BBA - Molecular Cell Research 1866 (2019) 409–417

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BBA - Molecular Cell Research

journal homepage: www.elsevier.com/locate/bbamcr

Review

Cell size homeostasis: Metabolic control of growth and cell division T

Mikael Björklund
Zhejiang University-University of Edinburgh (ZJU-UoE) Institute, Zhejiang University School of Medicine, International Campus, 718 East Haizhou Rd., Haining, Zhejiang
314400, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Joint regulation of growth rate and cell division rate determines cell size. Here we discuss how animal cells
Cell size control achieve cell size homeostasis potentially involving multiple signaling pathways converging at metabolic reg-
Metabolism ulation of growth rate and cell cycle progression. While several models have been developed to explain cell size
Growth rate control, comparison of the two predominant models shows that size homeostasis is dependent on the ability to
Cell size sensing
adjust cellular growth rate based on cell size. Consequently, maintenance of size homeostasis requires that larger
cells can grow slower than small cells in relative terms. We review recent experimental evidence showing that
such size adjustment occurs primarily at or immediately before the G1/S transition of the cell cycle. We further
propose that bidirectional feedback between growth rate and size results in cell size sensing and discuss potential
mechanisms how this may be accomplished.

1. Introduction results in a quite substantial 33% increase in cell volume. A further

How cell size is determined and regulated is an important yet poorly
understood question in biology. While it appears that cells attempt to
maintain their characteristic size, the mechanisms have remained elu-
sive [1,2]. This review will concentrate on how proliferating animal
cells control their size as research has primarily focused on how to
achieve size homeostasis through regulation of cell growth and cell
division. This joint regulation of cell size is perhaps best illustrated
through selective inhibition of cell cycle progression. Under such con-
ditions cells continue to grow largely uninterrupted [3,4] and may
reach sizes that would not be attainable under physiological conditions.
Therefore, both cell growth and normal cell cycle progression are re-
quired for the maintenance of cell size homeostasis in proliferating
cells. The principles of how non-dividing cell types maintain their size
are less well established [1], which is unfortunate as non-dividing cells
constitute the great majority of cell types in the animal body.
Before going into any details of cell size regulation, it is important to
realize that many different parameters are used as a measure for cell
size, including cell length, area, volume, total cell mass, dry mass and
total protein content. Typically, which one is used depends on selected
techniques, available instrumentation and even individual preferences.
In particular, cell length, area and volume are often used almost in-
terchangeably without consideration to the geometric implications. For
example, while many investigators might overlook the significance of a
10% increase in the diameter of a spherical-shaped cell, this actually
complication is that the term ‘growth rate’ is often used to describe the
increase in cell number per unit time. Instead, a more relevant measure
for cell size studies, and also used in this review, is to define growth rate
as the cell volume or mass increase per time (e.g., femtoliter/hour or
picogram/hour).
Current understanding is that cell size is determined by a number of
internal and external factors, e.g., genome size, nutrients and growth
factors [1,2], which affect cellular signaling pathways and metabolic
activity. Changes in cell size are typically observed when the balance
between the growth rate and cell division rate is altered. This balance is
a key determinant of cell size across different organisms from uni-
cellular bacteria to multicellular animals. However, an important con-
ceptual problem arises from this observation. Namely, if cell size is
determined by growth rate and cell cycle progression, it is easy to view
cell size a spandrel, an evolutionary byproduct [5]. Nevertheless, it is
obvious from our macroscopic world that size has a huge impact on
organisms [6], e.g., just compare the structural and functional differ-
ences between mice and elephants. Does size also matter for cells?
Experiments suggest that cell size is important for cellular fitness and
function [7,8] and altered cell size appears to have diverse effects at the
whole animal level from brain function to aging [9,10]. Loss of cell size
homeostasis is also commonly seen in chronic diseases such as cancers
[1,2].
Many general processes are size dependent, including transcription
[11,12], translation [13] and metabolism [12]. Additionally, specific

Abbreviations: SV, surface-to-volume; mTOR, mechanistic target of rapamycin; MAPK, mitogen-activated protein kinase
E-mail address: mikael.bjorklund.lab@gmail.com.

