Download Cell size: a consequence of growth and division?

2927
Journal of Cell Science 113, 2927-2934 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1047
COMMENTARY
Do growth and cell division rates determine cell size in multicellular
organisms?
Carmen M. Coelho1 and Sally J. Leevers1,2,*
1Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, UK
2Department of Biochemistry and Molecular Biology, University College London, Gower
Street, London WC1E 6BT, UK
*Author for correspondence (e-mail: [email protected])
Published on WWW 9 August 2000
SUMMARY
Studies in yeast have provided some clues to how cell size
might be determined in unicellular eukaryotes; yet little
attention has been paid to this issue in multicellular
organisms. Reproducible cell sizes might be achieved in
the dividing cells of multicellular organisms by the
coordination of growth with cell division. Recently,
mutations in genes encoding homologues of components
of the mammalian insulin/phosphoinositide 3-kinase
signalling pathway have been shown to affect organ growth
and cell size during Drosophila melanogaster imaginal disc
development. The data suggest that signalling through this
pathway alters cell size because it primarily affects the
growth of these organs (i.e. their increase in mass) and does
INTRODUCTION
The determination of cell size in multicellular organisms is
intimately linked to the relationship between growth and cell
division. The term growth has been widely used in biology
to describe processes including progression through a
developmental programme, cell differentiation, cell division
and increase in mass. Here, we use the term growth solely to
indicate mass increase. Growing tissues increase in mass, while
the cells within them increase, decrease or remain constant in
size. The increase in size of individual cells during a single cell
cycle is a process that is difficult to monitor in intact organisms.
When this increase in cell size is discussed, we have used the
term cell growth to describe it.
It is a long-standing observation that the size of cells varies
according to their biological context but is highly reproducible
within the same species, tissue and developmental stage. The
control of entry into and passage through the cell division cycle
has been analysed extensively and is now understood in some
depth (Sherr, 1996). Less attention has been paid to the
regulation of growth. The signalling pathways activated by
insulin and insulin-like growth factors (IGFs) in vertebrates
have been implicated in the regulation of various processes,
including growth and metabolism (Efstratiadis, 1998;
Shepherd et al., 1998). Certain components of these pathways
not have a proportional impact on cell division. These
observations are in keeping with the hypothesis that growth
and cell division are regulated independently, and that cell
size is just a consequence of the rate at which tissues grow
and the cells within them divide. However, signalling
through this pathway can affect cell cycle phasing and at
least influence cell division. These interactions may provide
a means of coordinating growth and cell division, such that
cells divide only when they are above a minimum size.
Key words: Cell size, Growth, Cell division, Imaginal disc,
Drosophila, Insulin/PI 3-kinase signalling, Growth regulation
are conserved in invertebrates and have recently been shown
to modify growth and cell size in the fruit fly, Drosophila
melanogaster (Edgar, 1999; Leevers, 1999; Lehner, 1999).
Here, we review the phenotypic observations from Drosophila
and their impact on our understanding of how cell size is
determined.
The experiments in Drosophila investigate the size of
dividing cells, and are the focus of this commentary. The
regulation of cell size in differentiating cells that have exited
the cell cycle or in germ cells undergoing meiosis is not
discussed. In addition, the alterations in cell size that
accompany increased DNA ploidy or the induction of cell
swelling (Nurse, 1985; Waldegger et al., 1997) are not covered.
THE RELATIONSHIP BETWEEN GROWTH, CELL
DIVISION AND CELL SIZE
To understand how the size of mitotic cells is determined, we
have to establish the nature of the relationship, if any, between
growth (mass increase) and cell division. To aid this discussion,
we would like to suggest four simplistic models (Fig. 1). In
models one and two, cell size results from the balance between
growth-promoting signals and signals that induce cell division.
