Current Biology, Vol. 14, R1014–R1027 December 14, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.11.027
How Cells Coordinate Growth and
Division
Paul Jorgensen1,2 and Mike Tyers1,2
Size is a fundamental attribute impacting cellular
design, fitness, and function. Size homeostasis
requires a doubling of cell mass with each division.
In yeast, division is delayed until a critical size has
been achieved. In metazoans, cell cycles can be
actively coupled to growth, but in certain cell types
extracellular signals may independently induce
growth and division. Despite a long history of study,
the fascinating mechanisms that control cell size
have resisted molecular genetic insight. Recently,
genetic screens in Drosophila and functional
genomics approaches in yeast have macheted into
the thicket of cell size control.
Introduction
“‘The first thing I’ve got to do,’ said Alice to herself, as
she wandered about in the wood, ‘is to grow to my
right size again...’” — Lewis Carroll, Alice’s Adventures
in Wonderland (1865).
The size of cells in nature varies over an enormous
range, with protists like Amoeba proteus being many
millions of times larger than the smallest Mycoplasma
[1]. A cell’s size is a fundamental attribute that contributes to function in the context of multicellular
organisms and to fitness in the context of unicellular
organisms. Size imposes constraints on cellular
design. For instance, as cells grow larger, passive diffusion may limit intracellular transport and the
decreased surface area to volume ratio may make
nutrient uptake limiting for cell growth. The first
insights into cell size control were made around 100
years ago, when Boveri, Hertwig, and their colleagues
[2] observed a fundamental correlation between ploidy
and cell volume. There is a revitalized interest in this
classic problem in cell biology, and, while we certainly
aren’t ‘out of the woods’, recent years have been
witness to key genetic advances (Figure 1). Various
aspects of the cell size problem have been recently
covered in several excellent reviews and books [3–9].
Here, we focus on how size homeostasis is
achieved in proliferating cells, that is, the coordination
of cell growth and division. It is clear that this coordination is an active process in a great variety of unicellular organisms ranging from bacteria to protists.
Indeed, the highly diverged budding and fission yeasts
each possess specific regulatory networks dedicated
to converting the accrual of sufficient biomass into a
stimulus for cell cycle progression. Just how cells
1Department
of Medical Genetics and Microbiology,
University of Toronto, Toronto ON, Canada M5S 1A8; 2Samuel
Lunenfeld Research Institute, Toronto, Ontario, Canada M5G
1X5. E-mail: [email protected]; [email protected]
Review
convert steady increases in size into a switch-like
decision to enter the cell cycle is a fascinating question in biological engineering. While it is clear that cell
growth and division must be correlated in metazoans,
it remains a contentious issue whether such cells
actively and cell-autonomously couple growth and
division. Size control has inspired much research over
the past century, yet, relative to other aspects of cell
biology, this problem has been recalcitrant to conventional genetics and biochemistry.
In thinking about cell size control, it is important to
distinguish cell growth, the cell cycle, and the mechanisms that coordinate the two. As it is still not certain
whether cells measure volume, mass, and/or biosynthetic capacity, we use the general term ‘size’ as a
catch-all descriptor. Biosynthetic activity obviously
drives increases in cell size, and, in the absence of
division, it is reasonable to view cell size as the sum of
past cell growth. Cell size can also be altered by
osmotic pressure or by autophagy, but for the sake of
brevity we ignore these effects here.
Trends in Cell Size Homeostasis
“The constant, which we must accept as something
given and not at present further analyzable, is the fixed
proportion between nuclear volume and protoplasmic
volume, namely, the karyoplasmic ratio.” — Theodor
Boveri, 1905 [2].
Despite constantly growing and dividing, there is
usually a limited and stereotypical size variation for
any given cell over successive generations. Correspondingly, proliferating cell populations show characteristic size distributions. When the normal size
distribution is disrupted by some insult, it is usually
restored quickly following removal of the insult,
further suggesting that cell size is under homeostatic control [10,11]. At its most basic level, cell size
homeostasis in proliferating cells requires a coordination of growth with division, such that on average
each cell division is accompanied by a doubling in
cell mass. In post-mitotic cells, such as neurons, the
maintenance of cell size requires that no net cell
growth occurs.
Although exceptions are common, a few general
trends concerning cell size control can be inferred.
The most established, but least well understood, trend
is that cell volume increases with ploidy. This correlation has been observed in a wide variety of eukaryotic
cells from yeast to mice [2,12–16]. On an organismal
level, developmental processes can be incredibly
robust when challenged with altered cell volume
(Figure 1A) [16–18]. Increased ploidy could exert its
effects by increasing nuclear volume, chromatin
content, or the expression of unknown genes, any one
of which might provide a metric against which cytoplasmic volume is somehow measured. Ploidy
increases are apparently required to prevent genomic
DNA from becoming limiting for cell growth [19]. In
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somatic cell lineages, organisms exploit the large cell
size conferred and permitted by increased ploidy to
create specialized cell types and to pattern tissues,
such as the larval salivary gland in Drosophila, and
muscle fibers, megakaryocytes, and giant trophoblast
cells in mammals [19]. Although variations in the body
size of animals are generally accounted for by
changes in cell number, cell size can affect body size
and its evolution [2,3,20,21].
The nucleo-cytoplasmic ratio plays a critical role in
metazoan embryonic development [22,23]. In many
animals, fertilization is followed by a series of rapid
and synchronous cleavage divisions that section the
huge zygote into thousands of smaller cells. After a
certain number of divisions that is characteristic for
each organism, cell cycle times lengthen and become
asynchronous. This mid-blastula transition occurs
when cells reach a particular nucleo-cytoplasmic ratio.
Thus, haploid embryos compensate for their
decreased nucleo-cytoplasmic ratio by going through
exactly one extra cleavage division [22]. Similarly, artificially increasing the nucleo-cytoplasmic ratio results
in fewer cleavage divisions [22,23].
Cell growth and the cell cycle are coordinated but
separable processes [10,24]. In principle, this coordination can be attributed to one or more of four simple
mechanisms: the dependency of cell cycle progression on growth, the dependency of growth on cell
cycle progression, the coordinate control of growth
and cell cycle progression, or the complete intertwining of growth and cell cycle progression [25]
(Figure 2A). Of these possible mechanisms, the
dependency of cell cycle progression on growth
appears to be utilized by many types of proliferating
cells. Blocking cell growth in eukaryotes by nutrient or
growth factor deprivation results in a cell cycle arrest,
usually in G1 phase [26–28]. Indeed, cell growth is typically limiting such that nutrient deprivation or treatment with translation inhibitors leads to a lengthening
of the cell cycle in G1 phase [28]. Similarly, abundant
nutrients or overactivation of growth regulation pathways, for instance the PI(3)K pathway, can impel cell
cycle progression, typically abbreviating the length of
the G1 phase [4,5,29].
