Ring-Shaped Assembly of Microtubules Shows Preferential

Jul 29, 2008 - Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. Biomacromolecules , 2008, 9 ...
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Biomacromolecules 2008, 9, 2277–2282

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Communications Ring-Shaped Assembly of Microtubules Shows Preferential Counterclockwise Motion Ryuzo Kawamura, Akira Kakugo, Kazuhiro Shikinaka, Yoshihito Osada,† and Jian Ping Gong* Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Received June 12, 2008; Revised Manuscript Received July 14, 2008

In this paper, we reveal that microtubules (MTs), reconstructed from tubulin in vitro in the presence of guanosine5′-triphosphate (GTP), have a ring or spiral shape on a motor protein-fixed surface, and these MTs show biased motion in the counterclockwise direction. By cross-linking these MTs during the sliding motion, we obtained large ring-shaped MT assemblies, 1∼12.6 µm in diameter. The ratio of the rings rotating in the counterclockwise direction to those rotating in the clockwise direction was approximately 3/1. Under optimized conditions, the ratio was as high as 14/1. Thus, we successfully obtained aggregated MTs with a large hierarchic structure that shows a preferential motion, through a dynamic process in vitro.

Introduction Establishment of a left-right asymmetrical structure to obtain biased function is a challenging task. In nature, cytoskeletal proteins induce handedness-related asymmetry through a dynamic process. Inhibition of the functions of these proteins reverses or even destroys the asymmetry.1-3 These phenomena motivated us to build an asymmetric structure in vitro from microtubules (MTs) through a dynamic process. MTs are one of the components of cytoskeletal proteins with polarity and handedness in their lattice structure and are involved in many cellular functions, including structural functions and organelle transportation. An MT is a hollow, cylindrical protein filament formed by the self-assembly of R- and β-tubulin heterodimers (tubulins). In vivo, an MT is usually composed of 13 protofilaments (PFs) in which heterodimers are arranged head-to-tail in a polar fashion. These PFs are aligned parallel to the longitudinal axis of the MT.4 However, when MTs are reconstructed from tubulin in the presence of guanosine-5′-triphosphate (GTP) in vitro, they consist of various numbers of PFs.5,6 With the number of PFs different from 13, the MTs are known to have left- or right-handed supertwist in PFs arrangement.7-9 As a consequence, the MTs assembled in vitro have distributions in the handedness of PFs configuration depending on the condition of preparation. Kinesin is a motor protein that moves unidirectionally on an MT surface, powered by adenosine triphosphate (ATP) hydrolysis. It is reported that the MTs show the rolling motions along the supertwist pitch of PFs when they are driven on a kinesin-fixed surface, as shown in Figure 1.6 This indicates that kinesin follows the paths of the PFs. When the movement of the front end of an MT on the kinesin-fixed glass surface is * To whom correspondence should be addressed. Tel./Fax: +81-11-7062774. E-mail: [email protected]. † Present address: RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.

Figure 1. Schematic illustration of a single MT in motion and the possibility to induce a biased rotational motion due to the handedness of PFs. An MT moves with its minus end as the front end on a kinesincoated surface. For an MT in translational motion, the supertwist structure causes a rolling motion (red) that is superimposed on the linear motion (blue). The inhibition of motion at the front end of the MT generates a torque (violet) on the MT and leads to bending of the front segment of MT, which can be spread to following segments by relaxing the local bending stress during the sliding motion on kinesin-fixed surface. To be specific, the left-handed supertwist of MTs with 14 PFs causes leftward bending of front end is shown in the figure. This bending leads to the rotation in a counterclockwise direction (green) of the MT.