In model one, these stimuli are independent of each other, but
2928 C. M. Coelho and S. J. Leevers
1
2
3
4
Growth and division
regulated
independently
Growth and division
regulated by
common signal
Growth
triggers
division
Division
triggers
growth
Growth
Division
Division
Growth
Division Growth
Division
Growth
REPRODUCIBLE CELL SIZE
Fig. 1. The relationship between growth, cell division and cell size. Four possible ways in which growth and cell division might be regulated to
give reproducible cell size are shown. An explanation of the models shown is given in the text.
their ratio is normally constant. In scenario two, a common
signal drives growth and division. Models three and four
invoke a cell-size-sensing mechanism. In model three, growth,
dRBF
dE2F-dDP
String
CDK1−
Cyclin-A/B
Other
inputs
M
dRBF
G1
dE2F-dDP
G2
S
CDK2−
Cyclin-E
which increases cell size past a particular threshold, triggers
division. In model four, division, which decreases cell size past
a particular threshold, triggers cell growth. In each of the above
cases, growth and cell division are regulated such that when a
cell enters mitosis it has achieved the appropriate size. These
mechanisms are not all mutually exclusive. For example,
models one and three could co-exist if the signal that promotes
cell division in each model is necessary but not sufficient for
Fig. 2. The activities of some of the known cell cycle regulators in
Drosophila (for a review see Edgar and Lehner, 1996). As in other
higher eukaryotes, the G1-S and G2-M transitions are regulated by
cyclin-dependent-kinases (CDKs). Specific cyclins associate with
and positively regulate specific CDKs. The G1-S transition requires
the activity of CDK2–cyclin-E, whereas the G2-M transition requires
the activity of CDK1–cyclin-A/B. dE2F promotes the G1-S transition
by inducing cyclin E transcription, leading to CDK2–cyclin-E
activation. The activity of CDK1–cyclin-A/B is inhibited by
phosphorylation and is activated upon dephosphorylation by the
cdc25 homologue, String. During the later phases of imaginal disc
development, string transcription is regulated by multiple inputs that
coordinate tissue pattern and growth, including dE2F and its cofactor
dDP (Lehman et al., 1999). Thus, dE2F can influence both G1-S and
G2-M. dE2F is negatively regulated by the Drosophila homologue of
the retinablastoma gene produce, dRBF (Neufeld et al., 1998).
Cell size: a consequence of growth and division? 2929
Fig. 3. The effect of cell cycle
deregulation on growth, cell division and
cell size in the developing imaginal disc.
Modifying cell cycle progression by
overexpressing cell cycle regulators in
clones of cells marked with green
fluorescent protein (GFP) has shown that
altering cell cycle progression does not
alter net clonal growth. This figure depicts
the effects of overexpressing dE2F, which
accelerates cell division without affecting
growth, and hence reduces cell size, and
dRBF, which inhibits cell division without
affecting growth, and hence increases cell
size (Neufeld et al., 1998).
Control
+E2F
+RBF
GFPexpressing
clone
Rest of
imaginal
disc
division to occur. Thus, cell division can occur only once both
signals are provided.
STUDYING GROWTH AND CELL SIZE IN
DROSOPHILA IMAGINAL DISCS
Drosophila imaginal discs are simple epithelial structures in
which growth is accompanied by cell division. They grow and
are patterned during larval stages, and then differentiate
during the pupal period, giving rise to most of the adult
epidermis (Cohen, 1993). Imaginal discs grow to highly
reproducible sizes, and classical experiments have shown that
their growth is influenced by factors that are extrinsic of and
intrinsic to the disc (Bryant and Simpson, 1984; Milan et al.,
1997).
Recent experiments have investigated the regulation of the
disc cell cycle at the molecular level, and the relationship
between the cell cycle and disc growth (Neufeld et al., 1998;
Weigmann et al., 1997; Duronio, 1999; Su and O’Farrell, 1998;
see Fig. 2 for a summary of key cell cycle regulators in
Drosophila). Overexpression of the cell cycle regulator dE2F
during imaginal disc development accelerates the cell cycle
without affecting net growth (see Fig. 3). This results in many
more cells of a smaller size (Neufeld et al., 1998). Similarly,
overexpression of dRBF, an inhibitor of dE2F, slows cell
division without affecting net growth. This results in fewer,
larger cells. These important results indicate that the cell
Fig. 4. The insulin/PI 3-kinase signalling pathway
Vertebrates
Flies
in vertebrates and flies. In vertebrates, insulin or
insulin-like growth factors (IGFs) bind to and
Insulin/IGFs
Nutrients
activate the intrinsic tyrosine kinase activity of
insulin and IGF receptors (Rs). Once activated, the
receptors phosphorylate insulin receptor substrate
Insulin/IGF-Rs
INR
proteins (IRSs), leading to the recruitment and
activation of Class IA PI 3-kinases (PI3Ks) and
subsequently, activation of the serine/threonine
CHICO
IRSs
kinases PDK1, p70S6kinase (p70S6K) and Akt
(IRS)
(also known as protein kinase B and RAC).
