Myosin V is a left-handed spiral motor on the right-handed actin helix

Ali, M. Yusuf; Uemura, Shunsuke; Adachi, K.; Itoh, Hiroyasu; Kinosita, Kazuhiko; Ishiwata, Shin’ichi

doi:10.1038/nsb803

Cited by 135 publications

(128 citation statements)

References 28 publications

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“…We failed to observe any processive myosin X runs in this experimental geometry, but this may be because of the extremely low density of filaments on the surface. To force myosin X motor interactions, we used an optical trap to position motor-decorated beads near suspended filaments in a geometry that was described by Ali et al (22). We identified beads that moved processively on surface-attached fascin-actin bundles, then positioned these beads near suspended single filaments.…”

Section: Resultsmentioning

confidence: 99%

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A myosin motor that selects bundled actin for motility

Nagy

Ricca

Norstrom

et al. 2008

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

131

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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Section: Resultsmentioning

confidence: 99%

“…The chamber also contained surface-attached 1-m-diameter streptavidin beads. Biotinylated actin filaments (17) were attached to these beads by solution flow, as described in (22). We trapped a motor bead and identified one that moved processively on a fascin actin bundle.…”

Section: Methodsmentioning

confidence: 99%

A myosin motor that selects bundled actin for motility

Nagy

Ricca

Norstrom

et al. 2008

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

131

152

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“…In some cases, one single molecule experiment can provide a dramatic direct visualization of a picture that was painstakingly constructed from many ensemble experiments. The rotation of the F 1 F 0 -type ATP synthase [17,18] and the movement of the motor proteins myosin and kinesin [14,[19][20][21] are excellent examples.…”

Section: Why Single Molecules?mentioning

confidence: 99%

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Yang

Musser

2006

Methods

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The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

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“…The processive stepping is a hand-over-hand motion (6,7) and has recently been reported to involve a ''wiggly'' movement around actin (8). This is in contrast to myosin V, arguably the best understood myosin motor (9), which takes a 36-nm step size (10), in a hand-over-hand fashion (11), tilting its large lever arm approximately 70° (12,13), in a relatively straight fashion, with some twisting about the actin axis (13)(14)(15).…”

mentioning

confidence: 99%

Myosin VI undergoes a 180° power stroke implying an uncoupling of the front lever arm

Reifenberger

Toprak

Kim

et al. 2009

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

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'wiggly'' walking on actin. Instead, we propose that for the two heads of myosin VI to coordinate their processive movement, the lever arm of the lead head must be uncoupled from the converter until the rear head detaches. More specifically, intramolecular strain causes the myosin VI lever arm of the lead head to uncouple from the motor domain, allowing the motor domain to go through its product-release (phosphate and ADP) steps at an unstrained rate. The lever arm of the lead head rebinds to the motor and attains a rigor conformation when the rear head detaches. By coupling the orientation and position information with previously described kinetics, this allows us to explain how myosin VI coordinates its heads processively while maintaining the ability to move under load with a (semi-) rigid lever arm.FIONA ͉ fluorescence ͉ motility ͉ single molecule assay ͉ unconventional myosin D espite fairly intensive study, how myosin VI moves remains elusive. It is known that myosin VI moves toward the minus end of actin in contrast to all other myosins (1). Myosin VI also has a large and variable step size of Ϸ30 Ϯ 12 nm with occasional back steps of approximately 13 Ϯ 8 nm (2, 3), which is made possible by a combination of a short calmodulin-containing lever arm (4) and a lever-arm extension that is created by the unfolding of a threehelix bundle (5). The processive stepping is a hand-over-hand motion (6, 7) and has recently been reported to involve a ''wiggly'' movement around actin (8). This is in contrast to myosin V, arguably the best understood myosin motor (9), which takes a 36-nm step size (10), in a hand-over-hand fashion (11), tilting its large lever arm approximately 70°(12, 13), in a relatively straight fashion, with some twisting about the actin axis (13-15).The atomic structure of myosin VI has been revealed in the post-power stroke states (16) and a truncated form in the pre-power stroke states (17). It is largely the same as myosin V, but myosin VI has two inserts, one near the nucleotide pocket, which allows it to fine-tune its response to ADP and ATP, and a second unique insert at the end of the converter domain, where the lever arm connects to the motor domain, which repositions the lever arm and reverses the power stroke (18,19).While it is unclear as to the exact degree of rotation that the power stroke undergoes, it is undoubtedly large. Ménétrey et al., based on the crystal structures, suggested it was nearly 180°(16, 17). Bryant et al., based on a series of single-headed constructs in an optical trap, also concluded that the lever arm undergoes a 180°r edirection during the power stroke (20). Sun et al., based on single-molecule fluorescence angular changes, stated that their data were most consistent with variable lever arm positions, with the majority involving an approximate 180°reorientation of the lever arm (8). Furthermore, they observed ''chaotic left-right wiggling'' of the lever arm as the myosin VI moves along the actin filament.The aspect of the myosin VI processive mechanism that had b...

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Myosin V is a left-handed spiral motor on the right-handed actin helix

Cited by 135 publications

References 28 publications

A myosin motor that selects bundled actin for motility

A myosin motor that selects bundled actin for motility

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Myosin VI undergoes a 180° power stroke implying an uncoupling of the front lever arm

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