hindered by obstacles such as inactive kinesins, a torque is generated that bends the MT into a spiral or even a ring shape and leads to rotation in the plane of the surface.10 Although some reports showed that the rotation is almost in symmetry, that is, the clockwise and the counterclockwise direction appear in a same probability, no detailed analysis on the rotation direction have been performed.11 We consider that the MTs should show preferential rotations in clockwise or counterclockwise direction according to the handedness of PFs. For example, MTs with the left-handed supertwist of PFs might bend into ring that shows counterclockwise rotation when its front end sticks to the surface (Figure 1). Therefore, the probability to form rings should depend on the inhibition frequency of the

10.1021/bm800639w CCC: $40.75  2008 American Chemical Society Published on Web 07/29/2008

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front end motion of MTs and their rigidity, while the bias in motion should depend on the supertwist structure of PFs in MTs. In this paper, we investigate the motion of MTs and show that MTs assembled in vitro are sometimes have ring or spiral shape when they are driven on a kinesin-fixed surface. These MTs show biased motion which seems to be related to the handedness of PFs in MTs. Taking advantage of this biased motion of MTs, we further try to assemble them into large structures, while keeping the biased motion.

Experimental Section Tubulin Purification. Tubulin was purified from porcine brain by using a high concentration PIPES buffer (1 M PIPES, 20 mM EGTA, 10 mM MgCl2; pH adjusted to 6.8 using HCl); high-molarity PIPES buffer (HMPB) and Brinkley BR buffer 1980 (BRB80) were prepared using dipotassium salt (PIPES-2K; Sigma), and the pH was adjusted using HCl.12 Kinesin Purification. A GFP-fused kinesin-1 consisting of the first 560 amino acids (K560-GFP) was prepared, as described in previously published papers, by partially modifying the expression and purification methods.13 Biotin Labeling and Stoichiometric Estimation. Biotin-labeled tubulin was prepared using biotin-XX-SE (Invitrogen) according to standard techniques.14 The labeling stoichiometry was approximately two per tubulin, which was estimated by spectrometric titration using a 2-(4′-hydroxyphenylazo)benzoic acid (HABA) dye (Dojindo).15 Rhodamine Labeling and Stoichiometric Estimation. Rhodaminelabeled tubulin was prepared using tetramethylrhodamine succinimidyl ester (TAMRA-SE; Invitrogen) according to standard techniques.16 Rhodamine-labeled tubulins were obtained by chemical cross-linking. Dimly and brightly-labeled rhodamine tubulins were obtained and their labeling ratios were 0.03 and 1.2, respectively; these ratios were determined by measuring the absorbance of the protein at 280 nm and the absorbance of tetramethylrhodamine at 555 nm. PFs Number Observation. Rhodamine-labeled MTs were reconstructed from dimly labeled rhodamine tubulin (34 µM) in polymerization buffer (80 mM PIPES-2K, 1 mM EGTA, 5 mM MgCl2, 1 mM GTP, 5% DMSO; pH adjusted to 6.8 using HCl) at 37 °C for 30 min; this solution was then diluted 70-fold in aging buffer (80 mM PIPES2K, 1 mM EGTA, 1 mM MgCl2, 1% DMSO, 10 µM paclitaxel; pH adjusted to 6.8 using HCl) and incubated for 0-24 h at 25 °C. The cross-sectional images of MTs were obtained according to a standard method with several modifications as follows; the paclitaxel-stabilized MTs were centrifuged at 115000 g for 1 h at 30 °C, and the resulting pellets were fixed with fixing buffer (4% glutaraldehyde and 2% tannic acid in 10 mM phosphate buffer pH 7.0), and the fixed pellets were dehydrated with ethanol and embedded to Quetol-651 resin.17 The sliced sections with a thickness of ∼70 nm were stained with uranyl acetate and lead citrate and observed by transmission electron microscope (TEM; H-7650, Hitachi) To determine the distribution of PFs number, only clearly ring-shaped objects were counted. Motility Assay of Single MTs. Rhodamine-labeled microtubules that were diluted into aging buffers, as described above, were used in this analysis. Flow-cells were prepared by placing a coverglass (18 × 18 mm2; MATSUNAMI) on a slideglass (26 × 76 mm2) equipped with a pair of spacers to form a chamber of approximately 4 × 18 × 0.1 mm3 (W × L × H) in dimension. A single layer of Parafilm was used to fix the spacer-separated glasses by heating. The flow-cell was filled with 0.2 mg mL-1 anti-GFP antibody (Invitrogen) for 15 min, followed by a wash with 48 µL of casein solution (80 mM PIPES-2K, 1 mM EGTA, 1 mM MgCl2, ∼0.5 mg mL-1 casein; pH adjusted to 6.8 using HCl). After incubating for 5 min with casein solution to mask the remaining glass surface, 24 µL of 63 nM K560-GFP solution (∼80 mM PIPES-2K, ∼40 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL-1 casein, 1 mM DTT, 4.5 mg mL-1 D-glucose, 50 U mL-1 glucose oxidase, 50 U mL-1 catalase, 10 µM paclitaxel, ∼1% DMSO; pH 6.8)