Conversely, cell growth does not typically rely on
cell cycle progression. In a great variety of cell types,
when cell cycle events are blocked with chemicals or
genetic lesions, growth continues unchecked
[18,24,30–36]. Correspondingly, increasing the rate of
cell cycle progression does not accelerate mass
accumulation, thus resulting in abnormally small cell
size [18,35,37–41].
Although a strict dependency of division on growth
(Figure 2A) often appears to coordinate these two
processes, growth and the cell cycle can be regulated independently by distinct extracellular signals,
for instance during embryonic patterning. Blocks to
cell cycle progression do not typically prevent
growth [10,24], but direct interactions between cell
cycle regulators and growth regulators suggest that
the two processes may be controlled by partially
overlapping networks in metazoans (Figure 2A).
These possibilities are discussed further below.
A
B
C
D
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Figure 1. Genetic alterations that dramatically affect cell size.
(A) In a series of polyploid salamander larvae, Fankhauser [17]
observed that while cell size increases proportionally with
ploidy, the overall size of the animal and its organs did not
change. These cross-sections of pronephric tubules demonstrate the remarkable robustness of the developmental process
in response to cell size changes. (Reproduced with permission
from [17].) (B) Budding yeast lacking the SFP1 gene are very
small (left panel) while overexpression of SFP1 leads to huge
cells (right panel). (C) Loss of dAkt or dS6K in Drosophila
reduces cell size. A clone of dAkt−/− rhabdomeres (white box)
exhibits very small cell size relative to twin-spot cells and dAkt/+ heterozygote cells.(Reproduced with permission from
[195].)Flies lacking dS6K have cells that are ∼30% smaller than
wild-type, resulting in small body size (right, wild-type is left).
(Reproduced with permission from [177].) (D) Ectopic overexpression of the c-Myc gene in murine hepatocytes leads to
hypertrophy (right panel, wild-type is left panel), as well as
engorged nuclei and nucleoli. (Reproduced with permission
from [160].)
Measuring up Size Thresholds
The dependency of the cell cycle on growth is thought
to be established by size requirements for major cell
cycle transitions. In eukaryotes, such critical cell size
thresholds are imposed at the G1/S phase transition
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Proliferation
signal(s)
A
(1)
(4)
(2)
Growth/
cell cycle
(3)
B
Growth
Cell cycle
Cell cycle
Growth
Symmetric
cytokinesis
Growth
Cell cycle
Critical cell
size threshold
G1 length
S-phase
Asymmetric
cytokinesis
G2/mitosis (constant length)
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Figure 2. Coordination of cell growth and division.
(A) Mechanisms that could in principle couple cell growth and
the cell cycle (adapted from [25]). See text for details. (B) A
single critical cell size requirement suffices to couple growth
and division. Whether cytokinesis is symmetric or asymmetric
with respect to cell mass, daughter cells must grow past a cell
size threshold in order to enter S phase. The growth requirement results in variable G1 lengths while the S/G2/M phase is
constant.
and/or the G2/M phase transition. They thereby
govern the time spent by cells in G1 phase and/or G2
phase. In a classic experiment, Hartmann [42,43] prevented a growing amoeba in G2 phase from entering
mitosis indefinitely by periodically resecting a portion
of its cytoplasm, thereby preventing the attainment of
a presumed critical cell size. Following the pioneering
studies of Killander and Zetterberg on mouse fibroblasts [44,45], strong evidence for critical cell size
thresholds has been found in numerous organisms
and cell types, including: bacteria [46], the acellular
slime mold Physarum polycephalum [47], the protists
Stentor and Tetrahymena [48,49], fission yeast [37],
budding yeast [31], Xenopus blastomeres [50], unicellular algae [51], and avian erythroblasts [52].
Conceptually, critical cell size thresholds are a
simple and effective way of coordinating cell growth
and division (Figure 2B). By definition, size thresholds
enforce a minimal cell size. It has been observed in
many cell types that smaller cells delay cell cycle progression until they have grown to a certain size
[11,31,50,52–57]. Conversely, size thresholds correct
for overgrowth in the previous cell cycle by speeding
up division relative to growth in the next cycle. Oversized daughter cells arise frequently in proliferating
populations due to cell cycle delays (e.g., upon checkpoint activation following DNA damage) and unequal
segregations of mass during cytokinesis. As all cell
cycles have minimal time requirements, depending on
the extent of the overgrowth, it may take several
minimal cycles before convergence to wild-type size
[11]. Acceleration of cell division following cell overgrowth has been observed in many organisms and
cell types [30,49,52,53,58–62].
Critical cell size thresholds entail a ‘sizing’ mechanism. As noted above, cell size is a general term and
it is not clear whether cells measure their volume,
mass, and/or biosynthetic status. The latter parameter, more specifically the rate of protein synthesis, is
generally proposed to be what cells actually assess.
The rate of protein synthesis might be a universal
proxy that reflects upstream events, including nutrient
status and growth factor signaling, thereby integrating
many disparate signals into the size threshold [63].
Protein synthesis rate is thought to be relayed to the
cell cycle by unstable ‘translational sizers’, whose
abundance reports translation rates and whose activity is rate-limiting for cell cycle transitions (see Box 1).
In E. coli, the activated form of the replication initation
factor DnaA appears to be such a translational sizer
[8]. In eukaryotic cells, cyclins, which combine with
cyclin-dependent kinases (CDKs) to drive cell cycle
transitions, and other CDK activators, like the Cdc25
phosphatase, have been proposed to be translational
sizers [4].
A Good Place to Start: Budding Yeast
The importance of cell size control is particularly
obvious in the budding yeast Saccharomyces cerevisiae. Cytokinesis is asymmetric with respect to cell
mass, producing a large mother cell and a smaller
daughter cell. As defined by Hartwell [64] and colleagues, coordination between cell growth and the cell
cycle occurs at Start, a short interval in late G1 phase
during which the yeast commits to division. Passing
Start requires that cells first obtain a critical cell size,
such that large mother cells traverse a minimal G1
phase while small daughter cells spend a long time in
G1 phase growing to the threshold size [31,65].
Although ipso facto larger than the critical cell size,
mother cells do arrest prior to Start in response to
nutrient starvation, mating pheromones, or translation
deficiencies [63,65]. Following Start, the cell cycle progresses until the subsequent G1 phase even if cells
are subjected to nutrient starvation, mating
pheromones (if haploid), and signals that initiate
meiosis (if diploid) [64]. Therefore, in addition to maintaining average size over the generations, the size
requirement at Start ensures that the yeast possesses
enough resources to complete the crucial processes
of genome duplication and segregation.
