Controlling the Bias of Rotational Motion of Ring-Shaped Microtubule

Dec 4, 2014 - Study of active self-assembly using biomolecular motors. Arif Md. Rashedul Kabir , Akira Kakugo. Polymer Journal 2018 50 (12), 1139-1148...
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Controlling the Bias of Rotational Motion of Ring-Shaped Microtubule Assembly Shoki Wada,†,‡ Arif Md. Rashedul Kabir,‡,§ Ryuzo Kawamura,∥ Masaki Ito,† Daisuke Inoue,† Kazuki Sada,†,§ and Akira Kakugo*,†,§ †

Graduate School of Chemical Sciences and Engineering and §Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan Department of Chemistry, Saitama University, Saitama 338-8570, Japan

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ABSTRACT: Biomolecular motor system microtubule (MT)kinesin is considered a building block for developing artificial microdevices. Recently, an active self-organization method has been established to integrate MT filaments into ring-shaped assembly that can produce rotational motion both in the clockwise and in the counterclockwise directions. In this work, we have investigated the effect of parameters such as MT and kinesin concentration, length, and rigidity of MT and type of kinesin (structure of tail region) on the preferential rotation of the ring-shaped MT assembly produced in an active selforganization. We elucidated that these factors can significantly affect the bias of rotation of the ring-shaped MT assembly, which seems to be related to the fluctuation of leading tip of moving MT filaments. This new finding might be important for designing handedness regulated artificial biomachine using the ringshaped MT assembly in future.

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MT tends to rotate R-MTs in CCW direction.22 In the present work, we investigated the role of some other factors that are yet to be explored, for example, length of MT and type of kinesin (structure of tail region), concentration of MT and kinesin, and rigidity of MT in controlling the bias of the rotational motion of R-MTs. We found that flexibility of MT’s movement in an in vitro motility assay, which can be tuned by changing above factors, plays an important role in determining the fraction of R-MTs rotating in the CW or in the CCW direction. This work thus offers a pathway to control the population of R-MT rotating in the CW or in the CCW direction, which in turn would be important for the development of artificial biomachines with regulated handedness.

INTRODUCTION Nowadays development of biomaterial-based artificial devices has been attracting much attention in nanotechnology.1−6 One of those biomaterials, namely, biomolecular motor system, for example, microtubule (MT)-kinesin, appeared to be a promising candidate as the building block of the artificial devices because of their several attractive features compared to manmade machines.1,3,4 Use of biomolecular motor system MT-kinesin in developing artificial devices mainly relies on the “in vitro motility assay” technique where MTs, in the presence of adenosine triphosphate (ATP), demonstrate sliding motion on a kinesin-coated substrate.7−10 Structural integration of the building block, that is, MT-kinesin system is a prerequisite for employing them in the development of artificial devices and to meet this requirement a method named active self-organization (AcSO) has recently been introduced based on the in vitro motility assay.11 Depending on the experimental conditions, AcSO provides bundle-, network-, and ring-shaped assemblies of MTs, which can generate translational, amoeboid, and rotational motion, respectively.12−15 Among these MT assemblies, the ring-shaped MT assembly (R-MT) is capable of storing a huge amount of mechanical energy11,16,17 and can produce continuous rotational motion both in clockwise (CW) and in the counterclockwise (CCW) directions without changing the position of the mass center.18−23 Therefore, RMTs have been attracting attention for future potential applications in nanotechnology where tuning the rotational direction might play a crucial role. So far, change in supertwist structure of MT was found effective in altering the rotational direction of R-MTs where left-handed supertwist structure of © 2014 American Chemical Society

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EXPERIMENTAL SECTION

Preparation of Tubulin and Kinesin. Tubulin was purified from porcine brain using a high-concentration PIPES buffer (1 M PIPES, 20 mM EGTA, and 10 mM MgCl2; pH 6.8). The high-concentration PIPES buffer and 80 mM PIPES brain reconstitution buffer (BRB80) were prepared using PIPES from Sigma, and the pH was adjusted using KOH.24 Recombinant kinesin-1 consisting of the 573 amino acid residues was prepared from the N-terminus of conventional human kinesin (kin573). This recombinant kinesin (kin573) fused with His-tag at the N-terminus was expressed in Escherichia coli and purified through the general method using Ni-NTA-agarose. Green fluorescent protein (GFP) fused kinesin-1 consisting of the first 560 amino acids Received: October 29, 2014 Revised: December 3, 2014 Published: December 4, 2014 374

