J. Cell Set. 54, 173-191 (1982)
Printed in Great Britain © Company of Biologists Limited 1982
173
CELL VOLUME AND THE CONTROL OF
THE CHLAMYDOMONAS
CELL CYCLE
R. A. CRAIGIE AND T. CAVALIER-SMITH
Department of Biophysics, King's College London, 26-20 Drury Lane, London
WCzB sRL, U.K.
SUMMARY
Chlamydomonas reinhardii divides by multiple fission to produce 2" daughter cells per
division burst, where n is an integer. By separating predivision cells from synchronous cultures
into fractions of differing mean cell volumes, and electronically measuring the numbers and
volume distributions of the daughter cells produced by the subsequent division burst, we have
shown that n is determined by the volume of the parent cell. Control of n can occur simply, if
after every cell division the daughter cells monitor their volume and divide again if, and only
if, their volume is greater than afixedminimum value.
In cultures synchronized by 12-h light/12-h dark cycles, the larger parent cells divide earlier
in the dark period than do smaller cells. This has been shown by two independent methods:
(1) by separating cells into different size fractions by Percoll density-gradient centrifugation
and using the light microscope to see when they divide; and (2) by studying changes in the
cell volume distribution of unfractioned cultures. Since daughter cells remain within the mothercell wall for some hours after cell division, and cell division causes an overall swelling of the
mother-cell wall, the timing of division can be determined electronically by measuring this
increase in cell volume that occurs in the dark period in the absence of growth; we find that
cells at the large end of the size distribution range undergo this swelling first, and are then
followed by successively smaller size fractions.
A simple model embodying a sizer followed by a timer gives a good quantitative fit to these
data for 12-h light/ 12-h dark cycles if cell division occurs 12-h after attaining a critical volume
of approximately 140 fitn'. However, this simple model is called into question by our finding
that alterations in the length of the light period alter the rate of progress towards division even
of cells that have attained their critical volume. We discuss the relative roles of light and cell
volume in the control of division timing in the Chlamydomonas cell cycle.
INTRODUCTION
The control of cell volume and of cell division are two fundamental and closely
interrelated problems. The idea that growth in cell volume beyond a certain point
in some way triggers division has been repeatedly discussed (Hertwig, 1903, 1908;
Fantes et al. 1975; Mitchison, 1977; Cooper, 1979) and has received experimental
support in Amoeba (Hartmann, 1928; Prescott, 1956), Escherichia colt (Maaloe &
Kjeldgaard, 1966; Donachie, 1968, 1974; Donachie, Jones & Teather, 1973; Davern,
1979), Stentor (Frazier, 1973), the yeasts Schizosaccharomyces pombe (Nurse, 1975;
Fantes, 1977, 1979; Fantes & Nurse, 1977, 1978; Thuriaux, Nurse & Carter, 1978;
Nurse & Mitchison, 1980) and Saccharomyces cerevisiae (Johnston, Pringle & Hartwell,
1977; Lorincz & Carter, 1979; Johnston, Singer, Sharrow & Slater, 1980), and the
slime mould Physarum (Sudbery & Grant, 1975, 1976), A radically different view
174
R. A. Craigie and T. Cavalier-Smith
(Smith & Martin, 1973, 1974; Minor & Smith, 1974) is that commitment to divide
is a purely random process, unrelated to cell volume. This random transition model
has received most support from studies of mammalian cell-cycle kinetics (Smith &
Martin, 1973, 1974; Minor & Smith, 1974; Shields & Smith, 1977; Shields, 1978),
but because of statistical difficulties (Nurse & Fantes, 1977; Wheals, 1977) and
alternative explanations of the data (Castor, 1980), the evidence for it is equivocal. Nurse
(1980) has suggested that deterministic and probabilistic models may describe
different aspects of a common cell-cycle control mechanism. This view is supported
by the finding (Wheals, 1980) that bud emergence in the yeast S. cerevisiae, although
probabilistic, is a monotonic increasing function of cell volume; the largest cells
have a constant high probability of bud emergence and therefore exhibit random
kinetics. There is also evidence in mammalian cells that entry into the postulated B
phase, or the length of the B phase (Smith & Martin, 1973, 1974) is volume-dependent
for cells smaller than a minimum size (Shields et al. 1978).