Vertebrate Akt/PKBs have many potential
p60/Dp110
Class IA PI3Ks
downstream targets (for a review see
Class
IA PI3K
Vanhaesebroeck and Alessi, 2000). An additional
DPTEN
PTEN
downstream target of this pathway is the regulation
of translation. Activated p70S6Ks phosphorylate
mTOR
PDK1
the ribosomal protein S6, leading to increased
translation of 5′ TOP mRNAs (Dufner and
Thomas, 1999); activation of eIF4E increases
Akt/PKBs
p70S6Ks
dAKT1
DS6K
4E-BP1
global translation initiation (Sonenberg and
Gingras, 1998). Both these processes are also
influenced by the nutrient-activated and
S6
eIF4E
rapamycin-sensitive protein kinase, mTOR. eIF4E
activity is regulated by direct phosphorylation and
is inhibited by the binding of a family of proteins,
the 4E-BPs. The best-studied 4E-BP, 4E-BP1, is
translation
translation
phosphorylated in response to insulin stimulation
of 5′ TOPs
initiation
in a PI-3-kinase-dependent and rapamycinsensitive manner (Pause et al., 1994; Fadden et al.,
1997; Sonenberg and Gingras, 1998). It is unclear how insulin influences 4E-BP1 phosphorylation, although the phosphorylation (and possible
activation) of mTOR by Akt provides a potential mechanism (Nave et al., 1999; Burnett et al., 1998). Flies possess one insulin/IGF receptor
(INR), one IRS (CHICO), one class IA PI3K (Dp110/p60), one AKT (dAKT1) and one p70S6K (DS6K). The downstream targets of dAKT1
and DS6K have not yet been characterised. The pathway is antagonised by the lipid phosphatases PTEN, in vertebrates, and DPTEN, in flies.
2930 C. M. Coelho and S. J. Leevers
division machinery can be activated independently of the
growth machinery. At face value, these experiments also
appear to rule out model four: division-triggered cell growth
(Fig. 1). However, model four invokes links between cell
growth and division that would have been disrupted in these
experiments. According to model four, the reduction in cell
size that results from division should trigger cell growth, but
E2F overexpression causes premature division, and so may not
allow time for adequate cell growth to occur.
MODULATION OF IMAGINAL DISC GROWTH AND
CELL SIZE BY THE INSULIN/PI 3-KINASE
SIGNALLING PATHWAY
Recently a flurry of papers has revealed that the mutation of
molecules on the insulin/PI 3-kinase signalling pathway in
Drosophila alters cell size and organism size (Leevers, 1999;
Edgar, 1999; Lehner, 1999). Many components of this pathway
are conserved in flies and mammals (Fig. 4). In addition, some
of the functions ascribed to this pathway in mammals are
consistent with its ability to regulate growth in Drosophila. For
instance, insulin/PI 3-kinase signalling has been implicated in
the regulation of protein synthesis, metabolism, cell division,
cell survival, and pre- and post-natal growth (Coffer et al.,
1998; Shepherd et al., 1998; Efstratiadis, 1998). Data
suggesting that this pathway can modulate the size of
mammalian cells are also beginning to emerge (Deleu et al.,
1999; Shioi et al., 2000).
What exactly are the effects of interfering with the activity
of this pathway in flies? Null mutations in the genes encoding
the Drosophila insulin receptor substrate (IRS), CHICO, and
the Drosophila p70S6kinase, DS6K, slow imaginal disc growth
and development, ultimately resulting in viable adults that are
small and are made up of small cells (Bohni et al., 1999;
Montagne et al., 1999). Null mutations in other identified
components of the pathway are lethal. However, weak loss-offunction mutations in the Drosophila insulin/IGF receptor
gene, Inr (Chen et al., 1996), and in the Drosophila homologue
of Akt, dAkt1 (E. Hafen and H. Stocker, personal
communication), also produce small flies that have small cells.