Start is a series of events that culminate in S phase
entry (Figure 3). The earliest known Start event is the
onset of transcriptional activation by the SBF
(Swi4–Swi6) and MBF (Mbp1–Swi6) complexes,
which drive expression of ~200 genes [66]. This
regulon contains numerous genes involved in DNA
synthesis and repair, but the key transcripts are the
G1 cyclins CLN1 and CLN2 and the B-type cyclins
CLB5 and CLB6 [67]. Cln1 and Cln2 bind to and activate Cdc28, the primary CDK that controls cell cycle
progression in budding yeast. Cln1/2–Cdc28 complexes trigger bud emergence and inactivate Sic1
and Cdh1, two key inhibitors of Clb–Cdc28 activity
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[67]. Once derepressed, Clb–Cdc28 complexes
immediately initiate DNA replication [67].
The onset of SBF and MBF mediated transcription
requires that cells attain a critical cell size. What
signals to SBF and MBF that the size requirement has
been met? The answer to this question remains
cloudy, but many of the genes involved have been
identified. A third G1 cyclin, Cln3, is a highly unstable,
rate-limiting activator of Start [38,68–70]. The WHI1-1
mutation was discovered in a screen for mutants with
small cell size [38], known as the whiskey (Whi) phenotype (see [71] for a rye explanation). WHI1-1 and a
similar whi mutation DAF1-1 were subsequently shown
to encode stabilized forms of Cln3 [68,69]. CLN3
appears to function in parallel to the BCK2 gene and
strains with deletions of both genes are inviable due to
permanent G1 arrest [72]. In contrast, CLN1 and CLN2
have no effect on the timing of the SBF and MBF transcriptional program [73,74]. Although CLN1 and CLN2
do not control SBF and MBF, they do control how long
the Start interval lasts, because critical concentrations
of Cln-Cdc28 activity are required for the phosphorylation of Cdh1, Sic1, and targets at the incipient bud
site, such as Cdc24 [73–77] (Figure 3A).
An obvious impediment to understanding Start has
been the dearth of key regulators. However, recent
application of reverse genetics in budding yeast has
yielded many new genes that influence Start [78–80].
Additional players have also emerged as interaction
partners of known Start factors [81,82]. One new regulator, Whi5, is the key G1 target of Cln3–Cdc28 and
is thus a linchpin in the Start hierarchy [78,82,83]. In
early G1 phase cells, Whi5 binds to and represses
promoter-bound SBF and MBF [82,83]. Multi-site
phosphorylation of Whi5 by Cln3–Cdc28 dissociates
Whi5 from SBF/MBF, drives Whi5 into the cytoplasm,
and induces the expression of Start specific transcripts [82,83]. In a sense, Whi5 is the long anticipated budding yeast analog of the retinoblastoma
(Rb) protein, which inhibits the E2F family of G1/S
transcription factors in metazoans [41,84]. While
Whi5 is clearly the critical target, Cln3–Cdc28 is likely
to phosphorylate additional substrates at SBF/MBF
promoters, including Swi6 [78,83,85]. The multi-site
phosphorylation of Whi5, and possibly Swi6, may
make SBF/MBF activation ultrasensitive to
Cln3–Cdc28 activity, thereby rendering Start entry
‘switch-like’ [76,86]. In contrast, Bck2 activates
SBF/MBF by an unknown mechanism that is independent of Cdc28 or Whi5 [72,78].
But what is the actual cell sizing mechanism?
Though not conclusive, there is evidence that budding
yeast assess their size by measuring the overall translation rate. This model unifies the volume, nutrient and
translation requirements for Start. In support of this
model, budding yeast growing in a stable nutrient
environment have an overall translation rate that correlates with cell volume [87]. Nutrient-sensing pathways control not only the rate at which ribosomes are
produced and the cytoplasmic ribosome concentration, but also the rate at which ribosomes function
[88–91]. Nutrient downshifts, therefore, can decrease
the translation rate per ribosome. It is probable that
Box 1
Translating growth into division.
It has been proposed for many years that translation rate
communicates cell size to the cell cycle [28,63]. The length of the
G1 phase in eukaryotic cells is particularly sensitive to sublethal
doses of cycloheximide or other factors that cripple translation
rate [28,63,65,142,144,197,198]. However, decreasing protein
synthesis could simply delay a necessary size accumulation. In
budding yeast, stronger evidence exists for translation rate as the
size proxy: sublethal doses of cycloheximide force the cell to
accumulate a bigger size (and more ribosomes) before passing
Start [92,93,95]. Similar, preliminary findings have been made in
fission yeast and plant meristematic cells [198,199].
There is an important distinction to be made between the overall
translation rate and the translation rate per ribosome or ribosome
activity. It is overall translation rates that correlate with cell size,
not ribosome activity. Growth factors stimulate ribosome activity
in mammalian cells, for instance by inactivating the 4E-BPs [5,6]. If
overall translation rates are the size proxy, increasing ribosome
activity could suffice to drive cell cycle transitions in the absence
of volume increases [28]. It is often suggested that Cln3 and
Cdc25 are intrinsically good sizers because their translation is
particularly sensitive to inhibition of translation initation [94,200].
While this potential non-linearity between ribosome activity and
Cln3 or Cdc25 translation is intriguing, it is not evidence that Cln3
and Cdc25 are necessarily good reporters of cell size as ribosome
activity is not a function of size [87].
But, overall translation rates may not always correlate well with
cell size. For instance, to what extent is the activity of translational
sizers influenced by the stochastic nature of protein expression
[201], and the resulting cell-to-cell variations in protein
concentrations? As demonstrated in the extreme by the giant
Beggiotoa bacterium [202], vacuoles and other organelles with no
protein synthetic activity can be dominant cell components.
Despite occupying up to a quarter of a budding yeast's volume, it
is not known what effects the vacuole has on cell size and Start.
Surprisingly, Cln3 may alter vacuolar size and biogenesis in
budding yeast [203]. The possibility that overall translation rates
are not how all cells measure size must be kept in mind. For
instance, experimentally lowering the nucleo-cytoplasmic ratio in
Stentor coeruleus or Physarum polycephalum suffices to induce
cycling in starved cells [48,204].
even cells larger than the critical cell volume (e.g.,
mother cells) do not pass Start upon starvation
because a critical translation rate cannot be attained
[63]. Indeed, passage through Start does require a
critical rate of translation, even in those cells that have
grown larger than the critical cell volume
[63,65,92–94]. As predicted, growing cells in sub-lethal
doses of cycloheximide increases the critical cell
volume [92,93,95].
As passage through Start is highly sensitive to Cln3
dosage, a critical translation rate of Cln3 might trigger
Start [38,68–70,96]. But the relative amount Cln3
protein does not appear to increase as cells approach
Start [70]. Instead, the relative abundance of Cln3
oscillates only weakly, peaking in early G1 when
daughter cells are smallest due to a transcriptional
induction [70,97]. Because Cln3 localizes to the
nucleus and is very unstable [98–100], the nuclear
concentration of Cln3–Cdc28 might reflect the overall
rate of Cln3 translation, which steadily increases as
cells grow larger and acquire more ribosomes [96]. An
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A
Budding
localization, must, therefore, be considered [102]. It
must also be remembered that despite the well
deserved attention it has received, CLN3 is not essential and cells lacking this gene do eventually pass Start.
