Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

Kovács, Mihály; Wang, Fei; Sellers, James R.

doi:10.1074/jbc.m500616200

Cited by 42 publications

(32 citation statements)

References 49 publications

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“…We expect that the motor, on its initial encounter with an actin bundle, samples several configurations before locking to the bundle in an orientation where both heads can engage. Consistent with this proposal, Kovács et al (35) have shown that single heads of myosin X populate the weak binding states of actin. Moreover, in our optical trapping results we have seen many examples of stepwise detachments (Fig.…”

Section: Structured Tail Domain Is Responsible For Bundle Selection Insupporting

confidence: 58%

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 Insupporting

confidence: 58%

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

Nagy

Rock

2010

Journal of Biological Chemistry

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“…One proposed that it is a motor with a high duty ratio (i.e., fraction of the motor ATPase cycle in the strongly bound state), potentially capable of processive runs (12). The other found a lower duty ratio, but with a significant population of myosin X weakly bound to actin (13). All of these in vitro studies used single actin filaments, rather than the native bundled actin filament assembly where myosin X has been shown to associate in vivo.…”

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confidence: 99%

A myosin motor that selects bundled actin for motility

Nagy

Ricca

Norstrom

et al. 2008

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

134

152

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Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.fascin ͉ motor navigation ͉ myosin X ͉ filopodia ͉ single-molecule fluorescence I n a dense mesh of cellular actin, if all filaments are functionally equivalent, then myosins will move in a random walk as they translate, detach, and reattach to new filaments. Alternatively, myosins may identify and walk along certain subpopulations of actin that lead to target locations, based on a set of common structural features. One possibility is that different myosin classes may partition among filaments based on actin isoforms. However, no significant difference between ␤␥-actin versus ␣-actin has been detected for myosin V, the only motor to be tested on both tracks (1). A second possibility is that actinassociated proteins, in particular tropomyosins, modulate the binding of myosin to actin. Indeed, class I myosins are excluded from tropomyosin-decorated actin in stress-fibers, whereas class II myosins are not (2, 3). In addition to this direct regulatory mechanism, tropomyosins may also direct myosin traffic by stabilizing particular actin tracks (4). However, tropomyosins are not typically found in regions of actively polymerizing, dynamic actin, such as the leading edge of the cell. Therefore, additional mechanisms likely exist for directing myosin traffic.To identify factors that could direct myosins to specific locations, we searched for myosin classes that are localized to limited populations of actin even in the presence of nearby dynamic actin. The class X myosin meets these criteria for a selective motor. Myosin X travels to and is highly concentrated at the distal tips of filopodia: long, slender projections often found at the leading edge of migrating cells (5-7). Filopodia are used for envi...

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“…Solution kinetic studies of truncated recombinant myosin-10, which resembles the soluble subfragment-1 produced by proteolytic cleavage of muscle myosin (i.e., S1-like), have shown that release of product, ADP, is slow relative to the overall ATPase cycle time, making it a high duty cycle ratio motor, spending a significant fraction of its ATPase cycle tightly bound to actin (13,14). Consistent with these kinetic studies, single molecule fluorescence imaging studies using artificially dimerized GFPtagged myosin-10 (15-19) constructs that resemble HMM (i.e., HMM-like) shows that it moves processively along filamentous actin (F-actin), presumably using its two heads to take one step after another.…”

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confidence: 99%

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

Takagi

Farrow

Billington

et al. 2014

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

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Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/ extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s −1 ) is similar to the maximum ATPase activity (∼12-14 s −1 ) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircaselike processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.actomyosin | stable single alpha-helix | optical trapping | myosin X | myosin-5a

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Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

Cited by 42 publications

References 49 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

A myosin motor that selects bundled actin for motility

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

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