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Alternate fast and slow stepping of a heterodimerickinesin moleculeKuniyoshi Kaseda1,2, Hideo Higuchi3 and Keiko Hirose1,4

A conventional kinesin molecule travels continuously along amicrotubule in discrete 8-nm steps. This processive movementis generally explained by models in which the two identicalheads of a kinesin move in a ‘hand-over-hand’ manner1–4.Here, we show that a single heterodimeric kinesin molecule (inwhich one of the two heads is mutated in a nucleotide-bindingsite) exhibits fast and slow (with the dwell time at least 10times longer than that of the fast step) 8-nm steps alternately,presumably corresponding to the displacement by the wild-type and mutant heads, respectively. Our results provide thefirst direct evidence for models in which the roles of the twoheads alternate every 8-nm step.

Various models have been proposed to explain the processive move-ment of a dimeric kinesin molecule. In the ‘hand-over-hand’model1–4, the two heads of a kinesin exchange their roles at every 8-nm step (Fig. 1a, left). In contrast, there are other models in whichone of the heads is always leading; for example, the ‘inchworm’model5,6 (Fig. 1a, right). We thought that the most direct way to dis-criminate between these models would be to make a heterodimerickinesin7 in which the mechanochemical cycle rates of the two heads

are different and to observe the time course of the displacement pro-duced by a single heterodimer through optical trapping nanometry.For the hand-over-hand model, we expect the dwell time (time inter-val between successive 8-nm steps) of every other step to be different;for typical inchworm models, however, there should be no systematicchanges in the dwell time (Fig. 1a).

To detect differences in the dwell time of every other step, if any, aheterodimeric kinesin in which the mechanochemical cycle rate ofone head is at least several times slower than the other is required. Inaddition, the heterodimer should move processively and produceenough force. To make such a heterodimer, mutant kinesins with analtered nucleotide-binding motif were constructed8. When Arg 14 ofa human conventional kinesin construct was mutated to alanine(R14A), the ATPase rate (1.5 ± 0.2 s−1 head−1, mean ± standard devi-ation) and the microtubule-gliding speed (36 ± 5 nm s−1) of themutant homodimer were considerably slower than the wild type(27.6 ± 4.8 s−1 head−1 and 636 ± 45 nm s−1; Table 1). In optical trap-ping experiments, latex beads sparsely coated with the R14A/R14Ahomodimers bound briefly to the microtubule (association time~200 ms; see Supplementary Information, Fig. S1) but did not showdetectable movement at the single-molecule level (see Methods). At a

1Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), and 2Japan Society for the Promotion of Science (JSPS),1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. 3Department of Metallurgy, Graduate School of Engineering and Center for Interdisciplinary Research, TohokuUniversity, Sendai, Miyagi 980-8579, Japan. 4Correspondence should be addressed to K.H. (e-mail: k.hirose@aist.go.jp)

Published online: 23 November 2003; DOI: 10.1038/ncb1067

Table 1 ATPase measurements, micotubule-gliding assays and optical trapping experiments

Construct ATPase assay Gliding assay Beads assay

ATPase rate Km(MT) Velocity Velocity Stall force

(s–1 head–1) (µM) (nm s–1) (nm s–1) (pN)

WT 27.6 ± 4.8 3.5 ± 2.0 636 ± 45 679 ± 156 5.4 ± 1.2

R14A/R14A 1.5 ± 0.2 0.1 ± 0.04 36 ± 15 58 ± 30* n.d.*

WT/R14A 2.9 ± 0.7 1.7 ± 0.7 86 ± 30 85 ± 33 5.8 ± 1.3

(2.8) (68) (106)

All values are shown as the mean ± standard deviation. If a hand-over-hand model is assumed, kcat and the velocity of the heterodimer is expected to be kh = 2/(1/kw + 1/km) and vh = 2/(1/vw+1/vm), respectively (kw, vw, km, vm; kcat and the velocities by the wild-type and mutant homodimers). The expected values(parentheses) are in good agreement with the experimental results. All measurements were performed in the presence of 1.5 mM ATP. *As R14A/R14A did notshow processive behaviour at the single-molecule level, the velocity at a multiple-motor level is shown. The stall force of R14A/R14A could not be determined forthe same reason.

