Journal of Cell Science 104, 1163-1173 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1163
Oscillator control of cell division in Euglena: cyclic AMP oscillations
mediate the phasing of the cell division cycle by the circadian clock
Isabelle A. Carré1 and Leland N. Edmunds, Jr*
Department of Anatomical Sciences, State University of New York, Stony Brook, New York 11794, USA
1Present
address: Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901, USA
*Author for correspondence
SUMMARY
The achlorophyllous ZC strain of Euglena gracilis
exhibits a circadian rhythm of cell division in constant
darkness (DD). Mitosis occurs during a restricted part
of the circadian cycle, corresponding to the dark intervals in a light-dark cycle comprising 12 h of light and
12 h of darkness. We have demonstrated that divisionphased cultures also exhibit bimodal, circadian changes
of cyclic AMP level. Maximum cyclic AMP levels
occurred at the beginning of the light period (CT (circadian time) 00-02), and at the beginning of darkness
(CT 12-14). These variations persisted in cultures that
had been transferred into DD and appeared to be under
the control of the circadian oscillator rather than to be
cell division cycle (CDC)-dependent, since they continued in cultures that had reached the stationary phase
of growth. In the experiments reported in this paper,
we tested for the possible role of this periodic cyclic
AMP signal in the generation of cell division rhythmicity by examining the effects of exogenous cyclic AMP
signals and of forskolin, which permanently increased
the cyclic AMP level, on the cell division rhythm.
Perturbations of the cyclic AMP oscillation by exogenous cyclic AMP resulted in the temporary uncoupling
of the CDC from the circadian timer. The addition of
cyclic AMP during the subjective day resulted in delays
(up to 9 h) of the next synchronous division step. In contrast, mitosis was stimulated when cyclic AMP was
administered in the middle of the subjective night. Measurement of the DNA content of cells by flow cytometry indicated that cyclic AMP injected at CT 06-08
delayed progression through S phase, and perhaps also
through mitosis. When added at CT 18-20, cyclic AMP
INTRODUCTION
Cells that grow with a generation time greater than 24 h
often exhibit circadian rhythms of cell division. Typically,
mitosis occurs at a certain phase of the circadian cycle, at
times (subjective nights) frequently corresponding to the
dark intervals in a synchronizing LD:12,12 cycle (the alter-
accelerated the G2/M transition. The circadian oscillator was not perturbed by the addition of exogenous
cyclic AMP: the division rhythm soon returned to its
original phase. On the other hand, the permanent elevation of cyclic AMP levels in the presence of forskolin
induced a rapid loss of cell division rhythmicity. These
findings are consistent with the hypothesis that cyclic
AMP acts downstream from the oscillator and that the
cyclic AMP oscillation is an essential component of the
signaling pathway for the control of the CDC by the circadian oscillator.
The receptors for cyclic AMP in Euglena have been
shown to be two cyclic AMP-dependent kinases (cPKA
and cPKB). Pharmacological studies using cyclic AMP
analogs suggested that cPKA mediates cyclic AMP
effects during the subjective day, and cPKB during the
subjective night. On the basis of these results, we propose a model for the control of the CDC by the circadian clock.
Abbreviations: DD, continuous darkness; LD, light-dark cycle;
LD:x, y, a repetitive light-dark cycle comprising x hours of light
and y hours of dark; τ, average period of a free-running rhythm
under constant conditions, here DD; CT, circadian time (CT 00
indicates the phase-point of a free-running rhythm that has been
normalized to 24 h and that corresponds to the identical phasepoint that occurs at the onset of light in a reference LD:12,12
cycle; the onset of cell division in a dividing culture occurs at
approximately CT 12).
Key words: cyclic AMP, cell division cycle, circadian rhythm,
Euglena, oscillator
nance of 12 h of light and 12 h of darkness). This phenomenon, which has been described in unicellular algae,
fungi and protozoa, as well as in mammalian cells in vivo
(Edmunds and Laval-Martin, 1984; Edmunds, 1988), is
thought to reflect an interaction between an autonomous circadian oscillator and the cell division cycle (CDC).
Circadian cell division rhythmicity in the algal flagellate
1164 I. A. Carré and L. N. Edmunds, Jr
Euglena gracilis has been studied extensively in our laboratory (Edmunds and Laval-Martin, 1984). We are interested in elucidating the biochemical basis of the circadian
clock and the signal transduction pathway(s) that mediate(s)
the control of the cell division cycle by this oscillator. In
order to circumvent the dual use of light (for photosynthesis, and as a time cue for the circadian clock), we have
made use of an achlorophyllous strain (the ZC mutant) that
has been shown to exhibit cell division rhythms that persist in constant darkness (DD) for up to 7 days with a period
(τ) that only approximates 24 h (Carré et al., 1989a).
We have hypothezised that second messengers, which
control many cellular activities, may play a role in the phasing of mitosis and of other processes by the circadian oscillator. We found (Carré et al., 1989b) that the cyclic AMP
level in the ZC mutant exhibits bimodal circadian oscillations, which are independent of cell cycle progression (since
they are also observed in nondividing cells), and which persist in DD with a period similar to that of the cell division
rhythm. Peak cyclic AMP levels occurred at circadian time
(CT) 00-02 (at a time when most cells were in the G1 phase
of the CDC) and at CT 12-14 (corresponding to the onset
of mitosis). Transient surges of cyclic AMP levels, which
were correlated with the initiation of DNA synthesis and
with the onset of mitosis, have also been observed during
the division cycle of Euglena cells that had been blocked
in S phase in the absence of vitamin B12, then released synchronously from this restriction point following the addition of the vitamin (Carell and Deardfield, 1982).