https://doi.org/10.1016/j.bbamcr.2018.10.002
Received 9 August 2018; Received in revised form 25 September 2018; Accepted 3 October 2018
Available online 11 October 2018
0167-4889/ © 2018 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M. Björklund
processes including actin filament and focal adhesion organization,
nuclear shape and cell contractility have also been shown to be influ-
enced by cell size [14]. All this data implies that cells adjust their
function to match their size and thus are somehow aware of their
physical dimensions. Such ‘size sensing’ could be an important me-
chanism for the maintenance of appropriate growth rate and/or cell
cycle progression [15,16]. Alternatively, size sensing could be directly
required for the control of cell size to maintain optimal cellular fitness
and function as suggested by the loss of cell size uniformity in many
cancers [1,2]. These explanations may not be mutually exclusive and
both interpretations imply that cell size matters.
2. How do animal cells grow? The linear vs. exponential growth
debate
In order to understand how cells maintain their size, it is important
to study how cells grow. In recent years, research efforts have con-
centrated on understanding cell size control mechanisms at a phe-
nomenological level and not so much at the level of molecular me-
chanisms [17,18]. This largely stems from the decades old debate
whether cell growth is exponential or linear during the cell cycle
[4,19]. While this might sound trivial, there is a solid theoretical ar-
gument why this is important and experimental reasons why this debate
continues to date. Theoretically, if cell growth is linear (larger and
smaller cells grow at a similar rate), population size homeostasis can be
maintained without paying specific attention to cell size. However, if
growth is exponential (larger cells grow faster than smaller cells) then
cell size needs to be regulated in order to maintain size homeostasis.
This is because exponential growth causes size variance in the cell
population to increase resulting in a loss of a stable cell size distribu-
tion. Therefore, it is thought that exponential growth requires specific
cell size checkpoints, which ensure that only cells that have reached a
certain minimal size may progress uninterrupted through the cell cycle.
One of the practical reasons why the exponential or linear growth
debate has continued so long is that it is still technically challenging to
reliably measure the difference between linear and exponential growth
as these models differ maximally by less than 6% during a single cell
cycle [20,21]. While such a high resolution is now achievable in single
cell mass measurements [21,22], other factors affecting experimental
variability, such as heterogeneity in growth rate of individual cells, are
often larger than this. Furthermore, the kinetics of cell growth appears
more complex than anticipated by these reductionistic models. Ex-
periments with animal cells have shown than growth rate is not con-
stant during the cell cycle [16,20,23], and is not purely linear or ex-
ponential [22]. Such cell-cycle dependent changes in growth rate are
also evident in both budding yeast and fission yeast [24,25].
Growth rate in animal cells can also be adjusted conditionally, de-
pending for example on cell size at birth (the cell size immediately after
cell division) [26]. By studying single cell growth trajectories, Cadart
et al. recently found that while overall growth is exponential in G1
phase of the cell cycle, further growth is adjusted in S-G2 phases in the
cell cycle so that there is a negative correlation between added volume
in G1 and S-G2 [23]. Interestingly, this finding may relate to the ex-
periments conducted by Conlon et al. [3,4], which revealed linear
growth in Schwann cells, a neuronal cell type that produces the myelin
sheath around axons. However, Conlon and Raff used cells arrested in S
phase using a DNA replication inhibitor aphidicolin. Consequently, if
growth rate changes observed by Cadart et al. are applicable to
Schwann cells, the use of aphidicolin would have resulted in missing
the exponential growth phase in G1. This realization may therefore
undermine some of the strongest evidence supporting linear growth.
Interestingly, these findings also suggest that the increased amount of
DNA is not accelerating growth, indicating that DNA template amount
is not limiting growth during normal cell cycle progression. The high
