Cytoskeletal Interactions of Dystrophin

PROJECT SUMMARY/ABSTRACT The long-term objective of this project is to fully define the functions of dystrophin in striated muscle to understand how its absence or abnormality leads to the pathologies observed in Duchenne and Becker muscular dystrophies, and to inform on the potential for miniaturized dystrophins or utrophin to substitute for dystrophin in a therapeutic context. In the current project period, we generated a new line of transgenic mdx mice that expresses dystrophin lacking in vitro microtubule binding activity, but which surprisingly presented with a fully rescued microtubule lattice. We also analyzed microtubule organization in existing lines of transgenic mdx mice expressing different truncated dystrophin constructs and found that two different micro- dystrophins were less effective than two mini-dystrophins in restoring microtubule organization in unstressed mdx muscles. Exciting new preliminary data identified a second region required for microtubule organization and showed that the microtubule lattice of transgenic mdx mice expressing mini- or micro-dystrophins is rapidly disrupted by eccentric contraction through a mechanism involving reactive oxygen species. Thus aim 1 is to generate and characterize two new lines of transgenic mdx mice that will more definitively confirm the regions of dystrophin necessary for microtubule lattice organization. Aim 1 will also investigate the relationship between eccentric contraction and reactive oxygen species in disrupting microtubule organization in mdx muscles expressing mini- or micro-dystrophins. Our recently published atomic force microscopy data suggest that utrophin may be much stiffer than dystrophin and functionally less equivalent than previously thought. We also have acquired exciting preliminary data showing that the cellular system used to express a model utrophin fragment significantly impacts its mechanical properties. In aim 2, we will extend these studies to measure the mechanical properties of a previously studied dystrophin construct expressed in bacteria, eukaryotic insect cells, and mammalian myoblasts. We will also analyze the recombinant proteins for post- translational modifications that can account for the measured differences in mechanical behavior. In aim 3, we will carry out the first mechanical characterization of single, full-length dystrophin molecules for comparison with our utrophin data. We will also investigate how internal truncations affect the mechanical behavior of therapeutically-relevant miniaturized dystrophins. Our proposed studies will provide new understanding into the functions of dystrophin and utrophin in healthy muscle, and will inform on the potential for miniaturized dystrophins and utrophin to functionally replace dystrophin as therapeutic approaches for Duchenne muscular dystrophy.