Torque generating properties of Tetrahymena ciliary three-headed outer-arm dynein

Eukaryotic cilia/flagella are cellular bio-machines that drive the movement of microorganisms. Molecular motor axonemal dyneins in the axoneme, which consist of an 9 + 2 arrangement of microtubules, play an essential role in ciliary beating. Some axonemal dyneins have been shown to generate torque coupled with the longitudinal motility of microtubules across an array of dyneins fixed to the coverglass surface, resulting in a corkscrew-like translocation of microtubules. In this study, we performed three-dimensional tracking of a microbead coated with axonemal outer-arm dyneins on a freely suspended microtubule. We found that microbeads coated with multiple outer-arm dyneins exhibited continuous right-handed helical trajectories around the microtubule. This unidirectional helical motion differs from that of other types of cytoplasmic dyneins, which exhibit bidirectional helical motility. We also found that, in an in vitro microtubule gliding assay, gliding microtubules driven by outer-arm dyneins tend to turn to the left, causing a curved path, suggesting that the outer-arm dynein itself is able to rotate on its own axis. Two types of torque generated by the axonemal dyneins, corresponding to the forces used to rotate the microtubule unidirectionally with respect to the long and short axes, may regulate ciliary beating with complex waveforms.

www.nature.com/scientificreports/ motion of microtubule gliding. Both OAD and IADs showed right-handed corkscrewing pitch within a range of 0.2-0.8 μm. Cytoplasmic dynein (CD), which belongs to a different class from axonemal dyneins, is responsible for intracellular transport processes, such as cargo/organelle transport 20 , and also drove gliding microtubules to rotate unidirectionally in an in vitro microtubule gliding assay 21 . In contrast, a microbead coated with CD molecules showed bidirectional helical movement around the microtubule longitudinal axis, suggesting the flexibility of CD to transport cargo in dense cellular environments 22 . However, whether bidirectional helical motion is common to both axonemal and cytoplasmic dyneins remains unknown because the three-dimensional (3D) trajectories of axonemal dyneins have not been examined.
In this study, we tracked the 3D motion of microbeads coated with T. thermophila OAD (22S dynein) molecules around a suspended microtubule to examine helical motion. We found that OAD molecule-coated microbeads moved helically in a right-handed manner around the suspended microtubule, which differs from the Scientific Reports | (2022) 12:16722 | https://doi.org/10.1038/s41598-022-21001-0 www.nature.com/scientificreports/ bidirectional helical motion of CD molecule-coated microbeads 22 . Moreover, in an in vitro classical microtubule gliding assay, where OAD molecules were fixed to the glass coverslip surface, paths of gliding microtubules were circular as observed for Chlamydomonas IAD-d or -g driven microtubule gliding motility 23 . The unique motile properties of axonemal dyneins may be involved in a specific function of the dyneins for cilia and flagella beating.

Results
Quantification of helical motion of microbeads coated with OAD molecules around a microtubule surface using 3D tracking. To gain insights into the torque generated during the minus-end-directed movement of axonemal dynein, the 3D motion of a fluorescent microbead (0.1 µm in diameter) driven by multiple OAD molecules was examined. To this end, three-headed OAD (22S dynein) molecules were extracted from T. thermophila ciliary axonemes and attached to microbeads. In an experimental geometry where microtubules are suspended away from the glass coverslip surface, microbeads bound to multiple dynein motors were allowed to move freely over the microtubule surface (Fig. 1a). 3D movements of the microbeads around the microtubule surface were tracked using a three-dimensional prismatic optical tracking (tPOT) microscope (Fig. 1a), originally developed by us to track single fluorescent molecules 24 , quantum dots 25 , microbeads 26 , or cells 27 . Our tPOT achieved nanometer-order accuracy in 3D positioning of a microbead fixed on the suspended microtubule (see supplementary Fig. 1 in Maruyama et al. 28 ). To quantify the motion of OAD molecules along the microtubule, the xyz positions of the OAD-coated microbeads were determined. The x and y positions of the microbeads were calculated from the average of the two positions of the microbeads in each image split by prism, and the z position of the microbeads was obtained from the difference between the two y-positions of the microbeads ( Fig. 1b and Supplementary Movie 1). Both x-y ( Fig. 1c pink) and x-z trajectories (Fig. 1c blue) of a microbead coated with OAD molecules at low ATP concentration (10 μM ATP) displayed oscillations. These trajectories with a phase lag between the sinusoidal oscillations indicated that the microbeads moved helically around the longitudinal axis of the microtubule. The corresponding y-z trajectory was circular (Fig. 1d), and the handedness of the helical motion was right-handed. Helical handedness was also confirmed by a 3D plot (Fig. 1e). For the microbead in Fig. 1b, the helical pitch, longitudinal, and rotational velocities of the helical motion were 0.62 μm, 0.50 μm s −1 and 0.82 rev s −1 , respectively (Fig. 1c, f, g).
