Tobacco BY-2 Cells as an Ideal Material for Biochemical Studies of Plant Cytoskeletal Proteins

  • Seiji Sonobe
  • Etsuo Yokota
  • Teruo Shimmen
Part of the Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 53)


Microtubules (MTs) and actin filaments (AFs) are the major cytoskeletons in plant cells. The former functions in cellular morphogenesis and cell division, while the latter functions in intracellular transport including cytoplasmic streaming and positioning of organelles. To accomplish such functions, cytoskeletons are organized into a variety of ordered structures, and to organize such structures, a variety of regulatory proteins are thought to be involved. Therefore, identification and characterization of all regulatory proteins are necessary to understand the mechanism of cellular functions performed by cytoskeletons. For this purpose, tobacco BY-2 cells have significantly contributed to physiological and biochemical approaches (Nagata et al. 1992; Shibaoka et al. 1995; Sonobe 1996). Here, we will introduce plant cytoskeletal components including microtubule-associated proteins (MAPs) and actin binding proteins (ABPs), which have been found biochemically by our group using tobacco BY-2 cells.


Pollen Tube Cytoplasmic Streaming Preprophase Band Cytoplasmic Strand Characean Cell 
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  1. Asada T, Kuriyama R, Shibaoka H (1997) TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells. J Cell Sci 110: 179–189PubMedGoogle Scholar
  2. Bokros CL, Hugdahl JD, Hanesworth VR, Murthy JV, Morejohn (1993) Characterization o f the reversible taxol-induced polymerization of plant tubulin into microtubules. Biochemistry 32: 3437–3447Google Scholar
  3. Burgess J, Lawrence W (1985) Studies of the recovery of tobacco methophyll protoplasts from an evacuolation treatment. Protoplasma 126: 140–146CrossRefGoogle Scholar
  4. Chan J, Rutten T, Lloyd C (1996) Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus. Plant J 10: 251–259PubMedCrossRefGoogle Scholar
  5. Chan J, Jensen CG, Jensen LCW, Bush M, Lloyd CW (1999) The 65-kDa carrot microtubuleassociated protein forms regularly arranged filamentous cross-bridges between microtubules. Proc Natl Acad Sci USA 96: 14931–14936PubMedCrossRefGoogle Scholar
  6. Cyr RJ (1994) Microtubules in plant morphogenesis: role of the cortical array. Annu Rev Cell Biol 10: 153–180PubMedCrossRefGoogle Scholar
  7. Cyr RJ, Palevitz BA (1989) Microtubule-binding proteins from carrot. Planta 177: 245–260CrossRefGoogle Scholar
  8. Fakhrai H, Haq H, Evans PK (1988) Enucleation of protoplasts derived from suspension cultures of winged bean and from a crown gall cell line of Parthenocissus tricuspidata. Biol Plant (Praha) 30: 401–408Google Scholar
  9. Friederich E, Pringault E, Arpin M, Louvard D (1990) From the structure to the function of villin, an actin-binding protein of the brush border. BioEssays 12: 403–408PubMedCrossRefGoogle Scholar
  10. Giddings TH, Staehelin LA (1988) Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp. Planta 173: 22–30CrossRefGoogle Scholar
  11. Gibbon BC, Staiger CJ (2000) Profilin. In: Staiger CJ, Baluska F, Volkmann D, Barlow PW (eds) Actin: a dynamic framework for multiple plant cell functions. Kluwer Academic Publishers, The Netherlands, pp 45–66Google Scholar
  12. Hamada T, Shimmen T, Sonobe S (2002) A 200-kDa microtubule-binding protein isolated from tobacco BY-2 cells. Plant Cell Physiol 43 (Suppl): 572Google Scholar
