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Reconstitution and mechanistic dissection of the human microtubule branching machinery

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 a...

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Detalles Bibliográficos
Autores principales: Zhang, Yaqian, Hong, Xing, Hua, Shasha, Jiang, Kai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9129923/
https://www.ncbi.nlm.nih.gov/pubmed/35604367
http://dx.doi.org/10.1083/jcb.202109053
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author Zhang, Yaqian
Hong, Xing
Hua, Shasha
Jiang, Kai
author_facet Zhang, Yaqian
Hong, Xing
Hua, Shasha
Jiang, Kai
author_sort Zhang, Yaqian
collection PubMed
description 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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spelling pubmed-91299232023-01-04 Reconstitution and mechanistic dissection of the human microtubule branching machinery Zhang, Yaqian Hong, Xing Hua, Shasha Jiang, Kai J Cell Biol Article 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. Rockefeller University Press 2022-05-23 /pmc/articles/PMC9129923/ /pubmed/35604367 http://dx.doi.org/10.1083/jcb.202109053 Text en © 2022 Zhang et al. https://creativecommons.org/licenses/by-nc-sa/4.0/http://www.rupress.org/terms/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/).
spellingShingle Article
Zhang, Yaqian
Hong, Xing
Hua, Shasha
Jiang, Kai
Reconstitution and mechanistic dissection of the human microtubule branching machinery
title Reconstitution and mechanistic dissection of the human microtubule branching machinery
title_full Reconstitution and mechanistic dissection of the human microtubule branching machinery
title_fullStr Reconstitution and mechanistic dissection of the human microtubule branching machinery
title_full_unstemmed Reconstitution and mechanistic dissection of the human microtubule branching machinery
title_short Reconstitution and mechanistic dissection of the human microtubule branching machinery
title_sort reconstitution and mechanistic dissection of the human microtubule branching machinery
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9129923/
https://www.ncbi.nlm.nih.gov/pubmed/35604367
http://dx.doi.org/10.1083/jcb.202109053
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