a. Example trajectory of a dynein-driven bead oscillated ± 4.9 pN in a square wave pattern without ATP. b. Example trajectory of a dynein-driven bead oscillated with + 6 pN and −1.5 pN in a square wave pattern at 2.5 Hz. Even though dynein was pulled more strongly backward, it moved towards the MT minus-end in the absence of ATP. c. The bead position was subtracted from the trap position to determine the force exerted on the bead during force oscillations. Due to the thermal relaxation of the bead and the rotational freedom of the bead–motor linkage, the bead–trap separation reaches near zero when the bead is moved between forward and backward positions. To determine how force affects velocity, this “dead” period t1 is omitted from total elapsed time and t2 is taken as half period of oscillations. (Left) To switch between assiting and hindering forces, the trap was first moved 250 nm and then with proportional feedback-controlled increments every 10 ms until the desired force was reached. (Right) The trap was first moved 500 nm and then with feedback-controlled increments every 10 ms until the desired force was reached. An increase in this “overshoot” distance decreased t1.