Research Summary

The goal of our research is to obtain a detailed molecular understanding of the regulatory circuits that control the transitions from one cell cycle stage to the next. In particular, we are focusing on how chromosome segregation and the anaphase to G1 transitions of the cell cycle are regulated and how the machinery responsible for triggering these events is modulated during a specialized cell cycle, the meiotic cell cycle.

Control of exit from mitosis.

Regulation of the protein phosphatase Cdc14
Exit from mitosis is triggered by the inactivation of mitotic cyclin-dependent kinases (CDKs), which is brought about by the conserved protein phosphatase Cdc14 (Visintin et al., 1998). Cdc14 is regulated by an inhibitor Cfi1/Net1 that binds to and sequesters Cdc14 in the nucleolus during G1, S phase, G2 and metaphase (Shou et al., 1999; 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 (Shou et al., 1999; Visintin et al., 1999).

Figure 1:
The FEAR network and the MEN promote the release of Cdc14 from its inhibitor.

The FEAR network promotes Cdc14 release from its inhibitor during early anaphase, the Mitotic Exit Network further promotes the release of Cdc14 and maintains the phosphatase in its released state during late anaphase.

The Mitotic Exit Network (MEN) resembles a Ras-like signaling cascade and includes a RAS-like GTP binding protein Tem1, a putative Guanine Nucleotide Exchange Factor (GEF) Lte1, a GTPase activating protein (GAP) complex Bub2-Bfa1and several protein kinases. The analysis of the subcellular localization of these MEN components revealed a signal controlling the MEN: the position of the nucleus within the cell. The MEN component 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 (Bardin et al., 2000). 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. Recently we identified the protein kinase Kin4 as a potential regulator of Bub2-Bfa1 when the nucleus is mis-positioned in the cell (D'Aquino et al., 2005). Currently we are investigating how Kin4 affects the GAP as well as determine whether and how Kin4 is regulated by nuclear position.

Analysis of Cdc14's functions during chromosome segregation. In addition to studying how Cdc14 is regulated, we investigated the function of this phosphatase during mitosis. Cdc14's main role is to reverse CDK phosphorylation events thereby triggering exit from mitosis. Our recent studies showed that Cdc14 also regulates the segregation of repetitive DNA (D'Amours et al., 2005, Sullivan et al., 2005).

Chromosome segregation requires the Separase-dependent removal of cohesin complexes, which link sister chromatids (reviewed in Nasmyth, 2001). Our findings show that the segregation of specialized chromosomal domains requires mechanisms in addition to cohesin removal. Late segregating DNA elements, such as the telomeres and the rDNA, also require Cdc14 activated by the FEAR network for proper segregation. Cdc14 promotes the enrichment of condensins, which are protein complexes required for chromosome condensation and rDNA segregation, at this locus (D'Amours et al., 2004). The fact that Cdc14 promotes the partitioning of late-segregating DNA regions as well as exit from mitosis provides a mechanism for ensuring that sister-chromatid separation is completed before cells exit from mitosis.

Regulation of a specialized cell cycle - the meiotic cell cycle.

Since many of the proteins that govern mitotic cell cycle progression have been identified, we began to analyze their roles during specialized cell division cycles. The meiotic cell cycle is a specialized cell cycle that is employed to generate gametes and consists of a single S-phase that is followed by two consecutive nuclear divisions. During meiosis I, separation of homologous chromosomes occurs, segregation of sister chromatids takes place during meiosis II (reviewed in Marston and Amon, 2004). For the meiotic chromosome segregation program to succeed, several meiosis-specific events need to take place. Among them are the stepwise loss of cohesin complexes, which hold sister chromatids together, from chromosome arms during meiosis I and from centromeric regions during meiosis II and the way in which microtubules attach to kinetochores. Construction of pole-kinetochore attachments via microtubules, so that sister kinetochores attach to microtubules emanating from the same pole (co-orientation) during meiosis I to facilitate homolog separation and then attach to microtubules emanating from opposite poles (bi-orientation) during meiosis II, to separate sister chromatids in anaphase II.

