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Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle
The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecula...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Company of Biologists Ltd
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450317/ https://www.ncbi.nlm.nih.gov/pubmed/28351835 http://dx.doi.org/10.1242/bio.022905 |
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author | Chen, Jack W. C. Chen, Zhuo A. Rogala, Kacper B. Metz, Jeremy Deane, Charlotte M. Rappsilber, Juri Wakefield, James G. |
author_facet | Chen, Jack W. C. Chen, Zhuo A. Rogala, Kacper B. Metz, Jeremy Deane, Charlotte M. Rappsilber, Juri Wakefield, James G. |
author_sort | Chen, Jack W. C. |
collection | PubMed |
description | The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of in vivo Augmin from Drosophila and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and γ-TuRC. We tested these predictions biochemically and in the Drosophila embryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the γ-TuRC protein, Dgp71WD, and are required for the accumulation of γ-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation. |
format | Online Article Text |
id | pubmed-5450317 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | The Company of Biologists Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-54503172017-06-01 Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle Chen, Jack W. C. Chen, Zhuo A. Rogala, Kacper B. Metz, Jeremy Deane, Charlotte M. Rappsilber, Juri Wakefield, James G. Biol Open Research Article The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of in vivo Augmin from Drosophila and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and γ-TuRC. We tested these predictions biochemically and in the Drosophila embryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the γ-TuRC protein, Dgp71WD, and are required for the accumulation of γ-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation. The Company of Biologists Ltd 2017-03-28 /pmc/articles/PMC5450317/ /pubmed/28351835 http://dx.doi.org/10.1242/bio.022905 Text en © 2017. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/3.0This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. |
spellingShingle | Research Article Chen, Jack W. C. Chen, Zhuo A. Rogala, Kacper B. Metz, Jeremy Deane, Charlotte M. Rappsilber, Juri Wakefield, James G. Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
title | Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
title_full | Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
title_fullStr | Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
title_full_unstemmed | Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
title_short | Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
title_sort | cross-linking mass spectrometry identifies new interfaces of augmin required to localise the γ-tubulin ring complex to the mitotic spindle |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450317/ https://www.ncbi.nlm.nih.gov/pubmed/28351835 http://dx.doi.org/10.1242/bio.022905 |
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