The big question of cell size
Wallace F Marshall
For well over 100 years, cell biologists have been wondering what determines the size
of cells. In modern times, we know all of the molecules that control the cell cycle
and cell division, but we still do not understand how cell size is determined. To
check whether modern cell biology has made any inroads on this age-old question, BMC
Biology asked several heavyweights in the field to tell us how they think cell size
is controlled, drawing on a range of different cell types. The essays in this collection
address two related questions - why does cell size matter, and how do cells control
it.
Why do cells care how big or small they are? One reason cell size matters is that
the basic processes of cell physiology, such as flux across membranes, are by their
nature dependent on cell size. As a result, changes in cell volume or surface area
will have profound effects on metabolic flux, biosynthetic capacity, and nutrient
exchange. A second reason is that the basic machinery of cell division in eukaryotes
relies on microtubules, both to form the mitotic spindle and position it properly
relative to the cortex. Because of the dynamic properties of microtubules, they are
able to probe a limited range of lengths, and if cells get too big or too small, the
mitotic apparatus may have difficulty working. Very small cells could not form a proper
spindle, and very large cells could not coordinate their divisions during cleavage.
This idea is elaborated in essays by Frankel and by Kimura, who discuss the apparent
upper and lower limits on cell size with respect to cell division machinery. Finally,
in both animals and plants, cells must fit together like puzzle pieces to form tissues
and organs, and that means that a cell has to have a size appropriate to its position
within the overall tissue, a topic discussed by Wallingford in the context of animal
development.
Given that cell size is important, how can a cell control how big it is? In terms
of 'design principles' for a size control system, the most fundamental question is
whether cells need to know how big they are in order to regulate size. The simplest
model is one in which cell mass grows at some rate determined by biosynthetic reactions
(the rate could be dependent on cell size or not), and as they are growing, the cells
divide at some constant frequency set by the cell cycle clock. Such a scheme would
not require cells to ever actually know how big they are, but as discussed by Swaffer,
Wood, and Nurse for yeast cells, experimental evidence rejects this simple model and
suggests instead that cells can measure their own size and regulate the timing of
cell division accordingly. This leads to the idea that cells can measure size, possibly
by reading out intracellular gradients. But as discussed by Young and by Qu and Roeder,
mechanical properties of the cell surface and of cytoskeletal elements can also play
a role in determining size.
At the end of the day regulation of cell size may prove to be the combined result
of several mechanisms operating in parallel, and that may be one reason it has been
hard to study.
Bacteria: appearances matter!
Kevin D Young
The most obvious characteristic of bacteria is that they are small. Really small.
As in requiring microscopes of high magnifying and resolving power to see them. So
it surprises people to learn that the volume of these normally tiny cells can differ
by as much as 106- to 108-fold, from the tiniest 0.2 μm cells of the Pelagibacter
SAR11 clade that fills the oceans [1] to the monstrous genera Thiomargarita and Epulopiscium
in which some species measure over 600 to 700 μm in length or diameter and are visible
to the naked eye [2-4]. Of course, large bacteria are an extreme minority, with most
known bacteria falling somewhere between 0.4 and 2 μm in diameter and 0.5 and 5 μm
in length (though many grow as filaments that can be tens or hundreds of times this
long). Another conceit is that bacteria are boring, at least in morphological terms.
But this is just because most of us rarely encounter bacteria outside of what are
usually brief episodes of disease, and the shapes of these common bacteria are admittedly
pretty lame, being, as they are, no more than tiny cylinders. However, on a more global
scale, bacterial shapes range from the plain (rods, spheres, strings) to the outlandish
(branched, curved, coiled, spiraled, star-shaped), to the truly bizarre (fluted and
tentacled) [5]. Given this range of possibilities, what determines the morphology
of any one bacterium?
The first determinant is, as always, evolutionary. Bacteria cope with at least six
fundamental selective forces that have some degree of control over the size that will
best suit them to survive in particular environments. Specifically, bacteria adopt
certain sizes and shapes so they can import nutrients most efficiently, meet requirements
imposed by cell division, attach themselves to external surfaces, take advantage of
passive dispersal mechanisms, move purposefully to pursue nutrients or avoid inhibitors,
or avoid predation by other organisms [5,6]. Fundamental to all these considerations
is that bacteria must accumulate nutrients that reach them by diffusion alone [7].
A basic tenet is that for such cells to exist the ratio of their surface area to cytoplasmic
volume has to be quite high. Therefore, to maximize this ratio, most bacteria produce
cells in the 0.2 to 10 μm size range and some organisms extrude long, exceedingly
thin appendages to harvest nutrients present in low concentrations [8]. Because of
this reliance on diffusion, those bacteria that reach near-millimeter size do so by
employing clever morphological tricks. For example, some deploy their cytoplasm as
a thin film around the outer rim of a large internal vacuole, creating a cell that
looks very much like the skin of a balloon [2,9]. Others localize tens of thousands
of chromosomes around the periphery of their cytoplasm, in near contact with the cell
surface, so that each genomic equivalent 'governs' a volume approximately equal to
that of a more normal, smaller cell [4]. Where a particular bacterium will eventually
land in this size universe depends on other selective forces, which basically revolve
around a bacterium's need to put itself in position to reach any nutrients at all
versus the need to defend itself against becoming a nutrient for others.
The second determinant of bacterial morphology is mechanical, a factor that encompasses
the biochemical mechanisms that do the heavy lifting of constructing cells of defined
sizes and shapes. The current consensus is that morphology is determined primarily
by molecular machines that synthesize the rigid cell wall. Three major types of machines
are available. One, directed by the protein FtsZ, is responsible for nucleating the
process of cell division and is shared by all bacteria, while the other, directed
by the protein MreB and its homologues, is responsible for cell elongation in rod-shaped
bacteria [10-13]. The third, first recognized by the activity of the CreS (crescentin)
protein of Caulobacter crescentus, is responsible for creating the curved cells of
this organism and the more regular shapes of other bacteria [14,15]. In a series of
conceptual surprises, it was realized that FtsZ is a homologue, and perhaps progenitor,
of the eukaryotic cytoskeletal protein tubulin [16,17], that MreB is a homologue of
actin [18,19], and that CreS and its relatives are homologues of intermediate filaments,
a third class of eukaryotic cytoskeleton proteins [14,15]. Though the structural similarities
are clear, these proteins have been co-opted to perform different functions in bacteria.
One last curiosity deserves mention: some classic metabolic enzymes also moonlight
as cytoskeletal filaments that affect bacterial shape, a discovery with potentially
far-reaching implications [20,21]. Finally, these basic tools can be modified, supplemented
or differentially regulated to create morphologies from the simple to the quite complex.
There is room here to give only three brief examples of how rod-shaped bacteria control
their overall size by varying cell length. The first involves Escherichia coli, a
plain cylindrical rod that is normally about 1 μm in diameter and 2 μm long. In this
organism, the future division site is determined by at least two mechanisms, each
of which inhibits the polymerization or function of FtsZ and thus regulates when and
where cell division occurs. First, driven by the MinD and MinE proteins, the MinC
inhibitor oscillates back and forth between the two polar ends of the cell, taking
approximately 1 to 2 minutes per cycle [22,23]. This behavior creates a time-averaged
MinC concentration gradient that is highest at the poles and lowest near mid-cell.
As the cell elongates, the concentration near the cell's center is reduced until it
becomes so low that FtsZ can polymerize and initiate cell division. Therefore, cell
size (as measured by length) is determined by the amount of MinC - larger amounts
produce longer cells. Conceptually, this is eerily similar to the mechanism that regulates
cell length in rod shaped fission yeast, as described by Swaffer et al. in this Forum
article (below). Though there are biochemical differences, in this eukaryote cell
length is regulated by a concentration gradient of Pom1 that is highest at the poles
of a growing cell. Division is therefore inhibited until the cells become long enough
so that the concentration of Pom1 at the cell center drops low enough to allow division.
The second way E. coli regulates cell length is by a 'nucleoid occlusion' mechanism
[24]. Here, the SlmA protein binds to specific DNA sequences, and the SlmA-DNA complex
prevents cell division by inhibiting FtsZ. Interestingly, SlmA binding sites are distributed
around the chromosome except near the area where DNA replication terminates. During
chromosomal segregation the two origins are pulled to either pole, and the two termination
regions remain near the cell center, where they are the last to be replicated and
separated. This means that as replication ends and when segregation is almost complete
there will be a dearth of SlmA near mid-cell, at which time FtsZ will no longer be
inhibited and can trigger division. Again, note how similar this is to the kind of
mechanism that may explain how chromosomal ploidy determines cell length in yeast
(see the contribution from Swafer et al. in this Forum article, below).
