Releasing the brake: how kinetochore localized phosphatases allow silencing of the spindle-assembly checkpoint and consequently mitotic exit upon chromosome bi-orientation.

• Applicant: Dr. Debora Bade
• Subject Area: Cell Biology
• Term: from 2015 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 268630125

Genomic instability is a major contributor to solid tumor formation. Chromosomal instability, one of the modes of genomic instability, is the main cause for aneuploidy, a deviation from the diploid chromosome set. Aneuploidy occurs in 70% of all solid tumors. It is the consequence of chromosome segregation errors. As the spindle assembly checkpoint (SAC) monitors chromosome-spindle interactions and prevents mitotic progression before all chromosomes are attached, it protects against aneuploidy. Accordingly, several studies emphasized the importance of SAC proteins in tumor suppression. Importantly, two strategies that involve targeting the SAC have shown promise for anti-cancer therapy. The first is chronic activation by microtubule poisons, a strategy already in clinical use. The second is preventing mitotic exit, which was proposed to be more efficient in cancer cell killing than the microtubule poison. It will therefore be valuable to uncover mechanisms of SAC silencing required for timely/efficient mitotic exit.I will investigate how SAC silencing is achieved on a molecular level. I will specifically concentrate on how regulated dephosphorylation triggers SAC silencing and mitotic progression. Three aims will be pursued. First, I will identify the targets of the two phosphatases required for anaphase onset, namely PP1 and PP2A-B56. For this, I will use BioID and APEX purification approaches. In parallel, I will perform quantitative phospho-proteomics from cell lines in which localization of the phosphatases to kinetochores is specifically disrupted. In the second aim, I will address how dephosphorylation of the uncovered targets promotes SAC silencing. I will therefore use RNAi techniques and also work with the corresponding phospho-mimetic mutants. Finally, I will study how the phosphatases themselves are regulated in space and time. Together, these insights will contribute to fundamental understanding of the spatiotemporal control of SAC silencing that ensures timely and error-free chromosome segregation.

• DFG Programme: Research Fellowships
• International Connection: Netherlands
• Host: Professor Dr. Geert Kops