Microtubule Gel

  • Yoshihito Osada
  • Ryuzo Kawamura
  • Ken-Ichi Sano


MT gel is obtained by simply cross-linking MTs [1]. This design is to utilize the intrinsic unique nature of MTs in a form of hydrogel. Protocols to prepare the MT gels are also harnessing the unique property of MT polymerization and depolymerization. Expecting that this introduction help readers to prepare customized MT gels with modifications in the method, the protocols will be introduced from basics. Here, we describe about tubulin purification, the cross-linking of polymerized MTs for gelation, and fluorescent label modification of tubulin for visualization by microscopy.


Bovine Spongiform Encephalopathy Ethylene Glycol Tetraacetic Acid Motility Assay Ethylene Glycol Tetraacetic Acid Ethylene Glycol Tetraacetic Acid 
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  1. 1.
    Sano, K., Kawamura, R., Tominaga, T., Nakagawa, H., Oda, N., Ijiro, K., Osada, Y.: Thermoresponsive microtubule hydrogel with high hierarchical structure. Biomacromolecules 12, 1409–1413 (2011). doi: 10.1021/bm101578x CrossRefGoogle Scholar
  2. 2.
    Weisenberg, R.C., Timasheffl, S.N.: Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry 9, 4110–4116 (1970)CrossRefGoogle Scholar
  3. 3.
    Shelanski, M., Gaskin, F., Cantor, C.R.: Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. U. S. A. 70, 765–768 (1973)CrossRefGoogle Scholar
  4. 4.
    Borisy, G.G., Marcum, J.M., Olmsted, J.B., Murphy, D.B., Johnson, K.A.: Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann. N. Y. Acad. Sci. 253, 107–132 (1975)CrossRefGoogle Scholar
  5. 5.
    Castoldi, M., Popov, A.V.: Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88 (2003)CrossRefGoogle Scholar
  6. 6.
    Drechsel, D.N., Hyman, A.A., Cobb, M.H., Kirschner, M.W.: Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol. Biol. Cell 3, 1141–1154 (1992)CrossRefGoogle Scholar
  7. 7.
    Szasz, J., Yaffe, M.B., Elzinga, M., Blank, G.S., Sternlicht, H.: Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin. Biochemistry 25, 4572–4582 (1986)CrossRefGoogle Scholar
  8. 8.
    Rees, D.A.: Structure, conformation, and mechanism in the formation of polysaccharide gels and networks. Adv. Carbohydr. Chem. Biochem. 24, 267–332 (1969)CrossRefGoogle Scholar
  9. 9.
    Petka, W.A., Harden, J.L., McGrath, K.P., Wirtz, D., Tirrell, D.A.: Reversible hydrogels from self-assembling artificial proteins. Science 281, 389–392 (1998)CrossRefGoogle Scholar
  10. 10.
    Yoshida, R., Uchida, K., Kaneko, Y., Sakai, K., Kikuchi, A., Sakurai, Y., Okano, T.: Comb-type grafted hydrogels with rapid de-swelling response to temperature changes. Nature 374, 240–242 (1995)CrossRefGoogle Scholar
  11. 11.
    Jeong, B., Kim, S.W., Bae, Y.H.: Thermosensitive sol-gel reversible hydrogels. Adv. Drug Deliv. Rev. 54, 37–51 (2002)CrossRefGoogle Scholar
  12. 12.
    Garbern, J.C., Hoffman, A.S., Stayton, P.S.: Injectable pH- and Temperature-Responsive Poly(N-isopropylacrylamide-co-propylacrylic acid) Copolymers for Delivery of Angiogenic Growth Factors. Biomacromolecules 11, 1833–1839 (2010)CrossRefGoogle Scholar
  13. 13.
    Pampaloni, F., Lattanzi, G., Jonas, A., Surrey, T., Frey, E., Florin, E.L.: Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length. Proc. Natl. Acad. Sci. U. S. A. 103, 10248–10253 (2006)CrossRefGoogle Scholar
  14. 14.
    Rosales, A.M., Murnen, H.K., Kline, S.R., Zuckermann, R.N., Segalman, R.A.: Determination of the persistence length of helical and non-helical polypeptoids in solution. Soft Matter 8, 3673–3680 (2012)CrossRefGoogle Scholar
  15. 15.
    Yang, Y.L., Lin, J., Kaytanli, B., Saleh, O.A., Valentine, M.T.: Direct correlation between creep compliance and deformation in entangled and sparsely crosslinked microtubule networks. Soft Matter 8, 1776–1784 (2012)CrossRefGoogle Scholar
  16. 16.
    Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., Mitchison, T.: Preparation of modified tubulins. Methods Enzymol. 196, 478–485 (1991)CrossRefGoogle Scholar
  17. 17.
    Peloquin, J., Komarova, Y., Borisy, G.: Conjugation of fluorophores to tubulin. Nat. Methods 2, 299–303 (2005)CrossRefGoogle Scholar
  18. 18.
    Hitt, A.L., Cross, A.R., Williams, R.C.: Microtubule solutions display nematic liquid crystalline structure. J. Biol. Chem. 265, 1639–1647 (1990)Google Scholar
  19. 19.
    Janmey, P.A., Euteneuer, U., Traub, P., Schliwa, M.: Viscoelastic properties of vimentin compared with other filamentous biopolymer networks. J. Cell Biol. 113, 155–160 (1991)CrossRefGoogle Scholar
  20. 20.
    Lin, Y.C., Koenderink, G.H., MacKintosh, F.C., Weitz, D.A.: Viscoelastic properties of microtubule networks. Macromolecules 40, 7714–7720 (2007)CrossRefGoogle Scholar
  21. 21.
    Lieleg, O., Claessens, M., Bausch, A.R.: Structure and dynamics of cross-linked actin networks. Soft Matter 6, 218–225 (2010)CrossRefGoogle Scholar
  22. 22.
    Symmons, M.F., Martin, S.R., Bayley, P.M.: Dynamic properties of nucleated microtubules: GTP utilisation in the subcritical concentration regime. J. Cell Sci. 109, 2755–2766 (1996)Google Scholar
  23. 23.
    Oosawa, F., Kasai, M.: A theory of linear and helical aggregations of macromolecules. J. Mol. Biol. 4, 10–21 (1962)CrossRefGoogle Scholar
  24. 24.
    Fygenson, D.K., Braun, E., Libchaber, A.: Phase diagram of microtubules. Phys. Rev. E 50, 1579–1588 (1994)CrossRefGoogle Scholar
  25. 25.
    Gaskin, F., Cantor, C.R.: Turbidmetric studies of the in vitro assembly and disassembly of porcine neurotubules. J. Mol. Biol. 89, 737–758 (1974)CrossRefGoogle Scholar
  26. 26.
    Howard, J., Hyman, A.A.: Dynamics and mechanics of the microtubule plus end. Nature 422, 753–758 (2003)CrossRefGoogle Scholar
  27. 27.
    Vale, R.D., Reese, T.S., Sheetz, M.P.: Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39–50 (1985)CrossRefGoogle Scholar
  28. 28.
    Kuznetsov, S.A., Gelfand, V.I.: Bovine brain kinesin is a microtubule-activated ATPase. Proc. Natl. Acad. Sci. U. S. A. 83, 8530–8534 (1986)CrossRefGoogle Scholar
  29. 29.
    Hackney, D.D.: Isolation of kinesin using initial batch ion-exchange. Methods Enzymol. 196, 175–181 (1991)CrossRefGoogle Scholar
  30. 30.
    Svoboda, K., Block, S.M.: Force and velocity measured for single kinesin molecules. Cell 77, 773–784 (1994)CrossRefGoogle Scholar
  31. 31.
    Kojima, H., Muto, E., Higuchi, H., Yanagida, T.: Mechanics of single kinesin molecules measured by optical trapping nanometry. Biophys. J. 73, 2012–2022 (1997)CrossRefGoogle Scholar
  32. 32.
    Case, R.B., Pierce, D.W., HomBooher, N., Hart, C.L., Vale, R.D.: The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain. Cell 90, 959–966 (1997)CrossRefGoogle Scholar
  33. 33.
    Kawamura, R., Kakugo, A., Shikinaka, K., Osada, Y., Gong, J.P.: Ring-shaped assembly of microtubules shows preferential counterclockwise motion. Biomacromolecules 9, 2277–2282 (2008)CrossRefGoogle Scholar
  34. 34.
    Gittes, F., Mickey, B., Nettleton, J., Howard, J.: Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape. J. Cell Biol. 120, 923–934 (1993)CrossRefGoogle Scholar
  35. 35.
    Kawamura, R., Kakugo, A., Osada, Y., Gong, J.P.: Selective formation of a linear-shaped bundle of microtubules. Langmuir 26, 533–537 (2010)CrossRefGoogle Scholar
  36. 36.