Figure 2. PFs number observation. (a) The cross-sectional TEM images of MTs: scale bar, 20 nm. (b, c) Distributions of PF numbers in MTs with 0 h (b, n ) 120) and 24 h aging (c, n ) 99). The average of PFs number was 13.8 (b) and 13.6 PFs (c). The majority of MTs consist of 14 PFs in both cases. Assuming that the 14 PFs have lefthanded supertwist and 13 PFs has parallel configuration to MT axis, and the majority of MTs have left-handed supertwist of PFs.

was introduced and incubated for 10 min to bind the kinesins to the antibody. The flow-cell was washed with 32 µL of motility buffer (80 mM PIPES-2K, 1 mM EGTA, 2 mM MgCl2, 0.5 mg mL-1 casein, 1 mM DTT, 4.5 mg mL-1 D-glucose, 50 U mL-1 glucose oxidase, 50 U mL-1 catalase, 10 µM paclitaxel, and ∼1% DMSO; pH 6.8). A diluted solution (24 µL) of MTs (9.6 nM in motility buffer) was then introduced and incubated for 5 min, followed by washing with 50 µL of motility buffer again. Finally, the motility assay was initiated by applying 24 µL of ATP solution (motility buffer supplemented with 5 mM ATP). Motility Assay of MT Bundles. Biotin- and rhodamine-labeled MTs were obtained by polymerizing biotin-tubulin, brightly-labeled rhodaminetubulin, and native tubulin (molar ratio, 45:10:45; final concentration, 24 µM); the solution containing the MTs was then diluted 50-fold with the aging buffer and incubated for 0-24 h at ∼25 °C. The following procedure to perform motility assay was the same as that of single MTs except for a streptavidin-FITC binding steps described as follows. The partial coverage of biotins on the MT surface with streptavidinFITC (ST; Wako) was done by incubating the MTs with ST solution (9.6 nM in motility buffer) for 10 min after fixing the MTs on the kinesins, then the system was initiated by adding ATP solution. The flow-cell was washed before and after this incubation. Microscopic Image Analysis of Motility Assays. For MT motility assays, rhodamine-labeled MTs were illuminated with a 100 W mercury lamp and visualized by epifluorescence microscopy using a PlanApo 60×/1.40 objective (Olympus). The images were captured using a cooled-CCD camera (Cascade II, Nippon Roper) connected to a PC. The motions of the MTs were analyzed using image analysis software (Image Pro Plus 5.1J, Media Cybernetics). Although the inner surface of the coverglass was observed through the glass, the captured images correspond to those observed from the reverse of flow-cell (Figure 3). The clockwise and counterclockwise directions were discussed based on the captured image.

