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

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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Figure 1: Initiation of cortical microtubules as branches in tobacco cells.
Figure 2: Extant microtubules are essential for microtubule assembly in the cell-free system.
Figure 3: γ-tubulin is essential for microtubule nucleation on microtubules.
Figure 4: Recruitment of cytosolic γ-tubulin for microtubule nucleation.
Figure 5: Model for nucleation of cortical microtubules in plants.

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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).

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Correspondence to Takashi Murata.

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Murata, T., Sonobe, S., Baskin, T. et al. Microtubule-dependent microtubule nucleation based on recruitment of γ-tubulin in higher plants. Nat Cell Biol 7, 961–968 (2005). https://doi.org/10.1038/ncb1306

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