Similar effects on organ size and cell size are induced by the
expression of transgenes that modulate the activity of dAKT1
or the Drosophila Class IA PI 3-kinase, Dp110, during eye and
wing development (Verdu et al., 1999; Weinkove et al., 1999;
Leevers et al., 1996). More recently, it has been shown that this
pathway is antagonised by DPTEN, the Drosophila homologue
of the human tumour suppressor PTEN (Huang et al., 1999;
Goberdhan et al., 1999; Gao et al., 2000). PTEN is a lipid
phosphatase that removes the phosphate added to
phosphoinositides by PI 3-kinases (Di Cristofano and Pandolfi,
2000). Consistent with this is the finding that mutation of
DPTEN increases cell size whereas overexpression of PTEN
produces small flies that contain small cells (Goberdhan et al.,
1999; Gao et al., 2000). Hence, here is a signalling pathway
that, at least at the gross phenotypic level, affects net growth
and alters cell size.
During tissue development, growth can occur in dividing cell
populations and in differentiating cells that have exited the cell
cycle. Therefore, one question immediately raised by the above
adult fly phenotypes is whether the size of cells in dividing
imaginal disc cell populations is altered. Several groups have
addressed this question by using immunohistochemistry and
flow cytometry to investigate what is happening when imaginal
discs are growing and their cells are dividing. These techniques
allow comparison of cell size in mitotic clones of mutant cells
and their wild-type sister clones or ‘twin-spots’, and analysis
of cell size in clones of cells overexpressing different
transgenes (Neufeld et al., 1998). Such experiments have
definitively shown that modulating signalling through the
insulin/PI 3-kinase pathway does alter the size of dividing
cells. For example, removal of CHICO or DS6K, inhibition of
the activity of dAKT1 or Dp110, or overexpression of DPTEN,
reduces dividing cell size. In contrast, removal of DPTEN or
overexpression of Dp110 or dAKT1 increases cell size (Verdu
et al., 1999; Weinkove et al., 1999; Montagne et al., 1999;
Bohni et al., 1999; Gao et al., 2000).
DOES THE INSULIN/PI 3-KINASE SIGNALLING
PATHWAY ALTER CELL NUMBER?
Another important issue is whether the insulin/PI 3-kinase
signalling pathway alters cell number and hence influences the
amount of cell division that occurs during imaginal disc
development. Wings from flies that completely lack chico
possess fewer cells than do wild-type wings (Bohni et al.,
1999). Similarly, overexpression of Dp110 in a large area and
during an extended period of wing development* can result in
a mild increase in cell number (Leevers et al., 1996). From
these observations, it is unclear whether this pathway directly
alters the activity of cell cycle regulators to influence cell
division. The effect on cell number could be indirect and
merely reflect the fact that, ultimately, inadequate biosynthesis
will not allow the normal number of cell divisions to occur
whereas increased biosynthesis might indirectly allow extra
cell divisions.
Other experiments have shown that clones of imaginal disc
cells overexpressing Dp110 or dAKT1 are larger in area than
but contain the same number of cells as control clones
(Weinkove et al., 1999; Verdu et al., 1999)‡. This observation
indicates that increasing signalling via the insulin/PI 3-kinase
pathway can promote growth without having a proportional
impact on cell division. Edgar and co-workers have obtained
similar results by overexpressing dMYC (Johnston et al.,
1999) or activated dRAS1 (Prober and Edgar, 2000),
although whether these molecules promote growth by
interacting with the insulin/PI 3-kinase signalling pathway
has not yet been addressed. In addition, flies without DS6K
have the same number of wing cells as do wild-type flies,
which suggests that DS6K modulates imaginal disc growth
and cell size without directly influencing the number of cell
divisions that occur during development (Montagne et al.,
1999).
The above observations appear in keeping with models one
and two in Fig. 1, in which a change in the ratio of signals
carried to the growth machinery and cell division machinery
*The MS10-96 GAL4 enhancer trap was used to drive expression in this experiment. This
driver is thought to be active for extended periods during larval and pupal life (Capdevila
and Guerrero, 1994)
‡Dp110 clones were observed 43 hours after induction and dAkt-1 clones 48 hours after
induction.
Cell size: a consequence of growth and division? 2931
alters cell size. The insulin/PI 3-kinase signalling pathway
would carry the signal to the growth machinery. However,
these models do not explain the subtle effects of this pathway
on cell number. Perhaps a minor component of the signal is
diverted towards the cell division machinery. Or, can the
growth signal synergise with other signals that drive cell
division?
