A new ultradian rhythm in mammalian cell dry mass observed by holography

Abstract

We have discovered a new 4 h ultradian rhythm that occurs during the interphase of the cell cycle in a wide range of individual mammalian cells, including both primary and transformed cells. The rhythm was detected by holographic lens-free microscopy that follows the histories of the dry mass of thousands of single live cells simultaneously, each at a resolution of five minutes. It was vital that the rhythm was observed in inherently heterogeneous cell populations, thus eliminating synchronization and labeling bias. The rhythm is independent of circadian rhythm, and is temperature-compensated. We show that the amplitude of the fundamental frequency provides a way to quantify the effects of, chemical reagents on cells, thus shedding light on its mechanism. The rhythm is suppressed by proteostasis disruptors and is detected only in proliferating cells, suggesting that it represents a massive degradation and re-synthesis of protein every 4 h in growing cells.

Figure 1

A new ultradian rhythm in mammalian cells. (a) Schematic view of the experimental approach (see “Materials and methods” for details). (b) Individual histories of the dry mass of two individual mouse embryonic fibroblast (MEF) cells are shown during the cell cycle interphase. (c) Fourier amplitudes of the dry mass traces as a function of frequency averaged over 702 MEF cells. First sharp peak (asterisk) of the dominant frequency of 0.004 min⁻¹ along with its higher harmonics is clearly distinguished from the background 1/f noise. (d) Reconstruction of the signal of dry mass (pg) by means of an inverse Fourier transform (two possible solutions). (e) The 4 h rhythm observed in various mammalian cell lines. The inserts show the distributions of the cell cycle lengths, the median cell cycle length is indicated in the insert.

Figure 2

Effect of the cell cycle manipulation on the rhythm. (a) Synchronization of HeLa cells using a double thymidine block does not affect the rhythm. The inserts show cell growth curves for the control and synchronized cells plotted using the data from direct cell counts by lens-free microscopy. (b) Comparison of HeLa cells at 34 °C (top) and 39 °C (bottom). The characteristic frequency of the rhythm does not change despite the significant change in the cell cycle length (inserts). (c) Serum starvation eliminates the 4 h rhythm. The inserts show cell counts in control (top) and serum starvation (bottom) conditions.

Figure 3

Relationship between the rhythm, cell cycle and circadian clock. (a) Cell cycle arrest by siKIF11 treatment in HeLa cells suppresses the 4 h rhythm. Western blots (in insert) confirm the inhibition of KIF11 expression (the effect of two specific siRNAs is compared to non-specific siCTR). (b) Incomplete cell cycle arrest by siKIF11 in MEF cells does not suppress the 4 h rhythm. Quantitative PCR analysis (in insert) shows the inhibition of KIF11 expression by two specific siRNAs in MEF. To the right of (a) and (b) we show the distributions of the cell cycle lengths corresponding to the two data sets. (d) Effects of cell cycle inhibition in temperature-sensitive CHO-K1 mutants at 39 °C, compared to the permissive 34 °C temperature (c). Cell cycle arrest in temperature-sensitive CHO-K1 mutants at 39 °C suppresses the 4 h rhythm. To the right of (c) and (d) we show the distributions of the cell cycle lengths for the three cell types. (c) Effect of circadian rhythm inhibition by siCLOCK in (e) MEF and (f) U2OS. Knocking down CLOCK does not eliminate the rhythm in MEF and U2OS. The Western blots (see inserts) show the inhibition of CLOCK expression by siCLOCK 1 and 2 in MEF and U2OS cells. To the right of (e) and (f) we show the corresponding distributions of the cell cycle lengths.

Figure 4

Effect of different inhibitors on the rhythm. (a) Effect of different inhibitors on the rhythm measured by the amplitude of the fundamental peak at 0.004 min⁻¹ in Hela (first row) and MEF (second row). (b) Inhibition of the 4 h rhythm by MG132 is reversible. Compared to control conditions (left panel), treatment of HeLa cells with 0.45 μM of MG132 for 24 h completely suppressed the rhythm (middle panel). Subsequent removal of the MG132 by rinsing restored the oscillations with increased amplitude (right panel). The inserts show the distributions of the cell cycle lengths.