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

Rios, Manolo U.; Stachera, Weronika E.; Familiari, Nicole E.; Brito, Cláudia; Surrey, Thomas; Woodruff, Jeffrey B.

doi:http://dx.doi.org/10.1242/jcs.264121

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

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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

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.