Letter pubs.acs.org/NanoLett
Transporting a Tube in a Tube Jieling Li, Yi Jia,* Weiguang Dong, Xiyun Feng, Jinbo Fei, and Junbai Li* Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, CAS Key Lab of Colloid, Interface, and Chemical Thermodynamics, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: LbL-assembled tubes were employed for micro/nanoscale cargo transportation through the kinesin-microtubule system. Selectively modified with kinesins onto the inner tube walls through Ni−NTA complexes, these tubes can work as channels for microtubules. A motility assay shows the smooth movement of microtubules along the tube inner wall powered by the inside immobilized kinesins. It could be envisioned that cargoes with different sizes can be transported through these tubular channels with little outside interruption.
KEYWORDS: LbL-assembly, nanotube, kinesin, microtubule, unidirectional movement
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assembled tubes are modified here to serve as channels for cargo transport because of their intrinsic tubular structure. Protein molecular motors such as kinesins are naturally evolved nanosized machines that are responsible for cell division and movement, transport of vesicles, and muscle contraction.6 In vivo, kinesins walk along microtubules to realize the intracellular transport.7 The designed integration of the motor proteins kinesins in nanodevices is of great prospect for the manipulation of nanoscale materials. In past decades, the microtubule−kinesin system was successfully reconstructed in vitro to build nanodevices for biosensors,8 cargo transportation,9 or molecular sorting;10 however, the random movement of microtubules remains a major obstacle in the construction of kinesin-powered nanodevices. Attempts were made to solve this problem including the use of photolithographic tracks,11 or electric/magnetic fields.10,12 Recently, the concept of employing linear tubes as tracks for microtubules seems to be a more simple and effective way to direct the movement of microtubules. Teizer et al. used the exterior surfaces of carbon nanotubes as tracks and successfully guided the microtubules moving on nonplanar nanotube surfaces.13 To further avoid any derailing from the exterior surface of the nanotube, Linke and co-workers ingeniously employed the inner space of Al2O3 hollow nanowires as tracks and achieved an unidimensional motion of filaments inside the nanotubes.14 In this work, another nonplanar surface guiding approach, exploitation of the inner surfaces of versatile LbL tubes, was studied to guide the movement of microtubules. As shown in
n the macroscopic world, we often use tubes for directed transport and to confine and protect the transported goods against unwanted interaction with the outside. This is also an issue for microscopic and living systems, hence it is most promising to mimic this in corresponding devices. The challenge to prepare micron sized channels with defined inner surfaces enables the transport of rod-like objects along the inner lumen. A straightforward approach to build microscopic tubular channels is the layer-by-layer (LbL) assembly template method, which was well-developed to fabricate multilayer tubes. The LbL assembly template method refers to the alternate deposition of different materials onto templates to form multilayer nanostructures. If the planar templates are porous, such as anodic aluminum oxide (AAO) and polycarbonate (PC) membranes, the tubular structure of these templates could be duplicated by the assembled multilayer films. After the templates are dissolved, free-standing and well-defined multilayer tubes can be obtained. The length, diameter, and wall thickness of these tubes could be easily adjusted by changing the templates and the number of assembled layers. Other factors such as temperature, pH, or ionic strength of the solutions can also be used to regulate the nanostructures and properties of these tubes. Because different forces such as electrostatic bonds,1 hydrogen bonding,2 and covalent bonds3 can be used to drive the assembling process, this method allows a wide range of materials, especially biocompatible materials such as proteins and phospholipids as assembled wall materials. Multifunctional tubes can be obtained through different treatment of the inner and outer surfaces.4 These tubes have been used for molecular capturing, virus trapping, and as bioreactors for micro/nanoreactions.5 Other than simply being used as carriers or bioreactors, these © 2014 American Chemical Society
Received: June 20, 2014 Revised: October 3, 2014 Published: October 10, 2014 6160
