Communication pubs.acs.org/Biomac
Controlled Clockwise−Counterclockwise Motion of the Ring-Shaped Microtubules Assembly Akira Kakugo,*,†,‡ Arif Md. Rashedul Kabir,§ Natsuki Hosoda,§ Kazuhiro Shikinaka,⊥ and Jian Ping Gong*,† †
Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan § Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan ⊥ Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei 184-8588, Japan ‡
S Supporting Information *
ABSTRACT: The microtubule (MT)−kinesin system has been proposed as the building block of biomolecular motor based artificial biomachines. Considerable efforts have been devoted to integrate this system that produced a variety of ordered structures including the ring-shaped MT assembly which is being considered as a promising candidate for the further development of the biomachines. However, lack of proper knowledge that might help tune the direction of motion of ring-shaped microtubule assembly from counterclockwise to clockwise direction, and vice versa, significantly restricted their potential applications. We report our success in controlling the direction of rotational motion of ring-shaped MT assembly by altering the preparation conditions of microtubules. The change in the direction of rotation of MT rings could be interpreted in terms of the accompanied structural rearrangement of the MT lattice. For achieving handedness-regulated efficient biomachines having tunable asymmetric property, our study will be significantly directive.
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involved in organelle transportation serving as tracks for the motors. To demonstrate unidirectional motion of the motors along MTs, in vitro motility assay system has been developed where molecular motors are fixed to a substrate surface to give a relative motion of the MTs and vice versa. MTs propelled by substrate fixed motor proteins have shown translational motions along their left- or right-handed supertwist pitch of PFs.4 Because of diversified in vivo functions, the MT−motor system had been proposed as the building block of artificial nano- or microactuators.8,9 Recently, an active self-organization (AcSO) method has been demonstrated to integrate MTs into ordered structures to quest for emergent functions as nature does, by employing a specific streptavidin (St)−biotin (Bt) interaction during the sliding motion of MTs on a kinesi-coated surface in the presence of ATP.10 This method brought a wide variety of MT assemblies in size or shape in response to the preparation conditions, i.e., tubulin concentration, degree of biotin labeling, and St/Bt ratio.11 Ring-shaped MT assemblies obtained through the AcSO also showed preferential counterclockwise (CCW) rotation. 12 These ring-shaped MTs assemblies would be promising candidates for further development toward artificial bioactuators, where free tuning of CCW
INTRODUCTION Recently, it has been revealed that the left−right asymmetrical structures in living organisms are attributed to the dynamic behavior of their cytoskeletal components such as actin filaments and microtubules (MTs). For example, Lymnaea stagnalis, a kind of snail, shows dextral−sinistral physical arrangement in their adult state which is considered to be originated from the change in the dynamic behavior of monomer actins.1 Left- or right-handed twisted growth of Arabidopsis thaliana, a member of the family brassicaceae, has also been reported caused by a particular mutation in tubulins that is the constituent of filamentous protein MTs.2 These reports motivated us to design for achieving the functionally biased asymmetric structures using the cytoskeletal components, in vitro. MTs are hollow and cylindrical filaments with walls composed of tubulins (α- and β- tubulin heterodimers) arranging head-to-tail in a polar fashion to form protofilaments (PFs). In vivo MTs are usually regulated to consist of 13 PFs, parallelly aligned along its longitudinal axis.3 However, when MTs are reconstructed from tubulins in the presence of GTP in vitro, they consist of various numbers of PFs depending on the conditions of treatment and the presence of additives as reported elsewhere.4 When the number of PFs differs from 13, the MTs are known to have left- or right-handed supertwist in their PFs arrangement, which results in the distribution in the handedness of PFs configuration.5−7 Coupling to the MTs associated motor proteins, such as kinesin and dynein, MTs are © 2011 American Chemical Society
Received: June 17, 2011 Revised: July 27, 2011 Published: September 9, 2011 3394