Recently a third, and surprising, mechanism was discovered by which cell length is
tied to the metabolic status of the cell. Bacillus subtilis, a rod shaped bacterium
about 1 to 2 μm in diameter and 5 to 10 μm in length, is longer when incubated in
a nutrient-rich medium and shorter when nutrients are limited. Although it sounds
simple, the question of how bacteria accomplish this has persisted for decades without
resolution, until quite recently. The answer is that in a rich medium (that is, one
containing glucose) B. subtilis accumulates a metabolite that induces an enzyme that,
in turn, inhibits FtsZ (again!) and delays cell division. Thus, in a rich medium,
the cells grow just a bit longer before they can initiate and complete division [25,26].
These examples suggest that the division apparatus is a common target for controlling
cell length and size in bacteria, just as it may be in eukaryotic organisms.
In contrast to the regulation of length, the MreB-related pathways that control bacterial
cell width remain highly enigmatic [11]. It is not just a question of setting a specified
diameter in the first place, which is a fundamental and unanswered question, but maintaining
that diameter so that the resulting rod-shaped cell is smooth and uniform along its
entire length. For some years it was thought that MreB and its relatives polymerized
to form a continuous helical filament just beneath the cytoplasmic membrane and that
this cytoskeleton-like arrangement established and maintained cell diameter. However,
these structures seem to have been figments generated by the low resolution of light
microscopy. Instead, individual molecules (or at the most, short MreB oligomers) move
along the inner surface of the cytoplasmic membrane, following independent, almost
perfectly circular paths that are oriented perpendicular to the long axis of the cell
[27-29]. How this behavior generates a specific and constant diameter is the subject
of quite a bit of debate and experimentation. Of course, if this 'simple' matter of
determining diameter is still up in the air, it comes as no surprise that the mechanisms
for creating even more complicated morphologies are even less well understood.
In short, bacteria vary widely in size and shape, do so in response to the demands
of the environment and predators, and create disparate morphologies by physical-biochemical
mechanisms that promote access to a huge range of shapes. In this latter sense they
are far from passive, manipulating their external architecture with a molecular precision
that should awe any contemporary nanotechnologist. The techniques by which they accomplish
these feats are just beginning to yield to experiment, and the principles underlying
these abilities promise to provide valuable insights across a broad swath of fields,
including basic biology, biochemistry, pathogenesis, cytoskeletal structure and materials
fabrication, to name but a few.
The puzzling influence of ploidy
Matthew Swaffer, Elizabeth Wood, Paul Nurse
Cells of a particular type, whether making up a specific tissue or growing as single
cells, often maintain a constant size. It is usually thought that this cell size maintenance
is brought about by coordinating cell cycle progression with attainment of a critical
size, which will result in cells having a limited size dispersion when they divide.
Yeasts have been used to investigate the mechanisms by which cells measure their size
and integrate this information into the cell cycle control. Here we will outline recent
models developed from the yeast work and address a key but rather neglected issue,
the correlation of cell size with ploidy.
First, to maintain a constant size, is it really necessary to invoke that passage
through a particular cell cycle stage requires attainment of a critical cell size?
If cells grow linearly - that is, the rate at which they accumulate mass in unit time
is constant regardless of the mass of the cell - and if the time between successive
cell divisions is maintained by a fixed timer, then cells will maintain size homeostasis.
In successive generations all cells will slowly tend towards an average size [30].
However, work from both fission and budding yeast has shown this not to be the case
[31]. Firstly, the variation in sizes at division of both yeast species is too small
to be accounted for by such a process [31]. Secondly, cell cycle arrest of fission
yeast results in enlarged cells that exhibit significantly shortened subsequent cycles
[32]. This rapid reversion to the original cell size indicates the presence of a size
correction mechanism. Similarly, in budding yeast, cells born smaller than normal
spend longer in G1 until they reach a critical size [33]. There is also no evidence
to suggest that yeast cells accumulate mass in a simple linear way for extended periods
of time [34-37]. Therefore, there is a mechanism that monitors cell size and uses
this information to regulate progression through events of the cell cycle. In the
case of fission yeast this occurs primarily during G2 [38,39] but can operate in G1
[39,40], and for budding yeast it occurs during G1 [33,41].
Two different molecular mechanisms for size control have been proposed for the two
yeasts. In fission yeast, Cdc2 (Cdk1) kinase activity drives entry into mitosis and
thus determines the length of G2 [42]. Wee1 catalyzes inhibitory phosphorylation of
Cdc2 on Tyr15 and is antagonized by the phosphatase Cdc25 [42-46]. Cell size information
is transduced via Cdr1 and Cdr2, inhibitors of Wee1 that localize to cortical nodes
at the center of the cell [47]. Pom1 is a kinase that inhibits Cdr1/Cdr2, thus alleviating
inhibition of Wee1 [47,48]. A gradient of Pom1 emanating from the cell tips inhibits
G2/M until cells reach a critical length, and as cells are rod-shaped this is correlated
with cell size. Pom1 is delivered to and associates with the plasma membrane at the
cell ends. Pom1 autophosphorylation results in membrane dissociation, generating the
Pom1 gradient, with a high concentration at the tips decreasing towards the cell center
[49]. In a small early G2 cell there is sufficient Pom1 at the cortical nodes to inhibit
Cdr1/Cdr2, preventing mitotic entry (Figure 1a). As cells grow and elongate, Pom1
concentration at the medial site becomes progressively lower, Wee1 is eventually inhibited
by Cdr1/Cdr2, and cells commit to mitotic division [47,48] (Figure 1b). In this way
the size of the cell regulates mitotic entry. However, this is unlikely to be the
whole story because in cells where Cdc2 Tyr15 phosphorylation is prevented from occurring
(thus bypassing Pom1-mediated regulation) cell size homeostasis is maintained, albeit
with a broader size distribution [50]. This indicates other unknown mechanisms operate
to measure size and integrate this information into cell cycle control.
Figure 1
The Pom1 gradient model for length sensing in fission yeast. (a) An early G2 cell.
Pom1 protein emanating from the cell tips (purple) inhibits Cdr1/Cdr2 in the cortical
nodes (light blue circles). Wee1 is therefore active and carries out inhibitory phosphorylation
of Cdc2. (b) A late G2 cell. Pom1 concentration at the medial site is decreased. At
a critical threshold, Cdr1/Cdr2 are no longer inhibited (dark blue circles) and so
the Wee1 inhibition of Cdc2 is lifted. The active CDK drives mitotic entry.
In budding yeast, size control operates at 'start', a G1 event that commits the cell
to cycle at a given size [33,41]. This is thought to operate by a protein synthesis-rate
sizer mechanism involving the G1 cyclin Cln3 [31,51,52]. Cln3 is a dose-dependent
activator of start [53-55], and is rapidly degraded [56]. Its high turnover rate means
that the amount of Cln3 should be a direct reflection of the current rate of protein
synthesis within the cell [57]. Since the number of ribosomes is indicative of cell
size, protein synthesis rate will correlate with cell size. Therefore, only once a
certain cell size is reached will there be sufficient Cln3 to drive the transition
through start. This is thought to involve the activation of the transcription factor
SBF by Cln3-CDK-mediated inhibition of Whi5, an SBF inhibitor [58,59]. A major problem
with using protein synthesis rate as a proxy for cell size is that as the cell gets
larger and more Cln3 is produced, the corresponding increase in cell volume should
dilute out the protein, keeping it at a constant concentration. This appears to be
the case for Cln3, as its relative abundance does not significantly change as cells
grow during G1 [55]. To overcome this problem it has been proposed that Cln3 import
into a nucleus of fixed size would allow the cell to 'measure' the absolute amount
of Cln3 [51]. However, it has been shown for both budding and fission yeast that nuclear
volume increases with the size of the cell [60,61], so this particular model remains
incomplete and still requires a fixed 'standard' against which to measure Cln3.
Both of these models are interesting but, at least in their simplest form, they cannot
account for a close to universal aspect of cell size: that is, the almost directly
proportional increase in size at division that is observed as ploidy increases [62,63].
This relationship holds within a ploidy series of a single species, as well as across
species [60,64]. This observation indicates that somehow cells can monitor their ploidy
and integrate this information into the cell size-monitoring mechanisms. In general
terms we envisage two types of model by which this might operate: either the cell
makes a specific amount of a critical component according to ploidy, or it measures
the amount of a given factor against ploidy.
An example of the former model would be if the transcript of the critical component
were produced as a single pulse at a specific time in the cell cycle. The size of
this burst of transcription would be a direct function of copy number, and as such
a reflection of ploidy. If the gene product is stable and acts to inhibit division,
as a cell grows, this fixed amount will be diluted down. Below a certain threshold
concentration, its inhibitory effect is alleviated and division is permitted [65].