  13. Hasezawa S, Nagata T (1993) Microtubule organizing centers in plant cells: localization of a 49 kDa protein that is immunologically cross-reactive to a 51 kDa protein from sea urchin centrosomes in synchronized tobacco BY-2 cells. Protoplasma 176: 64–74CrossRefGoogle Scholar
  14. Higashi-Fujime S, Ishikawa R, Iwasawa H, Kagami O, Kurimoto E, Kohama K, Hozumi T (1995) The fastest actin-based motor protein from the green algae, Chara, and its distinct mode of interaction with actin. FEBS Lett 375: 151–154PubMedCrossRefGoogle Scholar
  15. Higashiyama T, Sonobe S, Murofushi H, Hasezawa S (1996) Identification of a novel 70 kDa protein in cultured tobacco cells that is immunologically related to MAP4. Cytologia 61:229– 233Google Scholar
  16. Hussey PJ, Hawkins TJ, Igarashi I, Kaloriti D, Smertenko AP (2002) The plant cytoskeleton: recent advances in the study of the plant microtubule-associated proteins MAP-65, MAP- 190 and the Xenopus MAP215-like protein, MOR1. Plant Mol Biol 50: 915–924PubMedCrossRefGoogle Scholar
  17. Igarashi H, Vidali L, Yokota E, Sonobe S, Hepler PK, Shimmen T (1999) Actin filaments purified from tobacco cultured BY-2 cells can be translocated by plant myosin. Plant Cell Physiol 40: 1167–1171CrossRefGoogle Scholar
  18. Igarashi H, Orii H, Mori H, Shimmen T, Sonobe S (2000) Isolation of a novel 190-kDa protein from tobacco BY-2 cells: possible involvement in the interaction between actin filaments and microtubules. Plant Cell Physiol 41: 920–931PubMedCrossRefGoogle Scholar
  19. Ishizaki Y, Mikawa T, Ebashi S, Yokota E, Hosoya H, Kuroda K (1988) Preparation of tubulin from Caulerpa, a marine green alga, using casein as a protective agent against proteolytic degradation. J Biochem 104: 329–332PubMedGoogle Scholar
  20. Jiang CJ, Sonobe S (1993) Identification and preliminary characterization of a 65 kDa higher-plant microtubule-associated protein. J Cell Sci 105: 891–901Google Scholar
  21. Jiang CJ, Sonobe S, Shibaoka H (1992) Assembly of microtubules in a cytoplasmic extract of tobacco BY-2. Plant Cell Physiol. 33: 497–501Google Scholar
  22. Kakimoto T, Shibaoka H (1988) Cytoskeletal ultrastructure of phragmoplast-nuclei complexes isolated from cultured tobacco cells. Protoplasma (Suppl)2: 95–103Google Scholar
  23. Kamiya N (1959) Protoplasmic streaming. Protoplasmatologia, VIII ( 3a ). Springer, Berlin Heidelberg New YorkCrossRefGoogle Scholar
  24. Kamiya N, Kuroda K (1956) Velocity distribution of the protoplasamic streaming in Nitella cells. Bot Mag Tokyo 69: 544–554Google Scholar
  25. Kashiyama T, Kimura N, Mimura T, Yamamoto K (2000) Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world. J Biochem 127: 1065–1070PubMedCrossRefGoogle Scholar
  26. Kinkema M, Schiefelbein J (1994) A myosin from a higher plant has structural similarities to class V myosins. J Mol Biol 239: 591–597PubMedCrossRefGoogle Scholar
  27. Kinoshita K, Habermann B, Hyman AA (2002) XMAP215: a key component of the dynamic microtubule cytoskeleton. Trends Cell Biol 12: 267–273PubMedCrossRefGoogle Scholar
  28. Kohno T, Shimmen T (1988a) Mechanism of Ca2+ inhibition of cytoplasmic streaming in lily pollen tubes. J Cell Sci 91: 501–509Google Scholar
  29. Kohno T, Shimmen T (1988b) Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+. J Cell Biol 106: 1539–1543PubMedCrossRefGoogle Scholar
  30. Kohno T, Okagaki T, Kohama K, Shimmen T (1991) Pollen tube extract supports the movement of actin filaments in vitro. Protoplasma 161: 75–77CrossRefGoogle Scholar
  31. Kohno T, Ishikawa R, Nagata T, Kohama K, Shimmen T (1992) partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay. Protoplasma 170: 77–85Google Scholar