The role of cyclic AMP in the CDC is a subject of controversy, since exogenous cyclic AMP has been found to
stimulate mitosis in some cell types, and to inhibit it in
others (Dumont et al., 1989). Interestingly, however, transient increases of the cyclic AMP level are correlated with
cell cycle transitions at both the G1/S and the G2/M boundaries in many cell types (for reviews, see Boynton and
Whitfield, 1983; Whitfield et al., 1985, 1987). In Saccha romyces cerevisiae, genetic evidence indicates that cyclic
AMP is essential for cells to initiate DNA synthesis (Matsumoto et al., 1983). Cyclic AMP also has been found to
inhibit MPF (mitosis promoting factor), an activity that is
found in the cytoplasm of mitotic cells and is characterized
by its ability to induce nuclear envelope breakdown and
cell division upon injection into maturing Xenopus oocytes
(Maller, 1985).
On the basis of this evidence, we hypothesized that cyclic
AMP may play a role in the cell cycle of Euglena, to regulate the transition through the G1/S or the G2/M boundaries, or both. Thus, periodic cyclic AMP signals, under the
control of a circadian oscillator, may act to initiate certain
portions of the CDC at specific phases of the circadian
cycle. To test for this possibility, we examined the effect
of exogenous cyclic AMP signals, and of agents that permanently increased the cellular cyclic AMP level, on the
cell division rhythm in the ZC mutant.
We show that exogenous cyclic AMP perturbs cell cycle
progression in a manner that depends on the circadian time
of its addition. Cyclic AMP appears to act downstream from
the circadian oscillator, in the output pathway for the control of the CDC, since in most experiments the cell division rhythm quickly returned to its original phase (before
the perturbation). Finally, we investigated the role of the
two cyclic AMP-dependent kinases previously identified in
the ZC mutant of Euglena (Carré and Edmunds, 1992),
using cyclic AMP analogs that have been shown to specifically activate either of the two kinases.
MATERIALS AND METHODS
Organism and culture conditions
The achlorophyllous ZC mutant of Euglena gracilis Klebs (strain
Z) was obtained from Dr R. Calvayrac (Laboratoire des Membranes Biologiques, Université Paris VII, France). It was derived
from the wild-type strain by action of 2.5×10-5 M diuron (DCMU)
in a 33 mM lactate medium (pH 3.5) under illumination and anoxia
(Calvayrac and Ledoigt, 1976).
Axenic, aerated, magnetically stirred 4 l batch cultures were
grown at 16.5(±0.5)°C in environmental chambers, on a modified
Cramer and Myers’ (1952) medium supplemented with vitamins
B1 and B12 (as previously described by Edmunds and Funch,
1969), and containing ethanol (0.1 to 0.4%, v/v) as a carbon
source. Cysteine and methionine (10-5 M), were added to the
experimental cultures in order to improve cell division rhythmicity. Sulfur-containing coumpounds have been shown to allow a
better coupling between the CDC and the underlying circadian
oscillator in other chloroplastidic mutants of E. gracilis (Edmunds
et al., 1976). Illumination (3000 lx) was provided by clock-programmed, cool-white fluorescent bulbs. Cell number was monitored every 2 h by a miniaturized fraction collector and a Coulter Electronic Particle Counter (Edmunds, 1964).
Cultures growing in the infradian mode (g >24 h) were obtained
at 16.5(±0.5)°C. Cell division could then be entrained to a 24 h
period by imposition of LD:12,12. Typically, the cells divided
during the dark intervals, and the onset of mitosis occurred at the
onset of darkness (12 h after light onset). The circadian rhythm
of cell division persisted after transfer of the culture to DD, with
a period (τ) of 25±2 h (Carré et al., 1989a). The onsets of cell
division were taken as phase reference points and were considered to fall at CT 12, which corresponds to the onset of darkness
in a LD:12,12 reference cycle.
Methodology for deriving phase-response curves
Cultures that had been pre-entrained in LD:12,12 were placed into
DD. These ‘free-running’ cultures were perturbed by the addition
of cyclic AMP (or of other drugs) to the culture medium at different CTs (corresponding to different phase points of the CDC)
during the second day in DD. Advances or delays of the CDC
were measured as the difference between the onset of the subsequent cell division step and the predicted one, projected from the
last unperturbed cycle with a period of 25 h. The steady-state
phase shifts (∆φ), indicative of possible perturbations of the circadian oscillator itself, were also measured after transients had
subsided (in 2 or 3 days). Phase advances were arbitrarily designated +∆φ and phase delays −∆φ (Pittendrigh, 1965). In all cases,
both CT and data for ∆φ were normalized to 24 h in order to facilitate comparison among cell cultures which had different free-running periods ( τ). A plot of the ∆φ (if any) engendered by a cyclic
AMP signal as a function of the CT at which the pulse was administered yielded a phase-response curve for the rhythm of cell division (see Edmunds et al., 1982).
Flow cytometry analysis of DNA content
For each time point, approximately 2×106 cells were harvested by
centrifugation (3000 g for 10 min) and resuspended in 5 ml of
70% ethanol. Cells were washed once in 5 ml of 70% ethanol and
Cyclic AMP and circadian control of cell division 1165
stored in the same medium for up to 10 days at 4°C. On the day
of the assay, cells were treated with RNAse A and stained with
propidium iodide (50 µg/ml), as described by Yee and
Bartholomew (1988, 1989). The cell suspension was then filtered
through Nytex screens (60 µm pores) and analyzed with a Becton
Dickinson FACS flow cytometer. Total cellular DNA content also
was estimated colorimetrically by the diphenylamine reaction
(Burton, 1955).
