Overview

Cell division is the fundamental process by which an organism is built. Deciphering the regulatory networks that govern cell division is therefore vital to understanding both normal cell division and the abnormal cell division that is a hallmark of cancer. We study the mechanisms that control the transitions from one cell-cycle stage to the next using budding yeast as a model system. We focus on how the anaphase - G1 transition, also known as exit from mitosis, is regulated and integrated with other cell cycle events and on how a specialized cell cycle, the meiotic cell cycle is established. Furthermore, we study the consequences of aneuploidy on cell growth and proliferation in yeast and mammals.

Research Summary

Regulation of exit from mitosis. Exit from mitosis is triggered by the inactivation of mitotic cyclin-dependent kinases (CDKs). In 1998 we showed that this inactivation of CDKs is brought about by the conserved protein phosphatase Cdc14. We also found that Cdc14 is regulated by an inhibitor Cfi1/Net1 that binds to and sequesters Cdc14 in the nucleolus during G1, S phase, G2 and metaphase. During anaphase, Cdc14 is released from its inhibitor and spreads throughout the nucleus and cytoplasm, where it dephosphorylates its targets.

Subsequently, 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 and the Mitotic Exit Network (MEN) maintains Cdc14 in its released state during late stages of anaphase. The Mitotic Exit Network (MEN) resembles a Ras-like signaling cascade and its activity is controlled by nuclear position. This was revealed by the analysis of the subcellular localization of MEN components. The MEN GTPase Tem1 localizes to the SPB destined to migrate into the daughter cell during anaphase. The MEN activator and putative GEF Lte1 localizes to the bud as soon as it is formed. This spatial segregation of Tem1 and Lte1, which persists until the nucleus moves into the bud during anaphase, ensures that exit from mitosis does not occur until the nucleus has been partitioned between the mother and daughter cell. Tem1’s GAP also contributes to restraining mitotic exit when the nucleus is not partitioned between mother and daughter cell. It appears that the MEN inhibitor and protein kinase Kin4, which localizes to the mother cell cortex, in some way activates the GAP when the nucleus is mis-positioned in the cell. Currently we are investigating how Kin4 affects the MEN as well as determine whether and how Kin4 activity itself is regulated by nuclear position and develop methods to study the MEN biochemically.

Regulation of a specialized cell cycle - the meiotic cell cycle. Meiosis is a specialized cell cycle that leads to the formation of gametes. Defects in meiotic chromosome segregation are the leading cause of miscarriages and one of the leading causes of birth defects in humans. During the meiotic cell cycle a single S-phase is followed by two consecutive nuclear divisions. During meiosis I, separation of homologous chromosomes occurs; segregation of sister chromatids takes place during meiosis II. For the meiotic chromosome segregation program to succeed, protein complexes known as cohesin complexes that hold sister chromatids together must be lost from chromosomes in a step-wise manner; from chromosome arms during meiosis I and from centromeric regions during meiosis II. Furthermore, kinetochore orientation changes during meiosis. Sister kinetochores attach to microtubules so that they face the same spindle pole (co-orientation) during meiosis I. During meiosis II sister kinetochores attach to microtubules emanating from opposite poles (bi-orientation). We study how the step-wise loss of cohesins and kinetochore orientation are regulated during meiosis.

Stepwise loss of cohesins during meiosis: The step-wise loss of cohesins is essential for meiotic chromosome segregation. Loss of cohesins from chromosome arms is necessary for homologous chromosomes to segregate during meiosis I, retention of cohesins around centromeres is essential for accurate meiosis II chromosome segregation. A screen aimed at discovering genes required for the stepwise loss of cohesins identified IML3, CHL4 and SGO1. All three proteins localize to centromeric regions suggesting that these proteins are intimately involved in maintaining cohesins around centromeres during meiosis I. We furthermore showed that phosphorylation of the cohesin subunit Rec8, the cohesion protector Sgo1 and meiotic recombination function together to bring about the stepwise loss of cohesins from chromosomes. Currently, we are determining how Sgo1 singles out cohesins around kinetochores to prevent their removal during meiosis I and how the protein is itself regulated.

Sister kinetochore orientation during meiosis: Kinetochores of sister chromatids attach to microtubules emanating from the same pole (co-orientation) during meiosis I and to microtubules emanating from opposite poles (bi-orientation) during meiosis II. We recently found that the Aurora B kinase Ipl1 regulates kinetochore - microtubule attachment during both meiotic divisions and that a complex known as the monopolin complex ensures that the protein kinase co-orients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore co-orientation during mitosis provided insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins. We propose that this function of the monopolin complex helps Aurora B to co-orient sister chromatids during meiosis I.

By generating meiosis-specific loss-of-function alleles we were also able to characterize the role of the polo kinase Cdc5 in sister kinetochore co-orientation. We found that in the absence of CDC5, sister kinetochores attach to microtubules emanating from opposite poles rather than the same pole. In addition, proteins required for proper kinetochore orientation, such as Mam1, were mis-localized in Cdc5-depleted cells. Currently, we are addressing the molecular mechanisms whereby the monoploin complex is targeted to kinetochores and how it fuses sister kinetochores.

The effects of aneuploidy on cell proliferation. The importance of understanding the effects of aneuploidy on cell growth and proliferation is highlighted by the observation that aneuploidy is frequently found in tumor cells. Recently, we have begun to investigate what happens to yeast cells that acquired extra chromosomes and hence are aneuploid. We have created a collection of haploid yeast strains that each bear an extra copy of one or more of almost all of the yeast chromosomes. Their characterization revealed that aneuploid strains share a number of phenotypes, including defects in cell cycle progression, increased glucose uptake and sensitivity to conditions interfering with protein synthesis and folding. These phenotypes are observed only in strains carrying additional yeast genes indicating that they reflect the consequences of additional transcription and translation as well as the resulting imbalances in cellular protein composition. We conclude that aneuploidy causes not only a proliferative disadvantage but also a set of phenotypes that is independent of the identity of the individual extra chromosomes. Currently, we are in the process of identifying the mechanisms that cause these phenotypes and isolate mutants that allow yeast cells to tolerate aneuploidy or that specifically kill aneuploid cells. As with our work on chromosome segregation we will also test whether the mechanisms that select against aneuploidy in yeast operate in mammalian cells. It is our hope that these studies will shed light on how aneuploidy is tolerated in tumor cells.

Selected Publications

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.

D’Aquino, K. E., Monje-Casas, F., Paulson, J., Reiser, V., Charles, G. M., Lai, L., Shokat, K. M., and Amon, A. (2005). The protein kinase Kin4 inhibits exit from mitosis in response to spindle position defects. Mol. Cell 19, 223-234.

Hochwagen, A., Tham, W. H., Brar, G. A. and Amon A. (2005). The FK506 binding protein Fpr3 counteracts protein phosphatase 1 to maintain meiotic recombination checkpoint activity. Cell, 122, 861-873.

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.

Lee, B.H., and Amon, A. (2003). Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation. Science 300, 482-486.

Marston, A. L., Tham, W.-H., Shah, H. and Amon, A. (2004). A genome-wide screen identifies genes required for centromeric cohesion. Science 303, 1367-1370.

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.

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