The M-Line Protein Obscurin in the early Development of Drosophila Flight Muscle

Abstract

Obscurin is a 475 kDa protein in the M-line of Drosophila muscles. The protein is made up of Ig and Fn3 domains, a Rho-GEF domain near the N-terminus and two kinase domains near the C-terminus. The expression of obscurin was reduced by a P-element insertion, or by RNAi. Knockdown of obscurin by RNAi was targeted to all muscles, or specifically to the flight muscle (IFM). In P-element mutants and RNAi lines, embryo, larva and pupa developed normally; adults could walk and jump, but were flightless. In the wild-type pupa, obscurin in the IFM appeared in striations at 30 hours after puparium formation, when kettin (a Z-disc protein) and myosin were in amorphous strands. Thus, obscurin in the M-line precedes the regular assembly of the Z-disc and A-band. In the IFM of flies with reduced obscurin, the sarcomere length was normal but the M-line was missing and H-zone irregular. Isolated thick filaments were asymmetrical with the bare zone shifted from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depended on the position of the bare zone in adjacent thick filaments. Thus, the early expression of obscurin nucleates the assembly of a symmetrical thick filament, which leads to thin filaments of uniform length. Ligands of the kinase domains are: ball (another kinase) binding to kinase 1 and MASK (an ankyrin-repeat protein) binding to kinase 2. Confirmation of these interactions by injecting embryos with kinase constructs will be described. The abnormalities in the IFM sarcomere in RNAi lines with reduced ball or MASK were similar to those in obscurin knockdown flies. The expression of obscurin was normal in RNAi lines of both ligands, demonstrating the importance of obscurin, ball and MASK to sarcomere assembly.