell line and tools such as pharmacological agents, cDNA constructs, and shRNA constructs for knocking down specific proteins. ? ? Previously, we proposed that the reorganization of the Golgi complex during muscle differentiation depends on its constant recycling through the endoplasmic reticulum (ER) and on changes in the ER exit sites. In order to test this model we have been using drugs that specifically affect microtubules (nocodazole) or Golgi complex (brefeldin A). We have established conditions that maintain muscle differentiation, manifested by myogenin expression, in the presence of these drugs. We have found that all changes were affected by nocodazole or brefeldin A, except the redistribution of microtubule-organizing centers which is then identified as the essential and initial step. The redistribution of microtubule-organizing centers appears affected by LiCl and other drugs that inhibit the kinase GSK3-beta, suggesting that one of the signaling pathways involving this kinase may be involved in the early steps.? ? Since LiCl and GSK3-beta inhibition are known to affect microtubule stability, we wanted to independently examine how affecting microtubules without a complete depolymerization would affect muscle differentiation. Therefore we expressed constructs of the microtubule plus end protein EB1. The results suggested that EB1 is involved in elongation and fusion of muscle cells. When we used shRNAs to knock down EB1, we found that some cells differentiated but failed to fuse, but some cells completely failed to differentiate. The difference seems to result from the presence of a previously unknown EB1 isoform. We have also shown that EB1 which, in fibroblasts, is necessary and sufficient for microtubule stabilization, in muscle cells is neither. We have therefore identified a new isoform and a new role for EB1 in muscle development.? ? To study the organization of microtubules and Golgi complex in muscle in vivo, we carry out immunofluorescence and other microscopy of rodent muscle fibers. We take advantage of transgenic animals to evaluate the role of other cytoskeletal or structural proteins in the organization of microtubules and Golgi complex in muscle pathologies.? ? Following the discovery in a proteomics screening by Jill Humston and Jim Ervasti (U. Minnesota) that tubulin binds dystrophin directly, we decided to examine the status of microtubules in the mouse models of muscular dystrophy, i.e. mdx, utrophin-negative, and double knock-out mice. Staining of whole muscle fibers has confirmed the interaction by showing that the microtubule cytoskeleton organization is abnormal in mdx and double knock-out mice but is normal in utrophin-negative mice. Differences can already be observed in 3 week-old mice, in which the wave of muscle regeneration characteristic of mdx has not taken place, ruling out that the microtubules are only affected by the regeneration process. We are now examining the distribution of the Golgi complex in these transgenic mice. A functional defect in the organelle could compound the damage to the muscle fibers of dystrophin-deficient mice since the Golgi complex is important for assembly and targeting of several proteins of the dystrophin complex.? ? We have also continued to investigate the role of the intermediate filament protein desmin in the organization of microtubules and Golgi complex in muscle fibers. We are now successful at growing primary muscle cultures from slow and fast adult mouse muscles. This will allow us to determine if the damage that we observe mostly in slow muscles of desmin-null mice can be observed from the earliest stages of differentiation and might give us a handle to understand fiber type differences in microtubule and Golgi complex organization in muscle.
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