biosynthetic cost for genome duplication could at least in part explain
this growth rate adjustment in S-G2. Altogether, analysis of the
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BBA - Molecular Cell Research 1866 (2019) 409–417
increasing amount of single cell data clearly points to the conclusion
that the linear vs. exponential growth debate should finally put to an
end and replaced by more realistic, albeit more complex, models on
how cells grow.
3. The main models for the maintenance of size homeostasis are
not mutually exclusive
3.1. ‘Sizer’, ‘timer’ and ‘adder’ models
While the linear vs. exponential models have been used to argue for
and against the need for cell size control, several models have been
developed to explain how cell size homeostasis is maintained. These
include the ‘sizer’, ‘timer’ and ‘adder’ models. The ‘sizer’ assumes that a
cell needs to reach a critical size before it can divide, whereas the
‘timer’ suggests that cells grow for a certain time prior to dividing. The
‘adder’ model proposes that cells add a constant volume during each
cell cycle regardless of their size [27]. The ‘timer’ model maintains size
homeostasis if cells grow linearly for a constant amount of time.
However, this model is conceptually the most challenging one as it
assumes that cells have a mechanism to measure time. For this reason,
the ‘timer’ model has lost much of its shine and will not be discussed
further. However, it has been reported that growth during S and G2
phases of the cell cycle can resemble timer behavior in some cell lines
[23].
The ‘sizer’ model proposes that cells need to reach a minimal size in
order to progress through G1/S transition or mitosis (Fig. 1A). This has
lead a number of groups to propose that cells from bacteria to animal
cells can sense their size [15,16,28,29]. Recent work by Ginzberg et al.
showed that HeLa and retinal pigment epithelial (RPE) cells adjust their
growth rates twice in each cell cycle [16]. Specifically, growth rate was
shown to be selectively increased in small cells and reduced in large
cells in order to maintain cell size homogeneity. It is important to note
that many biological phenomena, including cell growth and metabo-
lism are multiplicative in nature, and consequently proportional rather
than absolute changes are more noteworthy [30]. Therefore, growth
rate changes discussed in this review should be understood in relative
(the change per cell volume/mass) rather than absolute terms.
The strongest adjustment of cell size variability [16] roughly cor-
responds to the G1/S transition, consistent with classical models of
growth rate regulation at G1/S transition or immediately before at so
called restriction point [31]. The authors pointed out that cell size
variance can only decrease if small cells grow faster than large cells
[16], a condition which counteracts the size homeostasis destabilizing
effect of exponential growth. Analysis of cell size variability as a
function of cellular DNA content as measured by flow cytometry sup-
ports the data from single cell imaging (Fig. 2). Finally, Ginzberg et al.
concluded that the cell's target size may be achieved through regulation
of cyclin dependent kinase CDK4, a key regulator of cell cycle pro-
gression in G1 phase, highlighting the tight connection between cell
size and cell cycle progression. The requirement for CDK4 in this pro-
cess is consistent with the recent suggestion that CDK4 regulates G1
length [32]. Interestingly, while CDK4 was required for setting the
target size, cell size variance was found to be regulated by p38 MAPK
kinase activity [33]. While this suggests that these processes are in-
dependently regulated, p38 MAPK has also been linked to direct reg-
ulation of CDK4 activity [34]. These encouraging results show how
growth rate may be adjusted in conjunction with G1 length regulation.
Identification of further molecular details should help to reveal me-
chanisms how cells can measure their size. For example, p38 MAPK
pathway is regulated by various signals including growth factors as well
as oxidative and osmotic stress. Which of these generates the signal
which p38 MAPK uses for regulation of cell size variance?
Accumulation of extensive single-cell time-lapse imaging data from
bacteria to mammalian cells has also resulted in resurrection and re-
finement of the ‘adder’ or ‘incremental’ model as it was initially called
M. Björklund BBA - Molecular Cell Research 1866 (2019) 409–417

