The objective of this project is to determine the cytoskeletal interactions of the dystrophin-glycoprotein complex in the skeletal muscle to understand how its absence or abnormality leads to Duchenne (DMD) and Becker (BMD) muscular dystrophies and some forms of cardiomyopathy. Rather than just simply serving to anchor its associated glycoprotein complex to the cortical actin, our previous studies lead us to hypothesize that dystrophin also plays an important role in stabilizing the cortical cytoskeleton through an extended lateral association with actin filaments. We further hypothesize that the dystrophin homologue utrophin is missing an actin binding suite important for F-actin stabilization. These hypotheses will be tested, both in vitro and in vivo, through the pursuit of 3 complementary specific aims. The F-actin binding properties of full-length and truncated forms of recombinant dystrophin and utrophin will be measured by established biochemical and spectroscopic procedures (Aim 1). Completion of this aim will yield the first direct structure/function comparison for dystrophin and utrophin up-regulation to effectively compensate for dystrophin deficiency. Recombinant dystrophin/utrophin will be visualized alone and in complex with actin filaments using electron microscopy combined with three-dimensional reconstruction techniques (Aim 2). These studies will yield important new information about the shape, dimensions and flexibility of dystrophin and utrophin and will independently determine how much (and which sub-domains) of dystrophin lie in close apposition with F-actin. Analysis by three-dimensional reconstruction will also identify changes in actin monomer and filament structure that may lead to more stable association of other costameric proteins with F-actin. Finally, we will relate the in vitro features of the dystrophin/F-actin interaction with its role in stabilizing costomeric actin in vivo (Aim 3). Sarcolemmal membranes will be mechanically isolated from muscles of transgenic mdx mice expressing dystrophin constructs deleted in different domains and the status of costameric actin determined by confocal microscopy. We will also determine whether the absence of dystrophin results in an unstable sarcolemmal association of other costameric actin binding proteins. Completion of these aims will result in a highly detailed and integrated understanding of dystrophin's role in stabilizing the muscle membrane cytoskeleton through its interaction with cortical actin.
| Lindsay, Angus; McCourt, Preston M; Karachunski, Peter et al. (2018) Xanthine oxidase is hyper-active in Duchenne muscular dystrophy. Free Radic Biol Med 129:364-371 |
| Strakova, Jana; Kamdar, Forum; Kulhanek, Debra et al. (2018) Integrative effects of dystrophin loss on metabolic function of the mdx mouse. Sci Rep 8:13624 |
| Le, Shimin; Yu, Miao; Hovan, Ladislav et al. (2018) Dystrophin As a Molecular Shock Absorber. ACS Nano : |
| McCourt, Jackie L; Talsness, Dana M; Lindsay, Angus et al. (2018) Mouse models of two missense mutations in actin-binding domain 1 of dystrophin associated with Duchenne or Becker muscular dystrophy. Hum Mol Genet 27:451-462 |
| Nelson, D'anna M; Lindsay, Angus; Judge, Luke M et al. (2018) Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins. Hum Mol Genet 27:2090-2100 |
| Lindsay, Angus; Schmiechen, Alexandra; Chamberlain, Christopher M et al. (2018) Neopterin/7,8-dihydroneopterin is elevated in Duchenne muscular dystrophy patients and protects mdx skeletal muscle function. Exp Physiol 103:995-1009 |
| Belanto, Joseph J; Olthoff, John T; Mader, Tara L et al. (2016) Independent variability of microtubule perturbations associated with dystrophinopathy. Hum Mol Genet 25:4951-4961 |
| McCourt, Jackie L; Rhett, Katrina K; Jaeger, Michele A et al. (2015) In vitro stability of therapeutically relevant, internally truncated dystrophins. Skelet Muscle 5:13 |
| Talsness, Dana M; Belanto, Joseph J; Ervasti, James M (2015) Disease-proportional proteasomal degradation of missense dystrophins. Proc Natl Acad Sci U S A 112:12414-9 |
| Filareto, Antonio; Rinaldi, Fabrizio; Arpke, Robert W et al. (2015) Pax3-induced expansion enables the genetic correction of dystrophic satellite cells. Skelet Muscle 5:36 |
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