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grease after the addition of ATP. All of the above procedures were conducted at room temperature. Microscopic Image Analysis of Motility Assays. For monitoring the MT motility assays, rhodamine-labeled MTs were illuminated with a 100 W mercury lamp and visualized by epifluorescence microscopy using a Plan Apo 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 an image analysis software (Image Pro Plus 5.1J, Media Cybernetics). Although the inner surface of the cover glass was observed through the glass, the captured images correspond to those observed from the reverse of flow cell. The clockwise/counterclockwise motion was discussed based on the captured image. In fluorescence microscopic images, 1 pixel corresponds to 264 nm as a resolution. Measurements of velocity were conducted with the software by tracking the inhomogeneities, such as defects where MTs were partly winded down or difference in the fluorescence intensity of the MTs ring image manually (see Supporting Information Figure 1). Observation of the Number of PFs. Images of the cross section of MTs were obtained according to a standard method with several modifications as follows: the paclitaxel stabilized MTs were centrifuged at 115000g 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 of pH 7.0). The fixed pellets were dehydrated with ethanol and embedded to Quetol-651 resin.17 Subsequently, the samples were cut into slices approximately to thickness 70 nm using an ultramicrotome (EM UC7; Leica Microsystems, Germany) equipped with a diamond knife (Sumitomo Denki Kogyo, Japan). These sections were applied to carbon-coated 300-mesh copper grids (Nisshin EM Co., Tokyo). The mounted sections were stained with 2.5% (w/v) gadolinium triacetate for 10 min and then with 4% (w/v) lead citrate for 2 min, which was prepared according to a previously described method.18 The specimens were observed under a transmission electron microscope (JEM-2100; JEOL Ltd., Japan) at an acceleration voltage of 200 kV. The digital TEM data were obtained using a slow-scan CCD camera (Gatan USC1000, Gatan Inc.) and converted into images with a frame size of 1024 × 1024 pixels. A coldfinger and a cold trap cooled with liquid nitrogen were used to prevent sample contamination by the electron beams. To determine the distribution of PFs number, only clearly observed protofilaments were counted.
rotation to clockwise (CW) direction, and vice versa, is an important underlying issue yet to be addressed. In this paper, we report our findings that the preferential rotation of the ringshaped MTs assembly obtained through AcSO process is significantly affected by the incubation time for MTs preparation. We also found that change in the incubation time brought change in the size and the velocity of rings, associated with the rearrangement of the PFs in MTs lattice. These results would not only reveal the obscure mechanism that governs the formation of such ring-shaped organized structures and their preferential rotation but also provide an insight in understanding the handedness observed in highly ordered and complex assemblies of natural systems.
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EXPERIMENTAL SECTION
Tubulin Purification. 3X-tubulin (tubulin) was purified from porcine brain by using a high-concentration PIPES buffer. Highmolarity PIPES buffer (HMPB) and BRB80 buffers were prepared using dipotassium salt (PIPES-2K) (Sigma), and pH was adjusted to 6.8 using HCl.13 Kinesin Purification. GFP-fused kinesin-1 construct 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.14 Biotin Labeling and Stoichiometric Estimation. Biotin-labeled tubulin was prepared using biotin-XX-SE (Invitrogen) according to standard techniques.15 The labeling stoichiometry was ∼2 per tubulin, which was estimated by spectrometric titration using a 2-(4′hydroxyphenylazo)benzoic acid (HABA) dye16 (Dojindo). MT Preparation. Biotin and rhodamine-labeled MTs were obtained by polymerizing biotin−tubulin, brightly labeled rhodamine−tubulin, and native tubulin (molar ratio, 45:10:45; final concentration, 15−34 μM) in polymerization buffer (80 mM PIPES2K, 1 mM EGTA, 5 mM MgCl2, 1 mM GTP, 0.38−0.90 M DMSO; pH adjusted to 6.8 using HCl) without or with equimolar concentration of paclitaxel (taxol) to tubulin at a given temperature (25−45 °C). After the prescribed incubation time (30 min−48 h), the MTs was diluted to make the tubulin concentration 100 nM in a buffer (80 mM PIPES-2K, 1 mM EGTA, 1 mM MgCl2, 0.13 M DMSO, 10 μM taxol; pH adjusted to 6.8 using HCl) to allow to perform the following motility assay. Formation of Ring-Shaped MT Assembly by AcSO. Flow cells were prepared by placing a cover glass (18 × 18 mm2; Matsunami) on a slide glass (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 spacerseparated glasses by heating. The flow cell was filled with 0.2 mg mL−1 anti-GFP antibody (Invitrogen) and incubated 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 taxol, ∼0.13 M DMSO; pH 6.8) 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 ∼0.13 M 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. The partial coverage of biotins on the MT surface with streptavidin−FITC (ST) (Wako) was done by incubating the MTs with ST solution (10 nM in motility buffer) for 10 min after fixing the MTs on the kinesins. Finally, the motility assay was initiated by applying 24 μL of ATP solution (motility buffer supplemented with 5 mM ATP). To avoid drying, the flow cell was sealed with silicone
