Simultaneous observation of tail and head movements of myosin V during processive motion

Processive stepping of myosin Va (myoV) has been tracked by monitoring either the tail position (center of mass) or the position of one or both heads. Here, we combine these two approaches by attaching a quantum dot to one of the motor domains and a bead to the tail. Using laser trapping and total internal reflection microscopy, the position of one head and the tail are observed simultaneously as myoV moves processively on an actin filament bundle against the resistive load of the laser trap. The head moves one step (73 ± 10 nm) for every two steps of the tail (35 ± 9 nm). One tail step occurs concurrently with quantum dot-labeled head movement, whereas the other occurs with movement of the unlabeled head, consistent with a hand-over-hand model. Load increases the probability of the motor taking a back step. The back step is triggered by the motor taking a shorter forward step (head step, 68 ± 11 nm; tail step, 32 ± 10 nm), likely one actin monomer short of its preferred binding site. During a back step, the motor reverses its hand-over-hand motion, with the leading head detaching and reattaching to one of multiple actin sites behind the trailing head. After a back step, the motor can correct its mistake and step processively forward at resistive loads <0.7 piconewton or stall or detach at higher loads. Back stepping may provide a mechanism to ensure efficient cargo delivery even when myoV encounters obstacles within the actin cytoskeletal meshwork or when other motors are attached to the same cargo.