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bioRxiv · 10.1101/2025.02.20.639226

In vitro reconstitution of minimal human centrosomes

Rios, M. U.·Stachera, W.·Familiari, N. E.·Brito, C.·Surrey, T.·Woodruff, J. B.
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Abstract

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 ({gamma}-TuRCs) which, in the presence of /{beta} tubulin, generated microtubule asters. We found that F75 in CDK5RAP2 is partially needed to recruit {gamma}-TuRC yet is indispensable for {gamma}-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 /{beta} 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.

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Rios, M. U., Stachera, W., Familiari, N. E., Brito, C., Surrey, T., Woodruff, J. B.. 2025-02-23. In vitro reconstitution of minimal human centrosomes. https://doi.org/10.1101/2025.02.20.639226

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