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Microscopic Image Capture. To study the motility of MTs, the samples were illuminated with a 100 W mercury lamp and visualized by an epifluorescence microscope (Eclipse Ti, Nikon) using an oilcoupled Plan Apo 60 × 1.40 objective (Nikon). UV cutoff filter blocks (GFP-HQ: EX455−485, DM495, BA500−545; Nikon) were used in the optical path of the microscope. These filter blocks allowed visualization of samples but eliminated the UV portion of the radiation, thus, minimizing the harmful effect of UV radiation on the samples. Images were captured using a cooled-CMOS camera (NEO sCMOS, Andor) connected to a PC. To capture a field of view for more than several minutes, a ND filter (ND4, 25% transmittance) was inserted into the illumination light path of the fluorescence microscope to avoid photobleaching of the Alexa 488-labeled MTs. Image Analysis of Motility Assays. Movies of the motility assays of MTs captured by the fluorescence microscope were analyzed using image analysis software (ImageJ).

(kin560) was prepared as described in a previous report by partially modifying the expression and purification method.25 Preparation of Labeled Tubulin. Alexa 488-labeled tubulin was prepared using Alexa Fluor 488 succinimidyl ester (Alexa Fluor 488SE; Invitrogen) according to the standard technique.26 The labeling ratio of Alexa 488- modified tubulin was 1.0. This ratio was determined by measuring the absorbance of the protein and Alexa 488 at 280 and 495 nm, respectively. Biotin (Bt) Labeling and Stoichiometric Estimation. Bt-labeled tubulin was prepared using biotin-XX-SE (Invitrogen) according to the standard technique.27 The labeling stoichiometry was approximately 0.76 per tubulin heterodimer, which was estimated by spectrometric titration using 2-(4-hydroxyphenylazo) benzoic acid (HABA) dye (Wako).28 Preparation of Microtubule (MT). For polymerization of GTPMT using guanosine triphosphate (GTP), 70 μM of tubulin mix (Alexa tubulin:biotin tubulin = 1:1 in molar ratio) in a polymerization buffer (80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 5% DMSO, 1 mM GTP; pH ∼ 6.8) was incubated at 37 °C for 30 min. The solution containing the MTs was then diluted with motility buffer (80 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 0.5 mg mL−1 casein, 1 mM DTT, 10 μM paclitaxel, and ∼1% DMSO; pH 6.8). For polymerization of GMPCPP-MT using guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP), 40 μM of tubulin mix (Alexa tubulin:biotin tubulin = 1:1 in molar ratio) in a polymerization buffer containing 1 mM GMPCPP (Jena Bioscience, Jena, Germany) and 5 mM MgCl2 was incubated at 37 °C. After 10 min of incubation, the polymerized MTs were diluted 1000-fold into an elongation buffer containing 1 μM tubulin mix, 5 mM MgCl2, and 1 mM GMPCPP and incubated 4 h. For preparing length tuned MT, a Hamilton syringe (inner diameter: 2.06 mm) and a PEEK tube (nominal inner diameter: 0.26 mm) were used to shear MTs. A total of 30 μL of MT solution was passed backand-forth through the syringe-mounted PEEK tube by manual operation of the syringe.29 Change in MT length before and after shearing treatment was manually measured using the image analysis software “ImageJ”. Active Self-Organization (AcSO) of MTs. Flow cells with dimensions of 9 × 2.5 × 0.45 mm3 (L × W × H) were assembled from two cover glasses of sizes (9 × 18) mm2 and (40 × 50) mm2 (MATSUNAMI) and double-sided tape was used as the spacer. First, the flow cell was filled with 5 μL of 0.1 mg mL−1 anti-GFP antibody (Invitrogen) or 5 μL of casein buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2 and ∼0.5 mg mL−1 casein; pH adjusted to 6.8). The flow cell coated with antibody was then washed with casein buffer. After incubation of both types of flow cell for 2 min, K560 or K573 kinesin solution (∼80 mM PIPES, ∼40 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL−1 casein, 1 mM DTT, 10 μM paclitaxel, and ∼1% DMSO; pH 6.8) of prescribed concentrations were introduced into antibody coated and casein coated flow cell, respectively. The flow cells were incubated for 2 min to allow the kinesins to bind to the antibody or casein-coated glass surface. After washing the flow cells with 10 μL of motility buffer, 5 μL of 1000 nM MT solution (taxol stabilized GTP-MT or taxol stabilized GMPCPP-MT) was then introduced and incubated for 2 min, followed by washing with 5 μL of motility buffer (80 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 0.5 mg mL−1 casein, 1 mM DTT, 10 μM paclitaxel, and ∼1% DMSO; pH ∼ 6.8). Next, 5 μL of 100 nM streptavidin (St;Wako) solution was introduced so as to maintain the cross-linker ratio (St/Bt) at 1/3.8 in the flow cell and allowed for 2 min incubation, followed by washing with 5 μL of motility buffer. Finally, AcSO was initiated by applying 5 μL of ATP solution (motility buffer supplemented with 5 mM ATP). The time of the ATP addition was set as 0 h. The aforementioned experiments were performed at room temperature in the inert chamber system (ICS).15,30 The flow cell was placed inside the inert chamber after the addition of ATP, as described in previous reports.15,30 Humid nitrogen gas was continuously passed through the chamber to remove existing oxygen from the chamber. The first microscopic observation was performed after passing the nitrogen gas for 60 min. In this work ∼60 min was found to be the time by which the ring-shaped MT assemblies attained a stable state.