Although unicellular algae are especially well suited for cell cycle studies because
of the ease of synchronizing them by light/dark cycles (Lien & Knutsen, 1979),
previous studies of their cell cycle, mainly in Chorella (Wanka, 1965; McCullough &
John, 1972) and Chlamydomonas (Jones, 1970; Mihara & Hase, 1971, 1975; CavalierSmith, 1974; Howell, Blaschko & Drew, 1975; Lien & Knutsen, 1979; Spudich &
Sager, 1980) have not directly tackled the problem of volume control over cell
division. In the experiments reported here we have studied the relationship between
cell volume and the timing of cell division in the green alga Chlamydomonas reinhardii. By measuring cell volume distributions electronically, and comparing cell
volumes with division times in synchronized cultures and in different size fractions
separated from such cultures by density-gradient centrifugation, we have obtained
firm evidence that cell volume is a major determinant of the timing of cell division
in Chlamydomonas. Preliminary evidence derived from manipulation of the light
regime suggests, however, that attainment of a critical cell volume alone is insufficient
to explain the timing of division under all conditions. We discuss possible explanations of these extra complexities.
Chlamydomonas is especially suited to these studies because it is possible to measure
the spread of cell division times in a semi-synchronous population without the use
of time-lapse photography. This is because daughter cells remain inside the mother
cell wall for some hours after cell division itself. This makes it easy to distinguish
divided and undivided cells microscopically, and even to determine the time of
division of specific size fractions of the population by electronic cell-sizing (because,
as we show here, cell division causes a swelling of the mother cell wall).
Cell division in Chlamydomonas is by multiple fission rather than the binary fission
found in most organisms. Since multiple fission in the absence of cell growth is
widespread in protists (Cavalier-Smith, 1980), and also occurs in egg cleavage in
animals (Mitchison, 1971), its mechanism is of considerable interest. C. reinhardii is
probably the best model system for such studies, because of the ease of obtaining
synchrony, the possibility of genetic analysis (Lewin, 1976), and the availability of
cell cycle mutants (Cavalier-Smith, unpublished; Howell & Naliboff, 1973; Howell,
Chlamydomonas cell cycle
175
1974; Warr & Quinn, 1977). Two problems are of particular interest: the mechanism
that determines the number of daughter cells in multiple fission, and the nature of the
similarities and differences between multiple and binary fission cell cycles. Our
results show that the number of daughters is controlled by the volume of the mother
cells, and suggests that the mechanisms controlling cell division in multiple fission
cell cycles are fundamentally similar to those seen in normal binary fission cycles.
However, unlike the first DNA replication and division of the multiple fission burst,
subsequent rounds of replication and division appear to involve no special initiation
process and proceed automatically and in quick succession until daughter cells are
smaller than a threshold cell volume.
MATERIALS AND METHODS
Culture methods
C. reinhardii (32C strain from the Cambridge Culture Collection) were grown in minimal
medium (Cavalier-Smith, 1974) sterilized by filtration through a 0-22 fim Millipore filter. This
also removed particles that would have interfered with Coulter counting.
'Feeder' cultures were prepared by inoculating 100 ml volumes of medium from agar
slants. Feeder cultures (concentration range 5 x io'~3 x 10' cells/ml) were used to inoculate
1-litre volumes of medium in 2-litre Eilenmeyer flasks to an initial concentration of 5 x io*
cells/ml. These batch cultures were aerated with filtered air, and incubated at 20 °C under
alternating 12 h light/12 h dark cycles and at a light intensity of approximately 10000 lux.
Experiments were conducted during the 4th dark period following a full 12 h light period, by
which time the cell concentration was approximately 5 x io4 cells/ml.
Scoring of cell division
A portion (0-5 ml) of a solution of 4 % (w/v) iodine, 6 % (w/v) potassium iodide in distilled
water was added to 9-5 ml of cell suspension. After mixing, the cells were pelleted using a
benchtop centrifuge. The pellet was resuspended in 1 ml of supernatant by gently bubbling
air through a Pasteur pipette. Samples were examined under phase-contrast using a Zeiss
x 16 objective. A total of 1000 cells was examined from each sample and the percentage of
undivided cells recorded. The above method gave good preservation of the parent cell wall,
at least over a period of several days, even for cells fixed just prior to the onset of zoospore
release.
