pubs.acs.org/Langmuir © 2009 American Chemical Society
Selective Formation of a Linear-Shaped Bundle of Microtubules Ryuzo Kawamura,‡,† Akira Kakugo,‡,§ Yoshihito Osada,‡,† and Jian Ping Gong*,‡ ‡
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, § PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. † Present address: RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan Received June 18, 2009. Revised Manuscript Received August 26, 2009
By using rigid microtubules (MTs) prepared by polymerization with guanylyl-(R, β)-methylene-diphosphonate GMPCPP, giant straight-shaped MT bundles were selectively obtained through a dynamic self-assembly process. We demonstrate the effect of the rigidity on the shape and motility of MT bundle composed of GMPCPP-polymerized MTs (GMPCPP-MTs) compared with control MTs that were polymerized with GTP and stabilized with paclitaxel.
Introduction Motor proteins, such as the actin-myosin system and microtubule-kinesin system, have been proposed as the building blocks of ATP-fueled biomachines.1-3 The greatest difficulty with them lies in how to integrate their sophisticated functions to obtain results as high as those in cells and tissues. In a previous paper, we reported that large linear-shaped actin bundles, which consist of several tens of filament actin (F-actin), can be obtained through an electrostatic interaction with synthetic polymers carrying positive charges.4-8 In the presence of adenosine triphosphate (ATP), these bundles show motility on a surface coated with myosin. Recently, a method to assemble microtubules (MTs) into ring shaped structure on a kinesin-coated surface has been developed by employing a streptavidin-biotin interaction during the sliding motion of MTs in the presence of ATP.1 This method will bring the variety of motor protein assembly in size or shape. Following this method, we previously reported that large ringshaped microtubule (MT) bundles can be reproduced by integrating and simultaneously cross-linking MTs prepared in the presence of guanosine-50 -triphosphate (GTP) during the sliding motion on a surface coated with kinesin.9 Remarkable finding in this paper is that these ring-shaped MT bundles with diameter of 1-12 μm show a preferential rotation depending on the number of protofilaments (PFs) in MT. The ratio of the rings rotating in the counterclockwise (CCW) direction to those rotating in the clockwise (CW) direction reached 14/1 (nccw/ncw=222/16) when the majority of the MTs composed of 14 PFs (PFs) were *Author to whom correspondence should be addressed. E-mail: gong@ sci.hokudai.ac.jp. Telephone/FAX: þ81-11-706-2774.
(1) Hess, H.; Clemmens, J.; Brunner, C.; Doot, R.; Luna, S.; Ernst, K.-H.; Vogel, V. Nano Lett. 2005, 5, 629–633. (2) Diehl, M. R.; Zhang, K.; Lee, H. J.; Tirrell, D. A. Science 2006, 311, 1468– 1471. (3) Suzuki, H.; Oiwa, K.; Yamada, A.; Sakakibara, H.; Nakayama, H.; Mashiko, S. Jpn. J. Appl. Phys. 1995, 34, 3937–3941. (4) Kakugo, A.; Sugimoto, S.; Gong, J. P.; Osada, Y. Adv. Mater. 2002, 14, 1124–1126. (5) Kakugo, A.; Shikinaka, K.; Matsumoto, K.; Gong, J. P.; Osada, Y. Bioconjugate Chem. 2003, 14, 1185–1190. (6) Kwon, H. J.; Kakugo, A.; Shikinaka, K.; Osada, Y.; Gong, J. P. Biomacromolecules 2005, 6, 3005–3009. (7) Kwon, H. J.; Tanaka, Y.; Kakugo, A.; Shikinaka, K.; Furukawa, H.; Osada, Y.; Gong, J. P. Biochemistry 2006, 45, 10313–10318. (8) Shikinaka, K.; Kwon, H. J.; Kakugo, A.; Furukawa, H.; Osada, Y.; Gong, J. P.; Aoyama, Y.; Nishioka, H.; Jinnai, H.; Okajima, T. Biomacromolecules 2008, 9, 537–542. (9) Kawamura, R.; Kakugo, K.; Shikinaka, K.; Osada, Y.; Gong, J. P. Biomacromolecules 2008, 9, 2277–2282.