OADs display unidirectional right-handed helical motions around a suspended microtubule. Based on the plot of revolution versus x-displacement, the trajectories of the microbeads coated with OAD molecules were always right-handed helical paths (n = 32 microbeads; Fig. 2a). The handedness of the helical motions of microbeads driven by OAD molecules was the same as that of the corkscrewing microtubule driven by OAD 18 , IAD 14S dyneins 15 , IAD dynein-a 29 , and CD 21 in microtubule corkscrewing assays. The handedness of helical motions of microbeads driven by OAD molecules was unidirectional for each microbead, which differs from the bidirectional helical motions of microbeads driven by CD molecules around the microtubule longitudinal axis 22 . However, the trajectories were found to have a wide zone (helical pitch; + 0.3 to + 1.9 μm, the plus sign refers to a right-handed helical pitch), as observed in the trajectories of CD molecules. The mean helical pitch of OAD-coated microbeads was estimated to be + 0.65 ± 0.36 μm (mean ± standard deviation, SD, n = 32). From the time course of longitudinal displacement and rotation along a microtubule for individual microbeads, the longitudinal and rotational velocities for microbeads that made > ~ 2 revolutions were determined (Fig. 2b, c). The distribution in longitudinal and rotational velocity varied between 0.04-0.64 μm s −1 , and between 0.09-1.62 rev s −1 , with a mean longitudinal velocity of 0.32 ± 0.17 μm s −1 (mean ± SD, n = 32) and a mean rotational velocity of 0.55 ± 0.33 revolutions s −1 (mean ± SD, n = 32), respectively. An increase in the longitudinal velocity, with a subsequent increase in the rotational velocity, was observed (Fig. 2d). However, there was only a small effect on helical pitch (Fig. 2e). This result indicates that both longitudinal and rotational velocities were increased to the same extent, with the ratio of longitudinal to rotational velocity remaining constant.  corkscrewing assay, OAD has been shown to drive the right-handed corkscrewing motion of microtubules with a corkscrewing pitch of + 0.76 μm 18 , which is consistent with the right-handed unidirectional helical pitch of microbeads coated with OAD ( Fig. 2a). Such observations of unidirectional corkscrewing motions and helical motions driven by OAD molecules imply that the stepping of the motor head of a three-headed dynein tends, on average, to bind a right-forward tubulin of the microtubule, producing a unidirectional lateral force perpendicular to the longitudinal axis of the microtubule. Such a unidirectional transverse force might allow gliding of the microtubules to drive the lateral transition. Given that a microtubule (circumference of 0.08 μm) rotates once for every 0.76 μm it advances, it is estimated that a sideward displacement is ~ 1 μm for every ~ 10 μm it advances 15 .