Counting the daughter cells was greatly facilitated by squashing them gently between the
slide and coverslip, and quickly sealing the coverslip with nail varnish to prevent evaporation.
Cell counts and size distributions
Cell counts were obtained using a model ZBi Coulter counter, using an orifice diameter of
100 fim. A reciprocal amplification setting of 2 or 4, a reciprocal aperture current of 2 and a
lower threshold setting of 5 were used. The upper threshold was set at infinity.
Cell volume distributions were determined using a Coulter model C-1000 Channelyzer; the
edit function was used throughout to minimize the effect of coincidence, variation in transit
times and oblique particle trajectory (Harvey, 1968). Latex spheres of known mean diameter
were used for calibration, and to check that no drift occurred during experiments. Size distribution data were stored on punch cards for future analysis.
Fractionation of cells by volume
Percoll (Pharmacia Fine Chemicals) was diluted 1:3 (v/v) with filtered medium. Gradients
were formed in situ by centrifugation for 30 min at 20000 rev./min in a Beckman L275B
ultracentrifuge using an SW41 rotor. A total of 800 ml of culture was harvested by centrifugation
R. A. Craigie and T. Cavalier-Smith
i76
2
4
Time into dark period (h)
2
4
6
Time into dark period (h)
Fig. i. A. Timing of the ist (#), 2nd (A) and 3rd ( • ) divisions during a normal
12 h light/12 h dark cycle. B. Effect on the timing of the ist (#), 2nd (A) and 3rd ( • )
divisions of replacing the final dark period by a light period. The culture, which is
the same as for A, was divided into two at the end of the light period. Half was incubated in darkness (A) and half in continuous light (B). The higher percentage of cells
that divide to produce 8 daughters in the light-incubated culture is due to the extra
increase in cell volume (see text).
at 5000 rev./min in an MSE high-speed 18 centrifuge (MSE 6 X 250 ml rotor) at 200 C
for 20 min (includes time for acceleration). The pellets were resuspended in a few ml of
supernatant.
Two ml of concentrated cell suspension was layered onto the preformed gradients (equilibrated at 20 °C), and then spun in a benchtop centrifuge at 200 g for 5 min. This includes the
time taken for acceleration (65 s) but excludes that for deceleration (45 s). Samples (1 ml) from
the top, middle and bottom of the gradient were inoculated into 200 ml volumes of sterile
medium in 500 ml screw-top flasks at 20 CC (no aeration). Harvesting and separation together
took between 40 and 45 min.
RESULTS
General properties of the Chlamydomonas cell cycle
C. reinhardii divides by multiple fission, 2 n daughters being produced per division
burst, where n is an integer. Since the daughter cells remain enclosed by the parent
cell wall for a period after division, n can be readily determined by microscopy.
Under the 12 h light/12 h dark regime for inducing synchrony, cell division is confined to the dark period (Jones, 1970; Mihara & Hase, 1971; Cavalier-Smith, 1974).
Under our conditions most cells produced 4 daughters per division burst, but some
produced 2, 8 or 16. Fig. 1 A is a typical plot of the proportion of cells that have divided
to produce 2, 4 and 8 daughters at successive times in the dark period. Breakdown
Chlamydomona8 cell cycle
177
Time into dark period (h)
Fig. 2. Effect of the siie fractionation procedure on the timing of cell division. Half
the culture was harvested at the start of the dark period, sedimented in a Percoll
gradient, re-mixed and diluted with fresh medium. The riming of cell division in
this culture (A) is compared with the unfractionated control (A).
of the mother cell wall to release the daughters (zoospore release) occurs near the
end of the dark period, several hours after the cells have divided and is thus quite
distinct from cell division. As previously reported (Mihara & Hase, 1971, 1975;
Cavalier-Smith, 1974), the synchrony of zoospore release was greater than that of
cell division.