  32. Kovar DR, Staiger CJ (2000) Actin depolymerizing factor. In: Staiger CJ, Balusška F, Volkmann D, Barlow PW (eds) Actin: a dynamic framework for multiple plant cell functions. Kluwer Academic Publishers, The Netherlands, pp 67–88Google Scholar
  33. Kumagai F, Hasezawa S, Takahashi Y, Nagata T (1995) The involvement of protein synthesis elongation factor 1a in the organization of microtubules on the perinuclear region during the cell cycle transition from M phase to G1 phase in tobacco BY-2 cells. Botanica Acta 108:467– 473Google Scholar
  34. Ledbetter MC, Porter KR (1963) A “microtubule” in plant cell fine structure. J Cell Biol 19:239–250 Lee YRJ, Giang HM, Liu B (2001) A novel plant kinesin-related protein specifically associates with the phragmoplast organelles. Plant Cell 13: 2427–2439Google Scholar
  35. Liu B, Marc J, Joshi HC, Palevitz BA (1993) A y-tubulin related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner. J Cell Sci 104: 1217–1228PubMedGoogle Scholar
  36. Lloyd CW (1987) The plant cytoskeleton: the impact of fluorescence microscopy. Annu Rev Plant Physiol 38: 119–139CrossRefGoogle Scholar
  37. Lorz H, Paszkowski J, Dierks-Ventling C, Potrykus I (1981) Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells. Physiol Plant 53: 385–391CrossRefGoogle Scholar
  38. Maekawa T, Ogihara S, Murofushi H, Nagai R (1990) Green algal microtubule-associated protein with a molecular weight of 90 kD which bundles microtubules. Protoplasma 158: 10–18CrossRefGoogle Scholar
  39. Marc J, Sharkey DE, Durso NA, Zhang M, Cyr RJ (1996) Isolation of a 90-kD microtubuleassociated protein from tobacco membranes. Plant Cell 8: 2127–2138PubMedGoogle Scholar
  40. McCurdy DW, Staiger CJ (2000) Fimbrin. In: Staiger CJ, Baluska F, Volkmann D, Barlow PW (eds )Google Scholar
  41. Actin: a dynamic framework for multiple plant cell functions. Kluwer, Dordrecht, pp 87–102 Mehta AD, Rock RS, Rief M, Spudich JA, Mooseker MS, Cheney RE (1999) Myosin-V is a processive actin-based motor. Nature 400:590–593Google Scholar
  42. Mineyuki Y (1999) The preprophase band of microtubules: its function as a cytokinetic apparatus in higher plants. Int Nat Rev Cytol 187: 1–49CrossRefGoogle Scholar
  43. Mizuno K (1985) In vitro assembly of microtubules from tubulins of several higher plants. Cell Biol Int Rep 9: 13–21PubMedCrossRefGoogle Scholar
  44. Mizuno K, Koyama M, Shibaoka H (1981) Isolation of plant tubulin from azuki bean epicotyls by ethyl N-phenylcarbamate-sepharose affinity chromatography. J Biochem 89: 329–332PubMedGoogle Scholar
  45. Morejohn LC, Fosket DE (1982) Higher plant tubulin identified by self-assembly into microtubules in vitro. Nature 297: 426–428PubMedCrossRefGoogle Scholar
  46. Morejohn LC, Fosket DE (1984) Taxol-induced rose microtubule polymerization in vitro and its inhibition by colchicine. J Cell Biol 99: 141–147PubMedCrossRefGoogle Scholar
  47. Morimatsu M, Nakamura A, Sumiyoshi H, Sakaba N, Taniguchi H, Kohama K, Higashi-Fujime S (2000) The molecular structure of the fastest myosin from green algae, Chara. Biochem Biophys Res Comm 270: 147–152PubMedCrossRefGoogle Scholar
  48. Nagata T, Okada K, Takebe I, Matsui C (1981) Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol Gen Genet 184: 161–165Google Scholar
  49. Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLâ cell in the cell biology of higher plants. Int Rev Cytol 132: 1–30CrossRefGoogle Scholar
  50. Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz AM, Meehl JB, Staehelin LA (1999) Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121: 1127–1141PubMedCrossRefGoogle Scholar