Cyclic AMP measurements
Approximately 10×106 cells were harvested by centrifugation (10
min, 7700 g) of the cell suspension. The pellet was resuspended
in 0.7 ml of distilled H2O, and was extracted in 7.5% (final concentration, w/v) TCA for 20 min at 4°C. Pelletable material was
eliminated after centrifugation (10 min, 39000 g). The supernatant
was then extracted 5 times with an equal volume of water-saturated diethylether. The remaining ether was boiled off at 80-90°C
until bubbling stopped. The extracts were frozen in liquid nitrogen and were stored at −70°C until the day measurements were
performed.
The amounts of cyclic AMP were measured by a competitive
protein-binding assay, modified from Gilman (1970) and Døskeland and Ogreid (1988). Assays were carried out in test tubes kept
in iced water, into which were successively injected: 50 µl of cell
extracts (or of standard solutions of cyclic AMP), then 100 µl of
an incubation mixture containing 40 µg of protein kinase inhibitor
(Sigma) and 3 pmol (60000 d.p.m.) of [3H]cyclic AMP (Amersham) in sodium acetate (50 mM, pH 4.0). The binding reaction
was started by the addition of either 50 µl of a protein mixture
(containing approximately 10 µg of protein kinase (Sigma) and
40 µg BSA) or of 50 µl H 2O (for the measurement of nonspecific
binding). The samples were incubated at 0°C for 90 min, and the
reaction was stopped by dilution with 1 ml of 80% saturated
ammonium sulfate and 0.05 mM Hepes (pH 7.0). The reaction
mixture was filtered through cellulose ester Millipore filters
(HAWP025). The filters were rinsed three times with 3 ml of 60%
saturated ammonium sulfate and 0.05 mM Hepes (pH 7.0) and
then were transferred to scintillation vials. 1 ml of 2% SDS was
added in order to solubilize the proteins bound to the filters, prior
to the addition of scintillation fluid (Scintiverse, Fisher) and counting for 10 min in a Packard Tri-Carb liquid scintillation spectrometer (Model 3320). The cyclic AMP concentrations per 106
cells were extrapolated from a standard curve that was obtained
simultaneously by exactly the same procedure applied to known
quantities of cyclic AMP (ranging from 0.5 to 20 pmol).
RESULTS
Perturbation of the cell division rhythm by cyclic
AMP
To facilitate the understanding of the relationship between
circadian time (CT) of the rhythm of cell division in a population of Euglena and the discrete phases of the cell division cycle (CDC), we show (Fig. 1) as a reference one division step of a culture in LD:12,12 (see Edmunds, 1964).
The plateau of the growth curve lasts for approximately 10
h (from CT 02 to CT 12). Cellular DNA content starts to
increase earlier in the day (approximately CT 04). The rate
of DNA synthesis reaches a maximum at (or just before)
the beginning of the night (CT 10-12). The onset of cell
division in the culture corresponds approximately to CT 12.
The maximum division rate is observed at the end of the
night (CT 18 to CT 24), in parallel with a sharp drop in
Fig. 1. Circadian rhythm of cell division in a culture of the ZC
mutant of Euglena entrained by LD:12,12. Changes in the average
total DNA content (measured by the assay of Burton, 1958) are
indicated by open circles, and variations in cell concentration by
filled circles.
cellular DNA content. After the cultures are transferred to
constant darkness (DD), CTs are determined by projection
from the onset of cell division, taken as a phase-reference
point for CT 12.
To ask whether the periodic cyclic AMP signals generated by the circadian oscillator may play a role in phasing
cell division, we assayed for the effect of exogenous cyclic
AMP signals on the timing of the subsequent cell division
steps in cultures of the ZC mutant that were dividing rhythmically in DD with a period (τ) of 25 h. The incubation
the of ZC mutant for 1 h in the presence of exogenous
cyclic AMP increased the cellular cyclic AMP content in a
concentration-dependent manner (not shown). We chose to
test first the effect of 250 µM cyclic AMP, which after 1
h increased cellular cyclic AMP concentration from 20 to
approximately 60 pmol per 106 cells. We found that cyclic
AMP added to synchronously dividing cultures induced
advances or delays of the following burst of cell division,
depending on the CT at which the drug was given. Four
typical experiments are shown in Fig. 2. The injection of
cyclic AMP at CT 12 caused a 5 h delay of the subsequent
division burst (Fig. 2A). Although cyclic AMP was added
in a permanent manner (tonically), its effect on the cell division rhythm was only transient. The following period was
shortened, bringing the rhythm back to its original phase,
an indication that the underlying circadian oscillator had
not been perturbed by the cyclic AMP signal. The same
experiment repeated at CT 13.9 did not induce any perturbation, even transiently, of the cell division rhythm (Fig.
2B). Injection of cyclic AMP between CT 18 and CT 22
had dramatic effects on the oscillation. The plateau following the cyclic AMP signal was shortened (indicating the
advanced division of some of the cells); the pattern of cell
division then became arrythmic (Fig. 2C,D). In two experiments only, rhythmicity was restored after 36 h (as in Fig.
2C,D), and the cell division rhythm appeared to have been
phase-shifted by 10 to 11 h.
The free-running rhythm of cell division exhibited by the
ZC mutant was systematically scanned by cyclic AMP signals, given at different CTs during the second free-running
1166 I. A. Carré and L. N. Edmunds, Jr
period in DD. Advances or delays of the cell division steps
were measured as described for Fig. 2 (see Materials and
Methods). From these data, two phase-response curves were
generated, for (a) the immediate perturbations of the first
division step following the addition of cyclic AMP (which
reflect the effect of cyclic AMP on cell cycle progression),
Fig. 2. Perturbation of the
circadian rhythm of cell
division by cyclic AMP in the
achlorophyllous ZC mutant of
Euglena. Cultures that had
been synchronized by
LD:12,12 were transferred to
DD. Cyclic AMP was injected
into the culture during the
second circadian cycle in DD
at the circadian time (CT)
indicated by the open arrow.