Cell volume control of the number of daughter cells per division burst
It seemed likely that the volume of the parent cell would control the number of
daughters produced by each division burst. This was tested by separating predivision
cells into fractions of different mean cell volume, by centrifugation in a Percoll
density gradient. Percoll itself had no detectable effect on cell volume, or on cell
motility, even at higher concentrations and for longer exposures than used. The
diluted fractions were incubated in the dark at 20 °C to allow cell division and release.
It was necessary to extend the dark period to 14 h to ensure that all the cells were
released from the parental walls. The volume distribution and concentration of
daughter cells were recorded.
Fig. 2 shows that the separation procedure caused a small, though significant,
delay in the onset of cell division for cells that were to divide early. However this
delay gradually decreased for cells that divided later, and after 5 h into the dark
period the division curve was indistinguishable from that of the control (Fig. 2).
Direct comparison of cell concentrations before and after division showed that
fractions containing larger cells produced more daughters per division burst than
R. A. Craigie and T. Cavalier-Smith
i78
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Z
200
400
600
Cell volume (/im3)
E
z
100
150
200
250
Cell volume (jim'l
Fig. 3. Relation between parent and daughter-cell volume. Parent cells were separated
into 3 size classes at the start of the dark period: their volume distributions, designated
small (V), medium ( ^ ) and large (El) are shown in A. The daughter-cell volume
distributions produced by the small (V), medium ((}) and large (13) parent classes are
shown in B. Cells were separated by size at the start of the dark period and incubated in
darkness for 14 h, by which time all the daughter cells were released. The median
volume of each fraction was recorded immediately after fractionation (parent cells)
and after 14 h incubation in the dark (daughter cells).
Chlamydomonas cell cycle
160
180
200
220
240
179
260
Median parent-cell volume (pm3)
Fig. 4. Relation between parent and daughter-cell volume. The graph, which summarizes the pooled results of several experiments as described in the legend to Fig. 3,
shows that daughter-cell volume is essentially constant and independent of parent-cell
volume.
those containing smaller cells. In one experiment where the average number of
daughters produced per parent in the unfractionated culture was 4-4, the mean
numbers produced by the various size fractions shown in Fig. 3 were: 5-5 for the
large cells, 3-4 for the medium ones and 2-6 for the small ones. Fig. 3 shows that the
majority of the daughter cells were similar in volume irrespective of whether they
were produced by large, medium or small parent cells, which implies that in multiple
fission successive divisions continue until the daughter cells have reached a minimum
volume. However, a small minority of cells may exceptionally fail to divide to yield
daughters of the minimum size as is shown by the trimodal size distribution of the
daughter cells produced by the smallest parent cell size class in Fig. 3B. The small
peak at the right of this curve is due to a small number of cells remaining undivided.
The middle peak results from cells dividing into only 2 daughters, and the largest
peak at the left of the trimodal curve is due to division into 4 daughters. The size of
the small parental cell class shown in Fig. 3 A was the smallest obtained in these
experiments, and the only one that showed evidence of some cells not having divided.
In all other experiments, the daughter-cell size distributions produced by the small
parent-cell size classes were unimodal, with a shoulder due to some division into
only 2 daughter cells, i.e. similar to the medium daughter-cell size class in Fig. 3B.
The largest parent-cell size category in Fig. 3 A produced some of the smallest daughter
cells, because some of them divided into eight.
Fig. 4. shows the pooled results of several such experiments; the median cell
volume of populations fractionated at the start of the dark period is compared with
the median daughter-cell volume produced by each size category. Over the range
studied there is little or no positive correlation between parent and daughter-cell
volume. This shows that large cells must, on average, produce more daughters per
division burst than do small cells, and that the number of daughters produced is so
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R. A. Craigie and T. Cavalier-Smith
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Fig. 5. Influence of cell volume on the timing of the ist division of the cell cycle.
At the start of the dark period cells were fractionated into 2 sixe classes, having
median volumes of aoo/im* (A) and 260/tm1 (#), respectively, and the timing of
cell division was followed in each by microscopy.
