Persistent Cell-Autonomous Circadian Oscillations in Fibroblasts Revealed by Six-Week Single-Cell Imaging of PER2::LUC Bioluminescence

Tanya L Leise,Connie W Wang,David K Welsh,Paula J Gitis,Shin Yamazaki

doi:10.1371/journal.pone.0033334

Abstract

Biological oscillators naturally exhibit stochastic fluctuations in period and amplitude due to the random nature of molecular reactions. Accurately measuring the precision of noisy oscillators and the heterogeneity in period and strength of rhythmicity across a population of cells requires single-cell recordings of sufficient length to fully represent the variability of oscillations. We found persistent, independent circadian oscillations of clock gene expression in 6-week-long bioluminescence recordings of 80 primary fibroblast cells dissociated from PER2::LUC mice and kept in vitro for 6 months. Due to the stochastic nature of rhythmicity, the proportion of cells appearing rhythmic increases with the length of interval examined, with 100% of cells found to be rhythmic when using 3-week windows. Mean period and amplitude are remarkably stable throughout the 6-week recordings, with precision improving over time. For individual cells, precision of period and amplitude are correlated with cell size and rhythm amplitude, but not with period, and period exhibits much less cycle-to-cycle variability (CV 7.3%) than does amplitude (CV 37%). The time series are long enough to distinguish stochastic fluctuations within each cell from differences among cells, and we conclude that the cells do exhibit significant heterogeneity in period and strength of rhythmicity, which we measure using a novel statistical metric. Furthermore, stochastic modeling suggests that these single-cell clocks operate near a Hopf bifurcation, such that intrinsic noise enhances the oscillations by minimizing period variability and sustaining amplitude.

Highlights

Circadian clocks are intracellular timekeeping devices found in organisms from cyanobacteria to humans [1]
We developed a new test for circadian rhythmicity, described below, to study how the proportion of rhythmic cells depends on the interval length
Fibroblasts exhibit heterogeneity in a novel metric for strength of rhythmicity To quantify strength of rhythmicity, we develop a novel metric based on the observation that PER2::LUC bioluminescence recordings of arrhythmic Bmal12/2 suprachiasmatic nucleus (SCN) cells from Ko et al [19] exhibit characteristics consistent with Brownian noise, having a power spectrum proportional to the reciprocal of the frequency squared (Fig. 6A), quite different from the assumption of white

Summary

Introduction

Circadian (ca. 24 h) clocks are intracellular timekeeping devices found in organisms from cyanobacteria to humans [1]. 24 h) clocks are intracellular timekeeping devices found in organisms from cyanobacteria to humans [1]. These clocks orchestrate daily temporal programs of physiology and behavior, anticipating environmental light/dark transitions and persisting even under constant conditions. Tissue organization and cellular interactions are important for clock function, in the SCN, individual cells such as SCN neurons or fibroblasts contain autonomous circadian clocks [3]. After delays associated with transcription, translation, formation of molecular complexes, and nuclear translocation, the products of Per and Cry genes feed back to inhibit transcription of their own genes. The inhibition is relieved by protein turnover, allowing the cycle to begin anew

Methods

Results

Conclusion