Abstract
With the emergences of engineered devices at microscale and nanoscale dimensions, there is a growing need for controlled actuation and transport at these length scales. The kinesin–microtubule system provides a highly evolved biological transport system well suited for these tasks. Accordingly, there is an ongoing effort to create hybrid nanodevices that integrate biological components with engineered materials for applications such as biological separations, nanoscale assembly, and sensing. Adopting microtubules for these applications generally requires covalent attachment of biotin, fluorophores, or other biomolecules to tubulin enable surface or cargo attachment, or visualization. This review summarizes different strategies for functionalizing microtubules for application-focused as well as basic biological research. These functionalization strategies must maintain the integrity of microtubule proteins so that they do not depolymerize and can be transported by kinesin motors, while adding utility such as the ability to reversibly bind cargo. The relevant biochemical and electrical properties of microtubules are discussed, as well as strategies for microtubule stabilization and long-term storage. Next, attachment strategies, such as antibodies and DNA hybridization that have proven useful to date, are discussed in the context of ongoing hybrid nanodevice research. The review concludes with a discussion of less explored opportunities, such as harnessing the utility of tubulin posttranslational modifications and the use of recombinant tubulin that may enable future progress in nanodevice development.
Similar content being viewed by others
References
Agarwal A, Hess H (2010a) Biomolecular motors at the intersection of nanotechnology and polymer science. Prog Polym Sci 35:252–277
Agarwal A, Hess H (2010b) Molecular motors as components of future medical devices and engineered materials. J Nanotech Eng Med 1:1–9
Albrecht C, Blank K, Lalic-Multhaler M, Hirler S, Mai T, Gilbert I, Schiffmann S, Bayer T, Clausen-Schaumann H, Gaub HE (2003) DNA: a programmable force sensor. Science 301:367–370
Bachand GD, Rivera SB, Boal AK, Gaudioso J, Liu J, Bunker BC (2004) Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins. Nano Lett 4:817–821
Bachand GD, Rivera SB, Carroll-Portillo A, Hess H, Bachand M (2006) Active capture and transport of virus particles using a biomolecular motor-driven, nanoscale antibody sandwich assay. Small 2:381–385
Bachand GD, Hess H, Ratna B, Satir P, Vogel V (2009) “Smart dust” biosensors powered by biomolecular motors. Lab Chip 9:1661–1666
Boal AK, Tellez H, Rivera SB, Miller NE, Bachand GD, Bunker BC (2006) The stability and functionality of chemically crosslinked microtubules. Small 2:793–803
Brown TB, Hancock WO (2002) A polarized microtubule array for kinesin-powered nanoscale assembly and force generation. Nano Lett 2:1131–1135
Brunner C, Wahnes C, Vogel V (2007) Cargo pick-up from engineered loading stations by kinesin driven molecular shuttles. Lab Chip 7:1263–1271
Carroll-Portillo A, Bachand M, Greene AC, Bachand GD (2009) In vitro capture, transport, and detection of protein analytes using kinesin-based nanoharvesters. Small 5:1835–1840
Cassimeris LU, Walker RA, Pryer NK, Salmon ED (1987) Dynamic instability of microtubules. Bioessays 7:149–154
Castoldi M, Popov AV (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32:83–88
Cheng LJ, Kao MT, Meyhofer E, Guo LJ (2005) Highly efficient guiding of microtubule transport with imprinted CYTOP nanotracks. Small 1:409–414
Clemmens J, Hess H, Lipscomb R, Hanein Y, Bohringer KF, Matzke CM, Bachand GD, Bunker BC, Vogel V (2003) Mechanisms of microtubule guiding on microfabricated kinesin-coated surfaces: chemical and topographic surface patterns. Langmuir 19:10967–10974
Clemmens J, Hess H, Doot R, Matzke CM, Bachand GD, Vogel V (2004) Motor-protein “roundabouts”: microtubules moving on kinesin-coated tracks through engineered networks. Lab Chip 4:83–86
Davies DR, Padlan EA, Sheriff S (1990) Antibody-antigen complexes. Annu Rev Biochem 59:439–473
