Cell cycle phases under continuous illumination
The advantage of a single-cell cultivation method is that it enables to conveniently study and record changes in the size of individual cells as well as the duration of their cell cycle phases without the need for synchronous cell cultivation. Bright-field optical microscopy with a ×100 objective lens reveals the shapes of the cells with a spatial resolution of 0.2 μm, which is sufficient for identifying cell cycle phases, see Figure 2. We divided the Chlamydomonas cell cycle into two phases determined by changes in their outer shape: interdivision and division phases (Figure 3).
The interdivision phase consisted of two subphases called the 'ready-for-hatch' subphase and the 'growth-after-hatching' subphase (Figure 3). In the first subphase, the measured volumes of each of the daughter cells immediately after hatching appeared to be more than a quarter of their mother's final (maximum) cell volume (just before the mother cell entered the division phase). Therefore, daughter cells seem to have started their growth immediately following the mitosis of their mother cell. For example, when the final volume of a mother cell was 277 μm3 (a quarter of which is 69 μm3), the measured volume of each hatched daughter was 87 μm3. Being unable to measure daughter cell volumes immediately following the mitosis, we used the value equal to one quarter of the mother cell maximum volume as the daughter's initial cell volume.
The division phase consisted of five subphases called the 'shrinkage', 'rotation', 'first-mitosis', 'second-mitosis', and 'mitosis-completion' (Figure 3). After cell growth ceased, cells detached their cell membrane from their cell wall and shrunk to form a spherical shape (the shrinkage subphase) [18]. They then rotated within their cell wall (the rotation subphase). After the mother cell divided twice, the shape of the four daughter cells changed from spherical to rod-like (the mitosis-completion subphase).
Effect of illumination intensity on cell cycle phase duration
We examined the effect of phothsynthetically active radiation (PAR) on the duration of each phase at a single-cell level (Figure 3). The amounts of (PAR) used for continuous illumination were 200 (N = 26), 100 (N = 26), 40 (N = 9), 20 (N = 8), and 10 (N = 10) μmol m-2 s-1. Duration of the interdivision phase was inversely related to PAR, increasing by a factor of 8 as PAR decreased by a factor of 20 (Figure 4A). The standard deviation (SD) also differed depending on the light intensities, but the coefficients of variation (CV, a normalized measure of dispersion) were similar at each intensity setting (27%, 23%, 38%, 21%, and 31% at 200, 100, 40, 20, and 10 μmol m-2 s-1 respectively).
In contrast to the interdivision phase, duration of the subphases in the division phase did not change significantly with PAR (Figure 4B) or were independent on the cell volume (data not shown). We conclude therefore that PAR affects duration of the interdivision phase but not that of the following division phase.
Effect of illumination intensity on cell growth during the interdivision phase
We examined the time course of changes in cell volume during the interdivision phase under different conditions of uninterrupted continuous light exposure. We found that the rate of increase in cell volume increases with PAR (Figure 5). Cells cultured at all PARs greater than 0.2 μmol m-2 s-1 entered the division phase when they grew to ~ 4.1 times their initial volume (white arrowheads in Figure 5). Although the cells cultured under 0.2 μmol m-2 s-1 neither grew nor divided, changes in PAR between 10 and 200 μmol m-2 s-1 affected the rate at which cell volume increased but did not affect the ratio of the final cell volume to the initial cell volume.
Exponential growth model of cell volume during the interdivision phase
In order to quantify the rate of cell volume increase, we cultured daughter cells in a microchamber under continuous illumination at 200 μmol m-2 s-1 (Figures 6A and 6B). The volume V(t) of individual Chlamydomonas cells increased exponentially:
(2)
where V(0) is the initial cell volume, just after the completion of mitosis. The rates of cell volume increase (μ) were calculated for various light intensities:
(3)
where T is the time from the completion of mitosis to the beginning of the division phase (interdivision phase duration) and V(T) is the cell volume just before entering the division phase (final cell volume), see Figure 6C. The rates of cell volume increase with PAR and reach a plateau when PAR reaches approximately 300 μmol m-2 s-1. Duration of the interdivision phase (T) and the specific cell volume growth rate (μ) were inversely proportional (Figure 6D);
Combining equations (4) and (2) yields:
(5)
This ratio determines a threshold value for cell size and is identical to the values measured experimentally (see Figure 5).