Results and Discussions Biased Motion of Single MTs. Because it is assumed that the handedness of MTs might induce a biased motion, we first determined the distribution of PFs numbers in MTs prepared in vitro according to the reported method.18 The PFs numbers were determined from the transmission electron microscope (TEM) images of the cross-section of MTs (Figure 2a).17 The distribution of PFs numbers is shown in Figure 2b. MTs were found to be mainly composed of 13 (14% of total), 14 (53%), and 15 (18%) PFs, and others (9-12, 16, 17 PFs) were less than 15% from a total observed MT number, n ) 120. As

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Figure 3. Schematic diagram of the system used for motility assay. The flow-cell was attached to an epi-fluorescence microscope equipped with a cooled-CCD camera. In the captured image, the direction of MT rotation (indicated by violet arrow) in the surface plane corresponds to that observed directly inside the flow-cell. In this paper, the clockwise and counterclockwise directions of movement were discussed based on the images captured. For example, the rotation of MTs in a counterclockwise direction is shown in this figure.

reported, generally, 13 PFs are aligned in parallel to the longitudinal axis of the MT, the same as that in vivo, and the 12 PFs are aligned in a right-handed and 14 PFs in a left-handed supertwist, while both right- and left-handed supertwist exist in 15 PFs, though the handedness could have minor irregularities, depending on buffer conditions.5-9 According to these reports, in our case, more than 50% of MTs should have lefthanded supertwist of PFs (14 PFs) and 14% of MTs have PFs without supertwist (13 PFs). Next, we observe the motions of MTs on kinesin-fixed glass surface and to elucidate if there is a biased motion toward the counterclockwise rotation due to the abundance of 14 PFs that are assumed to have left-handed supertwist structure. The motility assay was performed according to the method described in a published paper.13 A flow-cell was first coated with antigreen fluorescence protein (GFP) antibody, and GFP-fused kinesin was allowed to bind to the antibodies. The GFP-fused kinesin is composed of the first 560 amino acids of kinesin-1 with GFP fused at the C-terminus.13 Rhodamine-labeled MTs stabilized by paclitaxel, which is a reagent inhibiting MT disassembly, were introduced into the flow-cell and bound to the kinesins. Then, ATP was added to initiate the movement of MTs on the kinesin-coated glass surface, and the MTs were observed under a fluorescence microscope (Figure 3). The majority of MTs on the kinesin-coated surface were linear, and they showed translational motion with occasional undulation at a constant velocity of 0.36 ( 0.08 µm s-1 ((s.d.; n ) 181, MTs observed within 45 min after ATP addition); some MTs bent into spirals or rings and rotated around a fixed point when the movement of the front ends was inhibited. Typical rotations of MTs observed are shown in Figure 4a. Occasionally, arc-shaped MTs were also observed, the same as reported by Amos and Amos.10 These MTs moved in circles with a linear speed similar to that observed in translational motion. A single turn of the left-handed twist of a long spiralshaped MT is also shown in Figure 4a (bottom); this figure indicates that the sliding force at the tail has a left-handed torque

Figure 4. The images of MTs in rotation (a) and the numbers of MTs that show continuous rotation in the counterclockwise and clockwise directions (b); 0 h (left) and 24 h (right) correspond to the assays in which MTs were aged for 0 and 24 h, respectively. Images of MTs in rotation (a) are indicated as time sequences with an interval of 2.2 s: scale bar, 2 µm. The rotating directions were confirmed by observing the successive images of MTs at 1.1 s intervals for a total 82.5 s.

