Chapter 2 Overview of the cell cycle 2.1 The organisation of cell cycle in eukaryotes During the cell cycle, the typical eukaryotic cell goes through a series of well defined phases, to divide into two roughly identical daughter cells. While cell growth and replication of most cellular components is a continuous process, DNA replication occurs during the S phase (S for synthesis), along with synthesis of the histones necessary for the packaging of the new DNA. At the end of this process, chromosomes are duplicated in two sister chromatids held together by cohesin. Specialised structures known as centrosomes or spindle pole bodies are also replicated early in the cycle. Separation of the replicated genetic material occurs during M phase (M for mitosis), itself subdivided into several subphases. Chromosomes are condensed in prophase. In the course of metaphase, they are attached at the level of their centromeres to the mitotic spindle, a microtubular structure that stems from the kinetochores located at the opposite poles of the cell, and align at the spindle equatorial plane. The transition to the next phase occurs only when all chromosomes are properly attached to each pole and aligned. Chromosomes separate in anaphase, and are decondensed during telophase. M phase ends up with proper cell division, or cytokinesis (Figure 2.1). S and M phases are usually separated by two gap phases, G1 (between M and S) and G2 (between S and M). A fifth phase called G0 can be reached from G1, that corresponds to a quiescence state of the cell. Gap phases enable the cell to monitor its environment and internal state before committing into S or M phase. The cell cycle is highly regulated. Indeed, external and internal signals may halt the cycle at particular checkpoints (cf. Figure 2.2). An important checkpoint, called Start in yeast, or the restriction point in mammalian cells, controls the G1/S transition. This checkpoint integrates signals depending on cell size, the presence of nutrients, or contact with other cells, thereby coordinating cell proliferation with cell growth and the needs of the organism. In the course of the metaphase to anaphase transition, the spindle checkpoints monitors chromosome attachment to the microtubules, and their alignment on the metaphase plate (Decordier et al., 2008). Additional checkpoints monitor DNA damage at different points of the cycle (Hartwell and Weinert, 1989; Murray, 1992; Toettcher et al., 2009). What we have described is the canonical cell cycle. Specialised variants exist, that present significant differences with the classical G1-S-G2-M scheme. 29
30 CHAPTER 2. OVERVIEW OF THE CELL CYCLE Figure 2.1: The different stages of mitosis. From Molecular Biology of the Cell 5/e ( Garland Science 2008) In the early developmental stages of frog embryos, for example, the first divisions involve fast and synchronous successions of S and M phases, with no gap phases between them (Philpott and Yew, 2008). During Drosophila development (Figure 2.3), early divisions are also fast and synchronous, but further limited to nuclei within a large syncytium, until gap phases appear, along with true cellularisation, at cell cycle 13 (Vidwans and Su, 2001; Mazumdar and Mazumdar, 2002). Various specialised cell types in animals and plants undergo partial or complete endoreduplication cycles enabling various rounds of replication of (portions of) chromosomes without intervening nuclear division (Edgar and Orr-Weaver, 2001; Lilly and Duronio, 2005). Finally, meiosis can also be considered a specialised variant of the cell cycle that produces, in two rounds of division, haploid germ cells from diploid precursors (Morelli and Cohen, 2005). All these events involve a whole machinery of enzymatic complexes, molecular motors and cytoskeleton. Here, we focus on the delineation of the regulatory network controlling the canonical mitosis. 2.2 The cell cycle molecular engine The cell cycle is controlled by a complex network of interacting proteins known as the cell cycle engine (Murray, 1992). Regulatory components contributing to this molecular machinery control each other as well as a range of downstream processes necessary for cell duplication. These processes feed back on the engine, forming checkpoints able to halt the progression of the cycle and to ensure enough time to complete each crucial step. At the core of the cell cycle engine lies the MPF (Maturation or Mitosis Promoting Factor see Figure 2.4). Discovered in 1971 for its role in meiotic maturation of frog oocytes (Masui and Markert, 1971), MPF was later found to display oscillating activity, with a period coincident with that of the cell cycle (Wasserman and Smith,