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higher homodimer concentration, where ~60% of the beads showedattachment signals, ~10% of the beads moved continuously (seeSupplementary Information, Fig. S2), indicating that R14A/R14A-coated beads can move along a microtubule when more than onemolecule interacts with the microtubule. Michaelis-Menten fitting ofthe velocities at varying ATP concentrations showed that R14A/R14Ahas a markedly decreased affinity for ATP when compared with wildtype (Km(ATP) of ~5 mM and ~30 µM for R14A/R14A and WT/WT,respectively; see Supplementary Information, Fig. S3). In the presenceof 1.5 mM ATP, the velocity of R14A/R14A was 58 ± 30 nm s−1, lessthan 10% of the wild-type homodimer rate (679 ± 156 nm s−1).

As the velocity of the R14A mutant is slow enough when comparedwith the wild type, we made the heterodimeric kinesin, WT/R14A. Incontrast to R14A/R14A, beads coated with WT/R14A moved slowlybut continuously at the single-molecule level (Fig.1b). The velocity ofWT/R14A, calculated from the initial slope of the displacementtraces, was 85 ± 33 nm s−1, which is 12.5% that of the wild-typehomodimer. Microtubule-gliding assays gave similar results (Table 1).The averaged maximum force was as high as wild type (5.8 ± 1.3 pNand 5.4 ± 1.2 pN for WT/R14A and WT/WT, respectively). However,although the wild-type homodimer clearly showed 8-nm steps

(Fig. 1d), the step size of WT/R14A seemed to be 16 nm (Fig.1b).Closer inspection of each 16-nm step revealed a shoulder at approxi-mately 8 nm (Fig. 1c). The averaged height of the shoulder (∆X1 inFig. 2a) was 8.2 ± 1.5 nm (mean ± standard deviation), and the size ofthe step after the shoulder (∆X2) was 7.8 ± 1.4 nm. These two stepsizes are statistically indistinguishable (P = 0.05). The resultsdescribed here indicate that the observed 16-nm step is actually twosuccessive 8-nm steps; the second 8-nm (∆X2) step occurred after avery short dwell time.

The above results suggest that for the WT/R14A heterodimer, a stepfollowed by a short dwell time (here, we call it a fast step) may alter-nate with one followed by a long dwell time (slow step). Fig. 2b showsexamples of how the dwell time of successive steps changed. In con-trast to WT/WT, which showed no systematic changes in dwell time,WT/R14A took a step with a long dwell time (typically >60 ms) andone with a short dwell time (typically <30 ms) alternately in mostinstances (>94%), irrespective of the applied load. We also comparedthe distributions of the dwell times of steps that occurred directlyafter a short dwell time (<20 ms; Fig. 2c, red) with those after a longdwell time (>100 ms; Fig. 2c, blue). The majority of steps after a shortdwell time had a dwell time longer than 60 ms, whereas most of the

Hand-over-hand Inchworm

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Figure 1 Time course of the displacement of a single heterodimerickinesin molecule. (a) Two models explaining the processive movement ofkinesin and expected changes in the dwell time when a heterodimerkinesin is used. According to the hand-over-hand model (left), two headsare interchanged at stage 2, compared with stage 1, but not in theinchworm model (right). Therefore, the dwell times at stages 1 and 2 areexpected to be different only in the hand-over-hand model. Note that inpractice, the dwell time fluctuates because the cycle includes

stochastic processes. (b) A representative trace of the displacement of abead coated with R14A/WT in an optical trap. In most of the cases, thesteps seem to be 16 nm (horizontal solid gridlines). (c) An enlarged viewof the traces in b. These traces reveal a shoulder at ~8 nm (dottedlines), indicating that the observed 16-nm step consists of twosuccessive 8-nm steps. The far-left trace is an enlarged view of the areacircled in b. (d) Control experiments using the wild-type homodimersshow clear 8-nm steps.

