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Tobacco microtubule-associated protein, MAP65-1c, bundles and stabilizes microtubules

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Abstract

Three genes that encode MAP65-1 family proteins have been identified in the Nicotiana tabacum genome. In this study, NtMAP65-1c fusion protein was shown to bind and bundle microtubules (MTs). Further in vitro investigations demonstrated that NtMAP65-1c not only alters MT assembly and nucleation, but also exhibits high MT stabilizing activity against cold or katanin-induced destabilization. Analysis of NtMAP65-1c-GFP expressing BY-2 cells clearly demonstrated that NtMAP65-1c was able to bind to MTs during specific stages of the cell cycle. Furthermore, in vivo, NtMAP65-1c-GFP-bound cortical MTs displayed an increase in resistance against the MT-disrupting drug, propyzamide, as well as against cold temperatures. Taken together, these results strongly suggest that NtMAP65-1c stabilizes MTs and is involved in the regulation of MT organization and cellular dynamics.

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Abbreviations

MAPs:

Microtubule-associated proteins

PIPES:

Piperazine-N,N′-bis (2-ethanesulfonic acid 1,4-piperazinediethanesulfonic acid)

EGTA:

Ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

References

  • Caillaud MC, Lecomte P, Jammes F, Quentin M, Pagnotta S, Andrio E, Almeida Engler J, Marfaing N, Gounon P, Abad P, Favery B (2008) MAP65–3 microtubule-associated protein is essential for nematode-induced giant cell ontogenesis in Arabidopsis. Plant Cell 20:423–437

    Article  CAS  PubMed  Google Scholar 

  • Castoldia M, Popov A (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32:83–88

    Article  Google Scholar 

  • 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 10:251–259

    Article  CAS  PubMed  Google Scholar 

  • Chan J, Jensen CG, Jensen LCW, Bush M, Lloyd CW (1999) The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. Proc Natl Acad Sci USA 96:14931–14936

    Article  CAS  PubMed  Google Scholar 

  • Chang HY, Smertenko AP, Igarashi H, Dixon DP, Hussey PJ (2005) Dynamic interaction of NtMAP65–1a with microtubules in vivo. J Cell Sci 18:3195–3201

    Article  Google Scholar 

  • Ehrhardt DW, Shaw SL (2006) Microtubule dynamics and organization in the plant cortical array. Annu Rev Plant Biol 57:859–875

    Article  CAS  PubMed  Google Scholar 

  • Gaillard J, Neumann E, Van Damme D, Stoppin-Mellet V, Ebel C, Barbier E, Geelen D, Vantard M (2008) Two microtubule-associated proteins of Arabidopsis MAP65s promote antiparallel microtubule bundling. Mol Biol Cell 19:4534–4544

    Article  CAS  PubMed  Google Scholar 

  • Geelen DNV, Inze DG (2001) A bright future for the bright yellow-2 cell culture. Plant Physiol 127:1375–1379

    Article  CAS  PubMed  Google Scholar 

  • Hamada T (2007) Microtubule-associated proteins in higher plants. J Plant Res 120:79–98

    Article  CAS  PubMed  Google Scholar 

  • Hussey PJ, Hawkins TJ, Igarashi H, Kaloriti D, Smertenko A (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 50:915–924

    Article  CAS  PubMed  Google Scholar 

  • Hyman A (1991) Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence. J Cell Sci 14(Suppl):125–127

    CAS  Google Scholar 

  • Ishida T, Thitamadee S, Hashimoto T (2007) Twisted growth and organization of cortical microtubule. J Plant Res 120:61–70

    Article  CAS  PubMed  Google Scholar 

  • Jiang CJ, Sonobe S (1993) Identification and preliminary characterization of a 65 kDa higher-plant microtubule-associated protein. J Cell Sci 105:891–901

    CAS  Google Scholar 

  • Kaloriti D, Galva C, Parupalli C, Khalifa N, Galvin M, Sedbrook JC (2007) Microtubule associated proteins in plants and the processes they manage. J Integr Plant Biol 49:1164–1173

    Article  CAS  Google Scholar 

  • Li H, Yuan M, Mao TL (2007) AtMAP65–1 binds to tubulin dimers to promote tubulin assembly. J Biochem Mol Biol 40:218–225

    CAS  PubMed  Google Scholar 

  • Li H, Zeng X, Liu ZQ, Meng QT, Yuan M, Mao TL (2009) Arabidopsis microtubule-associated protein AtMAP65–2 acts as a microtubule stabilizer. Plant Mol Biol 69:313–324

    Article  CAS  PubMed  Google Scholar 

  • Mao TL, Jin LF, Li H, Liu B, Yuan M (2005a) Two microtubule-associated proteins of the Arabidopsis MAP65 family function differently on microtubules. Plant Physiol 138:654–662

    Article  CAS  PubMed  Google Scholar 

  • Mao GJ, Chan J, Calder G, Doonan JH, Lloyd CW (2005b) Modulated targeting of GFP-AtMAP65–1 to central spindle microtubules during division. Plant J 43:469–478

    Article  CAS  PubMed  Google Scholar 

  • McNally KP, Buster D, McNally FJ (2002) Katanin-mediated microtubule severing can be regulated by multiple mechanisms. Cell Motil Cytoskeleton 53:337–349

    Article  CAS  PubMed  Google Scholar 

  • Mollinari C, Kleman JP, Jiang W, Schoehn G, Hunter T, Margolis RL (2002) PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone. J Cell Biol 157:1175–1186