A C
'Sizer' Volume Growth rate
increase

S= x
Cell volume

Size Cell cycle length

ADDER MODEL SIZE SENSING MODEL

Size added is constant Target size is constant

Large cells grow
slower and/or their cell
cycle is faster Size control
achieved by joint regulation of
growth and cell cycle
Division and/or
Birth
Time G1-S transition

B D
'Adder' Volume
increase

Metabolic rate Mass3/4

Cell volume

KLEIBER'S LAW

Metabolic activity is proportional to mass

Expected to reduce relative growth rate in larger

cell consistent with both adder and size sensing model
Relative growth rate of
large cells is slower
and/or cell cycle is faster

Birth Division
Time
Fig. 1. The ‘sizer’ and ‘adder’ models. (A) With ‘sizer’ cells need to reach certain size before they can start DNA synthesis (move through G1/S transition) or divide
and therefore cells that are initially of different sizes show different volume increase. (B) In the ‘adder’ model, cells add the same volume before cell division, meaning
that the relative growth rate of larger cells is slower or cell cycle is faster. Note that in both ‘sizer’ and ‘adder’ models the time to reach the critical cell cycle transition
may also vary, larger cells typically reaching G1/S or mitosis faster. (C) Size is jointly regulated by growth rate and cell cycle length. Here I use the expression by
Ginzberg et al. [16], but cell size could also be more generally expressed as a joint function of growth rate and cell cycle time (S = f(υ,τ)). Consequently, cell size can
be altered if growth rate or cell cycle length is altered. On the other hand, if cell size is constant, growth rate increase must be accompanied by decrease in cell cycle
length. (D) Kleiber's law indicates that metabolic activity is proportional to mass at organismal level. Provided that this is also true for individual cells, this formula
could potentially explain reduced growth rate of larger cells which is a requirement for robust cell size control in both ‘sizer’ and ‘adder’ models.

[27]. ‘Adder’ model postulates that both small and large cells add the from mean reversion as the daughter cells correct their size towards the
same volume during each cell cycle thus resulting in a stable cell size typical cell size. The size adjustment may take several cell divisions
distribution (Fig. 1B). The ‘adder’ model has been covered elsewhere in before the final target cell size is achieved. In contrast to the ‘sizer’
great detail [18,35], but the key point is that size homeostasis emerges model, the ‘adder’ model represents a more passive approach to

A B
CV of cell size
Cell count
Cell size

DNA content DNA content

Fig. 2. (A) Animal cells preferentially adjust their size at cell cycle transitions. Heatmap showing DNA content vs. cell size in Drosophila Kc167 cells shows reduction
in cell size variability at G1/S transition (white arrows). (B) The DNA content histogram depicting the cell cycle and median cell size variability (CV = coefficient of
variation; red dots) for the cells shown in (A). The cells were stained with Nuclear-ID Red DNA binding dye and analysed by flow cytometry. Cell size was measured
as forward scatter (FSC-A). The cell cycle stages G1, S and G2/M are shown along subG1 fraction (apoptosis/dead cell fraction).