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steps after a long dwell time had a dwell time shorter than 30 ms.These results can only be explained by models in which the roles ofthe two heads alternate every 8 nm.

The ATPase rate (2.9 ± 0.7 s−1 head−1) and the velocity(86 ± 30 nm s−1 in microtubule-gliding assays and 85 ± 33 nm s−1 inbeads assays) of WT/R14A were also consistent with models in whichthe two heads work alternately (see Table 1 legend). This is differentfrom our previous heterodimers with microtubule-binding defects,which showed a simple average of the ATPase rates from wild-typeand mutant homodimers7. These results indicate that WT/R14A,unlike our previous weak-binding heterodimers, retains the tight cou-pling mechanism.

The simplest interpretation for the above observations is that thefast steps are caused by the wild-type head and the slow steps arecaused by the mutant head. The force dependency of the averageddwell times (see Methods) of the fast steps (τf), the slow steps (τs) andthose of the wild-type homodimer (τw) are shown in Fig. 2d. At allforce levels, τs was significantly longer than τw. We could not measurethe dwell time of the R14A/R14A homodimer, because it did notmove processively. The maximum velocity of R14A/R14A at a multi-ple motor level (58 nm s−1) gives a rate of ~140 ms per 8-nm step at alow load. In addition, the association time of the R14A/R14A homod-imer was ~200 ms (see Supplementary Information, Fig. S1). Theserates are similar to τs at a low load (150–200 ms at 1–2 pN), in agree-ment with the idea that the R14A mutant head of a heterodimer isresponsible for the slow step. At a low load (1–3 pN), the dwell time of

the fast step (τf) was similar to τw, supporting the idea that the wild-type head of WT/R14A is responsible for the fast step. However, τf

showed lower load dependency than the wild type. At a higher load(>4 pN), τf was significantly shorter than τw.

We wondered how the fast step of a heterodimer could be even fasterthan the steps of the wild-type kinesin. With the wild type kinesins,previous biochemical studies indicated that ATP binding or hydrolysisby one of the heads triggers binding of the second head to tubulin3,9,10,followed by ATP binding/hydrolysis by the second head. However, asR14A is mutated in the site that is thought to interact with the adeninering, the ATP-binding rate of the R14A head is likely to be slower thanthat of the wild type. In fact, Km(ATP), determined from the velocity ofR14A/R14A at a multiple molecular level, was >100 times higher thanthat of the wild-type molecule (see above). Thus, it is possible that thewild-type head in WT/R14A can bind and/or hydrolyse a new ATPmolecule during the long waiting time, before the R14A head does so.Once the mutant head is ready and the slow step is complete, thealready active, wild-type head could produce the next step quickly,resulting in a step with a shorter dwell time11. The probability of thewild-type head entering the active state during the waiting time shouldbe greater at a high load, because τs becomes longer. This could explainwhy τf was significantly shorter than τw at a high load, but more simi-lar to τw when the force was small.

Recently published work using single-molecule fluorescence polariza-tion12,13 demonstrated that the fluorescently labelled light-chain regionof one of the heads of myosin V shows two different, well-defined angles

≈ ≈

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Figure 2 Dwell time analysis. (a) A displacement trace showing how stepsizes (∆X1, ∆X2) and dwell time (τ) were measured. (b) The dwell time ofsuccessive steps recorded for several beads and plotted logarithmicallyagainst bead displacement. The corresponding force is also shown on theupper axis. WT/R14A, but not WT/WT, showed long and short dwell times,alternately. (c) Distribution of the dwell time of the step directly after a step

with a long (>100 ms) dwell time (blue), and those following a step with ashort (<20 ms) dwell time (red). (d) Force dependency of the averaged dwelltime (mean ± s.e.m.). Dwell times increased with load. The dwell time of theslow step (τs; blue square) of WT/R14A was at least 10 times longer thanthat of the fast step (τf; black triangle) at all force levels. τf was clearlyshorter than the dwell time of WT/WT (τw; red circle) at a high load.