Data from other experimental systems suggest that PI 3kinase can increase the activity of cell cycle regulators, but that
PI 3-kinase activation alone is not sufficient to promote
division (Klippel et al., 1998; Brennan et al., 1997, 1999). For
example, in rat 3Y1 cells, expression of activated PI 3-kinase
under low-serum conditions activated E2F, but the cells
arrested in mid-S-phase and died by apoptosis. These effects
were rescued by higher concentrations of serum, which lead to
uncontrolled proliferation and growth*, presumably because PI
3-kinase-induced E2F activation synergises with other
signalling pathways to promote 3Y1 cell division (Klippel et
al., 1998). Similar factors might come into play when Dp110
is overexpressed in Drosophila imaginal discs. This artificial
situation will not precisely mimic the context in which Dp110
is usually activated. During normal development, the insulin/PI
3-kinase signalling pathway is likely to synergise with other
signalling pathways, which are activated in parallel, to
influence cell division. A major regulator of the G2-M
transition in Drosophila imaginal discs is the homologue of the
cdc25 phosphatase, String. String is regulated largely at the
transcriptional level (Milan et al., 1996), and its overexpression
is sufficient to induce imaginal disc cell division (see Fig. 2).
The string gene has a large and complex promoter that might
provide a convergence point for synergism, given that its
transcription is influenced by several signalling pathways and
by dE2F (see Fig. 2; Neufeld et al., 1998; Johnston and Edgar,
1998; Lehman et al., 1999).
DOES THE INSULIN/PI 3-KINASE SIGNALLING
PATHWAY AFFECT THE RATE OF CELL CYCLE
PROGRESSION?
In contrast to the inability of the insulin/PI 3-kinase signalling
pathway to influence cell division directly, several observations
demonstrate that insulin/PI 3-kinase signalling affects the rate
at which cells progress through the cell cycle in Drosophila.
DS6K and chico mutant larvae are developmentally delayed
and grow more slowly, and yet the resulting flies have normal
or reduced numbers of cells (Bohni et al., 1999; Montagne et
al., 1999). This indicates that the cell cycle is lengthened in
these organisms‡. In addition, the analysis of cell number in
clones, a short period after their induction§, indicates that cells
with reduced DS6K or Dp110 activity divide more slowly
(Weinkove et al., 1999; Montagne et al., 1999)¶. Note that, for
*In these experiments, increase in biomass was not directly measured, but cell density on
tissue culture dishes increased (see Klippel et al., 1998).
‡Another interpretation of these observations is that in the mutant discs cell division is
increased, but is accompanied by increased cell death. However, in the case of chico, no
increase in cell death was observed (Bohni et al., 1999).
§Dp110 clones were observed 43 hours after induction and dAkt-1 clones 48 hours after
induction.
¶Cell death might also be increased in these experiments. However, when cell death was
blocked by co-expression of the baculovirus caspase inhibitor, p35, inhibition of Dp110
still slowed cell division (S. J. Leevers, unpublished observations).
technical reasons, some caution should be applied when one is
interpreting the results of these experiments*.
The analysis of cell cycle profiles by flow cytometry has
suggested that altering signalling via the insulin/PI 3-kinase
signalling pathway disproportionately influences progression
through different phases of the cell cycle. Overexpression of
Dp110 or loss of DPTEN reduces the proportion of cells in G1
and increases the proportion of cells in S phase and at G2-M,
without affecting cell doubling time (Weinkove et al., 1999;
Gao et al., 2000). These observations suggest that increased
signalling via Dp110 is sufficient to hasten entry into S phase.
Although similar results have been obtained by modulating
dRAS1 and dMYC activity (Prober and Edgar, 2000; Johnston
et al., 1999), effects on cell cycle phasing have not been
observed when the activity of other insulin pathway
components is altered. The slowing down of the cell cycle in
chico and DS6K mutant cells is accompanied by a proportional
extension of all phases of the cell cycle (Bohni et al., 1999;
Montagne et al., 1999). Thus, the observation that Dp110
overexpression or loss of PTEN can hasten S phase entry might
reflect the fact that these interventions somehow have a stronger
impact on the signalling pathway than do the other experimental
interventions examined. Alternatively, the pathway might
branch and be less linear than is depicted in Fig. 4. Note that
the effect on growth and the cell cycle of INR, which is
upstream of Dp110, has not yet been characterised in any detail.
