Key Points
- Characterize the kinetics of cross-bridge detachment, reattachment, and force generation in insect fibrillar flight muscle fibers.
- Applied laser pulse photolysis to release caged ATP (100 µM to 1 mM) and caged inorganic phosphate (Pi) inside single glycerol-extracted insect flight muscle fibers.
- Measured mechanical tension transients under differing calcium concentrations (absence to ~30 µM Ca2+), Pi levels (up to 10 mM), and mechanical step stretches.
- ATP-mediated detachment of rigor cross-bridges proceeded with an apparent second-order rate constant of 5 × 10^4 to 2 × 10^5 M^-1s^-1, insensitive to Ca2+, Pi, or initial rigor tension.
- Cross-bridges exhibited transient reattachment after detachment, with tension recovery kinetics indicating that reattachment into force-generating states depends on filament lattice strain.
- Elevated inorganic phosphate (10 mM) suppressed transient reattachment and stretch activation, while photolysis of caged Pi during stretch activation abruptly terminated tension generation.
Structured PICO
PPopulationSingle glycerol-extracted fibers of insect fibrillar flight muscle
IInterventionLaser pulse photolysis of caged ATP (yielding 100 microM-1 mM ATP) and caged inorganic phosphate (Pi)
OOutcomeKinetics of the cross-bridge cycle (rate constant for detachment of rigor cross-bridges by ATP)surrogate
Photolysis of caged ATP and Pi in insect flight muscle fibers demonstrates that the proportion of myosin cross-bridges reattaching into force-generating states depends on filament lattice stress, and supports a linkage between Pi release and force production.