Figure 3.

Desynchrony of Primary Fibroblasts

(A) Individual primary fibroblasts expressed diverse circadian periods. Above is a histogram of circadian period values for luminescence rhythms of 178 primary fibroblasts cultured from mPER2::LUC-SV40 knockin mice. Periods averaged 25.65 hrs, but ranged widely from 22.4 to 29.7 hrs. Below is a raster plot showing two cells with clearly different periods. In the raster plot, time of day is plotted left to right, and successive days down the page, such that vertically adjacent points are 24 hrs apart. Each row is extended to 48 hrs, duplicating data in the next row, so that patterns crossing midnight can be appreciated. One cell with a period > 24 hrs is plotted in red, another cell with a period < 24 hrs is plotted in blue, and thick bars designate times when the luminescence for a cell was above the mean for each row. Due to different circadian periods, the cells’ phase relationship changes over time.

(B) Phases of fibroblast luminescence rhythms were significantly clustered at the start of each experiment, but randomly distributed by the end. Each blue triangle represents the phase of one cell, following the convention that 0˚ is the phase of the fitted peak of the luminescence rhythm. The radial line indicates the average start phase (295˚, or 4.3 circadian hrs before peak), and the arc indicates the 95% confidence interval.

(C) Damping population rhythms emerge from undamped single cell rhythms, as illustrated by plots of bioluminescence over the course of an 11 day experiment. Medium was changed just prior to starting the experiment. Plots are of two representative fibroblasts (#6,10), the sum of all 25 cells monitored, and the entire microscope field. Damped luminescence rhythmicity previously recorded from the same culture dish in the luminometer is also plotted for comparison. Note first that the two individual cells begin with similar phases, peaking ~4-6 hrs after the start of the experiment, but that due to differences in period, the cells have drifted completely out of phase by the end. As cells become desynchronized, they begin to cancel out one another’s rhythms, and this is reflected in the rapidly damped oscillations seen in the arithmetic sum of luminescence from all 25 cells monitored, as well as in the luminescence from the entire microscope field, and finally also in the luminometer recording from the entire dish.