Cell cultivation under various continuous light (LL, Light-Light) conditions
When cells were cultivated under the continuous but variable light exposure conditions (LL, Light-Light), with the PAR ranging from 10 to 200 μmol m-2 s-1, their volume increased exponentially (see Figure 7A) but the product of the volume increase rate μ
L
and the duration of interdivision phase T
L
remained constant (μ
L
T
L
= 1.4 ± 0.2, mean ± SD, see Figure 8A). Because μ
L
T
L
is dependent on the ratio of final cell volume to initial cell volume:
(6)
and if μ
L
T
L
= 1.4, we conclude that a mother cell is destined to enter the division phase to produce four daughter cells when it had grown to 4.1 times its initial volume. These results suggest that the time at which a Chlamydomonas cell enters the division phase is regulated by a 'sizer'.
Cell cultivation under discontinuous illumination (LD, Light-Dark) conditions
We then examined whether cells would enter the division phase under discontinuous illumination (LD) conditions, i.e. if the cells are devoid of light before their volume increased to the critical threshold level of 4.1 times their initial volume (Figure 7B). If the time at which the division phase is entered were regulated only by their size, cells would stop growing and their cell cycles would stop progressing in absence of any illumination. If cells could enter the division phase in the absence of illumination and growth, they should have another mechanism to regulate the timing of cell division. Under our experimental settings cells stopped growing when illumination (200 μmol m-2 s-1) was switched off, they maintained their volume for some time, but eventually entered the division phase at the time indicated by the white arrowhead in Figure 7B. There V(T
L
)/V(0) is the ratio of the cell volume measured at the time of switching the light off to the initial cell volume, T
L
is the duration of the light exposure. V(T
LD
)/V(0) is the ratio of the final cell volume to the initial cell volume, T
LD
is the interdivision phase duration under the LD condition, and μ
L
is the rate of cell volume increase during the light exposure.
Cell exposure to light was stopped at various times as shown in Figure 7B. Different PARs were tested for cell cultivation, including 200 μmol m-2 s-1 (N = 27), 100 μmol m-2 s-1 (N = 7) and 10 μmol m-2 s-1 (N = 3). Cells devoid of light always stopped growing but eventually entered the division phase even though they had not grown to 4.1 times their initial volume (V(T
L
)/V(0) < 4.1). The product of cell volume growth rate and the duration of interdivision duration μ
L
T
LD
was 1.4 ± 0.2, regardless of illumination timing and PAR as long as V(T
L
)/V(0) > 2, see Figure 8B. These results suggest that the timing of entering the division phase is controlled not only by a 'sizer' but also by another mechanism that is sensitive to the rate of cell growth (rate of cell volume increase). This mechanism triggers a cell to enter the division phase at an interdivision time T = 1.4/μ. We call this new interdivision control mechanism the 'interdivision timer'.
Regulation of the interdivision phase by a volume-based 'interdivision timer'
We also investigated whether re-exposing cells to light would have an effect on the duration of the interdivision phase by the 'interdivision timer'. This was examined using a Light-Dark-Light sequence (LDL conditions). As shown in Figure 7C, re-illuminated cells restarted their growth from the point they had reached before the illumination (200 μmol m-2 s-1) stopped, and eventually entered the division phase. Similarly to the earlier introduced ratio V(T
L
)/V(0), where TL was the duration of the first light period, we can define V(T
LDL
)/V(0) as the ratio of the final cell volume to the initial cell volume, where T
LDL
is the duration of the interdivision phase under the LDL conditions. We found that the rates of cell volume increase μ
L
during the light periods before and after the dark period were virtually the same. We tested various illumination stop and restart points, with the darkness periods varying between 0.8 and 4.0 hours, see Figure 8C. The product of the volume increase rate μ
L
and interdivision phase duration T
LDL
was constant at 1.4 ± 0.2. The re-exposure of cells to light and the timing of darkness periods had little effect on the regulation of the duration of interdivision phase by the 'interdivision timer' as long as V(T
L
)/V(0) > 2. The initial cell volume was 79.7 ± 12.6 μm3 (mean ± SD).
We also examined the effect of reducing PAR during cell growth at continuous lighting conditions (L1L2) on the duration of the interdivision phase by the 'interdivision timer'. As illustrated in Figure 7D, cell growth slowed when PAR was reduced. Similarly to the earlier introduced ratio V(TL)/V(0), where TL is the duration of the first light period, we can define V(T
L1
)/V(0) as the ratio of cell volume at the end of the L1 period to the initial cell volume, where T
L1
is the duration of the first light period. Similarly, V(T
L1L2
)/V(0) is the ratio of final cell volume to initial cell volume, where T
L1L2
is the duration of the interdivision phase (the end of the L1L2 period). A number of different T
L1
and T
L1L2
settings were tested (N = 12), see Figure 8D. We found that the product of the rate of cell volume increase during the first light period, μ
L1
, and the duration of the interdivision phase T
L1L2
remains constant at 1.5 ± 0.2. Changes to the PAR had little effect on the 'timer' when V(T
L1
)/V(0) > 2.
