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 Kd of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼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.

This study was designed to define the molecular epitopes of dystrophin-actin interaction and to directly compare the actin binding properties of dystrophin and utrophin. According to our data, dystrophin and utrophin both bound alongside actin filaments with submicromolar affinities. However, the molecular epitopes involved in actin binding differed between the two proteins. In utrophin, the amino-terminal domain and an adjacent string of the first 10 spectrin-like repeats more fully recapitulated the activities measured for full-length protein. The homologous region of dystrophin bound actin with low affinity and near 1:1 stoichiometry as previously measured for the isolated amino-terminal, tandem (CH) domain. In contrast, a dystrophin construct including a cluster of basic spectrin-like repeats and spanning from the amino terminus through repeat 17, bound actin with properties most similar to full-length dystrophin. Dystrophin and utrophin both stabilized preformed actin filaments from forced depolymerization with similar efficacies but did not appear to compete for binding sites on actin. We also found that dystrophin binding to F-actin was markedly sensitive to increasing ionic strength, although utrophin binding was unaffected. Although dystrophin and utrophin are functionally homologous actin-binding proteins, these results indicate that their respective modes of contact with actin filaments are markedly different. Finally, we reassessed the abundance of dystrophin in striated muscle using full-length protein as the standard and measured greater than 10-fold higher values than previously reported.Dystrophin and utrophin are homologous proteins with similar interacting partners. By providing a link between the actin cytoskeleton and the extracellular matrix, dystrophin functions to maintain the integrity of the cell membrane during muscle contraction. Consequently, genetic ablation of dystrophin leads to increased fragility in the muscle membrane and results in the pathologies observed in Duchenne and Becker muscular dystrophies and some forms of cardiomyopathy. The functional role of utrophin is not completely understood. However, utrophin overexpression in the dystrophin-deficient mdx mouse has been shown to correct all known parameters of the dystrophic phenotype (1). Notably, utrophin overexpression rescued the mechanical linkage between costameric actin and the sarcolemma of the dystrophin-deficient mdx mouse muscle (2).Like other members of the spectrin superfamily of proteins, both dystrophin and utrophin interact with actin via the amino-terminal tandem calponin homology (CH) 2 actin-binding domain (3-6). Additionally, in both dystrophin and utrophin, the spectrin-like repeats of the rod domain have been shown to contribute to actin binding. According to our recent study, the first 10 spectrin-like repeats of utrophin increase the affinity and capacity of its amino-terminal domain for actin (7). The actin binding activity of the homologous region of dystrophin has not been investigated yet. However,...

Iowa, where she has taught introductory and advanced chemistry courses. She has also taught Inquiry into Physical Science, an inquiry-oriented introduction to physics and chemistry for elementary education majors, for the Department of Science Education. Jill is also the Projects Coordinator for the Iowa Mathematics and Science Education Partnership. She worked as the Director of the Young Scientists' Camp at UNI before joining the IMSEP team. While in graduate school, she volunteered with Expanding Your Horizons to encourage middle and junior high school girls' interest in mathematics and science. She received her Ph.D. in Molecular and Cellular Pharmacology from the University of Wisconsin-Madison and her B.S. in Chemistry from the University of Northern Iowa.