CDK5RAP2/CEP215 is a key pericentriolar material (PCM) protein that recruits microtubule-nucleating factors at human centrosomes. Using an in vitro reconstitution system, we show that CDK5RAP2 is sufficient to form micron-scale scaffolds around a nanometer-scale nucleator in a PLK-1-regulated manner. CDK5RAP2 assemblies recruited and activated gamma tubulin ring complexes (γ-TuRCs) which, in the presence of α/β tubulin, generated microtubule asters. We found that F75 in CDK5RAP2 is partially needed to recruit γ-TuRC yet is indispensable for γ-TuRC activation. Furthermore, our system recapitulated key features of centrosome-amplified cancer cells. CDK5RAP2 scaffolds selectively recruited the molecular motor KifC1/HSET, 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 centrosome assembly and function. This minimal centrosome model offers a powerful tool for studying centrosome biology and dysfunction in human health and disease.
In vitro reconstitution of minimal human centrosomes.
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作者:Rios Manolo U, Stachera Weronika E, Familiari Nicole E, Brito Claudia, Surrey Thomas, Woodruff Jeffrey B
期刊: | bioRxiv | 影响因子: | 0.000 |
时间: | 2025 | 起止号: | 2025 Feb 20 |
doi: | 10.1101/2025.02.20.639226 |
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