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RESULTS AND DISCUSSION The lattice structure of the reconstructed MTs in vitro has been known to be strongly affected by taxol, DMSO, pH of the medium, and incubation time for MTs preparation. Transition in number of PFs is frequently observed during rapid MTs growth19 which is associated with the nucleation kinetics of MTs formation. Enhanced lateral interaction among MTs protofilaments seems to initiate the shift of MTs supertwist structure toward a larger number of PFs.20,21 It has been revealed that longitudinal interactions between tubulin subunits is basically hydrophobic, and lateral inter-PFs interaction is electrostatic due to the polar interactions of amino acids. 22 The former one, an entropy driven process, is affected by the temperature and pH change whereas the latter one by the change of dielectric constant of medium. The incubation time for polymerizing MTs is also considered as an essential factor to allow the MTs to reach their most energetically favorable configuration although no direct data were available in the literature.5,22 Left- or right-handed supertwist in PFs arrangement is finally determined by the number of the PFs settled, minimizing the restoring force in the MTs lattice. To demonstrate transition of the motion of ring-shaped MTs assembly from CCW to CW direction with the change of incubation time, experiments were performed according to the 3395
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scheme shown in the Figure 1. 34 μM tubulin monomers (biotin−tubulin, rhodamine−tubulin, and white tubulin at a
Figure 2. Representative ring-shaped MTs assembly formed through active self-organization (AcSO) on a kinesin-fixed surface (left) and an enlarged image of an MT ring (right). MTs were prepared by 30 min incubation at 37 °C in the presence of 0.64 M DMSO without taxol. Brightness and contrast were adjusted to visualize the ring-shaped MTs assembly after the capture using Image Pro 5.1J and Image J 1.41 software.
Figure 1. Schematic representation of the investigation on the effect of incubation time for MTs preparation on the preferential rotation of MTs ring formed through active self-organization (AcSO) on a kinesin-fixed surface.
molar ratio of 45:10:45) kept at 0 °C were polymerized to form MTs in the presence of 0.64 M DMSO incubating at 37 °C for a prescribed period of time (30 min, 3 h, 6 h, 9 h, 24 h, 48 h). MTs thus obtained after different incubation time were diluted (340 times) in a buffer containing 10 μM taxol to stabilize them and prevent further formation of new MTs. MTs then were used for AcSO after adsorption on a kinesin-coated glass surface in a flow cell. Under a molar ratio of Bt to tubulin at 1:1 and Bt to St at 10:1, the AcSO was initiated by the addition of ATP. After ∼3 h of the AcSO, preferential rotation of the obtained ring-shaped MTs assembly was evaluated (see the Experimental Section for details). The typical fluorescence microscopic image of the ring-shaped MTs assemblies is presented in the Figure 2. A drastic transition of the preferential rotation of the MTs ring from CCW direction to CW direction was observed (percentage in the population of rings rotating in CCW/CW direction was changed) with the prolongation of the incubation time for MTs polymerization, as shown in the Figure 3. For a short incubation time of 30 min, MTs rings rotated predominantly in the CCW direction (71% out of 55 rings). After 48 h incubation, surprisingly MTs rings exhibited preferential rotation in the CW direction, and the percentage of rings rotating in the CW direction increased to 87.5% (16 rings) from 29% (55 rings) found due to 30 min incubation of MTs. We also found that this effect of incubation time can be reset. If the MTs incubated for 24 h (CW rotation dominant) were disassembled into tubulin monomers by cooling on ice for 3 h and then followed by reassembly of those tubulins to MTs again at 37 °C for 30 min incubation, the MTs rings formed from these repolymerized MTs showed rotation dominant in
Figure 3. Effect of incubation time of MTs preparation on the preferential rotation of MTs ring formed by active self-organization (AcSO). MTs ring rotating in counterclockwise (CCW) and clockwise (CW) directions are shown as percentage of total number of rings. The rotating directions were confirmed by observing the real time images with naked eyes and the successive images of rings at 5 s intervals for at least 15 s. MTs were prepared from 34 μM tubulin without using taxol and incubating for different time at 37 °C in the presence of 0.64 M DMSO. “n” stands for the number of MTs ring considered for analysis. “After reset” means the depolymerization and subsequent repolymerization of MTs already incubated for 24 h and preferential rotation was evaluated after 30 min incubation followed by AcSO.
