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Protoplasma

, Volume 248, Issue 3, pp 493–502 | Cite as

TMBP200, a XMAP215 homologue of tobacco BY-2 cells, has an essential role in plant mitosis

  • Hiroki YasuharaEmail author
  • Yuki Oe
Original Article

Abstract

TMBP200 from tobacco BY-2 cells is a member of the highly conserved family of microtubule-associated proteins that includes Xenopus XMAP215, human TOGp, and Arabidopsis MOR1/GEM1. XMAP215 homologues have an essential role in spindle assembly and function in animals and yeast, but their role in plant mitosis is not fully clarified. Here, we show by immunoblot analysis that TMBP200 levels in synchronously cultured BY-2 cells increased when the cells entered mitosis, thus indicating that TMBP200 plays an important role in mitosis in tobacco. To investigate the role of TMBP200 in mitosis, we employed inducible RNA interference to silence TMBP200 expression in BY-2 cells. The resulting depletion of TMBP200 caused severe defects in bipolar spindle formation and resulted in the appearance of multinucleated cells with variable-sized nuclei. This finding indicates that TMBP200 has an essential role in bipolar spindle formation and function.

Keywords

TMBP200 MOR1 XMAP215 family Mitosis Tobacco BY-2 cells Inducible RNAi 

Notes

Acknowledgements

We thank Dr. M. Curtis (University of Zürich, Switzerland) for providing pMDC7 and Dr. Y. Niwa (Shizuoka University, Japan) for the 35S-sGFP(S65T) vector. This work was supported by grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (no. 15770033 to H.Y.).

Conflicts of interest statement

The authors declare that they have no conflict of interest.

Supplementary material

709_2010_189_MOESM1_ESM.mov (3 mb)
Supplemental Movie 1 Time-lapse observation of the dynamics of microtubule localization during mitosis and cytokinesis in a living YTUB-TMRNAi-1 cell (an uninduced control cell) Images were recorded every 1 min. An image showing nuclear envelope breakdown set at 0 min. The progression of time before or after the breakdown is shown in the upper right corner of the images. (MOV 3036 kb)

Supplemental Movie 2 Time-lapse observation of the dynamics of microtubule localization during the inhibition of bipolar spindle formation in a living YTUB-TMRNAi-1 cell (an RNAi induced cell) Images were recorded every 1 min. An image showing nuclear envelope breakdown is set at 0 min. The progression of time before or after the breakdown is shown in the upper right corner of the images. (MOV 7177 kb)

709_2010_189_Fig7_ESM.gif (24 kb)

Supplemental Figure 1 Induction of RNAi silencing of YFP-fused β-tubulin did not affect the levels of TMBP200 and mitosis. YTB-GFPRNAi-1 cells were cultured for 3 days and then further cultured in the absence or presence of 5 μM β-estradiol to induce RNAi expression. At the indicated times after induction of RNAi expression, cells were harvested for protein extraction and also for staining with DAPI. Top Western blot with PAb200C, with anti-GFP antibodies, which also crossreact with YFP, and with ant-α-tubulin antibody as a loading control. Bottom Percentages of cells with a single nucleus (1), mitotic cells (mitotic), binucleated cells (2), and multinucleated cells (multi.). (GIF 24 kb)

709_2010_189_MOESM3_ESM.tif (14.1 mb)
High resolution image (TIFF 14416 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and BioengineeringKansai UniversitySuitaJapan

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