Download Introduction Cell Cycle

Chapter 1
Introductory remarks on cell cycles
Contents
1.1. Growth and division
1.2. Early observations on the cell cycle
1.3. Experiments with bacteria
1.4. Experiments with yeasts
1.5. Experiments with mammalian cells
1.6. “Synthesis” between cytological, biochemical and genetic findings
1.7. The central control system
1.8. Overview of cell cycles
1.9. Simulation of the bacterial and eukaryotic cell cycle
1.10. References
Questions
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1.1. Growth and division
The “dream” of every cell is to become two cells (Jacob, 1971). Or, in less
anthropocentric words, the fate of a cell is to duplicate. Living organisms have
developed from inanimate systems by selection for the function of duplication
(Hartwell et al., 1999). By continuous dissipation of energy harvested from its
environment, a cell can exist in a state far from equilibrium. In this state the cell
will grow and will eventually divide when it has duplicated all its components
and doubled its size. In the growth process most macromolecules are
synthesized continuously, causing a gradual increase in mass, surface and
volume. When the cell reaches some critical size, however, this gradual process
is suddenly “disturbed” by the initiation of DNA replication. This intriguing
discontinuity is followed by other discontinuous events like chromosome
segregation (mitosis) and finally cell division.
Individual cells supplied with nutrients will adapt to their environment. If richer
nutrients are added to the growth medium (“shift-up”), the change is detected
as an external signal, which is transduced to the chromosome to change gene
expression. In this adaptive response, the population growth-rate will increase,
whereas it will decrease when nutrients start to deplete (Fig. 1.1A). If the
environment remains constant, the population will eventually reach a certain
steady-state of growth (see Section 5.4). In this condition, every individual cell in
the population will perform the same cycle of growth and division except for
statistical variations (Fig. 1.1B). A steady-state population is therefore
characterized by an average cell with an average mass and an average
generation time, both constants. Apparently, depending on the nutritional
condition, cells can reach many steady states in which their energy dissipation is
at a non-equilibrium, thermodynamic minimum.
How is this constancy of an average cell, this so-called homeostasis achieved? It
must involve, in every individual cell, a coupling between mass growth and the
process of DNA replication leading to cell division. The mechanism of coupling
differs in the cell cycle of bacteria and eukaryotic cells because of differences in
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scale and structure. But the self-maintaining nature of the system seems to be
similar: internal signals are generated by the growing cell inducing initiation of
DNA replication at a constant average mass.
Fig. 1.1. (A) A cell population increases its total biomass under different conditions at
different growth rates. (B) An individual cell will grow and divide. In this sequence of
three consecutive cell cycles, the statistical variation in cell mass and cycle time is
evident; the cells maintain a constant average size. (C) Linear representation of the
canonical cell cycle of eukaryotes (compare with Fig. 1.10). (D) In this circular
representation of a eukaryotic cell cycle, the cycle continuity and the alternation of the
phases of DNA replication (S) and segregation (M) are emphasized.
The term “cell cycle” encompasses all chemical reactions and structural changes
which take place between birth and division of a growing cell. The “logic of the
cell cycle” expresses that every cell has to perform the same four processes: it
must grow, it must replicate and segregate its DNA and it must divide between
the segregated DNA daughter strands. When comparing the huge size
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differences as shown in Fig. 1.2, it is remarkable how these same tasks are being
performed by the simple bacterial cell just as well as by the much more complex
eukaryotic cells consisting of many more genes and proteins and of many
intracellular compartments and structures.
In Fig. 1.1.B and C, cell cycles are depicted as linear sequences, emphasizing the
cell-to-cell variability, resulting in a different “history” of individual cells.
Although the successive divisions follow each other in a linear sequence (as
depicted in Fig. 1.1.B), a single interdivision period is often depicted in the form
of a circle as in Fig. 1.1.D. The circle emphasizes the alternation of cycle periods
and the cyclic behavior of cells. But it suggests wrongly that each cycle starts the
same way, just as a clock starts at time zero; and it disregards the “history” of a
cell and the cell-to-cell variations in mass and cycle time (cf. Fig. 1.1 B).
Because the main events, DNA replication, segregation and cell division, have
always to occur in a strict order, cell cycle studies have concentrated on the
determination of cycle periods and on structural changes during the transitions
between cell cycle phases.