The onset of cell division was
used as a phase marker
(vertical dotted line) and
compared to the theoretical
phase of the rhythm (black
markers) in an unperturbed
control culture, projected from
the last division step before
the perturbation with a period
of 25 h, a value which
corresponded to the freerunning period of the rhythm
in DD. Phase differences are
given here in real time. The
period length (interval
between successive division
bursts) is indicated in a circle.
The step size (factorial
increase in cell concentration,
from plateau to plateau) is
given on the left of the
corresponding step.
Cyclic AMP and circadian control of cell division 1167
and (b) the steady-state effects of cyclic AMP on the cell
division rhythm (which might indicate perturbations of the
underlying circadian timer).
Fig. 3A shows the phase-response curve for the perturbation of the cell cycle by cyclic AMP in the ZC mutant.
Cyclic AMP injected between CT 06 and CT 12 caused
delays (4 to 6 h) of the following burst of cell division,
whereas the same signal given between CT 18 and CT 22
induced some of the cells to divide before the following
CT 12 time-point. The advance was estimated to be as great
as 8 h. Cyclic AMP had no effect when injected at CT 0002 and at CT 12-14 (when the endogenous cyclic AMP
level is at its peak). These results indicate that cyclic AMP
signals may modulate cell cycle progression in Euglena.
To test for a possible role of cyclic AMP as an element
or ‘gear’ of the circadian oscillator, we also looked for permanent phase-shifting effects of cyclic AMP on the cell
division rhythm. Fig. 3B shows the steady-state resetting
effects of cyclic AMP on the cell division rhythm. No significant lasting phase shifts were observed at any circadian
time (with the exception that in two experiments apparent
phase delays of 10 to 12 h surprisingly were elicited by
cyclic AMP administered at approximately CT 20; see Fig.
2C,D). Similarly, no steady-state phase shifts were obtained
when synchronously dividing cultures of the ZC mutant
were exposed to 500 µM cyclic AMP for 1 h, then diluted
50-fold, thus reducing the cyclic AMP concentration down
to 10 µM (Fig. 3B, open symbols).
Flow cytometry analysis of DNA content
In order to determine whether the perturbations seen on the
growth curve reflected real changes in the rates of cell cycle
transitions, rather than changes in cell motility or settling,
we measured cellular DNA content in control Euglena cultures and in cultures that had been perturbed by cyclic
AMP, using flow cytometry techniques.
A typical DNA histogram from a nonsynchronized culture of the ZC mutant growing exponentially in constant
light is shown in Fig. 4A. Approximately 41% of the cells
had a single (1C) DNA content and constituted the G0/G1
A
population. An estimated 37% of the cells had a double
(2C) DNA content and thus were either in G2 or in mitosis. Cells having an intermediate DNA content (approximately 22%) constituted the S phase population. The mean
fluorescence of the G2 peak was only 184% of that of the
G1 peak, due to the poor accessibility of DNA for dye in
G2 cells, as previously described in Euglena (Bonaly et al.,
1987). The high percentage of cells with a 2C DNA content (normally half of that of the percentage of cells in G1
in rapidly cycling cultures) suggested a blockage point in
G2. The existence of such an arrest point was confirmed by
examining the DNA content in cells from stationary phase
cultures (Fig. 4B). No cells were found with an intermediate DNA content, a finding that indicated that cells had
stopped cycling. Cells were found arrested with either a 1C
or a 2C DNA content (59% and 41%, respectively). Thus,
in Euglena, major cell cycle control points appear to operate in both the G1 and the G2 phases of the CDC.
To test for the effect of cyclic AMP on cell cycle progression, cultures of the ZC mutant were synchronized by
LD:12,12 and then were transferred to DD. At the time of
the experiment, the cultures were divided into two halves,
one of which received cyclic AMP or a cyclic AMP analog,
while the other half was used as a control. Fig. 5A shows
the effect of cyclic AMP (1 µM) injected at CT 06 (plateau
of the growth curve) on cell cycle progression. In the control culture, from t=0 to t=12 h, the G1 peak decreased,
while the G2/M peak increased, an observation indicating
the progression of cells from G1 into S and G2. By t=22 h
(CT 04), the G1 peak was markedly increased, a finding
that indicated that a large portion of the cell population had
just completed mitosis. We noticed the slower accumulation of cells with a 2C DNA content (visible from t=4 to
t=12) in the presence of cyclic AMP (1 µM, added at CT
06), results that were indicative of an inhibition of the progression of cells through S phase. By t=22 h, most cells
had left G1 and were in late S phase or in G2, at a time
when cells from the control experiment were already going
back to the G1 phase. Thus, the delay of cell division
observed in growth curves after the addition of cyclic AMP
at CT 06-08 was due to an inhibition of DNA synthesis. In
B
Fig. 3. Phase-response curves for the perturbation of the free-running rhythm of cell division by cyclic AMP in the achlorophyllous ZC
mutant of Euglena. The curves were derived from 17 experiments similar to those shown in Fig. 2. (A) Advances or delays of the first cell
division step following cyclic AMP injection into the culture medium. (B) Steady-state phase shifts, measured (when possible) 3 to 4 days
after the perturbation. Open symbols: the cell suspension was diluted 50-fold with fresh medium after a 1 h exposure to cyclic AMP.
Filled symbols: the culture was not diluted. Triangles, 500 µM cyclic AMP:, circles, 250 µM cyclic AMP:, squares, 100 µM cyclic AMP.
1168 I. A. Carré and L. N. Edmunds, Jr
CDC. This observation was consistent with the advance of
cell division observed in growth curves (Fig. 2C,D).
