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 cortical 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 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, the first goal of 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 stable microtubule lattice organization. Aim 1 will then elucidate the relationship between eccentric contraction and reactive oxygen species in disrupting the microtubule lattice in mdx muscles expressing mini- or micro-dystrophins. Finally, aim 1 will delineate the interplay between the dystrophin-glycoprotein complex and cytoplasmic actins in effecting stable cortical microtubule organization in mature skeletal muscle. We have also recently published atomic force microscopy data suggesting that utrophin may be much stiffer than dystrophin and functionally less equivalent than previously thought. We 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 our preliminary studies in bacteria and insect cells to measure the mechanical properties of dystrophin and utrophin constructs expressed in mammalian myoblasts. We will then carry out the first mechanical characterization of single, full-length dystrophin molecules for comparison with our published utrophin data. And finally, in aim 2 we will investigate how internal truncations affect the mechanical behavior of the most 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.