Branching microtubule (MT) nucleation is mediated by the augmin complex and γ-tubulin ring complex (γ-TuRC). However, how these two complexes work together to promote this process remains elusive. Here, using purified components from native and recombinant sources, we demonstrate that human augmin and γ-TuRC are sufficient to reconstitute the minimal MT branching machinery, in which NEDD1 bridges between augmin holo complex and GCP3/MZT1 subcomplex of γ-TuRC. The single-molecule experiment suggests that oligomerization of augmin may activate the branching machinery. We provide direct biochemical evidence that CDK1- and PLK1-dependent phosphorylation are crucial for NEDD1 binding to augmin, for their synergistic MT-binding activities, and hence for branching MT nucleation. In addition, we unveil that NEDD1 possesses an unanticipated intrinsic affinity for MTs via its WD40 domain, which also plays a pivotal role in the branching process. In summary, our study provides a comprehensive understanding of the underlying mechanisms of branching MT nucleation in human cells.
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4 July 2022
Article|
May 23 2022
Reconstitution and mechanistic dissection of the human microtubule branching machinery
Yaqian Zhang,
Yaqian Zhang
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
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Xing Hong,
Xing Hong
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
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Shasha Hua,
Shasha Hua
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
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Kai Jiang
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
Correspondence to Kai Jiang: jiangkai@whu.edu.cn
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Yaqian Zhang
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
Xing Hong
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
Shasha Hua
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
1
The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
2
Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
Correspondence to Kai Jiang: jiangkai@whu.edu.cn
Received:
September 15 2021
Revision Received:
March 07 2022
Accepted:
May 02 2022
Online Issn: 1540-8140
Print Issn: 0021-9525
Funding
Funder(s):
National Natural Science Foundation of China
- Award Id(s): 31871356,32070705
Funder(s):
Fundamental Research Funds for the Central Universities
© 2022 Zhang et al.
2022
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
J Cell Biol (2022) 221 (7): e202109053.
Article history
Received:
September 15 2021
Revision Received:
March 07 2022
Accepted:
May 02 2022
Citation
Yaqian Zhang, Xing Hong, Shasha Hua, Kai Jiang; Reconstitution and mechanistic dissection of the human microtubule branching machinery. J Cell Biol 4 July 2022; 221 (7): e202109053. doi: https://doi.org/10.1083/jcb.202109053
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