Fig. 4. Flow cytometry analysis of DNA content in the ZC mutant
of Euglena, grown at 17°C on a mineral medium supplemented
with ethanol. Cells were fixed in 70% ethanol, treated with
RNAse A, then stained with propidium iodide immediately prior
to analysis in a fluorescence-activated cell sorter (FACS).
(A) Dividing culture, growing exponentially with a generation
time of 30 h. (B) Stationary-phase culture. The G0/G1 peak,
corresponding to cells with a single DNA content, was found at
channel 228, and the G2/M peak, corresponding to a fully
replicated DNA content, at channel 420. Cells in the S phase of
the cell cycle have an intermediate DNA content. Cells in
stationary phase were found with both single and double DNA
contents, an observation indicating that major restriction points for
the CDC operate in both the G1 and the G2 phases.
the experiment shown in Fig. 5A, we were unable to detect
any effect of cyclic AMP on the progession of cells through
mitosis. In another experiment (not shown), however, in
which 8-BZA-cAMP (8-benzylamino-cyclic AMP) was
used, we observed the accumulation of cells with a fully
replicated DNA, a finding that indicated an inhibitory effect
of 8-BZA-cAMP on the G2/M transition.
Fig. 5B shows the results of a similar experiment performed at CT 20 (in the middle of the division phase). In
the control culture, the G1 peak increased from t=0 to t=8
h, an observation demonstrating that cells proceeded through
mitosis. At the same time, cells began to escape from G1
into S phase. From t=12 h to t=22 h, the accumulation of
cells with a 2C DNA content was found. The effect of 1
µM cyclic AMP injected at CT 20 was clear 12 h after the
cyclic AMP signal: the proportion of cells with a 2C DNA
content was markedly decreased as compared to the control
experiment. The progression of cells through G1 and S
phases, however, did not seem to be affected since the proportion of cells in early S phase increased normally between
hour 8 and hour 12. Thus, the decreased cell population with
a double DNA content was due to a shortening of the G2
phase rather than to a blockage in an earlier phase of the
Permanent elevation of cyclic AMP level in the
presence of forskolin causes the loss of cell
division rhythmicity
We have demonstrated that exogenous cyclic AMP signals
cause lengthenings or accelerations of the CDC, depending
on the CTs when the drug is added. We still needed to
determine whether the endogenous circadian variations of
cyclic AMP levels have sufficient amplitude to have similar effects on cell cycle progression. Our approach was to
test the effect of drugs that may reduce the amplitude of
the cyclic AMP oscillation, or that keep cyclic AMP at a
level such that all cyclic AMP receptors should be saturated in a permanent manner. We would expect such drugs
to prevent the expression of cell division rhythmicity.
Forskolin, which has been shown to stimulate adenylate
cyclase in Euglena at times when the activity of the enzyme
is minimal (Tong et al., 1991), would be expected to have
such an effect on the cyclic AMP oscillation. When
forskolin was injected into a culture of the ZC mutant that
was maintained in LD:12,12, the cyclic AMP level was
increased by as much as 3-fold, as compared to an untreated
control (Fig. 6). Maximum increases of cyclic AMP level
corresponded to times when cyclic AMP in the control culture was minimum; forskolin had little effect at times when
cyclic AMP was at its maximum. As a result, the cyclic
AMP level in the presence of forskolin never exceeded the
range of cyclic AMP concentrations in control cultures, but
oscillated with a reduced amplitude, and at a higher level.
When forskolin was added to a free-running culture of the
ZC mutant (previously synchronized by LD:12,12) at the
time of its transfer to DD (at approximately CT 12), we
observed a rapid desynchronization of the cell population
(Fig. 7A). The cells appeared to go through one synchronous division step, then resumed nearly exponential growth.
Very low-amplitude waves of cell division were still visible, however, for a minimum of 3 more days, a sign that
that the circadian timer was running unaffected. Such a
rapid loss of cell division rhythmicity sometimes occurs
after transfer of control cultures to DD, but heretofore has
never been observed in cultures that had been maintained
in 24-h LD cycles. When forskolin was added to a culture
that was entrained by LD:12,12, a rapid damping of the cell
division rhythm was also observed (Fig. 7B). This effect
was not phase-dependent: similar results were obtained
when the drug was added at CT 12 (Fig. 7A,B) or at CT
04, 08, 15, or 00 (not shown). These results suggest that
the amplitude of the cyclic AMP oscillations determine the
amplitude of the cell division rhythm.
The opposite effects of cyclic AMP observed at CT
06-08 and at CT 18-20 are mediated by two
different cyclic AMP-dependent kinases
We have demonstrated earlier (Carré and Edmunds, 1992)
that two cyclic AMP-binding proteins are present in the ZC
mutant of Euglena, which comigrated with two peaks of
cyclic AMP-dependent kinase activity (cPKA and cPKB)
during DEAE-cellulose chromatography. The distinct cyclic
AMP analog-specificity of the two cyclic AMP-dependent
Cyclic AMP and circadian control of cell division 1169
Fig. 5. Effects of cyclic AMP on cell cycle
progression. Cultures of the ZC mutant
synchronously dividing in DD were divided into two
halves, one of which received cyclic AMP (white
histograms), while the other was used as a control
(black histograms). Cells were harvested every 4 h
following the perturbation and treated as described
for Fig. 4. (A) The addition of cyclic AMP at CT 06
delayed the progression of cells through S phase.
(B) The addition of cyclic AMP at CT 20 stimulated
mitosis (notice the decrease of the G2 peak at t=12 h).
