9728868 Vigoreaux Lay Abstract The goal of this project is to determine the functional properties of flightin, a protein associated with the thick filaments of insect flight muscle. This protein is interesting because it may be involved in some of the special properties of this muscle, namely its very rapid beating. Unlike a typical vertebrate muscle cell which contracts and relaxes in a cycle coordinated with a rise and then fall in the concentration of intracellular calcium, insect flight muscle undergoes continuing rapid cycles of contraction and relaxation while the calcium concentration remains high. Flightin is found tightly associated with the myosin thick filaments of the insect cells. Flightin may simply stabilize the highly organized filament architecture in the cells or may be involved in a fundamentally different type of contraction control during the interaction of actin and myosin. Flightin or a relative may also be found in other muscle cell types where it may have very different or related functional properties. The project will address three specific questions to understand flightin function in the fruit fly, Drosophila melanogaster, chosen for its many advantages as a powerful model system. (1) Are mutant flight muscle cells that lack flightin less stable than normal cells when these cells contract? This would be expected if flightin plays a basic structural role to stabilize the highly ordered arrays of actin and myosin filament assemblies? (2) What is the precise way flightin interacts with the myosin filament? Detailed analysis of binding should elucidate how flightin might stabilize filament structure or control actin-myosin interaction since many details of the actin and myosin structures are already known. (3) What role does phosphorylation of the flightin molecule play in controlling its activity? Since the molecule can exist in phosphorylated and non-phosphorylated states, by analogy to other phosphorylated proteins whose state of phosphorylation c an be enzymatically controlled, it is very likely that these different states are also functionally different. The principal investigator, who was instrumental in the discovery of flightin, will purify flightin to study its binding properties, sites of phosphorylation, and other biochemical properties. He and his co-principal investigator will integrate results from a variety of other techniques and approaches, including electron microscopy, molecular and classical genetics, and physiological techniques, to provide clear and coherent answers to these questions. ***