These results bear comparison with observations on growing
yeast cultures. Budding yeast pass rapidly through G1 when
nutrients are abundant, and growth is rapid, but pass slowly
through G1 when nutrient conditions are poor (Jagadish and
Carter, 1977; Johnston et al., 1977). These observations support
the idea that entry into S phase is regulated by growth, and that
high rates of growth hasten S phase entry. Thus, it would be
interesting to investigate whether the same is true in flies and
whether the ability of Dp110 and DPTEN to influence G1-S is
dependent on their ability to modulate growth. Furthermore, this
pathway may be regulated in flies, as it is in other organisms, by
nutrition (Edgar, 1999; Bohni et al., 1999, Weinkove et al., 1999).
Since expression of Dp110, dRAS1 or dMYC can promote
growth and S phase entry but not division, their ability to
increase cell size might diminish if entry into M phase could
be hastened. Consistent with this hypothesis is the observation
that when dRAS1 or dMYC were coexpressed with String (to
accelerate progression into M phase), net growth together with
cell number increased, and cell size returned to more normal
levels (Prober and Edgar, 2000; Johnston et al., 1999).
The data discussed so far demonstrate that the insulin/PI
3-kinase pathway promotes growth without proportionally
stimulating cell division. These observations are consistent
with cell size being determined through scenarios one and two
in Fig. 1. However, modulating the activity of this pathway can
*Extensive studies have shown that slow-growing mutant disc cells face competition when
they are surrounded by fast-growing wild-type cells. This means that they contribute to
less of the final organ than when both the clones and the surrounding cell s are mutant
and slow growing. Thus the lengthened cell cycle time observed when Dp110 was
inhibited might be exaggerated by cell competition. Indeed when chico−/− clones were
made in discs in which the non-clonal cells were also under a growth disadvantage because
of decreased function of a ribosomal (Minute) gene, the number of cells in chico−/− clones
increased (Bohni et al., 1999). In the DS6K experiments, cell cycle times was measured
in entirely mutant animals; hence cell competition is unlikely to have influenced the result.
However, DS6k causes developmental delay, making it difficult to stage and compare
control and mutant animals in these experiments.
2932 C. M. Coelho and S. J. Leevers
affect cell cycle progression (i.e. by slowing cell division or
altering cell cycle phasing). These effects on cell cycle
progression may be best-explained by the ability of this
pathway to promote growth and are more in keeping with
scenario three (Fig. 1). The linking of growth with cell cycle
progression might at least ensure that cells cannot progress
through the cell cycle without reaching a minimum size. In the
following sections, we discuss data from other experimental
systems that demonstrate how the impact of the insulin/PI 3kinase pathway on translation might increase growth in the
imaginal discs and coordinate cell growth with passage through
the different phases of the cell cycle.
HOW MIGHT THE INSULIN/PI 3-KINASE
SIGNALLING PATHWAY AFFECT GROWTH?
In order for a tissue to grow, increased biosynthesis must occur,
for which increased protein synthesis is likely to be critical. The
observation that dividing cells have an increased translational
capacity whereas quiescent cells have a reduced translational
capacity is consistent with this requirement for increased protein
synthesis (Terada et al., 1995; Agrawal and Bowman, 1987).
How are these alterations in translational capacity in response to
growth factor stimulation achieved? Translation initiation is
thought to be rate limiting in protein synthesis and is regulated
in multiple ways (Hentze, 1995), some of which are influenced
by insulin signalling (Campbell et al., 1999).
One way in which insulin signalling can increase translation
initiation is by activating the eukaryotic translation initiation
factor eIF4E (Dufner and Thomas, 1999). eIF4E binds to the
5′ cap structure of all eukaryotic mRNAs and is a subunit of
the eukaryotic translation initiation factor eIF4F. eIF4F activity
brings the 5′ end of mRNAs to ribosomes (reviewed by
Sonenberg and Gingras, 1998). In addition, eIF4F possesses
RNA helicase activity that can help dissolve mRNA secondary
structures, thereby enabling translation. The regulation of
eIF4E activity is complex (see Fig. 4) and mediated both by its
association with a family of eIF4E-binding proteins (the 4EBPs) and by phosphorylation. One of the effects of insulin/PI
3-kinase signalling is to promote 4E-BP1 phosphorylation
(Pause et al., 1994). This results in the disassociation of 4EBP1 from eIF4E, allowing eIF4E to bind to the other sub units of
eIF4F (Fadden et al., 1997).