component that induces the left-handed rotation of the MT around its longitudinal axis. The number of moving MTs was counted by observing the views (view area was 135 × 135 µm2) of various places of the kinesin-coated surface, where each view was observed for 82.5 s. Only those that rotated continuously for more than one turn are counted as rotation. As a result, among 187 MTs observed, 173 showed translational motion, and only 14 rotated. Of these 14 MTs, six were ring-shaped, two were spiral-shaped, five were arc-shaped, and one transformed its shape from spiral to arc; they all rotated in the counterclockwise direction. To ensure if the rotational motion is only by counterclockwise, we made further observation, only focused on the MTs that are in rotation. A total of 23 additional MTs in rotation were found besides the initial 14 MTs, among them 20 in counterclockwise and 3 in clockwise direction. As a result, 34 MTs rotated in the counterclockwise and 3 in clockwise directions out of 37 MTs in rotation. This result is in agreement with the prediction that left-handed supertwist might favor counterclockwise motion. To further elucidate the elementary process of the preferential motion of MTs in the counterclockwise direction, we analyzed the undulation of MTs, all of them in sliding motion. Because the MTs in rotation are often pinned at the front ends to the kinesin-fixed glass surface, we traced the tail end of them to analyze the motion. The angle fluctuation θ of the sliding MTs, defined as the angle formed by the tail ends between two successive directions at 5.5 s intervals, was measured for 82.5 s, as shown in Figure 5a. A negative angle was defined as the leftward deflection of the tail end and a positive angle, as the rightward deflection of the tail. Figure 5b shows the statistics

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Figure 5. Analysis of MTs in motion. (a) Method to trace the undulation of the tail ends of MTs. The redirecting angle θ was defined by the angle, left indicates negative and right indicates positive, formed by the tail ends between two successive directions at 5.5 s intervals. (b, c) Histograms of the angle fluctuations of MTs (left) and the corresponding magnified expression in the vertical axis (right). Total counts were 1792 (b) and 2357 (c). The aging times of MTs were 0 h (b) and 24 h (c).

of the 187 samples. As shown in Figure 5b, the main peak in the histogram is around θ ) 0°, indicating that the majority of MTs moved linearly with a symmetric angle fluctuation, mostly within 40°. The angle fluctuation of MTs appears to be suppressed more significantly in longer MTs than in shorter ones. However, we observed a slight asymmetry in the angle distribution for angles larger than 40° (Figure 5b, right), and therefore, the overall average of angle fluctuation was slightly negative at -3.67 ( 41.01° (n ) 1792), which favors leftward movement. The preferential motion of MTs in the counterclockwise direction might be associated with this preferential motion in the negative angle. We also found that some events were distributed around +180°, indicating that some of MTs were deflected by more than 180° in the counterclockwise direction (confirmed by observing the sequence of sliding images with a high-time resolution of 1.1 s). Interestingly, we found unexpectedly an aging effect, that is, both the number of the ring-shaped MT bundles and the preferential rotation depended on the storage time of MTs in the aging buffer before being placed on the kinesin-coated surface for motility assay. By increasing the aging time from 0 to 24 h, the total number of MTs showing the continuous rotation decreased from 37 (in 53 view areas) to 15 (in 63 view areas; Figure 4b); those counting were done for ∼40 min after starting the motility assay by ATP addition. Furthermore, the ratio of counterclockwise to clockwise rotation (CCW/CW) decreased from 34/3 ()11.3) to 11/3 ()3.7, one ring-shaped MT was indistinguishable about its rotating direction). Also, we observed that, while the MTs in continuous rotations decreased, the MTs rotated only temporally and then changed to translational motion increased by aging.

