We study multiple aspects of cell growth and division such as how macromolecule biosynthesis is coordinated with cell division and how chromosome segregation is controlled during mitosis and meiosis. We also investigate the consequences of chromosome mis-segregation on cell and organismal physiology and how the condition leads to cancer and aging.
Control of mitosis by nuclear position.
Correctly positioning the nucleus within the cell is essential for accurate chromosome segregation. If the site of nuclear division is not coordinated with the site of cytokinesis anucleate and multinucleate cells are produced. In budding yeast control of the Mitotic Exit Network (MEN) is central for ensuring accurate partitioning of the nucleus during mitosis. The Mitotic Exit Network is a Ras-like GTPase signaling cascade that triggers the final cell cycle transition, called exit from mitosis. It is known as the Hippo tumor-suppressor pathway in mammals.
Our studies showed that the localization of Mitotic Exit Network components is essential for coordinating the site of nuclear division with the site of cytokinesis. MEN constituents localize to the cytoplasmic face of spindle pole bodies (SPBs), the budding yeast equivalent of centrosomes (Figure 1; i.e. Chan et al., 2010). Our studies further show that the cell is divided into a MEN inhibitory zone in the mother cell, where the MEN inhibitor Kin4 resides, and a MEN activating zone in the bud, where the MEN activator Lte1 resides (Figure 1). The MEN component carrying SPB functions as the sensor of these zones. Only when the MEN bearing SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud during anaphase where the MEN activator Lte1 resides can exit from mitosis occur.
We also study how signaling through the MEN occurs. We find that it is mediated by an unusual two-step process (i.e. Rock et al., 2013). The MEN kinase Cdc15 first phosphorylates the scaffold Nud1. This creates a phospho-docking site on Nud1, which the effector kinase complex Dbf2-Mob1 binds to via a novel phosphoserine/threonine binding domain, in order to be activated by Cdc15. This novel mechanism of Hippo pathway activation has important implications for signal transmission through kinase cascades and might well represent a general principle in scaffold-assisted signaling.
The consequences of aneuploidy on cell physiology and its disease impact.
Aneuploidy results in an unbalanced genome in which chromosomes(s), or pieces of chromosomes are missing or supernumerary. The condition has a profound impact on cellular and organismal fitness. In humans, aneuploidy is the leading cause of mental retardation and spontaneous abortions and a key characteristic of cancer, with an estimated 70–90% of all solid human tumors harboring aneuploid genomes. Given the dramatic impact of aneuploidy on organismal health it is important to understand how the aneuploid condition impacts cell physiology. We study the effects of aneuploidy on normal cell physiology and its role in cancer.
We have developed several systems to study the effects of aneuploidy on cells. We created 20 yeast strains carrying one or two additional chromosomes as well as primary mouse embryonic fibroblasts (MEFs) carrying four different trisomies (trisomy 1, 13, 16 or 19; Torres et al., 2007; Williams et al., 2008). In addition we engineered yeast and mouse cell lines which mis-segregate chromosomes at a high rate and hence generate cells that harbor multiple random aneuploidies.
Our analyses of aneuploid yeast, mouse and human cells suggest that aneuploidy causes chromosome-specific effects that are elicited by the increased (or decreased) number of copies of individual genes and/or combinations of a small number of genes present on the aneuploid chromosome. Our data further revealed a suite of phenotypes shared by many different aneuploidies (Figure 2). We call these phenotypes the “aneuploidy-associated stresses”. They include a cell cycle delay in G1 (i.e. Torres et al., 2007), metabolic alterations (i.e. Williams et al., 2008), a gene expression signature characteristic of slow growth and stress (i.e. Torres et al., 2007; Sheltzer et al., 2012), genomic instability (Sheltzer et al., 2011) and proteotoxicity (Torres et al. 2007; Torres et al., 2010; Tang et al. 2011; Oromendia et al. 2012). Currently, we are focusing our characterization of aneuploidy on its effects on proteostasis, genome stability, cell proliferation and metabolism.
