Figure 1.

Rat-1 Cell Rhythms

(A) Long-term luminometer recording of luminescence in a 35 mm culture dish of Rat-1 cells acutely transfected with the mBmal1::luc circadian reporter plasmid. Cells were transfected one day before the recording began, and treated with a serum shock (50% serum × 2 hrs) just prior to recording. Note the initial circadian oscillations which damp out quickly in the first few days, followed by a slow increase in luminescence to a broad peak at 2-3 weeks after transfection. Even when the luminescence begins to decline, changing medium restores circadian oscillations.

(B) Phases of individual Rat-1 cell luminescence rhythms were significantly clustered at the start of each experiment, but randomly distributed by the end. Each blue triangle indicates 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 (127˚, or 15.7 circadian hrs before peak), and the arc indicates the 95% confidence interval.

(C) Plots of bioluminescence over the course of a 2 week experiment beginning 5 days after transfection. Medium was changed just prior to starting the experiment. Plots are of two representative Rat-1 cells (#22, 48), the sum of all 100 cells monitored in the experiment, and the entire microscope field. Note that the two individual cells begin with similar phases, peaking ~15 hrs after the start of the experiment, but that due to differences in period and phase instability the cells have drifted completely out of phase by the end of the experiment. The amplitudes of the individual cell rhythms are variable but do not damp significantly. Cell 22 actually increases its amplitude toward the end of the experiment. A movie of Cell 48 can be seen in Figure S1. As the 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 100 cells monitored, as well as in the luminescence from the entire microscope field. Finally, in accordance with the expected increase in plasmid expression at 5-10 days post-transfection (see A), there is an upward sloping baseline evident in the ensemble rhythms.