Reconstitution of circadian clock in synthetic cells reveals principles of timekeeping

Abstract

The cyanobacterial circadian clock maintains remarkable precision and synchrony, even in cells with femtoliter volumes. Here, we reconstitute the KaiABC post-translational oscillator (PTO) in giant unilamellar vesicles (GUVs) to investigate underlying mechanisms of this fidelity. We show that our encapsulation methodology replicates native protein variability. With long-term, single-vesicle tracking of circadian rhythms using fluorescent KaiB and confocal microscopy, we find that oscillator fidelity decreases with lower protein levels and smaller vesicle sizes. KaiB membrane association, observed in cyanobacteria, was recapitulated in GUV membranes. A mathematical model incorporating protein stoichiometry limitations suggests that high expression of PTO components and associated regulators (CikA and SasA) buffers stochastic variations in protein levels. Additionally, while the transcription-translation feedback loop contributes minimally to overall fidelity, it is essential for maintaining phase synchrony. These findings demonstrate synthetic cells capable of autonomous circadian rhythms and highlight a generalizable strategy for dissecting emergent biological behavior using minimal systems.

Fig. 1. Schematics of the experimental workflow for preparation, imaging, and analysis of PTO-GUVs.

Kai proteins that comprise the post-translational oscillator (PTO) are encapsulated into GUVs using the PAPYRUS with diffusive loading method (PAPYRUS-wDL) forming PTO-GUVs with varying sizes and PTO concentrations (red circles with green shading). The PTO-GUVs are tethered to a glass coverslip through streptavidin-biotin interactions for timelapse imaging over 4+ days with a confocal microscope. The images are then segmented to identify individual PTO-GUVs, and the time traces of the normalized fluorescence intensity (Norm. FI) is obtained.

Fig. 2. PAPYRUS-wDL produces GUVs encapsulating functional proteins with cell-like distributions of protein concentration.

A Histograms of protein concentration distribution in GUVs prepared with loading concentrations of 0.88 μM, 1.75 μM, 2.63 μM, and 4.50 μM FITC-BSA. The bin widths are 0.2 μM. Each bar is an average of N = 3 independent repeats (black dots), and the error bars are one standard deviation from the mean. The distribution of protein concentrations can be described well with a gamma distribution (thick solid lines). The inset numbers, Gamma (α,θ) is the best-fit gamma distribution with shape parameter, α, and scale parameter, θ, and n is the total number of GUVs from the three independent samples for each concentration. B A one-sided ANOVA test shows that the mean coefficient of variation (CV) of the encapsulated protein did not vary with loading concentration (p = 0.36, not significant (N.S.)). C The mean concentrations of protein in the lumen of the GUVs is linearly correlated with the loading concentrations. The black line is a linear regression (f(x) = x, R² = 0.95). For (B) and (C), the middle black bar shows the mean value of the N = 3 independent repeats and the error bars show ±1 standard deviation. D Violin plots showing the distribution of encapsulated protein concentrations versus the diameter of the GUVs. GUV diameters were binned so each integer value includes GUVs with diameters ±0.5 μm of the integer value. A Kruskal-Wallis (KW) ANOVA test shows that the encapsulated protein concentrations in the lumen is independent of the diameter of the GUVs (X²(6, 617) = 2.01, p = 0.92). The data shown is for a loading concentration of 1.75 μM FITC-BSA. The horizontal black and red lines are the mean and median values of the distributions respectively.

Fig. 3. PTO-GUVs show circadian oscillations, though some do not oscillate.

A A representative confocal image of PTO-GUVs prepared with a loading concentration of 1× PTO. The fluorescently-labeled GUV membranes are false-colored red and the KaiB-6IAF in the lumens are false-colored green. Scale bar 10 μm. B–D Top images: still images of the PTO-GUVs labeled V1 through V3 (green) at 12-h intervals. Bottom images: intensity color-mapped images to emphasize the changes in mean fluorescence intensity in the lumens. The color mapping is linear, and the range was adjusted to the maximum intensity of each image. Top plots show the time traces of the normalized mean fluorescence intensity (Norm. FI) for each PTO-GUV. The bottom plots are the single-sided amplitude spectrum of the respective traces. The position of the peak in the amplitude spectrum in (B) and (C) are marked with an open-faced red circle. The inset text is the period corresponding to the peak. The sample temperature was maintained at 30 °C. We show additional representative images at the other mean PTO concentrations in Supplementary Fig. 2. Experiments were repeated at least two times with similar results.

Fig. 4. The PTO in GUVs show different behavior from the PTO in the bulk.

