Class VI Myosin Moves Processively along Actin Filaments Backward with Large Steps

Nishikawa, So; Homma, Kazuaki; Komori, Yasunori; Iwaki, Mitsuhiro; Wazawa, Tetsuichi; Iwone, Atsuko Hikikoshi; Saito, Junya; Ikebe, Reiko; Katayama, Eisaku; Yanagida, Toshio; Ikebe, Mitsuo

doi:10.1006/bbrc.2001.6142

Cited by 161 publications

(172 citation statements)

References 31 publications

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“…A large stroke size is probably essential to the processive stepping mechanism of dimeric myosin VI, which moves along actin with Ϸ36-nm strides, matching the pseudorepeat of the actin helix (4,5). Unlike myosin V, myosin VI has not used multiple IQ repeats to achieve a large stroke.…”

Section: Discussionmentioning

confidence: 99%

“…A new puzzle arose when the stepping behavior of dimeric myosin VI was characterized by using single molecule techniques (4,5). Myosin VI, like myosin V, walks processively along actin by using a long stride that approximately matches the Ϸ36-nm pseudorepeat of the actin filament.…”

Section: A Long Stride and A Long Strokementioning

confidence: 99%

“…Class VI myosins are highly specialized (Ϫ)-end-directed motors involved in a growing list of functions in animal cells, including endocytosis, cell migration, and maintenance of stereociliar membrane tension (2). Initial biophysical characterization (3)(4)(5) of myosin VI raised two important questions concerning the structural basis of its unique motor properties. First, how does the motor achieve reverse directionality?…”

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confidence: 99%

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The power stroke of myosin VI and the basis of reverse directionality

Bryant

Altman

Spudich

2007

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

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Myosin VI supports movement toward the (؊) end of actin filaments, despite sharing extensive sequence and structural homology with (؉)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120°redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Mé né trey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005) Nature 435:779 -785]. However, this redirection alone is insufficient to account for the large (؊)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (؉)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (؉)-end-directed motion, whereas longer constructs move toward the (؊) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (؊) end. We suggest that the lever arm rotates Ϸ180°between pre-and poststroke conformations.actin ͉ molecular motor ͉ pointed end ͉ swinging cross-bridge T he basic actomyosin motor has been embellished, altered, and reused many times through the evolution of diverse members of the myosin superfamily (1). Class VI myosins are highly specialized (Ϫ)-end-directed motors involved in a growing list of functions in animal cells, including endocytosis, cell migration, and maintenance of stereociliar membrane tension (2). Initial biophysical characterization (3-5) of myosin VI raised two important questions concerning the structural basis of its unique motor properties. First, how does the motor achieve reverse directionality? And second, how does dimeric myosin VI take long steps along actin, matching the stride of myosin V without the apparent benefit of a long lever arm?A Backward-Moving Myosin Myosin VI attracted attention as a candidate for a (Ϫ)-enddirected motor after the identification of a class-specific insert following the converter domain. In the swinging cross-bridge model of actomyosin function, the converter domain transmits motion from the myosin head to the C-terminal lever arm, generating a directed power stroke. Wells et al. (3) hypothesized that a structure inserted between the converter domain and the lever arm could redirect the stroke and lead to backward movement. The predicted reverse directionality of myosin VI was experimentally confirmed (3), but the precise mechanism of this remarkable adaptation remained unclear, including whether the unique insert was necess...

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Section: Discussionmentioning

confidence: 99%

Section: A Long Stride and A Long Strokementioning

confidence: 99%

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confidence: 99%

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The power stroke of myosin VI and the basis of reverse directionality

Bryant

Altman

Spudich

2007

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

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“…Nishikawa et al observed that myosin VI heads attach to neighboring actin monomers, which never occurs in any intermediates of the hand-over-hand mechanism. 45 2. Hua et al performed microtubule gliding assay with conventional kinesin.…”

Section: The Challengementioning

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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“…(These correspond to residues C278-A303 and P774-Y812, respectively and are called insert-1 and insert-2 hereafter.) Single molecules of dimeric myosin VI, like myosin V, are capable of taking multiple steps (processive movement) of 30-36nm via a hand-over-hand mechanism along actin filaments 11,12 . This large step size is surprising within the context of the lever arm hypothesis, in that myosin VI binds only two calmodulins per head 13 , while myosin V binds six.…”

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The structure of the myosin VI motor reveals the mechanism of directionality reversal

Menetrey

Bahloul

Wells

et al. 2005

Nature

205

234

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We have solved a 2.4Å structure of a truncated version of the reverse direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulins. The structure reveals only minor differences in the motor domain as compared to plus-end directed myosins, with the exception of two unique inserts. The first insert is near the nucleotide-binding pocket, and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a previously unseen binding motif within the insert. This serves to redirect the effective "lever arm" of myosin VI, which includes a second calmodulin bound to an "IQ motif," towards the pointed (−) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to fully explain the movements of myosin VI.The myosin superfamily is composed of eighteen classes of molecular motor proteins, the vast majority of which traffic toward the barbed (+) end of actin filaments 1 . Class VI myosins were the first of the superfamily identified to traffic toward the pointed (−) end of the actin filament 2 . They function in a number of critical intracellular processes such as vesicular membrane traffic, cell migration, maintenance of stereocilia and mitosis [3][4][5][6] .The current view of how myosin motors couple ATP hydrolysis and actin binding to movement is known as the lever arm hypothesis 7 . In essence the proposed mechanism is that nucleotide binding, hydrolysis and product release are all coupled to small movements within the myosin motor core. These movements are amplified and transmitted via a region that has been termed the "converter" domain to a lever arm consisting of a target helix and associated light chains/ calmodulins. The lever arm further amplifies the motions of the converter domain into large directed movements. Consistent with the lever arm hypothesis, the stroke size has been shown to be proportional to the lever arm length 8,9,10 . In the absence of actin, ATP hydrolysis occurs, but product release is slow, thus trapping the lever arm in a primed or pre-powerstroke position.Correspondence should be addressed to either: Anne Houdusse, Structural Motility, UMR 144 Institut Curie-CNRS, 26 rue d'ULM, 75248 Paris cedex 05, France,, Anne.Houdusse@curie.fr or H. Lee Sweeney, Dept. of Physiology, University of Penn., A700 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6085, Tel. 215-898-8727, Fax. 215-573-2273, Lsweeney@mail.med.upenn.edu Binding to actin causes release of products, plus-end-directed movement of the lever arm, and force generation concomitant with formation of strong binding between myosin and actin.Perhaps because of their reverse directionality, myosin VI motors have a number of additional unusual features. First, the motor domain itself contains two inserts that are unique within...

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Class VI Myosin Moves Processively along Actin Filaments Backward with Large Steps

Cited by 161 publications

References 31 publications

The power stroke of myosin VI and the basis of reverse directionality

The power stroke of myosin VI and the basis of reverse directionality

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

The structure of the myosin VI motor reveals the mechanism of directionality reversal

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