Insulin can also stimulate translation by activating
p70S6kinase (p70S6K). p70S6K phosphorylates the ribosomal
protein S6 and thereby increases the incorporation into
polysomes of a class of mRNAs containing a polypyrimidine
stretch in their 5′ untranslated regions (5′ TOPs, reviewed by
Dufner and Thomas, 1999). These mRNAs represent 20–30%
of total cellular mRNAs and mostly encode components of the
translational apparatus, including ribosomal proteins and
translation elongation factors (Jefferies et al., 1994). Insulin
treatment promotes the translation of these mRNAs by
inducing p70S6K phosphorylation and activation. Some of the
p70S6K-activating phosphorylation events are PI 3-kinase
dependent. In addition to their regulation by PI 3-kinase,
activation of both eIF4E and p70S6K is dependent on the
rapamycin-sensitive kinase mTOR (for mouse target of
rapamycin, also known as RAFT (for rapamycin and FKBP
target) and FRAP (for FKBP-rapamycin associated protein)).
In addition, insulin-stimulated PI 3-kinase activation can
activate the translation initiation factor eIF2 (Dufner and
Thomas, 1999). Insulin/PI 3-kinase signalling can thus increase
rates of translation initiation in vertebrates through several
mechanisms. Although this pathway is likely to have similar
effects on translation in Drosophila, this has not yet been
proven. The ability of insulin/PI 3-kinase signalling to promote
protein synthesis provides a mechanistic explanation for its
ability to increase growth and again is consistent with scenarios
one and two in Fig. 1. However, as discussed above, the activity
of this pathway also influences cell cycle progression. Is this
regulation of cell cycle progression also mediated by effects on
translation? Below we consider how increasing global
translation might provide the optimal cellular environment for
the synthesis of key cell cycle regulators and whether alterations
in the relative levels of translation of different classes of
transcripts might affect cell cycle progression.
REGULATION OF CELL CYCLE PROGRESSION BY
PROTEIN TRANSLATION
The G1 cyclins must accumulate to critical cellular levels in
order to promote cell cycle progression (Evans et al., 1983).
Cyclin levels are highly dynamic and are regulated through
modulation of their synthesis, stability and targeted
degradation (Minshull et al., 1989; Sonenberg, 1993), which is
consistent with their roles as cell cycle regulators. Excitingly,
experiments in Saccharomyces cerevisiae suggest that
regulated translation of the G1 cyclin, Cln3p, provides a
mechanism that couples growth to cell cycle progression
(Polymenis and Schmidt, 1999). The cln3 mRNA has a long
5′ untranslated region (5′ UTR) that contains many short open
reading frames (uORFs), which reduce access to the start
codon of the cln3 message (Polymenis and Schmidt, 1997).
Polymenis and Schmidt have proposed that increasing
translation initiation rates facilitates leaky scanning of these
uORFs and thereby allows initiation from the actual start
codon. This would mean that levels of Cln3p sufficient to
trigger the G1-S transition accumulate only when translation
initiation (and hence growth) rates are high. Consistent with
this hypothesis is the observation that overexpression of Cln3p
uncouples growth from S phase entry, triggering premature S
phase entry and reducing cell size. In contrast, reducing Cln3p
levels delays S phase entry and increases cell size (Polymenis
and Schmidt, 1999).
Might a similar mechanism for linking changes in cell size
with cell cycle progression exist in multicellular organisms?
The mRNA encoding the vertebrate G1 cyclin, cyclin D1, is
predicted to contain complex secondary structures likely to
render it a poor target for the translational machinery. Increasing
translation initiation by overexpressing eIF4E in NIH-3T3
increases the polysomal fraction of cyclin D1 mRNAs. In
contrast, the polysomal fraction of more simple mRNAs, such
as those encoding actin and cyclin A, is unchanged (Rousseau
et al., 1996). Furthermore, the overexpression of eIF4E in these
cells is sufficient to induce uncontrolled proliferation and
growth (Lazaris-Karatzas et al., 1990).
What about G1 cyclins in Drosophila imaginal discs? As is
the case for cln3, the mRNA encoding one of the Drosophila
G1 cyclins, cyclin E, contains uORFs (Richardson et al., 1993).