411
M. Björklund
maintain cell size homeostasis, as there is no acute requirement for
active size monitoring.
3.2. ‘Sizer’ and ‘adder’ – more similarities than differences?
Despite the key conceptual differences in these models, there are
many similarities supported by the increasingly accurate experimental
measurements. For example, both the ‘adder’ and ‘sizer’ models agree
that cell size homeostasis is maintained by joint regulation of growth
rate and cell cycle duration [16,23]. This is exemplified by a mathe-
matical representation of how size relates to growth rate and cell cycle
duration. Ginzberg et al. [16] proposed that size is simply the product
of growth rate and cell cycle length (Fig. 1C). To maintain constant size,
one cannot modify growth rate without changing cell cycle length and
vice versa. This is consistent with common observation that cell cycle
length in G1 is inversely proportional to cell size, i.e., small cells spend
more time in G1 before they start DNA replication. This equation may
equally well be expanded to relate to the ‘adder’ model as size in this
equation could be either the constant target size (as in the ‘sizer’ model)
or the constant volume added per cell cycle (as in the ‘adder’ model)
(Fig. 1C). This simple mathematical representation how cell cycle
length, growth rate and cell size are interconnected will hopefully help
to link the ‘adder’ and ‘sizer’ models with the molecular mechanisms
responsible for growth and cell cycle regulation, including metabolism
and CDK regulation (see chapter 4).
Further similarities between the ‘adder’ and the ‘sizer’ models sug-
gest that these are not mutually exclusive, but rather different inter-
pretations of the same underlying biological mechanisms. Varsano et al.
reported that cultured mammalian cells that are roughly average size
display ‘adder’-like growth pattern, whereas abnormally small cells
display growth pattern that requires reaching a certain size before
transition to S phase, consistent with ‘sizer’ model [26]. Similar findings
have been reported for E. coli growth [36] and also suggested to apply
to budding yeast [37]. Specifically, Wallden et al. found that DNA re-
plication initiations in E. coli occur at integer multiples of a fixed vo-
lume, thus invalidating pure ‘timer’ and ‘adder’ models. They further
proposed a model based on timing of DNA replication initiation to ex-
plain this mode of growth where ‘adder’ becomes ‘sizer’ at slow growth
[36]. On the other hand, the model by Delarue et al. explains this two-
tier behavior by titration of an activator protein [37]. Overall these
results indicate that if the growth rate of the cells is sufficient, the (still
elusive) cell size checkpoints are not particularly strict and allow cells
of certain size range rather than a specific predetermined size to pass
through these inspections points.
Yet another shared feature of both the ‘adder’ and the ‘sizer’ models
is that the robustness of size control in these models is dependent on the
cell's ability to modulate growth rate and/or cell cycle progression in a
cell-size dependent manner. Even when cells add a constant volume
each cell cycle as in the ‘adder’ model, the growth rate is not constant
but is reduced in larger cells [23]. Currently the reason for such a size
dependent growth rate can only be speculated. Kleiber's law states that
the relative metabolic activity is reduced when organisms increase in
size (Fig. 1D). As metabolic activity is proportional to growth rate and
theoretical work suggests that metabolic allometry, the relationship
between metabolism and size, is universal across all scales of life [38],
this is potentially a useful framework for investigating size-dependent
growth of cells [8]. Notably, such reduced growth in larger cells con-
flicts with the purely exponential growth model (larger cells grow
faster), but is supported by experimental observations [7,16,23,39].
4. From models to molecules and mechanisms: metabolic
regulation of cell size
4.1. Growth signaling converges at metabolism
Genome-scale screens have identified many individual genes and
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BBA - Molecular Cell Research 1866 (2019) 409–417
cellular processes influencing cell size [40–42], although it has been
challenging to identify which mechanisms are direct. For example, a
large number of cyclin dependent kinases, their inhibitors and activa-
tors regulate normal cell cycle progression and many of these cell cycle
proteins influence cell size [42]. It is also well established that animal
cells can regulate their growth through multiple signaling pathways,
the PI3K-AKT-mTOR and RAS-MAPK pathways being the best char-
acterized examples [43].
Early work on bacterial growth established a relationship between
growth rate and cell size in a quantitative manner [44]. Essentially,
these analyses showed that faster growth rate in a richer culture
medium results in larger cell size, establishing a connection between
nutrient uptake, metabolic activity and cell size. While this is an in-
tuitive finding, it is much less clear how differences in cell size among
different cell types are achieved under identical culture conditions,
particularly in animal cells. By inference, such size differences could
also be related to differences in nutrient uptake and metabolic effi-
ciency in converting these nutrients to biomass. A large body of ex-
periments in single cell organisms has established that growth rate and
nutrient availability are the primary determinants of cell size and also
influence cell cycle timing. Some of the mechanisms on how specific
nutrients such as glucose act have been identified. For example, cell
division rate in budding yeast appears to depend on glucose influx
while volume growth is largely set by external glucose levels [45].
Other experiments point out that the nutrients (carbon source) have
clear effects on both duration of mitosis and cell size in yeast [46].
Similarly, work in bacteria has identified specific nutrients such as
uridine diphosphate glucose (UDP-glucose) [47] or metabolic enzymes
such as NAD-dependent glutamate dehydrogenase [48] as key meta-
bolic coordinators of cell size and cell division. Furthermore, genetic
evidence links nitrogen metabolism and pyruvate production to bac-
terial cell division, although the molecular mechanisms are yet to be
determined [49].
In contrast to these findings from unicellular organisms, there is
paucity of information regarding the role of specific nutrients and
metabolic enzymes in animal growth control. Most of our knowledge is
derived from experiments involving growth factor and metabolic sig-
naling pathways rather than the nutrients themselves, which may be
related to the more complex nutritional requirements of animal cells. In
proliferating animal cells growth factors and growth factor signaling
pathways are major regulators of nutrient uptake and metabolic ac-
tivity, and these pathways also influence cell cycle progression [43].
Although these growth factor signaling pathways have multiple out-
puts, regulation of metabolism to support cellular biosynthesis is re-
quired to achieve cell growth [43,50] (Fig. 3).
The mechanistic target of rapamycin (mTOR) is a good example
illustrating the multifaceted role of signaling pathways in modulating
cell growth and proliferation. The mTOR pathway is a key integrator of
cellular growth and metabolism with external inputs such as nutrients
and growth factors [51]. This pathway appears to influence virtually
every aspect of metabolism and has been directly linked to cell growth
and metabolism primarily via the mTORC1 complex as well as pro-
liferation primarily via the mTORC2 complex [51]. However, this dis-
tinction is not necessarily so clear-cut as for example recent work in
budding yeast indicates that both cell size and growth rate are modu-
lated via mTORC2 complex [52]. Manipulation of mTOR activity in-
fluences animal cell size in many model systems [53,54]. Despite its
many roles in metabolic regulation, experiments with rapamycin and
other mTOR inhibitors indicate that this kinase is not directly involved
in cell-size sensing [16] or setting the cell size dependent mitochondrial
activity [7]. Therefore, despite the extensive understanding of the
molecular details on mTOR signaling pathway [51], its true contribu-
tion to cell size regulation remains unresolved.
The central carbon metabolism where sugar is converted to meta-
bolites and energy via glycolysis and tricarboxylic acid cycle in mi-
tochondria forms the essential core for cellular metabolic networks and
M. Björklund BBA - Molecular Cell Research 1866 (2019) 409–417

