(A) One case of a force gliding assay is presented where three kinesins labeled with 705-nm QDs,marked 1, 2 and 3, move a labeled microtubule (green). Raw images at 0, 1.4, 5.2 and 14 sec taken every 0.1 sec (see Video 1) are shown. Yellow arrows show equilibrium kinesin positions. White arrows point along the direction of the force generated by kinesins on microtubule as they are displaced from their equilibrium positions. Red arrow shows the direction of microtubule movement. (B) Plots showing the microtubule kymograph and velocity, and kinesin positions over time. Time points 0, 1.4, 5.2, and 14 sec (corresponding to A) are marked with yellow vertical lines. Microtubule velocity (middle panel) starts from 0 nm/s at t = 0 sec, and increases to ~850 nm/s at frame 1.4 sec, when kinesin #2 starts driving (negative kinesin displacement). At 5.2 sec, kinesin #1 joins kinesin #2 to drive the microtubule, while kinesin #3 starts resisting. At 14 sec, kinesin #2 is resisting, kinesin #3 is driving and kinesin #1 is in equilibrium position. The fluctuation in kinesin displacement results from both Brownian motion and tracking error. Driving and resisting cycles are zoomed in for detail. Points 1’, 2’ and 3’ are transition regions. At positive/ negative slope of 1’ region, kinesin slow down (for resisting cycle) or speed up (for driving cycle) with respect to microtubule. At the zero slope of the 2’ region (plateau region), kinesin’s speed is the same as that of microtubule as DNA is fully stretched. At the 3’ region, kinesin returns to equilibrium. (C) The average number of transitions per minute. The major transition is from driving to driving.