|
 |
|
EMBO reports 4, 8, 757–760 (2003)
doi:10.1038/sj.embor.embor895
Coupling the cell cycle to cell growth
A look at the parameters that regulate cell-cycle events
Erik Boye1 & Kurt Nordström2
|
|
|
|
1 Department of Cell Biology,
Institute for Cancer Research, Montebello, 0310
Oslo, Norway
2 Department of Cell and
Molecular Biology, Uppsala University Biomedical Center, Box 596,
S-751 24 Uppsala, Sweden
To whom correspondence should be addressed
Erik Boye Tel: +47 22934256; Fax: +47 22934580;
eboye@labmed.uio.no
Received 25 February 2003; Accepted 26 May 2003.
|
|
|
|
Abstract
In order to multiply, both prokaryotic and eukaryotic cells go through
a series of events that are collectively called the cell cycle. However, DNA
replication, mitosis and cell division may also be viewed as having their own,
in principle independent, cycles, which are tied together by mechanisms
extrinsic to the cell cycle—the checkpoints—that maintain the order
of events. We propose that our understanding of cell-cycle regulation is
enhanced by viewing each event individually, as an independently regulated
process. The nature of the parameters that regulate cell-cycle events is
discussed and, in particular, we argue that cell mass is not such a
parameter.
EMBO reports 4, 8, 757–760 (2003)
doi:10.1038/sj.embor.embor895
|
|
|
|
Introduction
The classical way to study what has become known as the cell cycle is
to observe, in a continuously growing culture of cells, the successive
occurrence of chromosome replication, chromosome separation and cell division
(Maaløe & Kjeldgaard, 1966). During
exponential growth in a steady-state situation, each event is repeated at
regular intervals, corresponding to the doubling-time of the culture.
In contrast to biochemical cycles such as the trichloroacetic acid
(TCA) cycle, in which the product of one reaction is the substrate for the
next, the cell cycle is composed of a series of biochemically unrelated
reactions. In other words, the product of one round of the cell cycle (a newly
divided cell) cannot be considered the substrate for the first event of the
next (the initiation of DNA replication). Also, whereas a biochemical cycle is
totally arrested if one of the reactions is inhibited, the obstruction of cell
division does not preclude a new round of genome replication (see below),
suggesting that the cell cycle is a composite of distinct and heterogeneous
successive events. Finally, the 'substrate' and 'product' of most of these
events are the same: one genome begets two genomes and one cell becomes two.
Early investigators clearly made these distinctions between the cell cycle and
biochemical cycles (Hartwell & Weinert, 1989),
but the former was originally termed a cycle (Howard &
Pelc, 1951; Hartwell, 1978;
Lee & Nurse, 1988) because, normally, DNA
replication is followed by chromosome segregation and not by another round of
DNA replication. Similarly, only under special circumstances does a cell divide
twice without an intervening round of DNA replication.
Perturbations of the cell cycle
It is the exceptions to the norm, in other words the conditions
under which the perturbation of cell growth fails to lead to the inhibition of
later events, that give us insights into the differing natures of the cell
cycle and biochemical cycles. For example, whereas the addition of penicillin
to a culture of growing Escherichia coli cells inhibits cell division,
the cells continue to grow and elongate into filaments, DNA replication
continues, and the proper segregation of the daughter chromosomes is not
affected (Fig. 1A). Similarly, Schizosaccharomyces
pombe cells can go through mitosis without a preceding S phase, which means
that they can divide without distributing a full genomic complement to each
daughter cell (Fig. 1B). These observations suggest that
the cell cycle is composed of at least two separate 'cycles': a DNA-replication
cycle and a cell-division cycle for E. coli, and a DNA-replication cycle
and a mitotic cycle for S. pombe. This conclusion is further supported
by the following findings. There are conditional mutants in Saccharomyces
cerevisiae that are unable to enter S phase but continue to initiate
synchronous rounds of budding in the absence of DNA replication and mitosis
(Hartwell, 1971). Moreover, in S. pombe,
repeated rounds of uninterrupted DNA replication are observed on overproduction
of the initiation protein Cdc18 (Nishitani & Nurse,
1995). Likewise, treatment of human cells with the protein kinase
inhibitor staurosporine induces several consecutive rounds of chromosome
replication (Stokke et al., 1997). In
meiosis, two consecutive reductive cell divisions, which entail carefully
orchestrated chromosome pairing and segregation events, take place without
intervening DNA replication, a process that is essential for the natural
reproduction of almost all eukaryotic organisms, including humans.