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undesired adsorbed films at the top and bottom of the PC membranes, these membranes were immersed into N,Ndimethylformamide (DMF) solvent to release the assembled tubes. As seen in Figure 1, all images (scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM)) demonstrate that these tubes have a uniform diameter of about 3 μm and a length of 23 μm, which is similar to the pore size and thickness of the PC template. The wall thickness is estimated to be 80 nm (see the inserted image in Figure 1c). These images also show that our assembled tubes are truly qualified as channels because of their smooth inner wall surfaces, monodispersity, wellopened terminals, and large-enough space through which nanocargoes to be transported. We found that 30 polymeric bilayers are thick enough for the tubes to maintain their hollow tubular structure in a liquid environment or under high vacuum (see Figure S1, Supporting Information), and they are stable for at least three months. For immobilization of active motor protein, the Ni−NTA (nitrilotriacetic acid) complex was chosen. This benign protein complex can specifically and firmly interact with the His-tagged C-terminal of kinesins, while it leaves the N-terminal free for microtubule binding;15 it has been confirmed as a highly efficient way to immobilize the His-tagged kinesins.16 Since the LbL assembly template technique permits the assembling of tubes with different interior and exterior surface properties, we selectively modified the tube inner walls with the Ni−NTA complex by assembling the NH 2 −NTA (Nα,Nα-bis(carboxymethyl)-L-lysine hydrate)-modified sodium alginate (ALG) (denoted as ALG−NTA) as the innermost layer of the tubes (Scheme 2). The fluorescence analysis, CLSM analysis, and ultraviolet analysis (see Figures S5 and S6, Supporting Information) all proved that our Ni−NTA modified tubes could effectively bind the His-tagged kinesins. Next, we tested the properties of the assembled tubes as tubular channels for microtubules. For substrates without kinesins modification, owing to the lack of driving force, microtubules tended to swing in situ and could hardly approach the assembled tubes (movie S2, Supporting Information). Hence, to facilitate microtubules with their entrance of the assembled tubes, we first modified the substrate with kinesins at low density, but sufficient to support the movement of microtubules. Once the leading head of the microtubule drives into the tube, it could be captured by the inner immobilized kinesins, pulled into the assembled tube, and move unidirectionally along the tube wall (movie S3, Supporting Information). The mean gliding speed was determined to be 0.61 ± 0.15 μm s−1 (mean ± standard error, n = 25) (Figure S7, Supporting Information), which is consistent with the reference value (0.8 μm s−1).17 This suggests that the immobilized motor proteins have well retained their bioactivity. To prove the necessity of the Ni−NTA complex for transport, tubes with and without Ni−NTA modification in the inner walls were investigated. In the hollow nonfunctionalized tubes, the microtubule suspended and swung out from the assembled tube (Figure 2A(a−c); movie S4, Supporting Information); while in the Ni−NTA functionalized tube (Figure 2A(d−f); movie S3, Supporting Information), the microtubule grasped tightly to the tube wall and moved smoothly through the tubular channel. Figure 2, panel B shows the kymographs of the leading heads of microtubules within the tubes. As can be seen, in the nonfunctionalized tube (Figure 2B(a)), the microtubule showed no regular movement, but
Scheme 1, multilayer tubes were fabricated through the LbL assembly technique. Then with directional immobilization of Scheme 1. Illustration of LbL-Assembled Tubes as Channels To Guide the Motion of Microtubules. (A) Preparation of LbL-Assembled Tubes: Arrows a1−a3 Illustrate the Whole Tube Fabrication and Modification Process, and Arrow b Illustrates the Concrete Steps of the Formation of LbLAssembled Tubes. (B) The Microtubule Moves Unidirectionally along the Tube Inner Wall under Propulsion by the Inner Immobilized Kinesins. (Sizes of Kinesins, Microtubules, and Assembled Tubes are Not to Scale)
motor proteins onto the inner tube walls, the assembled tubes successfully worked as linear tracks to guide microtubules from random motion to unidirectional movement (movie S1, Supporting Information). Besides guiding the motion of microtubules, these tubes can play a role as channels for cargo transport at the same time. This rather simple technique allows control of the channel size and the coupling with biomolecules as well as selective modification with kinesins, so that both the inner and outer tube surfaces can be employed as tracks for microtubules. In this report, we mainly introduce the modification of the inner surface with kinesins to obtain a better control of the movement of the microtubule and avoid derailing. To our knowledge, this is the first time that the movement of a microtubule could be controlled in an LbLassembled tube. For the implementation of this method, PC membranes with a diameter of 3 μm and thickness of 23 μm were used as templates. Poly(allylamine hydrochloride) (PAH) and dextran sulfate sodium (DSS) were alternatingly deposited into the membranes. After treatment with oxygen plasma to remove the 6161
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Figure 1. Characterization of the assembled tubes: (a) SEM image of the tubes with both sides of the template treated with oxygen plasma; (b) SEM images of the tubes with only one side of the template treated with oxygen plasma before dissolution; (c) TEM images of the assembled tubes; and (d,e) CLSM images of the tubes.