Fig. 3. (A) Cells use nutrients such as glucose, glutamine and

A other amino acids as building blocks and also secrete waste
Glucose metabolites such as lactate and glutamate. Utilization of
nutrients by glycolysis, glutaminolysis and mitochondrial
Amino acids TCA cycle leads to generation of a wide variety of metabo-
lites for biosynthetic needs along energy (ATP) and redox
cofactors such as NADH and NADPH to support growth and
Lactate proliferation. In animal cells, metabolic activity is strongly
influenced by growth factors and the intracellular signaling
pathways such as PI3K-AKT-mTOR and RAS-MAPK path-
ways. (B) While glucose and glutamine are by far the most
Glutamate consumed nutrients, the carbon from these molecules con-
TCA CYCLE tributes relatively little to cellular biomass. Instead, amino
Glutamine acids other than glutamine make the biggest contribution to
cell mass [55]. The source of the remaining ~40% of carbon
has not been reliably identified. Part of this is derived from
lipids, especially palmitate and oleate [55].

e.g., mTORC1 e.g., mTORC2

Growth Proliferation

B
Glucose Glutamine Amino acids

Carbon up to up to up to
contribution ~10% ~10% ~40%
to biomass

is also essential for cell growth [43,50]. However, while the sugar-de-
rived metabolites can be used to make biomass, experimental data from
cultured mammalian cells indicates that the majority of cell mass de-
rives from amino acids rather than sugars [55] (Fig. 3B). Further
complexity in the system arises from the findings that metabolite up-
take is co-operative as in the presence of limiting amounts of a single
nutrient (e.g., glucose), cells immediately adjust the uptake of other
nutrients [56]. Understanding these complex metabolic requirements
and interdependencies for growth and cell division is likely to be es-
sential for developing effective therapeutic measures in cancers. It is
well established that many cancer cell types increase glucose and glu-
tamine uptake and reduce their dependency on mitochondrial oxidative
metabolism. The widespread use of this metabolic rewiring suggests
that this provides a growth advantage not only to cancer cells but also
to other highly proliferative cell types, including activated lympho-
cytes, thymocytes and embryonic cells.
Cellular metabolic activity and mitochondria in particular, not only
directly influences growth rate but also has an impact on cell cycle
progression through CDK and E2F activity [57]. The impact of meta-
bolism on proliferation has been demonstrated with several in vivo
models, including stem cells in Drosophila [58] and hepatocyte re-
generation in metabolic disorders [59,60]. Thus, metabolism is highly
interconnected with both growth and cell cycle progression and per-
haps even the cause for the observed coupling between growth, cell
cycle and cell size (Fig. 3A).
4.2. Size-scaling of cell cycle regulators and metabolism
As discussed earlier, cell-size dependent growth rate regulation is
important for cell size homeostasis. Size-scaling of macromolecule
synthesis and/or metabolism could in part explain the relative reduc-
tion of growth rate in larger cells. Overall, mRNA transcript abundance
increases with cell size due to increased global transcription in larger
cells [11,12]. Similarly, the proteome scales with cell volume [13] and
displays a uniform scaling across different subcellular compartments
and organelles across the cell cycle [7]. While overall macromolecular
biosynthesis appears to follow cell size changes closely, there are in-
dividual mRNAs and proteins that deviate from this general trend. Of
particular interest are cell cycle activators and inhibitors, which could
couple cell size to cell cycle progression. While the general principle for
the accumulation a critical amount of a specific protein was proposed
already 40 years ago [31,61], such proteins are sought after even today.
For example, cell size could influence cell cycle transitions if size-
scaling of an inhibitor and activator are different. In budding yeast, the
G1 cyclin Cln3 displays uniform size scaling [62]. In contrast, the levels
of Cln3 inhibitor Whi5 are diluted when cells grow in G1 phase. This
results in a threshold cell size where Whi5 levels cannot any more in-
hibit Cln3 activity allowing the yeast cells to progress in cell cycle [62].
An alternative cell cycle protein scaling has been proposed for cultured
Drosophila cells [63]. This model involves the mitotic kinase CDK1 and
its inhibitors, which are membrane bound Myt1 and nuclear Wee1. In
small cells, surface area and nuclear volume to cell volume ratios are