|
|
Figure 1
Examples of independence of cell-cycle events. (A) S phase
can be independent of cytokinesis. Escherichia coli cells were grown
exponentially in rich medium (left) or treated with ampicillin (right) for 90
min, fixed in ethanol and stained with DAPI (blue) for visualization of
nucleoids. The images were obtained by phase-contrast and fluorescence
microscopy. (B) Mitosis can occur independently of S phase.
Schizosaccharomyces pombe cdc10ts rad3 mutant cells were
incubated for 6 h at the non-restrictive temperature for
cdc10ts, a treatment that arrests the cells before S
phase. Four different cells are shown. In the absence of the checkpoint protein
Rad3 (the founding member of the ATR family of proteins), the cells proceed to
mitosis and attempt to divide a single nucleus. The DNA is stained by DAPI
(light foci located at the septum). Note the unequal amounts of DNA distributed
to the two daughter cells. ATR, ataxia-tengiectasia mutated and Rad3-related;
cdc10ts, cell division cycle 10 temperature-sensitive
mutant.
|
|
|
On the basis of this evidence, we argue that the cell cycle should
be treated as a series of distinct events (DNA replication, DNA segregation and
cell division) that only seem to be parts of an obligatory cycle because of how
they are tied together (Nordström et al.,
1991). Moreover, these events run in parallel (Fig.
2A), possibly even being initiated at roughly the same time (Hartwell et al., 1974; Murray
& Hunt, 1993; Nordström & Dasgupta,
2001). For an illustration of this point, consider the use of the
length of the G1 phase as a parameter by which to measure the effects of some
treatment (mutation or treatment with chemicals) on the regulation of S-phase
initiation. This implies that the treatment used does not affect the regulation
of any cell-cycle event other than S-phase entry, particularly cell division,
and this is usually difficult to verify. In such studies, S-phase entry should
instead be compared to a general parameter, such as cell mass (which does not
imply that mass is regulating the cell cycle (see below); mass is only a
convenient general parameter for comparisons between cells in the same
culture). Viewing cell-cycle events relative to a common parameter, rather than
to each other, makes it possible to see connections and mechanisms of
regulation more clearly. It is also important to realize that regulating a
particular event of the cell cycle (in this case, DNA replication) does not
automatically mean regulating the cell cycle as a whole. An interesting
illustration of the independence of cell-cycle events can be derived from
bacteria. At high growth rates, E. coli cell cycles are actually
overlapping, so that the DNA replication of one cell cycle may occur before the
cytokinesis event of the previous cycle (Cooper &
Helmstetter, 1968).
|
|
Figure 2
Representation of the cell cycle as being composed of three linear
and repetitive series of S phase (S), mitosis (M) and cytokinesis (C).
(A) In this simplified model, the three series of events would be
regulated individually and coupled to cell growth in an unknown manner.
(B) In reality, they are coupled to one another more directly through
checkpoint mechanisms. Cues are given to inhibit another process (blocked
arrows) or allow it to continue (arrows).
|
|
|
Checkpoints
What makes the events of the cell cycle occur in a given order?
Under constant growth conditions, the coupling between cell growth and
individual cell-cycle events is sufficient to preserve their order. In most
cases, the obstruction of one event leads to inhibition of a downstream event
by activation of a checkpoint (Hartwell & Weinert,
1989). There is no need for checkpoints in a biochemical cycle,
because a given reaction cannot start until the previous one, which provides
the substrate, has finished.