Our findings indicate that cancer cells must overcome the adverse effects of aneuploidy in order to outgrow euploid cells and take advantage of potential benefits that arise from the aneuploid condition such as phenotypic variation. Reasoning that identifying such mutations will shed light on tumorigenesis we begun to isolate second site suppressors of the proliferation defect of aneuploid yeast cells (Torres et al., 2010). Their molecular characterization revealed strain-specific genetic alterations as well as mutations shared between different aneuploid strains demonstrating the existence of aneuploidy-tolerating mutations.
The discovery that aneuploidy causes a decrease in cellular fitness, also prompted us to search for genetic and chemical perturbations that exhibit synthetic lethality with the aneuploid state either by exaggerating the adverse effects of aneuploidy or interfering with pathways essential for the survival of aneuploid cells. Especially, compounds with such properties could provide the basis for the discovery of new tumor treatments. We have begun to screen chemical libraries for compounds that preferentially inhibit the proliferation of aneuploid mouse cells (Tang et al., 2011). Our results suggest that compounds that interfere with pathways essential for the survival of aneuploid cells could serve as a new treatment strategy against a broad spectrum of human tumors, especially when combined with traditional chromosome mis-segregation inducing chemotherapeutics (i.e. taxanes).
Aging: How can we overcome it?
Intuitively, we all know that the aging clock must reset from one generation to the next. The children of a 70 year-old man have the same life expectancy as those of a 20 year-old. But how does this happen? Is the germ-line somehow protected from aging or is age reset from one generation to the next? Budding yeast is the ideal model system to test the latter hypothesis. Budding yeast cells have a finite life-span and individual cells can be followed during the aging process. So we asked a simple question. What is the life expectancy of gametes (spores in yeast) produced by an old cell? Are they young or are they old? The result was astounding in its clarity. All four gametes generated by an aged cell show the same replicative potential as gametes generated by a young cell (Unal et al., 2011; Figure 3). This resetting of lifespan that occurs during gametogenesis is accompanied by the elimination of age-induced cellular damage. We are now determining the mechanisms underlying this rejuvination process. It is our hope that determining which aspects of gametogenesis causes the resetting of lifespan will provide insights into the mechanisms of aging and could facilitate the development of strategies for longevity.
Figure 3: Sporulation resets lifespan.
Lifespan analysis of old cells prior to inducing gametogenesis (A) and after sporulation (B). Lifespan resets during sporulation.
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.
Miller MP, Unal E, Brar GA, Amon A. Meiosis I chromosome segregation is established through regulation of microtubule-kinetochore interactions. elife. 2012;1:e00117. PMCID: PMC3525924
Oromendia AB, Dodgson SE, Amon A. (2012). Aneuploidy causes proteotoxic stress in yeast. Genes Dev. 15, 2696-708.
Rock JM, Amon A. Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit. Genes Dev. 2011 Sep 15; 25(18): 1943-1954. PMCID: PMC3185966
Rock JM, Lim D, Stach L, Ogrodowicz RW, Keck JM, Jones MH, Wong CC, Yates JR 3rd, Winey M, Smerdon SJ, Yaffe MB, Amon A. Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes. Science. 2013 May 17;340(6134):871-5.
Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, Humpton TJ, Brito IL, Hiraoka Y, Niwa O, Amon A. Aneuploidy drives genomic instability in yeast. Science. 2011 Aug 19; 333(6045): 1026-1030. PMCID: PMC Journal In Process
Sheltzer JM, Torres EM, Dunham MJ, Amon A. (2012). Transcriptional consequences of aneuploidy. Proc Natl Acad Sci U S A. 109,12644-12649.
Siegel JJ, Amon A. New insights into the troubles of aneuploidy. Annu Rev Cell Dev Biol. 2012; 28: 189-214.
Tang YC, Williams BR, Siegel JJ, Amon A. Identification of aneuploidy-selective antiproliferation compounds. Cell. 2011 Feb 18; 144(4): 499-512.
Tang YC, Amon A. Gene copy-number alterations: a cost-benefit analysis. Cell. 2013 Jan 31; 152(3): 394-405. PMCID: PMC3641674
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]
Unal E, Kinde B, Amon A. Gametogenesis eliminates age-induced cellular damage and resets life span in yeast. Science. 2011 Jun 24; 332(6037): 1554-15547.
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.