Orientation of actin monomers in moving actin filaments

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.