The cyanobacterial circadian clock follows midday in vivo and in vitro
- The University of Chicago, United States
Figures
Figure 1 with 3 supplements
Phase of the cyanobacterial circadian rhythm scales linearly with day length.
(A) LED array device used to grow S. elongatus in programmable light-dark cycles. Cells grown in a 96-well plate on solid media (lower plate, green circles) are illuminated from above by LEDs (red …
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Figure 1—source data 1
Source data for Figure 1B.
- https://doi.org/10.7554/eLife.23539.004
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Figure 1—source data 2
Source data for Figure 1C.
- https://doi.org/10.7554/eLife.23539.005
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Figure 1—source data 3
Source data for bioluminescence trajectories in Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.23539.006
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Figure 1—source data 4
Source data for Kendall’s τ correlations in Figure 1—figure supplement 1.
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Figure 1—source data 5
Source data for Figure 1—figure supplement 2A–E.
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Figure 1—source data 6
Source data for Figure 1—figure supplement 3A, showing bioluminescence output from the purF repoter.
- https://doi.org/10.7554/eLife.23539.009
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Figure 1—source data 7
Source data for Figure 1—figure supplement 3B.
- https://doi.org/10.7554/eLife.23539.010
Figure 1—figure supplement 1
Bioluminescence recordings from PkaiBC::luxAB reporter in light-dark cycles.
(left) Selected bioluminescence traces (PkaiBC::luxAB, black) recorded from individual wells of the 96-well LED array device in conditions simulating day-night cycles of different day length (same …
Figure 1—figure supplement 2
The circadian rhythm of S. elongatus rapidly entrains to 24 hr diurnal cycles with 8–16 hr of daylight.
(A) Peak times of PkaiBC::luxAB reporter after release into LL from 1 to 7 LD cycles of different day length (LD 8:16, LD 12:12 or LD 16:8), as estimated by sinusoidal regression. Error bars …
Figure 1—figure supplement 3
Bioluminescence recordings from PpurF::luxAB reporter in light-dark cycles.
(A) Rhythms in bioluminescence in continuous light recorded from a dawn gene reporter (PpurF::luxAB) after entrainment to 24 hr light-dark cycles of different day length τ (LD 8:16, LD 10:14, LD …
Figure 2 with 1 supplement
Reconstitution of the seasonal clock response in vitro.
(A) Buffer exchange protocol to simulate metabolic driving of the clock. To mimic daytime in vitro, purified Kai proteins (green, blue and red symbols) were incubated in ‘day’ reaction buffer …
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Figure 2—source data 1
Source data for Figure 2B.
- https://doi.org/10.7554/eLife.23539.016
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Figure 2—source data 2
Source data for Figure 2C.
- https://doi.org/10.7554/eLife.23539.017
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Figure 2—source data 3
Source data for Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.23539.018
Figure 2—figure supplement 1
Validation of KaiB fluorescence polarization reporter against KaiC phosphorylation rhythm.
(top) Fluorescence polarization of KaiABC mixture probed with fluorescently labeled KaiB exhibits ≈24 hr rhythms (black and blue squares). (bottom) KaiC phosphorylation rhythm of the same reaction …
Figure 3 with 2 supplements
Clock responses to metabolic steps mimicking dawn (step up) and dusk (step down).
(A) Phase shift in fluorescence polarization (red curve) caused by a shift to a buffer that mimics the nucleotide pool at night ([ATP]/([ATP]+[ADP]) ≈ 25%, gray bar). The control reaction remained …
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Figure 3—source data 1
Source data for Figure 3A–B.
- https://doi.org/10.7554/eLife.23539.021
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Figure 3—source data 2
Source data for Figure 3C–D.
- https://doi.org/10.7554/eLife.23539.022
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Figure 3—source data 3
Source data for Figure 3E–F.
- https://doi.org/10.7554/eLife.23539.023
Figure 3—figure supplement 1
Example calculation of phase shifts in response to metabolic step transitions.
Phase shifts are computed from the difference in phase of the control reaction and the perturbed reaction evaluated at the time of the step. Phase of each reaction at the time of the step (green …
Figure 3—figure supplement 2
Experimentally measured step-response functions predict entrainment to driving periods near 24 hr.
(A) Entrainment simulations with driving periods 4–48 hr were performed for 1000 cycles, and phases at the end of nighttime (immediately before the action of ) of the last 920 cycles were plotted. …
Figure 4 with 3 supplements
Entrainment of the phase oscillator model to a driving cycle.