With an increase in ploidy there would be a requirement for a cell to be proportionately
larger before the threshold is reached. If any gene were to operate in such a copy
number-dependent inhibitory manner, it would be expected that a heterozygous diploid
of such a gene would produce half as much protein (with no compensation) and therefore
be the size of a haploid. It would be interesting to screen for genes behaving in
this manner, but to our knowledge no such gene has yet been described. This may suggest
that the model is an oversimplification, and such a mechanism might involve a number
of interacting factors resulting in greater redundancy and adding robustness to the
system.
An example of the second type of model would be a genomic titration mechanism [65,66].
This model invokes a protein that is maintained at a constant concentration, and which
binds sites in the genome. As the cell grows larger, the absolute amount of this factor
increases and thus more genomic sites become occupied. A critical threshold size is
reached when a certain number of sites are bound. The occupancy of these sites could
functionally drive a cell cycle transition - for example, by regulating transcription.
Alternatively, this threshold could be a point of saturation at which no more sites
are available to bind and the factor is free in the nucleoplasm or cytoplasm. The
unbound factor could execute a pro-division function, which could even involve binding
another DNA sequence for which it has lower affinity. A doubling in ploidy would be
accompanied by a doubling in the number of DNA sequences for the protein to bind.
This would impart the requirement on the cell to be twice the size before the occupancy
threshold is surpassed. These are merely examples of the two types of models that
could allow ploidy to regulate cell size and other variants are also possible [65].
Is it possible to modify the two proposed molecular mechanisms described above for
yeast size control to take account of the effects of ploidy? With respect to the fission
yeast Pom1 gradient model, perhaps the amount of a critical component in the network
could be determined by gene copy number. In principle this could be the inhibitor,
Pom1, although this is unlikely as deleting one copy of pom1 in a diploid does not
reduce cell size to that of a haploid (Jacqueline Hayles, personal communication).
Turning to the Cln3 activator model, it has recently been shown that Cln3 can bind
to SBF binding sites across the genome [67]. Introduction of additional SBF binding
sites increases cell size at 'start' in a Cln3-dependent manner [67]. It is plausible
that this allows the cell to measure the amount of Cln3 against the genome, so only
when a fixed number of sites are occupied is division permitted, as per the genome-titration
model discussed above. This provides a possible solution to the aforementioned problems
with the Cln3 protein synthesis rate sizing mechanism, as well as a means for ploidy
to regulate size control directly. Other mechanisms, operating outside of the known
size control network, that take account of ploidy could also be envisaged.
The universality of cell size scaling with ploidy means that ploidy should be taken
account of when considering cell size-sensing mechanisms. It may also imply that there
is conservation of the mechanisms involved, although whether this conservation exists
at the level of molecules or of control network architecture remains to be seen.
Physical limits of cell size for embryonic cell division in Caenorhabditis elegans
Akatsuki Kimura
Early embryos are a good model for studying the relationship between cell size and
intracellular organization. Blastomeres can exhibit various sizes during embryonic
cell division since cells divide without cell growth during this phase. In addition,
embryonic cells are generally large, which makes these cells useful models for microscopic
observation. Thus, transparent Caenorhabditis elegans embryos represent an ideal model
for investigating these relationships [68-71]. Here, I argue, based on prior studies
in C. elegans and other systems, that cell size may be limited by the physical properties
of the cell. In order to proliferate, the cell has to divide, and for faithful cell
division, molecular machinery, such as the mitotic spindle, must be constructed at
the right position and with the correct size. This may not be accomplished in extremely
large or small cells due to the physical properties of macromolecules, such as microtubules
and chromosomes.
Positioning of the mitotic spindle at the cell center is critical for symmetric cell
division, as it defines the origin of sister chromatid segregation and the position
of the cell division plane [72]. The centrosome is a major organizing center of the
microtubule cytoskeleton and in animal cells often includes the poles of the mitotic
spindle. Centrosomes have the ability to position themselves at the cell center [73],
enabling the mitotic spindle also to position at the cell center [74,75] (Figure 2).
This central positioning of the centrosome is accomplished through the function of
the microtubule cytoskeleton [76,77]. Several mechanisms have been proposed to describe
how microtubules bring centrosomes to the cell center, including pushing of the cell
cortex and pulling by motor proteins [74,77-88]. The mechanisms mediating centrosome
centration may differ among species, especially among species with different cell
sizes [83]. Recent studies have supported the idea that the cytoplasmic pulling force
is a major driving force for centrosome centration in animal cells [75,78,81,86,89].
Importantly, for all proposed mechanisms, microtubule-dependent centration of the
centrosome must be facilitated by microtubules, which grow from the centrosome and
span throughout the cytoplasm to find the geometrical center of the region [87,88].
Figure 2
Microtubules in C. elegans
. An image of microtubules in an embryonic cell - astral microtubules from the spindle
reach the cell cortex.
If the cell is too large compared to the length of microtubules, the centrosome will
not position at the cell center (Figure 3a, middle panels). Since the microtubules
grow and shrink in a stochastic manner known as dynamic instability [90], the mean
length of microtubules (nav
) is defined by the velocities of growth (v+
) and shrinking (v-
), the frequency of switching from growth to shrinking (f+-
) and vice versa (f-+
), as nav
≈ (v-v+
)/(v-f+-
- v+f-+
) [91]. According to this equation, mean lengths are estimated to be in the micron
range based on experimentally measured dynamic instability parameters in vitro [92]
and in vivo [93], which is consistent with observed in vivo microtubule lengths [91,94].
Interestingly, this length is comparable to the size of ordinary animal cells, suggesting
that the length scale of a microtubule is related to the size of the cell. Therefore,
the length of microtubules may define the upper limit of cell size. This idea has
been supported by experimental shortening of microtubule length. When cells were treated
with microtubule depolymerizing drugs or partial knockdown of microtubule polymerizing
molecules, centrosomes did not reach the cell center and the cell division plane was
positioned in an asymmetric manner [76,95,96]. Interestingly, studies have demonstrated
that centrosomes can find the cell center in extremely large cells, such as newly
fertilized frog embryos [97] (see the contribution from Dr Frankel in this Forum article,
below). In large embryos, a large microtubule aster that expands throughout the cell
is formed and centers the centrosome toward the cell center, possibly due to cytoplasmic
pulling forces [89]. In an in vitro centering experiment using microfabricated chambers,
it was demonstrated that efficient elongation of microtubules to reach the boundaries
of the chamber was critical for robust positioning of the microtubule aster at the
center of the chamber [88]. These studies collectively support the idea that cell
radius cannot exceed the maximum length of microtubules for cell proliferation.
Figure 3
Possible scenarios in which centrosome centering (a) and spindle elongation (b) set
the upper and lower limit of cell size. (a) If the cell exceeds the upper limit of
size, the centrosome, and consequently the mitotic spindle, cannot position at the
cell center, leading to nonsymmetrical cell division (middle panels versus left panels).
If the cell falls below the lower limit, the centrosome may not stably position at
the cell center due to the excess elastic forces of the microtubules (right panels
versus left panels). (b) If the cell exceeds the upper limit, astral microtubules
do not reach the cell cortex, potentially leading to insufficient spindle elongation.
If the cell falls below the lower limit, there may not be sufficient space for accurate
chromosome segregation compared to the size of the cell's chromosomes.
How are the lower limits of cell size defined? The centrosome may not correctly center
if the cell is too small compared to the length of the microtubules. Because of the
elastic properties of microtubules, short microtubules generate strong pushing forces
when the growing tips encounter obstacles such as the cell cortex. Due to the reactions
of these pushing forces, the centrosome will be subjected to strong forces from multiple
microtubules, which may destabilize the positioning of the centrosome (Figure 3a,
right panels). This idea is based on an elegant in vitro centering experiment using
microfabricated chambers combined with theoretical analyses [79,98,99]. This experiment
was performed in a cell-size chamber (a square with about 20 μm on a side), but the
length of the microtubules were long due to the lack of shrinking; many microtubules
reached the chamber boundaries, exerting pushing forces against the chamber walls.
Under these conditions, microtubule asters moved away from the center of the chamber
[79] or failed to reposition to the cell center after relocation [99]. This off-center
movement was restored by promoting microtubule shrinkage [99]. Shortening of the average
length of the microtubule by promoting shrinkage resulted in fewer microtubules reaching
the cortex, thereby generating less pushing force, and stably positioned the aster
at the geometrical center [99]. This experiment indicated that, in small cells where
many short (and thus rigid) microtubules reach the cell cortex, the centrosome cannot
stably position at the cell center.