Scheme 2. Illustration of the Kinesins Immobilization Through the Ni−NTA Complex. (Sizes for Kinesins and Assembled Tube are Not to Scale)
Brownian motion. On the contrary, in the Ni−NTA modified tube (Figure 2B(b)), the motion of the microtubule shows a linear trend, which is consistent with the orientation of the assembled tube. According to the statistical data, there are 51 times when microtubules entered the Ni−NTA modified tubes, which take a percentage of 64% in the total observation with Ni−NTA modified tubes (n = 80). For the 130 times of the total observation with nonfunctionalized tubes, we captured 60 times that microtubules entered the assembled tubes, which took a percentage of 46%. The microtubule moves smoothly along the inner tube wall in 49% of the total entry observations with Ni−NTA modified tubes, while nearly 0% with nonfunctionalized tubes (Table S1, Supporting Information). These different phenomena result from the different kinesins densities on the tube inner walls. In the nonfunctionalized tubes, motor proteins were trapped merely by physical adsorption. This weak, nonspecific adsorption captures fewer
motor proteins than does the specific absorption. Either the Nterminal or C-terminal of kinesins could be stuck onto the wall; thus, the bioactive sites of these motor proteins cannot be fully utilized. Hence, without enough bioactive motor proteins, Brownian motion is the main motional form for microtubules, and they tended to detach from the tube walls and float randomly out of the tubes. Oppositely, the Ni−NTA functionalized tubes can specifically interact with the Cterminal of the kinesins, while they leave the N-terminal free for microtubule binding.15c,d This stable and directional immobilization can provide enough bioavailable motor proteins to interact with microtubules. Thus, the microtubule adheres strongly to the inner wall, and with the power of kinesins it moves smoothly along the inner tube walls. Our result agrees with Jorge’s modeling analysis, which shows the crossover from Brownian fluctuation of microtubules to a more directed motion with the increase of kinesins density.18 Among the 6162
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Figure 2. (A) Time-lapse images of microtubule movement in the tubular channel: (a−c) microtubule suspended in the nonfunctionalized tube; and (d−f) microtubules move along the inner wall of the Ni−NTA functionalized tube (white arrows point to the leading head of the microtubule). (B) Kymographs of the leading head of microtubules within the assembled tubes: (a) microtubule in the nonfunctionalized tube; and (b) microtubule in the Ni−NTA modified tube (black arrows point to the moving direction of microtubules).
experiment and microtubule movement. This material is available free of charge via the Internet at http://pubs.acs.org.
kinesins that are in contact with microtubules, only a small fraction, which remain attached to microtubules for longer periods of time, perform the main job of propelling the microtubules. Clearly, a higher kinesins density can provide more kinesins that stay attached to microtubules for a longer time;18 however, it is noteworthy that the gliding velocity of the microtubules is independent of the kinesins density in the Ni− NTA modified tube, which is also consistent with the previous experimental19 and theoretical computation18 results. It would be ideal if the cargoes attached to the microtubules could be transported under protection through these kinesins-modified tubular channels. For this, we took streptavidin-modified magnetic polystyrene (PS) particles as example cargoes. Though we did not capture the whole process of microtubule loading with PS particles to move through the LbL-assembled tube, we caught moments of microtubule loading with PS particles swinging within the assembled tubes (Figure S8, Supporting Information). Other problems such as the fluid resistance and the size ratio between diameters of assembled tube and cargo still need to be solved before our system can be used in cargo transport. Nonetheless, this shows the prospect that our system can be exploited in cargo transport. In summary, the LbL-template technique can be employed to assemble nanotubes as channels to guide the movement of the microtubules and to protect cargoes to be transported. This technique can provide size tunable, multifunctional tubular channels for biomotor systems. Microtubules can move unidirectionally along the tube inner walls under propulsion of the immobilized kinesins. This tube orienting and confining method may be of great help in the fabrication of motor protein powered micro and nanodevices, and this whole system will have great potential for cargo transportation and separation.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Fax: +86 10 82612629. Phone: +86 10 82614087. *E-mail:
[email protected]. Author Contributions
Jieling Li and Junbai Li conceived and designed the experiments, and Jieling Li performed the experiments. Jieling Li and W.D. expressed and purified the full length kinesin-1 and copolymerized rhodamine-labeled microtubules. Jieling Li, Y.J., X.F., and J.F. analyzed the data. All authors discussed the results. Jieling Li, Y.J., and Junbai Li cowrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Stefan Diez for the assistance with kinesin purification. We also acknowledge the financial support from the National Nature Science Foundation of China (Project No. 91027045, 21320102004, 21321063, and 21303221) and the National Basic Research Program of China (973 program, 2013CB932800).
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ABBREVIATIONS AAO, anodic aluminum oxide; PC, polycarbonate; PAH, poly(allylamine hydrochloride); DSS, dextran sulfate sodium; DMF, N,N-dimethylformamide; NTA, nitrilotriacetic acid; NH 2 −NTA, Nα,Nα-bis(carboxymethyl)- L-lysine hydrate; ALG, sodium alginate; PS, polystyrene
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ASSOCIATED CONTENT
REFERENCES
(1) Ai, S. F.; Lu, G.; He, Q.; Li, J. B. J. Am. Chem. Soc. 2003, 125 (37), 11140−11141. (2) Tian, Y.; He, Q.; Cui, Y.; Tao, C.; Li, J. B. Chem.Eur. J. 2006, 12 (18), 4808−4812.