P P PP
Sic1 PP
P P P
Cdh1
P
P PP
Far1
SBF,MBF
activated
[Cln1-Cdc28]
[Cln2-Cdc28]
G1
G1
Start
G1
B
Start
S S
S
G2
M
Nutrients
Cell size
(Translation rate?)
Ras/PKA TOR
Sfp1 Sch9
Cln3Cdc28
Bck2
RP and Ribi regulons
Whi3
Ribosome synthesis
Swi6
Critical cell
size threshold
Whi5
SBF Swi4
G1/S
transcription
Ploidy
C
Nutrient modulation
(Ribosome biogenesis?)
Size
(Translation?)
Low size
threshold
High size Time
threshold
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Figure 3. Start in the budding yeast S. cerevisiae.
(A) Start is a short interval during which Cln1/2-Cdc28 activity
is elaborated. Phosphorylation of Far1, Cdh1, Sic1, and
Cdc24 allows DNA replication and bud emergence to be initiated. (B) A current model of Start entry. Activation of SBF and
MBF (not shown) results from the dissociation of the Whi5
repressor upon Cln3–Cdc28 phosphorylation of Whi5 and
perhaps Swi6. Cln3, and perhaps Bck2, abundance may be
proportional to translation rate and must overcome a poorly
understood size threshold. The size threshold is increased by
cell ploidy and rich nutrients, perhaps via Ras/PKA, Sfp1, and
Sch9 mediated control of ribosome biogenesis rates. (C) The
mechanism that sets the critical cell size threshold in
response to nutrients (possibly ribosome biogenesis rates)
appears to be distinct from the mechanism that actually
determines cell size (possibly the translation of an unstable
sizer). The latter increases over time as cells accumulate
biosynthetic capacity, whereas the former is determined by
current nutrient conditions and ploidy.
important requirement of this model is that the
nuclear volume remains constant during G1 phase,
because the nuclear volume would be the metric
against which the protein synthetic rate is measured.
It has recently been observed, however, that nuclear
volume increases as cells grow during G1 phase,
such that the nucleo-cytoplasmic volume ratio does
not appreciably decrease prior to Start [101] (P.J.,
M.T., B. Futcher, in preparation). This unexpected
result undermines simple models that connect cell
size to Cln3 translation. More complex models, such
as Whi3-dependent control of Cln3–Cdc28 nuclear
Supersized: Nutrient Modulation of Critical Cell
Size
The critical size threshold is large for budding yeast
growing in rich nutrients and small for those grown in
poor nutrients [103–105]. Such nutrient modulation of
size thresholds was first characterized in fission yeast
[106], but hints had been observed earlier in budding
yeast [107,108]. Given that overall translation rates
appear to report cell size to the Start machinery and
that yeast growing in rich nutrient conditions have
higher ribosome concentrations [89], one might
naively expect that yeast growing in rich nutrients
would achieve the critical translation rate (and pass
Start) with less cell volume than yeast growing in
poor nutrients. Because the opposite is true, the existence of a powerful nutrient repression of Start is
inferred. As the threshold is reset very quickly [103],
nutrients must exert a continual repression of Start
through G1 phase.
It is frequently argued that nutrients modulate the
critical cell size threshold via Cln3, as its abundance is
greatly diminished in cells growing in poor media due
to transcriptional and translational controls
[94,109–112]. This ‘Cln3 abundance model’ successfully explains the effects nutrients have on the length
of G1 phase, i.e., in poor nutrients yeast spend more
time in G1 phase. However, the model cannot explain
why in these conditions a smaller critical cell size is
required to pass Start. Slowing translation rate with
cycloheximide also extends G1 phase, but does not
lower the critical cell size setpoint, demonstrating that
an extended G1 phase does not necessarily lead to
Start entry at a smaller cell size [65,92,95]. Apparently,
there is no temporal integration of Cln3–Cdc28 activity (or other Start activators) over extended G1
phases, consistent with the extreme instability of Cln3
and the prompt resetting of the size threshold upon
nutrient shifts [98,103]. With respect to critical cell
size, the Cln3 abundance model of nutrient modulation breaks down: in poor nutrients, yeast have the
lowest levels of Cln3 yet pass Start with the least
amount of mass and translational capacity. Shifting
cells into glucose delays Start relative to cell size
despite
increases
in
Cln3
abundance
[103,104,109,111,112]. From this perspective, nutrient
modulation of the critical cell size threshold is even
more remarkable, as not only must the yeast growing
in poor nutrients enter Start with less translational
capacity but it does so with much less Cln3. Consistently, poor nutrient conditions partially suppress the
Cdc28 requirement at Start [113]. A final problem with
the Cln3 abudance model is that the size of WHI1-1
and cln3∆ cells responds appropriately to nutrient
modulation [68,95]. In fact, nutrient modulation
appears to be independent of all known upstream regulators of SBF/MBF [95]. These results suggest that
an uncharacterized pathway(s) signals from nutrients
to increase the critical cell size threshold.
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Nutrient modulation of Start entry appears to involve
at least three main conduits. Activation of the Ras/PKA
signaling pathway, either by glucose or mutation,
increases critical cell size and is accompanied by a
transient reduction in SBF-dependent transcription
[114–116]. A genome-wide size screen identified the
zinc-finger transcription factor Sfp1 and the Akt-like
kinase Sch9 as two other effectors of nutrient modulation [78,95]. Despite functioning largely in parallel,
when any of these three factors is crippled, cells are
small and impervious to carbon source control of their
size [78,95,114,115]. Significantly, Ras/PKA, Sfp1, and
Sch9 all activate the transcription of ribosomal protein
(RP) and ribosome biogenesis (Ribi or RRB) genes in
response to nutrient signals [78,95,117–121]. Along
with transcription of rRNA, transcription of the RP and
Ribi regulons is rate-limiting for ribosome synthesis in
budding yeast [88]. Furthermore, the same genomewide screen for cell size revealed that at least 15 Ribi
genes encode potential Start repressors [78]. All of
these observations can be unified by a model in which
the rate of ribosome biogenesis, which is proportional
to nutrient quality, represses SBF/MBF, thereby linking
nutrients to the critical cell size threshold (Figure 3B).
In this model, for any cell in G1 phase, the current rate
of ribosome biogenesis modulates the critical cell size
setpoint, while the overall translation rate (an integral
of all past ribosome biogenesis) reports cell size
(Figure 3C). This model has the appealing property that
future changes in overall translation rate are anticipated at an early juncture and the cell size threshold
can be reset accordingly. The unknown mechanism
that connects ribosome biogenesis and SBF/MBF may
be key to the mystery of critical cell size. Tantalizing
connections also exist between ribosome biogenesis
and replication initiation in yeast [122,123].
