The Role of Flightin in Thick Filament Assembly and Flight Muscle Mechanics

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Striated muscle is a remarkable tissue. Within the cytoplasm of each muscle cell is a highly organized cytoskeletal lattice where actin and myosin are assembled in perfectly ordered, interdigitating arrays of thin and thick filaments. The architecture of the sarcomere is extraordinarily well designed to generate force for work and locomotion, as evidenced by its strong evolutionary conservation across animals of many phyla. The way in which myosin motors assemble into bipolar filaments of specific and uniform length, a requirement for proper muscle function, has long been a topic of great interest but few concrete answers have been found. An important goal of this research is to address this fundamental question in muscle biology through theinvestigation of the functional properties of Drosophila flightin. Previous work from this lab has shown that Drosophila indirect flight muscle (IFM) thick filaments assemble to abnormally long lengths in the absence of flightin. This project will combine in vivo and in vitro approaches to establish the mechanism by which flightin participates in thick filament length determination during IFM development. A central thesis of this work is that flightin is a myosin binding protein. The first aim will be to test the hypothesis that flightin binds to two sites in the myosin rod region. Recombinant fragments representing distinct parts of the myosin heavy chain protein will be tested for flightin binding using four different biochemical assays. The second aim will be to explore the role of flightin phosphorylation in thick filament assembly. Competing hypotheses, that phosphorylation is or is not required for proper assembly, will be tested using transgenic flies that express phosphorylation-site mutant flightin. Electron microscopy will be used to assess the effect of mutant flightin on sarcomere and thick filament length. These studies will be accompanied by experiments in vitro that will test the effect of flightin on myosin polymerization.The combination of classical and molecular genetics with biochemistry, cell structure and physiology/biophysics will result in a coherent, integrated view of the functional roles of flightin and provide valuable insight into the mechanism of thick filament assembly and the mechanism of stretch activation.