We investigated the behavior of MTs with aging time. There was no significant effect of aging on the average length and the density of single MTs. For the aging times of 0 and 24 h, the average lengths were 7.8 ( 6.1 µm ((s.d; n ) 143) and 9.2 ( 6.1 µm (n ) 131), respectively, and the numbers of MTs per unit area were 871 ( 240 mm-2 ((s.d; n ) 10 view areas) and 805 ( 163 mm-2 (n ) 10), respectively. On the other hand, an aging effect was observed for the distribution in the PF numbers (Figure 2c). The fraction of the 13 PFs (straight alignment) increased, 15 PFs (in both right- and left-handed supertwist) decreased, and 14 PFs (left-handed) did not change by the aging. As a result, the total ratio of MTs in left-handed supertwist structure decreased by the aging. The aging effect was also observed in the histogram of the angle fluctuation of single MTs (Figure 5c). The center distribution became narrower, and furthermore, the average value of MT tail deflections decreased from -3.67 ( 41.01° to -1.84 ( 20.09° after a 24 h aging. The less asymmetry in angle fluctuation agrees well with the PFs number changes with aging. The increase in the fraction of 13 PFs, which may cause rotations in counterclockwise or clockwise direction by the same probability, leads to less-biased angle fluctuation. We discuss the above aging results in terms of two quantities: one is the ratio of CCW to CW rotations; another is the ratio of rotation to overall motion. The ratio of CCW/CW decreased with aging time, simultaneously the aging caused a shift in the distribution of PFs numbers and the MTs consisted of 13 PFs with straight alignment increased; therefore, we consider that the decrease in the ratio of CCW/CW is due to the decrease in the fraction of left-handed supertwist in the overall PFs. The ratio of rotation to overall motion should depend on the sticking frequency of the front end motion of MTs. The narrower fluctuation angle distribution after 24 h aging (Figure 5c) might be attributed to a less frequency of sticking events. We observed that the inhibition of motion at the front end was less frequent in the case of MTs aged for 24 h than in those aged for 0 h (data not shown). We assume that there is an unstable structure at front tip of MTs such as unclosed C-tubule reported by Kamimura and Mandelkow.19 The structure seems to inhibit the sliding motion at front and induce a ring formation reflecting the handedness of PFs in the rotation direction. The change in the sticking frequency upon aging might be due to the change in this unstable structure of front end in aging solution. As described in the Experimental Section, MTs were polymerized from tubulins in the polymerization buffer containing 5% DMSO and then were diluted 70 times by the aging buffer containing 1% DMSO. Because DMSO is known to stabilize the unstable structure of MTs during polymerization, the decrease of DMSO concentration from 5 to 1% enhanced the depolymerization of this unstable structure during the aging, as reported.19 Accordingly, by aging, the unstable structure became less and the sticking frequency decreased. This explanation is in agreement with the narrower center distribution in the fluctuation angle (Figure 5c). Other evidence seems also to support the interpretation that the unstable structure at the front end accounts for the bending and rotation: The bias toward the counterclockwise rotation was not observed in the literature that performed the experiment under a quite similar condition with the present work, except the absence of DMSO in the polymerization buffer. That is, in that case, there is a less unstable structure that exists at the front ends, therefore, the sticking and bending frequency was low.11 Thus, the above results strongly suggest that the biased motion toward the counterclockwise rotation is originated from the left-

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Figure 6. Size distribution and motility assay of aggregated MTs in ring-shape. (a) Images of ring-shaped MT bundles with various sizes and their size distribution: scale bar, 5 µm. (b) Numbers of rings that show rotation in the counterclockwise and clockwise directions; 0 h (left) and 24 h (right) correspond to the assays in which MTs were aged for 0 and 24 h, respectively. The rotating directions were confirmed by observing the successive images of rings at 3.1 s intervals for a total 12.4 s.

handed supertwist of PFs. Because the number of PFs cannot be a direct evidence to determine the handedness of PFs in MTs, a direct observation of the handedness is needed in the future study.8 Biased Motion of Aggregated MTs. In nature, macroscopic asymmetric structures are aggregated from molecular levels through asymmetrical structures or processes, whereupon the asymmetry is propagated through well-organized hierarchal structure of molecules. Now we attempt to aggregate MTs into large assemblies while preserving the preferential counterclockwise motion by taking advantage of the asymmetric motion of MTs. To accomplish this, we employed a method to cross-link MTs through streptavidin-biotin (ST-B) interaction.20 During the sliding motion of biotin-labeled MTs on a kinesin-fixed surface, MTs might be assembled into larger bundles with preferential polarity by adding ST. Cross-linking occurs only when the sliding MTs encounter each other during sliding to form a bundle of MTs. As a result, MT bundles, which might be composed of several tens of single MTs, were observed after 12 h of cross-linking. At this time point, most of the bundles were linear; however, a few bundles changed to a stable ring shape later on, presumably through an intermediate spiral state (see Supporting Information). During the dynamic aggregating process, some of the rotating MT bundles or the linear MT bundles slowed down due to aggregation of MTs moving in opposite directions. With the lapse of observation time, less and less MT bundles in translational motion remained in the observation area since the motion was terminated at the walls of the flow-cell, while those in rotation remains in the observation area for more than 14 h. We have observed 121 ring-shaped MT bundles of various sizes, ranging from ∼1 to 12.6 µm (mean diameter, 3.9 ( 2.2 µm; n ) 121; Figure 6a). Among these ring-shaped MT bundles, 86 (71%) rotated in the counterclockwise direction (Figure 6b), demonstrating that the biased motion of single MTs (counter-