In addition to the physical properties of microtubules such as the length and stiffness,
other properties may also define the limits of cell size. Elongation of the mitotic
spindle during anaphase is known to depend on cell size; the larger the cell, the
longer and faster the spindle elongates. To date, this trend has only been demonstrated
in the C. elegans embryo [68,69]; however, it may represent a general trend in other
cells. If a spindle elongates only for a short distance in small cells, the separation
of sister chromatids may not be enough to segregate them completely. In the yeast
Saccharomyces cerevisiae, artificially elongated chromosomes become more condensed
at anaphase to ensure complete segregation of the chromatids [100]. This observation
indirectly implies that the spindle must elongate to a certain distance in order to
segregate chromosomes with a certain size [101]. Within a given genome size, there
should be a lower limit to the size of condensed chromosomes, which may further define
the lower limit of the elongation of the mitotic spindle, thus defining the size of
the cell (Figure 3b, right panels).
Elongation of the mitotic spindle may define the upper limit of cell size as well.
Since elongation is partly driven by pulling astral microtubules from the cell cortex
[102,103], if astral microtubules do not reach the cell cortex in large cells, the
elongation of the spindle is impaired, potentially causing insufficient chromosome
segregation (Figure 3b, middle panels). To my knowledge, there has been no experimental
evidence to support this idea so far. However, when the cortical pulling force was
impaired in C. elegans embryos, we were able to shorten spindle elongation, albeit
not completely [69]. Chromosomes appear to manage segregation under these conditions.
The identification of genes responsible for the remaining elongation may allow us
to test whether impairing the interaction between astral microtubules and the cell
cortex leads to chromosome segregation defects.
In this text, I discussed a basic and simplified view of the relationship between
cell size and the material properties of macromolecules comprising the mitotic machinery.
As the biology always has to deal with diversity, an individual cell may have its
own unique mechanism to set cell size and to accomplish cell division. Nevertheless,
the diversity in cell size among species is far smaller than that in body size, suggesting
common constraints for the majority of cell types. I believe understanding such common
constraints will reveal the basic design principle of cell architecture.
The largest dividing cells: are they alike?
Joseph Frankel
'Physical extremes, in this case a very large cytoplasm, are always interesting in
biology' [97]
How can we frame a useful inquiry about the upper limits of cell size?
If one asks which cell is the largest, one comes up with a list of highly diverse
candidates. Those on the centimeter scale include the ostrich egg measured at 8 cm
by EB Wilson [104], the 3 to 5 cm unicellular stalked marine alga Acetabularia [105],
and various giant shelled (testate) amoeboid denizens of the deep-sea bottom, including
the 3 cm 'living fossil' Gromia sphaerica [106]. The multinucleate green alga Caulerpa
is, however, the champion unicellular organism, with a tubular stolon extending to
a length of one meter or more, as well as a remarkable degree of internal differentiation
and a propensity for rapid vegetative growth [107].
The modes of propagation of Acetabularia and Gromia are typical of most marine giant
single-celled organisms. They do not appear to undergo binary fission, but instead
become multinucleate for at least part of their life cycle, after which they produce
large numbers of flagellated gametes [108,109], each of which includes only a small
portion of the overall cytoplasmic mass of the large parent cell [105,108]. The same
is true for Caulerpa during its episodes of sexual reproduction [110]. Thus, these
giant cells can endow their progeny with DNA and elementary organelles such as mitochondria
and (in algae such as Acetabularia and Caulerpa) also chloroplasts [109,110], but
they almost certainly do not transmit any significant portion of their cytoplasmic
organization to their individual offspring.
All of these enormous cells have some device for escaping the consequences of their
large size when they reproduce, either by cleaving only a small portion of their mass,
as in reptilian or bird eggs, or by subdividing all or part of their large cell bodies
to produce swarms of diminutive progeny, as in centimeter- and meter-scale marine
unicellular organisms such as Acetabularia or Caulerpa. When producing reproductive
cells, such organisms probably do not need to make any global assessment of their
overall dimensions and organization. In my view, the interesting upper size limit
is the largest size at which a cell can carry out such a global assessment and then
make use of this assessment to perpetuate itself by dividing into two daughter cells
similar in form and structure to itself. We can then ask what this size limit actually
is.
An interesting 'test case' is provided by two amoebae, Amoeba proteus and Chaos chaos,
that are known to be very closely related [111]. Amoeba proteus is roughly 500 μM
in length when actively moving [112], is uninucleate, and goes through a fairly typical
process of mitosis and cytokinesis [113]. By contrast, the giant amoeba, Chaos chaos
(A.K.A. Chaos carolinensis, Pelomyxa carolinensis) measures 1 to 5 mm when extended
[112] and is multinucleate. While these nuclei undergo synchronous mitoses [114],
the subsequent cell division is very atypical: it simultaneously produces between
two and six daughters, and the several hundred nuclei of the parent cell are segregated
apparently at random among these division products [115]. This process, called 'plasmotomy'
by Kudo [114], is a far cry from the typical mitotic cell division found in the closely
related but much smaller uninucleate Amoeba proteus.
This example introduces a problem: large cells typically need more DNA than normal-sized
cells to support their metabolic and synthetic needs. In extreme cases, they may become
multinucleate, in which case the multinucleate condition itself may generate an obstacle
to normal cell division. Even more profoundly, the extensive endoreduplication of
DNA that is often found in large differentiating cells in plants is closely associated
with cessation of cell division [116] (also see the contribution from Qu and Roeder
in this Forum article).
Two distinct types of large cells have found different ways of circumventing these
obstacles to binary fission. The ciliates do so by possessing a single large polygenomic
macronucleus that maintains the vegetative functions of the cell and divides amitotically
together with the cell in which it resides. Cleaving eggs do not need multiple nuclei
or polytene chromosomes because they possess abundant stored maternal mRNA and (generally)
delay the onset of zygotic nuclear transcription, thereby allowing several initial
rounds of nuclear division and cytoplasmic cleavage to occur in rapid succession.
I shall here consider these two examples of the consequences of progressive enlargement,
in ciliates and in amphibian eggs, the first analyzed using a microsurgical approach
and the second following a comparative approach. Comparison of these two cell types
indicates that there is a fairly constant upper size limit to whatever organization
permits normal cell division, but that the specific organization appears to be markedly
different in these two types of cells.
A microsurgical approach: grafting Stentor
The paradigm for the microsurgical approach has been provided by the largest ciliate,
Stentor coeruleus, measured by Morgan [117] at 1.4 to 2.8 mm in length when fully
extended (Figure 4). Vance Tartar carried out a systematic program of intra-species
grafting to discover the limits of its recovery of normal form and of its capacity
for cell division. Comprehension of Tartar's findings requires awareness of three
basic facts about Stentor: first, 'that cell shape is an expression of the cortical
stripe pattern' ([118], p. 211), which in turn is closely associated with the pattern
of ciliary rows that are interdigitated among the stripes; second, that Stentor is
capable of maintaining one, two or three (but not four or more) parallel sets of major
cortical landmarks (oral structures and oral primordia) without severely compromising
its normal cell form; and third, that the consistency of its internal cytoplasm makes
it possible for an experimenter, with some skill and practice, to fuse whole stentors
together in any orientation, or to graft parts of one stentor onto another stentor
[118].
Figure 4
A descriptive diagram of Stentor coeruleus
. All of the features shown are on the cell surface, except for the macronuclear nodes,
the micronuclei, and the contractile vacuole, all three of which are located just
beneath the surface. The cortical fibrillar system, not shown in the diagram, is located
within the clear stripes between the granular (pigmented) stripes. From Figure 1 of
[118]. Image courtesy of Biodiversity Heritage Library. http://www.biodiversitylibrary.org
The overall result of Tartar's analysis of 272 combinations of whole stentors grafted
together in random orientations is best summarized in Tartar's own words: 'Fusion
masses of two to four stentors were generally capable of recovering fully or approximately
the monaxial, normal body shape and of dividing thereafter...to give single, doublet,
and triplet progeny. In the larger grafts (involving random fusion of more than four
whole stentors) both shape recovery and cell-division were lacking. ...the masses
were unable to even begin fission; only artificial cutting up of the masses into approximately
normal volumes produced normal singles' ([119], p. 564). These fusion masses (which
could not feed) 'lived for about the same period of time as starved controls' ([119],
p. 569) implying that their failure was most likely due to starvation resulting from
their inability to feed rather than to anoxia resulting from their increased mass
and reduced surface-to-volume ratio. This inability to feed was a consequence of the
inability of these large masses of artificially fused stentors to form normal oral
structures, for reasons to be explored below.
