1: Adv Exp Med Biol  1993;332:321-8; discussion 329 

Orientation of actin monomers in moving actin filaments.

Kinosita K Jr, Suzuki N, Ishiwata S, Nishizaka T, Itoh H, Hakozaki H, Marriott
G, Miyata H.

Department of Physics, Faculty of Science and Technology, Keio University,
Yokohama, Japan.

We have visualized, under an optical microscope, the orientations of actin
monomers in individual actin filaments undergoing Brownian motion in solution,
actively sliding past myosin molecules, or immobile on a surface. For the
visualization, two strategies have been adopted. One is to exploit the
fluorescence polarization of a fluorescent probe firmly attached to actin. Using
the probe phalloidin-tetramethylrhodamine, the fluorescence was clearly
polarized along the filament axis, showing alignment of the probe molecules
along the filament axis. Within our temporal resolution of 33 ms and spatial
resolution of better than 1 micron (average over approximately 10(2) actin
monomers), the orientation of the probe (hence of actin monomers) did not change
upon interaction of the filament with heavy meromyosin; myosin-induced
reorientation was estimated to be a few degrees at most. This first method,
while highly sensitive to small reorientations of monomers off or toward the
filament axis, does not report on reorientations around the axis. To detect
rotation around the filament axis, we adopted the second strategy in which we
attached small plastic beads to the actin filaments. Axial turns would be
immediately apparent from the movement of the beads. Preliminary observations
indicate that actin filaments can slide over a heavy meromyosin-coated surface
without axial rotations. Since rotations have been implicated in different
experiments, we are currently investigating the source of the apparent
discrepancy. The attached bead also serves as a handle through which we can
apply force, via optical tweezers, on the filament. By letting the sliding actin
filament pull the bead against the optical force, we were able to estimate the
sliding force and its fluctuation.

Publication Types:
Review
Review, Tutorial

PMID: 8109346 [PubMed - indexed for MEDLINE]