Cell division (Mitosis) is a fundamental process required for the generation of multicellular organisms, for tissue renewal and homeostasis. Mitosis drives the distribution of an identical genetic material into two daughter cells. Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. Consistently, unscheduled mitosis may contribute to genetic instability in numerous pathologies. Entry into mitosis is controlled by evolutionarily conserved serine/threonine kinases as well as counteracting phosphatases. How these kinase activities are regulated in space and time, and how they work in concert to trigger mitosis at the right time remain ill defined. Polo-like kinase (Plk1) is a highly conserved mitotic kinase instrumental for the accurate segregation of chromosomes during mitosis. Plk1 controls centrosome maturation, DNA condensation, chromatids separation, spindle assembly and cytokinesis. We recently determined that Plk1 is rapidly activated shortly before mitosis and is critically required for commitment to mitosis (Gheghiani et al. Cell Reports, in revision). We found that CyclinA2-Cdk, a S-promoting factor, is acting upstream of Plk1 activation. We also found that Plk1 is actively recruited to the nuclear envelope in prophase and contribute to Nuclear Envelope Break Down (NEBD) (Martino et al. Developmental Cell, in revision). Importantly, the mechanisms activating Plk1 in space and time to trigger a timely entry into mitosis are poorly defined. Likewise, the mechanism of Plk1 recruitment to the NE and its role in NEBD remain to be determined. Plk1 activation relies on the phosphorylation of a conserved residue (Thr210) in its activation segment (T-loop) by the Aurora-A kinase (AurkA). This process requires the conserved protein Bora, which stimulates Plk1 phosphorylation by AurkA. We determined that Bora phosphorylation by Cdk1/2 is critical for Plk1 activation in vitro and in vivo, both in C. elegans embryos and human cells. The identity of the Cyclin-Cdk complex involved, spatio-temporal regulation of Bora phosphorylation and the mechanistic by which Bora promotes AurkA-dependent Plk1 activation remain crucially unknown. Here, we aim to combine a multidisciplinary approach (cell biology, genetics, biochemistry and structural biology) in three model organisms (Xenopus egg extracts, human culture cells, C. elegans) to decipher the mechanistic of Plk1 activation and functions during mitotic entry. We believe that this project will provide a significant contribution in our understanding of the mechanisms that control a timely entry into mitosis to preserve the genetic integrity. We have the necessary complementary expertise in biochemistry, structural biology and cell biology as well as tools and methodologies to successfully achieve our objectives. • We will elucidate Plk1 activation mechanism by Bora and AurkA. Plk1 is a central component of cell division. Understanding its activation mechanism is of prime importance for the future development of strategies to interfere with its functions. Hence, this project will have significant societal and economic implications. Developing Bora inhibitors that will be used in combination with existing Plk1 inhibitors represents an attractive strategy to robustly inhibit Plk1 catalytic activity, which is deregulated in numerous diseases including cancers. • We will decipher the longstanding missing molecular link between CyclinA-Cdk2 and/or -Cdk1 and commitment to mitosis. Elucidating the role of CyclinA is essential to unravel how replication and mitosis are tightly coordinated during cell cycle. • We will decipher the unexplored role of Plk1 in nuclear envelope breakdown, a critical step for chromosome segregation and successful cell division.