Axial rotation of sliding actin filaments revealed by single-fluorophore imaging

Ichiro Sase, Hidetake Miyata, Shin'ichi Ishiwata, Kazuhiko Kinosita

Research output: Contribution to journalArticle

155 Citations (Scopus)

Abstract

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of ≃72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of ≃1 μm, which is much greater than the 72 nm pitch. Thus, myosin does not 'walk' on the helical array of actin protomers; rather it 'runs,' skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each 'kick' of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.

Original languageEnglish
Pages (from-to)5646-5650
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number11
DOIs
Publication statusPublished - 1997 May 27
Externally publishedYes

Fingerprint

Myosins
Actin Cytoskeleton
Protein Subunits
Actins
Actomyosin
Fluorescence Polarization
Optical Imaging
Glass
Observation
Proteins

Keywords

  • Epifluorescence microscopy
  • Fluorescence polarization
  • Force generation
  • In vitro motility
  • Molecular motor

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Axial rotation of sliding actin filaments revealed by single-fluorophore imaging. / Sase, Ichiro; Miyata, Hidetake; Ishiwata, Shin'ichi; Kinosita, Kazuhiko.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 11, 27.05.1997, p. 5646-5650.

Research output: Contribution to journalArticle

Sase, Ichiro ; Miyata, Hidetake ; Ishiwata, Shin'ichi ; Kinosita, Kazuhiko. / Axial rotation of sliding actin filaments revealed by single-fluorophore imaging. In: Proceedings of the National Academy of Sciences of the United States of America. 1997 ; Vol. 94, No. 11. pp. 5646-5650.
@article{5c458e7d37144fa38217a0bf82fd9750,
title = "Axial rotation of sliding actin filaments revealed by single-fluorophore imaging",
abstract = "In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of ≃72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of ≃1 μm, which is much greater than the 72 nm pitch. Thus, myosin does not 'walk' on the helical array of actin protomers; rather it 'runs,' skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each 'kick' of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.",
keywords = "Epifluorescence microscopy, Fluorescence polarization, Force generation, In vitro motility, Molecular motor",
author = "Ichiro Sase and Hidetake Miyata and Shin'ichi Ishiwata and Kazuhiko Kinosita",
year = "1997",
month = "5",
day = "27",
doi = "10.1073/pnas.94.11.5646",
language = "English",
volume = "94",
pages = "5646--5650",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "11",

}

TY - JOUR

T1 - Axial rotation of sliding actin filaments revealed by single-fluorophore imaging

AU - Sase, Ichiro

AU - Miyata, Hidetake

AU - Ishiwata, Shin'ichi

AU - Kinosita, Kazuhiko

PY - 1997/5/27

Y1 - 1997/5/27

N2 - In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of ≃72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of ≃1 μm, which is much greater than the 72 nm pitch. Thus, myosin does not 'walk' on the helical array of actin protomers; rather it 'runs,' skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each 'kick' of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.

AB - In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of ≃72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of ≃1 μm, which is much greater than the 72 nm pitch. Thus, myosin does not 'walk' on the helical array of actin protomers; rather it 'runs,' skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each 'kick' of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.

KW - Epifluorescence microscopy

KW - Fluorescence polarization

KW - Force generation

KW - In vitro motility

KW - Molecular motor

UR - http://www.scopus.com/inward/record.url?scp=0030902490&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030902490&partnerID=8YFLogxK

U2 - 10.1073/pnas.94.11.5646

DO - 10.1073/pnas.94.11.5646

M3 - Article

VL - 94

SP - 5646

EP - 5650

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 11

ER -