Myotilin cDNA has been cloned for the first time from chicken muscles and sequenced. Ectopically expressed chicken and human YFP-myotilin fusion proteins localized in avian muscle cells in the Z-bodies of premyofibrils and the Z-bands of mature myofibrils. Fluorescence recovery after photobleaching experiments demonstrated that chicken and human myotilin were equally dynamic with 100% mobile fraction in premyofibrils and Z-bands of mature myofibrils. Seven myotilin mutants cDNAs (S55F, S55I, T57I, S60C, S60F, S95I, R405K) with known muscular dystrophy association localized in mature myofibrils in the same way as normal myotilin without affecting the formation and maintenance of myofibrils. N- and C-terminal halves of human myotilin were cloned and expressed as YFP fusions in myotubes and cardiomyocytes. N-terminal myotilin (aa 1-250) localized weakly in Z-bands with a high level of unincorporated protein and no adverse effect on myofibril structure. C-terminal myotilin (aa 251-498) localized in Z-bands and in aggregates. Formation of aggregated C-terminal myotilin was accompanied by the loss of Z-band localization of C-terminal myotilin and partial or complete loss of alpha-actinin from the Z-bands. In regions of myotubes with high concentrations of myotilin aggregates there were no alpha-actinin positive Z-bands or organized F-actin. The dynamics of the C-terminal-myotilin and N-terminal myotilin fragments differed significantly from each other and from full-length myotilin. In contrast, no significant changes in dynamics were detected after expression in myotubes of myotilin mutants with single amino acid changes known to be associated with myopathies.

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