The path to visualization of walking myosin V by high-speed atomic force microscopy

The quest for understanding the mechanism of myosin-based motility started with studies on muscle contraction. From numerous studies, the basic frameworks for this mechanism were constructed and brilliant hypotheses were put forward. However, the argument about the most crucial issue of how the actin–myosin interaction generates contractile force and shortening has not been definitive. To increase the “directness of measurement”, in vitro motility assays and single-molecule optical techniques were created and used. Consequently, detailed knowledge of the motility of muscle myosin evolved, which resulted in provoking more arguments to a higher level. In parallel with technical progress, advances in cell biology led to the discovery of many classes of myosins. Myosin V was discovered to be a processive motor, unlike myosin II. The processivity reduced experimental difficulties because it allowed continuous tracing of the motor action of single myosin V molecules. Extensive studies of myosin V were expected to resolve arguments and build a consensus but did not necessarily do so. The directness of measurement was further enhanced by the recent advent of high-speed atomic force microscopy capable of directly visualizing biological molecules in action at high spatiotemporal resolution. This microscopy clearly visualized myosin V molecules walking on actin filaments and at last provided irrefutable evidence for the swinging lever-arm motion propelling the molecules. However, a peculiar foot stomp behavior also appeared in the AFM movie, raising new questions of the chemo-mechanical coupling in this motor and myosin motors in general. This article reviews these changes in the research of myosin motility and proposes new ideas to resolve the newly raised questions.