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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2022
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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. |
format | Online Article Text |
id | pubmed-9129923 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
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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