Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity

Abstract

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.

FIGURE 1

Myosin V processive run with heads labeled with different colored Qdots. Green and red open circles are the Qdot₅₆₅ and Qdot₆₅₅ positions, respectively, determined by Gaussian fits. Solid lines are the average Qdot positions between steps with the onset of steps determined by eye. Upper left are averaged Qdot images for steps labeled A–D, with red and green images offset by 12 pixels in y for clarity. The yellow lines connect Qdot centers emphasizing alternating relative head positions. Green arrow identifies substep. Lower right are histograms of interhead spacing and step size.