  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.