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employed. Meanwhile, when the MTs with increased 13 PFs are used, not only the ratio of CCW/CW but also the number of the ring-shaped MT bundles dramatically decrease. Thus, the preferential rotation of the MTs bundle is attributed to the lattice structure of the MT. These results also indicate that the functions and the morphologies of the MT bundles are potentially inherited from the nature (structure, mechanical property, and so on) of the single MTs. In this paper, we investigate the contribution of the rigidity of the MT to the patterns formed through this dynamic selfassembly process on a kinesin-coated surface. To study this, MTs with increased flexural rigidity, which can be prepared in the presence of GMPCPP, were employed as the building blocks for the MT bundles. GMPCPP, which is an analogue of GTP, is also incorporated into tubulins to form MTs in a similar way to GTP. However, the hydrolysis of GMPCPP in MTs is extremely slow compared to that of GTP, and this leads to a decrease in the dissociation rate of tubulin from the end of the MT. As a consequence, MT prepared in the presence of GMPCPP (GMPCPP-MT) is structurally stable. It is also reported that the flexural rigidity of the GMPCPP-MT is ∼2-fold higher than that of the GTP-MTs owing to a stronger lateral interactions between PFs, though the majority of the GMPCPP-MTs consist of 14 PFs as same as that in the MTs prepared in the presence of GTP (GTP-MTs) followed by stabilization with paclitaxel.10,11 Here, we demonstrate that large, straight MT bundles with preferential polarity are predominantly formed after the dynamic self-assembly process of the GMPCPP-MTs. The size and mobility of the MT bundles are also investigated before and after the dynamic self-assembly process.
Results and Discussion GMPCPP-MTs can be obtained by assembling tubulins in the presence of GMPCPP (see Experimental Section). In order to compare the MT bundles obtained from GTP-MTs during the dynamic self-assembly process, the GMPCPP-MTs were prepared by conditioning the incubation time and the tubulin concentration for elongation. The polymerized GTP-MT which was stabilized with paclitaxel and GMPCPP-MTs were bound to kinesin-fixed surface in flowcells without ATP. Parts a and d of (10) Vale, R. D.; Coppin, C. M.; Malik, F.; Kull, F. J.; Milligan, R. A. J. Biol. Chem. 1994, 269(38), 23769–23775. (11) Mickey, B.; Howard, J. J. Cell Biol. 1995, 130, 909–917.
Published on Web 10/08/2009
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Figure 1. MTs and MT bundles formed by the dynamic self-assembly process on the kinesin-fixed surface. (a-f) Fluorescent microscopy images: (a, d) before ATP addition; (b, e) 5 min after ATP addition; (c, f) 4 h after ATP addition. They are (a, b, c) for GTP-MTs, and (d, e, f) for GMPCPP-MTs. Brightness and contrast was adjusted to visualize both of the single MTs and the bundles after the capture using Image Pro 5.1J and ImageJ 1.41o. Scale bar: 20 μm. (g, h) Models for the interfilament interaction in which MTs which are encountering each other with obtuse angle. Blue arrows indicate driving forces by kinesins. Red points represent the cross-linking points of the two MTs through the biotin-streptavidin interaction. (g) Flexible MTs can deform their shape to cross-link with multiple points without breaking the biotin-streptavidin interactions, this leads the antiparallel configuration. (h) MT with higher rigidity gives a single interaction, and this prevents the antiparallel configuration.