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and two different sizes of displacement alternately, also supporting ahand-over-hand mechanism. Thus, alternate stepping might be acommon mechanism for the dimeric protein motors to move proces-sively. In the present study, we used optical trapping nanometry tech-niques to visualize elementary processes of an actively translocatingheterodimeric kinesin molecule. A single heterodimeric kinesinshowed a step with a long dwell time alternating with one with a shortdwell time. Our results provide the first direct evidence for a model inwhich the roles of the two heads of a kinesin molecule alternate as itdisplaces by 8 nm, such as the hand-over-hand model.

METHODSExpression constructs and protein purification. The procedures for obtainingmutant homodimeric and heterodimeric kinesins have been described indetail previously7. To make the R14A mutant, Arg 14 of K432 (amino acids1–432 of the human kinesin) was mutated to alanine using the QuickChangemutagenesis kit (Stratagene, West Cedar Creek, TX). The R14A/R14A homod-imer was prepared with an amino-terminal glutathione S-transferase (GST)tag and an additional carboxy-terminal sequence to be biotinylated. Theexpressed proteins were purified with a glutathione 4B–Sepharose resin(Amersham Pharmacia, Tokyo, Japan). To construct the WT/R14A het-erodimer, a DNA fragment encoding the wild-type kinesin with a C-terminalHis tag and a fragment encoding the R14A mutant kinesin with an N-terminalGST tag and a C-terminal biotinylation sequence were placed side by side in avector. The WT/R14A heterodimer was purified first with a glutathione resinto separate WT/WT, and then with a Ni-NTA–Agarose resin (Qiagen, Tokyo,Japan) to separate R14A/R14A. GST was removed by thrombin treatment(Sigma, St Louis, MO).

Optical trapping nanometry. The apparatus for the trapping nanometryexperiments was as described previously14. Streptavidin-coated 0.2-µm beadswere incubated with biotinylated kinesins, prepared as described above, andintroduced into an assay chamber. The kinesin concentration (<75 nM) wasdetermined so that the probability of kinesin-coated beads moving on amicrotubule was 0.15–0.30. Under this condition, it is considered that only asingle kinesin molecule is statistically involved in the movement of a bead15,16.Beads were optically trapped and positioned near a microtubule bounddirectly onto a coverslip. The bead positions were recorded at a sampling rateof 10 kHz using MacLab software11,14,17 (AD Instruments, New South Wales,Australia). Experiments were performed in 80 mM Pipes at pH 6.8, 2 mMMgCl2, 100mM NaCl, 1 mM EGTA and 1.5 mM ATP in the presence of an oxy-gen scavenging system at 24–26 °C. The trap stiffness (0.03 pN nm−1) was cal-ibrated from the amplitude of thermal diffusion11,18.

Analysis of step size and dwell time. Displacement traces were low-pass-fil-tered at 200 Hz. Step sizes (∆X1 and ∆X2 in Fig. 2a) were determined as thedifference between the plateau levels in the traces. For a short step, it was diffi-cult to measure the dwell time from the duration of the plateau. Therefore, thedwell time was defined as shown in Fig. 2a. From these measured dwell times,we classified the individual steps of the heterodimers into fast and slow steps.However, because of the fluctuation and force-dependency of the dwell time,we could not classify them simply by the length of the dwell time. Therefore,we plotted the dwell time of successive steps as in Fig. 2a and chose only thosepoints that could be definitively considered as fast or slow steps. For example,the point indicated by an arrow in Fig. 2b has a dwell time of ~500 ms and is

sandwiched between steps with much shorter dwell times (<20 ms). We cate-gorized this kind of a step as slow and then defined the steps directly before orafter it as fast. The second steps were considered to be slow steps. Occasionally,two successive steps had a dwell time shorter than 20 ms (or longer than 80ms) followed by a long (or short) step. We ignored these small percentages ofsteps (~2% for each). Within each population classified as above, the dwelltime/force data sets from different traces were grouped according to the force.At every 1.0 pN, the dwell time and force were averaged within the group. Theaveraged dwell time was plotted against the corresponding averaged force (Fig.2d). For the wild type, the dwell time/force data were similarly analysed butwere not classified into fast and slow steps.