  51. Nick P, Lambert A-M, Vantard M (1995) A microtubule-associated protein in maize is expressed during phytochrome-induced cell elongation. Plant J 8: 835–844PubMedCrossRefGoogle Scholar
  52. Prescott DM, Myerson D, Wallace J (1972) Enucleation of mammalian cells with cytochalasin B. Exp Cell Res 71: 480–485PubMedCrossRefGoogle Scholar
  53. Reichelt S, Kendrick-Jones J (2000) Myosins. In: Staiger CJ, Baluska F, Volkmann D, Barlow PW (eds) Actin: a dynamic framework for multiple plant cell functions. Kluwer Academic Publishers, The Netherlands, pp 29–44Google Scholar
  54. Rock RS, Rice SE, Wells AL, Purcell TJ, Spudich JA, Sweeney HL (2001) Myosin VI is a processive motor with a large step size. Proc Natl Acad Sci USA 98: 13655–13659PubMedCrossRefGoogle Scholar
  55. Rutten T, Chan J, Lloyd CW (1997) A 60-kDa plant microtubule-associated protein promotes the growth and stabilization of neurotubules in vitro. Proc Natl Acad Sci USA 94: 4469–4474PubMedCrossRefGoogle Scholar
  56. Sawano M, Shimmen T, Sonobe S (2000) Possible involvement of 65 kDa MAP in elongation growth of Azuki bean epicotyls. Plant Cell Physiol 431: 968–976CrossRefGoogle Scholar
  57. Seagull RW, Falconer MM, Weerdenburg CA (1987) Microfilaments: dynamic arrays in higher plant cells. J Cell Biol 104: 995–1004PubMedCrossRefGoogle Scholar
  58. Shibaoka H (1991) Microtubules and the regulation of cell morphogenesis by plant hormone. In: Lloyd CW (ed) The cytoskeletal basis of plant growth and form. Academic Press, New York, pp 159–168Google Scholar
  59. Shibaoka H (1993) Regulation by gibberellins of the orientation of cortical microtubules in plant cells. Aust J Plant Physiol 20: 461–470CrossRefGoogle Scholar
  60. Shibaoka H (1994) Plant hormone-induced changes in the orientation of cortical microtubules: alterations in the cross-linking between microtubules and the plasma membrane. Annu Rev Plant Physiol Plant Mol Biol 45: 527–544CrossRefGoogle Scholar
  61. Shibaoka H, Asada T, Yamamoto S, Sonobe S (1995) The use of model systems prepared from tobacco BY-2 cells for studies of the plant cytoskeleton. J Microsc 181: 145–152CrossRefGoogle Scholar
  62. Shimmen T, Yokota E (1994)Physiological and biochemical aspects of cytoplasmic streaming. Int Rev Cytol 155: 97–139Google Scholar
  63. Shimmen T, Ridge RW, Lambiris I, Plazinski J, Yokota E, Williamson RE (2000) Plant myosins. Protoplasma 214: 1–10Google Scholar
  64. Smertenko A, Saleh N, Igarashi H, Mori H, Hauser-Hahn I, Jiang CJ, Sonobe S, Lloyd CW, Hussey P (2000) A new class of microtubule-associated proteins in plants. Nat Cell Biol 2: 750–753PubMedCrossRefGoogle Scholar
  65. Sonobe S (1990) Cytochalasin B enhances cytokinetic cleavage in miniprotoplasts isolated from cultured tobacco cells. Protoplasma 155: 239–242CrossRefGoogle Scholar
  66. Sonobe S (1996) Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells. J Plant Res 109: 437–448CrossRefGoogle Scholar
  67. Tominaga M, Yokota E, Vidali L, Sonobe S, Hepler PK, Shimmen T (2000) The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis. Planta 210: 836–843PubMedCrossRefGoogle Scholar
  68. Tominaga M, Kojima H, Yokota E, Orii H, Nakamori R, Katayama E, Anson M, Shimmen T, Oiwa K (2003) Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity. EMBO J 22: 1263–1272PubMedCrossRefGoogle Scholar
  69. Twell D, Park SK, Hawkins TJ, Schubert D, Schmidt R, Smertenko A, Hussey PJ (2002) MOR1/ GEM1 has an essential role in the plant-specific cytokinetic phragmoplast. Nat Cell Biol 4:711– 714Google Scholar