The principal questions have been:
(1) How does the growing cell trigger the initial transition? In other words,
how is the initiation of DNA replication coupled to cell growth?
(2) How does the cell coordinate the order of the subsequent discontinuous
transitions, DNA segregation and cell division?
(3) How does the cell separate sister chromatids (DNA daughter strands),
move chromosomes or nucleoids and position the division site?
1.2. Early observations on the cell cycle
Early light microscope observations indicated that cell division was preceded by
the division of the nucleus (mitosis). In the period between these divisions,
called the interphase, the cells appeared to increase in mass. In 1953, Howard
and Pelc observed by autoradiographic measurements that the chromosomes
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are duplicated during a relatively short period in which the DNA is synthesized,
the S phase. Gaps in DNA synthesis divided the interphase in the periods G1, S
and G2 (Fig. 1.1 C,D).
Fig. 1.2. Comparison of the volume growth curves of a bacterial cell (Td = 45 min; V ≈ 1
µm3), a yeast cell (Td = 140 min; V ≈ 50 µm3) and a mammalian HeLa cell (Td = 10 h;
V ≈ 1000 µm3). See Table 1.1 for differences in cell cycle parameters between these cells.
Note that most mammalian cells are much larger than a HeLa cell as indicated by the
radius R = 30 µm (V ≈ 100,000 µm3).
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Fig. 1.3. A. Continuous and discontinuous processes. The growth process is drawn as a
circle to depict its continuity. The different sizes of the circles represent different growth
rates. The discontinuous processes like DNA replication, segregation and cell division
cause the cell to “cycle”. Taken from Cooper (1982). This cartoon also illustrates that
cell division is the end of the discontinuous processes, but not necessarily their
beginning. B. The growth process is drawn as a size/time diagram for two different
growth rates also indicated in A. See text for explication of the inhibition arrows.
In his classical book “The Biology of the Cell Cycle”, Mitchison (1971) makes the
distinction between (i) the “DNA-division cycle”, consisting of markers or
discontinuities caused by DNA replication, segregation and division, and (ii) the
“growth cycle”, consisting of macromolecular synthesis and volume increase.
This distinction is depicted in Fig. 1.3 in which an important behavior of a
cycling cell is indicated: if the continuous processes of growth (e.g. protein
synthesis) are inhibited (arrow 1 in Fig. 1.3 A, B) the ongoing discontinuous
processes will continue and finish, but new discontinuous processes (DNA
synthesis) will not be initiated. If, however, a discontinuous process is
specifically inhibited (arrow 2 in Fig. 1.3), there is in principle no effect on cell
growth: The ribosomes will continue the synthesis of proteins and cell mass will
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continue to increase (for some time) in the absence of DNA replication and cell
division. We will see how this behavior has been used to study the coordination
and timing of cell cycle processes. At this point, the important conclusion is that
under normal growth conditions (in the absence of inhibitors), a cycling cell is a
growing cell and a normally growing cell will progress through the cell cycle.
This is the reason why we have depicted the cell cycle as a size/time diagram
(cf. Fig. 1.1B) in most of the figures that follow.
1.3. Experiments with bacteria
The cycle of growth and division does not only hold for all eukaryotic cells but
also applies for the much smaller bacteria. In spite of the fact that bacteria are
about 1000x smaller than animal cells and 50x smaller than bakers yeast (Fig.
1.2), it was thought for a long time (until about 1965) that the bacterial cell cycle
must resemble that of eukaryotes. The observation (Abbo and Pardee, 1960) that
bacteria can synthesize their DNA continuously throughout the cell cycle was
therefore unexpected. For a long time it remained a puzzle that was eventually
solved in 1968 by Cooper and Helmstetter. Using age-synchronized bacteria (see
Chapter 2 and 5) they found that the duration of the DNA replication period (Cperiod) was constant, i.e. independent of the growth rate of the population. As
will be discussed in Chapter 2 this can be understood by considering that a
polymerization reaction on a single template is always slower than the fueling
reactions that can be performed by many enzymes. Thus, even under poor
growth conditions, the rate of DNA polymerization can be maintained at its
maximum. The same experiments showed that the period between replication
and division (D-period) was also constant. This means that the discontinuous
processes in bacteria occur in a constant period of C+D min.