The evolutionary rationale for nutrient modulation
of size in microorganisms in general, bears consideration. Experiments with E. coli show that cell size is
optimized for fitness under given nutrient conditions
[124]. A plastic size threshold may enable the
budding yeast to best compete for limited and fluctuating resources. In nutrient rich conditions, growth
is exponential (i.e., proportional to size) and limited
only by biosynthetic capacity. Under these conditions, cell size appears to have little effect on doubling time, as demonstrated by the nearly wild-type
doubling times of cells lacking either WHI5 or CLN3,
which differ in volume by a factor of two [68,69,78].
Therefore, cells in rich nutrients can afford to stockpile resources without increasing their doubling time.
When suboptimal nutrients limit growth rates, cells
may attenuate the size requirement at Start to minimize doubling times.
Look Twice Before You Divide: Fission Yeast
The budding yeast S. cerevisiae and the fission yeast
Schizosaccharomyces pombe diverged approximately
one billion years ago [125]. S. pombe was the first
organism in which mutant alleles were identified that
caused cells to advance their cell cycle relative to their
growth [37]. The wee1 and wee2/cdc2-1w mutants
were characterized by Nurse, Fantes, Thuriaux, and
Nasmyth in the Mitchison laboratory in the mid-1970s
[37,126–128]. The cell cycle kinetics of wee1 strains
revealed that there are actually two critical cell size
thresholds in the fission yeast cell cycle, the first
imposed at the G1/S phase transition and the second
prior to the G2/M phase transition [37,127]. Such a
scenario may also exist in budding yeast, as some
initial evidence suggests a subtle bud size control
mechanism at the G2/M transition [129]. When fission
yeast are propagated in nutrient rich conditions, cells
exiting mitosis are already sufficiently large to initiate S
phase and do so immediately, before entering a long
G2 phase during which they grow to reach the critical
cell size required for the G2/M transition [106,127]. In
contrast, fission yeast raised in poor nutrient sources
are much smaller at the end of mitosis and consequently must grow to a minimum size in G1 phase
before initiating S phase (Figure 4) [37,106,127].
The reason why fission yeast reared in poor nutrient
sources are smaller as they exit from mitosis is that
the critical cell size threshold prior to G2/M is elastic
and greatly reduced in poor nutrient sources [106].
The lengthening of the G1 phase in poor nutrients is
advantageous, because it allows time for an important
response to starvation, i.e. the mating of two G1
phase cells and the subsequent meiosis and generation of resilient spores [106,130]. In contrast, rapidly
proliferating fission yeast spend the majority of their
time in G2 phase, presumably because the preferred
haploid state has co-selected for repair of DNA
damage in G2 when a sister chromatid template is
present [126]. Therefore, the modulation of cell size
thresholds allows the fission yeast to tune its cell
cycle to the environment.
The wee1 and wee2/cdc2-1w mutations proved
crucial in elucidating the molecular basis for the G2/M
transition in fission yeast, a process that is highly conserved in eukaryotic cells [130]. In brief, control of the
G2/M transition centers around the phosphorylation
status of a conserved tyrosine (Tyr15) on Cdc2, the
homolog of budding yeast Cdc28 [4,130]. wee2 is a
hypermorphic allele of cdc2+, while wee1+ encodes a
protein kinase that phosphorylates and inhibits Cdc2,
thus preventing the Cdc13–Cdc2 (cyclin–CDK)
complex from propelling cells into M phase (Figure 4B)
[4,130]. The phosphorylation of Tyr15 is removed by
the protein phosphatase Cdc25 prior to mitotic entry
[4,130]. In all eukaryotic cells, the G2/M transition is
thought to be very switch-like due to positive feedback loops; once unleashed in mid-to-late G2, Cdc13Cdc2 further activates its activator Cdc25 and may
also repress its repressor Wee1 [4,130–132].
The key question is how cell size, or some parameter correlated with cell size, initiates the positive feedback loop that propels cells into mitosis [131,132].
Cdc25 is a dosage dependent activator of mitotic
entry and clearly mediates size control at G2/M
[133,134]. The cellular concentration of Cdc25 protein
(and mRNA) increases steadily as cells grow in G2
phase [130]. Additionally, the concentration of Cdc25
increases during G1 and S phase arrests, suggesting
that the accumulation is related to cell growth, not
cell cycle position [135]. Because Cdc25 protein
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accumulates more rapidly than its transcript, Cdc25
appears to be a translational sizer [135]. How ploidy
and nutrients modulate the G2/M critical cell size
threshold is unknown, but nutrients appear to
impinge on Wee1 and not Cdc25 activity
[134,136–138].
Culture Wars: Size Control Mechanisms in Animal
Cells
There is an obvious difference in the rationale for coupling cell growth and division in unicellular versus
multicellular organisms. Unicellular organisms are
limited by the nutrients in the environment and usually
divide as often as growth permits. In sharp contrast,
cells in multicellular organisms are typically immersed
in excess nutrients such that cell proliferation is regulated by limiting extracellular signals, namely growth
factors, which drive cell growth, and mitogens, which
drive cell division [3]. Cell growth and division are
strictly controlled to shape, pattern and maintain
tissues and organs [139]. These extra layers of developmental control may obscure the more primordial
mechanisms that couple cell growth and division.
Indeed, in many tissues of embryos, cell size gradually
changes in response to the patterning signals that
regulate the timing of cell divisions [139].
The first strong indication that cell cycle progression
was dictated by cell size in metazoan cells came from
influential work by Killander and Zetterberg [44,45],
who observed that the variability in size of mouse
fibroblasts that had just entered S phase was less than
the variability in the size of cells that had just exited
mitosis. Since then, many groups have demonstrated
that cell size and/or protein synthetic rate help determine the length of the cell cycle in many types of cultured cells, almost always by influencing the length of
G1 phase [5,28,52,54,55,57,59,61,140–144]. However,
on occasion others have found no such relationship
[35,145,146]. Unlike in budding yeast, the size requirement may not be imposed at cell cycle commitment,
termed the Restriction Point [26,27]. For example, in
Swiss 3T3 cells and human diploid fibroblasts the time
from cytokinesis to the Restriction Point in mid-G1
phase is relatively constant, whereas the time from the
Restriction Point to S phase entry is highly variable
[147]. Entry into S phase is thus likely to be the point
at which the size requirement is imposed, at least in
these cell types.
In many metazoan cell types, growth can be uncoupled from the cell cycle. For instance, glial growth
factor (GGF) propels cultured Schwann cells through
the cell cycle but does not increase growth rates [35].
