An investigation into the effects of dystrophin on the lateral mobility of muscle membrane components.

Abstract

Dystrophin is the product of the Duchenne Muscular Dystrophy gene locus, whose absence results in progressive skeletal muscle breakdown. Despite considerable work on the localisation of dystrophin and its associated complex, its role in muscle function remains unclear. In the light of the structural and mechanical instability of the dystrophic membrane, the idea was tested that dystrophin might impart membrane integrity and strength by anchoring membrane proteins and/or delineating the surface into specialised subcellular functional domains. Specifically, because dystrophin shows high sequence, structural and spatial similarities to the cytoskeletal protein spectrin; and because spectrin is proven to sterically restrict protein lateral diffusion through a subplasmalemmal network; the capacity of dystrophin to act as a 'molecular fence' to membrane diffusion was studied by comparing lateral mobility of membrane glycoproteins by fluorescence photobleach recovery in mdx and normal tissue. Secondly, as dystrophin has been proven to interact directly with proteins of the dystrophin associated glycoprotein complex in vivo, experiments addressed whether specific binding and immobilisation of the complex by dystrophin at the membrane was essential for function. Finally, given the homology of dystrophin and spectrin, the presence of dystrophin at the neuromuscular junction, and the importance of spectrins in immobilisation of voltage gated sodium channels in the nervous system, the role of dystrophin in regulating voltage gated sodium channel distribution at the neuromuscular junction was investigated. The results show that membrane glycoproteins were immobile in the presence and absence of dystrophin, suggesting dystrophin is not an essential molecular fence component. Alternatively, viability may have been the major influence on protein and lipid diffusion in these fibres and suggestions are made as to how this may be recognised and overcome for subsequent investigation. Three novel exon specific anti-dystrophin peptide antibodies were generated during the work that will be useful for studies into Duchenne muscular dystrophy in general, and dystrophin revertant fibres in particular.