Several cell-cycle checkpoints have been discovered. They include
the inhibition of DNA replication when DNA is damaged, inhibition of mitosis
when DNA has not been replicated or is damaged, and inhibition of cytokinesis
when mitosis has not been properly executed (for a review, see
Elledge, 1996). The first checkpoint to be
discovered was the E. coli SOS response, which involves the inhibition
of cell division in the presence of DNA damage (Defais et
al., 1971). The checkpoints are the active gatekeepers of the
cell cycle and they preserve the order of events even in the face of
perturbations of any of the processes involved. We argue that a linear
representation of the different events that take place during growth, with
checkpoints serving as cross-checks between them (Fig.
2B), is a true reflection of the cell cycle, and is more appropriate
than the common circular representation.
Regulation
In a culture growing at steady state, the key events in each cell
occur at a defined pace or frequency that is specified by the generation time
(Maaløe & Kjeldgaard, 1966). However,
this is simply a consequence of steady-state growth and does not yield any
information about the mechanisms of regulation. The system is regulated by the
conditions under which the cells are grown; that is, the growth medium
determines the growth rate of a culture, which in turn dictates the frequency
of all cell-cycle-related events (Jensen & Pedersen,
1990; Marr, 1991). This is trivial; to
grow with a doubling time of x minutes, the cells must perform each
required step once every x minutes.
It is not straightforward to identify the parameters regulating
cell-cycle events. The finding that a change in one parameter also changes the
cell cycle is not sufficient to prove that this parameter normally regulates
the cell cycle. Frequently, cell mass is identified as such a regulator (for
example, see Rupes, 2002), but we argue that this
is based on a logical flaw. Let us take a closer look at the steady-state
growth of a culture. All cell constituents double in amount from mitosis to
mitosis, from one initiation of DNA replication to the next, or from
cytokinesis to cytokinesis. Fig. 3 is a schematic,
two-parametric representation of cells in such a culture. The ordinate shows
DNA content per cell and the abscissa could be any general growth parameter,
such as cell mass, cell size, number of ribosomes, cell surface area, number of
messenger RNA molecules for a specific gene, and so on. Parameter values for
cells in a given steady-state culture will always fall within the red area.
Newborn cells appear at the bottom left and continue to increase in terms of
this parameter throughout G1 phase. At a certain point, DNA replication (S
phase) commences, and the DNA content is doubled. Thereafter, the parameter
increases in G2 phase until the cells divide. Therefore, cells at a given point
in the cell cycle have compositions identical to those of cells at the same
point in a previous passage. The culture is not at steady state unless this
criterion is fulfilled. It follows that in any given culture, the cells contain
the same amount of any constituent that one might care to measure (the abscissa
in Fig. 3) when they start DNA replication, and it is not
surprising that all the cells initiate DNA replication or divide at a fixed
mass, whether they are bacterial, yeast or mammalian cells. Although this has
been observed repeatedly, this constancy is a tautology, and the interpretation
of such observations to mean that cell mass has a regulatory function is
misguided.
|
|
Figure 3
Two-parametric histogram of a cell culture growing under
steady-state conditions. The cell cycle is composed of a G1 phase (G1), S phase
(S), G2 phase (G2), mitosis (M) and cytokinesis (C).
|
|
|
Indeed, it is important to discriminate between the correct
observation that mass is constant during a given event of the cell cycle and
the incorrect conclusion that mass is regulating this event. A correlation is
distinct from a causative relationship. We argue that the numerous publications
showing a constancy of mass at a certain point in the cell cycle under a given
set of conditions do not provide evidence for mass as a regulatory parameter.