Note: Supplementary Information is available on the Nature Cell Biology website.

ACKNOWLEDGEMENTS We thank L. Amos for comments on the manuscript. This work was aided bysupport from the Human Frontier Science Program (H.H. and K.H.) and the JapanSociety for the Promotion of Science (K.K.).

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

Received 25 July 2003; accepted 20 October 2003Published online at http://www.nature.com/naturecellbiology.

1. Howard, J. The mechanics of force generation by kinesin. Biophys J. 68,S245–S253; S253–S255 (1995).

2. Block, S. M., Goldstein, L. S. & Schnapp, B. J. Bead movement by single kinesinmolecules studied with optical tweezers. Nature 348, 348–352 (1990).

3. Hackney, D. D. Evidence for alternating head catalysis by kinesin during micro-tubule-stimulated ATP hydrolysis. Proc. Natl Acad. Sci. USA 91, 6865–6869(1994).

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6. Hua, W., Chung, J. & Gelles, J. Distinguishing inchworm and hand-over-hand processivekinesin movement by neck rotation measurements. Science 295, 844–848 (2002).

7. Kaseda, K., Higuchi, H. & Hirose, K. Coordination of kinesin’s two heads studiedwith mutant heterodimers. Proc. Natl Acad. Sci. USA 99, 16058–16063 (2002).

8. Vale, R. D. Switches, latches, and amplifiers: common themes of G proteins andmolecular motors. J. Cell Biol. 135, 291–302 (1996).

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10. Ma, Y. Z. & Taylor, E. W. Interacting head mechanism of microtubule-kinesinATPase. J. Biol. Chem. 272, 724–730 (1997).

11. Kojima, H., Muto, E., Higuchi, H. & Yanagida, T. Mechanics of single kinesin mole-cules measured by optical trapping nanometry. Biophys J. 73, 2012–2022 (1997).

12. Forkey, J. N., Quinlan, M. E., Shaw, M. A., Corrie, J. E. & Goldman, Y. E. Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polar-ization. Nature 422, 399–404 (2003).

13. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

14. Nishiyama, M., Muto, E., Inoue, Y., Yanagida, T. & Higuchi, H. Substeps within the8-nm step of the ATPase cycle of single kinesin molecules. Nature Cell Biol. 3,425–428 (2001).

15. Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation ofkinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993).

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Fig. S1 Optical trapping nanometry of R14A/R14A-coated beads. At a singlemolecule level, ~20% of the beads attached to a microtubule but none ofthem showed significant movement. ?Inset shows an example of thedisplacement traces. Attachment to a microtubule is detected as reducedvibration, as indicted by the solid line. Histogram of the association timeshowed exponential decay with the rate constant of 4.9 s-1 (the associationtime of 204 ms).

Fig. S2 Movement of R14A/R14A-coated beads at a multiple molecularlevel. The R14A/R14A-coated beads showed continuous movement whenmore than one molecule are thought to interact with the microtubule. In thisexample (with 15 mM ATP), the initial velocity was 195 nm/s. Clear stepsare not seen in the displacement trace, because the movement is caused bymultiple motors.

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Fig. S3 Michaelis-Menten fitting of the velocities (mean ± SE) at varyingATP concentrations. Km(ATP) of R14A/R14A (4.6 ± 1.9 mM; open squares)was markedly higher than that of the wild type (26 ± 4 µM; closedtriangles). Note that the scales of the x-axes are different for the wild type(top) and for R14A/R14A (bottom).

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