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clockwise preferred to clockwise rotation) was preserved in the aggregated MT bundles. The angular velocity of the ring-shaped MT bundles depended on their size. The line velocity of the rotation remained almost constant, independent of the ring size. For example, the line velocity of counterclockwise rotation was found to be 28 ( 14 nm s-1 ((s.d; n ) 5), independent of the size, and was comparable to that of single MTs (28 ( 23 nm s-1, n ) 19); here we found only five ring-shaped MT bundles in the movies to measure velocity and all of them were in the counterclockwise direction. These values are much less than that of the single MTs without ST modification (360 nm s-1). Because the velocities of aggregated MTs with ST labeling were observed at 12 h after ATP addition, this can be attributed to consumption of ATP during 12 h of cross-linking and ST labeling that cause steric hindrance. Occasionally, the aggregation processes were observed when the density of MTs was high enough to have frequent collisions of MTs. When the front of a sliding microtubule in linear shape encounters a rotating ring, it was spooled into the ring, the same as reported.20 The ringshaped MT bundles was stabilized by the cross-linking reaction by streptavidin-biotin that were sparsely existed on the surfaces of MTs. We assume that, in general, once an MT bundle encounters a single MT along the direction of preferential undulation, the merged bundle might bend to form a ring in the same direction as that in which the single MT bends. As mentioned above, since the number of single MTs moving in the counterclockwise direction was more than that in the clockwise direction, the ringshaped MT bundles rotating in the counterclockwise direction became preferential. No distinct tendency toward a preferential rotation was observed in case of ring-shaped MT bundles on increasing or decreasing the concentration of the cross-linker (streptavidin), except that the rotational lifetime dramatically decreased with an increase in streptavidin concentration due to a disturbance of the interaction between kinesin and MTs. On the other hand, the CCW/CW increased with an increase in the concentration of MT or kinesin. At 4 h after ATP addition, a maximum CCW/ CW of 222/16 (≈14/1) was obtained at 240 nM MT and 63 nM kinesin. The aging effects were also observed in the case of aggregated MTs. The total number of ring-shaped MT bundles decreased from 121 to 43, and the ratio CCW/CW decreased from 86/30 ()2.9) to 25/18 ()1.4), as is shown in Figure 6b. It is natural to consider that the decrease in the preferential rotation ratio CCW/CW with aging time for aggregated MTs is also due to aging effect on single MTs. The observed process in which the MT in translational motion spooled into the rotating MTs supports this interpretation.