The time elapsed since the addition of cyclic AMP to
the culture medium is indicated on the right of the
corresponding histograms, and the circadian time is
given on the left.
kinases identified in Euglena extracts enabled us to ask
whether only one or both of these enzymes play a role in
cell cycle control. Thus, we tested the effect of different
concentrations of cyclic AMP, 8-BZA-cAMP, 8-CPTcAMP (8-(4-chlorophenylthio)-cyclic AMP), and 6-MBTcAMP, (N6-monobutyryl-cyclic AMP), at times (CT 06-08
and CT 18-20) when 250 µM cyclic AMP induced maximum perturbations of the cell division rhythm.
We found that a 2- to 5-fold increase in cyclic AMP or
in cyclic AMP analog concentration was sufficient to elicit
maximum perturbation of division rhythmicity. Table 1
gives the minimum concentrations of cyclic AMP and of
cyclic AMP analogs that induced advances or delays of cell
cycle progression that were comparable to those previously
obtained with 250 µM cyclic AMP. Surprisingly, different
results were obtained at CT 06-08 and at CT 18-20. Thus,
8-CPT-cAMP, which specifically activated cPKB in vitro
(Carré and Edmunds, 1992), had no effect on cell cycle pro-
gression when given at CT 06-08, up to a 1 µM concentration; in contrast, very low concentrations (0.1 nM) of the
same analog were able to accelerate mitosis when given at
CT 18-20. Furthermore, 8-BZA-cAMP (which specifically
activates cPKA; Carré and Edmunds, 1992) caused large
delays of cell division when added to the culture at CT 0608 at a 100 nM concentration, but was ineffective up to a
1 µM concentration at CT 18-20. Finally, the minimum concentrations of cyclic AMP and cyclic AMP analogs that
caused perturbations of the cell division rhythm at CT 0608 were correlated with the Ka values of cPKA for these
ligands (compare columns 1 and 2, Table 1), and the concentrations that were effective at CT 18-20 corresponded
to the Ka values of cPKB for these ligands (columns 3 and
4, Table 1). These results suggested that the delaying effects
of cyclic AMP observed during the subjective day were
mediated by cPKA, and the accelerating effects of cyclic
AMP during the subjective night by cPKB.
1170 I. A. Carré and L. N. Edmunds, Jr
ing cell division step, depending on the CT at which the
signal was given (Fig. 2). Maximum delays were obtained
at CT 06-08 and maximum advances at CT 18-20, both
times at which the endogenous cyclic AMP level is minimal (Carré et al., 1989b). Cyclic AMP did not perturb the
cell division rhythm at times when its endogenous level was
at its peak (CT 00-02 and CT 12-14), a finding that suggests that the cellular cyclic AMP concentration was
already saturating.
Fig. 6. Effect of forskolin on the cyclic AMP oscillation in a
culture of the ZC mutant entrained by LD:12,12. Forskolin (an
activator of adenylate cyclase) was added at the beginning of the
experiment (CT 16), and cellular cyclic AMP content was
followed for 12 h (filled circles). The variation in cyclic AMP
level in a control culture is indicated by the open circles. Each
point is the average of triplicate assays:, the error bars indicate the
range of the values obtained for the cyclic AMP concentration.
DISCUSSION
We have investigated the possible role of the bimodal circadian oscillation of cyclic AMP levels, previously demonstrated in the ZC mutant of Euglena (Carré et al., 1989b),
in the generation of cell division rhythmicity. Our rationale
was that if the periodic cyclic-AMP signal does play a role
in phasing cell division, perturbations of the cyclic AMP
oscillation by drugs that affect cyclic AMP metabolism
should change the timing of the following burst of cell division. We showed that the permanent addition of cyclic AMP
to the cultures of the ZC mutant, synchronously dividing
in DD, induced transient advances or delays of the follow-
Cyclic AMP is unlikely to be an element of the
circadian oscillator itself
In most experiments, the perturbation of the cyclic AMP
oscillation by exogenous cyclic AMP did not induce permanent phase shifts of the cell division rhythm (Fig. 3B),
an indication that the circadian oscillator was unaffected by
the signal. A complete loss of cell division rhythmicity was
observed following cyclic AMP injection at CT 18-20, however, a finding that suggests a profound disturbance of the
coupling pathway for the control of the CDC by the circadian oscillator (Fig. 2C,D). In two cultures, the cells
resumed rhythmic cell division 48 to 72 h after the cyclic
AMP signal, with a 10-12 h phase shift. It is possible that
cyclic AMP is only able to interact with the circadian oscillator at precisely this time and brings it close to its singularity point. Another hypothesis is suggested by the bimodal
nature of the cyclic AMP oscillation: since cyclic AMP signals occur at 12 h intervals, it is possible that two solutions
exist (180° out of phase with each other) for recoupling the
CDC to the circadian oscillator. This would explain the fact
that only 10-12 h phase shifts were obtained.
We believe that it is unlikely that cyclic AMP serves as
an element of the circadian oscillator in Euglena. If the
pacemaker consists of a biochemical feed-back loop, one
would indeed expect exogenous cyclic AMP signals to
induce both permanent advances and delays of the cell division rhythm. It could be that some of the targets of cyclic
Fig. 7. Effect of forskolin on the cell
division rhythm in the ZC mutant. Cell
division was synchronized by LD:12,12
prior to the addition of forskolin.
(A) The culture was transferred to DD
at the time of the addition of the drug
(indicated by the open arrow). (B) The
culture was maintained in LD:12,12.
Both experiments show a rapid loss of
cell division rhythmicity, but the
generation time was unaffected.