Only when comparing individual cultures grown under different conditions can we
critically test whether a parameter is constant at a certain point in the cell
cycle, and such experiments have revealed that cell mass can be excluded as a
regulator of S-phase entry: when the growth conditions of bacterial, yeast and
mammalian cells are varied to give different average cell sizes, cell mass at
S-phase entry is indeed variable (Churchward et al.,
1981; Rønning et al.,
1981; Wold et al., 1994;
Carlson et al., 1999). In addition, crucial
experiments have identified factors that promote the cell-cycle progression of
Drosophila wing-tissue cells and rat cells in vitro without
promoting cell growth (Neufeld et al.,
1998; Conlon et al., 2001). Not
only is it counterintuitive that a cell uses a crude and chemically ill-defined
parameter such as cell mass to regulate its progress through the cell cycle,
but it seems likely that a central molecular parameter—or set of
parameters—that reflects the growth state of the cell is used for such
vital and flexible regulatory processes. Candidates for such regulators include
cell-cycle proteins themselves and low-molecular-weight signalling molecules
(see below). Intuitively, it is likely that the onset of the key cell-cycle
events is governed by the availability of many such components (Bernander, 1994; Tyson et al.,
1995).
Primary and secondary regulators of the cell cycle
Genetic approaches have identified a large number of genes in
different systems whose protein products are involved in the execution of the
key cell-cycle processes. These key processes may be inhibited by checkpoint
mechanisms, which mainly function in the case of perturbations. However, no
proteins have been proven to be the initial, or primary, regulators of
cell-cycle events under normal conditions. For example, in S. pombe
cells, overproduction of the DNA replication initiation protein Cdc18 promotes
several reinitiations in rapid succession (Nishitani &
Nurse, 1995), and the initiator protein DnaA does the same in E.
coli (Løbner-Olesen et al.,
1989). Nonetheless, it is not obvious that the two proteins are
rate-limiting for S-phase entry in vivo; they may simply be mediators of
the regulatory signal. Thus, it is important to discriminate between the
primary regulators (the parameters that normally regulate cell-cycle
progression) and the secondary regulators (parameters that either can be made
to regulate or that are actually involved in executing cell-cycle
progression).
Another potential regulator is the Cdc2 protein of S. pombe,
a cyclin-dependent kinase (CDK) that is involved in different protein
phosphorylation events that are required to trigger both DNA replication and
mitosis (Stern & Nurse, 1996). The kinase
activity of Cdc2 is regulated by its phosphorylation, by the availability of
its cyclin partners and by proteins that inhibit its activity, and there is
good evidence that mitosis is initiated when the kinase activity reaches a
certain level. Indeed, CDK activity has been referred to as the 'cell-cycle
engine' and it is now firmly established that members of the CDK family
choreograph cell-cycle changes in eukaryotes (for example, see
Nigg, 1995). However, as in the case of Cdc18, it
is not clear whether, under steady-state growth conditions, the Cdc2 protein
has a regulatory function or is simply a transmitter of an upstream regulatory
signal. In other words, there is as yet no direct evidence that Cdc2 activity
couples cell-cycle events to cell growth.
In E. coli, factors that are thought to regulate DNA
replication, chromosome partitioning or cell division are also directly
involved in the execution of these processes. An effect caused by modulating
the activity of such a factor may be due either to changes in a regulatory
function or to interference with the process itself. In such cases, it is
particulary difficult to distinguish between secondary and primary regulators.
Other candidates for regulators of initiation of DNA replication in E.
coli are the small signalling molecules guanosine tetraphosphate (ppGpp)
and cyclic AMP, which provide links between the cell cycle and the general
nutritional status of the cells (for a review, see Vinella
& D'Ari, 1995). In another bacterium, Caulobacter
crescentus, the CtrA protein has functions that may classify it as a
regulator. CtrA controls several events, including the initiation of DNA
replication, DNA methylation, cell division and flagellar biogenesis. In
addition, it is a member of the response-regulator family of two-component
signal-transduction systems and is activated by phosphorylation and inactivated
by proteolysis (Domian et al., 1997). This
complex regulation of CtrA at the levels of synthesis, phosphorylation and
degradation has parallels to the regulation of Cdc2 kinase/cyclin activity in
eukaryotes, a form of regulation that may supply the system with the periodic
oscillation believed to be necessary for the onset of the key cell-cycle
events. However, even this does not reveal a clear link to cell growth.
