The Cell Cycle
The cell cycle is an ordered series of events happening in eukaryotic cells to grow and divide into two new cells. Basically, cells must complete four major events during the cell cycle: growth (G1 and G2 phases), replicate the DNA (S phase), segregate the chromosome (M phase) and divide (cytokinesis). How eukaryotes, including human, inherit their nuclear genome is a fundamental question in biology. It also has direct clinical implications as chromosome missegregation is a leading cause for miscarriages and birth defects, and is tightly linked to malignant tumour progression. The cell cyle in budding yeast is very similar to the cell cycle in humans and is regulated by the same proteins that are conserved through evolution.
Mitosis is an intricately coordinated set of events that ensures the accurate inheritance of genetic information from one cell generation to the next. Entry into mitosis occurs when the Cdk-cyclinB complex reach a peak of kinase activity. In metaphase the chromosomes are condensed, aligned in the metaphase plaque and attached to the mitotic spindle. Proper attachment of the chromosomes will lead to the activation of the anaphase-promoting complex (APC) by its co-activator Cdc20. APC Cdc20 is an ubiquitin ligase that ubiquitilates securin, an inhibitor of the protease separase. Thereby, securin is degraded by the proteasome and separase is activated. Sister chromatid separation at anaphase onset is triggered when the Scc1 subunit of cohesin is cleaved by separase to destroy the cohesin complex. At this time, APC Cdc20 also targets the cyclin Clb2 for degradation promoting the reduction of Cdk activity. However, cyclin destruction by APC Cdc20 is not sufficient to completely remove all Cdk activity in order to exit from mitosis. For this reason, the activation of the mitotic phosphatase Cdc14 becomes essential. Cdc14 phosphatase directly counteracts the Cdk activity by dephosphorylating the Cdk targets. On the other hand, Cdc14 also contributes to the downregulation of the Cdk activity by dephosphorylating a second co-activator of APC, Cdh1, that complete destruction of all mitotic cyclins, and the Cdk inhibitor Sic1.
In metaphase, Cdc14 is kept inactive in the nucleolus by binding to the nucleolar protein Net1 (also called Cfi1). During anaphase, Cdk-dependent phosphorylation of Net1 release active Cdc14. Phosphorylated Net1 shows reduced affinity for Cdc14 and loose its ability to inhibit Cdc14 in vitro. Two different regulatory pathways are essentials for the Cdc14 release from the nucleolus. During early anaphase the FEAR pathway (Cdc f ourteen e arly a naphase r elease) initiates the Cdc14 release and is kept active later in anaphase by a G protein signalling cascade, the mitotic exit network (MEN). A number of proteins including, Cdk, Slk19, Spo12, Fob1 and separase have been implicated in this early anaphase Cdc14 release. Several mutants in the FEAR show a delay in the Cdc14 release from the nucleolus. Nevertheless, an essential role in Cdc14 activation and mitotic exit for separase has been recently described (Queralt et al., 2006) . The FEAR dependent Cdc14 release requires Net1 phosphorylation at Cdk consensus sites. PP2A Cdc55 phosphatase keeps Net1 under-phosphorylated in metaphase (Queralt et al., 2006) . Separase-dependent PP2A Cdc55 downregulation initiates the Cdk-dependent Net1 phosphorylation specifically in anaphase, when mitotic kinase activity starts to decline. The mechanistic basis for separase-dependent PP2A Cdc55 downregulation remains to be elucidated. Later in anaphase, when the Cdk activity is low, the MEN kinases mantain Net1 phosphorylated and Cdc14 release.