Figure 1 show the typical fluorescence microscope images of the GTP-MT and GMPCPP-MTs, respectively (in order to visualize MT, it was partially labeled with rhodamine), before the dynamic self-assembly has been initiated. The average length of the GMPCPP-MTs was 12.9 ( 7.8 μm (n = 352) on the kinesinfixed surface, which is approximately the same length as that of the GTP-MTs (11.0 ( 8.3 μm [n = 303]). The histograms in Figure 2, parts a and d, show the length distributions of the GTP-MTs and GMPCPP-MTs, respectively. As shown in the histogram, some of the MTs unexpectedly aggregated presumably due to the shear flow to introduce the sample solutions into the flowcell before applying the dynamic self-assembly process (shaded). We tried to integrate MTs into large bundles with linear shape during the dynamic self-assembly process, as described previously.1,9 The dynamic self-assembly process was initiated using 5 mM ATP solution. In this study, both GTP-MTs and GMPCPP-MTs were modified with biotin prior to the polymerization of the MT. Biotin is a vitamin H that binds to streptavidin with a very high binding constant (K=∼1014); the cross-linking during the sliding motion of the biotin-labeled MTs on a kinesin-fixed surface was made by adding streptavidin.12 A concentration of the GTP-MTs at 240 nM (tubulin concentration) and an equimolar solution of streptavidin were chosen after optimization for the cross-linking process; the same conditions were employed for the GMPCPP-MTs. After 4 h of the dynamic self-assembly process, an increased fluorescence intensity caused by the integration of the single MTs was observed (Figure 1, parts c and f). In the case of GMPCPP-MT, we observed that most of the MT bundles exhibited a linear-shaped structure (no ring-shaped GMPCPP-MTs bundles were observed in the randomly selected view fields); this observation was contrary to structure of bundle obtained by using GTP-MTs (Figure 1c), wherein ∼4% of the GTP-MTs bundles were ring-shaped. The length distributions of the GTP-MTs bundles and GMPCPP-MTs bundles are shown (12) Green, N. M. Methods Enzymol. 1990, 184, 51–67.
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in Figure 2, parts b, c, e, and f. After 4 h of the dynamic selfassembly process a large number of single GMPCPP-MTs were incorporated into bundles compared to the situation after 5 min of the process though there is a possibility that some of the MTs detached from the surface during the sliding motion. By assuming that the number of the detached GMPCPP-MTs is negligible, we can roughly estimate the number of single GMPCPP-MTs composing the bundles by measuring the sum length of the GMPCPP-MTs before and after the dynamic self-assembly process. The sum length of the GMPCPP-MTs before and after the process were 144 mm-1 (=sum length/unit area) and 42 mm-1, respectively. From this observation, it can be considered that the GMPCPP-MTs bundles are composed of approximately 3.4 GMPCPP-MTs. This estimation of the MTs number composing the bundle is also supported by observation result of end-labeling procedure after the bundle formation (Supporting Information, movie S2). After 4 h of the dynamic self-assembly process, the long GMPCPP-MTs bundles were observed, which are longer than 40 μm (compare Figure 2, parts d, e, and f). The average length of the GMPCPP-MTs bundles was 26 ( 19 μm (n=56), which was slightly longer than that of the unexpectedly observed GMPCPP-MTs aggregates (23 ( 7 μm; n=56) before adding ATP. The longest GMPCPP-MTs bundle that we obtained in this system was 94 μm. In this study, we also evaluated the motility performance of the GMPCPP-MTs bundles. The average velocity of the GMPCPP-MTs bundles, calculated from the mean displacement in 5.1 s, was 0.162 ( 0.040 μm s-1 (n=20) at 4 h of the dynamic self-assembly process, which is the same as that of GMPCPP-MTs (0.165 ( 0.043 μm s-1, n=38, only single MTs) calculated at 15 min of the process, which is the stage that bundles formation is scarcely observed. Moreover, the velocity of the GMPCPP-MTs bundles was independent of their filament length; this is presumably due to the small load for the motility in this system. Further, the average velocities of the GTP-MTs (i.e., single MTs) at 15 min and their bundles at 4 h of the process were 0.165 ( 0.053 μm s-1 (n=53) and 0.189 ( 0.048 μm s-1 (n=12), respectively. (In this study, mean velocities of the GMP-MT Langmuir 2010, 26(1), 533–537
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Figure 2. Histograms of the length of MTs (black) and MT complex or bundles (shaded) formed by the dynamic self-assembly process by adding ATP: (a, b, c) before, after 5 min, and after 4 h, respectively, of adding ATP for GTP-MTs, where the sample numbers were n=303, 330, and 241, respectively, for (a, b, c); (d, e, f) before, after 5 min, and after 4 h, respectively, of adding ATP for GMPCPP-MTs, where the sample numbers were n=352, 195, and 212, respectively, for (d, e, f). Right column: The histograms were magnified along the vertical axis to make the “shaded” fractions visible.
