Actin Structure-Dependent Stepping of Myosin 5a and 10 during Processive Movement

Bao, Jianjun; Huck, Daniel M; Gunther, Laura K.; Sellers, James R.; Sakamoto, Takeshi

doi:10.1371/journal.pone.0074936

Cited by 17 publications

(32 citation statements)

References 52 publications

(95 reference statements)

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“…We found little evidence of processive movements when probing M10HMM in the optical trap under our usual protocol, which uses a trap stiffness of ∼0.03 pN/nm. However, on lowering the trap stiffness, short processive runs could then be seen, showing both forward and backward steps of ∼35 nm, similar to the center of mass translocation value obtained via single molecule TIRF imaging assays [∼34 (18) and ∼31 nm (19)], but significantly longer than values obtained by Nagy et al (15) and Ricca and Rock (17), who found steps of 18 nm on F-actin bundles and a mixture of 10-to 20-nm steps on single surface-immobilized F-actin filaments. Nagy and Rock (16) also carried out optical tweezers experiments using F-actin bundles and found a load-dependent step size that was 18 nm at higher loads and 12 nm at lower loads.…”

Section: Discussionsupporting

confidence: 68%

“…Notably, the chimeric myosin-5a/SAH HMM construct used by Baboolal et al (23) also released ADP in a monoexponential manner. Because both the myosin-5a/SAH-containing HMM (23) and M10HMM (15,(17)(18)(19) constructs exhibit processive motion at low load, we conclude that strong kinetic gating of ADP release is not a prerequisite for processivity (23).…”

Section: Discussionmentioning

confidence: 80%

“…Each ATP-driven step taken by such truncated chimeras of myosin-10 has been reported to be in the range of 18-34 nm long (16)(17)(18)(19). Furthermore, live cell fluorescence imaging studies (20, 21) using a full-length GFP-tagged myosin-10 construct showed that single molecules move in a highly directed manner within the filopodia.…”

mentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 80%

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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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“…The results show that myosin 6 moves along actin with a much more variable step size and that myosin 7a takes somewhat shorter steps than myosin 5a. FIONA measurements of myosin 10 are somewhat controversial with a range of values reported [75–77]. The longer step size of 36 nm is surprisingly similar in magnitude to that of myosin 5a as the lever arm of myosin 10 binds only three calmodulins.…”

Section: 6 Super-resolution Tracking Of Moving Myosinsmentioning

confidence: 99%

Use of Fluorescent Techniques to Study the In Vitro Movement of Myosins

Toepfer

Sellers

2014

Experientia Supplementum

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Myosins are a large superfamily of actin-dependent molecule motors that carry out many functions in cells. Some myosins are cargo carriers that move processively along actin which means that a single molecule of myosin can take many ATP-dependent steps on actin per initial encounter. Other myosins are designed to work in large ensembles such as myosin thick filaments. In vitro motility assays are a powerful method for studying the function of myosins. These assays in general use small amounts of protein, are simple to implement, and can be done on microscopes commonly found in many laboratories. There are two basic versions of the assay which involve different geometries. In the sliding actin in vitro motility assay, myosin molecules are bound to a coverslip surface in a simply constructed microscopic flow chamber. Fluorescently labeled actin filaments are added to the flow chamber in the presence of ATP, and the movement of these actin filaments powered by the surface-bound myosins is observed. This assay has been used widely for a variety of myosins including both processive and nonprocessive ones. From this assay, one can easily measure the rate at which myosin is translocating actin. The single-molecule motility assay uses an inverted geometry compared to the sliding actin in vitro motility assay. It is most useful for processive myosins. Here, actin filaments are affixed to the coverslip surface. Fluorescently labeled single molecules of myosins (usually ones with processive kinetics) are introduced, and the movement of single molecules along the actin filaments is observed. This assay typically uses total internal reflection fluorescent (TIRF) microscopy to reduce the background signal arising from myosins in solution. From this assay, one can measure the velocity of movement, the frequency of movement, and the run length. If sufficient photons can be collected, one can use Gaussian fitting of the point spread function to determine the position of the labeled myosin to within a few nanometers which allows for measurement of the step size and the stepping kinetics. Together, these two assays are powerful tools to elucidate myosin function.

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“…Actin filament assembly and bundling can also be mediated by the combined properties of Enabled (Ena, a member of the Ena/vasodilator-stimulated phosphoprotein family) and fascin-1 [20]. Once assembled, fascin-1/Factin bundles provide surfaces for the motion of myosin motors that promote specific motion and stepping characteristics [21][22][23]. This property may be important for cargo transport dynamics within filopodia [24].…”

Section: Fascin-1: Molecular Functionsmentioning

confidence: 99%

Fascin-1 as a biomarker and prospective therapeutic target in colorectal cancer

Adams

2014

Expert Review of Molecular Diagnostics

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Fascin-1 is a filamentous actin-binding protein that crosslinks actin microfilaments into tight, parallel bundles. These bundles are important for the extension of microspikes, filopodia and invadopodia from cell surfaces and for the functionality of these protrusions in cell migration and/or dynamic sensing of the local microenvironment. Fascin-1 is absent in normal colonic epithelium, but its upregulation in colorectal adenomas and adenocarcinomas and its correlation with an aggressive clinical course has piqued the interest of many laboratories with research interests in cancer metastasis. This report summarizes current knowledge of the molecular interactions of fascin-1 in relation to its activities and mechanisms of upregulation in colorectal carcinoma cells. The status and key questions surrounding investigations of fascin-1 as a novel, early prognostic biomarker in colorectal cancer are discussed. Ongoing pre-clinical research into new migration inhibitory and anti-metastatic compounds that alter the actin cytoskeleton, and the goal of targeting fascin-1, is also discussed.

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Actin Structure-Dependent Stepping of Myosin 5a and 10 during Processive Movement

Cited by 17 publications

References 52 publications

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

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

Use of Fluorescent Techniques to Study the In Vitro Movement of Myosins

Fascin-1 as a biomarker and prospective therapeutic target in colorectal cancer

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