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.
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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).
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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).
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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.
CHAPTER 2. OVERVIEW OF THE CELL CYCLE
36
Clb1/2
Clb5/6
Cln1/2
Cln3
Budding Yeast
Cdc13
cig2
-
Puc1
Fission Yeast
cyclin B and
B3
Cyclin A
DmCycE
Cyclin D
Drosophila
Cyclin B1, B2 and B3
Cyclin A1, A2 and A3
Nicta;CYCA3;2
Cyclin D1 to D7
Arabidopsis
Cyclin B1/2/3
CyclinA1 and
A2
Cyclin E
Cyclin D
Mammals
G1 progression
(Vidwans and Su, 2001; Vandepoele et al., 2002; Wang
et al., 2004)
Function
WEE1/Myt1
p21, p27Kip1
WEE1
Cdc25B
KRP1 to KRP7
Wee1
-
Cdc20
Rux, Dacapo
Wee1 and Mik1
Cdc25String
Cdc20, Ccd52B
Rum1
Swe1
Cdc25
Fizzy
Sic1
Mih1
Slp1
E2Fa, b, c
Rb
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)
Binds and inactivates E2F
(Hao et al., 1995; Costanzo et al., 2004; Ahmed et al., 2004)
(Visintin et al., 1997; Vidwans and Su, 2001; Fülöp
et al., 2005)
Activator of the APC, active in late mitosis and G1
Kinase, inhibits Cyclin B
(Vandepoele et al., 2002; Bähler, 2005)
Phosphatase, activates Cyclin B
(Vidwans and Su, 2001; Vandepoele et al., 2002)
Activator of the APC, active in mitosis
(Visintin et al., 1997; Fülöp et al., 2005; Bähler, 2005)
G1/S transition
(Hao et al., 1995; Yu et al., 2003; Wang et al., 2004)
S phase progression, G2/M transition
(Hao et al., 1995; Nieduszynski et al., 2002; Vandepoele
et al., 2002; Wang et al., 2004)
G2/M transition and intra-M control
(Nieduszynski et al., 2002; Vandepoele et al., 2002; Wang
et al., 2004)
Cdk inhibitors
(Vidwans and Su, 2001; Vandepoele et al., 2002; Barberis
et al., 2005)
Cdc20
DmE2F-1
Rb
Cdh1
Fizzy-related
MBF
Rb
Ccs52A1, Ccs52A2
Ste9
SBF, MBF
-
Cdh1 (HCT1)
Whi5
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).