CENP-W plays a role in maintaining bipolar spindle structure.

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipo...

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Autores principales:	Agnieszka Kaczmarczyk, Kevin F Sullivan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/07e9c5a63d22406aa8fc07cb7cafaf64
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spelling	oai:doaj.org-article:07e9c5a63d22406aa8fc07cb7cafaf642021-11-25T05:56:35ZCENP-W plays a role in maintaining bipolar spindle structure.1932-620310.1371/journal.pone.0106464https://doaj.org/article/07e9c5a63d22406aa8fc07cb7cafaf642014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0106464https://doaj.org/toc/1932-6203The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. 'Spindle free' nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.Agnieszka KaczmarczykKevin F SullivanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e106464 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Agnieszka Kaczmarczyk Kevin F Sullivan CENP-W plays a role in maintaining bipolar spindle structure.
description	The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. 'Spindle free' nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.
format	article
author	Agnieszka Kaczmarczyk Kevin F Sullivan
author_facet	Agnieszka Kaczmarczyk Kevin F Sullivan
author_sort	Agnieszka Kaczmarczyk
title	CENP-W plays a role in maintaining bipolar spindle structure.
title_short	CENP-W plays a role in maintaining bipolar spindle structure.
title_full	CENP-W plays a role in maintaining bipolar spindle structure.
title_fullStr	CENP-W plays a role in maintaining bipolar spindle structure.
title_full_unstemmed	CENP-W plays a role in maintaining bipolar spindle structure.
title_sort	cenp-w plays a role in maintaining bipolar spindle structure.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/07e9c5a63d22406aa8fc07cb7cafaf64
work_keys_str_mv	AT agnieszkakaczmarczyk cenpwplaysaroleinmaintainingbipolarspindlestructure AT kevinfsullivan cenpwplaysaroleinmaintainingbipolarspindlestructure
_version_	1718414321832165376

CENP-W plays a role in maintaining bipolar spindle structure.

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