Single–motor mechanics and models of the myosin motor

Yanagida, Toshio; Esaki, S.; Iwane, Atsuko H.; Inoue, Yuichi; Ishijima, Akihiko; Kitamura, Kazuhiro; Tanaka, Hiroto; Tokunaga, Makio

doi:10.1098/rstb.2000.0585

Cited by 48 publications

(41 citation statements)

References 31 publications

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“…However, it is apparent that this model could not be applied to the movement of one-head myosin-V because the net working stroke of one head should be 32 nm for producing successive 32-nm steps by one head. We propose a possible model based on a strain sensor mechanism (22) in Fig. 4.…”

Section: Discussionmentioning

confidence: 99%

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

Watanabe

Tanaka

Iwane

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large (Ϸ36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP-and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (Ϸ32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.C lass V myosin (myosin-V) is a linear motor protein that moves unidirectionally along an actin filament with successive large (Ϸ36-nm) steps (1-3). Myosin-V has two heads, each of which consists of a motor domain and a long neck domain. Based on this structural feature, a ''hand-over-hand'' mechanism has been proposed to explain a unidirectional and successive large stepping of myosin-V (4). The proposed mechanism is that the trailing head detaches upon ATP binding, and rapidly moves forward by Ϸ72 nm (5). Upon reattachment with actin, it becomes the new leading head. The partner and former leading head remains attached with the same actin monomer. Based on a ''lever-arm tilting model'' (6), it has been proposed that the tilting of the long neck domain of the attached leading head biases Brownian motion of the trailing head forward (7). Recently, it was demonstrated by using a single-molecule f luorescence polarization technique that the orientation of f luorophores on calmodulin light chain attached to the neck domain of myosin-V indeed alters before and after mechanical steps (8). This finding is consistent with the lever-arm tilting model. However, it is still unclear whether the observed tilting of the neck domain may not be the cause of steps but rather the effect of steps generated by other mechanisms. This uncertainty is because the time resolution of the f luorescence polarization was not sufficient to detect the change directly coupled to these steps.On the other hand, we have recently reported that a short (Ϸ4-nm) neck mutant of myosin-V can also generate successive large (Ϸ35-nm) steps (9). That observation poses a challenge to the lever-arm tilting model because the neck domain is too short to lead the trailing head to the next target site Ϸ72 nm distant from the original site, especially at high loads. In support of this finding, class-VI myosin, which has a naturally very short (Ϸ4-nm) neck domain, has been found to move processively with large steps as well as myosin-V (10, 11).A central issue for the multiple successive large steps according to the hand-over-hand mechanism based on the lever-arm tilting model is that the two-headed structure is indispensable. P...

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Section: Discussionmentioning

confidence: 99%

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

Watanabe

Tanaka

Iwane

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…10 and 11); and step size is an integer multiple of the actin-monomer spacing. This is true both in the isolated molecular system, where myosin molecules translate along actin [74,79] and in the intact sarcomere, where thin filaments translate past thick filaments [58,75]. In the latter experiments, the striated image of a single my-ofibril is projected onto a photodiode array.…”

Section: Muscle Contractionmentioning

confidence: 99%

Is the Cell a Gel-and Why Does It Matter?

Pollack

2001

JJP

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It has been long recognized that the cytoplasm is a gel, even before the classic book by Frey-Wyssling [1]. Textbooks continue to emphasize the cytoplasm's gel-like consistency, and several groups have recently gone on to describe the functional relevance of the gel-sol transition [2,3]. Researchers working on actin filaments in particular have focused on the cytoplasm's gel-like behavior [4], as have some groups concerned with cellular metabolism [5]. There can be little doubt that the cytoplasm has the consistency of a gel.Yet virtually all cell biological mechanisms build on the principle of free diffusion in aqueous solution. One merely needs to examine representative mechanistic pathways to note the many diffusional steps required in proceeding from stimulus to action. These steps invariably include: ions diffusing into and out of channels; ions diffusing into and out of pumps; ions diffusing through the cytoplasm; proteins diffusing toward other proteins; and substrates diffusing toward enzymes. A cascade of multiple diffusional steps underlies nearly every intracellular process-notwithstanding the cytoplasm's character as a gel, where diffusion can be extremely slow.Surrounding this aqueous intracellular "solution" is the impermeant cell membrane, which restricts the passage of ions and other solutes to flow largely through an array of channels and pumps. Well over 100 solute-specific channels have been identified, with new ones emerging regularly. The same goes for pumps: Since ion concentrations inside and outside the cell are rarely in electrochemical equilibrium, the observed intracellular concentrations are maintained by active pumping. For pump and channel systems to operate, ions require free access.These concepts are certainly familiar to most readers of this journal. For others, the text by Alberts et al.[6] provides a detailed review of this foundational paradigm-along with the manner in which this paradigm accounts for basic cell function. Key words: phase-transition, polymer gel, cell, structured water, cooperativity, movement.Abstract: That the cell is a gel is broadly acknowledged. Textbooks begin with this assertion-and then proceed with great abandon to derive mechanisms based on free diffusion, as though the gel concept were groundless and cell was an aqueous solution. This disconnect emerges in part because the behavior of gels is not well understood, particularly among most biologists. Recently, great strides have been made in the understanding of gel behavior. It has become clear, for example, that a central mechanism in gel function is the phase-transition-a qualitative structural change prompted by a subtle change of environment, not unlike the transition from ice to water. Phase-transitions are capable of doing work. If the cell is a gel, then a logical approach to understanding cell function is to understand gel function-especially whether some role may be played by the phase-transition. Here we pursue this approach. We first consider the dichotomy of the cell as a gel and the cell...