To examine whether microtubules translocate sideways during forward motion, we observed the movement of individual microtubules over a distance of 20-120 μm in the presence of 10 μM ATP in a classical microtubule gliding assay ( Fig. 3a and Supplementary Movie 2). The concentration of loaded microtubules was sufficiently low to prevent the microtubules from colliding with each other. We found that most microtubules moved forward smoothly and did not show lateral movement. This is consistent with previous reports using T. thermophila IAD (14S dynein mixture) 15 , Chlamydomonas IAD-d, or -g 23 , which also drove microtubule corkscrewing 15,19 . However, tracking the forward position of the microtubules revealed that gliding microtubules tended to orbit to the left (Fig. 3b, c). The paths of the microtubules every 60 s were also analyzed by measuring the angle at which the forward tip position of the microtubule in each frame changed relative to the forward tip position of the microtubule in the previous frame ( Fig. 3d top). The mean angular change of the forward tip position of the microtubules every 60 s was obtained from Gaussian fitting to the histogram of the angular change, giving values of 3.4° every 60 s (Fig. 3d bottom). This corresponds to a radius of curvature of ~ 235 μm, which is consistent with the value of a previous report on Chlamydomonas IAD-g 23 . Thus, OAD caused microtubules to turn to the left in our setup. Consistent with a previous report using IADs 23 , this indicates that the same extent of microtubule circular motion is caused by an intrinsic property of the motor activity of both IAD and OAD.

Discussion
Dyneins are classified into two classes, axonemal and cytoplasmic dyneins, which are structurally and functionally distinct. Axonemal dyneins drive the high-speed oscillatory beating of motile cilia/flagella, while cytoplasmic dyneins are responsible for various intracellular transport processes, such as the mitotic spindle function and trafficking of various organelles. Thus, elucidating the characteristic activities of these two types of dynein motors is important for understanding a variety of cellular processes. As one mode of motility, in the microtubule corkscrewing assay, in which microtubules move across an array of motors fixed to the glass surface, both axonemal and cytoplasmic dyneins generate torque, which, together with the microtubule gliding motility, causes right-handed corkscrewing microtubule motions 15,18,19,21 . In contrast, when dynein-coated microbeads were allowed to move over the whole surface of the microtubule suspended away from the cover glass surface, the microbeads showed helical motion along the microtubules. In such a microbead assay, OAD-driven microbeads exhibited unidirectional, right-handed helical motions (Fig. 1), whereas CD-driven microbeads exhibited bidirectional helical motions 22 . Although the assay geometries and the particular forces acting either in the microtubule corkscrewing assay or in the microbead assay differ, the most likely factor affecting motility in these two in vitro systems is the difference in the number of dynein molecules involved in the movement. In a microtubule corkscrewing assay, hundreds to thousands of dynein molecules are involved in the corkscrewing motion of a microtubule, whilst in the microbead assay, a small number of dynein molecules act on the helical movement of a microbead. In such a microbead assay using CD, fluctuations due to the small number of dynein molecules involved in movement are likely to be apparent in the left and right directions as the CD molecule moves forward, resulting in bidirectional helical motions. The ability of switching directions in their sideways movement in a highly crowded environment in cells might facilitate the transport of CD-driven cargo, as discussed previously 22,30 . Bidirectional off-axis motions of motor-coated microbeads have also been reported for other microtubule-based motors, such as kinesin-6 MKLP1-CYK4 complex 28 and kinesin-8 Kip1 31 , which work in mitotic spindles. In contrast, even with a small number of molecules where fluctuations in the step direction are likely to appear, microbeads coated with multiple OAD molecules displayed unidirectional right-handed helical motions along a suspended microtubule (Fig. 2a). Since the tail domains of OAD molecules are fixed to the A-tubule of nine peripheral microtubule doublets and their stalk head domains interact with the B-tubule in the axoneme, it may efficiently fill the sliding of doublet microtubules by stroking in a specific direction, which is suitable for functioning in a beating organelle. This will be clarified by analyzing the direction in which single molecules of OAD move by single-molecule assays, as has been done for CD 32 .
