To advance towards objective 1, we have been working with the flexor digitorum brevis muscle of the mouse, from which collagenase-dissociated fibers can be reliably and reproducibly prepared, treated with drugs, stained with antibodies and observed in immunofluorescence. In experimental conditions of microtubule growth, we observe typical """"""""asters"""""""" of short microtubules. In most cell types such asters originate from the centrosome, although a population of specialized microtubules appears to be centered on the Golgi complex. During muscle development the centrosomes are disassembled. In muscle fibers, the asters we observe are centered on Golgi complex elements. We have shown that the Golgi complex elements are part of the nucleating mechanism by demonstrating that microtubule asters are severely reduced in number and in size in fibers treated with Brefeldin A, a drug that collapses the Golgi complex into the endoplasmic reticulum. We are now investigating the role of specific Golgi complex proteins in this nucleation. The first candidate is the Golgi complex protein GM130, a central Golgi matrix protein. In preliminary experiments we have tested shRNAs and succeded in knocking GM130 down in C2 muscle cell cultures. We have also optimized techniques for injecting cDNAs into mouse FDB muscle. We should therefore be able to knock down GM130 in FDB muscle fibers and assess the effects on microtubule nucleation and Golgi distribution in vivo. In order to observe microtubule dynamics in muscle in vivo (objective 2), we have constructed and tested DNA plasmids encoding fragments or the whole of two microtubule-associated proteins: MAP4, and the ensconsin microtubule-binding domain (EMTB),linked to fluorescent proteins. This preliminary work is necessary because the most obvious microtubule marker, tubulin-GFP, does not fully incorporate in existing microtubules, and does not track microtubule dynamics faithfully in C2 cultures. At low degrees of overexpression, MAP4 and EMTB show dynamic behavior in myoblasts and myotubes and, satisfy all our criteria for usefulness for in vivo experiments. The next stage will be to introduce them into muscle fibers in vivo. To evaluate the mechanism and consequences of the microtubule defects in disease models (objective 3) we have observed microtubule nucleation in mdx and Pompe disease mice and have found profound defects which we are currently analyzing. In the past year we have finalized a study showing that the initial reorganization of centrosomal proteins during myogenesis in muscle cell cultures drives all the other steps and takes place independently of microtubule reorganization. We have also continued our collaboration with Drs. Plotz and Raben on the study of Pompe Disease. Our results on microtubule nucleation in muscle in vivo are turning around the way we understand microtubule-organelle relationships. They will help us understand muscle phenotype in several diseases as well as the difficulties encountered by enzyme replacement therapy in Pompe disease.
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