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Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array

Nature Cell Biology volume 12, pages 1064–1070 (2010)Cite this article

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Abstract

Microtubule nucleation in interphase plant cells primarily occurs through branching from pre-existing microtubules at dispersed sites in the cell cortex1,2,3. The minus ends of new microtubules are often released from the sites of nucleation, and the free microtubules are then transported to new locations by polymer treadmilling1. These nucleation-and-release events are characteristic features of plant arrays in interphase cells, but little is known about the spatiotemporal control of these events by nucleating protein complexes. We visualized the dynamics of two fluorescently-tagged γ-tubulin complex proteins, GCP2 and GCP3, in Arabidopsis thaliana. These probes labelled motile complexes in the cytosol that transiently stabilized at fixed locations in the cell cortex. Recruitment of labelled complexes occurred preferentially along existing cortical microtubules, from which new microtubule was synthesized in a branching manner, or in parallel to the existing microtubule. Complexes localized to microtubules were approximately 10-fold more likely to display nucleation than were complexes recruited to other locations. Nucleating complexes remained stable until daughter microtubules were either completely depolymerized from their plus ends or released by katanin-dependent severing activity. These observations suggest that the nucleation complexes are primarily activated on association with microtubule lattices, and that nucleation complex stability depends on association with daughter microtubules and is regulated in part by katanin activity.

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Figure 1: GCP2–GFP particles are preferentially recruited to cortical microtubules.
Figure 2: New microtubules are initiated and released from locations defined by GFP-labelled GCP2 and GCP3 particles in etiolated Arabidopsis hypocotyl cells.
Figure 3: Classification of GCP2–GFP or GCP3–GFP localization, and microtubule polymerization, at the cell cortex.
Figure 4: Daughter microtubule release and stability of GCP2–GFP and GCP3–GFP in katanin mutant cells.
Figure 5: Model for nucleation and release of cortical microtubules in plant interphase cells.

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Acknowledgements

We thank Y. Fukao and M. Fujiwara (The Plant Science Education Unit, NAIST) for the mass spectrometry analysis, T. Kato for providing the katanin alleles, and D. Moss and R. Gutierrez for helpful discussions. The Arabidopsis Biological Resource Center is acknowledged for providing the genomic clones. M.N. was supported by a JSPS Research Fellowship for Young Scientist. The work was partly supported by a grant (20370023) and Global COE Program in NAIST (Frontier Biosciences: strategies for survival and adaptation in a changing global environment), MEXT, Japan, to T.H., and by endowment funds of the Carnegie Institution for Science to D.W.E.

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Authors and Affiliations

  1. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 630-0192, Ikoma, Japan

    Masayoshi Nakamura & Takashi Hashimoto

  2. Department of Plant Biology, Carnegie Institution for Science, Stanford, 94305, CA, USA

    David W. Ehrhardt

  3. Department of Biology, Stanford University, Stanford, 94305, CA, USA

    David W. Ehrhardt

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  1. Masayoshi Nakamura
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All authors designed experiments. M.N. and D.E. performed the experiments, and D.E. and T.H. wrote the paper. All authors analysed the results and edited the manuscript.

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Correspondence to David W. Ehrhardt or Takashi Hashimoto.

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The authors declare no competing financial interests.

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Nakamura, M., Ehrhardt, D. & Hashimoto, T. Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array. Nat Cell Biol 12, 1064–1070 (2010). https://doi.org/10.1038/ncb2110

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