(1) Stepwise loss of cohesins during meiosis. To identify genes required for the stepwise loss of cohesins from chromosomes we performed a screen in which we analyzed the segregation pattern of a GFP marked chromosome III in a collection of yeast strains in which individual genes were deleted (yeast knock-out collection; Marston et al., 2004). The screen identified several genes required for retaining centromeric cohesins on chromosomes beyond meiosis I and three genes, IML3, CHL4 and SGO1 were analyzed further (Marston et al., 2004). Importantly all three proteins localize to centromeric regions (Katis et al., 2004; Kitajima et al., 2004; Marston et al., 2004; Rabitsch et al., 2004; Kiburz et al., 2005) suggesting that these proteins are intimately involved in maintaining cohesins around centromeres during meiosis I. The meiosis-specific factor Spo13 is also important for protecting centromeric cohesins from removal during meiosis I (Lee et al., 2004; Katis et al., 2004b). Furthermore, Spo13, when overexpressed inhibits cleavage of the cohesin subunit Rec8 (Lee et al., 2002) raising the interesting possibility that SPO13 protects Rec8 from Separase activity. How Iml3, Chl4, Sgo1 and Spo13 function to protect centromeric cohesins from removal during meiosis I will be addressed in the future.

Figure 2:
Modulations necessary to bring about the meiotic chromosome segregation program.

For meiotic chromosome program to succeed, three meiosis-specific events need to take place. (1) Reciprocal recombination between homologs creates physical linkages between homologs, called chiasmata, which allows homologs to stably align on the metaphase I spindle. (2) Cohesin complexes (shown as red bars) are lost in a stepwise manner during meiosis. Cohesins are removed from chromosome arms during meiosis I and from centromeric regions during meiosis II. (3) Sister kinetochores attach to microtubules emanating from the same pole (co-orientation; kinetochore orientation is indicated by gray triangles) during meiosis I to facilitate co-segregation of sister-chromatid pairs. Sister kinetochores then attach to microtubules emanating from opposite poles (bi-orientation) during meiosis II, which separates sister chromatids in anaphase II.

(2) Sister kinetochore orientation during meiosis. By generating meiosis-specific alleles of key cell cycle regulators, we were able to characterized the role of the polo kinase Cdc5 during the meiotic divisions and found that the protein kinase is required for kinetochore co-orientation during meiosis I (Lee and Amon, 2003). In the absence of CDC5, sister kinetochores attached to microtubules emanating from opposite poles rather than the same pole. In addition proteins required for proper orientation were mis-localized in Cdc5-depleted cells (Lee and Amon, 2003). SPO13 was also found to be required for kinetochore co-orientation during meiosis I and for Mam1 to localize to kinetochores (Katis et al., 2004; Lee et al. 2004). Thus, Spo13 and Cdc5, which are found in a complex, are critical regulators of sister kinetochore co-orientation during meiosis I.

(3) Regulation of the meiosis I - meiosis II transition. Meiosis is characterized by two successive chromosome-segregation phases without an intervening S phase. Thus, conditions have to be established at the end of the first division that allow a second round of chromosome segregation and simultaneously inhibit S phase. Our studies indicate that Cdc14 and the FEAR network play an instrumental part in this process. Cells impaired for FEAR network or CDC14 function fail to promote exit from the first meiotic division but meiosis II events continue to occur (Marston et al., 2003). Thus, Cdc14 and the FEAR network play a central role in creating two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.

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 loss. However, despite the existence of surveillance mechanisms that monitor chromosome segregation, chromosome mis-segregation occurs in 1 in 105 yeast cells (Esposito et al., 1982). Recently, we have begun to investigate what happens to yeast cells that, defying the odds, acquired extra chromosomes and hence are aneuploid. We observed that the presence of extra chromosomes delays cell cycle progression. The mechanisms that impose a proliferative disadvantage on yeast cells with an incorrect chromosome number are completely unknown. We are in the process of identifying these mechanisms. 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.

References:

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D'Amours, D., Stegmeier, F. and Amon, A. (2004). Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell, 117, 455-469.

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.

Katis VL, Galova M, Rabitsch KP, Gregan J, Nasmyth K. Maintenance of cohesin at centromeres after meiosis I in budding yeast requires a kinetochore-associated protein related to MEI-S332. (2004) Curr Biol. 14, 560-572.

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