Description
Myosin, the molecular motor, interacts with actin filaments in the presence of ATP to produce muscle contraction. Mutations in the human embryonic myosin heavy chain cause Freeman Sheldon Syndrome (FSS), which is characterized by multiple congenital muscle contractures affecting facial and limb skeletal muscles. Structural analysis of myosin heavy chain reveals that most of the FSS mutations lie near the groove between the ATP binding site and actin binding site. These mutations are predicted to create structural changes in the ATP binding site, disrupting the binding of nucleotide to myosin. I hypothesize that Y583S and R672C mutant myosin molecules disrupt the structure and function of the indirect flight muscles of Drosophila. This dysfunctional myosin will permanently bind to actin in the myosin-ADP state, leading to permanent contractures. This will result in biochemical, structural and functional defects in the indirect flight and jump muscles of Drosophila. Our overall aim is to identify the structural and functional defects caused by FSS myosin mutations, using Drosophila melanogaster as the model organism. In vitro mutagenesis was performed to produce two myosin transgenes with the Y583S and R672C mutations. Lines containing transgenes were crossed into the indirect flight and jump muscle endogenous myosin null background to obviate the masking effect of wild-type myosin. Lines with near to wild-type expression of myosin were chosen to perform further studies. The transgenic flies showed a drastic reduction in their flight and jump ability when compared to controls indicating that sarcomere structure is compromised. Immunofluorescence confocal microscopy of the young transgenic flies showed disorganization of myofibrils. Electron microscopy of the indirect flight muscles of young Y583S transgenic flies showed thickening of Z-discs, fraying of myofibrils and diverging myofibrils, which indicates sarcomere disruption. A more severe phenotype was observed in R672C young transgenic flies. The complete loss of M-lines and Z-lines in the sarcomere shows that the sarcomere structure is completely compromised. ATPase and in vitro motility assays will help in understanding the effect of the mutations on the rate of ATP hydrolysis during the chemomechanical cycle and the ability of mutant myosin to translocate actin in the presence of ATP respectively. Overall, this model will yield insights into the mechanistic basis of FSS and may allow us to identify therapeutics to ameliorate FSS symptoms.