Similarly, S phase entry can occur without mass accumulation, as when platelet derived growth factor
(PDGF) is added to Swiss-3T3 cells [146,147]. In these
contexts, GGF and PDGF are strictly mitogens, as they
stimulate cell cycle progression but not growth [3]. This
has led to the proposal that in metazoans the extent of
cell growth and the length of the cell cycle are only correlated, due to the maintenance of presumably quite
stable extracellular concentrations of growth factors
and mitogens (Figure 2A). Such ‘correlative models’
contrast with cell-autonomous mechanisms that
actively couple cell growth and division, as in yeast.
The existence of mitogens is, nevertheless, compatible
with the existence of cell-autonomous size control.
Similar to the effect of poor nutrients on yeast, mitogens could decrease a hypothetical critical cell size
threshold. Alternatively, mitogens might simply override
cell-autonomous controls.
Much of the cell proliferation that takes place during
development leads to progressive alterations in cell
size and is often completely uncoupled from cell
growth (Figure 2A) [139]. Consistently, patterning
signals do not always upregulate growth regulatory
pathways to drive growth-coupled cell division, but
can transcriptionally activate cell cycle regulators like
Cdc25/string [148]. The lessons from developmental
control of cell size should not be overwrought,
however, as it is clear that, in many metazoan cell
types, cell cycles depend cell-autonomously on size
and growth [5,28,52,54,55,57,59,61,140–143], even
during development [139]. Furthermore, strictly extracellular mechanisms cannot easily explain the seemingly universal correlation between cell size and
ploidy. For example, if extracellular concentrations of
mitogens and growth factors were solely responsible
for cell size control, tetraploid cells would have to
respond twice as well to growth factors, but not mitogens, as diploid cells.
An argument against a general need for size thresholds in metazoan cell proliferation is the claim for
linear as opposed to exponential growth in these cells
[35,149]. In budding yeast, the rate of mass increase
is proportional to the size of the cell [87,150,151]. A
simple thought experiment, originally applied to
fission yeast by Nurse and Fantes [128], demonstrates
the exponential nature of growth in budding yeast.
Despite having stable average volumes that differ over
a two-fold range, whi5∆, cln3∆, and wild-type cells
have about the same doubling time in glucose media
[68,69,78]. Therefore, cln3∆ cells must produce twice
the cytoplasm as whi5∆ cells in a 90 min period; that
is, in absolute terms, cln3∆ cells grow twice as fast as
whi5∆ cells. Whi5 and Cln3 have no known impact on
growth processes and appear to simply determine the
size at which cells pass Start [68,69,78]. In yeast,
therefore, absolute growth rates are proportional to
cell size (i.e., growth begets increased growth rates).
Exponential growth requires mechanisms that correct
for cell size, as otherwise cell size would become heritable. In contrast, linear growth rates do not strictly
require mechanisms that couple growth and division,
because on average small cells will grow proportionally more than large cells over a cell cycle [152]. Unfortunately, it is uncertain whether metazoan cell growth
is exponential or linear, as both claims have been
made repeatedly in the literature [7]. Linear growth
may indicate that the extracellular concentration of
growth factors, rather than biosynthetic capacity, is
limiting for growth rate. In any case, linear growth
rates do not preclude cell size checkpoints. For
example, Amoeba proteus does not grow exponentially but does have size thresholds [42,43,53]. Finally,
as noted, linear growth models fail to explain the
numerous examples in which growth rate and cell size
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do determine the timing of cell cycle transitions
[5,28,52,54,55,57,59,61,141–143].
That size control in at least some metazoan cell
types is essentially similar to that in yeast has been
demonstrated recently with avian erythroblast and
mouse mesenchymal fibroblast cell lines [52]. Threshold size requirements were established by demonstrating that cell overgrowth caused by retarding S
phase abbreviated the subsequent G1 phase; interestingly, the G1 phase could only be shortened so far
that even cells born above the critical cell size spent
5–6 h in G1 phase. This finding is in keeping with a
size-independent component being present in G1
phase [61,147,153]. Intriguingly, avian erythroblasts
were also similar to yeast in that their critical cell size
requirement was proportional to their growth rate [52].
Switching between two growth factor regimes led to a
rapid re-adjustment of cell size over one or two cell
divisions, just as in yeast.
An alternative mechanism to couple growth and
division in metazoan cells is the intertwining of the two
processes (Figure 2A). A number of recent reports
indicates that several cell cycle regulators also directly
control cell growth, including the tumor suppressors
Rb and p53 and the oncogenes Cyclin D and Myc. Rb
and p53 directly control ribosome biogenesis by
repressing RNA PolI- and RNA PolIII-dependent transcription in mammalian cells [154,155]. Unexpectedly,
Cyclin D–Cdk4 complexes regulate cell growth in
Drosophila and plants and activate RNA PolI transcription in mammalian cells [156,157]. Myc increases
the growth and size of many metazoan cell types,
probably by activating the transcription of rDNA and
numerous genes implicated in ribosome biogenesis
[158–161]. Although interdigitation of cell growth and
division might suffice to maintain cell size without size
checkpoints, again, a strict version of this model does
not easily explain the numerous examples where cell
cycle events are strongly influenced by cell growth
[5,28,52,54,55,57,59,61,141–143].
Importantly, continuous ribosome biogenesis is
essential for the G1/S transition in mouse and human
cells, even in the presence of high translation rates and
sufficient cell size [162,163]. Upon conditional deletion
of the ribosomal S6 protein in the mouse, hepatocytes
were able to re-grow after starvation using their existing pool of ribosomes, but upon partial hepatectomy
could not enter S phase [162]. Similarly, induction of a
dominant negative form of the ribosome biogenesis
factor Bop1 in tissue culture cells caused a p53dependent G1 phase arrest without affecting translation rates [163]. Furthermore, the Arf tumor suppressor,
a critical activator of p53, inhibits rRNA processing
[164]. In a radical departure from traditional models of
p53 function, it has been proposed that all stresses
that stabilize and activate p53 do so by disrupting the
nucleolus, and presumably ribosome biogenesis [165].
Disc Drive: Developmental Control of Cell Size in
Drosophila
Great strides have recently been made in elucidating
metazoan growth signaling networks, predominantly
through imaginative Drosophila genetics. The
A
High G2/M critical
cell size
G1/S critical
cell size
Excess nutrients
G1
S
G2
Low G2/M critical
cell size
G1/S critical
cell size
Nutrient limitation
G1
M
S
G2
M
B
Nutrients
Cell size
P
Y15
?
Cdc13
CDK
?
Cdr2
Wee1
Mik1
Cdc25
Y15
Cdc13
CDK
Mitosis
Nim1
?
Current Biology
Figure 4. Cell size control mechanisms in the fission yeast S.
pombe.