S Supporting Information *
Materials and supplementary methods; figures, schemes, and table of microtubule data; and supplementary movies of the 6163
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(3) Tian, Y.; He, Q.; Cui, Y.; Li, J. Biomacromolecules 2006, 7 (9), 2539−2542. (4) Mitchell, D. T.; Lee, S. B.; Trofin, L.; Li, N.; Nevanen, T. K.; Söderlund, H.; Martin, C. R. J. Am. Chem. Soc. 2002, 124 (40), 11864−11865. (5) Komatsu, T. Nanoscale 2012, 4 (6), 1910−1918. (6) (a) Svoboda, K.; Block, S. M. Cell 1994, 77 (5), 773−784. (b) Wang, M. D.; Schnitzer, M. J.; Yin, H.; Landick, R.; Gelles, J.; Block, S. M. Science 1998, 282 (5390), 902−907. (c) Coy, D. L.; Wagenbach, M.; Howard, J. J. Biol. Chem. 1999, 274 (6), 3667−3671. (7) (a) Chen, Q.; Li, D.; Oiwa, K. Biophys. Chem. 2007, 129 (1), 60− 69. (b) Skoufias, D. A.; Cole, D. G.; Wedaman, K. P.; Scholey, J. M. J. Biol. Chem. 1994, 269 (2), 1477−1485. (c) Vale, R. D.; Reese, T. S.; Sheetz, M. P. Cell 1985, 42 (1), 39−50. (8) Fischer, T.; Agarwal, A.; Hess, H. Nat. Nanotechnol. 2009, 4 (3), 162−166. (9) Goel, A.; Vogel, V. Nat. Nanotechnol. 2008, 3 (8), 465−475. (10) Van den Heuvel, M. G.; De Graaff, M. P.; Dekker, C. Science 2006, 312 (5775), 910−914. (11) (a) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1 (5), 235−239. (b) Moorjani, S. G.; Jia, L.; Jackson, T. N.; Hancock, W. O. Nano Lett. 2003, 3 (5), 633−637. (c) Hiratsuka, Y.; Tada, T.; Oiwa, K.; Kanayama, T.; Uyeda, T. Q. P. Biophys. J. 2001, 81 (3), 1555−1561. (12) (a) Van den Heuvel, M. G. L.; Butcher, C. T.; Lemay, S. G.; Diez, S.; Dekker, C. Nano Lett. 2005, 5 (2), 235−241. (b) Hutchins, B. M.; Platt, M.; Hancock, W. O.; Williams, M. E. Small 2007, 3 (1), 126−131. (13) Sikora, A.; Ramon, J.; Kim, K.; Reaves, K.; Nakazawa, H.; Umetsu, M.; Kumagai, I.; Adschiri, T.; Shiku, H.; Matsue, T. Nano Lett. 2014, 14 (2), 876−881. (14) Lard, M.; ten Siethoff, L.; Generosi, J.; Mansson, A.; Linke, H. Nano Lett. 2014, 14 (6), 3041−3046. (15) (a) Hochuli, E.; Döbeli, H.; Schacher, A. J. Chromatogr., A 1987, 411, 177−184. (b) Gentz, R.; Chen, C.-H.; Rosen, C. A. Proc. Natl. Acad. Sci. U. S. A. 1989, 86 (3), 821−824. (c) Ho, C. H.; Limberis, L.; Caldwell, K. D.; Stewart, R. J. Langmuir 1998, 14 (14), 3889−3894. (d) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68 (3), 490−497. (16) (a) Yu, T. Y.; Wang, Q.; Johnson, D. S.; Wang, M. D.; Ober, C. K. Adv. Funct. Mater. 2005, 15 (8), 1303−1309. (b) Bhagawati, M.; Ghosh, S.; Reichel, A.; Froehner, K.; Surrey, T.; Piehler, J. Angew. Chem., Int. Ed. 2009, 48 (48), 9188−9191. (17) Rietdorf, J.; Ploubidou, A.; Reckmann, I.; Holmström, A.; Frischknecht, F.; Zettl, M.; Zimmermann, T.; Way, M. Nat. Cell Biol. 2001, 3 (11), 992−1000. (18) Gibbons, F.; Chauwin, J.-F.; Despósito, M.; José, J. V. Biophys. J. 2001, 80 (6), 2515−2526. (19) (a) Hancock, W. O.; Howard, J. J. Cell Biol. 1998, 140 (6), 1395−1405. (b) Howard, J.; Hudspeth, A.; Vale, R. Nature 1989, 342 (6246), 154−158.
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