bundles and GMPCPP-MT bundles are obtained from those showing sliding motion. GMP-MT and GMPCPP-MT bundles which do not show sliding motion were not taken into account for the calculation of the mean velocities.) The mean velocity was consistent with the value reported in a previous paper and this decreased velocity was explained in terms of the effect of the modification of streptavidins through biotins on the surface of MT.1 Thus, through the bundle formation, the average velocity was almost constant in both GMPCPP-MT and GTP-MTs systems, with respect to those displaying the sliding motions. In a previous paper, we showed that there was a linear relationship between the polarity of the actin bundle and its sliding velocity.13,14 Therefore, it can be suggested that the MTs in these bundles are arranged in unipolar fashion. It would be expected to be slowed or stopped if the MT assembles would contain antiparallel MTs. After 4 h of the dynamic self-assembly process, 94% (n = 72) of the GMPCPP-MT bundles were still actively moving and their linear shape was maintained; this was also in contrast to the bundles formed with GTP-MTs (44%, n=27). Since most of the stopped MT bundles formed by GTP-MTs were serpentine, the increased flexural rigidity of the MTs seemed to play an important role in the production of highly motile MT bundles. If the MTs are rigid enough, driving force induced by kinesins can be effectively integrated to produce translational motion along the filament. This force integration prevents MTs from stacking to the kinesin-coated surface. The increased flexural rigidity of the MTs may also be favorable for the polarity sorting in the MT bundles. When sliding MTs are integrated each other with acute angle to form a bundle favorably, in this case, MTs in its bundle should be arranged in unipolar fashion. If MTs are encountered each other with obtuse angle as illustrated in Figure 1, parts g and h, formation of the antiparallel bundle will depend on the interfilament interaction (binding force) mediated by streptavidin-biotin interaction. Here, if MTs are flexible as GTP-MTs, it is expected that they can deform their shape to cross-link with multiple interaction points (Figure 1g). MTs incorporated into MT bundle in an antiparallel fashion will decrease its sliding velocity or deprive its mobility; this is indicated by the lower percentage (13) Kakugo, A.; Shikinaka, K.; Takekawa, N.; Sugimoto, S.; Osada, Y.; Gong, J. P. Biomacromolecules 2005, 6, 845–849. (14) Kakugo, A.; Shikinaka, K.; Gong, J. P.; Osada, Y. Polymer 2005, 46, 7759– 7770.
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(44%) of the motile bundles formed with GTP-MTs than that with GMPCPP-MTs (94%). In an actual system, MTs were often trapped by the MT bundle which was already lost its mobility. The increase in the flexural rigidity of the MTs will reduce the interfilament interaction points and this will make it difficult for MTs to be incorporated into the MT bundle in an antiparallel fashion (Figure 1h). This might be a possible mechanism for the preferential polarity sorting of the GMPCPP-MTs. We have performed additional experiments designed to visualize the plus end and minus end of MT in order to evaluate the polarity of the MT bundles (see Supporting Information, movie S2 and Figure S1). However the polarity of the MTs in a bundle was difficult to distinguish because of the presence of multiple ends. Although both GTP-MTs and GMPCPP-MTs were straight-shaped on kinesins before ATP addition (Figure 1, parts a and d), a distinct difference in the rigidity was observed for the GMPCPP-MTs after adding ATP (5 min, Figure 1, parts b and e). The preferential alignment of the MTs observed prior to ATP addition might be due to shear flow caused during the process to introduce the MTs into the flowcell and to wash the flowcell. To characterize the rigidity of the MTs and the MT bundles in the dynamic self-assembly process, the end-to-end lengths (Le) of the MTs were plotted against the contour lengths (Lc) (Figure 3a). The relation of Le and Lc is indicated by exponent R in the relation R=Le/Lc. R=1 corresponds to straight shape, which is indicated by the dotted line in Figure 3, parts b, c, f, and g. The plots distributed on the dotted line were observed in the case of the GMPCPP-MTs and GMPCPP-MT bundles (Figure 3, parts f and g), suggesting that the MTs and MT bundles move in a linear trajectory as it is confirmed in the following discussion. Dispersed distribution of the plot at the area of Lc > Le was detected in the case of GTP-MT and GTP-MT bundles (Figure 3, parts b and c). As described previously, GMPCPP-MT (Figure 3f) is stiffer than GTP-MT (Figure 3b)10,15 and the rigidity of GMPCPPMT was maintained in the larger bundle, while that of the GTP-MT bundle decreased through the dynamic self-assembly process. To characterize the linear motion of the GTP-MTs and the GMPCPP-MT bundles, the displacement (d) of MTs was (15) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1, 235–239.
