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

Takashi Murata^1, 2, Seiji Sonobe³, Tobias I. Baskin⁴, Susumu Hyodo⁵, Seiichiro Hasezawa⁶, Toshiyuki Nagata⁷, Tetsuya Horio⁸ & Mitsuyasu Hasebe^1, 2

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

²  The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan.

³  Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan.

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

⁵  Ocean Research Institute, The University of Tokyo, Tokyo 164-8639, Japan.

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

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

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

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 array^{1, 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 -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 -tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting -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, -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 -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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