Recently, a structural study of purified Tetrahymena OAD bound to doublet microtubules demonstrated that free OADs had the tendency to bind the doublet microtubule in an array via tail-to-head interactions 14 .
In our experiments, since a part of the OAD domains (probably a part of the tail domain) was non-specifically anchored to the microbead in a random position, it is unlikely that OAD arrays were formed on the microtubules. Moreover, the structural study demonstrated that each motor head of a three-headed OAD tended to bind to doublet microtubule protofilaments with specific inter-protofilament angles 14 . However, in our experiments, singlet microtubules were self-assembled in vitro from purified porcine brain tubulin dimers, so that angle differences between protofilaments would not form as in doublet microtubules. Around the lattice surface of freely suspended, singlet microtubules, OAD-coated microbeads could move to the neighboring protofilament on the right to achieve right-handed helical motions along the suspended microtubule ( Figs. 1 and 2). Unlike the behavior of OADs on singlet microtubules, on doublet microtubules in axonemes, each OAD motor head may track the same protofilament. In this case, the effect of the torsional strokes of individual heads of OAD whose tail region is anchored to the A-tubule can efficiently twist the microtubules to control complex ciliary beats. Further studies with three-dimensional and higher temporal observations of the in vivo dynamics of OAD and doublet microtubules are required to reveal the biological roles of torque generation driven by OAD.
Using an in vitro microtubule gliding assay for long-term observation of microtubule movement in a relatively wide field of view, we have shown that OAD has the ability to render gliding microtubule orbits counterclockwise in our setup, while OAD does not cause microtubules to translocate sideways (i.e., rolling) during forward motion (Fig. 3). Given that a similar microtubule circumferential orbital motion has been reported only in Chlamydomonas IADs 23 , which also drive right-handed corkscrewing microtubule motions 19 , this motility mode may be unique to specific axonemal dyneins (i.e., both IAD and OAD) that also have the ability to corkscrew microtubules. As the mechanism of OAD-driven microtubule corkscrewing motion without lateral (rolling) motion remains unclear, the mechanism by which OAD causes circular motion in gliding microtubules remains unclear as well. The possible explanations are as follows: the direction of the lateral force acting when OAD molecules drive the right-handed corkscrewing motion of the microtubule and the direction of the lateral force acting when OAD molecules drive the microtubule gliding in the direction are identical, so that the lateral force exerted only by OAD molecules interacting with the tubulins near the leading plus-end of the microtubule, which has high freedom of movement, causes the tip of the microtubule to bend slightly to the left; therefore, the gliding microtubule shows counterclockwise arc-shaped trajectories. Another possibility is that, in addition to the forces acting along or around the longitudinal axis of the microtubule, which cause a unidirectional corkscrewing motion of microtubules or helical motion of dynein-coated microbeads along microtubules (Figs. 1 and 2), OAD may have an inherent yawing force (another type of torque); in other words, OAD itself has the ability to rotate along its own axis, which can bend a microtubule at its point of attachment to OAD. Such torque may cause OAD molecules to drive the gliding of microtubules in unidirectional circular paths (Fig. 3). A similar possibility was discussed in a study by Kikushima and Kamiya using Chlamydomonas IADs 23 , although the direction of the yawing force was the opposite. The difference may be due simply to the different yaw characteristics or number of motor domains in one-headed IADs and three-headed OADs, or potentially due to different glass surface conditions. How this motion is achieved is unclear and requires further investigation, such as polarization www.nature.com/scientificreports/ measurements that can detect changes in the rotational direction in a single OAD molecule, and force measurement of yawing generated by a small number of OAD molecules. In summary, in contrast to CD, which produces bidirectional helical motions around the microtubule surface, we found that T. thermophila axonemal OAD molecule-coated microbeads exhibited unidirectional, righthanded helical motions. Despite the similarity of the 3D structures of motor domains of the two types of dyneins (axonemal and cytoplasmic dynein), their helical motion behavior differs, suggesting particular roles for OAD in driving axonemal beating. In contrast to previous reports 15 , we also found that T. thermophila OAD molecules were able to drive the counterclockwise, circular motility of the microtubules, which is similar to that previously reported for Chlamydomonas IAD. Further studies on torque generation by both OAD and IAD in the axoneme during beating in three dimensions are required to reveal the particular role of axonemal dynein that regulates cilia/flagella beating.