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Figure 4. Histograms of the βs of the MTs and MT bundles. The βs were calculated by fitting to the plots of the displacements against the traveling times (5.1, 10.3, 20.5, 41.0, 82.0, 164 s) of each MTs or MT bundles. Only the MTs and the MT bundles that could be continuously traced during the observation time for 164 s. (a) Histogram of β at the beginning (5 min) of the dynamic selfassembly process with GTP-MTs stabilized with paclitaxel. (b) MT bundles after 4 h of the process with GTP-MTs stabilized with paclitaxel. (c) MTs after 5 min of the process with GMPCPP-MTs. (d) MT bundles after 4 h of the process with GMPCPP-MTs. In the area of β < 0.5, the MT and the MT bundles were moved in circular trajectory. Mean values of β and the number of the MTs or MT bundles are shown in Table 1. Table 1. Mean Values of β for MTs and MT bundles GTP-MT (paclitaxel) MTs
Figure 3. Plot of the end-to-end lengths (Le) of the MTs and MT bundles against their contour lengths (Lc). (a) An example of the measurement of Le and Lc. One pixel is 0.265 μm. (b) GTP-MTs after 5 min of the dynamic self-assembly process. (n=139) (c) MT bundles of the GTP-MTs after 4 h of the process. (n = 21) (f) GTP-MTs after 5 min of the dynamic self-assembly process. (n=144) (g) MT bundles of the GMPCPP-MTs after 4 h of the process (n=25). (d, e, h, i) Histograms of the rs of MTs and MT bundles: (d) at the beginning (5 min) of the dynamic self-assembly process with GTP-MTs stabilized with paclitaxel, (e) MT bundles after 4 h of the process with GTP-MTs stabilized with paclitaxel, (h) MTs after 5 min of the process with GMPCPP-MTs, and (i) MT bundles after 4 h of the process with GMPCPP-MTs.
plotted as a function of the observation time (t=5.1, 10.3, 20.5, 41.0, 82.0, and 164 s) (data not shown). The displacement increases with the increase in the observation time and follows a power law as d µ tβ. From the exponent β, we can characterize the motion. β=0.5 corresponds to random motion, while β=1 indicates constant linear translational motion. The βs obtained from the fitting curves are shown in Figure 4. The βs lower than the value of 0.5 in Figure 4 represent the displacement of the MTs moving in a circular trajectory. After 5 min of the dynamic self-assembly process, which is beginning of the process, the mean β value for the GTP-MTs and the GMPCPP-MTs were 0.895 and 0.987, respectively (Table 1). Thus, the translational motion GMPCPP-MTs is more than that of the GTP-MTs, though some of the measured displacements were less than the length of the MTs due to the technical limit of the observation time. The mean β value for the GTP-MT bundles and the GMPCPP-MT bundles that were formed during the 4 h dynamic self-assembly process were 0.818 and 0.993 respectively (Table 1). 536 DOI: 10.1021/la902197f
β (average ( sd) 0.895 ( 0.175 number (n) 39
GMPCPP-MT
MT bundles
MTs
MT bundles
0.818 ( 0.363 10
0.987 ( 0.036 30
0.993 ( 0.011 18
As observed at the beginning of the dynamic self-assembly process of the MTs, the tendency of GMPCPP-MTs to move in a straight trajectory was preserved in the case of the MT bundles. Formation of MT bundles decreased the exponent β of the GTP-MTs and increased the exponent β of the GMPCPP. It should be noted that the standard deviation of the β for the GMPCPP-MT bundle (sd=0.011) is much smaller than that for the GTP-MT bundle (sd=0.363). There is an almost 30 times difference between them. This wider distribution of the β in the GTP-MTs bundles can be seen in Figure 4. To show the correlation between the shape of the filament contour and β, histograms of R values were also presented in Figure 3 as parts d, e, h, and i. As shown in the figures, the wider distribution of the R in the GTP-MT system and narrower distribution of that in the GMPCPP-MT system are observed. These trends are almost similar to that observed in the distribution of β in Figure 4: that is, the narrower the R is, the more translational the motion MT become. Thus, we have