Further analysis revealed that surgical manipulation of the cortical pattern itself
could influence the recovery of normal form. Microsurgical disarrangement of large
blocks of cortex resulting from grafts of large portions of stentors in unnatural
arrangements often brought about bizarre structural outcomes, whereas stentors demonstrated
'an astonishing capability...to regenerate and to reconstitute the normal, orderly
arrangement of the ectoplasmic pattern...after all of the complex ciliary, contractile,
conductive and other differentiations of the ectoplasm have been cut into tiny pieces
scattered at random' ([118], p. 224). Further, such thorough disorganization ('minceration')
of the cortex of Stentor increased the upper size limit for attainment of normal form:
a minced six-mass (a group of six stentors artificially fused together followed by
random slicing into the cell cortex of the fusion mass using a fine glass needle)
'had succeeded, as un-minced six-masses do not, in reconstituting a doublet with a
single-cell shape' ([120], p. 200) (Figure 5). Even a 25-mass (that is, 25 stentors
artificially grafted together), after minceration, could manage a partial recovery
of normal form, although with no oral differentiation (see below).
Figure 5
Random slicing into the cell cortex ('minceration') facilitates the integration of
a large fusion mass. (a) A graft of six whole stentors fused together, minced, plus
an implanted oral apparatus. (b) After two days, four small oral primordia were formed
at irregular sites. (c) By the sixth day, the fusion mass reconstituted a doublet
with normal oral structures and a cell shape resembling that of a normal single cell.
From Figure 12 of [120].
These observations appear paradoxical until one realizes that the graft-fusions were,
as Tartar pointed out, mostly random. Thus, an un-minced fusion complex had large
blocks of normally juxtaposed cortex rearranged in a coarse crazy-quilt disorder.
The minceration effectively made the pieces of the quilt much smaller. These small
pieces then could rotate on the fluid endoplasm, and later come into alignment with
other such pieces, with selection probably favoring homopolar alignments, thereby
enabling a gradual reconstruction of a coherent and fairly normal cortical pattern
[120].
But then what is the basis of the upper size limit? Here we need to introduce one
further element of Stentor lore. That is the notion of gradients. The German investigator
Gotram Uhlig, who carried out his own experimental analysis of Stentor morphogenesis
independently of Vance Tartar in the 1950s, explained most of his results on the basis
of two interacting gradients, one basal-apical and the other circumferential [121].
Tartar subsequently adopted Uhlig's basal-apical gradient to account for certain otherwise
inexplicable results of one of his own experiments [122]. The relevance of this postulated
gradient in the current context is that one of its principal expressions is the induction
of mouthparts at the posterior end of the oral apparatus of Stentor as it develops
within the primordium site shown in Figure 4.
Returning to the Stentor-masses resulting from the fusion of whole stentors in random
orientations, minced six-masses were able to form normal oral apparatuses with mouthparts
(oral pouch and gullet, Figure 4), whereas minced 25-masses were not; they instead
produced 'two garlands of adoral cilia without cytostomes' (that is, membranellar
bands lacking the oral pouch and gullet) superimposed on a rough approximation of
the normal Stentor form ([119], p. 559). Failure to form proper mouthparts within
such 'garlands' was typical for the large fusion-masses, which Tartar attributed to
'the presence of numerous cell axes running in random directions and canceling each
other in their polar influences' ([118], p. 215). But failure would also be expected
if the sheer size of the large Stentor-masses rendered them unable to reconstitute
a normal basal-apical morphogenetic gradient. Recalling that if the large masses were
cut into smaller pieces they could then regenerate normal single stentors, one may
ask whether the insuperable dilemma faced by these large masses is due to their excessive
structural complexity or to their large size or to some combination of the two. This
issue may be ripe for re-investigation with modern means for visualizing cytoskeletal
organization.
Vance Tartar did not supply scale-bars for his published drawings of operated stentors.
While he acknowledged his failure to obtain 'super-giant normal stentors' ([119],
p. 567), the normal-appearing doublet that emerged from a fused and minced six-mass
(Figure 5) must nonetheless have been at least transiently larger than a normal Stentor.
Assuming an extended length of a normal Stentor in the order of 2 mm, I would then
estimate a maximum linear (basal-to-apical) dimension for form regulation in Stentor
coeruleus at somewhere close to 3 mm.
A comparative analysis: early cleavages in amphibians that lay extra-large eggs
In animals, the largest dividing cells are eggs. The largest totally cleaving (holoblastic)
eggs are found among amphibians. The most familiar frogs (such as Rana pipiens and
Xenopus laevis) and salamanders (such as Ambystoma mexicanum and Triturus) lay eggs
with a diameter between 1 and just over 2 mm [123,124]. However, other species found
in both of the two major amphibian orders (anurans and urodeles) produce eggs that
range up to 10 mm in diameter. This large size of the egg is associated with direct
development and/or parental care [125,126]. Unlike the situation with Tartar's grafted
stentors, we know that even the largest eggs somehow manage to complete their development.
Nonetheless, we can still ask whether their early cleavages remain regular and orderly
as egg diameter increases. The simple answer is that they do not.
The pattern of cleavage has recently been investigated in two large-egged amphibian
species: one marsupial frog and one land-dwelling salamander. Early cleavages in the
marsupial frog Gastrotheca riobambae, which has an egg diameter of 3 mm, are holoblastic,
extremely slow, commonly asynchronous, and frequently asymmetric in that they do not
cut the egg into equal halves. Hence, when different eggs at the same cleavage stage
are viewed from above the animal pole, each one has a different pattern of blastomeres,
and the number of complete blastomeres at any given time is commonly not in the series
of 2n [127]. The plethodontid salamander Ensatina eschscholtzii, the largest appropriately
studied amphibian egg, has a 6 mm diameter, and has taken a further step in the direction
of meroblastic (partial) cleavage. Early cleavages (beyond the first two) are extremely
irregular, and 'cleavage initially occurs only in the animal pole, with no cleavage
furrow visible in the vegetal pole until about the 16-cell stage' ([124], p. 3). This
implies that progression of cleavage furrows from the animal into the vegetal region
is either very slow or delayed, probably due to the high concentration of yolk in
the vegetal hemisphere.
Collazo and Keller [124] discuss the modifications of cleavage in large-egg amphibians
in the light of a distinction proposed by SJ Gould, between 'historical' and 'formal'
developmental constraints. Historical constraints are ones that depend upon contingencies
of ancestry and descent. Formal constraints exist independently of ancestry and are
dictated by physical principles or restrictive structural relationships [128]. Collazo
and Keller attribute most of the modifications of early development found in large-egged
salamanders to historical constraints, because these are diverse in different lineages.
However, they make an exception for the effect of egg size on early cleavage patterns,
which they attribute to a formal constraint in the Gouldian sense. In their words,
'The fact that the asymmetries and asynchronies in early cleavage seen in these four
species (the three others are from early 20th Century descriptions of large-egged
salamanders) are qualitatively similar and that these species represent two disparate
salamander families suggests that large egg size and not phylogenetic relationship
accounts for the differences in development from amphibians with smaller eggs' ([124],
p. 8).
To a first approximation, the threshold in linear dimension between regularity and
irregularity of amphibian cleavage appears to be somewhere between 2 and 3 millimeters
- similar to the size threshold in capacity of Stentor-masses to regulate to the normal
Stentor form (and concomitantly the normal capacity to divide). This might be a general
size-limit in the capacity for a well-organized binary cell division.
Divergent organization: outside-in versus inside-out
Even if we accept that a similar upper size threshold exists for normal cell division
in Stentor and in amphibian (and fish) eggs, it could still be that the similarity
in these size thresholds is coincidental. This is especially likely because the respective
roles of the cortex and the endoplasm in the cell's accommodation to large cell size
appear to be opposite in the two types of cells.
In Stentor as in other ciliates, the cortical layer is structurally the most highly
organized part of the cell. That is where the granular pigment stripes and the intervening
cortical fibrillar system, including basal bodies, cilia, and accessory microtubular
bands, are located. The nodes of the macronucleus adhere to the cortical layer and
several small micronuclei are nearby [118] (Figure 4). An ultrastructural study of
a closely related ciliate (Blepharisma) has shown that the mitotic division of the
micronucleus is closed, with no trace of centrioles, centrosomes or astral fibers
[129]. Tartar was able to remove 'practically all the endoplasm [of Stentor] by vigorous
pipetting', after which the eviscerated stentors could 'regenerate and fill out the
cell shape within a day' ([118], p. 108). Cell division in Stentor is a process that,
to a large extent, is driven by the longitudinal growth and transverse segmentation
of the cortical pattern [130].