Conclusions We showed that single MTs, which seem to consist mainly of left-handed protofilament supertwist, are sometimes bent by kinesin-driven sliding into a ring which subsequently rotates counterclockwise. Furthermore, when the tip of a sliding microtubule encounters a rotating ring, it is deflected according to the direction of rotation of the ring, and can then be aligned with and incorporated stably into the ring using sparse streptavidin-biotin cross-links. Thus, kinesin-driven sliding can be harnessed to specify the formation of a microtubule ring, containing several parallel microtubules. By the above process, MTs aggregated into large bundles in ring-shape sized from 1 to 12.6 µm in diameter, with a preferential counterclockwise

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motion. We expect that this finding will provide new insights into the handedness of biological systems. For example, MT bundles with clockwise motion might be generated by assembling MTs containing mainly 12 PFs. Motor proteins have been proposed as the building blocks of ATP-fueled biomachines, where the assembly of the motor proteins into highly ordered structures with well-controlled polarity and handedness is crucial.20-22 The present work might find a clue to control the handedness of the motor protein assembly and thus overcomes the largest obstacle in constructing biodevices or biomachines with asymmetric motion. Acknowledgment. The plasmid construct of K560-GFP was generously provided by R. D. Vale and M. Tomishige. We appreciate the fruitful discussion with K. Sato, T. Itoh, K. Takiguchi, K. Sano, and H. Furukawa. Concerning the purification of K560-GFP, we thank A. Nakatomi and M. Takahashi for technical support. This research was financially supported by the Ministry of Education, Science, Sports and Culture Japan (Grant-in-Aid of Specially Promoted Scientific Research). Supporting Information Available. A movie in which a giant MT bundle is forming a ring-shape from linear MTs. This information is available free of charge via the Internet at http:// pubs.acs.org.

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(3) Thitamadee, S.; Tuchihara, K.; Hashimoto, T. Nature 2002, 417, 193– 196. (4) Amos, L. A.; Klug, A. J. Cell Sci. 1974, 14, 523–549. (5) Arnal, I.; Wade, R. H. Curr. Biol. 1995, 5, 900–908. (6) Ray, S.; Meyho¨fer, E.; Milligan, R. A.; Howard, J. J. Cell Biol. 1993, 121, 1083–1093. (7) Chre´tien, D.; Wade, R. H. Biol. Cell 1991, 71, 161–174. (8) Chre´tien, D.; Kenney, J. M.; Fuller, S. D.; Wade, R. H. Structure 1996, 4, 1031–1040. (9) Chre´tien, D.; Fuller, S. D. J. Mol. Biol. 2000, 298, 663–676. (10) Amos, L. A.; Amos, W. B. J. Cell Sci. 1991, 14 (Suppl), 95–101. (11) Vale, R. D.; Coppin, C. M.; Malik, F.; Kull, F. J.; Milligan, R. A. J. Biol. Chem. 1994, 269, 23769–23775. (12) Castoldi, M.; Popov, A. V. Protein Expression Purif. 2003, 32, 83– 88. (13) Case, R. B.; Pierce, D. W.; Hom-Booher, N.; Hart, C. L.; Vale, R. D. Cell 1997, 90, 959–966. (14) Hyman, A.; Drechsel, D.; Kellogg, D.; Salser, S.; Sawin, K.; Steffen, P.; Wordeman, L.; Mitchison, T. Methods Enzymol. 1991, 196, 478– 485. (15) Green, N. M. Methods Enzymol. 1970, 18, 418–424. (16) Peloquin, J.; Komarova, Y.; Borisy, G. Nat. Methods 2005, 2, 299– 303. (17) Scheele, R. B.; Bergen, L. G.; Borisy, G. G. J. Mol. Biol. 1982, 154, 485–500. (18) Brown, T. B.; Hancock, W. O. Nano Lett. 2002, 2, 1131–1135. (19) Kamimura, S.; Mandelkow, E. J. Cell Biol. 1992, 118, 865–875. (20) Hess, H.; Clemmens, J.; Brunner, C.; Doot, R.; Luna, S.; Ernst, K.H.; Vogel, V. Nano Lett. 2005, 5, 629–633. (21) Diehl, M. R.; Zhang, K.; Lee, H. J.; Tirrell, D. A. Science 2006, 311, 1468–1471. (22) Kakugo, A.; Sugimoto, S.; Gong, J. P.; Osada, Y. AdV. Mater. 2002, 14, 1124–1126.

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