B R I E F C O M M U N I C AT I O N
Imaging of single light-responsive clock cells reveals
fluctuating free-running periods
Amanda-Jayne F. Carr and David Whitmore
Zebrafish tissues and cell lines contain circadian clocks that
respond directly to light1,2. Using fluorescence-activated cell
sorting, we have isolated clonal cell lines that contain the
reporter construct, zfperiod4-luciferase3. Bioluminescent
assays show that oscillations within cell populations are
dampened in constant darkness. However, single-cell imaging
reveals that individual cells continue to oscillate, but with
widely distributed phases and marked stochastic fluctuations
in free-running period. Because these cells are directly light
responsive, we can easily follow phase shifts to single light
pulses. Here we show that light acts to reset desynchronous
cellular oscillations to a common phase, as well as stabilize the
subsequent free-running period.
Peripheral circadian clocks found in the tissues of Drosophila and
zebrafish are directly light responsive2,4. This is in contrast to the tissue
clocks of mammals, which require a functional retina in order to entrain
to a light–dark (LD) cycle5. In the case of zebrafish, this phenomenon can
be extended to embryonic cell lines, in which the circadian clock can be
rapidly entrained by light cycles in an incubator2,3. By transfecting these
cells with a zfperiod4-luciferase (a homologue of mouse per1) reporter
construct, using fluorescence-activated cell sorting (FACS) to generate genetically identical clonal cell lines, and then applying single-cell
imaging techniques, we have been able to address the following questions relating to clock function: do individual cellular clocks continue to
run after long periods (months) in the dark? How do single cells in the
population phase shift in response to a light pulse? Is circadian period
tightly regulated at the cellular level, or is there evidence for random,
stochastic changes in clock period?
Figure 1a shows high-amplitude period-luciferase rhythms from a
population of clonal cells on a LD cycle and as they free-run into constant darkness (DD), alongside data from cells maintained in DD for
several months, which at the population level show no oscillations in
gene expression. By imaging single cells in both of these populations, it
is clear that the phases of individual cells entering DD after light entrainment remain very close (Fig. 1b and see Supplementary Information,
Movie 1), whereas long-term DD cells show a randomly distributed
circadian phase (Fig. 1b and see Supplementary Information, Movie 2).
These data are strongly supported by circular statistical analysis (see
Supplementary Information, Fig. S1a). Thus, even after several months
in complete darkness, the clocks within single zebrafish cells continue to
oscillate. This finding is similar to that recently described in mammalian
fibroblasts, in which individual cells show persistent circadian oscillations, which can also be synchronized by serum shock treatments6,7.
Because the cells that were analysed in these experiments are genetically identical, we expected the free-running period of each cell to be
very similar, as has recently been reported in cyanobacteria8. However,
Fig. 1c shows that this is not the case, because cells entering DD from an
entrained state show a range of free-running periods varying from 24.5
to 28.3 h. This property cannot be due to variations in culture conditions
or the amount of integrated reporter gene construct, and was also seen
in re-sorted clonal populations of cells (see Supplementary Information,
Fig. S1b, c). This range of periods is even greater in cells maintained for
several months in the dark (Fig. 1c; F-test, F = 6.3306, P < 0.001). In
other words, the two DD conditions, immediate versus long-term, are
not equivalent, and the suggestion is that light has a stabilizing effect on
period that persists for several days into the DD condition. The molecular basis for this residual action of light is not yet clear.
If the circadian periods are compared for a given zebrafish cell from
day to day (see Supplementary Information, Fig. S2a, b), it is clear that
clock period is very unstable and fluctuates widely in a stochastic manner. These data fit well with period distributions that are predicted
by stochastic models of clock function at the cellular level, where the
number of molecules and molecular interactions are few9. So within the
clock mechanism of single zebrafish cells, it would seem that fluctuations in clock-related molecular events generate considerable ‘noise’ with
regard to free-running period.
If the clock mechanism contained within these cells is not capable of
generating a precise free-running period, how then is phase precision
achieved? The value of a circadian clock lies in its ability to time internal
events accurately relative to the environmental light–dark cycle. In mammals, this precision seems to be achieved through coupling mechanisms
University College London, Centre for Cell and Molecular Dynamics, Department of Anatomy and Developmental Biology, Rockefeller Building, 21 University Street,
London WC1E 6DE, UK.
Correspondence should be addressed to D.W. (e-mail: d.whitmore@ucl.ac.uk).
Published online: 1 March 2005, DOI: 10.1038/ncb1232
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Figure 1 Rhythmic oscillations persist within individual cells held in constant
conditions. (a) Bioluminescence from clonal cell populations maintained
in constant darkness (closed circles) and entrained to a LD cycle for 2 days
(open circles). Mean bioluminescence (±s.e.m.; n = 12 wells; c.p.s., counts
per second) is plotted. The bars at the top indicate the light regime (white,
light; black, dark). (b) Luminescent traces from individual clonal cells
entrained to a LD cycle (top panel) and maintained in constant darkness
(bottom panel). Grey and black bars indicate the prior entrainment cycle
(grey, light; black, dark). (c) Circadian period of light-entrained (open bars)
and DD-maintained (closed bars) individual cells, plotted in 1-h time bins.
between cells10, but so far we have little evidence for this in zebrafish
cells. However, the zebrafish circadian clock is strongly reset each day
by light, and possesses a high-amplitude, type 0 phase response curve3.