Overall, the results obtained under several illumination conditions (LL, LD, LDL, and L1L2) indicate that there is an 'interdivision timer' regulating the time at which Chlamydomonas cells enter the division phase and that the duration of that interdivision phase T is reverse proportional to the rate of cell volume growth and their product remains constant μT = 1.4.
Onset of the next cell cycle during the interdivision phase is regulated by the 'commitment sizer'
Under LD conditions cells did not enter the division phase for 48 hours following the onset of darkness if V(T
L
)/V(0) < 2 (Figure 8B). This suggests that there is a threshold cell volume ratio for entering the division phase and that cell can divide only if their volume at the end of the light exposure period was more than twice their initial volume.
Under LDL conditions, when V(T
L
)/V(0) < 2 and the duration of dark period was 9 hours, the value of μ
L
T
LDL
increased far in excess of 1.4 (see three open triangles in Figure 8C). However, under continuous illumination μ
L
T
LL
became nearly 1.4 (see three filled triangles in Figure 8C) where T
LL
is the sum of the durations of the first and second light periods. It is evident that a 1.5-h period of darkness introduced before Chlamydomonas cells commit to division does not increase the duration of the interdivision phase. This means that the regulation of the onset of division phase by μT model will not work if V(T
L
)/V(0) < 2.
Under L1L2 conditions, when the cell volume at the time of the change in PAR was less than twice its initial volume (V(T
L1
)/V(0)) < 2.1), the value of μ
L1
T
L1L2
increased above 1.5 (two filled triangles in Figure 8D). However, if the value of μ
L1
T
L1L2
is substituted with μ
L1
T
L1
+ μ
L2
T
L2
, where μ
L2
is the specific volume growth rate during the L2 period and T
L2
is the duration of the L2 period, this values becomes near to 1.5 (two open triangles on Figure 8D).
Overall, the results obtained under several illumination conditions (LD, LDL, and L1L2) indicate that there is a mechanism that decides whether to commit to the next cell cycle phase by determining if V(t)/V(0) > 2. We call this mechanism the 'commitment sizer'.
Regulation of division number by the 'mitotic sizer'
Under LL conditions mother cells grew up to 4.1 times its initial volume and produced four daughter cells, regardless of the PAR (Figure 5). We set out to investigate how many daughter cells would be produced if the volume of the mother cell has not reached 4.1 times the initial volume by the end of the illumination period. Under LD conditions the number of daughter cells (division number) was determined by the ratio of the final cell volume to the initial cell volume, V(T
LD
)/V(0) (Figure 9A). When the value of V(T
L
)/V(0) was below 1.8, the mother cell did not enter the division phase even if more than 48 hours passed. If the value of V(T
LD
)/V(0) was between 2.2 and 2.8, mother cells divided once and produced two daughter cells. When the value of V(T
LD
)/V(0) was above 3.1, mother cells divided twice and produced four daughter cells. Division number varied if the value of V(T
L
)/V(0) was between 1.8 and 2.2 (no division or two daughter cells) or between 2.8 and 3.1 (either two or four daughter cells). These results were the same under various PAR conditions (200, 100, and 10 μmol m-2 s-1).
Similarly to LD conditions, the ratio of the final cell volume to the initial cell volume, V(T
LDL
)/V(0), determined the number of daughter cells under LDL conditions (Figure 9B). Mother cells divided once and produced two daughter cells if this value was between 2.3 and 2.9; whilst if it was above 2.9, mother cells divided twice and produced four daughter cells. The timing and duration of light exposure and darkness periods had little effect on the 'mitotic sizer'.
Similarly to LD and LDL conditions, the ratio of the final cell volume to the initial cell volume, V(T
L1L2
)/V(0), also determined the number of daughter cells under L1L2 conditions (Figure 9C). If it was below 2.7, mother cells divided once and produced two daughter cells, whilst if above 2.7, mother cells divided twice and produced four daughter cells. PAR and the timing of the L1 and L2 periods had little effect on the 'mitotic sizer'.
Results obtained under several illumination conditions (LL, LD, LDL, and L1L2) thus suggest that there is a mechanism that monitors current cell volume and its increase over its initial value (the initial cell volume), and determines the division number accordingly. For consistency with previously used nomenclature, see e.g. Bisova et al.[26], we refer to this mechanism as the 'mitotic sizer'. Previously introduced definition of the 'mitotic sizer', however, is based on the absolute cell volume, whereas ours is based on the relative cell volume (a ratio of the current cell volume to the initial cell volume).