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M. Björklund
high, resulting in inhibitory activity towards Cdk1. When the cell
grows, the cytoplasm grows faster than the nucleus, and surface to
volume ratio decreases, resulting in weakened inhibition of CDK1 and
mitotic progression [63]. Mitotic regulation by cell size dependent ex-
pression of Cdc25 in fission yeast is yet another example how cells
couple their size and/or growth rate to cell division [64]. As Cdc25 is
the phosphatase opposing the activities of CDK1 inhibitory kinases
Wee1 and Myt1, this points out to the various ways how the master cell
cycle regulators such as Cdk1 can be regulated through size-scaling of
their inhibitors or activators. Based on these examples, it is not difficult
to envision that such differences in scaling of a cell cycle activator to
inhibitor could represent a general, and possibly highly redundant,
mechanism to ensure size-dependent passage through various stages of
the cell cycle. Such titration-based mechanism could also be achieved
through gene expression regulation. For example, if expression of a
specific DNA binding factor increases with cell size, this essentially
results in titration against a constant DNA amount, which would then
trigger transcription after a specific threshold point is achieved. An
example of such a mechanism is histone titration during midblastula
transition in frog oocytes [65].
Apart from scaling of individual cell cycle proteins, multiple mem-
bers of a particular signaling pathway and/or a specific cellular process
could display differential scaling with cell size. We have previously
found that the relative contribution of mitochondrial activity in mouse
liver is reduced when hepatocytes grow in size [12]. As mitochondrial
activity declines, hepatocytes are increasingly utilizing glycolysis and
other alternative biosynthetic pathways. Additional experiments with
cultured cells (both primary and immortalized) showed that mi-
tochondrial activity displays a nonlinear size-dependency [7], con-
sistent with observations of variable growth rate within the cell cycle
[16,20,23]. Mechanistically, the changes in mitochondrial activity were
found to be dependent on mitochondrial dynamics, the balance be-
tween mitochondrial fusion and fission [7]. As the connectivity of mi-
tochondrial network influences metabolic output [66,67], we specu-
lated that this could represent a mechanism to regulate cellular growth
rate and consequently cell size [8]. Mitochondrial fusion and fission are
regulated via multiple proteins, including mitofusins, OPA1, Fis1 and
Drp1 opening up possibilities to investigate their activity in regulation
of cell size and growth rate.
4.3. Growth rate and cell size – a two-way road
While specific details how cells adjust their metabolism and growth
rate to regulate size remain elusive, some general trends can be ob-
served. Most importantly, causal relationships are difficult to establish
due to the multidirectional relationship between metabolism, signaling
and the cell cycle [68–72]. For example, bacterial physiologists see that
cell size results from growth rate regulation [36,44]. While it may be
intuitive to claim that cell size is the product resulting from the reg-
ulation of growth rate and cell division, biophysical reasoning argues
against this causality. This is because larger cell size should constrain
nutrient uptake and waste metabolite disposal and influence growth
rate, thus reversing the causality. Large cell size therefore makes me-
tabolism less efficient due to increases diffusion times and reduction in
the cell surface area relative to cell volume (SV ratio; for a more ex-
tensive discussion, see [8]). This difference in SV ratio could be one of
the reasons for allometric scaling at the cellular level. In support to this
biophysical reasoning of size – growth rate causality, ecologists typi-
cally perceive that metabolic activity is a function of size as indicated
by Kleiber's law. An objective interpretation of all this is that growth
rate and cell size influence each other.
Interestingly, the influence of the SV ratio may extend beyond cell
size as cell shape induced local changes in SV ratio could influence
metabolic activity and result in localized growth. Cell shape related
differences in SV ratio are particularly evident in migrating cells as well
as neurons [73]. These shape-related local SV changes could potentially
414
BBA - Molecular Cell Research 1866 (2019) 409–417
cause local differences in cellular metabolism and signaling resulting in
targeted growth at specific cellular locations. While specific examples
are relatively scarce, such a mechanism could be important for example
for localized mTOR dependent activation of translation in axons [74].
For a more comprehensive discussion on cell shape induced local SV
ratio changes, see [73]. Importantly, local membrane growth could
result in further changes in local SV ratio further reinforcing the con-
clusion that the relationship between growth and size is bidirectional.
A logical conclusion based on the observed bidirectionality between
growth rate and cell size is that cells can sense their size. The cell size
dependent changes in metabolism, as observed for example in mouse
hepatocytes in vivo [12], suggests that metabolism, and consequently
growth rate, can be adjusted based on cell size. More generally, any
cellular behavior consistent with size dependent adjustment of growth
rate and/or cellular functions could be interpreted as a consequence of
cell size sensing. What could be the mechanism(s) enabling cells to
measure their size?
5. Mechanisms for (apparent) cell size sensing
I already mentioned that if cells are able to sense their size, this
could be an important mechanism for the maintenance of appropriate
growth rate and/or cell cycle progression. However, some people might
not agree that cells sense their size as the term ‘sensing’ implies active
monitoring. Nevertheless, it is helpful to think of different ways how
cells could measure their overall size or even the size of organelles or
other specific cellular compartments [75,76].
Experiments in unicellular model organisms have identified puta-
tive ‘rulers’ or ‘size sensors’, including Pom1 and Cdr2 kinases in fission
yeast [77–79] and FtsZ and DnaA proteins in bacteria (reviewed in
[28]). Definitive evidence is still lacking, partly because of the lack of a
clear consensus how a cell size sensor is defined. For example, it was
proposed originally that Pom1, a cell polarity protein kinase, acts as a
dose-dependent inhibitor of mitotic entry and forms a gradient from the
cell ends towards the middle, essentially acting as a sensor for cell
length (Fig. 4A). To investigate if Pom1 was an actual size sensor, Wood
and Nurse [15] used two criteria. First, deleting a gene involved in size
sensing should result in greater variability in cell size. Second, with the
size sensor deleted, the large and small cells should return less effi-
ciently to normal size. Based on these two criteria, they disputed that
Pom1 acts as a ruler to measure cell size [15].
Redundancy in biological systems, especially in those processes
which are important for cellular fitness and viability, may be the reason
why bona fide size sensors have been difficult to identify. In addition, as
with man-made biosensors, it is possible that a cell size sensor is
composed of multiple components, which are separately involved in
size measurement and the transmission of the physical information into
biochemical and molecular information. Thus, genetic loss-of-function
experiments may fail to identify such multi-modular cell size sensors or
sensor genes, which are essential for viability.
An additional mechanism for cell size sensing is via cell area or SV
ratio (Fig. 4A). Cell area has been proposed as an alternative me-
chanism to cell length measurement in fission yeast [79] and SV ratio in
bacteria and animal cells [12,80,81]. In case of bacteria, it was sug-
gested that availability of bacterial cell wall precursors impacts SV
homeostasis and consequently cell size. Similar mechanisms may also
be operational in animal cells as the mevalonate pathway that produces
cholesterol, strongly influences cell size [12,82].
It was proposed that the relative difference between surface and
volume growth rather than the SV ratio itself is the key determinant for
size control [80,81]. This further suggests that for geometric size sen-
sing, cells do not need to have a simple geometrical shape such as a
sphere or a rod. As long as cells are able to monitor excess external
surface component accumulation via any biochemical feedback me-
chanism, SV ratio monitoring can be accomplished. Whether the actual
SV ratio or the differential growth of surface and volume is important
M. Björklund BBA - Molecular Cell Research 1866 (2019) 409–417

