Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

A PP2A-B55-Mediated Crosstalk between TORC1 and TORC2 Regulates the Differentiation Response in Fission Yeast.

Extracellular cues regulate cell fate, and this is mainly achieved through the engagement of specific transcriptional programs. The TORC1 and TORC2 complexes mediate the integration of nutritional cues to cellular behavior, but their interplay is poorly understood. Here, we use fission yeast to investigate how phosphatase activity participates in this interplay during the switch from proliferation to sexual differentiation. We find that loss of PP2A-B55Pab1 enhances the expression of differentiation-specific genes and leads to premature conjugation. pab1 deletion brings about a transcriptional profile similar to TORC1 inactivation, and deletion of pab1 overcomes the repression of differentiation genes in cells overexpressing TORC1. Importantly, we show that this effect is mediated by an increased TORC2-AKT (Gad8) signaling. Under nutrient-rich conditions, PP2A-B55Pab1 dephosphorylates Gad8 Ser546, repressing its activity. Conversely, TORC1 inactivation upon starvation leads to the inactivation of PP2A-B55Pab1 through the Greatwall-Endosulfin pathway. This results in the activation of Gad8 and the commitment to differentiation. Thus, PP2A-B55Pab1 enables a crosstalk between the two TOR complexes that controls cell-fate decisions in response to nutrient availability.

Nutritional Control of Cell Size by the Greatwall-Endosulfine-PP2A·B55 Pathway.

Proliferating cells adjust their cell size depending on the nutritional environment. Cells are large in rich media and small in poor media. This physiological response has been demonstrated in both unicellular and multicellular organisms. Here we show that the greatwall-endosulfine (Ppk18-Igo1 in fission yeast) pathway couples the nutritional environment to the cell-cycle machinery by regulating the activity of PP2A·B55. In the presence of nutrients, greatwall (Ppk18) protein kinase is inhibited by TORC1 and PP2A·B55 is active. High levels of PP2A·B55 prevent the activation of mitotic Cdk1·Cyclin B, and cells increase in size in G2 before they undergo mitosis. When nutrients are limiting, TORC1 activity falls off, and the activation of greatwall (Ppk18) leads to the phosphorylation of endosulfine (Igo1) and inhibition of PP2A·B55, which in turn allows full activation of Cdk1·CyclinB and entry into mitosis with a smaller cell size. Given the conservation of this pathway, it is reasonable to assume that this mechanism operates in higher eukaryotes, as well.