INSECT FLIGHT MUSCLE HOT, COLD, PULLED AND PULLING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our program combines & compares X-ray diffraction (glycerinated native fibers) and thin-section EM tomography (fibers quick-frozen & freeze-substituted) to characterize structure, arrangement and dynamic choreography of myosin crossbridges (motor molecules of muscle) in a waterbug insect flight muscle (IFM) of unexcelled crystalline regularity. All X-ray experiments proposed will, whenever revealing, be matched by parallel EM experiments on fibers quick-frozen during the same state or maneuver, to directly image the structural changes.) X-ray patterns from IFM differ in important ways from those of other striated muscles (frog skeletal) studied by XRD, owing to differences in filament lattice packing, in thick filament helical geometry, and a full-overlap sarcomere scheme limited to ~3% length changes. X-ray reflections (to ~5 nm) attributable to myosin filaments, actin filaments, and both jointly, are as well worked out as in frog muscle, but not yet so well studied in dynamic (contraction) states. The present proposal is to continue our novel recent work on crossbridge braking actions, and to begin time-resolved perturbations of active IFM planned with members of Vincenzo Lombardi's lab. This present work will continue analysis to 4 nm and higher of muscle dynamic structural responses to relaxed, rigor, and active states, to temperatures 1 to 40¿ C (IFM requires preflight warm up to ~40¿C to enable flight), to rapid stretches that elicit either stretch-activation or crossbridge braking (depending on muscle state), to isometric calcium-activation versus cyclic stretch-activation, and to stretch/release protocols that probe filament elasticity and load-dependent crossbridge distortion.