P.P.6 09 Damage-induced dysferlin translocation in C2C12 myotubes requires the full length protein

Abstract

Recently, a novel pathomechanism, defective repair of the plasma membrane, was demonstrated in dysferlin deficient myofibres. Dysferlin is the protein involved in limb-girdle MD type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. So in dysferlinopathy it appears that rather than an inherent problem with the structural integrity of the plasma membrane, it is a defect in its maintenance process which is disease causing. Dysferlin is a homologue of the Caenorhabditis elegans fer-1 gene which mediates vesicle fusion in spermatids. It is characterised by the presence of six C2 domains which are known to be involved in vesicle trafficking and membrane fusion events. To investigate the role of dysferlin in membrane maintenance and its subcellular expression pattern in response to membrane damage, we established two in vivo wounding assays in differentiated C2C12 myotubes: (a) glass beads were dispersed on a monolayer of cells in the presence of a Texas Red conjugated dextran. These 10 kDa dextran molecules are excluded from cells under normal circumstances, but dispersion of beads leads to breaches in the myotube membranes which can be identified by intracellular uptake of fluorescing dextran. (b) A UV nitrogen laser was utilised to create membrane disruptions on single C2C12 myotubes. GFP fusion constructs coding for full length dysferlin, truncated dysferlin mutants coding for distinct C2 domains, caveolin 3 and a Golgi marker were transiently transfected into C2C12 cells and their cellular localisation identified via immunofluorescence in response to the different methods of membrane damage. Both wounding assays showed distinct translocation of full length dysferlin to the plasma membrane after injury. This translocation was dependent on the expression of all C2 domains of dysferlin and the presence of extracellular calcium. Translocation of dysferlin to the plasma membrane in C2C12 myotubes supports a role for dysferlin in plasma membrane maintenance. This response requires the presence of the full length protein and extracellular calcium.