📝 Abstract

The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (twirling) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (micron), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity.

💡 Analysis

The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (twirling) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (micron), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity.

📄 Content

1 “This un-edited manuscript has been accepted for publication in Biophysical Journal and is freely available on BioFast at http://www.biophysj.org . The final copyedited version of the paper may be found at http://www.biophysj.org .”

Title:
Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay

Running Title: Twirling of actin by myosins II and V

Authors: John F. Beausang* Harry W. Schroeder III# Philip C. Nelson* Yale E. Goldman§¶

Affiliations

• Department of Physics and Astronomy

Biochemistry and Molecular Biophysics

§ Department of Physiology
¶ Pennsylvania Muscle Institute

University of Pennsylvania Philadelphia, PA 19104

2 ABSTRACT The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (~ µm), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity.

3 INTRODUCTION The swinging lever arm model (1, 2) explains force production and movement between actin and myosin II in muscle contraction (3) and also applies to non-muscle myosins (4). Separate crystal structures of myosin and actin docked into cyro-EM maps of actomyosin (5) indicate that the lever arm swing is nearly parallel to the axis of the actin filament, thereby efficiently converting the free energy released from ATP hydrolysis into motion along the filament. Even small torque components around the filament axis, however, may have biological roles in muscle contraction ((6), and references therein) or regulation (7). For processive non-muscle motors, a torque may be desirable for navigating cargo around obstacles present in the crowded environment of the cell (8-10). Several studies have suggested off-axis components to the relative motion between actin and myosin. For example, decreased lateral spacing of the filaments of frog skeletal muscle in rigor compared with relaxation was attributed to radial forces between the thick and thin filaments (11). In a modified gliding assay, actin filaments that were selectively immobilized onto the slide at their pointed ends formed superhelices that suggested a right-handed component of torque generated by myosin II (12). In standard gliding assays where the filaments are free to translocate, a torque component of the cross-bridge force could result in rotation of the filament about its longitudinal axis, a motion we term “twirling.” Actin filaments with marker beads attached at their ends showed no twirling on HMM-coated slides (13) while gliding, but when the filaments were marked sparsely with fluorescently labeled actin monomers, simultaneous twirling and gliding of the filaments was observed by polarized fluorescence microscopy (14, 15). Symmetries in the fluorescence polarization technique prevented both of those studies from determining the handedness of the twirling motion.
A separate way of gauging sideways motions and possible components of torque between actin and processive myosins is to suspend the actin filament above the surface of the microscope slide and record the path of a bead being transported by myosin along the suspended filament (16, 17). Off-axis force causes the bead to travel in a helical path. In the suspended filament assay, myosins V (16) and VI (17) exhibited left-handed and right-handed helical paths, respectively.
Polarized epifluorescence microscopy (14) and polarized total internal reflection fluorescence (polTIRF) microscopy (15, 18, 19) typically use incident and/or detected light polarized along the x, y and z axes of the microscope. In such configurations, rotation of the fluorophore, but not its handedness, can be observed because orientations of the fluorophore reflected across any of the Cartesian planes give the same fluorescence intensities and thus are not distinguishable. Intermediate excitation or emission polarizations that break these symmetries enable the

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