Circadian clock regulation of the bimodal rhythm of cyclic AMP in wild-type Euglena

Cell-cycle traverse is associated with fluctuations in the cellular content of cAMP; artificial alterations of these levels phase-shift cell division in free-running cultures of achlorophyllous Euglena maintained in constant darkness (DD). The phase shifts observed, however, are only transient: the cell division rhythm rephases to that of unperturbed controls. This implies that the second messenger functions downstream of the circadian oscillator. Further, the level of cAMP is known to indicate carbon nutrient status and the competency of cells to traverse various restriction points in the cell cycle of other eukaryotes. We wished to determine the profile of cAMP content in free-running, dividing and non-dividing cultures of green, wild-type cells, which survive well during prolonged growth arrest. We monitored cAMP content in photoautothropic cultures of E. gracilis (strain Z) at 25 degrees C under either an entraining light-dark cycle comprising 12 h of light and 12 h of darkness (LD:12,12) or free-running (LD:1/2,1/2) regimes. cAMP content in rhythmically dividing, light-phased or free-running cells exhibited bimodality [peaks at CT (circadian time) 9-14 and CT 19-22). Expression of cAMP content on a per milligram total cellular protein basis caused the day trough (CT 1-3) to be even more distinct. Non-dividing, free-running, photoautotrophic cultures displayed a similarly phased bimodality in cAMP content. These findings in wild-type Euglena confirm that the bimodal rhythm of cAMP content is regulated by the circadian oscillator that underlies division rhythmicity but is not dependent on the cell division cycle. We will now determine the effect of the fluctuating cAMP levels on the phosphorylation status and activity of cell-cycle regulatory proteins.