Obscurin, A Large Modular Protein, Regulating Sarcomere Formation In Drosophila Muscle

Abstract

Drosophila obscurin is a modular muscle protein of ∼ 420 kDa. The sequence, derived from the genome, contains two C-terminal serine-threonine kinases, both of which are predicted to be inactive, as well as 21 Ig and two Fn3 domains. A Rho/GEF domain has been identified in the N-terminal region. There are four obscurin isoforms, two of which are exclusively expressed in the indirect flight muscle (IFM). Obscurin is in the M-line throughout IFM- development and in the adult fly. In Drosophila IFM, the protein is across the M-line, unlike the vertebrate, where obscurin is at the periphery of the myofibril. A P-element insertion in the first intron of the gene leads to severely reduced obscurin protein levels and a flightless phenotype in homozygous mutant flies. Myofibrils are thinner and electron microscopy shows a disrupted M-line and shifted H-zones. This phenotype was rescued by precise P-element excision using a transgene fly stock carrying a transposase. Non-flight muscles are not affected by the mutation. Obscurin RNAi lines driven with an IFM specific Gal4 driver lead to a flightless phenotype, and the specific reduction of obscurin IFM isoforms. Electron microscopy shows the phenotype is more severe than in the P-element mutant. Co-immunoprecipitation showed that obscurin is associated with myosin. It is likely that obscurin is needed for normal alignment and symmetry of thick filaments. In yeast two-hybrid screens, a 400 kDa protein, MASK, was identified as a binding partner of obscurin kinase 2. MASK co-localises with obscurin in the M-line. MASK RNAi lines show a flightless phenotype. A possible binding partner for obscurin kinase 1 is ball, a kinase of unknown specificity. MASK and ball can both be linked to signalling pathways involved in muscle development.