ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

Sheetz, Michael P.; Chasan, Rebecca; Spudich, James A.

doi:10.1083/jcb.99.5.1867

Cited by 122 publications

(71 citation statements)

References 25 publications

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“…The half-maximal velocity occurred at =50 ALM ATP. Although these results are quite different from the ATP concentration dependence of actin activation of heavy meromyosin ATPase activity, which shows halfmaximal activity at 6 puM ATP (21), the ATP concentration dependence of movement in this purified protein movement assay is similar to that of the Nitella-based assay (19).…”

Section: Resultscontrasting

confidence: 72%

“…Movement was substantially inhibited by 75 mM KCl and completely stopped in 100 mM KCl. MgCl2 was necessary for movement at concentrations greater than the concentration of ATP, as we found for the Nitella-based system (19). CaCl2 was not required for movement.…”

Section: Resultssupporting

confidence: 61%

“…2). The broad pH optimum was somewhat different from the pH dependence of myosin movement in the Nitella-based system (19) but resembled the pH dependence of actin activation of myosin ATPase (20). At pH 6.5 and below and pH 9.5 and above, filaments were observed to bind to the myosin-coated surface but failed to move.…”

Section: Resultsmentioning

confidence: 79%

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Fluorescent actin filaments move on myosin fixed to a glass surface.

Kron¹,

Spudich²

1986

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

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824

540

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Single actin filaments stabilized with fluorescent phalloidin exhibit ATP-dependent movement on myosin filaments fixed to a surface. At pH 7.4 and 240C, the rates of movement average 3-4 ,um/s with skeletal muscle myosin and 1-2 jim/s with Dictyostelium myosin. These rates are very similar to those measured in our previous myosin movement assays. The rates of movement are relatively independent of the type of actin used. The filament velocity shows a broad pH optimum between 7.0 and 9.0, and the concentration of ATP required for half-maximal velocity is 50 ,AM. Evidence was obtained to suggest that movement of actin over myosin requires at most the number of heads in a single thick filament. This system provides a practical, quantitative myosin-movement assay with purified proteins.The lack of a simple in vitro assay for myosin movement has hampered progress in elucidating the mechanism by which actin and myosin couple ATP hydrolysis to mechanical work. We have sought to develop a totally purified system that would permit quantitative determination ofthe rate of myosin movement along actin.Several systems for observing movement of purified actin and myosin have been reported (1-6). The first quantitative measurement of rates of movement of purified myosin along actin in vitro were made by using the Nitella-based movement assay of Sheetz and Spudich (7). In that assay, myosin filaments are attached to polystyrene beads, and these myosin beads are deposited on the cytoplasmic face of a dissected Nitella axillaris cell. These cells contain rows of chloroplasts attached to the inner face of the cell membrane. Bundles of actin filaments of uniform polarity lie on these chloroplast rows (8). Myosin beads that come into contact with the actin bundles move unidirectionally over long distances in the same direction as cytoplasmic flow in the unopened cell. The rates of movement of the beads can be determined by noting position and time elapsed at several points along the bead path. The Nitella-based assay yields reproducible and quantitative rate data with relatively small samples of purified myosin. Nonetheless, this assay is flawed because it depends on the biochemically undefined actin cables of the Nitella cell, which are stabilized by unknown factors and may be contaminated by components of the Nitella cytoplasm. Thus, we have worked to replace the Nitella-based assay with a movement assay using only purified proteins.The Nitella-based assay demonstrated a principle that we have exploited in designing movement systems composed of purified proteins. Although the myosin filaments are bipolar and are bound to the beads in random orientations (9), the beads move in only a single direction-that defined by the polarity of the actin filaments. This suggests that only those myosin cross-bridges oriented properly can interact productively with an actin filament. Those not oriented correctly presumably cannot recognize their binding site on actin. Thus, the direction of movement is defined by the polarity of the actin...

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Section: Resultscontrasting

confidence: 72%

Section: Resultssupporting

confidence: 61%

See 1 more Smart Citation

Fluorescent actin filaments move on myosin fixed to a glass surface.

Kron¹,

Spudich²

1986

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

Self Cite

824

540

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show abstract

“…A more interesting model, suitable for quantitative studies, was constructed by covalent attachment of myosin to small beads. The coated beads moved along polarized actin filament bundles obtained from Nitella (Sheetz & Spudich, 1983;Sheetz, Chasan & Spudich, 1984). The velocity of movement corresponded closely to the velocity of unloaded shortening of the sarcomeres of the muscles used as the source of the myosin.…”

Section: Constructing a Motile Systemmentioning

confidence: 72%

Cell Motility

Taylor

1986

Journal of Cell Science

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“…[38,41,42]). Such an assay comprises motors and filaments, and can provide data on the transport velocity of filaments.…”

Section: (A) Motor Calibrationmentioning

confidence: 99%

Beam finite-element model of a molecular motor for the simulation of active fibre networks

2016

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Molecular motors are proteins that excessively increase the efficiency of subcellular transport processes. They allow for cell division, nutrient transport and even macroscopic muscle movement. In order to understand the effect of motors in large biopolymer networks, e.g. the cytoskeleton, we require a suitable model of a molecular motor. In this contribution, we present such a model based on a geometrically exact beam finite-element formulation. We discuss the numerical model of a non-processive motor such as myosin II, which interacts with actin filaments. Based on experimental data and inspired by the theoretical understanding offered by the powerstroke model and the swinging-cross-bridge model, we parametrize our numerical model in order to achieve the effect that a physiological motor has on its cargo. To this end, we introduce the mechanical and mathematical foundations of the model, then discuss its calibration, prove its usefulness by conducting finite-element simulations of actin-myosin motility assays and assess the influence of motors on the rheology of semi-flexible biopolymer networks.

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ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

Cited by 122 publications

References 25 publications

Fluorescent actin filaments move on myosin fixed to a glass surface.

Fluorescent actin filaments move on myosin fixed to a glass surface.

Cell Motility

Beam finite-element model of a molecular motor for the simulation of active fibre networks

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