2.2. THE CELL CYCLE MOLECULAR ENGINE 31 Figure 2.2: Cell cycle checkpoints. Progression through the different phases of the cell cycle is controlled by a series of checkpoint mechanisms that monitor the internal state of the cell, as well as its environment, to ensure faithful reproduction of its genetic material. 1978). In the course of the 1980s, MPF has been resolved as a heterodimer of cyclin and CDK (for cyclin-dependent kinase) (Evans et al., 1983; Murray and Kirschner, 1989a; Murray et al., 1989). Oscillations of the regulatory cyclin subunit, driven by an alternation of synthesis and degradation phases, control the activity of the enzymatic cdk subunit. A combination of positive and negative feedback circuits (cf. Part 4.4) is responsible for these oscillations (Figure 2.5). Early work already showed that the cell cycle can be blocked in stable states of high or low MPF activity (Wasserman and Smith, 1978). The underlying multistable behaviour is ensured by various positive feedback mechanisms. On the one hand, MPF self-activates through a positive effect on cyclin synthesis, as well as via post-transcriptional modifications controlled by the homologs of the Wee1 kinase and the Cdc25 phosphatase. On the other hand, MPF inhibits its own inhibitors, sometimes called the G1 stabilisers: the CKI (Cdk inhibitors) and Cdh1, an activator of the APC (Anaphase Promoting Complex). CKI sequester Cyclin-Cdk complexes, thereby inactivating them. Cdh1 activates the degradation of the cyclin subunit through the APC, a ubiquitinating enzymatic complex. Thus, in the course of cell proliferation, states with low MPF and high CKI and Cdh1 alternate with states with high MPF and low CKI and Cdh1 activity. How does the cell switch from a state of low cyclin activity to a state of high cyclin activity, and vice versa? The cyclin protein identified by Evans in 1983 has later been related to a larger family of cyclins (cf. Figure 2.6), whose members peak at different time points in the cycle. G1 cyclins (Cln1 and Cln2 in budding yeast, Cyclin E family members in mammals) are active in late G1 and play a key role in the Start transition. Homologous members of the Cyclin A family (sometimes called the S phase cyclins) are activated at the G1/S transition and trigger DNA synthesis; their expressions last until mitotic entry. B-type cyclins are mitotic cyclins, that promote entry into mitosis and the formation of the mitotic spindle, and whose degradation triggers mitotic exit and cytokinesis. The cyclin responsible for MPF activity belongs to this family (Murray, 2004).
32 CHAPTER 2. OVERVIEW OF THE CELL CYCLE Figure 2.3: Cell cycle variants in Drosophila. (From Vidwans and Su, 2001)
2.2. THE CELL CYCLE MOLECULAR ENGINE 33 Figure 2.4: MPF oscillations in the frog oocite and early embryo. High levels of MPF coincide with metaphase. (Murray and Kirschner, 1989b) Figure 2.5: Minimal cell cycle model. At the heart of the cell cycle lies the positive feedback between Cyclin B and Cdh1. In G1, Cdh1 is active and inhibits Cyclin B. Transitions to and from the mitotic state are triggered by negative feedbacks. Growth signals trigger the activation of G1 cyclins, which inhibit Cdh1, allowing Cyclin B to rise. Cyclin B in turns inactivates the G1 cyclins, and triggers its own degradation through the activation of Cdc20. A similar minimal model has been proposed by several authors (Chen et al., 2004; Irons, 2009). Graphical conventions as in Figure 4.1; node colours emphasise homology relationships (cf. Part 8).
34 CHAPTER 2. OVERVIEW OF THE CELL CYCLE Figure 2.6: Cyclins expression along the cell cycle. Different cyclins are expressed at specific phases of the cell cycle. Cyclin E peaks around the G1/S transition. Cyclin A expression begins in S phase and lasts until early M phase. Cyclin B accumulates in G2 and its degradation coincides with mitotic exit. Cyclin D is expressed throughout the cycle. G1 and S cyclins play a major role in the transition from low to high MPF activity. Indeed, G1 cyclins are not inhibited by the G1 stabilisers (Amon et al., 1994). In budding yeast, G1 cyclins Cln3 and Cln2 first inhibit the CKI, allowing the activation of the S cyclins Clb5 and Clb6 (Chen et al., 2000, 2004). Together with the G1 cyclins, they inhibit Cdh1, allowing the accumulation of Clb1 and Clb2, the mitotic cyclins of budding yeast. Clb1 and Clb2 are sufficient to maintain their activity by triggering their own synthesis and inhibiting the G1 stabilisers. They further inhibit the G1 and S cyclins. The transition from high to low MPF state, which corresponds to mitotic exit, is regulated by another negative feedback circuit enabling mitotic cyclins to trigger their own degradation. Given the role of proteolysis in the inactivation of MPF activity, a factor triggering Cyclin degradation, under the control of the mitotic spindle, has been suspected early on (Murray and Kirschner, 1989b). It was not until the late 1990s that this factor has been identified as Cdc20, and its regulator as the checkpoint protein Mad2 (Visintin et al., 1997; Fang et al., 1999). The activation of Cdc20 by Cyclin B (Prinz et al., 1998; Rudner and Murray, 2000) completes the negative feedback circuit. This circuit had already been postulated and integrated