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Fig. 1.4. History of cell cycle studies. In 2001, Lee Hartwell, Paul Nurse and Tim Hunt
jointly received the Nobel prize for their discoveries of “key regulators of the cell cycle”.
p34 is a cyclin dependent protein kinase or CDK; pRB is the retinoblastoma protein; preRC is prereplicative complex. See for a discussion of the cytological fusion experiments
Stillman (1996).
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Fig. 1.5. Size/time diagram illustrating (i) a resting cell of minimal size that adapts to
its growth medium, (ii) a slower growing cell, (iii) a rapidly growing cell (nutritional
shift-up) and (iv) a cell of which growth is inhibited after it passed START, illustrating
the cell’s strategy that its mass at initiation of DNA replication is about twice the
minimal size of a cell to survive. In other words, cells undergoing growth inhibition after
initiation of DNA replication (arrow) will, upon division, produce two daughter cells of
minimal size, that are still viable.
Apart from these DNA-incorporation studies, the size of Escherichia coli cells
cultured at different growth rates had been acurately determined by Maaløe and
coworkers (see Schaechter et al., 1958). It appeared that the average cell mass
increased exponentially with growth rate. Combining the data on mass increase
with the constant C+D period measured by Cooper and Helmstetter, it was
found by Donachie (1968) that bacteria initiate their DNA replication at a
constant cell mass. What determines this critical cell mass in molecular terms?
This has long been a mystery. For bacteria the mechanism will be discussed in
Chapter 2. In general terms it has been observed that when protein synthesis (i.e.
cell growth) is inhibited in cells that have just passed this critical mass or “point
of commitment” (arrow 1 in Fig. 1.3), the cells will still be able to proceed to
division. This made Cooper (1991) suggest that it is perhaps the cell’s strategy
not to start the DNA replication, segregation and division processes before it has
reached a size which, upon inhibition of further growth , will give rise to two
daughter cells of sufficient size to survive (Fig. 1.5-iv). We will later see that in
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bacteria, the critical size for the start of DNA replication, is indeed twice the
minimum size which can be observed for newborn cells in the slowest growing
cultures (Chapter 2, section 2.2.3). We will also see that the critical mass at
initiation of DNA replication (START) and the constant period between
initiation and division causes an increased cell size upon a shift-up in growth
rate (Fig. 1.5 and 1.10).
The concept of a critical mass, Mi, at initiation of DNA replication
and a
constant duration of the processes, C+D, leading to cell division greatly
stimulated bacterial cell cycle studies (Nanninga and Woldringh, 1985).
Moreover, these global rules dictating that cell cycle progress depends on cell
growth became an important concept for understanding the eukaryotic cell cycle
(Nurse, 1975).
In the bacterial cell cycle the key events to consider are the start of the DNA
replication or C period and the start of the constriction or T period, leading to
cell division. The B and D periods are defined as the generation time minus the
C+D period and as the time between termination of DNA replication and
division, respectively. As we will see in Chapter 2, there is no separate phase for
DNA segregation (mitosis), which makes it possible that C-periods can overlap
(see below Fig. 1.10 B), a phenomenon not possible in eukaryotc cells.
In genetic studies many of the genes of E. coli involved in DNA replication and
in cell division were identified by the isolation of temperature-sensitive (Ts)
mutants that failed to perform these processes at the restrictive temperature (42
°C). Such genetic studies have revealed among other complex mechanisms like
DNA replication, chemotaxis, protein secretion and cell division, an important
aspect of cell cycle regulation: a surveillance mechanism for the detection of
DNA damage, called the “SOS-response”. Through this mechanism the cell cycle
is arrested by induction of an inhibitor (SfiA) of the FtsZ protein, which is
essential for cell division in all bacteria and archaea. This mechanism served as a
model for Hartwell and Weinert (1989) when they formulated the “checkpoint
control” in the eukaryotic cell cycle (Fig. 1.4).
