Processive Runs of Full Length Myosin VA Are Interrupted by Pauses and Dwells

Abstract

Full length myosin Va (FL-MyoVa) forms an inhibited, folded conformation at low salt, stabilized by interactions between the globular tails and the heads. High ionic strength disrupts this interaction, resulting in an extended, active processive motor. In vivo, it has been postulated that cargo binding disrupts the folded conformation and activates the motor. It is possible that splice variations in the tail (-B+D+F, melanocyte; +B-D-F, brain) could modify the ability of myosin Va to form the inhibited state. Two FL-MyoVa splice variants and an HMM-MyoVa, with biotin tags for Qdot labeling, were expressed in Sf9 cells. Sedimentation velocity experiments showed similar transitions from the folded-to-extended conformation for the two splice variants as a function of salt. TIRF microscopy was then used to observe processive runs on actin. The velocities of both FL-MyoVa splice variants were similar, and increased 270% (171-460nm/sec) with increasing KCl concentration (25-200mM). In contrast, the velocity of HMM-MyoVa increased by a more modest 50% (381-586nm/sec). The trajectories of the FL-MyoVa and HMM-MyoVa were also strikingly different. Both FL-MyoVa splice variants underwent processive runs that were interrupted by periods during which the motor dwelled at fixed points on the actin filament, presumably in the folded, inhibited state. At lower KCl concentration, FL-MyoVa dwelled approximately half of the total trajectory duration. Increasing ionic strength decreased duration of the dwells. HMM-MyoVa was fully active and maintained continuous processive movement at all KCl concentrations. The slower overall velocities for the FL-MyoVa splice variants, compared to HMM-MyoVa, results from inclusion of the dwell periods. We propose that during a processive run, a single FL-MyoVa can switch between an active and inhibited state without dissociating from actin, and that this phenomenon is independent of splice variations in the tail domain.