    Kawamura, R., Kakugo, A., Osada, Y., Gong, J.P.: Microtubule bundle formation driven by ATP: the effect of concentrations of kinesin, streptavidin and microtubules. Nanotechnology 21, 145603 (2010)CrossRefGoogle Scholar
  37. 37.
    Yoshida, R., Murase, Y.: Self-oscillating surface of gel for autonomous mass transport. Colloids Surf. B Biointerfaces 99, 60–66 (2012)CrossRefGoogle Scholar
  38. 38.
    Thorn, K.S., Ubersax, J.A., Vale, R.D.: Engineering the processive run length of the kinesin motor. J. Cell Biol. 151, 1093–1100 (2000)CrossRefGoogle Scholar
  39. 39.
    Howard, J.: Molecular mechanics of cells and tissues. Cell. Mol. Bioeng. 1, 24–32 (2008)CrossRefGoogle Scholar
  40. 40.
    Howard, J., Hudspeth, A.J., Vale, R.D.: Movement of microtubules by single kinesin molecules. Nature 342, 154–158 (1989)CrossRefGoogle Scholar
  41. 41.
    Sano, K.I., Kawamura, R., Tominaga, T., Nakagawa, H., Oda, N., Ijiro, K., Osada, Y.: Thermoresponsive microtubule hydrogel with high hierarchical structure. Biomacromolecules 12, 1409–1413 (2011)CrossRefGoogle Scholar
  42. 42.
    Riedel-Kruse, I.H., Hilfinger, A., Howard, J., Julicher, F.: How molecular motors shape the flagellar beat. HFSP J. 1, 192–208 (2007)CrossRefGoogle Scholar
  43. 43.
    Camalet, S., Julicher, F., Prost, J.: Self-organized beating and swimming of internally driven filaments. Phys. Rev. Lett. 82, 1590–1593 (1999)CrossRefGoogle Scholar
  44. 44.
    Vogel, S.K., Pavin, N., Maghelli, N., Julicher, F., Tolic-Norrelykke, I.M.: Self-organization of dynein motors generates meiotic nuclear oscillations. PLoS Biol. 7, 918–928 (2009)CrossRefGoogle Scholar
  45. 45.
    Sanchez, T., Welch, D., Nicastro, D., Dogic, Z.: Cilia-like beating of active microtubule bundles. Science 333, 456–459 (2011)CrossRefGoogle Scholar
  46. 46.
    Okeyoshi, K., Kawamura, R., Yoshida, R., Osada, Y.: Thermo- and photo-enhanced microtubule formation from Ru(bpy)(3)(2+)-conjugated tubulin. J. Mater. Chem. B 2, 41–45 (2014)CrossRefGoogle Scholar
  47. 47.
    Okeyoshi, K., Kawamura, R., Yoshida, R., Osada, Y.: Effect of microtubule polymerization on photoinduced hydrogen generation. Chem. Commun. (Camb.) 51, 11607 (2015)CrossRefGoogle Scholar
  48. 48.
    Liu, Y.F., Guo, Y.X., Valles, J.M., Tang, J.X.: Microtubule bundling and nested buckling drive stripe formation in polymerizing tubulin solutions. Proc. Natl. Acad. Sci. U. S. A. 103, 10654–10659 (2006)CrossRefGoogle Scholar
  49. 49.
    Guo, Y.X., Liu, Y.F., Oldenbourg, R., Tang, J.X., Valles, J.M.: Effects of osmotic force and torque on microtubule bundling and pattern formation. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 78, 041910 (2008)CrossRefGoogle Scholar
  50. 50.
    Okeyoshi, K., Kawamura, R., Yoshida, R., Osada, Y.: Microtubule teardrop patterns. Sci. Rep. 5, 9581 (2015)CrossRefGoogle Scholar
  51. 51.
    Fenn, W.O.: A quantitative comparison between the energy liberated and the work performed by the isolated sartorius muscle of the frog. J. Physiol. 28, 175–203 (1923)CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Yoshihito Osada
    • 1
  • Ryuzo Kawamura
    • 2
    • 3
  • Ken-Ichi Sano
    • 4
  1. 1.RIKENWako-shiJapan
  2. 2.Nakabayashi Laboratory Department of Chemistry Faculty of ScienceSaitama UniversityNaraJapan
  3. 3.Saitama University Department of ChemistrySaitama-shiJapan
  4. 4.Nagoya UniversityChikusa-kuJapan

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