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RESULTS AND DISCUSSION The AcSO of MTs was performed inside the inert chamber system by using the St−Bt interaction among sliding MTs in an

Figure 1. Effect of kinesin (kin560) concentration on the preferential rotation of R-MTGTP formed by active self-organization of GTP-MTs. Here, nCCW/ntotal (%) represents the percentage of ring-shaped MT assemblies rotating in CCW direction, where nCCW and ntotal stand for the number of ring-shaped MT assembly rotating in the CCW direction and the total number of ring-shaped MT assembly considered, respectively. Average length and concentration of MT filaments used in the AcSO were fixed at ∼17 ± 12.8 μm and 1000 nM, respectively. The rotational direction of R-MTGTP was confirmed by monitoring successive images of rotating R-MTGTP at 10 s intervals for at least 180 s. Error bar: standard error of mean.

in vitro motility assay as reported in our previous work.15,21 In this work the cross-linker ratio (St/Bt) was fixed at 1/3.8 that preferentially allowed the formation of ring-shaped MT assembly (R-MT).12,13 The rotational direction of R-MT was evaluated after 1 h of AcSO by which time the system was found to attain a stable state.21 For evaluating the rotational direction of R-MT, we arbitrarily considered an area of 13.4 × 104 μm2, and all the R-MTs available in this area were considered for analyses. First we have investigated the effect of kinesin concentration on the preferential rotational direction of R-MT. For this, MTs were prepared by polymerizing tubulin using GTP and are termed hereafter as GTP-MTs. Ring-shaped assemblies obtained from GTP-MTs will be termed hereafter as R375

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Figure 4. Effect of length of GTP-MT on the preferential rotation of R-MTGTP formed by active self-organization on a substrate coated with kin560 (closed circle) and Kin573 (open circle). Concentration of kinesin (in both cases of kin560 and Kin573) and MT used in these experiments were fixed at 100 and 1000 nM, respectively. Error bar: standard error of mean.

Figure 2. Effect of kinesin (kin560) concentration on the size (inner diameter) of R-MTGTP formed by the active self-organization (AcSO) of GTP-MTs. Inset shows the relationship between size (inner diameter) and the preferential CCW rotation of R-MT GTP. Representative fluorescence microscopy images (inset) show the RMTGTP formed when kinesin concentration used in the AcSO were 138, 1100 and 2200 nM. Scale bar: 10 μm. Error bar: standard error of mean (*P < 0.01, #P < 0.05 using student’s t test).

Figure 5. Effect of length of GTP-MT (square) and GMPCPP-MT (circle) on the preferential rotation of ring-shaped MT assemblies formed by active self-organization on a Kin573 coated substrate. Concentration of kin573 and MTs used in these cases were fixed at 100 and 1000 nM, respectively. Error bar: standard error of mean.

Figure 3. Effect of concentration of GTP-MT on the preferential rotation of R-MTGTP formed by active self-organization on a substrate coated with kin560. Inset shows the fluorescence microscopy images of GTP-MTs at different concentrations employed in these experiments. Scale bar: 20 μm; error bar: standard error of mean.

preferential CCW rotation of R-MTGTP. With the increase in kin560 concentration, percentage of R-MTGTP rotating in CCW was found to increase (∼90% at 275 nM). Further increase in kin560 concentration brought no substantial change in the preferential rotational direction of R-MTGTP. When kin560 concentration was varied, the size of R-MTGTP was also changed. As shown in Figure 2, average size (inner diameter) of R-MTGTP decreased from 7.8 μm ± 0.71 (average ± standard error of mean) to 3.4 μm ± 0.24 when the kin560 concentration was increased from 138 to 2200 nM. From the student’s t test, this change in size of R-MTGTP due to the change in kin560 concentration was found to be statistically significant. However, no clear correlation was observed between the size and biased rotation of R-MTGTP (Figure 2, inset).

