Misplaced Golgi Elements Produce Randomly Oriented Microtubules and Aberrant Cortical Arrays of Microtubules in Dystrophic Skeletal Muscle Fibers.

Differentiated mammalian cells and tissues, such as skeletal muscle fibers, acquire an organization of Golgi complex and microtubules profoundly different from that in proliferating cells and still poorly understood. In adult rodent skeletal muscle, the multinucleated muscle fibers have hundreds of Golgi elements (GE), small stacks of cisternae that serve as microtubule-organizing centers. We are interested in the role of the GE in organizing a peculiar grid of microtubules located in the fiber cortex, against the sarcolemma. Modifications of this grid in the mdx mouse model of Duchenne muscular dystrophy have led to identifying dystrophin, the protein missing in both human disease and mouse model, as a microtubule guide. Compared to wild-type (WT), mdx microtubules are disordered and more dense and they have been linked to the dystrophic pathology. GE themselves are disordered in mdx. Here, to identify the causes of GE and microtubule alterations in the mdx muscle, we follow GFP-tagged microtubule markers in live mdx fibers and investigate the recovery of GE and microtubules after treatment with nocodazole. We find that mdx microtubules grow 10% faster but in 30% shorter bouts and that they begin to form a tangled network, rather than an orthogonal grid, right after nucleation from GE. Strikingly, a large fraction of microtubules in mdx muscle fibers seem to dissociate from GE after nucleation. Moreover, we report that mdx GE are mispositioned and increased in number and size. These results were replicated in WT fibers overexpressing the beta-tubulin tubb6, which is elevated in Duchenne muscular dystrophy, in mdx and in regenerating muscle. Finally, we examine the association of GE with ER exit sites and ER-to-Golgi intermediate compartment, which starts during muscle differentiation, and find it persisting in mdx and tubb6 overexpressing fibers. We conclude that GE are full, small, Golgi complexes anchored, and positioned through ER Exit Sites. We propose a model in which GE mispositioning, together with the absence of microtubule guidance due to the lack of dystrophin, determines the differences in GE and microtubule organization between WT and mdx muscle fibers.

Persistent upregulation of the ß-tubulin tubb6, linked to muscle regeneration, is a source of microtubule disorganization in dystrophic muscle.

In healthy adult skeletal muscle fibers microtubules form a three-dimensional grid-like network. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), microtubules are mostly disordered, without periodicity. These microtubule defects have been linked to the mdx mouse pathology. We now report that increased expression of the beta 6 class V ß-tubulin (tubb6) contributes to the microtubule changes of mdx muscles. Wild-type muscle fibers overexpressing green fluorescent protein (GFP)-tubb6 (but not GFP-tubb5) have disorganized microtubules whereas mdx muscle fibers depleted of tubb6 (but not of tubb5) normalize their microtubules, suggesting that increasing tubb6 is toxic. However, tubb6 increases spontaneously during differentiation of mouse and human muscle cultures. Furthermore, endogenous tubb6 is not uniformly expressed in mdx muscles but is selectively increased in fiber clusters, which we identify as regenerating. Similarly, mdx-based rescued transgenic mice that retain a higher than expected tubb6 level show focal expression of tubb6 in subsets of fibers. Tubb6 is also upregulated in cardiotoxin-induced mouse muscle regeneration, in human myositis and DMD biopsies, and the tubb6 level correlates with that of embryonic myosin heavy chain, a regeneration marker. In conclusion, modulation of a ß-tubulin isotype plays a role in muscle differentiation and regeneration. Increased tubb6 expression and microtubule reorganization are not pathological per se but reflect a return to an earlier developmental stage. However, chronic elevation of tubb6, as occurs in the mdx mouse, may contribute to the repeated cycles of regeneration and to the pathology of the disease.

Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements.

Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that muscle MTs are dynamic, growing at the typical speed of ∼9 µm/min, and forming small bundles that build a durable network. We also show that static Golgi elements, associated with the MT-organizing center proteins γ-tubulin and pericentrin, are major sites of muscle MT nucleation, in addition to the previously identified sites (i.e., nuclear membranes). These data give us a framework for understanding how muscle MTs organize and how they contribute to the pathology of muscle diseases such as Duchenne muscular dystrophy.