Interestingly, a free-running oscillator that drives G1 events independently of
cyclin activity has been described (Haase & Reed,
1999), supporting the idea of parallel cell-cycle processes with
their own, separate controls.
In summary, although several proteins have the ability to regulate
the cell cycle under certain conditions, none of them have been shown to have a
crucial role in regulating the cell cycle in a normal, unperturbed situation.
Therefore, most of these proteins represent downstream effector molecules,
leaving the initiators of cell-cycle regulation to be identified.
Conclusions
The cell cycle is a complex, multi-faceted process, consisting of a
number of biochemically independent events that run in parallel. Checkpoints
ensure that the order of events is preserved. Regulatory mechanisms acting
positively or negatively on individual cell-cycle events have been identified,
but it is important to discriminate between primary and secondary regulators.
In particular, the parameter of cell mass can be excluded as an active
regulator of the cell cycle. In fact, the nature of the connection between the
cell-cycle events and cell growth has not been identified in any organism, and
it is unlikely to be any simple parameter of general cell growth.
|
|
Acknowledgements
We thank O. Nielsen, K. Skarstad and T. Stokke for stimulating
discussions. This work was supported by the Norwegian Cancer Society, the
Norwegian Research Council, the Swedish Cancer Society and the Swedish Natural
Science Research Council.
|
|
References
Bernander, R. ( 1994) Universal cell cycle regulation? Trends Cell Biol., 4, 76–79. | Article |
Carlson, C.R., Grallert, B., Stokke, T. & Boye, E. ( 1999) Regulation of the start of DNA replication in Schizosaccharomyces pombe. J. Cell Sci., 112, 939–946. | PubMed | ChemPort |
Churchward, G., Estiva, E. & Bremer, H. ( 1981) Growth rate-dependent control of chromosome replication initiation in Escherichia coli. J. Bacteriol., 145, 1232–1238. | PubMed | ChemPort |
Conlon, I.J., Dunn, G.A., Mudge, A.W. & Raff, M.C. ( 2001) Extracellular control of cell size. Nature Cell Biol., 3, 918–921. | Article | PubMed | ChemPort |
Cooper, S. & Helmstetter, C.E. ( 1968) Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol., 31, 519–540. | PubMed | ChemPort |
Defais, M., Fauquet, P., Radman, M. & Errera, M. ( 1971) Ultraviolet reactivation and ultraviolet mutagenesis of lambda in different genetic systems. Virology, 43, 495–503. | PubMed | ChemPort |
Domian, I.J., Quon, K.C. & Shapiro, L. ( 1997) Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle. Cell, 90, 415–424. | PubMed | ChemPort |
Elledge, S.J. ( 1996) Cell cycle checkpoints: preventing an identity crisis. Science, 274, 1664–1672. | Article | PubMed | ChemPort |
Haase, S.B. & Reed, S.I. ( 1999) Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle. Nature, 401, 394–397. | Article | PubMed | ChemPort |
Hartwell, L.H. ( 1971) Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J. Mol. Biol., 59, 183–194. | PubMed | ChemPort |
Hartwell, L.H. ( 1978) Cell division from a genetic perspective. J. Cell Biol., 77, 627–637. | PubMed | ChemPort |
Hartwell, L.H. & Weinert, T.A. ( 1989) Checkpoints: controls that ensure the order of cell cycle events. Science, 246, 629–634. | PubMed | ChemPort |
Hartwell, L.H., Culotti, J., Pringle, J.R. & Reid, B.J. ( 1974) Genetic control of the cell division cycle in yeast. Science, 183, 46–51. | PubMed | ChemPort |
Howard, A. & Pelc, S.R. ( 1951) Nuclear incorporation of 32P as demonstrated by autoradiographs. Exp. Cell Res., 2, 178–187. | ChemPort |
Jensen, K.F. & Pedersen, S. ( 1990) Metabolic growth rate control in Escherichia coli may be a consequence of subsaturation of the macromolecular biosynthetic apparatus with substrates and catalytic components. Microbiol. Rev., 54, 89–100. | PubMed | ChemPort |
Lee, M. & Nurse, P. ( 1988) Cell cycle control genes in fission yeast and mammalian cells. Trends Genet., 4, 287–290. | Article | PubMed | ChemPort |
Løbner-Olesen, A., Skarstad, K., Hansen, F.G., von Meyenburg, K. & Boye, E. ( 1989) The DnaA protein determines the initiation mass of Escherichia coli K-12. Cell, 57, 881–889. | PubMed |
Maaløe, O. & Kjeldgaard, N.O. ( 1966) Control of Macromolecule Synthesis. W.A. Benjamin, New York, USA.
