Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5–7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M·ADP·Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP → M·ADP·Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by Ϸ20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M⅐ADP⅐P i ) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M ؉ ATP 3 M⅐ADP⅐P i . This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.Muscle contraction results from relative sliding between the thick and thin filaments driven by chemical energy liberated by ATP hydrolysis. In the crossbridge model of muscle contraction (1, 2), globular heads of myosin, i.e., the crossbridges extending from the thick filament, attach to actin in the thin filament and change their angle of attachment to actin (powerstroke), leading to filament sliding or force generation until they are detached from actin. Each attachment-detachment cycle between a myosin head and actin is coupled with hydrolysis of one ATP molecule. Despite extensive studies to detect the change in angle between the myosin head and the thin filament, however, there is no decisive evidence that the myosin head powerstroke is associated with the myosin head rotation (3, 4).A most straightforward way for studying the mechanism of muscle contraction may be to observe directly the movement of individual myosin heads on the thick filament under an electron microscope with sufficiently high magnifications. Though cellular functions, such as development, growth, and differentiation, are very readily impaired by electron beam irradiation (critical electron dose, 10 Ϫ9 Ϫ10 Ϫ5 C͞cm 2 ), crystalline structures of various biomolecules are known to be resistant to much higher electron doses (5). This indicates the possibility of studying dynamic structural changes of living biomolecules in an electron microscope, using a gas environmental (hydration) chamber (EC), a device to keep the specimen in wet state in an electron microscope (5). In fact, Fukushima...

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.More than 60 years have passed since the monumental discovery that muscle contraction results from relative sliding between actin and myosin filaments, coupled with ATP hydrolysis 1,2 . It is now widely accepted that the myofilament sliding is caused by cyclic attachment-detachment between the myosin heads extending from myosin filaments and the sites on actin filaments 3 . Concerning performance of the myosin heads in producing the myofilament sliding, it is generally believed that a myosin head first attaches to actin, change their structure, i.e. power stroke, to produce myofilament sliding, and then detaches from actin A most effective approach to the structural changes of a myosin head may be to determine the unitary distance of myofilament sliding caused by a myosin head power stroke. In the contraction model of A.F. Huxley and Simmons 6 , each myosin head is assumed to have elastic element, while the myofilaments are rigid; when a muscle fiber contracts isometrically, its isometric tension results from the tension in stretched elastic element, which resides in individual myosin heads. The amount of extension of myosin head elastic element is therefore equal to the amplitude of myosin head power stroke (or step size) in the isometric condition, and can be estimated from the amount of quick decrease in fiber length to drop the

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Although a contracting muscle regulates its energy output depending on the load imposed on it (''Fenn effect''), the mechanism underlying the load-dependent energy output remains obscure. To explore the possibility that the mechanical efficiency, with which chemical energy derived from ATP hydrolysis is converted into mechanical work, of individual myosin heads changes in a load-dependent manner, we examined the auxotonic shortening of glycerinated rabbit psoas muscle fibers, containing ATP molecules almost equal in number to the myosin heads, after laser-f lash photolysis of caged calcium. Immediately before laser-f lash activation, almost all of the myosin heads in the fiber are in the state M⅐ADP⅐P i , and can undergo only one ATP hydrolysis cycle after activation. When the fibers were activated to shorten under various auxotonic loads, the length, force, and power output changes were found to be scaled according to the auxotonic load. Both the power and energy outputs were maximal under a moderate auxotonic load. The amount of M⅐ADP⅐P i utilized at a time after activation was estimated from the amount of isometric force developed after interruption of fiber shortening. This amount was minimal in the isometric condition and increased nearly in proportion to the distance of fiber shortening. These results are taken as evidence that the efficiency of chemomechanical energy conversion in individual myosin heads changes in a loaddependent manner.In 1923, Fenn found that the total energy (heat ϩ work) liberated from a contracting muscle was greater when it lifted a load to do mechanical work than when it was held isometric (1). This phenomenon, called the Fenn effect, indicates that a muscle regulates its energy output depending on the load imposed on it. Although it has been well established that muscle contraction results from relative sliding between the thick and thin filaments, which in turn is caused by alternate formation and breaking of cross-links between the myosin heads on the thick filaments and the sites on the thin filaments, the mechanism underlying the Fenn effect remains obscure. It is also well established that ATP hydrolysis is the immediate source of energy for the myofilament sliding, and that the efficiency with which a muscle converts free energy derived from ATP hydrolysis into mechanical work changes depending on the load (2). The mechanical efficiency versus load relation is bell-shaped, because the efficiency is maximal at a moderate load and tends to zero when the load is greatly reduced or greatly increased. In the light of the above energetics studies, the Fenn effect is associated with load-dependent mechanical efficiency in contracting muscle fibers.In the contraction model of A. F. Huxley (3) and also in that of Podolsky and Nolan (4), the number of myosin heads involved in muscle mechanical response changes in a load-(or velocity-) dependent manner. In these models, load-dependent efficiency is somehow coupled with changes in the number of the myosin heads, each operating ...

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