(A) Fission yeast has two cell size thresholds. In rich nutrient
conditions, cells are larger than the G1/S critical cell size
requirement after mitosis and immediately enter S phase.
Cells reared in limiting nutrients decrease their G2/M critical
cell size threshold and exit mitosis as small cells that must
then grow to the G1/S critical cell size threshold. (B) A positive
feedback loop existsat the G2/M transition in fission yeast.
Entry into mitosis requires growth to a critical cell size; size
appears to be gauged by Cdc25 [134]. Cdc25 phosphatase
activates Cdc2 (CDK) by dephosphorylating Tyr15, while
Wee1 kinase performs the opposite reaction. As Cdc13–Cdc2
kinase appears to induce Cdc25 activity [135] and may
repress Wee1 activity, a sufficient induction of Cdc13–Cdc2
impels a positive feedback loop that drives cells into mitosis.
Rich nutrients (e.g., nitrogen) may raise the critical cell size
threshold by activating Wee1.
IGF/PI3K signaling pathway as well as the closely
intertwined Tsc1, Tsc2, S6K, 4E-BP, Tor, Rheb, and
Foxo proteins regulate cell growth and size in
Drosophila and mammals [5,6,29,166–168] (Figure 5).
Genetic alteration of most of these network components can lead to abnormal cell size. In a parallel
pathway, the Myc transcription factor has also been
demonstrated to increase size by inducing cell growth
in Drosophila and mammals [139]. The ncl-1 gene in
Caenorhabditis elegans and its Drosophila homolog
brain tumor (brat) are negative regulators of ribosome
biogenesis and, intriguingly, the corresponding
mutants display cell-autonomous increases in nucleolar and cell size [169,170].
The rapid proliferation of cells in the imaginal discs
of the Drosophila larva has been exploited to explore
the coordination of cell growth and division in vivo. In
the wing imaginal disc, cell growth and division are
separable processes [18]. It is not certain whether
yeast-like critical cell size thresholds couple growth to
the cell cycle in Drosophila. If such a threshold does
exist in proliferating imaginal disc cells, it is likely to be
imposed at the G1/S transition. Cells defective for
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growth activators like dRas1, dMyc, dPI3K, and dTOR,
accumulate in G1 phase, whereas overactivation of
these pathways promotes passage through the G1/S
transition [5]. Cyclin E may function as a translation
sizer that couples cell growth and size to G1/S progression. Cyclin E abundance in wing discs is sensitive to growth drivers like dRas1 or dMyc in a
post-transcriptional manner and is rate-limiting for
G1/S progression [18,171]. Shortening the length of
G1 phase by activating growth pathways in imaginal
disc cells, however, is compensated for by lengthening of the S and G2 phases, such that the doubling
time is the same as in wild-type cells, a situation that
generates oversized cells [18]. In cells that cut G1
phase short, dE2F activity is curtailed and the dE2F
target gene Cdc25/string appears to become limiting
for the G2/M transition, leading to the observed compensation [18,172]. In the wing imaginal disc, growth
is thus required to permit the G1/S transition, but
cannot drive the cell cycle as a whole. Rather, control
of Cdc25/string by developmental patterning signals
appears to determine the rate of mitosis and division
in this tissue [139]. It should also be noted that when
larval development initiates, wing imaginal disc cells
increase their mass six-fold before entering the phase
of exponential proliferation, during which cell size
gradually decreases as disc size increases and
approaches its final differentiated state [18,139]. In
contrast, the frequency of endocycles in the polyploid
cell types that make up most of the Drosophila larva is
strictly determined by cell growth [173]. These examples demonstrate the complexity and plasticity of cell
size control in vivo.
Stretching the Analogy: Elasticity of Size
Thresholds
Lowering size thresholds to increase cell number in
advance of starvation may be a common evolutionary
strategy for unicellular organisms and may allow multicellular organisms to develop normally when nutrients
are scarce. Nutrient upshifts delay cell division in the
protist Tetrahymena and delay nuclear division in the
acellular slime mold Physarum polycephalum, while
nutrient downshifts advance cell division in Tetrahymena and perhaps Paramecium [174–176]. These
results can be interpreted as nutrient modulation of cell
size thresholds. Similarly, in Drosophila, rearing larvae
in scarce nutrient conditions results in perfectly patterned flies with small cells [139].
In budding yeast, common pathways appear to
mediate increased ribosome biogenesis and critical
cell size. There is some evidence for such a scenario in
Drosophila. For instance, ablation of dS6K slows proliferation and strongly reduces cell and body size
(Figure 1C) [177]. S6K is an effector of the PI3K and
TOR signaling networks and induces cell growth, possibly by the translational upregulation of RPs (Figure 5)
[6,178]. In contrast, Minute mutations that cripple
genes encoding ribosomal proteins result in slow proliferation without affecting cell size [18,177]. Given their
proliferation rates, cells lacking dS6K are very small,
suggesting that dS6K also impinges on the mechanism
coupling cell growth and division. Consistently, loss of
dS6K in proliferating imaginal discs does not alter the
cell cycle phase distribution and cell size is diminished
in all phases of the cell cycle [177]. Likewise, weak heteroallelic combinations of dTOR or hypomorphic
alleles of dMyc or dAkt reduce the size of proliferating
cells in imaginal discs but do not change cell cycle
phase distribution [158,179,180]. Because in
Drosophila imaginal discs division appears to require
achieving a critical cell size threshold at G1/S, loss of
dS6K and perhaps dAkt, dTOR, and dMyc may well
lower that threshold [18]. Again, perhaps not coincidently, all of these pathways appear to help set the
rate of ribosome biogenesis (Figure 5). The cell size
phenotypes in the imaginal discs of larvae bearing
hypomorphic mutations in ribosome biogenesis
factors, for instance the minifly mutation [181], deserve
exploration in this regard.
Similar relations between growth regulation and
critical cell size thresholds may exist in vertebrate
cells. As mentioned above, the critical cell size requirement at the G1/S transition of avian erythroblasts may
be set higher in cells with vigorous growth factor signaling [52]. Similarly, in human epitheliod cells, far less
mass was required to divide in 0.03% serum as
opposed to 30% serum, a result interpretable as evidence of a flexible size threshold [182]. Remarkably,
mice heterozygous for a hypomorphic and null allele
of Pdk1, which encodes an upstream activator of Akt,
are 35–50% smaller than wild-type mice [183]. This
phenotype appears to be due to decreased cell size.
