Over the past year we have completed a collaborative study of the interaction between the muscle protein dystrophin and the microtubule constituent protein tubulin (Prins et al., 2009). Dystrophin is a large protein whose mutations are responsible for the most prevalent muscle disease, Duchenne Muscular Dystrophy (DMD). Dystrophin was already known to interact with the actin cytoskeleton and with intermediate filament proteins, suggesting that it may act as a cytolinker, a protein that serves to link all constituents of the cell cytoskeleton. Using muscle extracts from control and from mdx mouse, the mouse model for DMD, our collaborator James Ervasti (U. Minnesota) and his laboratory obtained biochemical evidence supporting a direct interaction between dystrophin and tubulin. Amisha Mehta and, later, Victoria Tate in LI performed immunofluorescence staining of single whole muscle fibers from control and mdx mice, as well as from utrophin knockdown mice and mdx-utrophin double knockdown mice. Microtubules lost their normal organization in mdx and mdx-utrophin double knockdown muscles, but not in the utrophin knockdown muscles. In addition, we showed that microtubule defects are already present in young (3 week-old) mice which have not started undergoing the cycles of degeneration-regeneration characteristic of the disease. These experiments support a specific link between dystrophin and microtubules in vivo. We also showed that microtubules in fast muscles of a normal mouse course along transverse and longitudinal bands of dystrophin, confirming that dystrophin contributes to organizing microtubules. This work thus classifies dystrophin as a cytolinker in muscle fibers and may help to understand some of the consequences of the absence of dystrophin, in the mdx mouse and in DMD. We have also progressed towards our goal of understanding the organization of microtubules in mature muscle fibers. Victoria Tate has been using a culture system using muscle fibers detached from the flexor digitorum brevis mouse muscle by enzymatic digestion. She has determined conditions to depolymerize the dynamic microtubules from the live fibers and to then observe their repolymerization. These experiments are important because microtubules are typically nucleated at the centrosome, but several cell types, such as skeletal muscle, lack a typical centrosome. Some cell types also show microtubule nucleation at the Golgi complex, apart from that seen at the centrosome. In skeletal muscle, centrosomal proteins, microtubules, and Golgi complex are all redistributed, first during differentiation, and then further during maturation of muscle fibers. Differentiated muscle cultures show microtubules forming at the nuclear membrane but microtubule dynamics have never been explored in muscle fibers in vivo. Interestingly, nucleation of microtubules in muscle fibers appears to originate at the Golgi complex elements. Victoria has also examined the role of microtubules in the close association between Golgi elements and lysosomes (LAMP-1 positive structures) in muscle fibers. Most Golgi elements (75%) have juxtaposed lysosomes in control and nocodazole-treated fibers but cold and nocodazole, each, increase the fraction of Golgi-unassociated lysosomes. This suggests that both dynamic microtubules and stable, nocodazole-resistant, glutamylated microtubules are involved in Golgi-lysosome association. Our findings provide the foundation of a model to understand microtubule and Golgi complex organization in muscle fibers. They also extend Golgi complex-based microtubule nucleation to a highly differentiated tissue. The mechanisms may not be identical. A poster (Tate et al.) will be presented at the 2009 meeting of the American Society for Cell biology. In parallel to our work on muscle fibers, we have pursued our work on muscle cultures which have provided us with a basic understanding of the changes taking place during differentiation of muscle cells. Tan Zhang together with Kristien Zaal has been investigating the role of the microtubule-associated protein EB1 in muscle differentiation. In other mammalian cells, EB1 is necessary for microtubule stabilization. We hypothesized that EB1 may play a similar role during muscle development. Indeed, we found that dominant-negative constructs of EB1 affect myoblast elongation. Knocking down EB1 permanently prevents microtubule stabilization and, unexpectedly, prevents differentiation of the muscle cells (Zhang et al., 2009). In addition, Tan Zhang has now demonstrated that there is an EB1 pool associated with the Golgi complex and that it may be play a role in the integrity of the Golgi complex (manuscript in preparation). We have also continued to collaborate with Drs. Plotz and Raben on the study of Pompe Disease, a lysosomal storage disorder in which cardiac and skeletal muscles are the source of the pathology (Raben et al., 2008 &2009).
| Raben, Nina; Baum, Rebecca; Schreiner, Cynthia et al. (2009) When more is less: excess and deficiency of autophagy coexist in skeletal muscle in Pompe disease. Autophagy 5:111-3 |
| Zhang, Tan; Zaal, Kristien J M; Sheridan, John et al. (2009) Microtubule plus-end binding protein EB1 is necessary for muscle cell differentiation, elongation and fusion. J Cell Sci 122:1401-9 |
| Prins, Kurt W; Humston, Jill L; Mehta, Amisha et al. (2009) Dystrophin is a microtubule-associated protein. J Cell Biol 186:363-9 |
| Raben, Nina; Hill, Victoria; Shea, Lauren et al. (2008) Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease. Hum Mol Genet 17:3897-908 |
