Two-headed binding of a processive myosin to F-actin

Walker, Matthew; Burgess, Simon; Sellers, James R.; Wang, Fei; Hammer, John A.; Trinick, John; Knight, Peter J.

doi:10.1038/35015592

Cited by 288 publications

(290 citation statements)

References 23 publications

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“…The extended neck allows the myosin V to take long steps that match the pseudo-repeat of the actin filament (5,7,8). In doing so, myosin V should keep its cargo positioned above the cytoskeleton while stepping from one actin filament crossover point to the next.…”

Section: Discussionmentioning

confidence: 99%

“…Experiments using optical trapping nanometry, electron microscopy, and single molecule motility studies demonstrated that myosin V moves processively along actin filaments, taking many 36-nm steps before dissociating (5)(6)(7)(8). Kinetic analyses demonstrated that it is a high duty cycle motor that associates strongly with actin in the presence of physiological concentrations of ATP (9).…”

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confidence: 99%

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Neck Length and Processivity of Myosin V

Sakamoto

Wang

Schmitz³

et al. 2003

Journal of Biological Chemistry

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147

146

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Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 M or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.The myosin superfamily consists of at least 18 classes of actin-dependent molecular motors (1). Class V myosins transport cargos such as endoplasmic reticulum in neurons, melanosomes in melanocytes, and mRNA in yeast (2). Similar to other myosins, they are composed of a head that binds ATP and actin and a neck region consisting of calmodulin (CaM) 1 molecules bound to an ␣-helical segment of the myosin heavy chain. The C-terminal tail domain of myosin V has coiled-coil forming motifs that dimerize and create two-headed molecules but do not self-associate into filamentous structures. The IQ motifs of the heavy chain, implicated in the binding of CaM or light chain subunits, have the consensus sequence, IQXXXRGXXXR, where X is any amino acid (3). The neck of mouse myosin V has six IQ motifs, each of which binds CaM, making its neck longer than that of most myosins (4). The six IQ motifs of all myosin V superfamily members are separated by 25-23-25-23-25 amino acids.The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term "low duty cycle" motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpede...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Neck Length and Processivity of Myosin V

Sakamoto

Wang

Schmitz³

et al. 2003

Journal of Biological Chemistry

Self Cite

147

146

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“…To generate ADP †Vi molecules, 100 mM ATP and 50 mM sodium orthovanadate were added at 4 8C for 30 min 38 . The specimen was applied to carbon grids treated with ultraviolet light, as described 39 . Before staining with 1% uranyl acetate 39 , grids were rinsed with MMED buffer without KCl to improve staining.…”

Section: Methodsmentioning

confidence: 99%

“…The specimen was applied to carbon grids treated with ultraviolet light, as described 39 . Before staining with 1% uranyl acetate 39 , grids were rinsed with MMED buffer without KCl to improve staining. Micrographs were taken at a nominal magnification of £ 40,000 and calibrated using the paramyosin spacing of 14.4 nm 40 .A total of 4,900 ADP †Vi molecules and 3,057 apo-molecules were selected interactively and subjected to single-particle image processing using procedures written in the SPIDER suite of programs 31 .…”

Section: Methodsmentioning

confidence: 99%

Dynein structure and power stroke

Burgess

Walker

Sakakibara

et al. 2003

Nature

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478

552

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Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke.

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“…The linker of choice was (GSG) 2 as it has been previously used on anchoring domains on myosin VI (21). The introduction of flexibility into the system is motivated by myosin V, which contains a free swiveling joint between the two lever arms and before the coiled-coil domain, allowing for a spherical search for the most favorable binding site (1,22). These swivel mutants will disrupt any structurally rigid elements in myosin X and, if our model is valid, result in a motor domain that is allowed to freely search for the next binding site.…”

Section: Structural Properties Of the Tail Region Impart Fascin Bundlementioning

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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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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Two-headed binding of a processive myosin to F-actin

Cited by 288 publications

References 23 publications

Neck Length and Processivity of Myosin V

Neck Length and Processivity of Myosin V

Dynein structure and power stroke

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

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