A Bulk in vitro (PTO-Bulk) measurements of the fluorescence intensity (FI) of KaiB-6IAF across a range of mean post-translational oscillator (PTO) concentrations (0.5× to 2.5×, various colors) evaluated using 50 μL reaction volumes in a fluorescence plate reader. The normalized time trace of the FI (detrended and normalized by the mean intensity) over time is shown in the left panel and the amplitude spectrum from the fast Fourier transform (FFT) analysis is shown in the right panel. The text shows the amplitude (top left) and period (top right) obtained from the global peak of the amplitude spectrum. B Population-averaged KaiB-6IAF FI from PTO-GUVs 2 ± 0.5 µm in diameter for varying mean PTO concentrations (0.5× to 2.5×, various colors). The population-averaged and normalized PTO amplitude (detrended and normalized by mean intensity) over time is shown in the left panel. The inset text reports N = the total number of PTO-GUVs for each concentration. The amplitude spectrum is shown in the right panel. C Plot of the PTO fidelity versus the mean PTO concentration (0.5× to 2.5×) across varying PTO-GUV sizes (Ø: 2 μm to 10 μm). D Plot of the fidelity versus the surface area to volume ratio (SA/V) across varying PTO-GUV sizes (Ø: 2 μm to 10 μm). The straight lines are guides for the eye. E Histograms of the periods of oscillating PTO-GUVs. The red line shows the mean period for each distribution. The text inset reports the mean period, the coefficient of variation (CV) of the period, and N = the number of PTO-GUVs analyzed. F Histograms of the amplitude of oscillating PTOs. The text inset reports the mean amplitude, the coefficient of variation (CV) of the amplitudes, and N = the number of PTO-GUVs analyzed. The histogram in (E) and (F) includes PTO-GUVs of all diameters.

Fig. 5. KaiB binds to GUV membranes.

Multichannel confocal images of GUVs encapsulating KaiB-6IAF or FITC-BSA. Images of the membrane channel are false-colored red and images of the protein channel are false-colored green. The leftmost panels show a composite image of the two channels, the middle panels show the protein channel, and the rightmost panels show the membrane channel. The lower plots are strip-intensity profiles of the regions shown in the rectangular boxes with the dashed yellow lines. A For the KaiB-6IAF sample, the images show a localized bright ring around the membrane in the protein channel. The strip intensity profile shows a colocalized region of high KaiB-6IAF intensity (green line) at the membrane (gray line). The colocalized region is highlighted by the thick black arrows. B There is no corresponding bright ring in the FITC-BSA channel or region of high FITC-BSA intensity at the membrane. Experiments were repeated at least two times with similar results. Scale bars = 5 μm.

Fig. 6. Modeling of the PTO-GUVs.

A 3D scatter plot of the distribution of KaiA, KaiB, and KaiC protein concentrations (monomeric) in the simulated PTO-GUVs. The plot aggregates a range of clock protein concentrations (0.5× to 2.5×, various colors) and a range of GUV sizes (2 μm, 3 μm, 4 μm, 6 μm, 8 μm, and 10 μm, all ±0.5 μm). PTO-GUVs that were determined to be oscillating (Osc. [+]) are colored green. All non-oscillating GUVs (Osc. [-]) are color-coded by concentration as indicated in the legend. B Plot of the fidelity of simulated PTO-GUVs obtained from the model. C Histogram of the periods of the simulated oscillating PTO-GUVs obtained from the model. The text inset shows the mean period, the coefficient of variation (CV) of the period, and N = the number of oscillating simulated PTO-GUVs. D Histogram of the amplitudes of the simulated oscillating PTO-GUVs obtained from the model. The text inset reports the mean amplitude, the coefficient of variation (CV) of the amplitudes, and N = the number of oscillating simulated PTO-GUVs from the model. The histograms in (C) and (D) include PTO-GUVs of all diameters.

Fig. 7. Insights into PTO fidelity and synchrony under constant conditions.

A Prediction of the fidelity as a function of mean PTO concentration with SasA and CikA (PTO’ (SasA+CikA[+]), solid purple line) and without SasA and CikA (PTO (SasA+CikA[–]), dashed red line) in simulated cyanobacteria. B–D Plots of the synchronization index and polar plots of the phase (angular axis) and the amplitude (radial axis) for the perfect memory, no memory, and perfect memory + TTFL models. The synchronization index is plotted for 14 days and was calculated from the time traces of 5000 simulated cyanobacteria. The data are presented as the mean values ± 1 standard deviation. The upper polar plots show Day 1, middle polar plots show Day 4, and lower polar plots show Day 14. For clarity, each polar plot contains N = 500 data points randomly selected from the 5000 simulated cyanobacteria.