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

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  • dc.contributor.author Rios, Manolo U.
  • dc.contributor.author Stachera, Weronika E.
  • dc.contributor.author Familiari, Nicole E.
  • dc.contributor.author Brito, Cláudia
  • dc.contributor.author Surrey, Thomas
  • dc.contributor.author Woodruff, Jeffrey B.
  • dc.date.accessioned 2025-10-09T05:48:44Z
  • dc.date.available 2025-10-09T05:48:44Z
  • dc.date.issued 2025
  • dc.description.abstract 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.
  • dc.description.sponsorship 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.
  • dc.format.mimetype application/pdf
  • dc.identifier.citation Rios MU, Stachera WE, Familiari NE, Brito C, Surrey T, Woodruff JB. In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters. J Cell Sci. 2025 Jun 15;138(12):jcs264121. DOI: 10.1242/jcs.264121
  • dc.identifier.doi http://dx.doi.org/10.1242/jcs.264121
  • dc.identifier.issn 0021-9533
  • dc.identifier.uri http://hdl.handle.net/10230/71441
  • dc.language.iso eng
  • dc.publisher Company of Biologists
  • dc.relation.ispartof Journal of cell science. 2025 Jun 15;138(12):jcs264121
  • dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/951430
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PE/PID2022-142927NB-I00
  • dc.rights © 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.
  • dc.rights.accessRights info:eu-repo/semantics/openAccess
  • dc.rights.uri http://creativecommons.org/licenses/by/4.0
  • dc.subject.keyword CDK5RAP2
  • dc.subject.keyword Centrosome
  • dc.subject.keyword Microtubule
  • dc.subject.keyword PCM
  • dc.subject.keyword Scaffold
  • dc.subject.keyword Self-assembly
  • dc.title In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters
  • dc.type info:eu-repo/semantics/article
  • dc.type.version info:eu-repo/semantics/publishedVersion

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