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1.4. Experiments with yeasts
In 1974 Hartwell and coworkers made use of the many cytological markers
visible during the cell cycle of Saccharomyces cerevisiae (Fig. 1.6). They isolated
conditional (Ts) mutants defective in a gene whose product was necessary to
pass specific cell cycle events (landmarks), which he could monitor by phase
contrast microscopy. The temperature-sensitive arrest was considered as cellcycle specific if cells continued to grow and if there was a uniform terminal
phenotype as reflected by the budding index (i.e. the percentage of budded
cells), overall morphology (cell size), DNA content and microtubule, spindle
pole or nuclear morphology and localization (see Fig. 1.6 for landmarks). If this
was the case an indication was obtained of which process in the cell cycle was
defective.
Fig. 1.6. Landmarks and “dependency map” of the yeast cell cycle (adapted from Pringle
and Hartwell, 1981). Consecutive arrows indicate a dependency of events (e.g. DNA
synthesis depends on the START-event and nuclear division depends on DNA
replication). Parallel arrows indicate independent pathways (e.g. bud development is
independent of DNA synthesis or Spindle Pole Body duplication). The numbers indicate
cdc mutations, causing a defect in a particular process when the cells are grown at
restrictive temperature (37 °C). Many of the thus characterized genes coded for proteins
that have been conserved in evolution and are often considered to belong to a “central
control system”. BE, bud emergence; NM, nuclear migration; MRF, CRF, (actine) ring
formation; IDS, DS, initiation of DNA synthesis; mND, lND, medial and late nuclear
division; CK, CS, cytokinesis and cell separation; SPBD, SPBS, spindle body
duplication and separation.
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From a collection of 1500 Ts mutants, Hartwell picked up 50 so-called cdc (cell
division cycle) mutants. The existence of such genes indicated that events like
START (i.e. G1/S transition; compare with the bacterial Mi) and the initiation of
mitosis do not occur implicitly when a cell reaches a certain critical size, but are
regulated by specific proteins assumed to belong to a cell cycle control system.
From a similar collection of Ts mutants obtained in 1980 in the fission yeast,
Schizosaccharomyces pombe, Nurse identified an important gene, cdc2, whose
function was critical for the G2/M transition. It appeared that in both yeasts,
genes had been isolated that coded for a protein kinase (cdc28S.c. and cdc2S.p.) and
for cyclins (cln1,2,3 and cdc13S.p.). In addition, it was found that these genes,
cloned by complementation, had been conserved in evolution and that the
human equivalent could complement the defective function in the yeast mutant.
The two principles found for bacteria, i.e. a critical size at initiation of DNA
synthesis and a constant period until cell division (cf. Fig. 1.5), also seemed to
hold for eukaryotic cells (Fantes and Nurse, 1981). Applied to yeast cells it has
been called the “sizer and timer” model (Wheals, 1991), based on observations of
mass increase with growth rate and on a constant budding period. We will see
in Chapter 4 that the model can also explain the behavior of certain yeast
mutants.
1.5. Experiments with animal cells
Like in bacteria, it was expected that specific factors would trigger S phase or
mitosis in the animal cell cycle. In 1971 cell fusion studies (see Fig. 1.4)
suggested that an M-phase promoting factor (MPF) from M-phase cells could
induce chromosome condensation in nuclei of cells in G1, S or G2 phase,
whereas an S-phase promoting factor could induce DNA synthesis only in G1
nuclei. What were these factors?
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Fig. 1.7. Demonstration of a meiosis-promoting factor, MPF. The cytoplasm of
stimulated, secondary oocytes from marine invertebrates injected in full-grown, primary
oocytes is able to induce meiosis I and II, the stage of a mature, unfertilized egg.
When the cytoplasm of progesterone-stimulated secondary oocytes of the frog,
arrested in the metaphase of meiosis II, was injected into primary oocytes
arrested in G2, these oocytes (without progesterone stimulation) progressed
through meiosis I and II (Fig. 1.7). This maturation of the unfertilized eggs was
ascribed to the activity of a meiosis-promoting factor, MPF. Subsequently, it was
found that the same cytoplasmic extract of oocytes was able to stimulate
multiple cycles of mitosis in sperm nuclei (see Chapter 4). Thus, MPF stands for
both meiosis- and mitosis-promoting factor. These experiments led to the
development of a biochemical assay and finally to the purification of a protein
complex with protein kinase activity, responsible for promoting chromosome
condensation and nuclear envelope breakdown.
