Molecular genetic analysis of Drosophila melanogaster paramyosin in muscle development, structure, and function

Paramyosin is a major structural component of invertebrate muscles. It forms thick filament cores with other proteins. The motor protein myosin assembles on the surface of the core to form functional thick filaments. To investigate the roles of paramyosin in thick filament assembly as well as muscle contraction, I functionally knocked out the Drosophila melanogaster paramyosin gene by mobilizing a P element localized in the promoter region. I found that homozygous paramyosin mutants die at the embryo stage. Using electron microscopy and confocal microscopy, I analyzed the phenotypic defects of a functional null allele prm1. I observed that, in the absence of paramyosin, thick filaments of embryo body wall muscles are abnormal and the striated pattern of myobibrils is disrupted. These results indicate that paramyosin is essential for thick filament assembly and myofibril formation. Surprisingly, the muscle pattern of paramyosin mutant embryos is also abnormal. Mutation of paramyosin causes random losses of muscle fibers. Using a marker for founder myoblasts and the DMEF2 antibody which recognizes all myoblasts, I proved that the muscle fiber loss is not due to defects in myoblast differentiation. Rather, it is caused by abnomal myoblast fusion. Using a paramyosin specific antibody, I revealed that paramyosin functions as a cytoplasmic protein before myofibril formation and is important for myoblast fusion.; I further investigated the function of paramyosin phosphorylation in the NH₂-terminal non-helical domain. I made transgenic flies in which 1, 3, or 4 phosphorylatable serine residues in this domain are substituted with alanines. I observed that mutations of paramyosin at these residues do not affect the ultrastructure of myofibrils. However, mutant flies with substitution at some specific sites are flight impaired. Mechanical studies of indirect flight muscle fibers revealed that the flight impairment is caused by reduced fiber stiffness and power output. These results indicate that paramyosin phosphorylation in the NH₂-terminal domain is important for muscle contraction.