Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin · ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.Kinesin, a microtubule-activated ATPase, functions as a cytoplasmic motor to drive organelle translocation toward the plus ends of microtubules 1-3 . The principles governing the conversion of chemical energy from ATP hydrolysis to force production for the sliding of kinesin along microtubules may be similar to those for actomyosin and the axonemal dynein-microtubule ATPases 4,5 . However, the motility of single motor molecules in vitro suggests that mechanochemical coupling for kinesin must be somewhat different from actomyosin. For example, a single molecule of kinesin (2 heads) will promote translocation for several micrometres and at maximal rates 6 . In contrast, multiple skeletal myosin molecules are required for directed movement along an actin filament and the velocity increases as the number of myosin molecules is increased 7 . In addition, the non-hydrolysable ATP analogue AMP-PMP (β, γ-imidoadenosine 5′-triphosphate) causes dissociation of the actomyosin and microtubule-dynein complexes, but promotes stabilization of the microtubule-kinesin complex 5,8 .Here we describe mechanistic studies of the kinetics of indi|vidual steps in the ATPase cycle of the kinesin ATPase to explain the interactions of kinesin with the microtubule lattice responsible for movement. We have used the Drosophila kinesin motor domain expressed in Escherichia coli 9-11 . This protein, designated K401 and containing the N-terminal 401 amino acids, is a fully active, homogeneous preparation with the kinetic and structural properties expected of a native kinesin 9-11 . It has a very low ATPase activity in the absence of microtubules which is limited by the rate of ADP release (∼0.01 s −1 ). In the presence of microtubules, the steady-state rate increases to a maximum of 20 ± 2 s −1 . Furthermore, K401 is a dimer under our experimental conditions (J. J. Correia, S.P.G., M. L. Moyer and K.A.J., manuscript submitted).Chemical quench-flow experiments 11 established the rate of ATP hydrolysis at the active site to be significantly faster (100 s −1 ) than steady-state turnover; therefore, the rate-limiting step † Present address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. § To whom correspondence should be addressed. of the microtubule-activated ATPase must occur after ATP h...

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.Eg5, a Kinesin-5 (formerly known as BimC) family member, has an unusual homotetrameric protein structure that is formed by the anti-parallel arrangement of heavy chains, with pairs of heads situated at opposite ends of a common stalk 1 . In dividing cells, Eg5 is essential for the organization and maintenance of mitotic and meiotic spindles. Organized bipolar spindles contain microtubule arrays emanating from each of two focused poles, with interdigitated, antiparallel microtubules occupying the mid-zone. Immunodepletion of Eg5 leads to monoastral spindles with disorganized poles and to the disruption of preformed bipolar spindles 2 , and several small-molecule inhibitors of Eg5 have been identified 3, 4 .However, some clues about the function of Eg5 in spindle organization are emerging. Microtubule gliding-filament assays have shown that this motor is plus-end-directed and slow, with reported velocities ranging from 10-40 nm s −1 (refs 2 , 5-7 ). These velocities are similar 6 Correspondence should be addressed to S.M.B. (e-mail: sblock@stanford.edu). 5 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONS P.M.F. and M.T.V. designed, performed and analysed the single-molecule experiments and co-wrote the manuscript. T.C.K. and S.P.G. generated the Eg5 constructs and conducted the biochemical measurements. S.M.B. and S.P.G. supplied guidance, designed research and edited the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Despite recent advances, fundamental questions persist, particularly with regard to Eg5 processivity (the average number of sucessive enzymatic cycles carried out while bound to the microtubule substrate). Conventional kinesin is processive and single motors can transport cargo over large distances. In contrast, Eg5 is thought to work in small ensembles and need not be processive. A previous study used solution biochemical measur...

The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.