Background: Two principle surveillance mechanisms have evolved to guard against errors during chromosome segregation. The mitotic checkpoint delays mitosis until each and every sister chromatid has achieved stable attachment to spindle microtubules (via a protein complex known as the kinetochore), and the microtubule error-correction pathway guides this attachment process by ensuring it remains free of errors. In spite of these safeguards, the majority of tumours still manage to continually missegregate their genome. Understanding exactly how they manage to achieve this is a major goal in cancer biology because the resulting chromosomal instability (CIN) is thought to drive tumour initiation and/or progression. We have previously shown the main kinases that regulate these processes (Mps1 and Aurora B) are dependent on each other for their proper localisation to kinetochores, which allows rapid activation of both kinases at the onset of mitosis (1, 2). We have recently gone on to show that two phosphatases (PP1 and PP2A-B56) antagonise these kinase inputs to silence the mitotic checkpoint (3, 4).
Aims: This project will test investigate how this kinase-phosphatase feedback network regulates chromosome segregation and why it fails to do this properly in cancer cells.
Methods: This project will use established gene-editing techniques (CRISPR/Cas9 and AAV – 5, 6) to fluorescently tag endogenous kinases and phosphatases in mammalian cells. We have also established a chemical-genetic system that allows endogenous phosphatase localisation to be inducibly modified with small molecule drugs (termed rapalogs). These techniques, coupled with quantitative high-resolution live cell microscopy, will allow the localisation of endogenous phosphatase complexes to be visualised and modified live. This have never previously been achievable, which will pave the way to tackle many unanswered questions.
- Saurin AT, van der Waal MS, Medema RH, Lens SMA & Kops GJPL. Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis. Nature Communications (2011).
- *van der Waal MS, *Saurin AT, Vromans MJ, Vleugel M, Wurzenberger C, Gerlich DW, Medema RH, Kops GJ & Lens SM. Mps1 promotes rapid centromere accumulation of Aurora B. EMBO Reports (2012). *Equal contribution
- Nijenhuis W, Vallardi G, Teixeira A, Kops GJ, Saurin AT. Negative feedback at kinetochores underlies a responsive spindle checkpoint signal. Nature Cell Biology. 2014. Dec;16(12):1257-64.
- Vallardi G and Saurin AT. Mitotic kinases and phosphatases cooperate to shape the right response. Cell Cycle. 2015;14(6):795-6.
- Shaltiel IA, Aprelia M, Saurin AT, Chowdhury D, Kops GJPL, Voest EE, Medema RH. Distinct phosphatases antagonize the p53 response in different phases of the cell cycle. PNAS. 2014. 20;111(20):7313-8.
- Akopyan K, Silva Cascales H, Hukasova E, Saurin AT, Müllers E, Jaiswal H, Hollman DA, Kops GJ, Medema RH, Lindqvist A. Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the s/g2 transition. Molecular Cell. 2014 53(5); 843-853.