Structural differences between dynein and kinesin suggest a unique molecular mechanism of dynein motility. Measuring the mechanical properties of a single molecule of dynein is crucial for revealing the mechanisms underlying its movement. We measured the step size and force produced by single molecules of active cytoplasmic dynein by using an optical trap and fluorescence imaging with a high temporal resolution. The velocity of dynein movement, 800 nm͞s, is consistent with that reported in cells. The maximum force of 7-8 pN was independent of the ATP concentration and similar to that of kinesin. Dynein exhibited forward and occasional backwards steps of Ϸ8 nm, independent of load. It is suggested that the large dynein heads take 16-nm steps by using an overlapping hand-over-hand mechanism.microtubules ͉ motor protein ͉ optical tweezers ͉ step size ͉ nanotechnology D ynein is a molecular motor that moves along microtubules to the direction of the minus end. Cytoplasmic dynein transports cellular organelles toward the minus end of microtubules, whereas most kinesin molecules transport organelles toward the plus end (1-3). Hirakawa et al. (4) have reported that purified axonemal dynein produced maximum forces of 5 pN, and they also showed stepwise displacements of 8 nm. In contrast, another study (5) has demonstrated that single molecules of purified cytoplasmic dynein move with short steps (8 nm) at high loads (Ͼ0.8 pN) and long steps (16-32 nm) at low forces (Ͻ0.4 pN), and from these findings a molecular gear mechanism was proposed. The values for maximum force and the size of the steps differ between those two reports. Moreover, the very low velocity (Ͻ50 nm͞s) of movement of the purified cytoplasmic dynein (5) is in direct contrast to the high velocity (Ϸ1 m͞s) of dynein movement in a cell and in an in vitro motility assay (6-8). These discrepancies possibly result from inactive dynein molecules being included with the purified native dynein in the analysis.In this study a method of coating the beads with dynein was developed to keep dynein active and allow the force and step size produced by single molecules of dynein to be measured. The step size of active cytoplasmic dynein was 8 nm and was independent of both force and ATP concentration. The stall force was 7-8 pN. These values are very similar to those recorded for kinesin (9); however, the stepping manner was different from that of kinesin. ResultsActive Dynein Bound to Protein A-Coated Beads. The method used to purify cytoplasmic dynein without its accessory protein, dynactin, has been described (Fig. 1a, lane D) (10, 11). Dynein was further purified by allowing it to bind to the microtubules (Fig. 1a, lane AMP-PNP) (12). Dynein was then released from microtubules in the presence of 0.1-10 mM ATP (Fig. 1a, lane ATP).The method used to bind dynein to the coverslips for in vitro motility assay and beads for optical trap assay was modified from previously reported methods (5, 8). Beads coated with protein A were suitable for motility and force generation....