Fig. 6. Correlation between cell size at the start of the dark period and the timing of
cell division. Cell division in populations of cells fractionated by size at the start of the
dark period was followed by microscopy. In some experiments less that 30 % of the
cells in the smallest size classes divided before the onset of zoospore release. Therefore
we used the time into the dark period required for 20 % of the population to divide
as a measure of division timing, and the 80th percentile of each volume distribution as
a measure of cell size. Results from several experiments are pooled, and clearly show
that larger cells divide earlier.
regulated as to keep the average daughter-cell volume constant irrespective of parentalcell volume.
Large cells divide earlier in the dark period than do small cells
Two independent types of experiment demonstrate this. In the first, we fractionated
cells into size classes at the start of the dark period, and then at intervals during the
dark period we determined (by light microscopy) the percentage of cells that had
by then divided at least once. In the experiment shown in Fig. 5 the large cells
divided about 2-5 h before the small cells. The pooled results from several such
experiments, summarized in Fig. 6, clearly show a strong negative correlation between
cell volume and the time elapsing before division. The scatter between points is due
to variation between experiments; in each individual experiment the larger volume
fraction entered division first. The points in Fig. 6 are not corrected for the effect
of the separation procedure slightly delaying division of the cells that divide early
Chlamydomonas cell cycle
181
in the dark period (Fig. 2). If such a correction were made there would be an even
stronger correlation, because this factor will decrease, rather than increase, the
correlation between cell size and the timing of division.
Independent evidence that larger cells divide earlier comes from the division
kinetics of cells of different volume in unfractionated cell populations. It is possible
to study this directly using the Coulter counter, because we find that Chlamydomonas
cells, paradoxically, increase in volume when they divide (the decrease in size discussed above occurs only later when daughters are released). This increase in 'cell'
volume on division can be demonstrated by fractionating cells according to size at the
start of the dark period and correlating changes in the cell size distribution during
the dark period with the timing of cell division as determined by microscopy. Fig. 7
shows an example where at 4-5 h into the dark period 40% of the large cell fraction
have divided and a corresponding increase in the cell size distribution has occurred,
whereas in the small cell fraction where only 5% have divided there is very little
increase in cell volume. This volume increase, observed at division in all our experiments, can be explained by the spaces visible in the microscope between daughter
cells. If the total protoplasmic volume remains constant during division, the mother
cell wall must clearly expand to accommodate these new intercellular spaces. Since
the Coulter counter essentially measures the volume of displaced electrolyte, it is not
surprising that it measures the overall volume enclosed by the mother cell wall,
including any intercellular spaces.
Cell volume distributions were consistently log-normal during the light period and
prior to cell division during the dark period. The cell volume distribution at the
start of the dark period is therefore conveniently represented as a straight line plot
on logarithmic probability co-ordinates (Fig. 8). Since cell division is accompanied by
an increase in volume, it will alter the cell size distribution. If large cells divide earlier,
there should be a deviation from the straight line plot at those times when only part
of the population has divided. Fig. 9 shows clearly that large cells divide first, and
that smaller ones divide later in the dark period. Just prior to the onset of zoospore
release the size distribution is again log-normal with the same slope, except for a
slight curvature at the small end of the cell size distribution (Fig. 9), due to some of
the small cells remaining undivided at this stage of the dark period.
Timing of the second and subsequent divisions
Division curves for the ist, 2nd and 3rd divisions are parallel, with an interval of
1-1-5 h between each (Fig. 1 A, B). The timing of 2nd and subsequent divisions, when
they occur, is therefore determined only by the timing of the ist division.
Effect of shortened and extended final light period on cell division
Though not investigated in detail, the results obtained are summarized in Table 1.
As the light period is shortened the timing of division is progressively delayed and the
proportion of the population dividing is reduced from near 100% for a 12 h light
period to only 3-4% for a 6-5 h light period. Conversely, extending the light period
advances division and decreases its spread. Fig. 1 B indicates that although extending
R. A. Craigie and T. Cavalier-Smith
182
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425
850
Cell volume (pirn3)
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Cell volume (>im3)
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Cell volume (pm 3
Fig. 7-
850
Chlamydomonas cell cycle
120
0-1
30
50
70
183
90
99
999
Cumulative % of cells
Fig. 8. Cell volume distribution of an unfractionated population at the start of the
dark period. The logarithm of cell volume is plotted agpinst the cumulative percentage
of cells of that volume on probability co-ordinates. The resulting straight line indicates
that the logarithm of cell volume is normally distributed.