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“…Hence, the contractile machinery apparently executes steps not only of 2.7 nm but also of 5.4 nm and of larger size as well. The capacity to generate such larger steps is also seen in various single molecule experiments (10,13,15,21). It implies a paradoxical combination of deterministic and stochastic components: a component that produces steps that are exact integer multiples of 2.7 nm, n ϫ 2.7 nm, and a component that determines the value of n for each step, seemingly unpredictably, although no systematic attempt has been made to determine whether the size sequence might follow some subtle pattern.…”

Section: Discussionmentioning

confidence: 99%

“…

(10,21). We have carried out parallel measurements on single myofibrils from rabbit cardiac muscle and bumblebee flight muscle.

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mentioning

confidence: 99%

“…Although measurements in the past have yielded steps of broadly variable size, possibly because of challenging analytical obstacles (8,14,17), recent experiments with improved signals have shown steps on the order of 5-6 nm (20), and experiments on single myosin molecules translating along actin filaments have shown step size consistently of ϳ5.4 nm (10, 21). The value 5.4 nm is equal to the monomer repeat along a single actin strand (10,13,15,20,21).…”

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confidence: 99%

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Fundamental step size in single cardiac and skeletal sarcomeres

Yakovenko

Blyakhman

Pollack

2002

American Journal of Physiology-Cell Physiology

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(10,21). We have carried out parallel measurements on single myofibrils from rabbit cardiac muscle and bumblebee flight muscle. Activated specimens were released or stretched with a motorimposed ramp, and the time course of length of individual sarcomeres was measured by projecting the image of the striations onto a linear photodiode array and tracking the spacing between A-band centroids. We confirmed the 5.4-nm step. With subnanometer precision, however, we find that this value is two times that of a more fundamental step size of 2.7 nm.Step sizes were always integer multiples of 2.7 nm, whether the length change was positive or negative. This value is equal to the linear repeat of actin monomers along the thin filament, a result that ties dynamic events to molecular structure and places narrow constraints on any proposed molecular mechanism. single myofibril; actin; optical microscopy; quantal shortening IN APPROACHING THE MECHANISM of motility and contraction, attention has turned toward the elementary molecular translation step. Although measurements in the past have yielded steps of broadly variable size, possibly because of challenging analytical obstacles (8,14,17), recent experiments with improved signals have shown steps on the order of 5-6 nm (20), and experiments on single myosin molecules translating along actin filaments have shown step size consistently of ϳ5.4 nm (10, 21). The value 5.4 nm is equal to the monomer repeat along a single actin strand (10,13,15,20,21).We have carried out parallel measurements on single sarcomeres. The single sarcomere model is useful because it preserves the natural filament lattice, yet cooperativity assures that molecular translation steps are not obscured (19). Previous myofibril experiments have shown that activated sarcomere shortening occurred in steps that were integer multiples of 2.7 nm (2). Because of noise, however, detectability was limited to steps larger than 4-5 nm, so we could not establish whether the implied 2.7-nm quantum actually existed.In the present experiments, we employed a differentially based algorithm that could suppress noise contributions sufficiently to bring the detection limit to subnanometer levels (18). With this high-resolution algorithm, we measured steps in activated sarcomeres both during shortening and during imposed stretch. Although the 5.4-nm step seen in single molecule studies is confirmed in this study, the myofibril reveals a more fundamental step size of half that value, or 2.7 nm. METHODS Myofibril PreparationIsolated myofibrils were prepared from rabbit left ventricular trabecular muscles as described previously (12). Briefly, thin strips of muscle tissue were dissected for storage in rigor/glycerol solution (50/50 by volume) for a minimum of 5 days at Ϫ20°C. To obtain single myofibrils, glycerinated strips were minced, and the pieces were further skinned in a 4°C rigor solution containing 0.5% Triton X-100 for 30 min. After being washed with fresh rigor solution, the tissue pieces were homogenized in a blender (Sorv...

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Single–motor mechanics and models of the myosin motor

Cited by 48 publications

References 31 publications

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

A one-headed class V myosin molecule develops multiple large (≈32-nm) steps successively

Is the Cell a Gel-and Why Does It Matter?

Fundamental step size in single cardiac and skeletal sarcomeres

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