The consequences of this can be seen in Fig. 2a, in which a 15-min light
pulse, applied to a population of DD-maintained cells, resets the population to ZT2 (Zeitgeber Time 2, 2 h after dawn), from which they free-run
and subsequently dampen. This immediate, strong resetting of phase
occurs at the level of each individual cell (Fig. 2b and see Supplementary
Movie 3), and is confirmed by a highly significant change in the distribution of phase when analysed by circular statistics (see Supplementary
Figure 2 A light pulse tightens circadian period and resets desynchronized
cellular oscillations to a common phase. (a) A 15-min light pulse (arrow)
induces circadian oscillations in a cell population previously maintained in
constant darkness. Mean bioluminescence (±s.e.m., n = 10) is plotted; the
time of the light pulse is indicated by the white bar below the arrow.
(b) Luminescence traces are shown from individual cells before and after the
light pulse; the arrow and white bar indicate the time of the pulse. (c) The
period of cellular oscillations was calculated before (black bars) and after
(white bars) the light pulse and plotted in 1-h time bins.
Information, Fig. S3). Furthermore, the light pulse also had a significant
effect on the distribution width of free-running period (Fig. 2c; F-test,
F = 9.3007, P < 0.001). Following the light pulse, the range of periods is
reduced and cellular clocks run with more precise timing. Light, therefore, may induce molecules that have a long-term effect on the core
clock mechanism, or perhaps strengthen the coupling between potential
multiple oscillators within the cell.
We believe that the clock mechanisms observed in our zebrafish cell
lines represent well the changes that occur within peripheral tissues of
the animal itself11,12. It is our working hypothesis that circadian clocks
within peripheral tissues free-run with relatively imprecise periods in
constant conditions. However, every dawn, as animals are exposed to
the early solar light signal, clocks within these tissues are rapidly reset
to a common phase, which corresponds to the beginning of the day.
320
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B R I E F C O M M U N I C AT I O N
In this system, in which light plays such a dominant role, there may
be little selective pressure to produce a molecular clock that generates
an accurate and stable free-running period. These clonal clock cells
certainly lack this precision, but their exceptional light responsiveness
produces the required accuracy of phase in a rhythmic environment.
Note: Supplementary Information is available on the Nature Cell Biology website.
ACKNOWLEDGEMENTS
The authors wish to thank N.S. Foulkes for the kind donation of luciferase
reporter cell lines and many useful discussions; K. Allen and D. Davies for their
expert assistance with cell sorting; M. Pando for help with retroviral techniques;
K. Swann for essential input regarding imaging; J. H. Zhao for advice with circular
statistics; M. Straume for guidance with FFT-NLLS; and T. K. Tamai for many
useful suggestions. This work was supported by funds from The Wellcome Trust
and BBSRC.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
Received 25 November 2004; accepted 3 February 2005
Published online at http://www.nature.com/naturecellbiology.
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a
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Figure S1 Analysis of free-running phase and period in cells maintained
in constant darkness in comparison to cells previously entrained to a light
dark cycle. a) The phase of zfper4 bioluminescence from individual clonal
cells was analysed after maintenance in constant darkness (DD) and over a
3-day period after entrainment to a LD cycle (LD). The phase of individual
cellular bioluminescence was plotted in 80min time bins, with mean angle
and 95% confidence intervals. The phase of circadian oscillations in cells
maintained in DD was uniform (Rayleigh’s Uniformity Test, Z = 0.148,
P=0.864) with a Mean Vector Length of 0.06. The distribution of phase
in DD was significantly different from that observed in cells previously
entrained to a LD cycle (Mardia-Watson-Wheeler Test, W = 37.495,
p<0.001), whose distribution was directional (Rayleigh’s Uniformity Test,
Z = 33.81, p<0.001) with a Mean Vector Length of 0.943. b) Clonal cells
were subjected to a second round of FACS and sorted at 1 cell per well in a
96-well plate. The cells were expanded for 6 weeks in constant conditions.
The subsequent cell populations were entrained to a 12L:12D LD cycle for
2 days and free-run in DD for 6 days. The relative bioluminescence from 11
individual wells is plotted from the second LD cycle and for the following 6
days in DD. The black and white bars indicate the LD cycle. Free-running
period was calculated using the FFT-NLLS analysis software package and
plotted in 30min time bins. (c) There is a significant difference between
the distribution of period during entrainment (mean = 23.81hrs ± 0.12)
compared to the free-running period (mean = 24.39hrs ±0.56) as analysed
by F-Test (F= 5.7525, p<0.01)
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Figure S2 Period change in individual cells as a function of time.
Bioluminescence was recorded from entrained cells as they free ran into
constant darkness (a) and from cells maintained for many months in
constant darkness (b). The free-running period of each individual cell was
calculated over each 2 successive days using the FFT-NLLS analysis software
package and plotted as a function of time, where 1 = 0-48 hr, 2= 24-72 hr,
3 =48-96 hr, 4 = 72-120 hr and 5= 96-144 hr.