The division of a fertilized frog egg could hardly be more different. Frog eggs are
roughly spherical and have no obvious cortical differentiations, apart from the pigmented
cap on the animal hemisphere [131]. The division apparatus is entirely internal. The
mitotic spindle is small relative to the large size of the egg and becomes located
deep within the cell (Figure 6, left). The asters, on the other hand, are dynamic
structures that re-form at telophase of each of the early divisions, expand tremendously,
and while expanding become centered by dynein-mediated pulling forces that act on
the astral microtubules even before these microtubules touch the cell surface. Cell
division furrows then ingress 'where the interaction zones between telophase asters
touch the cortex' ([89], p. 2043). Thus, while the cortex is involved passively in
the determination of the location of the fission zone (and actively in its subsequent
ingression), the earlier dynamic processes, including the sensing of cell volume,
are all endoplasmic. These mechanisms, based on the balancing of pulling forces from
multiple locations in the cytoplasm rather than from the cortex, were proposed as
adaptations that would allow asters to function properly in large-sized amphibian
eggs [97], yet they also appear to function in the smaller eggs of the sea urchin
and of the nematode worm Caenorhabditis elegans [75,81,86] (also see the contribution
from Dr Kimura in this Forum article).
Figure 6
Accommodations must be made. The one cell Xenopus embryo (left) is over 1 mm across,
while cells within the embryo several hours later (right) are closer to 50 microns.
Mitotic spindles are shown in red and chromosomes in yellow. Complex mechanisms have
evolved to allow for cellular functions such as cytokinesis and mitosis to be effective
in cells of diverse sizes. Images are courtesy of Martin Wühr (Harvard).
These studies were carried out on the egg of Xenopus laevis, which at a diameter of
1.2 millimeters is large as a cell and even as an egg, yet smaller than the eggs of
many other frog species [126]. Therefore, one wonders whether the irregularities of
cleavage that emerge as amphibian eggs get larger are in some way related to size
limitations in the effective functioning of the centrosomal-centering and cleavage-site
determining mechanisms that operate so efficiently in the Xenopus egg. This problem
is discussed by Kimura in this Forum, and is illustrated in Figure 3. The postulated
'nonsymmetric cell division' resulting from the failure of centrosomes to reach the
cell center (Figure 3, top center) is reminiscent of the irregular cleavage patterns
observed in the very large eggs of the frog Gastrotheca riobambae [127] and the gigantic
eggs of the salamander Ensatina eschscholzii [124].
In view of these major differences, we can also wonder whether the size limits of
the two largest cleaving cell types, the large ciliate Stentor coeruleus and the even
larger amphibian eggs such as those of Ensatina eschscholtzii, have anything at all
in common. I think that they just might. In cleaving eggs, there are good reasons
to believe that size limits are based on properties of microtubules (see above, as
well as the contribution from Dr Kimura in this Forum article). While a consideration
of microtubular systems of ciliates is beyond the scope of this contribution, these
systems are known to be abundant and in part dynamic over the cell cycle, yet have
been little investigated in the largest ciliates such as Stentor. It is just barely
possible that there might be some underlying limit to the spatial extent over which
microtubule-based cytoskeletal systems can organize and then reorganize themselves
in the absence of internal cell boundaries. Further structural and molecular investigations
of large minced Stentor grafts and the largest amphibian eggs might yield some interesting
and unexpected insights into these and perhaps other unanticipated questions.
Size matters, but in animals so does shape
John Wallingford
I am nearly two meters tall, so the neurons linking my toes to my spinal cord are
quite enormous. These neurons are well over twice the size of those belonging to my
four year old daughter, even though her skin fibroblasts are probably about the same
size as mine. This anecdote, unscientific though it may be, serves to illustrate two
key facts. First, that in addition to the many problems of cell size control faced
by unicellular organisms, animals face the added challenge of establishing and maintaining
cell type-specific cell sizes. And second, that the need to control cell size over
developmental time presents an additional hurdle.
The control of cell size in animals is of course a wide-ranging topic, and it is no
surprise that key players in cell size control in unicellular organisms are also key
players in animal cell size control. Genetic studies in Drosophila have revealed the
key role of cell cycle regulators in controlling cell size, and the phosphoinositide
3-kinase pathways are also widely studied for their link to cell size in mammals [132,133].
However, given the deep conservation of such mechanisms, it is perhaps more interesting
in this forum to discuss some less well-known, cell type-specific problems that arise
at the interface of cell size control and development. In this respect, a consideration
of developing amphibians provides some illuminating vignettes.
Some of the pioneering studies for the link between cell size and cell proliferation
in animals were performed in salamanders, where Fankhauser noted that the increase
in cell size in heteroploid animals was compensated for by decreases in cell numbers.
Thus, he found that the salamanders and their constituent organs were all roughly
the same size, be they diploid, triploid or even pentaploid [134]. Such compensatory
effects are widespread in animals, as reflected by the more recent genetic studies
in Drosophila, for example [132].
Like most animals, amphibians develop externally and without ongoing maternal nutrition,
and so their eggs are packed with yolk. The enormous size of the one-cell frog embryo
(>1 mm across [135]) is therefore a crucial facet of its lifestyle, but it also presents
a problem. During cell division, these large cells must deploy specialized mechanisms
for generating the enormous amounts of new plasma membrane to build the >500 micron-long
nascent cleavage furrow [136]. Likewise, the mechanisms of mitosis have been modified
to achieve proper chromosome separation in such a gigantic cell [89,97], even though
the size of their spindles remains surprisingly small, capped apparently by an upper
physical limit [137] (Figure 6). During these early stages, cell division is uncoupled
from cell size [138], but these modifications - however crucial to the early embryo
- are quickly abandoned. By the 12th division, cells are a far more reasonable approximately
50 microns in diameter and links between cell size and cell division are put in place
[138].
At this same time, another developmental landmark serves to illustrate the importance
of cell size: after 12 divisions, Xenopus embryos engage the zygotic transcriptional
machinery for the first time [139]. As in many other animals, this onset is determined
by a nuclear-to-cytoplasmic volume ratio [140]. This ratio must necessarily be influenced
not only by cell size, but also by nuclear size, so it is noteworthy that nuclear
size, like organelle size generally, is not a simple reflection of overall cell size.
Rather, recent in vivo studies in frog embryos combined with in vitro studies exploiting
embryo extracts have identified factors in the cytosol that are crucial to the control
of nuclear size [141]. These cytosolic factors are even more crucial than is ploidy
[141], a result that in many ways parallels findings in yeast [60].
Similar experiments suggest that mitotic spindle length is also dependent on cytosolic
factors [142]. In the smaller cells of later stage embryos, spindle length scales
with cell size [137], and this scaling requires input from the actin cytoskeleton
[143]. In larger cells, spindle length does not scale with cell size, and even in
cytoplasm extracts in vitro, where spindles cannot be constrained by any physical
cue, there are spindle-intrinsic cues that set a strict upper limit on length [137,144].
Collectively, these results not only illustrate some of the recent advances in our
understanding of organelle and cell size in embryos, but they also highlight a general
gap in our understanding at the intersection of developmental and cell biology: we
have a fairly detailed picture now of many fundamental cell biological processes,
but much of this picture is drawn from studies of relatively few cell types, many
of which exist only in culture. Though comparatively sparse, in vivo studies consistently
show that these fundamental processes vary from cell type to cell type in animals,
but the factors controlling such cell type-specific modifications remain for the most
part poorly defined.
Finally, there is one issue of cell size control that may be unique to animals, and
that is the impact of cell size on cell movement. Large-scale movements of individual
cells are central to animal morphogenesis, and the last decade has seen huge leaps
forward in our understanding of the molecular control of force generation during animal
morphogenesis [145,146], but we know very little about how cell size influences these
processes. This fundamental question was articulated by Fankhauser himself, who noted
that radical changes in cell shapes were needed in order to generate normally shaped
organ structures out of the much larger cells in heteroploid animals. One example
he gave was the developing kidney tubule, where five or six cells of roughly columnar
shape spanned the circumference in normal diploid animals. Only two of the larger
cells in a pentaploid animal enclosed the tubule, and these cells were flattened and
curved such that tubule diameter remained similar to that in diploids (Figure 7).
This finding suggests that regulatory mechanisms are in place to sense cell size and
adjust cell morphology accordingly in order to maintain tissue structure [134]. Conversely,
mechanisms also exist to allow larger cells to form a larger but morphologically normal
kidney, a situation called compensatory renal hypertrophy [147]. Such compensation
commonly occurs in one kidney when the other is somehow compromised.
