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Description
Hereditary inclusion-body myopathy type 3 (IBM-3) is caused by a single amino acid Glu706Lys substitution in the SH1 helix of the myosin head. The SH1 domain has been proposed to play a key role in the conformational changes that occur in the myosin head during force generation which is coupled to ATP hydrolysis. We are using an integrative approach to study the structure-function relationship of the myosin SH1 domain in the Drosophila model system We constructed a gene encoding myosin with the single amino acid mutation and expressed it in place of wild-type myosin heavy chain by germline transformation and crossing into a line that lacks myosin in its flight and jump muscles.. Our results suggest that the E699K mutation (corresponding the E706K in humans) in the SH1 helix of the myosin head severely affects myofibril structure and function in homozygous flies. Myofibrillar disarray is detected in E699K indirect flight muscle at 2 hours of age, and more severe defects are seen at 3 weeks. Some vacuoles are observed, as found in the human disease. The E699K ultrastructure data agree with the locomotion assay, as the E699K flies are unable to beat their wings and a wings-up phenotype was present in 2 day old flies. The jump ability of young flies was also severely impaired compared to controls. The mutation depressed calcium as well as basal and actin-activated MgATPase (V[lower case max]) by _ 75% compared to wild-type PwMhc2 myosin. Also, the mutation decreases in vitro motility of actin filaments by__ 80%. Thus, the homozygous E699K MHC mutation severely affects muscle structure and function in Drosophila. In the heterozygous condition, the myofibril structure shows normal thick and thin filament packing in young and older flies. The jumping ability of heterozygotes is increased significantly when compared with the homozygotes, but not as good as that of the control. The indirect flight muscle of heterozygotes cannot support flight. These results suggest that one copy of E699K with one copy of wild-type Mhc in the Mhc__ background affects myosin function but not IFM myofibril assembly and structure.