the CCW direction similar to that observed for 30 min incubation at the first run (Figure 3). The average lengths of MTs before starting the AcSO were almost similar in both the cases of 30 min (8.07 μm in average) and 24 h (8.47 μm in average) incubation time, indicating that preferential rotation of the ring-shaped MTs was essentially independent of the length of MTs. After prolonged incubation of 24 h the average diameter of the MT ring increased to 5.8 μm (SD = 3.0 μm) from 4.5 μm (SD = 2.5 μm) observed after 30 min incubation, as shown in the Figure 4a,b. Increase in diameter of the MT rings with the prolongation of incubation time might be interpreted by the increase in the rigidity of MTs either due to an enhanced inter- or 3396
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Figure 4. Effect of the incubation time on the size and velocity of MTs rings obtained from MTs polymerized for different incubation time. Size distribution: (a) 30 min, (b) 24 h. Velocity distribution: (c) 30 min, (d) 24 h. Average ring size: (a) 4.56 μm (standard deviation: 2.53 μm) and (b) 5.82 μm (standard deviation: 2.98 μm). Average velocity of MT rings: (c) 0.20 μm/s (standard deviation: 0.040 μm/s) and (d) 0.24 μm/s (standard deviation: 0.044 μm/s). MTs were polymerized from 34 μM tubulin without using taxol and incubating at 37 °C in the presence of 0.64 M DMSO. Velocity measurements were conducted using the software Image Pro Plus 5.1 J Media Cybernetics, by tracking the inhomogeneity such as difference in fluorescence intensity or defect of the ring structure.
intrafilament close packing along lateral or longitudinal direction or due to an increase in number of PFs of the MTs during incubation (discussed later). At the same time, the average velocity of the MTs rings also increased with the prolongation of incubation time, as shown in the Figure 4c,d. Although the mechanism is unclear at this moment, this seems to be due to the change in bending stress in the MTs rings. Thus, the ring size and velocity might be closely interrelated (Supporting Information Figure 2). It has also been revealed that the size of MTs ring rotating in CCW direction (majority) was larger than that rotating in CW (minority) direction after 30 min incubation, but after 24 h incubation this observation was found to be reversed where the size of the MTs ring rotating in CW (majority) direction became larger than that of rotating in CCW direction (minority) (Supporting Information Figure 3). The drastic transition in the direction of rotation of the MT rings is assumed to be related to the possible structural rearrangement of the MTs lattice due to prolonged incubation. Hence, we further investigated the number of PFs through the observation of the cross section of MTs prepared by 30 min and 24 h incubation by using transmission electron microscopy (TEM). The result showed that, with the increase of incubation time, the number of PFs shifted toward larger value with a slight change in their distribution (Figure 5). This increase in number of PFs seems to be responsible for the transition of
Figure 5. Relation between incubation time and distribution of the number of protofilament of MTs. (a) Transmission electron microscopy (TEM) image of cross section of MTs prepared from 34 μM tubulin without using taxol but 0.64 M DMSO and incubating for 24 h at 37 °C. Scale bar: 25 nm. (b) Distribution of number of PFs of MTs prepared by 30 min incubation (shadow) (n = 50) and 24 h incubation (dark) (n = 55) at 37 °C without using taxol in the presence of 0.64 M DMSO. “n” stands for the number of MTs considered for analyses.