MTGTP. The kinesin employed here in the AcSO was a dimeric construct consisting of the first 560 amino acid residue of human kinesin-1 and will be termed as kin560. First we varied the concentration of kin560 in the AcSO and Figure 1 shows the consequent change in the preferential rotation of R-MTGTP with the change of kin560 concentration. From Figure 1, it could be observed that at ralatively low kin560 concentration (69 nM) majority of R-MTGTP (∼73%) rotated preferentially in the CCW direction. This preferential CCW rotation of R-MTGTP is in good agreement with that reported in our previous work,22 and hence, we conclude that left-handed supertwist structure of GTP-MT filaments was responsible for the observed 376

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Figure 6. Schematic illustration showing the origin of counterclockwise motion of R-MT where fluctuation of leading tip of moving MT filament with left handed supertwist plays an important role in determining the probability of counterclockwise rotation of R-MT (a). During the sliding of MT filament inhibition of movement at any point may enhance the lateral force shown by the red arrow and difference in extent of lateral force gives rise in different probability of rotational direction. For example at low kinesin concentration (b), fluctuation of the leading tip of moving MT might be higher than that at high kinesin concentration (c). Here Ck stands for kinesin concentration. Thickness of green arrow in (b) and (c) represents the probability of counterclockwise motion of moving MTs.

Next, fixing the kin560 concentration at 100 nM, we changed the concentration of MT to investigate the role of MT density on the bias of rotation of R-MTGTP. The result is shown in Figure 3, which reveals that the density of MT has negligible effect on the bias of rotational direction of R-MTGTP. In our previous work, we discussed that biased flactuation of the moving MT filament caused by its supertwist strucuture finally determined the preferential rotational direction of the RMTGTP.22 This argument next prompted us to investigate the effect of length of MT on the rotational direction of R-MTGTP. For this, GTP-MT with different lengths were prepared by applying shear stress using a micro syringe, as reported in literature.29 Figure 4 shows the effect of length of GTP-MT on the rotational direction of R-MTGTP. On decreasing the length of GTP-MT, the percentage of R-MTGTP rotating in the CCW direction was found to decrease. This result could be accounted for by increased fluctuation of leading tip of moving MT due to the decrease in MT length and rigidity, where flexural rigidity of MT is known to decrease with the decrease in the MT length.31 This interpretation is compatible with the result of the AcSO of MTs that produced linear-shaped assembly, where rigid MTs with less fluctuation produced highly polarized assembled structures.32 This argument might also be similarly applicable to the change in the bias of rotational direction of R-MTGTP when we varied the kin560 concentration, as discussed above. When kinesin concentration was decreased, the fluctuation of the leading tip of moving MTs might has increased and MTs showed pivoting like behavior.33 In the AcSO single MT filaments are known to produce MT-bundles first, which then form ring-shaped MT assemblies.17,21 Electron microscopic images of MT bundles and ring-shaped MT assembly17,34 revealed that a large number of single MT filaments are incorporated in each of these assembled structures. Prior to forming the ring-shaped MT assemblies, a metastable state was reported when colliding MT filaments formed coiled structures. Leading end of the coiled MT structure, that was associated with a kinked domain, therefore still seems to be able to interact with kinesin, which is primarily determined by the supertwist helical structure of MTs as supported by the transmission electron microscopy image of single MT filament.22