Studies of synchronously fertilized sea urchin eggs labeled with a radioactive
amino acid led to the discovery of a periodically synthesized protein called
cyclin, which appeared to be a subunit of MPF. How this factor together with
many other proteins plays a major role in cell cycle regulation will be discussed
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below. In Chapter 4 we will see that mutations in genes coding for
these
proteins can lead to a deregulation of the cell cycle and to the development of
cancer.
1.6. “Synthesis” between the cytological, biochemical and genetic findings
All the above-mentioned studies of yeast and animal cells combined into the
notion that the discontinuities of the G1/S and G2/M transitions are induced by
the activation of cyclin-dependent protein kinases (CDKs), often indicated as
“p34cdc2” to honour the first characterization of its gene in S. pombe by Paul
Nurse.
The protein kinase is only active when complexed with a cyclin as regulatory
subunit (Fig. 1.8A), which is broken down (proteolysis) at the end of mitosis.
Different combinations of CDKs and cyclins promote the alternation between S
phase and M phase through the phosphorylation (activation) of specific sets of
proteins: e.g. transcription factors necessary for the expression of DNA
replication genes and structural proteins (histone H1, nuclear lamina) involved
in chromosome condensation, nuclear envelope breakdown and spindle
formation.
The CDK/cyclin complexes act as a biochemical motor driving the transitions
from G1 to S and from G2 to M (Fig. 1.8 B). This “cell cycle engine” (Murray and
Hunt, 1993) is also involved in surveillance mechanisms able to arrest the cell
cycle at specific points by inhibiting the activity of the CDK/cyclin complexes.
These points were named “checkpoints” by Hartwell and Weinert (1989):
processes or biochemical signal transduction pathways that verify whether all
conditions to perform a discontinuous event or transition are fulfilled. If not,
progression through the cycle is arrested ensuring dependency of cell cycle
events. For instance, if there are not enough nutrients or growth factors, genes
involved in firing DNA replication will not be expressed and the cell will not
pass START: this is called G1-arrest and the inhibitory pathway is called the
“DNA replication checkpoint”. If DNA damage occurs, DNA repair enzymes
will be induced and the cell will not pass to mitosis: this is called G2-arrest and
the inhibitory pathway is called the “DNA damage checkpoint”. (In eubacteria
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like E. coli, the latter mechanism resembles the above mentioned “SOSresponse”; see also Chapter 2.4.2) If microtubules do not attach to all
kinetochores and chromosomes do not become well aligned in metaphase, the
metaphase-to-anaphase transition is blocked. The inhibitory pathway is called
the “spindle assembly checkpoint”.
Fig. 1.8. Components of the “cell-cycle control system”. A. Schematic representation of
the CDK or p34cdc2 protein, which consists of an amino-terminal lobe rich in betasheets and a larger carboxy-terminal lobe of mainly alpha-helices. Between the two lobes
is a deep cleft which represents the catalytic site of protein substrate binding. ATP binds
in the cleft after dephosphorylation of the tyrosine 15-phosphate. Cyclin binds to one side
of the cleft near an alpha-helix which contains the characteristic PSTAIRE sequence. The
T-loop, which blocks the entrance to the catalytic cleft in the inactive kinase, bends away
upon cyclin binding and phosphorylation of its threonine 160 (after Jeffrey et al., 1995).
B. Simplified view of the cell cycle: progression, cycle periods and the G1/S and G2/M
transitions are caused by the “biochemical motor” of CDKs and cyclins. The activity and
specificity of this “motor” is changed by phosphorylations and de-phosphorylations and
by the appearance of specific S- or M-cyclins. The complexes are both inactivated by
CDI’s (Cyclin Dependent protein kinase Inhibitor) and, at the end of the cycle, by
proteolysis of their cyclines.
In the early 90’s it thus seemed as if the eukaryoticcell cycle could be readily
explained in terms of CDKs and cyclins (Fig. 1.8).
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1.7. Two viewpoints
In the thinking about the cell cycle, the point of departure is generally that the
complicated cellular and molecular machinery requires mechanisms of
regulation, coordination and error correction. Only with such mechanisms all
the chemical and structural changes during the sequential steps of the cell cycle
can be performed in a proper order.
