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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.

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K(d) of 0.66 microM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, alpha- and beta-tubulin expression is increased approximately 2.5-fold in mdx skeletal muscle without altering the tubulin-MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.