We propose to continue systematic molecular genetic investigation of Drosophila muscle development and pathology, the broader goal being enhanced understanding of myopathies afflicting humans and domestic animals. During prior grant periods we isolated most Drosophila contractile protein genes and characterized muscle abnormalities caused by their mutant alleles. This work advanced our understanding of contractile proteins, illuminated fundamental aspects of myofibril assembly and function, and provided insights into the pathology of familial hypertrophic cardiomyopathy, nemaline myopathy, and Duchenne/Becker muscular dystrophies. During the next three years we intend to undertake five substantive projects. In the first we will investigate the causes of nemaline myopathy. Recently, a human TPM3 alpha-tropomyosin mutation was correlated with this syndrome, providing the first direct evidence that it is a sarcomeric disease. In order to confirm that this mutation causes dominantly inherited nemaline myopathy we will introduce it into transgenic Drosophila and examine resultant myofibrillar abnormalities. If nemaline rods form, then we will attempt to establish what tropomyosin properties confer the syndrome. In a second project we will characterize the gene that encodes tropomodulin, a protein that caps the pointed ends of actin filaments. Pointed end caps appear essential for thin filament length constancy, but the roles of the protein in myofibril assembly and maintenance are unknown and mutants would facilitate further investigating this interesting protein. In a third project we will characterize muscle-specific genes identified in recent enhancer trap screens in order to learn their roles in Drosophila myogenesis. Fourth, we will conduct a systematic search for P-element-induced mutants having myofibrillar perturbations. We will use polarized light microscopy and anti-myosin antibody decoration to recognize mutants having defective myofibrils. Since the locations of all contractile protein genes of Drosophila are known, this project will provide an opportunity to discover proteins having transient roles in myofibril assembly. Finally, we will complete the characterization of Drosophila actin and other contractile protein gene expression and use the corresponding promoters to construct ectopic expression vectors that will permit potentially myopathic gene mutations to be expressed within particular Drosophila muscles. Results of our investigation will improve our understanding of muscle cell biology and illuminate the bases of muscle cell dysfunction and degeneration, thus improving prospects for therapeutic interventions into muscle diseases.