Depending on their biochemical, genetical or cytological background scientists
have considered the cell cycle from different angles. In general, two viewpoints
or concepts exist (Nurse, 2000 a and b).
The first viewpoint can be said to be explicit, genomics-driven, has been called
the “clock-concept” and its elements have been compared with an automatic
washing machine (cf. Alberts et al., 1994, p.867). In this viewpoint it is assumed
that the cell cycle steps occur independently from each other and that a central
control system, clock, autonomous oscillator or key regulator, distinct from the
performing machinery, triggers each process in turn and in a specific sequence.
The problem of how the cell knows when a particular step has been completed
is solved by relative timing (which operates especially in embryonic cycles) or
by a feed back control mechanism that activates the clock when a process has
finished. In this way a dependency of processes is established by feedback
control loops, i.e. by regulatory proteins that have a signaling function and act
as rate-limiting steps for cell cycle progression; any error in a process will arrest
the machinery (checkpoint control) and thereby prevent a catastrophe.
The second viewpoint is implicit, structure-driven, has been called the dominoconcept and has been compared with the “hard-wired” self-assembly process of
phages as well as with the substrate-product relationship in metabolic
pathways. In this viewpoint it is rather assumed that within the existing
structural framework the cell cycle steps occur in a dependent sequence.
Dependency could be achieved not only by a substrate-product relationship or
by signal transduction, but also by the state of cellular structures. In other
words, there is no central or key regulator!
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The notion that the cell cycle is driven by a “biochemical motor” consisting of
CDKs and cyclins (Fig. 1.8) has appeared to be too simple. For instance, different
combinations can activate S phase in budding yeast and a typical M phase
promoting combination can also trigger S phase in animal G1-cells. Other events
like the meta-to-anaphase transition are obtained through proteolysis (Fig. 1.8B),
performed by non-CDK components like the anaphase promoting complex or
cyclosome (APC). Also the initiation of DNA replication by origin-firing at prereplicative complexes (preRCs) is rigidly controlled by other kinds of protein
(Nasmyth, 1996b).
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In other words, the CDK/cyclin combinations are not so specific that they alone
dictate the order of events. Ultimately this order is also determined by the state
of cell structures (substrates) on which they act. This makes it difficult to
identify the hypothetical, central “key regulator” or “master switch” assumed to
exist in the first viewpoint. In Chapter 4 we shall see that apart from the gradual
increase during the cell cycle of proteins phosphorylated by the CDK/cyclin
complexes and the abrupt ending of this process through cyclin proteolysis,
there is a second strategy. This consists of the two opposing activities that each
CDK/cyclin complex has. For instance, S-phase CDK/cyclin (S-CDK) triggers
initiation of replication from the origins at the pre-replicative complexes, but
inhibit at the same time the assembly of new pre-replicative complexes. In this
way, a fixed sequence of steps is maintained.
In bacteria, the cell cycle is performed with remarkable simplicity, without
cyclins and protein kinases! Here, on a smaller scale (see Fig. 1.2) the concept of
a “central control system” and of elaborate “checkpoints” vanishes. Cycling on
this scale the E. coli cell emphasizes the view that a sequence of events is
determined by the changing state of interacting proteins and chromosomes in
the growing cell. This notion, that not only the regulatory and activating system
of cyclins and CDKs but also the structural state of the cell is important in
determining the dependency of events, has been emphasized by Nasmyth
(1996b).
1.8. Overview of cell cycles
In both the bacterial and the eukaryotic cell cycle, the key events are the
initiation of DNA replication (G1/S transition), DNA segregation (G2/M
transition) and cell division.
The most fundamental difference between
prokaryotes and eukaryotes lies in the structure of the genome and in the way
the replicated DNA is segregated (Table 1.1): In eukaryotes the genome is
distributed over a number of different chromosomes in which, after replication,
the daughter strands (chromatids) remain aligned by cohesins until their
separation in a distinct phase, mitosis. Prokaryotes (Fig. 1.9A) have in most cases
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a single chromosome of which the replicated daughter strands separate and
become immediately segregated..
