ABSTRACT
Since the monumental discovery of sliding filament mechanism in muscle contraction, the mechanism of attachment-detachment cycle between myosin heads extending from myosin filaments and actin filaments has been the central object in research field of muscle contraction. As early as the 1980s,
we started to challenge electron microscopic visualization and recording of myosin head power stroke coupled with ATP hydrolysis, using the gas environmental chamber (EC), which enables us to observe myofilaments in wet, living state. In this chapter, we first explain historical background of our work, and then describe our experimental methods together with our findings, which can be summarized as follows: (1) the time-averaged position in individual myosin head does not change with time, indicating that they fluctuate around a definite equilibrium position; (2) In the absence of actin filaments, ATP-activated individual myosin heads move by ~7 nm at both distal and proximal regions in the direction away from the center of myosin filaments, indicating that myosin heads can perform recovery stroke without being guided by actin filaments; and (3) In the presence of actin filaments, ATP-activated individual myosin heads exhibit power stroke in nearly isometric condition, with the amplitude ~3.3 nm at distal region and ~2.5 nm at proximal region; (4) At low ionic strength, the amplitude of power stroke increases to >4 nm at both distal and proximal regions, being consistent with the report that the force generated by individual myosin heads increases approximately twofold at low ionic strength. Advantages of our methods over in vitro motility assay methods are discussed.
