title: Stepwise motion of an actin filament over a small number of heavy meromyosin molecules is revealed in an in vitro motility assay.
abstract: In order to determine the relative motions of an actin filament and a myosin molecule upon hydrolysis of one ATP, an in vitro motility assay, in which individual actin filaments slide over heavy meromyosin molecules bound to a substrate, was combined with an optical trapping technique. An actin filament, attached to a gelsolin-coated bead, was captured with an optical trap. The surface-bound heavy meromyosin molecules pulled the filament against the trapping force, which resulted in back and forth motions of the actin-bound bead. The number of heavy meromyosin molecules interacting with an actin filament (at most 1/micron filament) and the ATP concentration (< or = 0.5 microM) were chosen so as to facilitate detection of each "pull." Calculation of the centroid of the bead image revealed abrupt displacements of the actin filament. The frequency of such displacements was between 0.05 and 0.1 per 1 s per 1 micron actin filament, being consistent with calculated values based on the reported bimolecular binding constants of ATP and the actomyosin rigor complex. The distribution of the displacements peaked around 7 nm at a trapping force of 0.016 pN/nm, but it became broader, and some displacements were as large as 30 nm, when the trapping force was reduced to 0.0063 pN/nm, suggesting that the force generation due to the structural change of a myosin head may be insufficient to explain such displacements.
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, Kanagawa.
authors.forename: H
authors.initials: H
authors.lastname: Miyata
authors.forename: H
authors.initials: H
authors.lastname: Hakozaki
authors.forename: H
authors.initials: H
authors.lastname: Yoshikawa
authors.forename: N
authors.initials: N
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Kinosita
authors.forename: T
authors.initials: T
authors.lastname: Nishizaka
authors.forename: S
authors.initials: S
authors.lastname: Ishiwata
mesh_terms: D000199:Actins / Q000737:chemistry*; D000818:Animals; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008863:Microspheres; D009038:Motion; D015879:Myosin Subfragments / Q000737:chemistry*; D055095:Optics and Photonics; D011817:Rabbits
chemical_list: D000199:Actins; D015879:Myosin Subfragments