the light period advanced the 1st division, there was no detectable difference in the
time interval between the 1st and 2nd, or the 2nd and 3rd divisions compared to the
control (Fig. 1 A). There was, however, an increase in the proportion of cells that
divided to produce 8 daughters. During extended light periods it was not possible
to follow the division curves down to a lower percentage of undivided cells because
zoospore release began before all the cells had divided. We also noticed that in
normal 12 h light/12 h dark cycles, when samples were taken near the end of the
dark period, light from the Coulter counter optics stimulated zoospore release. These
observations confirm earlier reports (Schlosser, 1966) that light directly initiates cell
release.
DISCUSSION
Our results establish two fundamental features of the multiple-fission cell cycle
of C. reinhardii. One is that parental cell volume controls n, the number of divisions
per cell cycle. The other is that the timing of the first division in each cycle is under
Fig. 7. Effect of cell division on the volume enclosed by the parent cell wall. Small
( • ) and large (D) volume classes fractionated at the start of the dark period are shown
in A. After 4-5 h into the dark period s % of the small fraction and 40 % of the large
fraction had divided. The volume distribution of the small fraction at 5 h into the
dark period (<^) is shown in B, together with the original volume distribution (V) for
comparison. The volume distribution of the large fraction at this time (A) is shown
in c together with the original distribution (Q).
R. A. Craigie and T. Cavalier-Smith
184
o
10
50 70
30
Cumulative % of cells
90
999
Fig. 9. Changes in the volume distribution of unfractionated cells during the dark
period. Cell size distributions are represented after 2 % ( • ) , n % ( • ) , 71 % ( • )
and 92 % ( ^ ) division. After 2 % of the cells have divided, the volume distribution
is still log-normal. After 11% cell division there is a clear departure from lognormality at the large end of the size distribution, indicating that essentially only the
largest cells have increased their volume as a result of division. As the percentage of
divided cells increases, the volume increase spreads towards the lower end of the cell,
size distribution. After 92 % division ( • ) , the size distribution is again log-normal,
except for a slight curvature for the smallest 10 % of the population, because some of
these cells have still not divided.
Table 1. Effect of the length of the final light period on cell division
Length of final light
period (h)
6-5
9
12
Continuous light
Time from start
of light period
for 20 % cells
divided once
(h)
20-25 ±030
1475 ±030
I37S ±030
Time from start
of light period
for 40 % cells
divided once
(h)
Comments
4 % divided after 24-5 ± 0-3 h
34% divided after 22-0 ±0-3 h
17-5 ±0-30
15-5 ±
separate control from that of subsequent divisions: the timing of the first division is
strongly influenced by cell volume as well as by the light regime, whereas the timing
of the subsequent divisions appears not to be directly dependent on either of these
factors but to occur at fixed times after the first division.
Chlamydomonas cell cycle
185
2
4
6
Time into dark period (h)
Fig. 10. Comparison of the timing of the 1st cell division during the dark period with
that predicted by a simple critical size model. Assuming a critical volume of 140 /tm*
(see text) the cumulative percentage of cells larger than this size was recorded as a function of time into the light period. Assuming a constant interval of 12 h between reaching
the critical size and division itself, there is excellent agreement between the predicted
curve (—) and the experimental curve (A), recorded during the following dark period.
Control of the timing of the first division in each cycle
The correlation we observe between the timing of cell division and cell volume
could only be explained in terms of a purely random commitment to divide (Smith &
Martin, 1973, 1974), by postulating that the larger cells that divide early in each
cycle were produced earlier in the preceding dark period than the smaller cells that
divide later. This possibility is excluded by our finding that parent and daughter-cell
volumes are not in general positively correlated. A simple transition probability
model (Smith & Martin, 1973, 1974) (or any other purely time-dependent model)
therefore cannot explain our observations on the Chlamydomonas cell cycle. In
principle, the observed correlation between cell volume and the timing of division
can be explained by supposing either that cells must attain a critical volume before
they can enter some phase of the cell cycle, or that the rate of the preparations for
division itself depends on cell volume.