Dennis J, Howard J, Vogel V (1999) Molecular shuttles: directed motion of microtubules along nanoscale kinesin tracks. Nanotechnology 10:232–236
Diez S, Reuther C, Dinu C, Seidel R, Mertig M, Pompe W, Howard J (2003) Stretching and tranporting DNA molecules using motor proteins. Nano Lett 3:1251–1254
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Fischer T, Agarwal A, Hess H (2009) A smart dust biosensor powered by kinesin motors. Nat Nanotechnol 4:162–166
Freitas RA (2006) Pharmacytes: an ideal vehicle for targeted drug delivery. J Nanosci Nanotech 6:2769–2775
Gittes F, Meyhofer E, Baek S, Howard J (1996) Directional loading of the kinesin motor molecule as it buckles a microtubule. Biophys J 70:418–429
Greene AC, Trent AM, Bachand GD (2008) Controlling kinesin motor proteins in nanoengineered systems through a metal-binding on/off switch. Biotechnol Bioeng 101:478–486
Gupta ML Jr, Bode CJ, Dougherty CA, Marquez RT, Himes RH (2001) Mutagenesis of beta-tubulin cysteine residues in Saccharomyces cerevisiae: mutation of cysteine 354 results in cold-stable microtubules. Cell Motil Cytoskeleton 49:67–77
Hammond JW, Cai D, Verhey KJ (2008) Tubulin modifications and their cellular functions. Curr Opin Cell Biol 20:71–76
Helm CA, Knoll W, Israelachvili JN (1991) Measurement of ligand-receptor interactions. Proc Natl Acad Sci 88:8169–8173
Hess H, Vogel V (2001) Molecular shuttles based on motor proteins: active transport in synthetic environments. J Biotechnol 82:67–85
Hess H, Clemmens J, Qin D, Howard J, Vogel V (2001) Light-controlled molecular shuttles made from motor proteins carrying cargo on engineered surfaces. Nano Lett 1:235–239
Hess H, Clemmens J, Matzke CM, Bachand GD, Bunker BC, Vogel V (2002a) Ratchet patterns sort molecular motors. Appl Phys A 75:309–313
Hess H, Howard J, Vogel V (2002b) A piconewton forcemeter assembled from microtubules and kinesins. Nano Lett 2:1113–1116
Hess H, Bachand GD, Vogel V (2004) Powering nanodevices with biomolecular motors. Chemistry 10:2110–2116
Hess H, Clemmens J, Brunner C, Doot R, Luna S, Ernst KH, Vogel V (2005) Molecular self-assembly of “nanowires”and “nanospools” using active transport. Nano Lett 5:629–633
Hiller Y, Gershoni JM, Bayer EA, Wilchek M (1987) Biotin binding to avidin. Oligosaccharide side chain not required for ligand association. Biochem J 248:167–171
Hinterdorfer P, Baumgartner W, Gruber HJ, Schilcher K, Schindler H (1996) Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc Natl Acad Sci 93:3477–3481
Hirabayashi M, Taira S, Kobayashi S, Konishi K, Katoh K, Hiratsuka Y, Kodaka M, Uyeda TQ, Yumoto N, Kubo T (2006) Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability. Biotechnol Bioeng 94:473–480
Hiratsuka Y, Tada T, Oiwa K, Kanayama T, Uyeda TQ (2001) Controlling the direction of kinesin-driven microtubule movements along microlithographic tracks. Biophys J 81:1555–1561
Hiyama S, Gojo R, Shima T, Takeuchi S, Sutoh K (2009) Biomolecular-motor-based nano- or microscale particle translocations on DNA microarrays. Nano Lett 9:2407–2413
Hiyama S, Moritani Y, Gojo R, Takeuchi S, Sutoh K (2010) Biomolecular-motor-based autonomous delivery of lipid vesicles as nano- or microscale reactors on a chip. Lab Chip 10:2741–2748
Hutchins BM, Hancock WO, Williams ME (2006a) Magnet assisted fabrication of microtubule arrays. Phys Chem Chem Phys 8:3507–3509
Hutchins BM, Platt M, Hancock WO, Williams ME (2006b) Motility of CoFe2O4 nanoparticle-labelled microtubules in magnetic fields. Micro Nano Lett 1:47–52
Hutchins BM, Platt M, Hancock WO, Williams ME (2007) Directing transport of CoFe2O4-functionalized microtubules with magnetic fields. Small 3:126–131
Hyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T (1991) Preparation of modified tubulins. Methods enzymol 196:478–485
Jang MH, Kim J, Kalme S, Han JW, Yoo HS, Koo BS, Kim SK, Yoon MY (2008) Cloning, purification, and polymerization of Capsicum annuum recombinant alpha and beta tubulin. Biosci Biotechnol Biochem 72:1048–1055
Janke C, Kneussel M (2010) Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton. Trends Neurosci 33:362–372