successfully demonstrated that large linearshaped MTs bundles with preferential polarity are predominantly produced when the dynamic self-assembly process on the kinesincoated surface has been applied to the MTs with higher rigidity. It is remarkable that a factor of 2 on the MT rigidity could have a strong effect on the bundle shapes. In a previous paper, we have discussed that ring formation occurs as a consequence of the buckling of the MT and the probability to form rings would depend on the inhibition frequency of the front end motion of MTs, while the bias in motion would depend on the supertwist structure of PFs in MTs. According to Euler’s buckling equation, the force required to buckle a MT is proportional to its flexural Langmuir 2010, 26(1), 533–537
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rigidity. (Here, if the ends are free to pivot, the force required to buckle a beam is expressed as F ¼ π2
EI , L2
where L is the length of the beam, EI is the flexural rigidity of the beam. Flexural rigidity of GMPCPP-MT and GTP-MT has been estimated as EIGMPCPP= 62 10-24 Nm2 and EIGTP= 30 10-24 Nm2, respectively.11) Supposing that the length of the MT is 10 μm, the buckling forces can be estimated as 6.1 pN for GMPCPP-MT and as 3.0 pN for GTP-MTs. The maximum force per a kinesin head is estimated as ∼7 pN and this is almost comparable to that required to bend the GTP-MTs with 10 μm in length.16,17 Thus, it may be possible that the higher rigidity of the GMPCPP-MTs prevented buckling by breaking the inhibition of the motion at front end. The increased flexural rigidity of GMPCPP-MT elevates the counter force which is necessary for buckling and this may also decrease the chance to stack at the front end, though there is a possibility that the interaction between the GMPCPP-MTs and kinesins is less strong than that in the case of GTP-MTs. Once the sum of the driving force from the kinesins exceeds the critical force, the bending direction of MT is expected to follow the supertwist structure of PF. It has been reported that PFs in GMPCPP-MT still keep their lattice structure as same as in GTP-MT with 14 PFs, though PFs in GMPCPP-MT are less tightly supertwisted than those in GTP-MT.10
Conclusion In the present work, by using GMPCPP-MTs, we successfully obtained selectively straight-shaped MT bundles. 94% of these bundles were motile 4 h after ATP addition, and the velocity was almost the same as that of single GMPCPP-MTs just after the addition of ATP. From the highly motile property of these bundles, it was suggested that there could be a highly ordered structure with unipolarity. In future, in combination with the microfabrication technique, the application of this linearly shaped and highly motile MT bundle, which allows us to exploit translational motion, can contribute to the development of nano- and microtechnology. For instance, MTs were proposed as a molecular shuttle through the control of the propelling direction with a micropatterned wall.18 Our giant linear bundle can be applied to scale up this concept to a cellular shuttle.
Experimental Section Polymerization of MTs. Tubulin, rhodamine- or biotinlabeled tubulin, and green fluorescent protein-fused kinesin (GFP-kinesin) were prepared using the same methods described previously; tubulins were all purified from porcine brain.9 All solutions were prepared using the following buffer with appropriate modifications: 80 mM Pipes, 1 mM EGTA, 1 mM MgCl2, pH 6.8. For polymerization into GMPCPP-MT, 40 μM of RBNtubulin mix (rhodamine tubulin:biotin tubulin:native tubulin = 10:45:45 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, hereafter called “seed,” were diluted 1000-fold into an elongation buffer containing 1 μM RBN-tubulin mix, 5 mM MgCl2, and 1 mM GMPCPP. The solution, which contained a (16) Kojima, H.; Muto, E.; Higuchi, H.; Yanagida, T. Biophys. J. 1997, 73, 2012–2022. (17) Visscher, K.; Schnitzer, M. J.; Block, S. M. Nature. 1999, 400, 184–189. (18) Brunner, C.; Wahnes, C.; Vogel, V. Lab Chip. 2007, 7, 1263–1271.
