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

Abstract

The Drosophila M-line protein, obscurin (475 kD) has 21 tandem Ig domains, 3 Fn3 domains, a Rho-GEF signalling domain near the N-terminus and two kinase domains near the C-terminus. Obscurin is needed for the formation of a symmetrical sarcomere in the indirect flight muscle (IFM). Obscurin forms periodic striations in the IFM sarcomere 30 h after puparium formation, when kettin and myosin are still in unstructured strands and there are no Z-discs. Early expression of obscurin acts as a template for the formation of symmetrical thick filaments. Reducing expression by RNAi had no effect on sarcomere length in IFM, but the M-line was missing and the H-zone was shifted from the midline of the sarcomere. The length and polarity of thin filaments was determined by the position of the bare zone in adjacent thick filaments. Therefore obscurin is essential for the assembly of correctly overlapped thick and thin filaments in a symmetrical sarcomere. We have identified ligands of the two kinase domains in vivo. Kinase constructs with tags were injected into embryos and individual kinases with bound proteins were isolated from IFM. Kinase 1 bound Ball (an active kinase) and kinase 2 bound MASK (a 400 kD protein with ankyrin repeats). Reducing expression of these proteins by RNAi produced a phenotype in IFM that was similar to that produced by reducing obscurin, although obscurin itself was present at wild-type levels. Therefore Ball and MASK probably act downstream of obscurin. These proteins are linked via signalling pathways involved in the development of Drosophila muscle.