Figure S3 Circular histograms of phase distribution from cells held for many
months in constant darkness prior to, and after exposure to a 15-minute
white light pulse. Circular statistical analysis shows that the phase of
individual cellular oscillators is uniform prior to the light pulse (Mean length
of vector = 0.197, Rayleigh’s Uniformity Test p=0.32, Z=1.16). However,
subsequent to the pulse, there is a significant difference in the distribution
of phase (Mardia-Watson Wheeler Test, W = 30.924, p<0.01), which
becomes strongly directional (Mean length of vector = 0.758, Rayleigh’s
Uniformity Test p<0.01, Z=18.38) (Fig. 2c) as the phase of individual
cellular oscillations are immediately synchronised by light.
2
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Movie 1 - Free-running bioluminescence from single luminescent cells was recorded using an Imaging Photon Detector for 84 hrs after entrainment to a LD
cycle.
Movie 2 – Free-running bioluminescence from single luminescent cells maintained in DD was recorded using an Imaging Photon Detector for 84 hrs.
Movie 3 - Oscillations from DD maintained luminescent cells was recorded using an Imaging Photon Detector prior to (for 48 hr), and after a 15-min white
light pulse (for 72 hr). The timing of the light pulse is indicated in the movie by yellow frames.
SUPPLEMENTARY METHODS
Transfection of luminescent cells with GFP. GP2-293 cells were seeded at 20% confluency in DMEM and 10% fetal calf serum, and transfected with 15 µg of retroviral plasmid (pCLNCX – Retromax, Imgenex) containing GFP insert, 5 µg of the envelope plasmid (pMD.G) and 10 µg of carrier plasmid (pBSII SK-, Stratagene)
by calcium phosphate precipitation. Viral medium was collected and used to infect DAP20 (zfper4 luciferase reporter) cells seeded at 1x105cells/ml in 10 cm2 flasks at
25oC. The medium was removed and replaced with fresh viral medium every 12 hours for the next 2 days.
Fluorescent activated cell sorting of luminescent cells. Individual DAP20-GFP cells were isolated using Fluorescent activated cell sorting (FACS) based on GFP
expression. FACS was performed using a FACSVantage (BD Bioscience). Single cells were sorted into individual wells of a 96-well plate. Bioluminescent activity was
analysed using a TopCount NXT luminometer (Packard), and clonal lines with high luminescence reporter activity were trypsinised from the wells and expanded.
General cell culture. General maintenance of the clonal cell lines was performed as previously described3. Two months prior to luminescence recording cells were
maintained in constant darkness (DD) by wrapping individual flasks with foil. Consequently, sub-culturing and preparation of cells for all experiments was performed in darkness using IR-goggles.
Bioluminescence recording of clonal cell populations. Bioluminescence was assayed using a Packard TopCount NXT. During entrainment plates were exposed to
a 12-hour light: 12-hour dark LD cycle from a halogen light source (188µW/cm2) for 2 days. For the DD experiment, the plate remained inside the TopCount NXT
and bioluminescence was recorded automatically each hour over the course of 5 days. Bioluminescence was initially recorded for 48 hours with the plate inside the
luminometer for the light pulse experiment, after which the plate was ejected from the machine and subjected to a 15 min. white light pulse from a halogen lamp
(400 µW/cm2). Data from cell populations was analysed using Import and Analysis 2000 (Steve Kay, The Scripps Research Institute).
Bioluminescence recording of individual clonal cells. Luminescent cells were diluted 1:10 with PAC2 cells and seeded onto a glass bottomed WillCo dish (WPI,
Inc). For the LD experiment, plates were contained in a glass box and subjected to a 12:12 LD cycle in a temperature controlled water bath for 2 days. Luminescence
recording began at the end of the 2nd light period. For the light pulse experiment the cells were pulsed with white light (400 µW/cm2) from a halogen lamp for 15
minutes. Luminescence signals from individual cells were detected using an Imaging Photon Detector (IPD) (Sciencewares), consisting of a resistive anode imaging
photon detector (Photek Inc, UK) and a CCD camera (Dage-MTI, IN) mounted on an inverted microscope (Zeiss) with a heated stage set to 29oC. Images and count
rates for individual cells (photons/sec) were acquired over a 30 min integration window. The presence of single cells in the IPD photon image was confirmed by
examining GFP fluorescence at the end of each experiment.
Data Analysis. Variations in the profiles of luminescence were tested by one-way ANOVA followed by Tukey multi comparison test (SigmaStat, SPSS Inc). Phase and
period was calculated using the FFT-NLLS analysis software package (Marty Straume, University of Virginia). Differences in the distribution of free-running period
were analysed using the F-test (MedCalc, Belgium). Circular data was analysed and plotted using Oriana 2.0 (Kovach Computing Services, UK). The Mean Length of
Vector was calculated to indicate the degree of clustering in the samples. Clustering of data was analysed by Rayleigh’s Uniformity Test, and differences in the distribution of phase were evaluated by the Mardia-Watson-Wheeler Test.
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