A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin VI

Rock, Ronald S.; Ramamurthy, Bhagavathi; Dunn, Alexander R.; Beccafico, Sara; Rami, Bhadresh R.; Morris, Carl; Spink, Benjamin J.; Franzini‐Armstrong, Clara; Spudich, James A.; Sweeney, H. Lee

doi:10.1016/j.molcel.2005.01.015

Cited by 92 publications

(134 citation statements)

References 30 publications

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“…Generally, these regulated forms are considered undesirable in mechanical studies, so tail truncations or substitutions are employed to activate the motors. However, note that our GCN4 dimerization domain is insufficient to force dimerization under the extremely low concentrations used here (28,31). Therefore, we know that additional regions of the native myosin X sequence must contribute to the dimerization as well.…”

Section: Structured Tail Domain Is Responsible For Bundle Selection Inmentioning

confidence: 89%

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Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Nagy

Rock

2010

Journal of Biological Chemistry

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Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses.The essential role of cytoskeletal motor proteins in organizing cellular compartments and cargoes is widely accepted (1). However, many of the molecular details of the overall transport and organization process are poorly understood. These essential features include how motors engage cargoes at their source, how motors are activated once they engage cargo, how they choose the correct set of cytoskeletal tracks, how they disengage from cargo at their destination, and how they return to sources of cargo. Clearly, errors at any of these stages would lead to faults in cellular organization and misplaced cargoes.To begin to address these questions we have focused on a particular motor protein, myosin X, due to its ability to navigate to a precise location within the cell (2). Myosin X is found at the mitotic and meiotic spindle, where it sets the orientation of the spindle relative to the substrate and influences spindle length (3, 4). However, myosin X is more commonly found in interphase cells at the tips of filopodia (5). These long, slender projections at the leading edge of migrating cells are involved in cell motility and environmental sensing. Myosin X transports components such as Ena/VASP and integrins that are found in the filopodial tip complex (2, 6). The early work by Berg et al. (5) demonstrated that myosin X reaches filopodial tips under its own power. Recently, we found that myosin X identifies filopodia by recognizing the fascin-bundled actin filaments found in the filopodial core (7). Myosin X takes long processive runs along these tightly bundled actin filaments while largely ignoring isolated actin filaments found throughout the cell. In contrast, myosin V does not distinguish between single actin filaments and bundled actin filaments when taking processive runs.Here, we seek to identify the "selectivity module" that allows myosin X to recognize bundled actin filaments. Our first approach is to swap similar domains between the selective myosin X and the nonselective myosin V. In this approach, we are aided by the fact that both m...

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Section: Structured Tail Domain Is Responsible For Bundle Selection Inmentioning

confidence: 89%

“…To observe further the behavior of this motor along a bundle, a more sensitive three-bead assay was used (19,28). We constructed a dumbbell using fascin-actin bundles and lowered it onto a surface-attached myosin X.…”

Section: Optical Trapping Reveals Shortmentioning

confidence: 99%

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Nagy

Rock

2010

Journal of Biological Chemistry

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“…Optical Trapping. Suspended filament assays on antibody-coated platforms were performed as described (10,26) by using a custombuilt, dual-beam optical trap. Actin dumbbells were held taut (at typical tensions of 1.5-2.5 pN) to minimize attenuation of stroke size due to series compliance.…”

Section: Methodsmentioning

confidence: 99%

“…The predicted coiled coil region also has surprising properties: the first Ϸ80 aa of this region (dubbed the ''proximal tail domain'') do not appear to form a stable coiled coil. Instead, the proximal tail forms an unknown flexible structure that contributes to the length of the stride (10). A model emerged in which the unique insert and IQ domain form a lever arm that strokes to provide an initial bias for the myosin VI stride, followed by a diffusive component facilitated by the proximal tail domain.…”

Section: A Long Stride and A Long Strokementioning

confidence: 99%

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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“…In Reifenberfer et al 1 , the variable step size is dismissed by postulating that each molecule walks straight, rarely taking sideways steps, and the overall distance distribution is broad due to contributions from different molecules. This argument is fallacious for at least four reasons: a) if each molecule has a fixed step size, then certain molecules would need short steps and others long ones, both of these groups of molecules exhibiting strong helical (not straight) motion 10,11 ; b) All individual recordings presented in papers from the authors' lab 1,4,9 and others 7,12 show variable steps; c) Binding sites for myosin VI to a filament on the glass allow a broad distribution of azimuths (α) (Fig. 3 of Sun et al 2 ), but the α distributions (Fig.…”

Section: Introductionmentioning

confidence: 99%

Myosin VI Lever Arm Rotation: Fixed or Variable?

et al. 2010

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Two recent articles 1,2 addressed the power-stroke of myosin VI molecules during stepping. Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is fixed 1 or variable 2 . Here we discuss the causes of the discrepancy between the two studies and the implications for myosin VI processive motility.Introduction: Myosin VI, an unconventional myosin motor that walks toward the minus ends of actin filaments, has received great attention recently. Reifenberger et al. 1 have presented evidence from Defocused Orientation and Positional Imaging (DOPI) assays that myosin VI lever arms tilt by an angle very close to 180° on each step. This contrasts with results from Sun et al. 2 wherein we applied single molecule Polarized Total Internal Reflection Fluorescence (polTIRF) microscopy and found variable degrees of tilting both in the axial and azimuthal directions relative to actin. This variability is consistent with the known variable step size of myosin VI. There are several technical differences in the two papers that interested researchers should understand, and more importantly, substantive differences in the conclusions.

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A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin VI

Cited by 92 publications

References 30 publications

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

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

Myosin VI Lever Arm Rotation: Fixed or Variable?

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