Now, since length of kinesin is also known to affect the movement of MTs in an in vitro motility assay,35 we have also varied the strucuture of tail region of the kinesin at this point. Instead of kin560, we have next used a kinesin consisting of the 573 amino acid residue of conventional human kinesin, as discussed in the Experimental Section (kin573). Kin573 might be more flexible than the kin560 and thereby may affect the mechanical coupling with moving MT.35 As shown in Figure 5, when kin573 was employed in the AcSO, where we also varied the length of GTP-MT, majority of the R-MTGTP still rotated in the CCW direction, although the percentage was not as high as that observed in case of kin560 (Figure 4). In addition to this, length of GTP-MT was found to have negligible effect on the rotational direction of the R-MTGTP produced on the kin573 coated substrate, which is clearly different from that observed in the case of kin560 coated substrate. These results imply that not only the fluctuation of leading tip of moving MT, but also the extent of mechanical coupling with kinesin during movement of MT determines the preferential rotation of R-MTs. To further confirm, we next performed AcSO of MTs on the kin573 coated substrate by changing the rigidity of MT. Rigidity of MT was tuned by polymerizing tubulin in the presence of a GTP analogue, guanosine-5′-[(α,β)-methyleno] triphosphate (GMPCPP), instead of the GTP.36 The MTs prepared in the presence of GMPCPP (GMPCPP-MT) are structurally stable compared to the GTP-MTs and rigidity of the GMPCPP-MTs is ∼2-fold higher than that of the GTP-MT.36 In addition to this, a majority of the GMPCPP-MTs are known to consist of 14 protofilaments, providing the left-handed supertwist structure, similar to the case of GTP-MT prepared in our work.14,22 Since the ring-shaped assemblies of GMPCPP-MT (R-MTGMPCPP) were not found to form as frequently as in the case of R-MTGTP, all the R-MTGMPCPP available in an area of 33.5 × 104 μm2 were considered in our analyses. Figure 5 describes the result obtained from the AcSO of GMPCPP-MT where we also varied the length of GMPCPP-MT. As shown in this figure, employment of GMPCPP-MT in the AcSO brought no substantial change in terms of the preferential rotational direction of R-MTGMPCPP, that is, percentage of R-MTGMPCPP rotating in CCW was almost similar to that observed in the case of R-MTGTP. However, R-MTGMPCPP formed from relatively shorter GMPCPP-MTs showed a higher tendency to 377

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(8) Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Nano Lett. 2002, 2, 113−116. (9) Hess, H.; Bachand, G. D.; Vogel, V. Chem.Eur. J. 2004, 10, 2110−2116. (10) Kabir, A. M. R.; Kakugo, A.; Gong, J. P.; Osada, Y. Macromol. Biosci. 2011, 11, 1314−1324. (11) Hess, H.; Clemmens, J.; Brunner, C.; Doot, R.; Luna, S.; Ernst, K. H.; Vogel, V. Nano Lett. 2005, 5, 629−633. (12) Tamura, Y.; Kawamura, R.; Shikinaka, K.; Kakugo, A.; Osada, Y.; Gong, J. P.; Mayama, H. Soft Matter 2011, 7, 5654−5659. (13) Kawamura, R.; Kakugo, A.; Osada, Y.; Gong, J. P. Nanotechnology 2010, 21, 145603−1−145603−11. (14) Kawamura, R.; Kakugo, A.; Shikinaka, K.; Osada, Y.; Gong, J. P. Biomacromolecules 2008, 9, 2277−2282. (15) Kabir, A. M. R.; Inoue, D.; Kakugo, A.; Sada, K.; Gong, J. P. Polym. J. 2012, 44, 607−611. (16) Lam, A. T.; Curschellas, C.; Krovvidi, D.; Hess, H. Soft Matter 2014, 10, 8731−8736. (17) Liu, H.; Spoerke, E. D.; Bachand, M.; Koch, S. J.; Bunker, B. C.; Bachand, G. D. Adv. Mater. 2008, 20, 4476−4481. (18) Luria, I.; Crenshaw, J.; Downs, M.; Agarwal, A.; Seshadri, S. B.; Gonzales, J.; Idan, O.; Katira, J. P.; Pandey, S.; Nitta, T.; Phillpota, S. R.; Hess, H. Soft Matter 2011, 7, 3108−3115. (19) Kabir, A. M. R.; Wada, S.; Inoue, D.; Tamura, Y.; Kajihara, T.; Mayama, H.; Sada, K.; Kakugo, A.; Gong, J. P. Soft Matter 2012, 8, 10863−10867. (20) Idan, O.; Lam, A. T.; Kamcev, J.; Gonzales, J.; Agarwal, A.; Hess, H. Nano Lett. 2012, 12, 240−245. (21) Inoue, D.; Kabir, A. M. R.; Mayama, H.; Gong, J. P.; Sada, K.; Kakugo, A. Soft Matter 2013, 9, 7061−7068. (22) Kakugo, A.; Kabir, A. M. R.; Hosoda, N.; Shikinaka, K.; Gong, J. P. Biomacromolecules 2011, 12, 3394−3399. (23) Ito, M.; Kabir, A. M. R.; Inoue, D.; Torisawa, T.; Toyoshima, Y.; Sada, K.; Kakugo, A. Polym. J. 2014, 46, 220−225. (24) Castoldi, M.; Popov, A. V. Protein Express. Purif. 2003, 32, 83− 88. (25) Case, R. B.; Pierce, D. W.; Hom-Booher, N.; Hart, C. L.; Vale, R. D. Cell 1997, 90, 959−966. (26) Peloquin, J.; Komarova, Y.; Borisy, G. Nat. Methods 2005, 2, 299−303. (27) Hyman, A.; Drechsel, D.; Kellogg, D.; Salser, S.; Sawin, K.; Steffen, P.; Wordeman, L.; Mitchison, T. Methods Enzymol. 1990, 196, 478−485. (28) Green, N. M. Methods Enzymol. 1970, 18, 418−424. (29) Jeune-Smith, Y.; Hess, H. Soft Matter 2010, 6, 1778−1784. (30) Kabir, A. M. R.; Inoue, D.; Kamei, A.; Kakugo, A.; Gong, J. P. Langmuir 2011, 27, 13659−13668. (31) Pampaloni, F.; Lattanzi, G.; Jonás,̌ A.; Surrey, T.; Frey, E.; Florin, E. L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10248−10253. (32) Kawamura, R.; Kakugo, A.; Osada, Y.; Gong, J. P. Langmuir 2009, 26, 533−537. (33) Howard, J.; Hudspeth, A. J.; Vale, R. D. Nature 1989, 342, 154− 158. (34) Liu, H.; Bachand, G. D. Cell. Mol. Bioeng. 2013, 6, 98−108. (35) Bieling, P.; Telley, I. A.; Piehler, J.; Surrey, T. EMBO Rep. 2008, 9, 1121−1127. (36) Mickey, B.; Howard, J. J. Cell Biol. 1995, 130, 909−917. (37) Vale, R. D.; Coppin, C. M.; Malik, F.; Kull, F. J.; Milligan, R. A. J. Biol. Chem. 1994, 269, 23769−23775.

