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

Abstract

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.

Gene Expression Analysis of Yeast Strains with a Nonsense Mutation in the eRF3-Coding Gene Highlights Possible Mechanisms of Adaptation

“…Most cells that remain in mitosis for a long time undergo apoptosis, but some of them skip cytokinesis and enter G1 with non-segregated chromosomes. This process, called mitotic slippage, increases genome instability and results in aneuploidy [ 40 ]. If a similar process can occur in S. cerevisiae cells upon CDC20 downregulation, such slippage can provide a mechanistic basis for the observed amplification of sup35-n and sup45-n alleles [ 10 ].…”

Genome-wide profiling of the hierarchical control of autophagy dynamics using deep learning

“…Cell lysis and immunoblotting were performed as previously described 90 . For protein analysis, 2 x 10 8 cells were collected by filtration, washed with STOP buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM sodium azide) and frozen on dry ice before extraction.…”

“…RNA extraction and quantitative PCR (qPCR) were performed as previously described 90 . For qPCR analysis, 8 x 10 7 cells were collected by centrifugation, washed in DEPC-treated water and frozen in dry ice.…”