Cell size: a consequence of growth and division? 2933
When disc growth is increased by overexpression of dRAS1 or
dMYC, levels cyclin E protein, but not mRNA, increase
(Prober and Edgar, 2000). It is tempting to speculate that cyclin
E levels rose in these experiments as a result of increased
translation initiation, and that activation of the insulin/PI 3kinase pathway would have the same effect. However, at
present, there are no data to support such a hypothesis. In
addition, note that cells overexpressing Dp110 are bigger than
control cells both in G1 and in S phase (Weinkove et al., 1999).
This observation suggests that Dp110 boosts growth more than
it promotes S phase entry and that G1-S is only loosely coupled
to growth rates in Drosophila wing imaginal discs. However,
increasing Dp110 activity might also interfere with the
regulation of events in G1, such that the G1-S phase transition
does not respond adequately to the increased rate of growth.
As discussed above, insulin/PI 3-kinase signalling can alter
translation patterns specifically by increasing 5′ TOP translation.
Intriguing observations made in Xenopus laevis oocytes have
shown that inhibiting p70S6K activity not only reduces the
translation of 5′ TOPs but also induces the premature translation
of non-5′-TOP mRNAs, presumably because ribosomes are
freed from 5′ TOP translation (Schwab et al., 1999). In these
experiments, inhibiting p70S6K induced premature cdc25 and
mos translation, leading to premature oocyte maturation. A
similar mechanism might operate in the Drosophila imaginal
discs (Thomas, 2000). In this case, inhibiting p70S6K (and other
components of the insulin/PI 3-kinase pathway) would result in
premature translation of cell-cycle-promoting molecule(s) at the
same time as reducing growth; thus cells would divide at a
reduced size. However, it remains to be seen whether reducing
p70S6K activity enhances the translation of non-5′-TOP mRNAs
in other developmental systems.
CONCLUSIONS
In the first part of this review, we presented data that support
models one and two in Fig. 1, in which growth and cell division
are independently regulated and the balance between the two
signals in a developing tissue ensures appropriate cell size. In
the second part we have speculated on mechanisms that could
enable the insulin/PI 3-kinase signalling pathway to couple
changes in cell size with cell cycle progression as depicted in
model three. At present, there is no conclusive evidence in
favour of any of these models for determining cell size, and it
is difficult to design appropriate experiments that would
distinguish between them. However, the identification of
critical components of the cellular machinery downstream of
this pathway and characterisation of the effects of this pathway
on protein translation would be good places to start. We also
need to explore other avenues and find out more about how this
pathway stimulates growth – for example, through effects on
proteolysis, energy metabolism and lipid biosynthesis.
Can models one, two and three co-exist? In principle,
models one and two would allow the achievement of
reproducible cell sizes in a large variety of tissues and at
different developmental stages. An important pre-requisite for
these models is that the signals that drive growth and division
are limiting and, hence, critically regulated. There is good
evidence that extracellular signals are limiting in vivo, although
how this is achieved is rather unclear (Barres et al., 1996;
Conlon and Raff, 1999). Model three provides the opportunity
for cross-talk between cell growth and division, which could
ensure that cells do not divide until they are a minimum size.
The activity of p70S6K and eIF4F might define, at least in part,
the translational capacity of a cell and link cell growth to the
synthesis of specific cell cycle regulators. However, it is likely
that the balance between signals that drive growth and cell
division determines maximum cell size (as in models one and
two) and that cell division is regulated in a developmentalcontext-dependent manner. For example, in the imaginal discs,
transcription of the G2-M regulator string might be ratelimiting. Thus, once cells have achieved an adequate
translational capacity, the induction of string transcription in
response to other signals might trigger cell division and hence
determine maximum cell size. Other aspects of the regulation
of growth and cell division that are likely to receive attention
in the future are the roles played by synergy between different
pathways, and positive and negative feedback. These
mechanisms could influence the production of extracellular
signals and hence play a critical role in the determination of
cell size.
We thank Saif Alrubaie, Kathy Barrett, Lindsay MacDougall, Klaus
Okkenhaug, George Thomas, David Weinkove and Bart
Vanhaesebroeck for advice on the preparation of this article. In
addition, we thank George Thomas for sharing a model for the
regulation of cell size with us prior to publication.
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