We study the mechanisms that prevent chromosome missegregation and the consequences of aneuploidy on cell growth and proliferation. We focus on how chromosome segregation during mitosis is controlled, how growth and cell division are coordinated, and on how a specialized cell division, the meiotic cell division, is established. We also study what happens to cells in which the chromosome segregation quality controls fail, causing cells to become aneuploid.
Preventing Aneuploidy: Mechanisms That Ensure Accurate Chromosome Segregation During Mitosis and Meiosis
Regulation of exit from mitosis. Exit from mitosis is the final cell cycle transition when cells disassemble their mitotic spindles, re-form the nuclear envelope, and undergo cytokinesis. In budding yeast, this transition is triggered by the protein phosphatase Cdc14, whose activity in turn is controlled by a Ras-like GTPase signaling pathway known as the mitotic exit network (MEN). During the past 10 years, we have identified and characterized individual components of this signaling pathway and discovered signals that control its activity. Key among the MEN regulatory signals is spindle position. Our studies led to the identification of the components of spindle position control and an explanation for how spindle position is sensed in cells.
The mitotic spindle must be correctly positioned within a cell for accurate chromosome segregation. Thus mechanisms must exist that sense spindle position and convey this information to the cell cycle machinery. We found that the localization of MEN components and MEN regulators is at the heart of spindle position sensing: the MEN constituents localize to the cytoplasmic face of spindle pole bodies (SPBs), the budding yeast equivalent of centrosomes. The MEN activator Lte1 localizes to the bud; the MEN inhibitor Kin4 localizes to the mother cell. Thus, the cell is divided into a MEN inhibitory zone in the mother cell, where Kin4 resides, and a MEN-activating zone in the bud, where Lte1 resides (Figure 1).
The MEN component carrying SPB functions as the sensor. Exit from mitosis can occur only when the MEN bearing SPB escapes the MEN inhibitor Kin4 in the mother cell and moves during anaphase into the bud where the MEN activator Lte1 resides. Thus, spatial information is sensed and translated into a chemical signal by targeting activators and inhibitors of signal transduction pathways to specific cellular locations. Determining how Kin4 and Lte1 are targeted, respectively, to the mother cell and bud will be a focus of our future studies.
Establishing meiosis, a specialized cell division. 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 and how kinetochore-microtubule attachments are modified during meiosis I (Figure 2). In particular, we focus on the regulation of these processes by phosphorylation. We identified two protein kinases, the Polo kinase Cdc5 and cyclin-dependent kinases (CDKs), as key regulators of both events and determined how phosphorylation transforms the mitotic chromosome segregation pattern into the unique meiotic pattern. We hope to further dissect these meiosis-specific specializations and determine the molecular signals that control entry into the germ cell fate.
Effects of Gametogenesis on Aging
We are also interested in the relationship between aging and gametogenesis. All eukaryotic organisms age, a condition characterized by morphological and cellular deterioration. The detrimental age-associated traits are, however, not passed on to the progeny. How life span is reset from one generation to the next is not known. We find that in budding yeast, resetting of life span occurs during gametogenesis. Gametes, spores in yeast, generated by aged cells show the same replicative potential as gametes generated by young cells. This rejuvenation is associated with the reversal of age-induced cellular damage. Furthermore, we find that transient induction of a transcription factor essential for later stages of gametogenesis extends the replicative life span of aged cells. Our results suggest that gamete formation brings about rejuvenation by eliminating age-induced cellular damage. Determining which aspects of gametogenesis cause the resetting of life span may provide insights into the mechanisms of aging and could facilitate the development of strategies for longevity.
Effects of Aneuploidy on Cell Physiology and Tumorigenesis
What happens to cells in which the mechanisms that ensure accurate chromosome segregation fail? What impact does a chromosome missegregation event has on a cell or an organism? In all organisms analyzed to date, aneuploidy, the consequence of chromosome missegregation, is frequently associated with disease. In humans, aneuploidy is the leading cause of miscarriages and mental retardation. It is also a key characteristic of cancer. More than 90 percent of all solid human tumors are aneuploid. To begin to understand how aneuploidy causes diseases, we analyzed the effects of aneuploidy on normal cell physiology in yeast and in the mouse.
We created 20 yeast strains carrying one or two additional chromosomes and, subsequently, primary mouse embryonic fibroblasts (MEFs) carrying four different trisomies (trisomy 1, 13, 16 or 19). Our analysis revealed that aneuploidy causes 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 also 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.
From these studies we concluded that aneuploidy leads to a common cellular response. In this response cells engage protein-folding and degradation pathways in an attempt to correct protein stoichiometry imbalances caused by aneuploidy. This response leads to an increased burden on the protein quality control pathways and an increased need for energy. We are now studying how aneuploidy affects the protein quality control pathways of the cells and the importance of protein quality control pathways for the survival of aneuploid cells.
Because aneuploidy is deleterious for cells, cancer cells must overcome the adverse effects of aneuploidy to outgrow euploid cells and take advantage of potential benefits that arise from the aneuploid condition. Furthermore, cancer cells may be more sensitive to conditions that exaggerate the stresses associated with aneuploidy. In our studies in yeast we have begun to identify second-site suppressors of the proliferation defect of aneuploid yeast cells. This approach identified many genetic alterations, prominent among them mutations in the proteasomal degradation system. Several mutations improved the proliferative ability of multiple different aneuploid yeast strains. These results not only increase our understanding of the defects underlying the aneuploid condition but also may shed light on the evolution of tumors.
Genetic alterations that exhibit synthetic lethality with the aneuploid state—either by exaggerating the adverse effects of aneuploidy and/or by interfering with pathways essential for the survival of aneuploid cells—could provide the basis for the discovery of new tumor treatments. We have begun to screen for compounds that preferentially inhibit the proliferation of aneuploid mouse cells. This study has determined that the energy stress-inducing agent AICAR, the protein-folding inhibitor 17-AAG, and the autophagy inhibitor chloroquine exhibit this property. AICAR and 17-AAG, especially when combined, also show efficacy against aneuploid human cancer cell lines. 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. It is our hope that these efforts will pave the way for the development of new cancer treatments.
This work is supported in part by a grant from the National Institutes of Health.
As of May 30, 2012