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

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.

💡 Research Summary

The paper addresses a long‑standing question in the field of actomyosin mechanics: whether the interaction between actin filaments and myosin motors generates not only longitudinal force but also a lateral torque that can cause the filament to rotate about its own axis. To answer this, the authors adapted the classic actin gliding assay and combined it with polarized total internal reflection fluorescence (polTIRF) microscopy, a technique that can resolve the three‑dimensional orientation of sparsely incorporated rhodamine‑labeled actin monomers. By introducing two intermediate linear polarizations positioned between the conventional s‑ and p‑polarizations, they reduced the four‑fold ambiguity that normally limits orientation determination, allowing a precise measurement of the filament’s azimuthal angle as it moves.

Using this setup, they examined two motor proteins: muscle myosin II, which drives sarcomeric contraction, and the processive cargo‑transport motor myosin V. In both cases the actin filaments displayed a consistent left‑handed (counter‑clockwise when viewed from the motor side) helical trajectory. The measured pitch—the axial distance advanced per full rotation—was on the order of 1–2 µm, corresponding to roughly 30–60 actin subunits per turn. Importantly, this pitch was invariant across a wide range of experimental conditions: myosin surface density (0.1–1 µM), filament length (2–10 µm), and gliding velocity (0.2–1 µm s⁻¹).

These observations suggest that the torque is an intrinsic property of the motor–track system rather than a secondary effect of experimental parameters. The authors discuss two plausible mechanistic origins. First, the power stroke of a myosin head may be slightly tilted relative to the filament axis, producing a small off‑axis component that accumulates into a measurable rotation. Second, the inherent helical geometry of the actin filament (13 subunits per 6 turns) could bias the binding geometry of successive myosin heads, leading to a systematic handedness. The fact that both a non‑processive, ensemble motor (myosin II) and a highly processive, single‑molecule motor (myosin V) generate the same handedness supports a model in which the filament’s structural chirality plays a dominant role.

Beyond the mechanistic insight, the work has broader physiological implications. In muscle, a persistent microscopic twist could influence the elastic response of sarcomeres, contribute to energy dissipation, or affect the alignment of thick filaments during contraction. In intracellular transport, the rotational component of myosin V movement may help align cargoes, facilitate the navigation of crowded actin networks, or even be exploited by the cell to generate torque‑driven processes such as membrane remodeling.

The study also highlights methodological strengths and limitations. The polTIRF approach provides nanometer‑scale spatial resolution and sub‑degree angular precision, but it relies on low labeling density and careful calibration of polarization optics; any drift or misalignment can introduce systematic errors. Moreover, the evanescent field used in TIRF may exert an electric field on the filament, potentially altering its mechanical properties. Future work could integrate high‑resolution cryo‑EM structures of myosin‑actin complexes, perform molecular dynamics simulations of tilted power strokes, and extend the assay to other myosin classes (e.g., myosin I, VI) to test the universality of the observed left‑handed twist.

In summary, the authors provide the first direct, quantitative evidence that actin filaments twirl with a left‑handed pitch when propelled by either myosin II or myosin V, and that this behavior is robust to changes in motor density, filament length, and speed. The findings open new avenues for exploring how lateral forces and torques contribute to muscle physiology and intracellular transport, and they suggest that the chiral architecture of the actin filament itself is a key determinant of motor‑induced rotation.