Rios Manolo U, Stachera Weronika E, Familiari Nicole E, Brito Claudia, Surrey Thomas, Woodruff Jeffrey B
Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), 08003 Barcelona, Spain.
J Cell Sci. 2025 Jun 15;138(12). doi: 10.1242/jcs.264121. Epub 2025 Jun 27.
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.
CDK5RAP2（也称为CEP215）是一种关键的中心粒外周物质（PCM）蛋白，可在人类中心体招募微管成核因子。在此，我们利用体外重建系统表明，CDK5RAP2足以以一种受PLK-1调节的方式，利用纳米级成核剂形成微米级支架。CDK5RAP2组装体招募并激活γ-微管蛋白环复合物（γ-TuRCs），后者在α/β-微管蛋白存在的情况下生成微管星状体。我们发现，CDK5RAP2中的氨基酸F75有助于招募γ-TuRC，并且对于γ-TuRC的激活必不可少。此外，我们的系统重现了中心体扩增癌细胞的关键特征。CDK5RAP2支架招募分子马达HSET（也称为KifC1），后者增强了α/β-微管蛋白的浓度、微管聚合以及组装体的聚集。我们的结果突出了体外生成的CDK5RAP2支架的特异性和选择性，并确定了人类PCM组装和功能所需的一组最小成分。这个最小模型为研究人类健康和疾病中的中心体生物学及功能障碍提供了一个强大的工具。
