Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

Forkey, Joseph N.; Quinlan, Margot E.; Shaw, Michael; Corrie, John E. T.; Goldman, Yale E.

doi:10.1038/nature01529

Cited by 448 publications

(445 citation statements)

References 42 publications

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“…17,18 This domain likely acts as a lever arm that amplifies small nucleotidedependent conformational changes in the motor domain to a large displacement. 19,20 Myosin V walks toward the plus end of actin by taking ∼37 nm steps per ATP hydrolyzed. 21,22 Myosin V transports a wide variety of cargo including pigment granules, membranous organelles and secretory vesicles in vertebrates, and mRNA in yeast.…”

Section: Molecular Motorsmentioning

confidence: 99%

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

2005

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We introduce the technique of FIONA, fluorescence imaging with one nanometer accuracy. This is a fluorescence technique that is able to localize the position of a single dye within ∼1 nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit. We motivate the development of FIONA by unraveling the walking mechanism of the molecular motors myosin V, myosin VI, and kinesin. We find that they all walk in a hand-over-hand fashion.

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Section: Molecular Motorsmentioning

confidence: 99%

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

2005

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“…A crystal structure and electron micrographs have suggested the lever-arm tilts in conjunction with ATP hydrolysis cycle 12,13 , leading to the model assumes that the 'lever-arm swing' is the dominant process for force generation 14,15 . However, recent single-molecule nano-imaging and high-speed atomic force microscopy (AFM) have revealed that the two head domains alternately step 72 nm (hand-over-hand mechanism) [16][17][18] by combining the lever-arm swing 19 by the lead head with a 'diffusional Brownian search' by the rear head 17,20 . In general, the lever-arm swing is responsible for force generation and triggering the Brownian search following detachment of the rear head.…”

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Switching of myosin-V motion between the lever-arm swing and Brownian search-and-catch

et al. 2012

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motor proteins are force-generating nanomachines that are highly adaptable to their everchanging biological environments and have a high energy conversion efficiency. Here we constructed an imaging system that uses optical tweezers and a DnA handle to visualize elementary mechanical processes of a nanomachine under load. We apply our system to myosin-V, a well-known motor protein that takes 72 nm 'hand-over-hand' steps composed of a 'lever-arm swing' and a 'Brownian search-and-catch'. We find that the lever-arm swing generates a large proportion of the force at low load ( < 0.5 pn), resulting in 3 k B T of work. At high load (1.9 pn), however, the contribution of the Brownian search-and-catch increases to dominate, reaching 13 k B T of work. We believe the ability to switch between these two forcegeneration modes facilitates myosin-V function at high efficiency while operating in a dynamic intracellular environment.1 Soft Biosystem Group, Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. 2 Graduate School of Medicine, Osaka University, 1-3 Yamadaoka, Suita, 565-0871, Japan. switching of myosin-V motion between the lever-arm swing and Brownian search-and-catch

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“…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.…”

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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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Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

Cited by 448 publications

References 42 publications

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

Switching of myosin-V motion between the lever-arm swing and Brownian search-and-catch

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

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