    Article  CAS  PubMed  Google Scholar 

  • Müller S, Smertenko A, Wagner V, Heinrich M, Hussey PJ, Hauser MT (2004) The plant microtubule-associated protein AtMAP65–3/PLE is essential for cytokinetic phragmoplast function. Curr Biol 14:412–417

    Article  PubMed  Google Scholar 

  • Olmsted JB, Stemple DL, Saxton WM, Neighbors BW, Mclntosh JR (1989) Cell cycle-dependent changes in the dynamics of MAP2 and MAP4 in cultured cells. J Cell Biol 109:211–223

    Article  CAS  PubMed  Google Scholar 

  • Qiang L, Yu WQ, Andreadis A, Luo MH, Baas PW (2006) Tau protects microtubules in the axon from severing by katanin. Neurosci J 26:3120–3129

    Article  CAS  Google Scholar 

  • Sasabe M, Soyano T, Takahashi Y, Sonobe S, Igarashi H, Itoh TJ, Hidaka M, Machida Y (2006) Phosphorylation of NtMAP65–1 by a MAP kinase down-regulates its activity of microtubule bundling and stimulates progression of cytokinesis of tobacco cells. Genes Dev 20:1004–1014

    Article  CAS  PubMed  Google Scholar 

  • Schuyler SC, Liu JY, Pellman D (2003) The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. J Cell Biol 160:517–528

    Article  CAS  PubMed  Google Scholar 

  • Smertenko A, Saleh N, Igarashi H, Mori H, Hauser-Hahn I, Jiang C, Sonobe S, Lloyd C, Hussey P (2000) A new class of microtubule-associated proteins in plants. Nat Cell Biol 2:750–753

    Article  CAS  PubMed  Google Scholar 

  • Smertenko A, Chang H, Wagner V, Kaloriti D, Fenyk S, Sonobe S, Lloyd C, Hauser M, Hussey P (2004) The Arabidopsis microtubule-associated protein AtMAP65–1: molecular analysis of its microtubule bundling activity. Plant Cell 16:2035–2047

    Article  CAS  PubMed  Google Scholar 

  • Smertenko A, Kaloriti D, Chang H, Fiserova J, Opatrny Z, Hussey P (2008) The C-terminal variable region specifies the dynamic properties of Arabidopsis microtubule-associated protein MAP65 isotypes. Plant Cell 20:3346–3358

    Article  CAS  PubMed  Google Scholar 

  • Stoppin-Mellet V, Gaillard J, Vantard M (2002) Functional evidence for in vitro microtubule severing by the plant katanin homologue. Biochem J 365:337–342

    CAS  PubMed  Google Scholar 

  • Stoppin-Mellet V, Gaillard J, Timmers T, Neumann E, Conway J, Vantard M (2007) Arabidopsis katanin binds microtubules using a multimeric microtubule-binding domain. Plant Physiol Biochem 45:867–877

    Article  CAS  PubMed  Google Scholar 

  • Van Damme D, Van Poucke K, Boutant E, Ritzenthaler C, Inzè D, Geelen D (2004a) In vivo dynamics and differential microtubule-binding activities of MAP65 proteins. Plant Physiol 136:3956–3967

    Article  PubMed  Google Scholar 

  • Van Damme D, Bouget F, Van Poucke K, Inzè D, Geelen D (2004b) Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP-tagged proteins. Plant J 40:368–398

    Article  Google Scholar 

  • Wasteneys G, Galway M (2003) Remodeling the cytoskeleton for growth and form: an overview with some new views. Annu Rev Plant Biol 54:691–722

    Article  CAS  PubMed  Google Scholar 

  • Wicker-Planquart C, Stoppin-Mellet V, Blanchoin L, Vantard M (2004) Interactions of tobacco microtubule-associated protein MAP65–1b with microtubules. Plant J 39:126–134

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Prof. Ying Fu at China Agricultural University for generously providing the katanin construct. This research was supported by grants from the National Basic Research Program of China (2006CB100101 to M.Y.), the 111 Project (B06003), the National Natural Science Foundation of China (30770186 and 31070258 to T. M.; 30830058 and 30721062 to M.Y.) and Chinese Universities Scientific Fund (2009JS72 to T.M.).

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Correspondence to Tonglin Mao.

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Qiutao Meng and Jizhou Du have contributed equally to this paper and are considered as joint first authors.

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11103_2010_9694_MOESM1_ESM.jpg

NtMAP65-1c decreased the critical concentration (Cc) for tubulin polymerization. (a) Tubulins at concentration of 5 or 10 μM underwent polymerization in presence or absence of 2 μM NtMAP65-1c at 35°C for 30 min. After centrifuging at 100,000 g for 20 min, the supernatant and pellet were subjected to SDS–PAGE. Tubulins assembled without GTP were used a control. (b) The amount of MTs in the pellet of (a) was estimated following density scanning of gels. There was a notable increase in the amount of tubulin in the pellet in the presence of NtMAP65-1c, as compared to control. (JPEG 574 kb)

11103_2010_9694_MOESM2_ESM.jpg

NtMAP65-1a is capable of stabilizing MTs against in vitro cold treatment. Tubulins (20 μM) were assembled with NtMAP65-1a fusion protein at 35°C for 20 min and then incubated at 4°C for 20 min. The pellets were subjected to SDS–PAGE. As a positive control, paclitaxel (10 μM) was used in place of NtMAP65-1a. (JPEG 586 kb)

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Meng, Q., Du, J., Li, J. et al. Tobacco microtubule-associated protein, MAP65-1c, bundles and stabilizes microtubules. Plant Mol Biol 74, 537–547 (2010). https://doi.org/10.1007/s11103-010-9694-4

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