CCW motion of MT ring toward CW direction. In the literature, it was reported that the left-handed supertwist in MT lattice shifted toward energetically favorable right-handed 3397
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presence of taxol.11 We found that when the concentration of DMSO in the polymerization buffer was as low as 0.38 M, the percentage of the MT ring rotating in the CCW (65% out of 134 rings) direction was dominant. When the concentration of DMSO was increased to 0.64 M, the percentage of the MT ring rotating in CW direction was found to be almost equal to that rotating in the CCW direction. With the further increase in the DMSO concentration up to 0.9 M, the percentage of the ring rotating in the CW (81% out of 86 rings) direction exceeded that of rotating in the CCW direction (Supporting Information Figure 5). Thus, DMSO favored the rotation of MT ring to shift toward CW direction from CCW direction, although the difference between the percentage of ring rotating into CCW and CW direction was not as significant as that observed changing the incubation time. We also examined the effect of the polymerization temperature by using MTs prepared at 25, 37, and 45 °C to perform AcSO. Higher percentage of MT rings was found rotating in the CW direction with the elevation of polymerization temperature (Supporting Information Figure 6). As reported in the literature, the number of PFs of MTs tends to decrease with increasing the polymerization temperature of MTs.20 From this result we can make a correlation between change in the number of PFs and handedness. Decrease in PFs number from 14 to smaller PFs number as 12 also provides MTs with more chance to attain a right-handed supertwist structure.5 As it was previously assumed that MTs with lefthanded supertwist of PFs would bend into rings that produce CCW motion,9 this result supports the above assumption that the shift of CCW motion of MTs ring toward CW direction arose from the transition of MTs lattice structure occurred during prolonged incubation of MTs. A similar trend in the change of preferential rotation of MTs ring induced by the increase in DMSO concentration and temperature is very likely attributed to the hydrophobic and electrostatic interaction playing within MTs lattice structure. 28 It seems that change in number of PFs is induced by changing the extent of lateral interaction among PFs. Increase in the number of PFs of MTs at a higher DMSO concentration (0.9 M) was also experimentally confirmed (Supporting Information Figure 7). Increase in the number of PFs with the prolonged incubation time is presumed to be attributed to the thermodynamics controlled transition of MT structure toward more stable configuration, resulting in enhanced lateral interaction among PFs of MTs. The effect of taxol that brought about no change in the direction of rotation of MTs ring is, however, not elucidated clearly at this moment. We summarized the discussion in Supporting Information Figure 8, where relations among the experimental conditions, major number of PFs, and rotational direction of MTs ring are represented in a tabular form. Although the CCW/CW rotation of ring-shaped MTs assembly was previously reported to be originated from the left/right-handed supertwist of PFs, which in turn is related to the number of PFs,12 direct observation of handedness of MTs will be performed in the near future. In conclusion, we have shown that the direction of the rotational motion of ring-shaped assembly of MTs could be tuned from CCW to CW direction and vice versa by changing the initial conditions of MTs preparation. Especially the change in incubation time brought the most significant change in both the MTs structure and the resultant preferential motion of the MTs assemblies. These observations strongly suggest that MTs polymerized in vitro can switch from a configuration that seems
supertwist with increasing number of PFs from 14 to 15 or larger.5 The above result may support our argument that increase in MTs ring diameter on prolonging the incubation time might be caused by the increase in number of PFs, where bending moment is significantly influenced with the increase in the PF numbers following the second moment of inertia of the cross section, I, as shown in eq 1:23 (1)
Here, we envisage that an MT is a hollow cylinder assembled from n number of rods, each having a radius of r. When such a straight hollow cylinder is bent into a circular arc, the radius of its curvature, R, will be proportional to the second moment of inertia of the cross section, I, according to the following relation: (2)