Cyclic AMP and circadian control of cell division 1171
Table 1. Cyclic AMP-analog specificities of cyclic AMP-dependent kinases from Euglena and minimum doses of
cyclic AMP analogs that perturb the cell division rhythm at CT 06-08 or at CT 18-20 in the ZC mutant
Analog
Cyclic AMP
8-(4-chlorophenylthio)-cAMP
8-benzylamino-cAMP
N6-monobutyryl-cAMP
(1)
cPKA
(Ka,nM)
(2)
CT 06-08
(delays)
(3)
cPKB
(K a, nM)
(4)
CT 18-20
(advances)
5 (200%)
No effect
200 (600%)
5 (130%)
1
No effect
100
10
50 (200%)
2 (150%)
No effect
50 (130%)
50
0.1
No effect
ND
The magnitude of the stimulation of the kinases (% of basal activity) by the cyclic AMP analogs is given in parentheses (columns 1 and 3). The
maximum concentration tested was 1 mM.
The lowest concentrations of the cyclic AMP analogs that were found to reproduce the effects of 250 nM cyclic AMP applied at either CT 06-08
(column 2) or CT 18-20 (column 4) are given. The maximum concentration tested was 1 µM.
ND, not determined.
AMP or of cyclic AMP-dependent kinases in excitable tissues in which cyclic AMP does phase-shift the output
rhythm (Eskin et al., 1982; Prosser and Gillette, 1989) interact with the circadian oscillator. Such indirect resetting of
the clock by cyclic AMP – if, indeed, this be the case –
does not occur in Euglena (except possibly around CT 20).
Cyclic AMP controls cell cycle progression in
Euglena
The advances and delays of cell division steps observed in
growth curves following cyclic AMP injection reflected real
changes in the rate of cell cycle progression, as shown by
DNA flow cytometry (Fig. 5). Addition of the drug at CT
06 (Fig. 5A) caused a delay in the progression of cells
through S phase. This finding is reminiscent of results
obtained in mammalian cells, showing that, although a
cyclic AMP signal is necessary for G1/S transition, preventing it from subsiding blocks the initiation of S phase,
or prematurely terminates DNA replication (Boynton and
Whitfield, 1983). In another experiment, utilizing 8-BZAcAMP, cells also appeared to be blocked in the G2 phase
or in mitosis, since the accumulation of cells with a 2C
DNA content was observed 12 h after the addition of the
drug (not shown). A similar inhibitory action of cyclic AMP
and cyclic AMP-dependent kinase on cell cycle progression
late in the division cycle has been demonstrated in animal
oocytes arrested in the first prophase of meiosis. Reinitiation of cell division by fertilization in starfish, or by specific hormones in amphibian and in mammalian oocytes, is
accompanied by a drop in cyclic AMP concentration
(Meijer and Zarutskie, 1987; Maller, 1985; Schultz et al.,
1983; Maller and Krebs, 1977). Thus, cyclic AMP may play
a similar role in Euglena, possibly by mediating the inhibition of MPF activity (Maller, 1985).
A cyclic AMP signal given between CT 18 and 20 (Fig.
5B) had the opposite effect on synchronously dividing cultures: 12 h after cyclic AMP injection we observed a marked
decrease in the amount of cells with a 2C DNA content, as
compared to the control, unperturbed half of the culture. The
depletion of G 2 cells was due to an acceleration of mitosis,
rather than to an inhibition of DNA synthesis, since no delay
was observed in the progression of cells through S phase.
This result indicates that cyclic AMP also plays a stimulatory role during the cell cycle in Euglena. It has not yet been
demonstrated unambigously whether cyclic AMP plays such
a role in mitosis. It has been suggested, however, that cyclic
AMP-dependent phosphorylation plays a role in the regulation of mitosis through the regulation of spindle assembly
and (or) of microtubule function. The subunits of cyclic
AMP-dependent kinase have indeed been localized on the
cytoplasmic microtubules, on centrosomes, and on the
mitotic spindle (Browne et al., 1980; Tash et al., 1981; Nigg
et al., 1985; De Camilli et al., 1986). Furthermore, the
microinjection of the heat-stable specific inhibitor of the
cyclic AMP-dependent kinase into sea urchin oocytes 20 to
45 min after fertilization blocks the assembly of the mitotic
spindle, without preventing nuclear envelope breakdown
(Browne et al., 1990). In the ZC mutant of Euglena, cyclic
AMP did not seem to have any stimulatory effect on the
progression through G1 and S phases, although it does in
other systems (Boynton et al., 1983). It is possible, however, that our assay was not sensitive enough, or that the
degree of synchrony of the cell population was insufficient
to detect such an effect.
Do rhythmic cyclic AMP signals play a role in the
expression of cell division rhythmicity?
These results are consistent with the hypothesis that periodic
cyclic AMP signals act downstream from the circadian oscillator to control cell cycle progression (and other cellullar
activities). If this hypothesis holds true, however, permanent
increases in cyclic AMP level would be expected to have an
uncoupling effect, inducing a permanent loss of cell division
rhythmicity. Such a permanent effect was not observed following the permanent addition of cyclic AMP or of phosphodiesterase-resistant cyclic AMP analogs of such as (SpcAMP) to the culture medium. One possible explanation is
that the cells may have become desensitized (for example,
by becoming impermeable to the drug), thus allowing the
circadian oscillator to regain control of the CDC.
Nevertheless, we were able to reduce the amplitude of
the cyclic AMP oscillation by using forskolin (Fig. 6),
which activates adenylate cyclase in Euglena at CTs when
its activity is minimal (Tong et al., 1991). When added to
LD-synchronized, rhythmically dividing cultures of the ZC
mutant, forskolin (10 µM) also caused a rapid loss of cell
division rhythmicity, both in populations that were maintained in LD:12,12 and in those that were subsequently
transferred to DD (Fig. 7A,B). This desynchronizing effect
of forskolin on the cell division rhythm in Euglena strongly
1172 I. A. Carré and L. N. Edmunds, Jr
indicates that the bimodal, circadian oscillation of cyclic
AMP levels exhibited by the ZC mutant is necessary for
the phasing of cell division by the circadian oscillator.
