Fig. 3.

Load dependence of myosin-5B stepping signatures investigated with high-speed optical tweezers. a Schematic of ultrafast force-clamp spectroscopy applied to a processive myosin-5B motor. A single myosin-5B molecule is attached to a glass bead pedestal through a streptavidin-biotin link. A single actin filament is trapped by suspending it between alpha-actinin coated beads. Black arrows represent the force clamped on the right (F₁) and left bead (F₂), red arrow represents the net force (F) on the dumbbell. F is alternated back and forth to maintain the dumbbell within a limited oscillation range when myosin is not bound to actin. b Left: cartoon showing the position of the right bead when: (1) myosin is unbound and the dumbbell oscillates; (2) myosin attaches to actin; and (3) myosin moves towards the actin barbed end. Right: example trace showing displacement and force during the corresponding phases of dumbbell oscillation, myosin-5B attachment, and processive runs under assistive and resistive loads. c Position record showing myosin-5B processive runs and the step and run detection algorithm. Detected beginning and end of each run are indicated by green and cyan vertical lines, respectively. Red horizontal lines indicate the detected steps. [ATP] = 100 μM