Regulation of exit from mitosis
Exit from mitosis is the final cell cycle transition when mitotic cyclin-dependent kinases (CDKs) are inactivated, the mitotic spindle disassembles, chromosomes decondense and cytokinesis occurs. In budding yeast, the protein phosphatase Cdc14 is the central regulator of this cell cycle transition (Visintin et al., 1998). We study how this phosphatase is controlled and how it regulates the final stages of mitosis.
Cdc14 is regulated by an inhibitor Cfi1/Net1 that binds to and sequesters Cdc14 in the nucleolus during G1, S phase, G2 and metaphase (Visintin et al., 1999). During anaphase, Cdc14 is released from its inhibitor and spreads throughout the nucleus and cytoplasm, where it dephosphorylates its targets. We identified two pathways that control the association between Cdc14 and its inhibitor. The Cdc14 Early Anaphase Release Network (FEAR network) promotes Cdc14 release from the nucleolus during early anaphase (Stegmeier et al., 2002) and the Mitotic Exit Network (MEN) maintains Cdc14 in its released state during late stages of anaphase (Visintin et al., 1999). Currently we are determining how the individual MEN components are regulated and how their activity is integrated with other cell cycle events such as chromosome segregation and nuclear position (Chan and Amon, 2009; Chan and Amon, 2010).
Regulation of a specialized cell cycle - the meiotic cell cycle
Our insights into mitosis spawned our interest in how the molecular mechanisms that govern mitosis are modulated to bring about a specialized division, meiosis. Meiosis consists of two consecutive chromosome segregation phases. During meiosis I, separation of homologous chromosomes occurs; during meiosis II, segregation of sister chromatids takes place. We study how removal of cohesins, the proteins that hold sister chromatids together, is changed during meiosis (Kiburz et al., 2005; Brar et al., 2006) and how kinetochore – microtubule attachments are modified during meiosis I (Monje-Casas et al., 2007). Finally, we determine how meiosis-specific control of CDKs establishes the meiotic chromosome segregation pattern (Carlile and Amon, 2008). Unexpectedly, Clb3-CDK activity is restricted to meiosis II by 5’UTR-mediated translational control. Furthermore, our data indicate that this regulation is essential for establishing the meiotic chromosome segregation pattern. Currently, we are determining the mechanisms of CDK control and how the kinases themselves help establish the meiotic chromosome segregation pattern.
We have also initiated a new research area that addresses the question: Why does the ability to enter the meiotic program and chromosome segregation fidelity decrease with age? We find that aged yeast cells exhibit both these phenotypes (Boselli et al., 2009). Using the tools we established to study meiotic chromosome segregation we now employ to determine the molecular basis for these defects. We are also interested in determining the effects of meiosis on age in budding yeast. Specifically, we are investigating whether meiosis can reset the replicative life span in yeast.
The effects of aneuploidy on cell proliferation
Our studies on the mechanisms of chromosome segregation have provided insights into the processes that prevent aneuploidy due to chromosome mis-segregation. We also study what happens to yeast cells that, defying the mitotic quality controls, acquired extra chromosomes and hence are aneuploid. We discovered unanticipated commonalities among many different aneuploid yeast strains that we created. This encouraged us to extended our analysis to aneuploid primary mouse cells.
The exploration of the commonalities among cells that are aneuploid involved the creation of 20 yeast strains carrying one or two additional chromosomes and, subsequently, of primary mouse embryonic fibroblasts (MEFs) carrying four different trisomies (trisomy 1, 13, 16 or 19; Torres et al., 2007; Williams et al., 2008). Their analysis revealed that aneuploidy is deleterious at the cellular level, causing cell proliferation defects in both yeast and mouse. Perhaps most exiting was our discovery that aneuploid yeast and mouse cells share a number of phenotypes that are indicative of proteotoxic and energy stress. Our studies further showed that the genes located on the additional chromosomes are expressed and that the phenotypes shared by aneuploid strains are due to the proteins that are being produced from the additional chromosomes. Currently, we are focusing on characterizing the effects of aneuploidy on the protein quality control pathways of the cell in yeast and in the mouse.
From our studies we concluded that aneuploidy leads to a cellular response, which we term the “aneuploidy stress response”. In this response cells engage protein folding and degradation pathways in an attempt to correct protein stoichiometry imbalances caused by aneuploidy. Although the proteins that engage the protein degradation and folding machineries will be different for each additional chromosome, the necessity to degrade and fold excess protein, compromises the cell’s ability to fold and degrade proteins whose excess presence in the cell interferes with essential cellular processes. Thus, aneuploidy leads to an increased burden on the protein quality control pathways, an increased need for energy and contributes to the cell proliferation defect seen in aneuploids. Currently, we are identifying genetic alterations that suppress and enhance the adverse affects of aneuploidy (Torres et al., 2010). It is our hope that these studies will provide insights into how cancer cells evolve to tolerate the adverse effects of aneuploidy in order to take advantage of this genetic condition. Identifying compounds and genetic alterations that cause lethality in aneuploid but not euploid cells may provide new cancer treatments.
Boselli M, Rock J, Unal E, Levine SS, Amon A. (2009). Effects of age on meiosis in budding yeast. Dev Cell 16, 844-855.
Brar GA, Kiburz BM, Zhang Y, Kim JE, White F, Amon A. (2006). Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis. Nature, 441, 532-536.
Carlile TM, Amon A. (2008). Meiosis I is established through division-specific translational control of a cyclin. Cell 133, 280-291.
Chan LY, Amon A. (2009) The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4. Genes Dev. 23, 1639-49
Chan LY, Amon A. (2010). Spindle Position Is Coordinated with Cell-Cycle Progression through Establishment of Mitotic Exit-Activating and -Inhibitory Zones. Mol Cell 39, 444-454.
Kiburz B. M, Reynolds, D. B., Megee, P. C., Marston, A. L., Lee, B. H., Lee, T. I., Levine, S. S., Young, R. A. and Amon A. (2005). The core centromere and Sgo1 establish a 50-kb cohesin-protected domain around centromeres during meiosis I. Genes Dev. 19, 3017-3030.
Monje-Casas, F., Prabhu, VR., Lee, BH., Boselli, M. and Amon, A. (2007). Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex. Cell 128, 477-490.
Stegmeier, F., Visintin, R. and Amon, A. (2002). Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase. Cell 108, 207-220.
Torres EM, Sokolsky T, Tucker CM, Chan LY, Boselli M, Dunham MJ, Amon A. Effects of aneuploidy on cellular physiology and cell division in haploid yeast. (2007). Science 317, 916-924.
Torres EM, Dephoure N, Panneerselvam A, Tucker CM, Whittaker CA, Gygi SP, Dunham MJ, Amon A. Identification of Aneuploidy-Tolerating Mutations. Cell. 2010 Sep 15. [Epub ahead of print]
Visintin, R., Craig, K., Hwang, E. S., Prinz, S., Tyers, M. and Amon, A. (1998). The phosphataseCdc14 triggers mitotic exit by reversal of CDK-dependent phosphorylation. Mol. Cell 2, 709-718.
Visintin, R., Hwang, E. S. and Amon, A. (1999). Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature 398, 818-823.
Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA, Housman DE, Amon A. (2008). Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science 322, 703-709.