When mouse embryonic fibroblasts (MEFs) were isolated from these heterozygotes, the cultured cells
were ~35% smaller than wild-type but proliferated at
the same rate [183], demonstrating that Pdk1 can
control size cell-autonomously. Similar cellautonomous size defects are seen in fibroblast lines
lacking a Rho GTPase activating protein (p190-B
RhoGAP) that has been implicated in IGF/PI3K signaling [184]. In contrast, MEFs derived from mice heterozygous for a deletion within the RPL24 gene, grow
slowly but maintain wild-type cell size [144]. Assuming
critical cell size thresholds exist in these fibroblasts,
as suggested by the normal size of slowly growing
RPL24 +/- heterozygous MEFs [144], these cell size
and cell cycle phenotypes suggest that Pdk1 and
p190-B RhoGAP can raise the threshold setpoint,
much as nutrients do in budding yeast. But, such a situation does not appear to apply to PI3K signaling in all
cell types. The hyperactive PI3K signaling resulting
from PTEN deletion drives rampant proliferation [29].
In most cell types examined, however, this rapid proliferation is not accompanied by cell size increases,
suggesting that growth and division are properly
coupled in these cells [29]. The potential flexibility of
size thresholds in metazoan cells adds yet another
layer to the enigma of size control.
No Small Problem
Although a sophisticated model of the eukaryotic cell
cycle has been constructed, cell growth and cell size
remain relatively understudied phenomena [3]. One
fundamental restraint on the growth of cells is their
translational capacity and hence the number of
Current Biology
R1023
Figure 5. Two distinct cell growth pathways in
Drosophila and mammals.
A simplified dRas1 activation and effector
network is shown in purple. The PI3K/TOR activation and effector network is shown in blue
(activators) and orange (repressors). Proteins
that appear to directly induce growth by activating ribosome biogenesis, translation, and other
anabolic processes are shown in gray. Question
marks indicate uncertain interactions. When
hyperactivated, dRas1 can induce dPI3K activity, although this connection does not appear to
be important in wild-type cells [196]. It is uncertain whether Tsc1 and Tsc2 are downstream of
Akt. Similarly, it is uncertain if TOR is downstream of PI3K and Rheb or whether it lies in a
dominant, parallel pathway. Abbreviations: IR:
insulin receptor; ILPs: insulin-like peptides; IRS:
insulin receptor substrate; PtdIns: phosphatidylinositol; PI3K: class 1 phosphoinositide
3′′-kinase; PTEN: phosphoinositide 3′′phosphatase; PDK1: phosphoinositide-dependent
kinase 1; MAPK: mitogen activated protein
kinase module; EGF: epidermal growth factor;
EGFR: EGF receptor; 4EBP: eIF4E binding
protein. See recent reviews for further details
[5,6,166]. All of these proteins and interactions
appear to be conserved in mammals.
PDK1
Rheb
ILPs
Akt
PTEN
?
Tsc1
Tsc2
?
IR
PtdIns PtdIns
(4,5)P2 (3,4,5)P3
IRS
p60
FOXO
TOR
PI3K
4EBP
?
S6K
Ribosome synthesis
and growth
Myc
EGF
EGFR
Ras
Grb2
SOS
Raf/MAPK
Current Biology
ribosomes they possess [185]. Ribosome synthesis,
which can be viewed as a growing cell’s investment
in future growth, dominates a growing cell’s
economy, accounting for more than 50% of total transcription in budding yeast and mammalian cells
[88,186]. The mechanisms that control ribosome production in response to growth factors are highly
complex and need much further exploration [187].
Given its central role in cell growth, it is perhaps not
surprising that ribosome biogenesis also regulates
cell size, the cell cycle, and responses to stress
[78,122,123,155,162,163,165,188]. Mapping the pathways that converge on and emanate from ribosome
biogenesis will be crucial to understanding how
growth is coupled to division.
The highly complex nature of cell size control will
ultimately require mathematical modeling. Indeed,
there is a long history of modeling cell size, particularly as an aspect of the cell cycle [12,189–191].
Rather than just viewing cell growth as a single parameter within the cell cycle, however, dedicated size
models could incorporate variables such as division
rates, ribosome biogensis rate, metabolic flux, nuclear
and nucleolar volume, and critical cell size setpoints.
All of these parameters must be subject to stochastic
variation, especially as it is not clear how accurate
sizing mechanisms really are, even in the well characterized budding and fission yeast. Within limits, cell
volume and critical cell volume do vary considerably
and there is no reason to think that size homeostatic
mechanisms are, or need to be, blindingly accurate
[24,152,192]. Sloppiness should not, however, be mistaken for lack of control [11].
Discovering the mechanisms that couple cell
growth and division will continue to challenge the
experimentalist and the theoretician alike. Cell size is
easily influenced: systematic analysis has shown that
more than 10% of the viable deletion mutants in
budding yeast exhibit notable cell size defects, though
most of these genes do not control Start [78,79]. The
lack of sustained study of size thresholds in a single
mammalian cell type has precluded a focused molecular examination of the potential coupling mechanism(s). Systematic analyses of cell size responses to
RNAi knockdown of gene function promise to identify
many new regulators, as demonstrated recently in
Drosophila tissue culture cells [193]. Lymphocytes
would seem to be an excellent system for a sustained
analysis of mammalian cell growth and size [57,194].
A broad survey of metazoan cell types to discover
the pervasiveness of critical cell size thresholds and
alternative size control methods is also in order
[35,52]. Particularly in the context of development, it is
unclear how frequently the cell cycle is coupled to cell
growth and size. The generalization that metazoan cell
cycles are contingent on some aspect of cell growth
or size is largely based on work with cell lines in vitro.
Hematopoietic cells may be particularly inclined to
having cell autonomous, yeast-like size control mechanisms in vivo as many of these cell types (e.g., lymphocytes) must proliferate as rapidly as possible when
stimulated; additionally, the fluid environs these cells
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R1024
inhabit may limit the contact inhibition and paracrine
signaling extant in more fixed tissues. The potential for
multiple layers of size control must not be forgotten, a
reality established by Nurse [37] in the relatively
simple fission yeast nearly 30 years ago. For instance,
all metazoan cells may possess a primordial cell size
threshold that prevents division at dangerously small
sizes, but, due to additional non-cell-autonomous
controls, many cell types may have a large cell size at
which this primordial threshold is irrelevant.
At the end of a thoughtful review penned nearly 50
years ago, Swann stated that “The time has come
when it is doubtful whether experiments on proliferation that do not make a determined attempt to disentangle the many factors involved in cell growth and
cell division are worthwhile” [24]. Swann’s admonition
has been ignored for too long — cells care about how
large they are and so should we.
Acknowledgments
We apologize to the many authors whose work we
could not cite directly. We are grateful to Yoshio
Masui, Bruce Edgar, Bruce Futcher, Peter Sudbery,
Brenda Andrews, Martin Raff, Rob Rottapel, Ivan
Rupeš, Jeff Sharom, and Mike Cook for thoughtful
conversations about size control and to anonymous
reviewers for helpful comments. This work was supported by a Canadian Institutes of Health Research
(CIHR) grant to M.T., a CIHR Doctoral Award to P.J.
and a Canada Research Chair to M.T.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
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31.
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