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....

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments comigrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin-1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co-precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein-LIS1 complex on transportable MTs, which is a possibility supported by our data.

In neurons, microtubules (MTs) span the length of both axons and dendrites, and the molecular motors use these intracellular ‘highways' to transport diverse cargo to the appropriate subcellular locations. Whereas axonal MTs are organized such that the plus-end is oriented out from the cell body, dendrites exhibit a mixed MTs polarity containing both minus-end-out and plus-end-out MTs. The molecular mechanisms underlying this differential organization, as well as its functional significance, are unknown. Here, we show that kinesin-1 is critical in establishing the characteristic minus-end-out MT organization of the dendrite in vivo. In unc-116 (kinesin-1/kinesin heavy chain) mutants, the dendritic MTs adopt an axonal-like plus-end-out organization. Kinesin-1 protein is able to cross-link anti-paralleled MTs in vitro. We propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain.DOI: http://dx.doi.org/10.7554/eLife.00133.001

Dyneins and kinesins move in opposite directions on microtubules. The question of how the same-track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein-microtubule interactions. To address this issue, we studied dynein-microtubule interactions using the tip of the microtubule-binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo-electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 A, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain-like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH-kinesin head-microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule-binding site, and imply that binding at this site has an essential role for these motor proteins.