Purification and characterisation of the fission yeast Ndc80 complex

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that mult...

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Autores: Matsuo, Yuzy, Maurer, Sebastian, Surrey, Thomas, Toda, Takashi
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2017
País:España
Institución:Universitat Pompeu Fabra
Repositorio:Repositorio Digital de la UPF
OAI Identifier:oai:repositori.upf.edu:10230/35151
Acceso en línea:http://hdl.handle.net/10230/35151
http://dx.doi.org/10.1016/j.pep.2017.05.002
Access Level:acceso abierto
Palabra clave:Dis1
Fission yeast
Kinetochore
Ndc80 complex
Microtubule
TIRF microscopy
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spelling Purification and characterisation of the fission yeast Ndc80 complexMatsuo, YuzyMaurer, SebastianSurrey, ThomasToda, TakashiDis1Fission yeastKinetochoreNdc80 complexMicrotubuleTIRF microscopyThe Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1.Elsevier201820182017info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfapplication/pdfhttp://hdl.handle.net/10230/35151http://dx.doi.org/10.1016/j.pep.2017.05.002reponame:Repositorio Digital de la UPFinstname:Universitat Pompeu FabraInglésProtein Expr Purif. 2017 Jul;135:61-9© 2017 The Francis Crick Institute. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)http://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:repositori.upf.edu:10230/351512026-06-12T07:21:37Z
dc.title.none.fl_str_mv Purification and characterisation of the fission yeast Ndc80 complex
title Purification and characterisation of the fission yeast Ndc80 complex
spellingShingle Purification and characterisation of the fission yeast Ndc80 complex
Matsuo, Yuzy
Dis1
Fission yeast
Kinetochore
Ndc80 complex
Microtubule
TIRF microscopy
title_short Purification and characterisation of the fission yeast Ndc80 complex
title_full Purification and characterisation of the fission yeast Ndc80 complex
title_fullStr Purification and characterisation of the fission yeast Ndc80 complex
title_full_unstemmed Purification and characterisation of the fission yeast Ndc80 complex
title_sort Purification and characterisation of the fission yeast Ndc80 complex
dc.creator.none.fl_str_mv Matsuo, Yuzy
Maurer, Sebastian
Surrey, Thomas
Toda, Takashi
author Matsuo, Yuzy
author_facet Matsuo, Yuzy
Maurer, Sebastian
Surrey, Thomas
Toda, Takashi
author_role author
author2 Maurer, Sebastian
Surrey, Thomas
Toda, Takashi
author2_role author
author
author
dc.subject.none.fl_str_mv Dis1
Fission yeast
Kinetochore
Ndc80 complex
Microtubule
TIRF microscopy
topic Dis1
Fission yeast
Kinetochore
Ndc80 complex
Microtubule
TIRF microscopy
description The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1.
publishDate 2017
dc.date.none.fl_str_mv 2017
2018
2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10230/35151
http://dx.doi.org/10.1016/j.pep.2017.05.002
url http://hdl.handle.net/10230/35151
http://dx.doi.org/10.1016/j.pep.2017.05.002
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Protein Expr Purif. 2017 Jul;135:61-9
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Repositorio Digital de la UPF
instname:Universitat Pompeu Fabra
instname_str Universitat Pompeu Fabra
reponame_str Repositorio Digital de la UPF
collection Repositorio Digital de la UPF
repository.name.fl_str_mv
repository.mail.fl_str_mv
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