Marr, A.G. ( 1991) Growth rate of Escherichia coli. Microbiol. Rev., 55, 316–333. | PubMed | ChemPort |
Murray, A.W. & Hunt, T. ( 1993) The Cell Cycle. Oxford Univ. Press, Oxford, UK.
Neufeld, T.P., de la Cruz, A.F., Johnston, L.A. & Edgar, B.A. ( 1998) Coordination of growth and cell division in the Drosophila wing. Cell, 93, 1183–1193. | PubMed | ChemPort |
Nigg, E.A. ( 1995) Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays, 17, 471–480. | PubMed | ChemPort |
Nishitani, H. & Nurse, P. ( 1995) p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell, 83, 397–405. | PubMed | ChemPort |
Nordström, K. & Dasgupta, S. ( 2001) Partitioning of the Escherichia coli chromosome: superhelicity and condensation. Biochimie, 83, 41–48. | Article | PubMed | ChemPort |
Nordström, K., Bernander, R. & Dasgupta, S. ( 1991) The Escherichia coli cell cycle: one cycle or multiple independent processes that are co-ordinated? Mol. Microbiol., 5, 769–774. | PubMed |
Rønning, O.W., Lindmo, T., Pettersen, E.O. & Seglen, P.O. ( 1981) The role of protein accumulation in the cell cycle control of human NHIK 3025 cells. J. Cell Physiol., 109, 411–418. | PubMed |
Rupes, I. ( 2002) Checking cell size in yeast. Trends Genet., 18, 479–485. | Article | PubMed | ChemPort |
Stern, B. & Nurse, P. ( 1996) A quantitative model for the cdc2 control of S phase and mitosis in fission yeast. Trends Genet., 12, 345–350. | Article | PubMed | ChemPort |
Stokke, T., Smedshammer, L., Jonassen, T.S., Blomhoff, H.K., Skarstad, K. & Steen, H.B. ( 1997) Uncoupling of the order of the S and M phases: effects of staurosporine on human cell cycle kinases. Cell Prolif., 30, 197–218. | PubMed | ChemPort |
Tyson, J.J., Novak, B., Chen, K. & Val, J. ( 1995) Checkpoints in the cell cycle from a modeler's perspective. Prog. Cell Cycle Res., 1, 1–8. | PubMed | ChemPort |
Vinella, D. & D'Ari, R. ( 1995) Overview of controls in the Escherichia coli cell cycle. Bioessays, 17, 527–536. | PubMed | ChemPort |
Wold, S., Skarstad, K., Steen, H.B., Stokke, T. & Boye, E. ( 1994) The initiation mass for DNA replication in Escherichia coli K-12 is dependent on growth rate. EMBO J., 13, 2097–2102. | PubMed | ChemPort |
|
|
top  |
|
|
|
