The Function of Obscurin in Regulating Assembly and Symmetry of the Sarcomere in Drosophila Flight Muscle

Abstract

Obscurin is a 400 kDa protein in the M-line of insect flight muscle (IFM). The protein has 21 Ig domains, two Fn3 domains and two kinase domains near the C-terminus. The effect of reducing the expression of obscurin on the structure of the sarcomere was investigated. A P-element inserted into the first intron of the gene resulted in reduced protein expression and homozygotes were unable to fly. Sarcomere length was normal, but the M-line was missing and the H-zone shifted from the middle of the sarcomere. Obscurin RNAi mutants had a similar but more extreme phenotype. Electron micrographs of mutant sarcomeres showed that thin filament length and the polarity of crossbridges was dependent on the position of bare zones in adjacent thick filaments: shifted bare zones resulted in abnormally short or long thin filaments. Isolated thick filaments were asymmetrical with displaced bare zones and in RNAi mutants, some thick filaments were up to three times the normal length. The kinase domains of obscurin are predicted to be inactive. Ligands binding to the two domains have been identified. Kinase 1 binds ball, itself a kinase, and kinase 2 binds MASK, which contains ankyrin repeats. MASK is present in both Z- and M-lines. RNAi mutants of ball and MASK had IFM with abnormal sarcomere structure: H-zones were shifted, thick filaments asymmetrical, and Z-discs irregular. Surprisingly, expression of obscurin was normal in RNAi mutants of both ligands, and MASK was present in obscurin RNAi mutants. Therefore, correct assembly of the sarcomere needs obscurin, ball and MASK. Obscurin is likely to form a scaffold binding ball and MASK. The two ligands are genetically linked to the same signalling pathway in the development of muscles in Drosophila.