A very simple model for division control is that commitment to divide occurs when
cells reach a critical size, but that division itself takes place later after the elapse of a
time interval that is independent of cell volume. This model can be tested by predicting the course of cell division during the dark period from changes in the cell volume
distribution during the preceding light period. Since essentially all the cells divide
during each dark period, the critical size must be below that of the smallest cells at
the end of the light period. A lower limit to the critical size is set by the size of the
CEL 54
186
R. A. Craigie and T. Cavalier-Smith
largest cells at the end of the dark period: if any newly produced daughter cells were
larger than the critical size, a secondary division burst should occur before the main
division period; this was never observed. These two constraints limit the critical size
to 140+ 20/mi 8 under our experimental conditions. Fig. 10 shows that, for a critical
size of 140 /ira. and a time interval of 12 h between commitment and division, there is
excellent agreement between the predicted and experimental curves for the timing
of division in the dark period. The model can also account for our (unpublished)
observation that a reduced light intensity results in delayed onset and increased
temporal spread of cell division. Under these conditions cells grow more slowly
during the light period, so they take longer to reach the critical volume; the increased
spread of division is accounted for by the reduced rate at which cells reach the critical
size.
The above model fails, however, to account for the effect of shortening or extending
the final light period, for it predicts that cells that are larger than the critical size
when the light period is truncated will divide at the usual time. Thus the early part
of the division curve should be the same as for a 12 h light period, flattening off at a
point determined by the length of the light period. But this is not what we observed.
As Table 1 shows, shortening the final light period delays division and increases its
spread; moreover, shortening the light period by only a few hours results in a much
greater increase in the proportion of cells that remain undivided at the end of the
dark period than we predict from the above model. Conversely, extending the light
period beyond 12 h (Fig. I B ) advances division, also against the prediction of the
model.
The increase in the fraction of undivided cells when the light period is shortened
is in keeping with the observations of Spudich & Sager (1980) on light/dark synchronized C. reinhardii cultures: under their experimental conditions progression towards
division became independent of light about halfway through the light period. This
transition to light-independence must be separate from our postulated size threshold,
since we find that after a 6-5 h light period essentially no cells divide, even though a
substantial proportion are larger than the small cells that do divide after a 12 h light
period. Though the existence of a light requirement for division early in the cell cycle
(Spudich & Sager, 1980) explains the reduction in the proportion of cells dividing
when the light period is truncated, it does not explain the changes in the timing of
division caused by alterations in the length of the light period. These changes, however,
could be simply explained by supposing that the time interval between a cell's sizedependent commitment to progress towards division and division itself is shortened
by light; since shortening the final light period reduces the amount of light received
by all cells during this period their division will be delayed. However, the proportional
reduction in light is greater for cells that reach their critical size later in the light
period; the delay is therefore greater for cells that divide late during the following
dark period, resulting in an increased spread in the timing of division as we observed.
Conversely, extending the light period should advance division and reduce its spread,
as is also observed. The most serious objection to this explanation is the good agreement between our experimental division curves after a 12 h light period and those
Chlamydomonas cell cycle
187
predicted by assuming a constant time between reaching the critical size and division
(Fig. 10). If the length of this period is light-dependent, the predicted curve in Fig. 10
should be steeper than the experimental curve. Changing the assumed critical size
within the limits explained above does not significantly alter the spread of the predicted
division curve.
Further work is clearly needed to clarify the relationship between the volumedependent and light-dependent controls on the Chlamydomonas cell cycle. This should
make possible a unified model for the timing of division. Our experiments indicate
that, for autotrophic growth, a complete model must eventually incorporate:
(1) A threshold cell volume as a prerequisite for division.
(2) An interval between reaching the threshold volume and division that is influenced by light. (Since extra light causes growth in cell volume, we cannot say
whether this influence is entirely independent of cell volume, or is mediated by means
of an effect of cell volume on the rate of progression towards division after reaching a
threshold volume.)
(3) A minimum period or amount of light early in the cell cycle (also shown by
Spudich & Sager, 1980).