Jia L, Moorjani SG, Jackson TN, Hancock WO (2004) Microscale transport and sorting by kinesin molecular motors. Biomed Microdevices 6:67–74
Kato K, Goto R, Katoh K, Shibakami M (2005) Microtubule-cyclodextrin conjugate: functionalization of motile filament with molecular inclusion ability. Biosci Biotechnol Biochem 69:646–648
Keith CH, Feramisco JR, Shelanski M (1981) Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts. J Cell Biol 88:234–240
Kellogg DR, Mitchison TJ, Alberts BM (1988) Behaviour of microtubules and actin filaments in living Drosophila embryos. Development 103:675–686
Korten T, Diez S (2006) Setting up roadblocks for kinesin-1: mechanism for the selective speed control of cargo carrying microtubules. Lab Chip 8:1441–1447
Korten T, Mansson A, Diez S (2010) Towards the application of cytoskeletal motor proteins in molecular detection and diagnostic devices. Curr Opin Biotechnol 21:477–488
Kufer SK, Puchner EM, Gumpp H, Liedl T, Gaub HE (2008) Single-molecule cut-and-paste surface assembly. Science 319:594–596
Ledbetter MC, Porter KR (1963) A “microtubule” in plant cell fine structure. J Cell Biol 19:239–250
Leslie RJ, Saxton WM, Mitchison TJ, Neighbors B, Salmon ED, McIntosh JR (1984) Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching. J Cell Biol 99:2146–2156
Lin CT, Kao MT, Kurabayashi K, Meyhofer E (2008) Self-contained, biomolecular motor-driven protein sorting and concentrating in an ultrasensitive microfluidic chip. Nano Lett 8:1041–1046
Lowe J, Li H, Downing KH, Nogales E (2001) Refined structure of alpha beta-tubulin at 3.5 A resolution. J Mol Biol 313:1045–1057
MacDonald LM, Armson A, Thompson RC, Reynoldson JA (2003) Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli. Protein Expr Purif 29:117–122
MacRae TH (1997) Tubulin post-translational modifications—enzymes and their mechanisms of action. Eur J Biochem 244:265–278
Mandelkow E, Mandelkow E-M (1995) Microtubules and microtubule-associated proteins. Curr Opin Cell Biol 7:72–81
Minoura I, Muto E (2006) Dielectric measurement of individual microtubules using the electroorientation method. Biophys J 90:3739–3748
Muthukrishnan G, Roberts CA, Chen Y, Zahn JD, Hancock WO (2004) Patterning surface-bound microtubules through reversible DNA hybridization. Nano Lett 2004:2127–2132
Nedelec FJ, Surrey T, Maggs AC, Leibler S (1997) Self-organization of microtubules and motors. Nature 389:305–308
Nogales E, Whittaker M, Milligan RA, Downing KH (1999) High-resolution model of the microtubule. Cell 96:79–88
Oxberry ME, Geary TG, Winterrowd CA, Prichard RK (2001) Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects. Protein Expr Purif 21:30–39
Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem 47:1179–1188
Platt M, Muthukrishnan G, Hancock WO, Williams ME (2005) Millimeter scale alignment of magnetic nanoparticle functionalized microtubules in magnetic fields. J Am Chem Soc 127:15686–15687
Pucciarelli S, Ballarini P, Miceli C (1997) Cold-adapted microtubules: characterization of tubulin posttranslational modifications in the Antarctic ciliate Euplotes focardii. Cell Motil Cytoskeleton 38:329–340
Raab M, Hancock WO (2008) Transport and detection of unlabeled nucleotide targets by microtubules functionalized with molecular beacons. Biotechnol Bioeng 99:764–773
Ramachandran S, Ernst KH, Bachand GD, Vogel V, Hess H (2006) Selective loading of kinesin-powered molecular shuttles with protein cargo and its application to biosensing. Small 2:330–334
Reuther C, Hajdo L, Tucker R, Kasprzak AA, Diez S (2006) Biotemplated nanopatterning of planar surfaces with molecular motors. Nano Lett 6:2177–2183
Rios L, Bachand GD (2009) Multiplex transport and detection of cytokines using kinesin-driven molecular shuttles. Lab Chip 9:1005–1010
Ross JL, Shuman H, Holzbaur EL, Goldman YE (2008) Kinesin and dynein-dynactin at intersecting microtubules: motor density affects dynein function. Biophys J 94:3115–3125
Schiff PB, Fant J, Horwitz SB (1979) Promotion of microtubule assembly in vitro by taxol. Nature 277:665–667
Schmidt C, Vogel V (2010) Molecular shuttles powered by motor proteins: loading and unloading stations for nanocargo integrated into one device. Lab Chip 10:2195–2198