Figure 7
Accommodations must be made again. Animals have evolved mechanisms that maintain tissue
size in the face of changing ploidy. These images from Fankhauser illustrate this
point. Kidney tubules of the same size are constructed by larger and larger cells
as ploidy increases, and cells must change their shapes so that the tubule diameter
can remain constant. In the haploid animal, many nuclei can be observed around the
tubule circumference, and so these cells have a columnar morphology. In the pentaploid
animals, only one or two nuclei can be observed, and these much larger cells are flattened
and squamous in order to enclose the same tubule diameter.
Recent molecular studies also provide clues to the interaction between cell size control
and morphogenesis. For example, live imaging suggests that kidney tubule diameter
is controlled in part by cell rearrangements, and when these are disrupted, tubules
become dilated and cystic [148,149]. Similar cystic phenotypes have been linked to
the Hippo pathway, a key signaling mechanism governing cell division and organ size
control [150,151]. Likewise, the same genetic pathways that govern cell size control
in normal development also govern renal hypertrophy [152,153]. So here again, detailed
cell biological studies performed in vivo will be central to our efforts to understand
the tangled interactions between cell size and morphogenesis in animals.
Thinking inside the wooden box - classic views of cell size control in plants
Virginia Walbot
Historically, botanists quantified various cellular shape and volume parameters and
discovered a very tight correlation between nuclear DNA content (the C value), nuclear
volume, and cell volume in meristematic cells with small vacuoles [154]. Over a diverse
range of C values, including exemplar monocots with giant genomes, mid-range genome
size monocots and dicots, and Arabidopsis thaliana with a tiny genome, the log cell
volume is linearly related to log nuclear volume with a correlation of 0.99. There
are numerous 'ploidy' series within species (or very close relatives) in nature and
among horticultural derivatives. Ficus spp. trees, ubiquitous decorations in hotel
and airport lobbies, are diploid, tetraploid, or octoploid. These ploidy levels are
readily distinguished by comparing the size of the epidermal guard cell pairs or other
epidermal cells. From a few centimeters distant, however, the trees are indistinguishable
in architecture and leaf size (V Walbot, personal observation) despite the obvious
fact that octoploid leaves contain fewer, larger cells.
In the Introduction to this Forum, Wallace F Marshall opines that cell physiology
depends on cell size. This is true, perhaps, in cuboidal animal cells in which relative
surface area declines with volume and the cytoplasm is served by a 'smaller' surface
area. Because plant cells have vacuoles, however, gigantic diploid cells with very
large vacuoles will have a tremendous surface area, and such cells will actually experience
a much higher surface area per unit cytoplasm than a smaller cuboidal cell. In a ploidy
series or in an organ with cells of various ploidy levels, the higher ploidy cells
can have an even more favorable surface to cytoplasmic volume relationship.
Observations on living plants indicate that growth and most morphology do not depend
on the number of cells or their size within an organ. A very striking observation
concerns lethally irradiated seeds: upon germination the pre-existing cells of the
embryo enlarge, generating a seedling that is remarkably normal despite the lack of
cell division [155-157]. Not so surprising for the first leaf, which contained almost
the full complement of cells, but each successive leaf starts with fewer cells yet
achieves near normal morphology despite ridiculously large cells and abnormal anatomy.
In a separate study, the same researchers applied colchicine to inhibit cell division
in roots, yet single-celled lateral root primordia initiated in the normal location
and grew in a manner paralleling the normal developmental pattern of multicellular
lateral roots [158].
Yet, plant development is highly regular, and leaves and roots of similar size with
approximately equivalent numbers of cells are produced by a population of seedlings.
This regularity of developmental pattern is particularly high in reproductive organs
such as the anther, in which stereotyped cell division patterns establish the tissues,
and there is a high degree of similarity in cell numbers and volumes in different
anthers [159]. For example, anther developmental outcome in maize is extremely similar
in different inbred lines, although developmental mechanism may differ: in the inbred
W23 the secondary parietal layer makes a physically symmetric periclinal division
to establish the middle layer and tapetal cell layer while in inbred A619 this division
is highly asymmetric. At the conclusion of cell patterning and growth, however, the
differentiated middle layer and tapetal cells are virtually identical in these two
inbred lines. This regularity of development is the basis for genetic screens to identify
mutants with perturbations in cell enlargement or cell division. Despite the large
number of mutants available and many measurements of growth parameters, the relationship
between cell division, cell expansion, and their balance to establish cell size remains
mysterious. Moving up to the scale of a tissue or organ, the cellular composition
(number of cells and their sizes) can vary widely, depending on ploidy or treatments
that modulate cell division and expansion.
Emergent patterns during plant growth is a topic that fascinated Alan Turing. This
brilliant mathematician pondered the Fibonacci series intrinsic to spiral botanical
patterns (pinecones, petals, and so on), although unfortunately nearly all of his
botanical insights remained as unpublished manuscripts and notebooks. Thus, it is
gratifying that today in addition to cytological and genetic study of plant development,
mathematical modeling is enjoying a renaissance in plant biology, and the fundamental
problem of growth control is one attractive target. Improvements in cell imaging -
principally the use of confocal imaging and the use of in vivo fluorescent cell type
markers - has generated copious data on cell numbers, sizes, and changes in dimensions
over developmental time. These new datasets have sparked a renaissance in the application
of mathematical modeling to plant growth. The integration of observation, computation,
and simulation represents a new frontier in growth analysis, with cells at the heart
of all three ways of thinking about tissue growth. In the next section, our current
state of knowledge, tool kit, and challenges for these three paths are integrated
and critiqued.
Plant cell size control: all things considered
Xian Qu, Adrienne HK Roeder
Plant biologists have long wondered about the mysterious phenomenon called 'compensation':
when the cell number is decreased as the result of a mutation, the cell size increases,
leading to the production of organs with nearly normal area [160]. This phenomenon
raises two basic questions for plant biologists: how is the size of a plant organ
controlled and how is the size of plant cells controlled? Although these interrelated
puzzles have been extensively studied for many years, neither is fully understood.
Two processes contribute to size control during organogenesis: cell division and cell
growth. Cell size growth in plants is driven by increase in mass (reflecting macromolecular
synthesis) and increase in volume (primarily through expansion of the cell wall and
the central vacuole). The relationship between cell division, cell size, and organ
size remains controversial, as illustrated by the contradictory hypotheses put forward
in two recent computational models [161,162]. Here we will examine the contributions
of cell cycle regulation, ploidy, macromolecular synthesis of cytoplasm, cell wall
expansion, and developmental regulation in the control of plant cell size, focusing
on examples from Arabidopsis.
Changing cell cycle regulator activity can alter the length of time that cells spend
in growth phases G1 and G2 before dividing, thus affecting cell size. In Arabidopsis
leaves and cultured cell lines, overexpression of CYCLIN D3;1 (CYCD3;1) triggers a
quick transition into the mitotic cycle, reducing the proportion of cells in the G1
phase of the cell cycle, and decreasing cell size [163,164]. In contrast, slowing
division with a dominant negative form of the CYCLIN DEPENDENT KINASE A;1 (CDKA;1)
increases final cell size [165]. Likewise, expressing the CDKA;1 inhibitor KIP RELATED
PROTEIN1 (KRP1) under the control of an epidermis-specific promoter results in slower
epidermal division and somewhat increased epidermal cell size [162,166]. In contrast,
overexpressing either APC10 or CDC27, which are two subunits of the anaphase-promoting
complex/cyclosome (APC/C), increases cell division rates without decreasing the final
cell size [167,168]. Thus, cell size can be altered by some cell cycle regulators,
but is unaffected by others.
In many organisms, including plants, a strong correlation exists between ploidy, nuclear
size, and the volume of the cytoplasm [169-171]. In young plant cells with small vacuoles,
the cytoplasm fills the cell, so that ploidy correlates with cell volume. In contrast,
in mature plant cells, the large central vacuole fills the volume, and the cytoplasm
is restricted to a thin layer on the surface of the cell, such that the ploidy correlates
with the surface area [172].
Polyploid cells are found outside of polyploid plants; diploid plants commonly contain
polyploid cells produced through endoreduplication - a variant of the cell cycle in
which cells stop dividing and instead continue to grow and replicate their DNA [171].
Endoreduplication causes an increase in both cell size and ploidy [172] and is often
associated with specialized cell types. Endocycles and mitotic cell cycles can occur
simultaneously during development in neighboring cells [162] and endoreduplication
is a mechanism for cell enlargement. Many alterations inhibiting the cell cycle regulatory
machinery cause increased endoreduplication in organs. Although the exact trigger
that causes an individual cell to endoreduplicate is unknown, one speculation is that
the cell needs to exceed a prior cell size checkpoint. One finding supporting this
hypothesis is that overexpression of either of two D-type cyclins, CYCD2;1 or CYCD3;1,
induces smaller cell sizes and blocks endoreduplication [163,173]. Once the putative
cell size checkpoint is passed, endoreduplication can be switched on by various mechanisms.
