PLK1 promotes the mitotic surveillance pathway by controlling cytosolic 53BP1 availability

Mitosis is a highly regulated process leading to the formation of two genetically identical cells. The main events defining mitotic duration are Cyclin B/CDK1 activation and the activation of the anaphase-promoting complex/cyclosome (APC/C), with the former essential for mitotic entry and the latter responsible for mitotic exit. There are a lot of control mechanisms ensuring mitotic fidelity, among them, the spindle assembly checkpoint (SAC) delays the anaphase onset until all chromosomes are bi-oriented on the mitotic spindle. However, this arrest cannot last indefinitely, and SAC activity might not suffice to preserve mitotic fidelity. Indeed, an additional fail-safe mechanism was revealed: the mitotic surveillance pathway (MSP). This mechanism monitors the duration of cell division, inducing p53-dependent cell cycle arrest when a critical time threshold is exceeded. 53BP1, a protein playing a central role in bridging DNA repair and the p53-mediated stress response, functions as a key component of the mitotic surveillance pathway. 53BP1 has been reported to be associated with mitotic kinetochores. Furthermore, the interaction between 53BP1 and kinetochores seems to rely on mitotic duration, regardless of the SAC activation status. Whether the temporary binding and release of 53BP1 at kinetochores play a role in MSP activation was not clear. Herein, I present evidence demonstrating that Polo-like kinase 1 (PLK1) activity is vital for the time-dependent release of 53BP1 from kinetochores. Indeed, in prolonged mitosis, the inhibition of PLK1 results in forcing 53BP1 at the kinetochore. This leads to a reduction in the interaction with p53 in the cytosol, diminishing MSP efficiency. In addition, I show that the fibrous corona protein CENP-F directly interacts with 53BP1 and it is crucial for recruiting 53BP1 to kinetochores (KT). Using gene editing, I introduced a single amino acid substitution engineering the endogenous CENPF locus in human cells. Taking advantage of this mutant, I present evidence supporting the notion that the localization of 53BP1 at KTs is not essential for the functionality of the MSP as neither an unscheduled activation nor an inactivation of the pathway occur when 53BP1 recruitment at the KT is hindered. However, in contrast with the findings in WT cells, our CENP-F mutant cells show that cytosolic 53BP1 can still bind to p53 and support MSP activation, even in the absence of PLK1 activity. Collectively, these results suggest that PLK1 supports the MSP by generating a cytosolic pool of 53BP1. Although this is a step forward in the understanding of this pathway, the measurement of mitotic timing relies on an unknown cytosolic mechanism.