Materials and methods
Preparation of OAD (22S dynein). OAD was purified from T. thermophila (strain B-255) cilia as previously described 18  After sedimentation at 89,000g for 16 h at 2 °C in a centrifuge with a P28S ultracentrifuge rotor (CP70MX, Hitachi Koki, Tokyo, Japan), the gradient was separated into 20 fractions, and the fractions containing the OAD protein peak were pooled. The pooled samples were further purified using an anion-exchange column (Mono Q 5/50 GL, Cytiva, Marlborough, MA), and concentrated using an ultrafiltration unit (Amicon Ultra-100 K, Millipore, Billerica, MA). Purified OAD was aliquoted, flash-frozen, and stored in liquid nitrogen. The concentration of OAD was estimated by SDS-PAGE on 7.5% acrylamide gels using bovine serum albumin (BSA) standards (Thermo Fisher Scientific, Waltham, MA) loaded on the same gel. The gels were stained with Quick-CBB PLUS (Wako Pure Chemical, Osaka, Japan) and imaged using a CCD camera (CSFX36BC3, Toshiba-teli, Tokyo, Japan). The bands containing OAD and BSA standards were quantified using the Quantity One software (Bio-Rad, Hercules, CA).
Purification of tubulin and polymerization of microtubules. Tubulin was purified from porcine brain by three cycles of depolymerization and polymerization followed by phosphocellulose chromatography 34 . Purified tubulin was aliquoted and flash-frozen and stored in liquid nitrogen. For the microbead motility assay, biotinylated (biotin-(AC 5 ) 2 -Sulfo-OSu, Dojindo, Kumamoto, Japan) and Alexa 488-labeled (Alexa Fluor 488 succinimidyl ester, Thermo Fisher Scientific, Waltham, MA) microtubules were prepared by polymerization of a mixture of biotinylated, Alexa488-labeled, and non-fluorescent tubulin in a molar ratio of 15 : 1 : 30-50 in BRB80 buffer (80 mM PIPES, 1 mM MgCl 2 , 1 mM EGTA, pH 6.8) supplemented with 1 mM GTP and 1 mM MgCl 2 for 30 min at 37 °C, and then stabilized by adding 40 µM taxol (Sigma-Aldrich, St. Louis, MO) 28 . For the in vitro microtubule gliding assay, rhodamine-labeled (X-rhodamine succinimidyl ester, Thermo Fisher Scientific, Waltham, MA) microtubules were prepared by polymerization of a mixture of rhodamine-labeled and non-fluorescent tubulin in a molar ratio of 1 : 16 in BRB80 with 1 mM MgCl 2 and 1 mM GTP for 30 min at 37 °C and then stabilized by adding 20 µM taxol 35 . Preparation of dynein-coated beads. OAD coated 0.1-µm diameter carboxylate-modified polystyrene microbeads (FluoSphere, red fluorescent (580/605), Thermo Fisher Scientific, Waltham, MA) were prepared. First, 5 µL of 0.6-nM microbeads were incubated with 5 µL of 170 nM OAD solution in 50 K-Ace buffer (10 mM PIPES, 50 mM potassium acetate, 4 mM MgSO 4 , 1 mM EGTA, pH 7.4) for 2 min on ice, and then 0.5 µL of 11 mg mL −1 casein (Nacalai Tesque, Kyoto, Japan) was added to prevent further non-specific adsorption. After collection by centrifugation, the microbeads were re-suspended into the 50 K-Ace buffer (100 µL) containing 0.5 mg mL −1 casein, 10 µM ATP, 20 µM taxol, ATP regeneration (creatine kinase (Roche, Basel, Switzerland), creatine phosphate (Roche, Basel, Switzerland)) and oxygen scavenger systems (catalase (Roche, Basel, Switzerland), glucose oxidase (Sigma-Aldrich, St. Louis, MO) and glucose). The re-suspended microbeads were observed for approximately 30 min. In a preliminary screening for a condition that allows to observe stable motion of the microbeads along the microtubule, excessive amounts of OAD molecules required mixing with the microbeads in a molar ratio of 1 : 280. We estimated the maximum number of OAD molecules attached to each microbead to be approximately ten (half the surface area of a microbead / occupied area of OAD ≈ 1.6 × 10 4 nm 2 /30 × 50 nm 2 ), although we could not exactly determine the number of simultaneously interacting OAD molecules with a microtubule at any given time.