  70. Vantard M, Schellenbaum P, Fellous A, Lambert A-M (1991) Characterization of maize microtubule-associated proteins, one of which is related to tau. Biochemistry 30: 9334–9340PubMedCrossRefGoogle Scholar
  71. Vidali L, Yokota E, Cheung AY, Shimmen T, Hepler PK (1999) The 135 kDa actin-bundling protein from Lilium longiflorum pollen is the plant homologue of villin. Protoplasma 209: 283–291CrossRefGoogle Scholar
  72. Wallin A, Glimelius K, Eriksson T (1978) Enucleation of plant protoplasts by cytochalasin B. Z Pflanzenphysiol 87: 333–340Google Scholar
  73. Wasteneys GO (2002) Microtubule organization in the green kingdom: chaos or self-order? J Cell Sci 115: 1345–1354PubMedGoogle Scholar
  74. Whittington AT, Vugrek O, Wei KJ, Hasenbein NG, Sugimoto K, Rashbrooke MC, Wasteney GO (2001) MOR1 is essential for organizing cortical microtubules in plants. Nature 411: 610–613PubMedCrossRefGoogle Scholar
  75. Yamamoto K, Kikuyama M, Sutoh-Yamamoto N, Kamitsubo E (1994) Purification of actin based motor protein from Chara corallina. Proc Jpn Acad Ser B 70: 175–180Google Scholar
  76. Yasuhara H, Sonobe S, Shibaoka H (1992) ATP-sensitive binding to microtubules of polypeptides extracted from isolated phragmoplasts of tobacco BY-2. Cell 33: 601–608Google Scholar
  77. Yasuhara H, Muraoka M, SHogaki H, Mori H, Sonobe S (2002) TMBP200, a microtubule bundling polypeptide isolated from telophase tobacco BY-2 cells is a MOR1 homologue. Plant Cell Physiol 43: 595–603PubMedCrossRefGoogle Scholar
  78. Yokota E, Shimmen T (1994) Isolation and characterization of plant myosin from pollen tubes of lily. Protoplasma 177: 153–162CrossRefGoogle Scholar
  79. Yokota E, Shimmen T (1999) The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity. Planta 209: 264–266PubMedCrossRefGoogle Scholar
  80. Yokota E, Shimmen T (2000) Characterization of native actin-binding proteins from pollen. In: Staiger CJ, Balusška F, Volkmann D, Barlow PW (eds) Actin: a dynamic framework for multiple plant cell functions. Kluwer Academic Publishers, The Netherlands, pp 103–118Google Scholar
  81. Yokota E, McDonald AR, Liu B, Shimmen T, Palevitz BA (1995a) Localization of a 170 kDa myosin heavy chain in plant cells. Protoplasma 185: 178–187CrossRefGoogle Scholar
  82. Yokota E, Sonobe S, Igarashi H, Shimmen T (1995b) Plant microtubules can be translocated by a dynein ATPase from sea urchin in vitro. Plant Cell Physiol 36: 1563–1569Google Scholar
  83. Yokota E, Takahara K, Shimmen T (1998) Actin-bundling protein isolated from pollen tubes of lily. Biochemical and immunocytochemical characterization. Plant Physiol 116: 1421–1429Google Scholar
  84. Yokota E, Muto S, Shimmen T (1999a) Inhibitory regulation of higher-plant myosin by Ca2+ ions. Plant Physiol 119: 231–239PubMedCrossRefGoogle Scholar
  85. Yokota E, Vidali L, Tominaga M, Tahara H, Orii H, Morizane Y, Hepler PK, Shimmen T (2003) Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells. Plant Cell Physiol 44, in pressGoogle Scholar
  86. Yokota E, Yukawa C, Muto S, Sonobe S, Shimmen T (1999b) Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells. Plant Physiol 121: 525–534PubMedCrossRefGoogle Scholar
  87. Yokota E, Sonobe S, Orii H, Yuasa T, Inada S, Shimmen T (2001) The type and the localization of 175-kDa myosin in tobacco cultured cells BY-2. J Plant Res 114: 115–116CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2004

Authors and Affiliations

  • Seiji Sonobe
    • 1
  • Etsuo Yokota
    • 1
  • Teruo Shimmen
    • 1
  1. 1.Department of Life Science, Graduate School of Science, Himeji Institute of TechnologyHarima Science Park City, HyogoJapan

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