preferential rotation in the CCW direction than that formed from longer GMPCPP-MTs. This could be accounted for by the frequent transition of the protofilament number often observed in longer GMPCPP-MTs that can cancel the biased motion toward the CCW direction.37 This result is also consistent with the arugument that flexibility of kinesin can affect the rotational direction of R-MTGTP. Establishment of a new methodology to enhance the rigidity of MTs keeping the protofilament configuration unaltered might allow an effective production of R-MTs rotating in the CCW direction that would be addressed in future work.

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CONCLUSIONS In conclusion, we have investigated the effect of length of MT and type of kinesin, concentration of MT and kinesin, and rigidity of MT on the bias of the rotational direction of R-MTs without changing the supertwist structure of the MT filament. Among these factors, the kinesin concentration, length of MT, and type of kinesin were found to strongly affect the preferential rotational direction of R-MTs. Our work suggests that fluctuation of the leading tip of moving MTs plays an important role in determining the bias of the preferential rotational direction of R-MTs. As schematically described in Figure 6, less fluctuation during the movement of MTs on a kinesin coated substrate can efficiently bias the preferential rotational direction of R-MTs toward CCW direction. Tuning the fluctuation during MT’s movement in an in vitro motility assay by changing factors studied in our work offers means to regulate the biaseness of R-MTs rotating in a specific direction. Therefore, this work might be important in designing handedness regulated artificial biomachine using the ringshaped MT assembly.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: +81-11-706-3474. Author Contributions ‡

These two authors contributed equally to this work (S.W. and A.M.R.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by PRESTO (Japan Science and Technology Agency) and Grant-in-Aid for Scientific Research on Innovative Areas named “Molecular robotics” (Grant Number 24104004) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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REFERENCES

(1) van den Heuvel, M. G. L.; Dekker, C. Science 2007, 317, 333− 336. (2) Kakugo, A.; Sugimoto, S.; Gong, J. P.; Osada, Y. Adv. Mater. 2002, 14, 1124−1126. (3) Hess, H. Annu. Rev. Biomed. Eng. 2011, 13, 429−450. (4) van den Heuvel, M. G. L.; de Graaff, M. P.; Dekker, C. Science 2006, 312, 910−914. (5) Chan, V.; Asada, H. H.; Bashir, R. Lab Chip 2014, 14, 653−670. (6) Bachand, G. D.; Bouxsein, N. F.; VanDelinder, V.; Bachand, M. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2014, 6, 163−177. (7) Kron, S. J.; Spudich, J. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6272−6276. 378

dx.doi.org/10.1021/bm501573v | Biomacromolecules 2015, 16, 374−378