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Twirling of Actin by Myosins II and V Observed
via Polarized TIRF in a Modified Gliding Assay.
Biophys J. 95:5820-5831. 2008.
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 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
microscopy. 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 fourfold. We found that
whole myosin II and myosin V both twirl actin with a relatively
long (~1µm), left-handed pitch that is insensitive to
myosin concentration, filament length, and filament velocity.
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University of Pennsylvania