in mathematical models of the cell cycle (Novák and Tyson, 1993) prior to the discovery of its molecular components. Cell cycle progression is further constrained by checkpoint mechanisms that condition the activation and inactivation of key regulatory components to the completion of specific events. Activation of Cdc20 by Cyclin B is controlled by the spindle checkpoint to ensure that sisters chromatids are not separated before chromosomes are properly attached to the spindle and aligned on the metaphase plate (Decordier et al., 2008). Cdc20 promotes mitosis by triggering the degradation of cyclins as well as the separation of sister chromatids. Additional checkpoint mechanisms condition the completion of mitotic exit to the separation of sister chromatids by regulating the
2.2. THE CELL CYCLE MOLECULAR ENGINE 35 Figure 2.7: Just-in-time assembly of protein complexes. The cyclic activation of a complex may depend on a single cell-cycle regulated protein (de Lichtenberg et al., 2007). activation of Cdh1 and the CKI by Cdc14. A G2/M checkpoint monitors both DNA damage and unreplicated DNA, thereby ensuring that replication is complete before entering M phase. In budding yeast, the morphogenesis checkpoint conditions the activation of Clb2 to the formation of a bud (Ciliberto et al., 2003; Lew, 2003). Consistent with the crucial importance of cell division, cell cycle engine components are highly conserved among eukaryotes (Nasmyth, 1995). Table 2.1 presents the homology relationships existing between key regulatory components of the cell cycle control network in budding yeast, fission yeast, arabidopsis, drosophila and mammals. However, substantial differences exist between organisms in terms of precise wiring of the network, as well as of timing of expression and activity pattern of regulatory components (Jensen et al., 2006). In this respect, Jensen et al. recently showed that timing of expression of key players cyclins and Cdc20 in particular is relatively consistent between different organisms, but that the timing of expression of many other cell cycle-regulated proteins is poorly conserved (Jensen et al., 2006). Moreover, components that are cyclically expressed or post-transcriptionally modified in one organism do not appear to be regulated in others. However, Jensen et al. showed that such components often take part in molecular complexes involving other cycle regulated subunit(s) (a principle called just-in-time assembly (de Lichtenberg et al., 2005, 2007), cf. Figure 2.7). In brief, although the molecular details may differ, the general organisation of the regulatory network may still be conserved.
36 CHAPTER 2. OVERVIEW OF THE CELL CYCLE Budding Yeast Fission Yeast Drosophila Arabidopsis Mammals Function Cln3 Puc1 Cyclin D Cyclin D1 to D7 Cyclin D G1 progression (Vidwans and Su, 2001; Vandepoele et al., 2002; Wang et al., 2004) Cln1/2 - DmCycE Nicta;CYCA3;2 Cyclin E G1/S transition (Hao et al., 1995; Yu et al., 2003; Wang et al., 2004) Clb5/6 cig2 Cyclin A Cyclin A1, A2 and A3 CyclinA1 and A2 Clb1/2 Cdc13 cyclin B and B3 S phase progression, G2/M transition (Hao et al., 1995; Nieduszynski et al., 2002; Vandepoele et al., 2002; Wang et al., 2004) Cyclin B1, B2 and B3 Cyclin B1/2/3 G2/M transition and intra-m control (Nieduszynski et al., 2002; Vandepoele et al., 2002; Wang et al., 2004) Sic1 Rum1 Rux, Dacapo KRP1 to KRP7 p21, p27kip1 Cdk inhibitors (Vidwans and Su, 2001; Vandepoele et al., 2002; Barberis et al., 2005) Swe1 Wee1 and Mik1 Wee1 WEE1 WEE1/Myt1 Kinase, inhibits Cyclin B (Vandepoele et al., 2002; Bähler, 2005) Mih1 Cdc25 Cdc25String - Cdc25B Phosphatase, activates Cyclin B (Vidwans and Su, 2001; Vandepoele et al., 2002) Cdc20 Slp1 Fizzy Cdc20, Ccd52B Cdc20 Activator of the APC, active in mitosis (Visintin et al., 1997; Fülöp et al., 2005; Bähler, 2005) Cdh1 (HCT1) Ste9 Fizzy-related Ccs52A1, Ccs52A2 Cdh1 Activator of the APC, active in late mitosis and G1 (Visintin et al., 1997; Vidwans and Su, 2001; Fülöp et al., 2005) SBF, MBF MBF DmE2F-1 E2Fa, b, c E2F-1, -2, -3 Transcription factor, controls the G1/S transition (Hao et al., 1995; Hateboer et al., 1998; Vidwans and Su, 2001; Vandepoele et al., 2002; Bähler, 2005) Whi5 - Rb Rb Rb Binds and inactivates E2F (Hao et al., 1995; Costanzo et al., 2004; Ahmed et al., 2004) Table 2.1: Homology relationships between cell cycle regulatory components. Most of the components presented as homologs in this table share high sequence similarity with each other. However, in some cases, unrelated components may fulfill homologous functions. This is the case for example for Nicta;CYCA3;2 in plants (related to the Cyclin A family but functional homolog of Cyclin E), or Rb and E2F on the one hand, and Whi5 and SBF and MBF on the other. Not shown in the table, plants and, to a lesser extent, mammals display plethora of paralogs, whereas yeast usually have only one member of each family of component (cf. Wang et al., 2004, regarding cyclins in plants for example).