(A) Schematic of a phase-only oscillator that responds to dawn and dusk with instantaneous phase shifts. The oscillator runs at constant velocities during the day and at night (green lines), …
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Figure 4—source data 1
Source data for Figure 4C.
This file contains data from in vitro entrainment measurements shown as blue circles and squares.
- https://doi.org/10.7554/eLife.23539.027
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Figure 4—source data 2
Source data for Figure 4—figure supplement 2.
This file contains in vitro entrainment measurements shown as blue squares in Figure 4—figure supplement 2B.
- https://doi.org/10.7554/eLife.23539.028
Figure 4—figure supplement 1
Example simulation of a phase oscillator governed by one set of experimentally determined and functions and subjected to a driving cycle.
(A) The phase oscillator reaches stable entrainment within 3–5 light-dark cycles (τ = 10–14 hr) for a wide range of starting phases. Simulation parameters same as in Figure 4B; and as shown in …
Figure 4—figure supplement 2
Simulations of seasonal entrainment for a phase oscillator driven by linearized step-response functions ( and ).
(A) Simulated seasonal response of phase oscillators governed by the four possible combinations of nonlinear and step-response functions in Figure 3(C–D) (blue shaded areas) and their …
Figure 4—figure supplement 3
Dependence of the slope of entrained phase on the slopes of step response functions.
Heat map of slope m, describing the scaling of oscillator peak time with day length, as a function of the slopes l and d of linear and step response functions and oscillator frequencies in …
Figure 7
Phase oscillator entrainment.
https://doi.org/10.7554/eLife.23539.032
Figure 5 with 1 supplement
Phase oscillator model with linear phase shift functions predicts entrainment of the cyanobacterial clock to different light-dark (LD) patterns.
In all panels, error bars represent standard deviations (n = 4–8 technical replicates per point). Lines are fit globally to all three datasets in (A)-(C). See Computational methods for details. (A) …
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Figure 5—source data 1
Source data for Figure 5A–C.
- https://doi.org/10.7554/eLife.23539.034
Figure 5—figure supplement 1
Simulation of a phase-resetting curve.
(left) Simulated phase-resetting curve due to a 12 hr dark pulse for a phase oscillator governed by linear step-response functions and (right), as in in Figure 4—figure supplement 2. Colored …
Figure 6 with 3 supplements
Nearly linear step response functions can arise from the relative geometry of day and night limit cycles.
(A) Geometric model of oscillator phase resetting. During the day, the oscillator runs with constant angular velocity along the daytime orbit (yellow), which has unit radius and is centered at the …
Figure 6—figure supplement 1
Illustrations of limit cycle geometries that give rise to step-response functions and with different slopes, resulting in dusk-, dawn-, or midday-tracking entrainment.
In all schematics, the day orbit (yellow) is centered at the origin and has radius 1. The night orbit (black) has radius R and is displaced from the day orbit ( units). Light-dark () and …
Figure 6—figure supplement 2
The relative size (R) and center-to-center distance (X) of day and night limit cycles are major determinants of entrained behavior.
Heat maps of m, the slope of the approximately linear relationship between entrained phase and day length, are plotted as a function of X and R on the same color scale as in Figure 6E. See …
Figure 6—figure supplement 3
Interpretation of m, the slope of the approximately linear relationship between entrained phase and day length.
(A) The value of m dictates whether the circadian rhythm aligns to dawn (m = 0), dusk (m = 1), or an intermediate point of the day-night cycle (e.g. midday for m = 0.5). Orange and gray curves show …
Appendix 1—figure 1
Phase oscillator with step response framework.
(A) Schematic of the framework. In the light portion of the day, the oscillator runs along the ‘light’ limit cycle (orange) and accumulates phase at constant rate ; in the dark, the oscillator …
Tables
Table 1
Summary of biologically independent in vivo experiments measuring entrainment to 24 hr light-dark cycles of varying day length and corresponding estimates of m, the proportionality coefficient …
| Figure | Driving period T (hr) | Day length τ (hr) | Slope m ± SD of estimate |
|---|---|---|---|
| Figure 1C | 24 | 4, 6, 8, 9, 10, 11, 12, 13, 14, 16, 18, 20 | 0.55 ± 0.02 (sinusoidal fitting) 0.53 ± 0.01 (parabolic fitting) |
| Figure 1—figure supplement 2 | 24 | 8, 12, 16 | 0.47 ± 0.03 (sinusoidal fitting) 0.57 ± 0.02 (parabolic fitting) |
| Figure 5C | 22, 23, 24, 25, 26 | 8, 10, 12, 14 | 0.51 ± 0.11 (sinusoidal fitting) |