Seetharam R, Wada Y, Ramachandran S, Hess H, Satir P (2006) Long-term storage of bionanodevices by freezing and lyophilization. Lab Chip 6:1239–1242
Soto CM, Martin BD, Sapsford KE, Blum AS, Ratna BR (2008) Toward single molecule detection of staphylococcal enterotoxin B: mobile sandwich immunoassay on gliding microtubules. Anal Chem 80:5433–5440
Stracke R, Bohm KJ, Burgold J, Schacht H, Unger E (2000) Physical and technical parameters determining the functioning of a kinesin-based cell-free motor system. Nanotechnology 11:52–56
Stracke R, Bohm KJ, Wollweber L, Tuszynski JA, Unger E (2002) Analysis of the migration behaviour of single microtubules in electric fields. Biochem Biophys Res Commun 293:602–609
Strunz T, Oroszlan K, Schafer R, Guntherodt HJ (1999) Dynamic force spectroscopy of single DNA molecules. Proc Natl Acad Sci USA 96:11277–11282
Taira S, Du YZ, Hiratsuka Y, Konishi K, Kubo T, Uyeda TQ, Yumoto N, Kodaka M (2006) Selective detection and transport of fully matched DNA by DNA-loaded microtubule and kinesin motor protein. Biotechnol Bioeng 95:533–538
Tomishige M, Vale RD (2000) Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change. J Cell Biol 151:1081–1092
Tucker R, Katira P, Hess H (2008) Herding nanotransporters: localized activation via release and sequestration of control molecules. Nano Lett 8:221–226
Turner D, Chang C, Fang K, Cuomo P, Murphy D (1996) Kinesin movement on glutaraldehyde-fixed microtubules. Anal Biochem 242:20–25
Tyagi S, Kramer FR (1995) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308
Uppalapati M, Huang YM, Jackson TN, Hancock WO (2008a) Enhancing the stability of kinesin motors for microscale transport applications. Lab Chip 8:358–361
Uppalapati M, Huang YM, Jackson TN, Hancock WO (2008b) Microtubule alignment and manipulation using AC electrokinetics. Small 4:1371–1381
Uppalapati M, Huang YM, Shastry S, Jackson TN, Hancock WO (2009) In: Zahn JD, Lee LP (eds) Microtubule motors in microfluidics. Methods in bioengineering: microfabrication and microfluidics. Artech House, Boston
Vale RD, Malik F, Brown D (1992) Directional instability of microtubule transport in the presence of kinesin and dynein. J Cell Biol 119:1589–1596
van den Heuvel MG, Butcher CT, Smeets RM, Diez S, Dekker C (2005) High rectifying efficiencies of microtubule motility on kinesin-coated gold nanostructures. Nano Lett 5:1117–1122
van Oss CJ, Good RJ, Chaudhury MK (1986) Nature of the antigen-antibody interaction: primary and secondary bonds: optimal conditions for association and dissociation. J Chromatogr 376:111–119
Vassilev P, Kanazirska M (1985) The role of cytoskeleton in the mechanisms of electric field effects and information transfer in cellular systems. Med Hypotheses 16:93–96
Verma V, Hancock WO, Catchmark JM (2009) Nanoscale patterning of kinesin motor proteins and its role in guiding microtubule motility. Biomed Microdevices 11:313–322
Westermann S, Weber K (2003) Post-translational modifications regulate microtubule function. Nat Rev Mol Cell Biol 4:938–947
Wilchek M, Bayer EA (1989) Avidin-biotin technology ten years on: has it lived up to its expectations? Trends Biochem Sci 14:408–412
Williams RC Jr, Lee JC (1982) Preparation of tubulin from brain. Methods enzymol 85(Pt B):376–385
Wong J, Chilkoti A, Moy VT (1999) Direct force measurements of the streptavidin-biotin interaction. Biomol Eng 16:45–55
Yokokawa R, Takeuchi S, Kon T, Ohkura R, Edamatsu M, Sutoh K and Fujita H (2003) Transportation of micromachined structures by biomolecular linear motors. Proceedings Micro Electro Mechanical Systems, pp 8–11
Acknowledgments
J.L.M was supported by NSF grant MCB 0920911 and W.O.H was supported by NIH grant GM083297.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Malcos, J.L., Hancock, W.O. Engineering tubulin: microtubule functionalization approaches for nanoscale device applications. Appl Microbiol Biotechnol 90, 1–10 (2011). https://doi.org/10.1007/s00253-011-3140-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-011-3140-7