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

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of approximately 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 approximately 1 microm, 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.

[1]  D. Axelrod Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. , 1979, Biophysical journal.

[2]  M. Anson Temperature dependence and Arrhenius activation energy of F-actin velocity generated in vitro by skeletal myosin. , 1992, Journal of molecular biology.

[3]  A. Hudspeth,et al.  Movement of microtubules by single kinesin molecules , 1989, Nature.

[4]  I. Sase,et al.  Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. , 1995, Biophysical journal.

[5]  D A Winkelmann,et al.  Three-dimensional structure of myosin subfragment-1: a molecular motor. , 1993, Science.

[6]  H. Huxley Sliding filaments and molecular motile systems. , 1990, The Journal of biological chemistry.

[7]  W. Kabsch,et al.  Atomic model of the actin filament , 1990, Nature.

[8]  T. Yanagida,et al.  Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. , 1990, Journal of molecular biology.

[9]  K. Kinosita,et al.  A theory of fluorescence polarization decay in membranes. , 1977, Biophysical journal.

[10]  S. Ishiwata,et al.  Right-handed rotation of an actin filament in an in vitro motile system , 1993, Nature.

[11]  James D. Jontes,et al.  A 32° tail swing in brush border myosin I on ADP release , 1995, Nature.

[12]  S. Ishiwata,et al.  Preparation of bead-tailed actin filaments: estimation of the torque produced by the sliding force in an in vitro motility assay. , 1996, Biophysical journal.

[13]  R. Vale,et al.  Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia , 1988, Cell.

[14]  Kiwamu Saito,et al.  Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution , 1995, Nature.

[15]  B. Brenner Mechanical and structural approaches to correlation of cross-bridge action in muscle with actomyosin ATPase in solution. , 1987, Annual review of physiology.

[16]  R. Yasuda,et al.  Direct measurement of the torsional rigidity of single actin filaments. , 1996, Journal of molecular biology.

[17]  K. Sekimoto,et al.  Protein friction exerted by motor enzymes through a weak-binding interaction. , 1991, Journal of theoretical biology.

[18]  Edward D. Salmon,et al.  The Drosophila claret segregation protein is a minus-end directed motor molecule , 1990, Nature.

[19]  Kazuhiko Kinosita,et al.  Submicrosecond and microsecond rotational motions of myosin head in solution and in myosin synthetic filaments as revealed by time-resolved optical anisotropy decay measurements , 1984 .

[20]  S. Ishiwata,et al.  Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium , 1991, The Journal of cell biology.

[21]  T. Yanagida,et al.  Nano-manipulation of actomyosin molecular motors in vitro: a new working principle. , 1993, Trends in biochemical sciences.