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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nature. Author manuscript; available in PMC 2010 April 9.
Published in final edited form as:
PMCID: PMC2852185

Direct observation of single kinesin molecules moving along microtubules


Kinesin is a two-headed motor protein that powers organelle transport along microtubules1. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule2,3. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy4 in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a ~ 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin’s two force-generating heads operate by a hand-over-hand mechanism.

Kinesin is composed of two heavy chains, each consisting of a 340-amino-acid, N-terminal force-generating domain, a long α-helical coiled coil interrupted in the middle by a proline–glycine-rich hinge, and a small globular C-terminal domain that interacts with light chains5,6. To add a fluorescent label to kinesin in a manner that does not interfere with motor function, the ubiquitous human kinesin gene7 was truncated just before the hinge at amino-acid residue 560 and a short peptide containing a reactive cysteine8 was introduced at the C terminus (uhK560cys). uhK560cys was expressed in bacteria, purified, and reacted at its C-terminal cysteine with a maleimide-Cy3 fluorescent dye (Fig. 1 legend).

FIG. 1
Movement of a single fluorescently labelled kinesin molecule along an axoneme. a, Kinesin (uhK560cys) labelled at the C-terminal cysteine residue with Cy3 fluorescent dye was combined with Cy5-labelled axonemes in the presence of ATP. The specimen was ...

When uhK560-Cy3 (0.6 nM) was combined with 1 mM ATP and Cy5-labelled flagellar axonemes, fluorescent kinesin molecules could be seen binding to and moving unidirectionally along axonemal microtubules (Fig. 1). The velocity of movement was 300 ± 70 nm s−1 (mean ± s.d.; n = 60), which was similar to the gliding velocity of axonemes on uhK560-Cy3-coated glass surfaces (370 ± 40 nm s−1; n = 28).

To verify that the translocating objects were single kinesin molecules, the photobleaching characteristics and intensities of fluorescent spots were examined. The fluorescence of surface-adsorbed kinesin disappeared primarily in either a one-step (Fig. 2a) or a two-step process (Fig. 2b), as expected for photobleaching reactions of one or two dye molecules, respectively, bound to the uhK560cys dimer. The percentage of one-step, two-step, three-step and more-than-three-step photobleaching events was 74, 25, 2 and 0%, respectively (n = 355). Assuming labelling of only the C-terminal cysteine and random dimerization of two chains, these values agree with the percentages expected of single-dye (72.6%) and double-dye (27.4%) labelled kinesin molecules predicted from the labelling stoichiometry (0.43 dye/polypeptide). The fluorescence intensities of kinesin molecules randomly adsorbed onto a glass slide (Fig. 2c) were also similar to those of moving spots (Fig. 2d). Collectively, these results are consistent with the idea that single kinesin molecules can move along microtubules.

FIG. 2
Photobleaching properties and fluorescence intensities of fluorescently labelled kinesin. uhK560-Cy3 was adsorbed onto a fused-silica slide, and the intensities of individual fluorescent spots plotted against time. Typical examples of one-step (a) or ...

The distance travelled by single kinesin molecules was distributed exponentially (Fig. 3a), as described for bead motility assays9. Based upon the mean travel distance of 630 ± 30 nm and an 8-nm step per cycle for kinesin10, the probability of remaining bound to the microtubule per mechanical cycle is ~ 99% (e(−8/630)). If one ATP is hydrolysed per 8-nm step (which is consistent with uhK560 ATPase rates), then 40 ATP molecules on average would be hydrolysed by each kinesin motor domain per diffusional encounter with the microtubule, which is intermediate between two previous estimates2,3.

FIG. 3
Effects of ionic strength on the travel distance and association frequencies of uhK560-Cy3. The distances travelled by single uhK560-Cy3 molecules in motility buffer containing 0 M (a), 0.1 M (b), 0.2 M (c) or 0.3 M NaCl (d) are shown. The dots indicate ...

The effect of ionic strength on single kinesin molecule motility is shown in Fig. 3. Although the number of measurable associations events decreased dramatically with increasing salt concentration, the average travel distance and velocity (Fig. 3 legend) of kinesin molecules that had initiated movement were much less affected. The number of ATP molecules hydrolysed per microtubule encounter, however, was reported to decrease 10-fold when salt was raised from 0.05 to 0.15 M (ref. 3). This apparent discrepancy may reflect a difference between bulk ATPase measurement and observation of single molecule behaviour in a microscopic assay. At higher salt, kinesin may undergo transient microtubule interactions (too fast to be detected in our assay) that result in ATP hydrolysis or product release, but not processive movement. Once kinesin is bound in a manner that allows processive movement to begin, then the travel distance appears to be only modestly affected by ionic strength.

The mechanism by which kinesin moves for long distances on a microtubule without dissociating remains unresolved, although it has been suggested that the two heads of kinesin may operate in a hand-over-hand manner3,1113. To explore this idea, we prepared a series of truncated, monomeric Drosophila kinesin proteins12,14,15, as well as a longer kinesin molecule (dK440) that forms a dimer15,16 (Table 1). Consistent with previous studies12,17, even the monomeric motors generated movement in a gliding assay in which many motors interact simultaneously with the microtubule, although the velocity of dK340 was very slow. In the single-molecule fluorescence motility assay, dK440-Cy3 molecules moved along microtubules with a velocity and travel distance comparable to that described for uhK560-Cy3. In contrast to observations for dK440-Cy3, unidirectional movement by dK340-Cy3, dK350-Cy3 or dK365-Cy3 was not observed. Furthermore, association–dissociation reactions of these monomeric kinesins with the axoneme were seen less frequently than for dK440, suggesting that their association–dissociation cycle may be faster than can be detected by video analysis.

Motility of monomeric and dimeric kinesin

In conclusion, these results provide definitive evidence for the ability of a single kinesin molecule at physiological ionic strength to move along a microtubule even in the absence of attachment to a cargo. The travel distance reported here is only slightly lower than that of kinesin moving a bead of diameter 0.2 µm (1.4 µm)9, which probably reflects the smaller diffusion coefficient of a bead compared with a protein. Furthermore, kinesin molecules containing a single motor domain showed no detectable processive movement, although higher spatial resolution techniques10 will determine whether they undergo very short movements. In any event, these results and others12 argue that two kinesin force-generating heads are required for long-distance movements along a microtubule. Assays similar to those described here could be applied to other processive enzymes such as polymerases and helicases.


This work was supported, in part, by grants from the NIH. We thank C. McGee for discussion, T. Yagi for preparation of axonemes, N. Hom-Booher for technical assistance and C. Coppin and R. Cooke for comments on the manuscript.


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