Cell division cycles and circadian clocks : phase-response curves for light perturbations in synchronous cultures of euglena.

The cell division rhythm in Euglena gracilis Klebs (Z strain) freeruns with a circadian period (30.2 +/- 1.8 hours for 156 monitored oscillations) in aerated, magnetically stirred, 8-liter, axenic batch cultures grown photoautotrophically at 25 degrees C in LD: 3,3, (7,500 lux, cool-white fluorescent) 6-hour light cycles from the moment of inoculation. Cell number was measured at 2-hour intervals with an automatic fraction collector and Coulter Electronic Particle Counter. At different circadian times throughout the 30-hour division cycle, 3-hour light perturbations were imposed on free-running cell populations by giving light during one of the intervals when dark would have fallen in the LD: 3,3 regimen. Using the onset of division as the phase reference point, the net steady-state phase advance or delay (+/-Deltaphi) of the rhythm was determined after transients, if any, had subsided (usually in one or two days) relative to an unperturbed control culture. Both +Deltaphi and -Deltaphi were found, with maximum values of approximately +/-11 to 12 hours being obtained at circadian time (CT) 20 to 22 (the ;breakpoint'); little, if any phase shift occurred if the light signal was given between CT 6 and CT 12. The phase-resetting curve obtained by plotting new phase (phi') versus old phase (phi) was of the type 0 (;strong') variety. Light perturbations, no matter when imposed, engendered new phases which mapped to a relatively restricted portion (CT 6 to CT 13) of the circadian cycle.These data provide the first detailed phase-response curve for a circadian mitotic clock. The findings, therefore, not only further support the hypothesis that a circadian oscillator (perhaps exhibiting limit cycle behavior) can modulate cell division in eukaryotic cells, but also provide a useful basis for the dissection of the nature and extent of the coupling between cell division and circadian cycles.