Fig. 1.9. Schematic representation of the various cell cycles. Note the different cycle
times and the different terminologies with respect to the cell cycle phases. The bacterial
cell cycle (B, C, D) can be shortened until C periods overlap and DNA synthesis is
continuous (“T” stands for the duration of the constriction period in E. coli). The
eukaryotic cycle (G1, S, G2, M) can be shortened at the expense of the G1 phase and, in
embryonic cells, also of the G2 phase. No further shortening is possible because S and M
phases cannot overlap (see Fig. 1.10). G* signifies a G1 period in the previous cycle
where the cell is in a bi-nucleate state.
The cell cycle of yeasts is somewhat different from the usual eukaryotic pattern
as depicted in Fig. 1.9 D because mitosis and cytokinesis do not necessarily
coincide. In S. cerevisiae (Fig. 1.9 B), mitosis could be considered to coincide with
the S phase so that there is no clearcut G2 phase. This is because spindle pole
duplication and spindle assembly already start during S phase. The bi-nucleate
phase between mitosis and cell division in yeasts is called “G1pm”, for postmitotic G1 phase or G1* (Lord and Wheals. 1981).
In the fission yeast S. pombe, the situation is again different: Here mitosis occurs
relatively early, and because there is a relatively long G2 phase, the S phase
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may even start in the previous cycle (Fig. 1.9 C)! Under no condition, however,
is the generation time shorter than the “constant period” of S+G2+M.
Although it was stated above that a cycling cell is always a growing cell (as
depicted in Fig. 1.2), the embryonic cell cycle forms an exception. This is because
the primary oocyte of the frog, while remaining arrested in G2, increases its
volume by a factor of 10- to 100- thousand. After meiosis and fertilization, it
subsequently divides itself up through synchronous cycles consisting of
alternating S and M phases (Fig. 1.9 E). Although S-phase and M-phase CDKs
are present in large amounts and specific cyclins are formed and broken down,
no other regulatory proteins for checkpoint control seem to be present.
Especially in the animal cell cycle, the division process or cytokinesis is not
considered as a separate cell cycle phase like the bacterial T period, because
mitosis and division follow each other so quickly in time.
1.9. Simulation of the bacterial and eukaryotic cell cycle
In contrast to eukaryotic cells, the generation time in bacteria can be shorter
than their “constant period” (C+D) and even shorter than their replication
period (C). Bacteria can cope with this situation of overlapping cell cycle periods
by the mechanism of multifork replication (Cooper and Helmstetter, 1968) and
continuous and hierarchical segregation. In the eukaryotic nucleus the replicated
sister chromatids are first “glued” together by cohesion proteins, subsequently
attached to microtubuli and finally segregated, after proteolysis of the “glue”,
with the help of motor proteins by the process of mitotic segregation. The two
mechanisms for chromosome segregation are further discussed in Woldringh
(2010)
Fig. 1.10 summarizes the difference between eukaryotic and prokaryotic cell
cycles. These figures can be constructed for different durations of generation
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time and cell cycle periods by using the Object-Image program “Simulation Cell
Cycle”developed by Norbert Vischer.
See web site: http://simon.bio.uva.nl/Object-Image/CellCycle/index.html
The program conforms to the so-called “continuum model” proposed by Cooper
(1999) which is based on insights obtained from the bacterial cell cycle (see also
Fig. 1.3 A).
In this model the G1 phase is merely a period that appears when the generation
time is longer than the (S+G2+M)-period. In other words, there are no G1
specific events necessary for progress of the cell cycle. The synthetic processes
that trigger S phase (START), occur continuously, i.e. they can already occur
and can thus overlap with the (previous) S, G2 and M phase. Under conditions
of rapid growth the duration of the S, G2 and M phase (assumed to remain
constant) is sufficient for the synthesis of the necessary proteins for the next
cycle and the G1 phase vanishes (see Fig. 1.10 A).
