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Microtubule-dependent microtubule nucleation based on recruitment of γ-tubulin in higher plants

Nature Cell Biology volume 7, pages 961968 (2005) | Download Citation

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Abstract

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array1,2. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex3. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic γ-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain γ-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting γ-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, γ-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic γ-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.

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Acknowledgements

We greatly acknowledge T. Hashimoto for providing the GFP–tubulin line. We thank P. Wadsworth for incisive comments on the manuscript. Field-emission scanning electron microscopy was done at the University of Missouri's Core Facility for Electron Microscopy and we thank R. Johnson for his help there. This work was supported by grant-in-aids from Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science and Technology to T.M. and M.H., and in part by a grant to T.I.B. from the US Department of Energy (award No. 03ER15421).

Author information

Affiliations

  1. National Institute for Basic Biology, Okazaki 444-8585, Japan.

    • Takashi Murata
    •  & Mitsuyasu Hasebe
  2. The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan.

    • Takashi Murata
    •  & Mitsuyasu Hasebe
  3. Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan.

    • Seiji Sonobe
  4. Biology Department, University of Massachusetts, Amherst, MA 01003, USA.

    • Tobias I. Baskin
  5. Ocean Research Institute, The University of Tokyo, Tokyo 164-8639, Japan.

    • Susumu Hyodo
  6. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan.

    • Seiichiro Hasezawa
  7. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

    • Toshiyuki Nagata
  8. Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8503, Japan.

    • Tetsuya Horio

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Takashi Murata.

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DOI

https://doi.org/10.1038/ncb1306