Since C. reinhardii can grow and divide in the absence of light if supplied with
acetate (Jones, 1970), we suggest that the light-dependent effects on the cell cycle can
be explained purely in terms of its role as an energy source. Exactly the same cell
cycle controls may therefore operate under both photoautotrophic and heterotrophic
conditions, which would not be the case if light is a specific prerequisite for division,
independent of its role as an energy source.
Control of the second and subsequent divisions
When C. reinhardii divides into more than 2 daughters, a round of DNA synthesis
immediately precedes each mitosis (Kates, Chiang & Jones, 1968; Jones, 1970),
and the second mitosis is usually initiated after the start of the first cytoplasmic
cleavage (Jones, 1970; Mihara & Hase, 1971; Osafune, Mihara, Hase & Ohkuro,
1972). The parallel curves we obtain for the timing of the first, second and third
divisions (Fig. 1 A, B) suggest that the timing of the second and subsequent divisions
is determined only by the timing of the first division, with an interval of approximately 1-1-5 h between each. This interval, which is unaffected by light, probably
represents a constant time required for DNA replication, nuclear division and cytokinesis. Our results show that the number of daughters produced in a division burst
is determined by the parent cell volume. However daughter-cell number does not
increase continuously with cell volume: there is instead a sharp doubling in daughtercell number when parent-cell volume reaches 2 n times that of daughter cells (where n
is an integer), but no change in number for intermediate values. Extending the light
period increases the proportion of cells that divide into 4 and 8 daughters (Fig. IB).
Since these large cells divide early in the dark period, the size of the division burst is
not determined until or just prior to division.
We propose that following a division burst, daughter cells enter a state in which
DNA cannot be replicated, and that, in contrast to binary fission cell cycles in which
7-2
188
R. A. Craigie and T. Cavalier-Smith
DNA replication is immediately initiated at a threshold volume (see references in the
Introduction for evidence), nuclear DNA replication in the multiple fission cycle of
Chlamydomonas is not initiated until after the elapse of an interval after reaching a
threshold volume. Cycles, consisting of S+M phases only, then repeat, in quick
succession, until daughter-cell volume is below a threshold level. The number of
daughters produced in a division burst is therefore determined by the amount of cell
growth during the period between attainment of a threshold volume and the last
division of the division burst.
Mechanism and significance of division synchrony
Growth in C. reinhardii cultures under light/dark cycles in minimal medium ceases
during the dark period. If, as we propose, progression beyond some part of Gj
depends on cells first reaching a critical size, this, together with the possibly volumeindependent requirement for a minimum period, or amount, of light before progression
towards division becomes light-independent (Spudich & Sager, 1980), will result in
repeated dark periods synchronizing cell division. The maximum attainable synchrony within each dark period is limited by: (1) the spread in cell size of newly
produced zoospores; and (2) the rate of, and intercellular variation in, growth during
the light period. We predict that in minimal medium the degree of synchrony is
positively correlated with the growth rate during the light period, since the proportion
of the population passing the critical size per unit time is determined by this. Our
results suggest that the length of the period between attainment of a critical size and
division is light-dependent. The proportional difference in the length of this period
for the first and last cells to reach their threshold volume will also be least at high
growth rates, since cells will attain this volume earlier during the light period. Division synchrony should deteriorate if conditions allow newly produced zoospores to
grow as soon as they are produced, as observed (Mihara & Hase, 1971).
C. reinhardii can grow in the dark and may be synchronized by light/dark cycles if
supplied with acetate (Jones, 1970). This is expected provided the growth rate in the
light period is significantly greater than the growth rate of newly produced zoospores
during the dark period. If light/dark cultures are grown at various light intensities
and acetate concentrations, we expect poor division synchrony when the ratio between
the growth rate in the light and that in the dark is low. Synchrony should improve as
this growth-rate ratio increases.
It seems probable that in C. reinhardii, as in many other multiple fission systems
(Cavalier-Smith, 1980), the function of multiple fission is to facilitate the temporal
separation of growth and division. In natural conditions autotrophic growth cannot
occur at night. The period of approximately 12 h between the attainment of a threshold size and division, will ensure that division, which interrupts RNA synthesis
(Mitchison, 1971), will only occur at night and therefore not retard growth during
the day.
Chlamydomonas cell cycle
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(Received 24 July 1981)