Sethi Suruchi, Ghetti Sabrina, Cmentowski Verena, Guerriere Teresa Benedetta, Stege Patricia, Piano Valentina, Musacchio Andrea
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
Eradigm Consulting, 6-7 St Cross St, London, EC1N 8UB, UK.
Nat Commun. 2025 May 24;16(1):4823. doi: 10.1038/s41467-025-59970-1.
The spindle assembly checkpoint (SAC) ensures mitotic exit occurs only after sister chromatid biorientation, but how this coordination is mechanistically achieved remains unclear. Kinetochores, the megadalton complexes linking chromosomes to spindle microtubules, contribute to SAC signaling. However, whether they act solely as docking platforms or actively promote the co-orientation of SAC catalysts such as MAD1:MAD2 and BUB1:BUB3 remains unresolved. Here, we reconstitute kinetochores and SAC signaling in vitro to address this question. We engineer recombinant kinetochore particles that recruit core SAC components and trigger checkpoint signaling upon Rapamycin induction, and test their function using a panel of targeted mutants. At approximately physiological concentrations of SAC proteins, kinetochores are essential for efficient mitotic checkpoint complex (MCC) assembly, the key effector of SAC signaling. Our results suggest that kinetochores serve not only as structural hubs but also as catalytic platforms that concentrate and spatially organize SAC components to accelerate MCC formation and ensure timely checkpoint activation.
纺锤体组装检查点（SAC）确保有丝分裂退出仅在姐妹染色单体双定向之后发生，但这种协调在机制上是如何实现的仍不清楚。动粒是将染色体与纺锤体微管连接起来的兆道尔顿复合物，对SAC信号传导有贡献。然而，它们是仅仅作为对接平台，还是积极促进诸如MAD1:MAD2和BUB1:BUB3等SAC催化剂的共定向，仍未得到解决。在这里，我们在体外重建动粒和SAC信号传导以解决这个问题。我们设计了重组动粒颗粒，其在雷帕霉素诱导时募集核心SAC成分并触发检查点信号传导，并使用一组靶向突变体测试它们的功能。在大约生理浓度的SAC蛋白下，动粒对于高效有丝分裂检查点复合物（MCC）组装是必不可少的，MCC是SAC信号传导的关键效应器。我们的结果表明，动粒不仅作为结构枢纽，而且作为催化平台，浓缩并在空间上组织SAC成分以加速MCC形成并确保及时的检查点激活。