Fig. 4. (A) Cells may sense their size through multiple mechanism, including local or global balance between the concentration of cell cycle activators [a] and
inhibitors [i]. For example, when local threshold is achieved as in case of Pom1 [i] gradient mediated inhibition of Cdr2 [a] in fission yeast, local Cdr2 activation
induces cell size dependent mitotic entry. A global, cell volume-related balance between Cln3 [a] and Whi5 [i] in budding yeast results in G1/S transition.
Alternatively, cells may sense their surface-to-volume ratio by monitoring the excess production of cell wall (e.g., in bacteria or plants) and/or cell membrane
material, illustrated as m (such as cholesterol in animal cells). This requires that accumulation of excess membrane material results in feedback that reduces cellular
volume growth and is independent of cell shape. (B) Proposed architecture of a putative cell size sensor. Such a sensor could comprise of a metabolic module that
senses either the growth rate or physical dimensions through one or more metabolic parameters. The size sensor is assumed to continuously monitor the cell size,
growth rate or metabolic rate. The output module is likely a cell cycle related protein and would cause growth rate adjustment only at cell size transitions such as G1/
S or G2/M, at other times size-sensing would be non-productive.

could be potentially dissected using 3D micro niches where individual
cells fill a predetermined shape [14], thus allowing one to analyse the
effect of SV ratio on cellular functions in the absence of growth.
Because cellular growth rate is so closely associated with metabolic
changes, it would be logical for cells to have a metabolic size sensor
rather than a kinase-based ruler that measures cell length. Sensing the
differences in surface and volume growth, as in bacteria cells could be
one of the easiest ways to achieve metabolic size sensing [80,81]. Ex-
perimental data indicates that size is adjusted more strongly at the cell
cycle transitions in animal cells [16], but also in plants where the shoot
apical meristem cell size is regulated at both major cell cycle check-
points (G1/S and mitosis) [83]. However, what we do not know for sure
is if the molecular networks involved in size sensing are acting con-
tinuously or only triggered at these specific times during the cell cycle.
If the size sensing system is composed of two or more modules, the
sensing and information transmission could be decoupled so that cells
would only adjust their size in relation to cell cycle transitions, despite
continuous size monitoring.
Based on this, a cell size sensor could be composed of a metabolic
module responsible for sensing cellular geometry and/or biosynthesis
and a cell cycle trigger module as an output (Fig. 4B). Such a sensor
would therefore link cell size to cell cycle progression and explain why
size adjustment occurs primarily at cell cycle transitions even if size
monitoring would be continuous. While the quest for the cell size sensor
is ongoing, it is important to remember that the apparent size-sensing
could also be an emergent property of the system rather than active
cellular computation based on the measurement of a single parameter
related to cellular dimensions.
6. Concluding remarks
Recent single cell studies show that animal cell growth during the
cell cycle is not purely linear or exponential, necessitating the future
development of better and more realistic models. While the technical
measurements are now increasingly more accurate, it appears that one
of the main limitations to understand cell size control is the inter-
pretation of experimental results. This is exemplified by the extra-
polation of the observed linear growth in S phase [3,4] and also illu-
strated by the fact that more than one model can equally well fit the
same high quality data [23,80]. In addition, single cell studies have
highlighted the fact that despite substantial variability in growth rate at
single cell level, this translates into stable cell size distribution and a
more predictable average growth rate at the population level. Some-
what counterintuitively, comparison of the ‘adder’ and ‘sizer’ models
suggests that proper maintenance of size homeostasis requires that
larger cells grow slower than small cells in relative terms. How do cells
sense their size to modulate their growth rate accordingly? The mole-
cular mechanisms by which cells regulate their size remain poorly un-
derstood and are likely to be more complex than anticipated. We need
to clarify how different signaling pathways contribute to cell size reg-
ulation, particularly through regulation of metabolism. For example,
while manipulation of mTOR activity alters cell size, this kinase is not a
key contributor to cell-size sensing in animal cells [14]. Instead, the p38
MAPK pathway reduces cell size variability while coordinating cell size
and cell cycle progression [33]. Neither is mTOR activity involved in
setting the cell size dependent mitochondrial activity, whereas the
mevalonate/cholesterol pathway is involved [5]. How can these results
be reconciled? Does the whole question of cell size control need to be
subdivided into two or more processes? The role of CDK4 in regulating

415
M. Björklund
cell's target size also warrants further investigation as this may directly
link cell size regulation with cancer as specific CDK4 inhibitors have
remarkable activity particularly in breast cancer [84]. While many
questions remain open, the recent advances clearly imply that we are
now several steps closer in understanding cell size control and the
implications of cell size homeostasis for human health.
Conflict of interests
The author declares no conflict of interests.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgements
The author wants to thank Dr. T. Miettinen and Dr. K.Y. Chan for
critical reading of the manuscript. This work was supported by funds
from Zhejiang University.
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