3D-motility of ODA-coated microbeads around a suspended microtubule. A 3D prismatic optical tracking (tPOT) microscope was constructed as previously described 18,25,28 . The flow chamber was prepared according to the method described by Maruyama et al. 28  www.nature.com/scientificreports/ coverslips (130-170 μm thick; Matsunami Glass, Ind., Osaka, Japan) using two thin strips of tape (810-1-18, Scotch 3 M) placed with a 5 mm gap as spacers inside two parallel lines of grease (Apiezon M Grease, M&I Materials, Manchester, UK). Microgrooves on the patterned glass coverslip were fabricated using a standard photolithography technique 28,36 . Parallel walls on the patterned glass allowed microtubules to suspend away from the glass surface. First, the flow chamber was incubated with two chamber volumes of 5 mg mL −1 biotinylated-BSA (Sigma-Aldrich, St. Louis, MO) in 50 K-Ace buffer for 3 min, then rinsed with two chamber volumes of cold 50 K-Ace buffer, and incubated for 3 min with two chamber volumes of 2.5 mg mL −1 streptavidin (Sigma-Aldrich, St. Louis, MO) in 50 K-Ace buffer at room temperature. Following a rinse with two chamber volumes of W buffer (50 K-Ace buffer supplemented with 0.5 mg mL −1 casein and 10 µM taxol), two chamber volumes of 0.1 mg mL −1 Alexa488-biotin-labeled microtubules were loaded and incubated for 3 min at room temperature. The non-immobilized microtubules were washed away with two chamber volumes of W buffer. Finally, the chamber was loaded with two chamber volumes of OAD-coated microbead solution containing 10 μM ATP (BioXtra, Sigma-Aldrich, St. Louis, MO), ATP regeneration and oxygen scavenger systems, 1 mM dithiothreitol (DTT, Roche, Basel, Switzerland), and 20 μM taxol, sealed with grease, and mounted on the tPOT microscope to measure the microbead motion at room temperature (24 ± 1 °C). Fluorescence was passed through an appropriate filter set (for Alex488-labeled microtubules, GFP filter set, Semrock, Rochester, NY, and for fluorescent red microbead, G2A set, Nikon, Tokyo, Japan). Microbeads on a straight microtubule were tracked every 0.1 s with the tPOT system to track their 3D positions (x: parallel to the longitudinal axis, z: perpendicular to the imaging plane, y: perpendicular to both x and z; Fig. 1a). The position of the two optically separated images of a microbead from an identical microbead were determined by 2D Gaussian fitting as (x 1 , y 1 ) and (x 2 , y 2 ), and x, y, and z was calculated by (x 1 + x 2 )/2, (y 1 + y 2 )/2 and y 1 −y 2 , respectively. For calibration of the real z-axis position and y 1 −y 2 , a custom-built stable stage (Chukousya Seisakujo, Tokyo, Japan) equipped with a pulse motor (SGSP-13ACTR-BO, Sigma Koki, Tokyo, Japan) and controller (QT-CM2, Chuo Precision Industrial, Tokyo, Japan) was used to move the objective vertically while observing the stable microbead (0.1 µm in diameter, Thermo Fisher Scientific) bound to the suspended microtubule. The calculated z-position and actual z-position (as defined by the pulse motor) corresponded linearly over a range of ± 0.5 µm from the focal plane. We analyzed microbeads that exhibited stable motion (> 0.9 µm) along a stably suspended single microtubule. Those on fluctuating, bundled, or crossed microtubules, and those on a non-suspended part of a microtubule, that is, the segment bound on the patterned hilltop, were ignored. Microbeads that encountered other microbeads and those that