Further downstream along the transduction
pathway: role of cyclic AMP-dependent kinases
We have previously shown that the ZC mutant of Euglena
gracilis contains two types of cyclic AMP-dependent
kinases (cPKA and cPKB), which have different affinities
for cyclic AMP and for several cyclic AMP analogs (Carré
and Edmunds, 1992). A correlation between the potency of
a cyclic AMP analog in activating one type of kinase and
in causing physiological responses can provide evidence for
a role of this enzyme in mediating the effect of cyclic AMP
(Beebe et al., 1988). The differential activation of the two
kinases identified in Euglena extracts by these cyclic AMP
analogs provided us with a tool for the study of their respective roles in the control of cell cycle progression, and we
determined the mimimum doses of cyclic AMP or of cyclic
AMP analogs that caused perturbations of the cell division
rhythm during the subjective day, or during the subjective
night.
Interestingly, different analogs were effective at CT 0608 and at CT 18-20, results that suggest that the action of
cyclic AMP at the different CTs was mediated by two different cyclic AMP receptors. In addition, there was a correlation between the doses of cyclic AMP, 8-BZA-cAMP,
8-CPT-cAMP, 8-Br-cAMP, and 6-MBT-cAMP that caused
perturbations of the CDC at CT 06-08, and the Ka values
of cPKA for these analogs (Table 1), a finding that suggests that cPKA mediates the delaying effects of cyclic
AMP at those CTs. Similarly, there was a correlation
between the doses of the same nucleotides that caused perturbations of the CDC at CT 18-20 and the Ka values of
cPKB for these analogs (Table 1), an observation indicating that cPKB mediates the accelerating effects of cyclic
AMP at CT 18-20.
A model for the control of the CDC by the
circadian oscillator
These results have been incorporated into a model for the
coupling of the CDC to the circadian oscillator (Fig. 8).
Bimodal, out-of-phase variations in the activities of adenylate cyclase and phosphodiesterase cause the cellular cyclic
AMP level to oscillate (Tong et al., 1991), with peaks corresponding to CT 00-02 and CT 12-14 (Carré et al, 1989b).
We propose that the cyclic AMP surge at CT 00-02 delays
DNA synthesis and holds the cells at a restriction point in
G2, preventing cell division during the subjective day. The
cells are released from this blockage after the level of cyclic
AMP subsides, and the G2/M transition, or mitosis itself,
is accelerated by the second cyclic AMP peak, at CT 1214, so that division is phased to the subjective night. The
delaying effects of cyclic AMP on cell cycle progression
during the subjective day would be mediated by the activation of cPKA, and the stimulation of mitosis during the
subjective night by the activation of cPKB. Activation of
either of these kinases would cause the phosphorylation of
a different set of targets, and perturb different cell-cycle
control pathways. cPKA and cPKB may be expressed at
different phases of the CDC. Alternatively, the level of
Fig. 8. Model for the ‘gating’ of cell division by the circadian
oscillator. We propose that the cyclic AMP surge at CT 00-02
delays DNA synthesis (and, perhaps, holds the cells at a
restriction point in G2) to prevent cell division during the
subjective day. The cells are released from this (these) blockage(s)
after cyclic AMP levels subside, and mitosis is stimulated by the
second cyclic AMP peak (at CT 12-14) so that cell division is
phased to the subjective night. Opposite effects of cyclic AMP on
cell cycle progression are explained by its action through two
different cyclic AMP-dependent kinases (cPKA and cPKB),
which would be expressed at different stages of the CDC (or at
different phases of the circadian cycle) and which have different
sets of targets.
these enzymes might exhibit circadian variations, with
cPKA being expressed during the subjective day and cPKB
during the subjective night. Another possibility is that the
level of one of their downstream targets oscillates, so that
only cPKA activation has an effect on cell cycle progression during the subjective day, and cPKB during the subjective night. Future studies of the regulation of cyclic
AMP-dependent kinases during the CDC (or during the circadian cycle), and the identification of targets that are selectively phosphorylated by these enzymes, will be necessary
to ascertain this part of the model.
In contrast to the model of Homma and Hastings (1989)
for the circadian control of cell division in Gonyaulax poly edra, which supposes the existence of a circadian timer
within the CDC that controls the exit of cells from G1, we
show that the cell cycle and the circadian oscillator in
Euglena are two distinct mechanisms that can be temporarily uncoupled, as suggested by previous results
obtained for this unicell (Edmunds, 1964; Edmunds et al.,
1976; Edmunds and Laval-Martin, 1984). The model proposed here closely resembles the one proposed by Adams
et al. (1984), which formally proposes two different timers
(a circadian pacemaker and a cell cycle oscillator) that can
either run independently from each other or interact,
depending on the growth conditions. This paper, however,
is the first to suggest a biochemical basis for the interaction between these two complex processes.
The model that was presented may be of a more general
interest, since other cellular activities might be gated by the
circadian clock in a similar manner. The further investigation of the signal transduction pathway for the control of
the CDC by cyclic AMP signals may permit the identifi-
Cyclic AMP and circadian control of cell division 1173
cation of cell cycle regulators that modulate the timing of
cell division in response to signals from the circadian clock,
or to other signals, and should provide important information about the role(s) of cyclic AMP in the CDC.
This work was supported by National Science Foundation
grants DCB-8901944 and DCB-9105752 to L. N. Edmunds, Jr.
We thank Dr J. Tong for helpful discussion and J. Simone for
performing the flow cytometry analysis. Some of this work was
reported at the Third Meeting of the Society for Research on Biological Rhythms, 6-10 May 1992, Amelia Island, Jacksonville, FL,
USA (Abstract 99).
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(Received 20 July 1992 - Accepted, in revised form,
7 December 1992)