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Fig. 1.10. Coupling between cell growth and DNA replication/segregation/division by
two principles: (i) a constant period between initiation of DNA replication and cell
division, indicated by S+G2+M or C+D, and (ii) a critical mass (or volume) at initiation
indicated by “START”in (A) or “mi, 2xmi and 4xmi” in (B), indicating whether
initiation takes place at 1, 2 or 4 origins. Because of these two principles, more rapidly
growing cells are larger. (A) The eukaryotic (diploid) cell growing with a cycle time of
10 h is shifted up to a cycle time of 5 h. In this hypothetical example the shortening
occurs at the expense of the G1 period. In the new cell cycle the cells are slightly larger
and START occurs at birth, i.e. immediately after M phase. The minimal cycle time is
S+G2+M (compare embryonal cell cycle in Fig. 1.9 E) because the processes during S
and M cannot overlap. (B) The bacterial (haploid) cell, growing with a cycle time of 100
min is shifted up to a cycle time of 40 min. In the new cycle the B period has disappeared
and the C+D periods are overlapping allowing the cycle time to be shorter than the
constant C+D period of 60 min. This is only possible in bacteria because of their
immediate and continuous segregation of daughter strands, which allows for multifork
replication. The minimal cycle time is probably D min, in which period termination
triggers FtsZ-ring formation which starts the constriction process (T period). See further
Chapter 2.
23
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26
Questions on the cell cycle
1. The volume of an individual cell increases continuously during the cell cycle.
a. How can it be explained that the average volume of cells in a population
stays constant in time?
b. How can it be explained that cells increase in size when mitosis is
inhibited?
c. Why can it be stated that “a cycling cell is a growing cell”?
2. Studies of the bacterial cell cycle revealed that cells start DNA synthesis at a
critical cell mass which remains constant when the growth rate is changed.
This principle of constant initiation mass also holds for many eukaryotic cells.
a. Which other general principle of the cell cycle was detected studying
bacteria?
b. What is the effect of both principles on the size of cells if growth rate is
increased?
3. Yeast geneticists have isolated and characterized temperature-sensitive cell
division cycle mutants.
a. Mention two specific properties of yeast cells that made this isolation
possible.
b. According to which criteria did the geneticists consider ts-mutations to be
cell cycle specific?
4. Protein kinases and cyclins play an important regulatory role at two transitions
during the cell cycle.
a. Why are they also considered to play a role in the so-called “checkpoint
control”?
b. At which cell cycle transition do they not seem to play a role?
5. According to the “clock theory” a central control system in combination with
feed-back regulation can coordinate a sequence of cell cycle events.
a. What cellular processes would represent this clock?
b. What is the essential difference between the clock and the domino theory?
6. Rapidly growing bacteria demonstrate the phenomenon of “overlapping cell
cycle periods”.
a. What is meant by this statement and does it also occur in the eukaryotic cell
cycle?
b. Which cell cycle processes cannot overlap in either the bacterial or the
eukaryotic cell cycle?
c. What cell cycle phases determine the minimal generation time of cells?
27
Answers
1. a. The growth of each individual cell is coupled to division allowing for
homeostasis, i.e. the rate of mass increase equals the rate of division.
b. Protein synthesis is not inhibited (cf. Fig. 1.3).
c. Only a growing cell will reach the critical cell mass at START and proceed
through the cell cycle.
2. a. The principle of a constant cell cycle period between initiation of DNA
replication and cell division.
b. As a result of the two constants, cell size increases when cells grow more
rapidly(Fig. 1.5).
3. a. Yeast cells are easy to culture and they exhibit many morphological features
that can mark the progress through the cell cycle (landmarks).
b. If the mutation allowed growth to continue and if the cells showed an
uniform terminal phenotype (Fig. 1.6).
4. a. Signal transduction pathways that are induced by deficiencies, DNA
damage for instance will eventually inhibit the CDK/cyclin complexes
preventing the transition.
b. At the metaphase-anaphase transition (Fig. 1.7).
5. a. The clock could be represented by overall growth and size of the cell.
b. The clock theory assumes that all regulation is carried out by signalling
proteins (Fig. 1.8). In the domino theory, structural or physical changes may
represent the trigger.
6. a. It means that process(es) specific for a certain cell cycle period or phase can
coincide or overlap with processes specific for another cell cycle period or even
of the same phase preparing for a next cycle.
To a limited extent this can also occur in the eukaryotic cell cycle.
b. In the bacterial cell cycle the processes involved in cell constriction (D and T
period) cannot overlap with the same processes preparing the next cycle. In the
eukaryotic cell cycle the processes occurring in S+G2+M cannot overlap with the
same period preparing the next cycle.
c. The phases that cannot overlap, i.e. D in bacteria and S+G2+M in eukaryotes.