In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters

CDK5RAP2 (also known as CEP215) is a key pericentriolar material (PCM) protein that recruits microtubule-nucleating factors at human centrosomes. Here, using an in vitro reconstitution system, we show that CDK5RAP2 is sufficient to form micron-scale scaffolds using nanometer-scale nucleators in a PL...

Descripción completa

Detalles Bibliográficos
Autores: Rios, Manolo U., Stachera, Weronika E., Familiari, Nicole E., Brito, Cláudia, Surrey, Thomas, Woodruff, Jeffrey B.
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2025
País:España
Institución:Universitat Pompeu Fabra
Repositorio:Repositorio Digital de la UPF
OAI Identifier:oai:repositori.upf.edu:10230/71441
Acceso en línea:http://hdl.handle.net/10230/71441
http://dx.doi.org/10.1242/jcs.264121
Access Level:acceso abierto
Palabra clave:CDK5RAP2
Centrosome
Microtubule
PCM
Scaffold
Self-assembly
id ES_83542e26bc9b5f9013eaf71bf734fadb
oai_identifier_str oai:repositori.upf.edu:10230/71441
network_acronym_str ES
network_name_str España
repository_id_str
spelling In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule astersRios, Manolo U.Stachera, Weronika E.Familiari, Nicole E.Brito, CláudiaSurrey, ThomasWoodruff, Jeffrey B.CDK5RAP2CentrosomeMicrotubulePCMScaffoldSelf-assemblyCDK5RAP2 (also known as CEP215) is a key pericentriolar material (PCM) protein that recruits microtubule-nucleating factors at human centrosomes. Here, using an in vitro reconstitution system, we show that CDK5RAP2 is sufficient to form micron-scale scaffolds using nanometer-scale nucleators in a PLK-1-regulated manner. CDK5RAP2 assemblies recruited and activated γ-tubulin ring complexes (γ-TuRCs) which, in the presence of α/β-tubulin, generated microtubule asters. We found that amino acid F75 in CDK5RAP2 helps to recruit γ-TuRC and is indispensable for γ-TuRC activation. Furthermore, our system recapitulated key features of centrosome-amplified cancer cells. CDK5RAP2 scaffolds recruited the molecular motor HSET (also known as KifC1), which enhanced concentration of α/β-tubulin, microtubule polymerization and clustering of the assemblies. Our results highlight the specificity and selectivity of in vitro-generated CDK5RAP2 scaffolds, and identify a minimal set of components required for human PCM assembly and function. This minimal model offers a powerful tool for studying centrosome biology and dysfunction in human health and disease.J.B. Woodruff is supported by a Welch Foundation grant (V-I-0004-20230731), an R35 grant from the National Institute of General Medical Sciences (1R35GM142522), and the Endowed Scholars program at UT Southwestern. T. Surrey acknowledges the support of the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI/10.13039/501100011033) and the Generalitat de Catalunya through the CERCA program; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 951430); and from the Spanish Ministry of Science and Innovation (grant PID2022-142927NB-I00/AEI/10.13039/501100011033/FEDER, EU). C.B. was supported by a European Molecular Biology Organization (EMBO) long-term fellowship ALTF-883-2020 and Marie Curie fellowship TuRCReg. M.U.R. was supported by a National Research Service Award T32 (GM007062). Open Access funding provided by UT Southwestern Medical Center. Deposited in PMC for immediate release.Company of Biologists202520252025info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfapplication/pdfhttp://hdl.handle.net/10230/71441http://dx.doi.org/10.1242/jcs.264121reponame:Repositorio Digital de la UPFinstname:Universitat Pompeu FabraInglésJournal of cell science. 2025 Jun 15;138(12):jcs264121info:eu-repo/grantAgreement/EC/H2020/951430info:eu-repo/grantAgreement/ES/3PE/PID2022-142927NB-I00© 2025. Published by The Company of Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.http://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessoai:repositori.upf.edu:10230/714412026-06-12T07:21:37Z
dc.title.none.fl_str_mv In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
title In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
spellingShingle In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
Rios, Manolo U.
CDK5RAP2
Centrosome
Microtubule
PCM
Scaffold
Self-assembly
title_short In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
title_full In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
title_fullStr In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
title_full_unstemmed In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
title_sort In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
dc.creator.none.fl_str_mv Rios, Manolo U.
Stachera, Weronika E.
Familiari, Nicole E.
Brito, Cláudia
Surrey, Thomas
Woodruff, Jeffrey B.
author Rios, Manolo U.
author_facet Rios, Manolo U.
Stachera, Weronika E.
Familiari, Nicole E.
Brito, Cláudia
Surrey, Thomas
Woodruff, Jeffrey B.
author_role author
author2 Stachera, Weronika E.
Familiari, Nicole E.
Brito, Cláudia
Surrey, Thomas
Woodruff, Jeffrey B.
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv CDK5RAP2
Centrosome
Microtubule
PCM
Scaffold
Self-assembly
topic CDK5RAP2
Centrosome
Microtubule
PCM
Scaffold
Self-assembly
description CDK5RAP2 (also known as CEP215) is a key pericentriolar material (PCM) protein that recruits microtubule-nucleating factors at human centrosomes. Here, using an in vitro reconstitution system, we show that CDK5RAP2 is sufficient to form micron-scale scaffolds using nanometer-scale nucleators in a PLK-1-regulated manner. CDK5RAP2 assemblies recruited and activated γ-tubulin ring complexes (γ-TuRCs) which, in the presence of α/β-tubulin, generated microtubule asters. We found that amino acid F75 in CDK5RAP2 helps to recruit γ-TuRC and is indispensable for γ-TuRC activation. Furthermore, our system recapitulated key features of centrosome-amplified cancer cells. CDK5RAP2 scaffolds recruited the molecular motor HSET (also known as KifC1), which enhanced concentration of α/β-tubulin, microtubule polymerization and clustering of the assemblies. Our results highlight the specificity and selectivity of in vitro-generated CDK5RAP2 scaffolds, and identify a minimal set of components required for human PCM assembly and function. This minimal model offers a powerful tool for studying centrosome biology and dysfunction in human health and disease.
publishDate 2025
dc.date.none.fl_str_mv 2025
2025
2025
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/10230/71441
http://dx.doi.org/10.1242/jcs.264121
url http://hdl.handle.net/10230/71441
http://dx.doi.org/10.1242/jcs.264121
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Journal of cell science. 2025 Jun 15;138(12):jcs264121
info:eu-repo/grantAgreement/EC/H2020/951430
info:eu-repo/grantAgreement/ES/3PE/PID2022-142927NB-I00
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 Company of Biologists
publisher.none.fl_str_mv Company of Biologists
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
_version_ 1869412127404457984
score 15,811543

Similares en LA Referencia