Under the assumption that the bending moment, M, applied to the MT and Young’s modulus, E, of the cylinder are constant, we could evaluate the change (ratio) of the radius, R, of the MT ring with the change of the number of PFs. Here, if 14 and 15 are the major number of PFs observed after 30 min and 24 h of incubation, respectively, the ratio of the radius, R, was estimated as, R15PFs/R14PFs = 1.22. The calculated ratio was found to coincide well with the experimental value obtained above (R24 h/R30 min = 1.28). To further investigate the factors that might affect the rotation of MT rings, we next studied the effect of buffer condition on preferential rotation of MTs rings by using taxol and DMSO that have been widely used as additives for stabilizing MTs. Taxol, an MTs directed agent, is an important antitumor drug that has been shown to bind to MTs in a stoichiometric manner and stabilize MTs against depolymerization by decreasing the dissociation rate constant of tubulins from the ends of MTs.24 It has also been reported that in the presence of taxol MTs prefer to form 12 PFs with mainly a right-handed supertwist in PFs arrangement.11,25 Using MTs prepared by tubulin polymerization at 37 °C for 3 h in the presence of taxol (15 μM) and DMSO (0.64 M), AcSO was performed for ∼3 h and the rotation of ring-shaped MTs assembly was analyzed. As the result, CCW rotation was found to be dominant along with the percentage of rings, similar to the case of AcSO performed using MTs prepared without taxol (Supporting Information Figure 4). This result indicates that preferential rotation of ring-shaped MTs assembly was not affected by the presence of taxol during MT polymerization under the given condition. This was also observed in the case when MTs were prepared in the presence of a low DMSO concentration (0.013 M). Next we examined the effect of DMSO concentration using MTs obtained by the polymerization of 34 μM tubulin at 37 °C for 3 h without using any taxol. Because of the unfavorable interaction between DMSO, which is a solvent having dielectric constant lower than water, and the polar surface of the tubulin, inclusion of the DMSO in polymerization buffer lowers the critical concentration of tubulin to form nuclei and also decreases the dissociation rate constant, but it has no effect on the association rate constant of tubulin to MTs.26,27 In the presence of DMSO (0.64 M) MTs prefer to form 14 PFs with a left-handed supertwist, unlike the MTs prepared in the 3398
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(21) Pierson, G. B.; Burton, P. R.; Himes, R. H. J. Cell Biol. 1978, 76, 223. (22) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. Cell 1999, 96, 79. (23) Howard, J. Mechanics of Motor Proteins and the Cytoskeleton; Sinauer Associates: Sunderland, MA, 2001. (24) Schiff, P. B.; Horwitz, S. B. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 1561. (25) Andreu, J. M.; Bordas, J.; Diaz, J. F.; Ancos, J. G.; Gil, R.; Medrano, F. J.; Nogales, E.; Pantos, E.; Andrews, E. T. J. Mol. Biol. 1992, 226, 169. (26) Algaier, J.; Himes, R. H. Biochim. Biophys. Acta, Protein Struct. 1988, 954, 235. (27) Arakawa, T.; Kita, Y.; Timasheff, S. N. J. Biophys. Chem. 2007, 131, 62. (28) Amos, L. A.; Amos, W. B. J. Cell Sci., Suppl. 1991, 14, 95.
not to be the most stable one energetically to a more energetically favorable state both in terms of inter- and intraPFs interactions. This switching in configuration would be related to the handedness of PFs in MTs lattice, which in turn amplified to demonstrate the preferential rotation of the ringshaped MTs assemblies obtained through the AcSO, although we have no direct evidence right now that strongly support this speculation. Well-defined polarity and tunable handedness are prerequisites for the formation of efficient ATP fueled artificial biodevices for which the MTs−kinesin system has been proposed as the building block. Our findings thus offer a significant direction to achieve handedness-controlled artificial biomachines with tunable asymmetric property. At the same time, our findings would provide with a significant clue to the understanding of the handedness observed in complex biological systems.
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ASSOCIATED CONTENT
S Supporting Information *
Some additional figures (Figures 1−8). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] (J.P.G.); kakugo@sci. hokudai.ac.jp (A.K.). Phone: +81-11-706-2774.
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ACKNOWLEDGMENTS
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REFERENCES
This research was financially supported by the Ministry of Education, Science, Sports, and Culture of Japan (Grant-in-Aid of Specially Promoted Scientific Research) and PRESTO (Japan Science and Technology Agency).
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