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

TL;DR

This study uses polarized TIRF microscopy to observe actin filament twirling caused by myosin II and V, revealing a consistent left-handed rotation that is unaffected by various experimental conditions.

Contribution

It introduces a modified gliding assay combined with polarized TIRF to measure actin twirling and determines the handedness and pitch of the rotation induced by different myosins.

Findings

Both myosin II and V induce actin twirling with a long, left-handed pitch.

The twirling behavior is independent of myosin concentration, filament length, and velocity.

The method allows precise measurement of filament rotation and handedness.

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.