Circadian period is compensated for repressor protein turnover rates in single cells

Abstract

Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.

Keywords: circadian rhythms; fluorescence microscopy; metabolic compensation; protein degradation; single-cell imaging.

Fig. 1.

CRY2 and PER1 protein oscillation in single cells. (A and B) Fluorescent knock-in and wild-type (wt) U-2 OS cells transduced with shRNA targeting either CRY2 or PER1 or with a nonsilencing control shRNA (shNS1). (Scale bar: 20 µm.) (C and D) Montage of a CRY2-mSca (C) or PER1-mSca (D) knock-in cell nucleus recorded at indicated hours after dexamethasone (dex) treatment and time course of mean fluorescence intensity. (Scale bar: 5 µm.) (E) Time series of mean nuclear fluorescence of indicated knock-in clones after dex treatment. Only time series with ≥60 h are shown. Y-axis ticks mark every 10th cell. (F) Median fluorescence intensities for individual cells from all time points after background subtraction. Horizontal lines: median of all cells, red line: median nuclear autofluorescence. (G) Percentage of rhythmic cells as a function of P-value cutoff. Vertical dashed lines represent P-values of 0.05 and the more stringent value used here, respectively. See SI Appendix, Supplementary Note 1. (H and I) Relative amplitudes (H) and periods (I) of rhythmic time series. (J) Montage of a CRY1-mClo/CRY2-mSca double knock-in (DKI) cell nucleus imaged in the two fluorescence channels at the indicated hours after dex treatment, and time course of mean fluorescence intensities. (Scale bar: 5 µm.) (K) Histogram of phase difference (30-min bins) in unsynchronized CRY1/CRY2 DKI cells. P-value: Wilcoxon signed-rank test. (L) Sequence of mean nuclear peak expression in U-2 OS cells. PER1 timing is estimated from SI Appendix, Fig. S3 B and C.

Fig. 2.

Stability of repressor proteins and correlation with circadian dynamics. (A) Experimental setup: Unsynchronized single knock-in reporter cells transduced with shRNA were imaged 2 d prior and 1 d after CHX addition. Circadian parameters were extracted from days 1 and 2, and protein half-life from day 3. (B) Median periods of reporter cells transduced with the indicated shRNAs, each calculated from ≥10 rhythmic cells. The same colors (green: mClo clones, purple: mSca clones) represent the same clonal population from different experiments. P-value: Wilcoxon signed-rank test. (C and D) Median protein half-life of clonal populations transduced with control shRNA (C) or the indicated shRNAs (D), each calculated from ≥10 decay fits. Black lines (C) indicate the median. P-values: Mann–Whitney U test (C) and Wilcoxon signed-rank test (D). (E) Half-life of individual PER2-mSca cells. Shown are means ±95% CI (CI). P-value: Wilcoxon signed-rank test. (F) Correlation of CRY1 and CRY2 half-life in DKI cells (Spearman). (G) Highly rhythmic time series (SI Appendix, Supplementary Note 1) were filtered for reliable decay fits. (H–J) Correlation of measured half-lives with signal intensity (abundance, H), relative circadian amplitude (I), and circadian period length (J). Correlation coefficient (r) and P-value from Spearman correlation, line: linear regression.

Fig. 3.

Stability of repressor proteins is circadian phase dependent. (A) Harmonic regression analysis of protein half-life and circadian phase in which the half-life was measured. P-values: F-test. (B) Relative expression at time of CHX addition (mean ± SEM) for each 3-h phase window. (C) Protein half-life (median and 95% CI) and cell number for each 3-h phase window. Dashed lines represent the median, and * indicates a statistically significant (P < 0.05) difference from the median (one-sample Wilcoxon signed-rank test, corrected for multiple testing (Sidak–Holmes). (D) Harmonic regression analysis of protein half-life and circadian phase after knockdown of FBXL3. P-values: F-test. (E) Protein half-lives as in C after knockdown of FBXL3. (F) Increase in median protein half-life in FBXL3 knockdown cells compared to controls, number of cells as in C and E. *P < 0.05, Mann–Whitney U test, corrected for multiple testing (Sidak–Holmes).

Fig. 4.

Circadian period depends on repressor protein stability in a phase-dependent manner. (A–E) Three-dimensional plots of circadian period, CRY1 half-life, and circadian phase at half-life measurement. Data from indicated phase windows (A–D) or from all phases (E) are shown (see text for details). Colored lines represent linear regression (m: slope, r: Spearman correlation coefficient). (F–H) Spearman correlation coefficient and slope (m) of linear regression (median and 95% CI) for each 3-h phase window, and number of cells for each correlation. Red dashed line: n = 30 cells (minimum for correlation analysis).

Fig. 5.

Period is compensated for protein turnover rates: a mathematical model. (A) Simple schematic of how CRY1 stability differentially affects period length. (B) Architecture of the mathematical model (see text for details). x: CRY1 mRNA, y: “early”, nonrepressive CRY1, z1: CRY1 in high molecular weight complexes, z2: “late” monomeric, repressive CRY1. (C) Oscillation in the absolute abundance of the state variables for the default parameters. (D) Regression analysis of the period and total “pool half-life” (i.e., all CRY1 species y, z1, and z2) of a simulated population of single cells with stochastically varying turnover rates of early and late CRY1 for two indicated phases (n = 155). (E) Pool half-lives of the simulated population described in D. For each cell, decay is simulated at five random time points. (F and G) Effect of changes in early CRY1 (y) stability on period length (F) and expression level of early CRY1 protein (y) and CRY1 mRNA (x) (G). Stability = 1/dy, dz1 and dz2 are constant. (H) Effect of changes in late CRY1 (z2) stability on length of rising phase, falling phase, and total period (rising + falling). Stability = 1/dz2, dy is constant. (I) Heatmap of period changes for different combinations of y and z2 stabilities (1/y and 1/dz2, normalized to default values). The gray area shows a period length of ±0.5 h from that for default parameters. The white line shows where the degradation rate of y is equal to that of z2.

Fig. 6.

The effects of FBXL3 on circadian period is CRY1 dependent. (A) Average peak shape of CRY1 expression after normalization (Materials and Methods). (B) Histogram of trough-to-peak durations in CRY1 time series. Lines represent kernel density estimation. P-value: Mann–Whitney U test, n as in A. (C and D) Detrended bioluminescence time series of wt, Cry1^−/−, and Cry2^−/− Bmal1:Luc reporter cells after dexamethasone synchronization either transduced with nonsilencing or FBXL3-targeting shRNA (C) and treated with dimethyl sulfoxide (DMSO) [solvent control, (C) and (D)] or 1 µM KL001 (D). Mean ± SD of 5 to 8 replicates representative of three (C) and two (D) experiments. (E) Periods from the recordings shown exemplarily in C and D, n = 2 (KL001+shNS1) and 3 (DMSO+shNS1, DMSO+shFBXL3) independent experiments, respectively.