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total of 1.04 μM tubulin, was incubated for 4 h at 37 °C. Thereafter, the elongated GMPCPP-MTs were diluted to 480 nM, which is indicated as the tubulin concentration, and were stored at room temperature until use for the motility assay of the MTs. An MT was also prepared by a standard technique in which GTP and paclitaxel are used for polymerization and stabilization, respectively, according to previous methods; hereafter this MT is called “GTP-MT.” Motility Assay for Bundle Formation. Dynamic self-assembly of the GTP-MTs was performed as previously described.1,9 The flowcell for motility assay was assembled by attaching a coverglass and a slideglass with a pair of heat-melted Parafilm slips. To complete the motility assay system, solutions containing each component were introduced to the flowcell with a volume of ∼8 μL. The procedure to introduce the solutions is as follows. A 0.2 mg mL-1 sample of antiGFP antibody (Molecular Probes, Eugene, OR) was introduced into the vacant flowcell and incubated for 15 min. Unbound antibodies were removed by a washing buffer 1 (∼0.5 mg mL-1 casein). The flowcell was incubated for 5 min with the washing buffer to let the caseins bind to the glass surface where the anti-GFP did not cover it, and GFP fused kinesins were fixed to the antibodies with kinesin solution (63 nM GFPkinesin, ∼40 mM NaCl, ∼0.5 mg mL-1 casein, 10 μM paclitaxel, ∼1% (v/v) DMSO, 1 mM dithiothreitol (DTT)) with incubation for 10 min. With washing buffer 2 (2 mM MgCl2, ∼0.5 mg mL-1 casein, 10 μM paclitaxel, ∼1% (v/v) DMSO, 1 mM DTT, 4.5 mg mL-1 D-glucose, 50 U mL-1 glucose oxidase, 50 U mL-1 catalase), unbound kinesins were washed out. Next, microtubules were bound to the surface-fixed kinesins by introducing an MT solution (240 nM MT in washing buffer 2) and incubated for 5 min. After washing with washing buffer 2, the kinesin-attached MTs were modified with streptavidin by introducing streptavidin solution (240 nM streptavidin-FITC (Wako, Osaka, Japan) in washing buffer 2) and 10 min of incubation. Before starting the motility assay, it was verified that the MTs were attached to the surface kinesins after being washed with washing buffer 2 using fluorescent microscopy. Finally, the motility assay system was started by adding ATP solution (5 mM ATP in washing buffer 2). To avoid drying, the flowcell was sealed with silicone grease after the addition of ATP. All of the above procedures were conducted at room temperature (∼25 °C). For the GMPCPP-MTs, the dynamic self-assembly processes were performed without paclitaxel and DMSO in all the preparation procedures. Observation and Image Analysis. The sliding motions of MTs on the kinesin-fixed surface were observed under an epifluorescent microscope (BX-50, Olympus, Tokyo, Japan) equipped with a 60 (oil) objective lens and a cooled-CCD camera (Cascade II, Nippon Roper, Tokyo, Japan). The image capture and analysis were conducted using the software Image Pro 5.1J or ImageJ 1.41o. The contour lengths of the MTs and the MT bundles were measured by tracing them manually. In fluorescent microscope images, one pixel corresponds to 260 nm as a resolution. Measurement of velocities was conducted with the software by tracing the tail end of the MTs manually. The length the movie analyzed was 251 s with a frame rate of 5.1 s.
Acknowledgment. The plasmid construct for kinesin-GFP fusion protein expression was generously provided by M. Tomishige and R. D. Vale. This research is financially supported by the Ministry of Education, Science, Sports, and Culture, Japan (Grant-in-Aid of Specially Promoted Scientific Research). Supporting Information Available: Movie S1, in which giant linear MT bundles are showing translational motions on a kinesin-fixed surface, movie S2 showing movement of endlabeled MT bundles on a fixed surfcae, text giving discussions about the polarity of the MT bundles and the number of the GMPCPP-MTs composing the bundles, and Figure S1, showing preparation of end-labeled MT on a kinesin-fixed surface. This material is available free of charge via the Internet at http:// pubs.acs.org. DOI: 10.1021/la902197f
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