Monitoring Chk1 kinase activity dynamics in live single cell imaging assays.

The ATR/Chk1 pathway is an important regulator of cell cycle progression, notably upon genotoxic stress where it can detect a large variety of DNA alterations and induce a transient cell cycle arrest that promotes DNA repair. In addition to its role in DNA damage response (DDR), Chk1 is also active during a non-perturbed S phase and contributes to prevent a premature entry into mitosis with an incompletely replicated genome, meaning the ATR/Chk1 pathway is an integral part of the cell cycle machinery that preserves genome integrity during cell growth. We recently developed a FRET-based Chk1 kinase activity reporter to directly monitor and quantify the kinetics of Chk1 activation in live single cell imaging assays with unprecedented sensitivity and time resolution.

Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome.

In human cells, ATR/Chk1 signaling couples S phase exit with the expression of mitotic inducers and prevents premature mitosis upon replication stress (RS). Nonetheless, under-replicated DNA can persist at mitosis, prompting chromosomal instability. To decipher how the DNA replication checkpoint (DRC) allows cells to enter mitosis over time upon RS, we developed a FRET-based Chk1 activity sensor.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity.

AURKA destruction is decoupled from its activity at mitotic exit but is essential to suppress interphase activity.

Activity of AURKA is controlled through multiple mechanisms including phosphorylation, ubiquitin-mediated degradation and allosteric interaction with TPX2. Activity peaks at mitosis, before AURKA is degraded during and after mitotic exit in a process strictly dependent on the APC/C coactivator FZR1. We used FZR1 knockout cells (FZR1) and a novel FRET-based AURKA biosensor to investigate how AURKA activity is regulated in the absence of destruction.

Re-investigating PLK1 inhibitors as antimitotic agents.

Polo-like kinase 1 (PLK1) plays key roles during mitosis, prompting the development of PLK1 inhibitors for anticancer therapy. We recently determined that PLK1 is crucially required for entry into mitosis. Hence, we discuss the potential and limitations of PLK1 inhibition strategies to promote mitotic arrest and death of cancer cells.