Co2 production after induction synchrony of the fission yeast Schizosaccharomyces pombe: the origin and nature of entrainment
Authors: Novak B. , Mitchison J. M. 
Published: April 2021

ABSTRACT
Earlier work has shown that there is a periodic change in the rate of production of CO2 during the cell cycle of fission yeast and that this periodicity persists after a block to the DNA-division cycle and also after a block to protein synthesis. It appears that there is a periodic control or ‘oscillator’ affecting CO2 production that is normally closely entrained to the cell cycle, but which can ‘free-run’ after a block. In this paper, we examine what events in the DNA-division cycle can generate the entrainment signals and what is the nature of such signals.
In the first set of experiments, CO2 production was measured by manometry during induction synchrony produced by blocking the DNA-division cycle in an asynchronous culture for a period and then releasing the block. Synchronous cell division occurs after the release with cell cycles shorter than normal. After release from a block imposed by shifting up the mutant cdc2.33 to the restrictive temperature, oscillations in CO2 production started rapidly and remained closely entrained to the division cycles (with slightly different patterns and timing from those after selection synchrony). This showed that there was an entrainment signal but did not show whether it came from start, the S period or mitosis. A similar experiment with cdclO.129 showed that an early signal came from either start or the S period, as did an experiment with release from N-starvation. The results with cdc25.22 were similar to those with cdc2.33. After a block with hydroxyurea, there was entrainment but with no signs of the early signal that occurred with cdclO. This showed that the early signal came from start and not from the S period.
In a second set of double-block experiments, the first block was followed by a second different block. With cdc25.22 followed by MBC (an inhibitor of nuclear division) the cells passed through a narrow window of the cell cycle between the transition point of cdc25.22 and mitosis. This was sufficient to start the oscillations, showing that an entrainment signal could be generated at about the time of mitosis. The results from using hydroxyurea followed by cdc2.33 showed no genuine oscillations, confirming the conclusion from the single hydroxyurea block. The results from using hydroxyurea followed by cdclO.129 confirmed the existence of a mitotic signal. The conclusion from the two sets of experiments is that in induction synchrony an entrainment signal can come either from start or from mitosis but not from the S period itself.
Further experiments using a selection synchronised culture of cdc2.33 with different periods of block at the restrictive temperature confirmed the strength of the entrainment signal and showed that it could reset the CO2 oscillator whatever its phase.
Measurements with cdc!3.117 showed that a culture blocked at 35 °C (but not at 37 °C) leaked through a mitotic block and accumulated with condensed chromatin and septa. In these conditions, CO2 acceleration fell to zero without any major changes in the rate of synthesis of protein and rRNA. This suggests that the entrainment signal at mitosis is a ‘stop acceleration’ signal that is reversed shortly afterwards in normal cells but not in this mutant at 35 °C.
The S period is earlier and Gi is probably reduced in the large cells produced by a cell cycle block. There is a size control of DNA synthesis which is not cryptic in normal cells but its effect is weaker in large cells than in small cells.
We suggest that the entrainment signals from start and from mitosis also operate in the normal cell cycle and that the nature of the signal (at any rate at mitosis) is to stop CO2 acceleration and then to recommence it It is possible that the signal involves protein phosphorylation.