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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...

In accord with a set of prespecified principles of cell synchrony induction, a three-step procedure was developed to arrest cells reversibly in the G2 phase of the cell cycle. Cultures of Chinese hamster ovary (CHO) cells were presynchronized in early S phase by sequential treatment with isoleucine deficiency and hydroxyurea blockades; then they were switched to medium supplemented with either of two agents that inhibit DNA topoisomerase II activity by different mechanisms, Hoechst 33342 at 7.5 ,Lg/ml for 12 hr or VM-26 at 0.5 ,gg/ml for 8 hr. Up to 95% of the cells accumulated in G2 phase under those conditions. After switch of Hoechst 33342-treated cells to drug-free medium, the cells divided as a highly synchronized cohort of cells within 3 hr. Up to 85% of the cells in a culture of human diploid dermal fibroblasts (HSF-55 cells) could be accumulated in G2 phase by placing cells presynchronized in early-S phase in medium containing Hoechst 33342 at 0.1 jug/ml for 10 hr. Reversal of G2 arrest in the HSF-55 cultures resulted in cells dividing sychronously over 3.5 hr. By varying the concentration of Hoechst 33342 and the duration of the treatment period, it was possible to alter the position within G2 phase at which cells accumulated. This synchronization protocol should greatly facilitate study of G2/M biochemical events in mammalian cells, in particular, those associated with cdc2 gene regulation of the onset of mitosis.Studies of events within the mammalian cell cycle are greatly facilitated by the use of highly synchronized populations in which all or most cells perform the same biochemical operations simultaneously. Among factors to be considered when using or developing a synchronization protocol are the following: (i) avoidance of conditions that force cells into a state of "biochemical imbalance" (1) in which the ratios of major macromolecules are grossly perturbed; (ii) assurance that the synchronization protocol is completely reversible; and (iii) recognition that the synchrony is rapidly lost (2, 3) to the extent that no single synchronization protocol is suitable for study of events throughout the entire cell cycle.In this manuscript, these principles of synchrony induction have been applied to the development of a protocol to arrest mammalian cells reversibly in G2 phase. The need for such populations stems from the recent rekindling of interest in studies of the cell cycle, attributable to the development of techniques for elucidating the mechanisms responsible for genetic regulation of cell proliferation. In particular, studies with a variety of biological systems, predominantly involving the lower eukaryotes, have revealed the existence of a series of cdc2-like genes shown to play a crucial role in regulating the initiation of mitosis (4-11). These studies are currently focusing on the molecular interactions between the proteins encoded by the cdc2 gene and cellular constituents that trigger the transition from G2 phase into mitosis.Such studies previously were difficult to do with mamma...

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