One such mechanism is reduction of CDKA;1/CYCLIN B activity to a level that fails
to initiate mitosis but is still able to drive replication of DNA. For example, mitotic
CDKA;1 activity can be reduced by elevated activity of cyclin-dependent kinase inhibitors
of the KRP and SIAMESE families [174,175]. The SIAMESE protein has CYCLIN and CDKA;1
binding sites, and the protein can inhibit mitosis and stimulate endoreduplication
in the Arabidopsis hair cells (trichomes) [176,177]. A second trigger of endoreduplication
is ectopic DNA replication. CDC6 catalyzes assembly of the ORIGIN OF REPLICATION (ORC)
complex, enabling DNA replication and overexpression of CDC6 increases endoreduplication
in Arabidopsis [178].
In plants, the ratio of the ploidy to the cytoplasm remains constant, suggesting that
cytoplasm can influence cell size as well. Cytoplasm increases through macromolecular
synthesis. For example, Arabidopsis EBP1 is related to human ErbB-3, an epidermal
growth factor receptor binding protein that enhances translation. Overexpression of
the EBP1 gene in Arabidopsis increases cell growth [179], implying that increased
protein synthesis correlates with increased cell size. Similarly, inhibiting protein
degradation by mutation of the 26S proteasome subunit REGULATORY PARTICLE AAA-ATPASE
2a (RPT2a) [180] produces larger cells in Arabidopsis leaves [181,182], showing that
optimizing proteasome activity levels is important for cell size control. As in many
other organisms, macromolecular synthesis is regulated in Arabidopsis by the TARGET
OF RAPAMYCIN (TOR) pathway [183]. The pathway regulates cell growth and metabolism
in response to growth factors, nutrients, energy, and environmental conditions in
yeast and mammals [184,185]. The Arabidopsis genome encodes only one TOR gene, and
it regulates cell size, translation initiation, and ribosomal RNA synthesis [183,186].
Cell wall expansion is a major mechanism controlling plant cell size. The plant cell
wall greatly confines the enlargement of plant cells because it consists of a rigid
mesh of complex polysaccharides and a few structural proteins [187]. The cell wall
grows by repeated stretching and polymer reconnection (termed stress relaxation):
turgor pressure constantly exerts a stretching force on the cell wall, and the force
is alleviated when polysaccharide polymers rearrange their interconnections to take
on the new shape [188]. To maintain cell wall integrity, new polymers are synthesized
simultaneously and added to the growing wall. Thus, proteins that modify the interconnections
between polysaccharide polymers regulate plant cell growth. The EXPANSIN (EXP) family
is one class of plant proteins that are thought to loosen cell walls by weakening
the binding of polysaccharide polymers to one another [189]. In fact, adding active
EXPANSIN proteins to dead cell walls is sufficient to cause their rapid extension
[190]. Overexpression of EXP10 in Arabidopsis under the control of its own promoter
results in larger leaves containing larger cells [191]. The extensibility of the cell
wall is not uniform in all directions, and is limited by the reinforcement of the
cellulose microfibrils embedded in the wall. The primary direction of expansion, and
ultimately the shape of the cell, depend on the orientation and alignment of the cellulose
microfibrils, which are controlled by the cytoskeleton just inside the plasma membrane
of the cell. In vascular plants, a cellulose microfibril is synthesized by a cellulose
synthase complex of integral plasma membrane proteins [187]. The cellulose synthase
complex tracks through the plasma membrane along the cortical microtubules in such
a way that microtubules determine the orientation of the new cellulose microfibrils
in the cell wall, and thus control the direction of structural reinforcement [192].
In Arabidopsis, both microtubule- and microfilament-associated proteins facilitate
normal cell expansion in different organs [193-195]. The cell wall presents a second
challenge to plant cell growth in that the walls of neighboring cells are tightly
connected. Because plant cells generally do not slip relative to one another, the
growth of neighbors is coordinated along their adjoining walls. Nonetheless, heterogeneity
of cell growth is not only still possible but common in individual cells, because
portions of the cell wall in contact with different neighbors can grow with different
rates [196].
The relationship between organ growth through turgor-induced cell wall expansion and
organ growth through cytoplasm biosynthesis and cell division is unclear. As mentioned
above, plant cells have a tremendous ability to increase their cell wall expansion
to compensate for decreased cell division, resulting in an organ with normal size
but extremely large cells. However, compensation fails when too few cells are available.
Perhaps this was best illustrated by experiments in which cell division was completely
blocked in wheat seedlings by gamma irradiation of the grains. Remarkably, the leaf
primordia of these gamma plantlets still grow and produce first foliage leaves with
the correct shape [155,197]. However, after ten days, the gamma irradiated plant leaves
are only about 15% as long as unirradiated plants, despite greatly increased cell
expansion [197]. These experiments indicate that there is a limit to cell wall extensibility
in living plants and some cell division is required for organs to reach their normal
size.
All of these cell size control pathways can be developmentally regulated with temporal,
tissue and spatial specificity to produce a variety of different cell sizes, often
corresponding to specialized cellular functions. Cell size is regulated by transcription
factors and co-activators such as GROWTH-REGULATING FACTOR (GRF) [198,199] and ANGUSTIFOLIA3
(AN3) [200,201]. Furthermore, cell size is actively patterned in different plant organs.
For example, the Arabidopsis sepal epidermis has a broad diversity of cell sizes,
from small cells with one-hundredth the length of the sepal to giant cells with one-fifth
the length of the sepal (Figure 8a,b). This pattern forms as a result of the stochastic
entry of cells into endoreduplication at different times [162]. The pattern is also
regulated by the epidermal specification pathway, which promotes the formation of
giant cells [116]. Giant cells are found on the back (abaxial) epidermis of Arabidopsis
sepals and leaves, whereas the epidermal cells in the petal blade do not endoreduplicate
and consequently they have a uniform small size.
Figure 8
The diversity of cell sizes in the Arabidopsis sepal epidermis. (a) A scanning electron
micrograph (SEM) of the sepal epidermis shows large giant cells are interspersed between
smaller cells in a range of sizes. (b) A confocal maximum intensity projection of
the sepal epidermis in which the cells are outlined in red by the plasma membrane
dye FM4-64 revealing the variation in cell sizes. The nuclear size shown in green
(ML1::H2B-mYFP) corresponds with the ploidy. The correlation between cell size and
ploidy is evident: large cells have large nuclei indicating that they have undergone
endoreduplciation whereas small cells have small nuclei indicating that they have
remained diploid. Scale bars represent 50 μm.
In summary, cell size control in plants is a highly dynamic and complicated process
involving multiple biological pathways (Figure 9). Although many individual cell size
regulators in each of these pathways have been discovered, a future challenge will
be determining how these pathways integrate to form a complete cell size control network.
Two recent advances are likely to guide the construction of such a network [202].
First, live imaging of cells in developing organs will allow us to determine the exact
timing and detailed mechanism through which each player affects cell division, cell
growth, and cell expansion. For example, the transcription factor JAGGED (JAG) regulates
proliferation to control lateral organ shape and size in Arabidopsis sepals and petals
[203,204]. Recently, by using three-dimensional live imaging, Schiessl et al. [205]
discovered that JAG regulates the transition between tight coordination of cell volume
with initiation of S phase of the cell cycle in the stem cells of the meristem and
loose coordination in the initiating organ primordium. Ectopic expression of JAG in
the floral meristem stem cells bypasses this tight cell size checkpoint, resulting
in smaller cells entering S phase [205]. Second, computational modeling will allow
us to predict the cumulative effect of multiple pathways acting simultaneously and
feeding back on one another [206]. For example, while a diagram can be used to conceptualize
the increase in cell size caused by one cell entering endoreduplication earlier than
its neighbors [207], a computational model can expand this analysis to about 1,400
cells entering endoreduplication or dividing at stochastic times to pattern the entire
sepal [162]. Such an integrated understanding may finally show how plant cells increase
in size to compensate for decreased cell numbers to achieve consistent organ size.
Figure 9
Overview of cell size control in plants. Plant cell size is determined by the growth
of the cell versus its division. Cell growth is determined by macromolecular synthesis
as well as expansion of the cell wall. Future challenges include untangling the regulatory
network between the various pathways regulating the cell cycle, endoreduplication,
the cell wall, and synthesis of cytoplasm, to elucidate how this crosstalk determines
the ultimate cell size.