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

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.

生物振荡器（biological oscillators）因分子反应的随机特性，天然会在周期与振幅上呈现随机波动。要精准测定受噪声干扰的振荡器的精度，以及细胞群体中节律周期与节律强度的异质性，需采用时长充足的单细胞记录，以充分覆盖振荡的全部变异特征。我们对80株取自PER2::LUC小鼠、经体外（in vitro）培养6个月的原代成纤维细胞（primary fibroblast cells）开展了为期6周的生物发光记录（bioluminescence recordings），观测到持续且独立的生物钟基因（clock gene）表达昼夜节律振荡（circadian oscillations）。由于节律的随机本质，被判定为具有节律性的细胞比例会随检测区间长度增加而升高；当采用3周时长的分析窗口时，所有细胞均被检出存在节律性。在整个6周的记录周期内，平均周期与平均振幅始终保持极高稳定性，且测量精度随时间推移逐步提升。针对单个细胞而言，其周期与振幅的精度与细胞大小及节律振幅呈显著相关，但与周期本身无关；同时，周期的逐周期变异系数（coefficient of variation, CV）仅为7.3%，远低于振幅的37%。本次时间序列的时长足以区分单个细胞内的随机波动与细胞间的固有差异，据此我们得出结论：细胞群体在节律周期与节律强度上确实存在显著异质性，我们通过一种全新的统计指标（statistical metric）完成了该异质性的量化评估。此外，随机建模结果显示，这些单细胞生物钟的运作状态接近霍普夫分岔（Hopf bifurcation）：内在噪声可通过最小化周期变异、维持振幅的方式，强化振荡特性。