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GTP-dependent formation of straight oligomers leads to nucleation of microtubules

Rie Ayukawa, Seigo Iwata, Hiroshi Imai, Shinji Kamimura, Masahito Hayashi, Kien Xuan Ngo, Itsushi Minoura, Seiichi Uchimura, Tsukasa Makino, Mikako Shirouzu, Hideki Shigematsu, Ken Sekimoto, Benoît Gigant, Etsuko Muto
doi: https://doi.org/10.1101/2020.03.05.979989
Rie Ayukawa
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
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Seigo Iwata
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
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Hiroshi Imai
2Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan
7Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan
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Shinji Kamimura
2Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan
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Masahito Hayashi
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
8Department of Frontier Bioscience, Hosei University, Tokyo, Japan
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Kien Xuan Ngo
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
9WPI Nano Life Science Institute, Kanazawa University, Kanazawa, Japan
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Itsushi Minoura
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
10Product Development Division, Goryo Chemical, Inc., Sapporo, Japan
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Seiichi Uchimura
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
11CPI Company, DAICEL Corporation, Hyogo, Japan
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Tsukasa Makino
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
12Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
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Mikako Shirouzu
3Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan
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Hideki Shigematsu
3Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan
13Life Science Research Infrastructure Group, RIKEN SPring-8 Center, Hyogo, Japan
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Ken Sekimoto
4Matière et Systèmes Complexes (MSC), CNRS UMR 7057, Université de Paris, Paris, France
5Gulliver, CNRS UMR 7083, ESPCI Paris and PSL University, Paris, France
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  • For correspondence: etsuko.muto@riken.jp benoit.gigant@i2bc.paris-saclay.fr ken.sekimoto@espci.fr
Benoît Gigant
6Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
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  • For correspondence: etsuko.muto@riken.jp benoit.gigant@i2bc.paris-saclay.fr ken.sekimoto@espci.fr
Etsuko Muto
1Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan
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  • For correspondence: etsuko.muto@riken.jp benoit.gigant@i2bc.paris-saclay.fr ken.sekimoto@espci.fr
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Abstract

Microtubule (MT) nucleation is essential for cellular activities, but its mechanism is not known because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. In cells, MTs are nucleated at tubulin concentrations significantly lower than those required for spontaneous nucleation in vitro. The high efficiency of nucleation is due to the synergistic effects of various cellular factors, but the underlying mechanism has not been clarified yet. Here, combining negative stain electron microscopy and kinetic analysis, we demonstrate that the formation of single-stranded straight oligomers with critical size is essential for nucleation in vitro. While the single-stranded oligomers of GTP-tubulin that form prior to MT nucleation show variable curvatures including a few straight oligomers, only curved oligomers are observed among the GDP-bound counterparts. The Y222F mutation in β-tubulin increases the proportion of straight oligomers and drastically accelerates MT nucleation. Our results support a model in which GTP binding causes a small shift in the distribution of oligomer curvature, generating a minor population of straight oligomers compatible with lateral association and further growth to MTs. Our study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

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Posted March 06, 2020.
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GTP-dependent formation of straight oligomers leads to nucleation of microtubules
Rie Ayukawa, Seigo Iwata, Hiroshi Imai, Shinji Kamimura, Masahito Hayashi, Kien Xuan Ngo, Itsushi Minoura, Seiichi Uchimura, Tsukasa Makino, Mikako Shirouzu, Hideki Shigematsu, Ken Sekimoto, Benoît Gigant, Etsuko Muto
bioRxiv 2020.03.05.979989; doi: https://doi.org/10.1101/2020.03.05.979989
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GTP-dependent formation of straight oligomers leads to nucleation of microtubules
Rie Ayukawa, Seigo Iwata, Hiroshi Imai, Shinji Kamimura, Masahito Hayashi, Kien Xuan Ngo, Itsushi Minoura, Seiichi Uchimura, Tsukasa Makino, Mikako Shirouzu, Hideki Shigematsu, Ken Sekimoto, Benoît Gigant, Etsuko Muto
bioRxiv 2020.03.05.979989; doi: https://doi.org/10.1101/2020.03.05.979989

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