exhibited a helical trajectory whose radius was too large for some unknown reason (> 250 nm, ~ twice as large as the sum of the radii of the microtubule and the microbead) were also excluded. Finally, we obtained 32 trajectories. All these showed helical motion around the microtubules. The observed pitch (P obs ) comprised two helical elements: the helical motion driven by dyneins (P mot ) around the microtubule surface, and the supertwist of the microtubule depending on the number of the protofilaments (P MT , 7-8 µm for the 14-protofilament microtubule, but none for the 13-protofilament microtubule). Based on the well-known relationship between them, P mot −1 = P obs −1 − P MT −121, 22,28,37 . According to our previous study, microtubule polymerization with 1 mM GTP in BRB80 buffer formed a 5 : 3 mixture of 13-and 14-protofilament microtubules 25 . The difference between P obs and P mot in proportion of the 13-protofilament microtubules in the mixture to the 14-protofilament microtubules of our preparation was less than 10%. Thus, the values of the observed pitch without correction were used in this study. xyz trajectories were determined using measurements from at least three independent assays. Microtubule gliding assay. Microtubule gliding assays were performed in flow chambers assembled from two coverslips attached together using double-sided tape (NW-25, Nichiban, Tokyo, Japan). The chamber was first incubated with one chamber volume of 0.03 μM dynein solution for 3 min. The chamber was filled with four chamber volumes of 1 mg mL −1 casein in 50 K-Ace buffer for 2 min to coat the glass surface and then rinsed with four chamber volumes of 50 K-Ace buffer. Two volumes of rhodamine-labeled microtubules (~ 1 μg mL −1 ) in BRB80 buffer supplemented with 20 μM taxol and 0.4 mg mL −1 casein were added and incubated for 2 min, and then rinsed with two chamber volumes of 50 K-Ace buffer. Finally, two chamber volumes of 50 K-Ace buffer containing 10 μM ATP, ATP regeneration and oxygen scavenger systems, 1 mM DTT, and 20 μM taxol were introduced to the chamber. The chamber was sealed with a nail varnish. Assays were performed at room temperature (24 ± 1 °C). Microtubule gliding was observed using an inverted fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan) with a custom-built stable stage (KS-N) and a stage controller (QT-CM2-35), using illumination from a mercury lump (Intensilight, Nikon, Tokyo, Japan), 100× /1.49 NA, Plan-Apochromat objective lenses (Nikon, Tokyo, Japan), and G2A filter set. A reduction lens (0.6×) was inserted into the camera port on the right to widen the field of view. Images of microtubule gliding driven by OAD molecules coated with the lower cover glass composing the flow chamber were recorded using an EMCCD camera (iXon X 3 DU-897E-COO-#BV, Andor Technology, Belfast, UK) via the Solis software (Andor Technology, Belfast, UK) with a 5-s exposure for a 60-s imaging cycle. The positions of the leading tip of the microtubule were obtained manually using tracking software (Mark2, kindly provided by Dr. Ken'ya Furuta, NICT) 38 . The positions of the tip of the gliding microtubules were determined using measurements from at least three independent assays.

Data availability
All samples used in this study are available from the corresponding authors on reasonable request. The source data for graphs in the figures during the current study are provided as Supplementary Data 1.