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Abstract
The achlorophyllous ZC strain of Euglena gracilis exhibits a circadian rhythm of cell division in constant darkness (DD). Mitosis occurs during a restricted part of the circadian cycle, corresponding to the dark intervals in a light-dark cycle comprising 12 h of light and 12 h of darkness.
We have demonstrated that division-phased cultures also exhibit bimodal, circadian changes of cAMP level. Maximum cAMP levels occurred at the beginning of the light period (CT (circadian time) 00-02), and at the beginning of darkness (CT 12-14). These variations persisted after tranfer of the cultures into DD and appeared to be under the control of the circadian oscillator rather than to be CDC-dependent, since they continued in cultures that had reached the stationary phase of growth. On the basis of the observation that cAMP regulates cell cycle progression in many cell types, we hypothesized that the periodic changes of cAMP level may act as signals to restrict some of the stages of the CDC to specific phases of the circadian cycle.
Perturbations of the cAMP oscillation by exogenous cAMP resulted in the temporary uncoupling of the CDC from the circadian timer. The addition of cAMP during the subjective day resulted in delays (up to 9 h) of the next synchronous division step. In contrast, mitosis was stimulated when cAMP was administered in the middle of the subjective night. Following the DNA content of the cells by flow cytometry indicated that cAMP injected at either CT 06-08 delayed progression through S phase, and perhaps also through mitosis. When added at CT 18-20, cAMP accelerated the G2-M transition.
The circadian oscillator was not perturbed by the addition of exogenous cAMP: the division rhythm soon returned to its original phase. On the other hand, the permanent elevation of cAMP levels in the presence of forskolin induced a rapid loss of cell division rhythmicity. These findings are consistent with the hypothesis that cAMP acts downstream of the oscillator, and that the cAMP oscillation is an essential component of the signalling pathway for the control of the CDC by the circadian oscillator.
The receptors for cAMP in Euglena were found to be two cAMP-dependent kinases (cPKA and cPKB). Pharmacological studies using cAMP analogs suggested that cPKA mediates cAMP effects during the subjective day, and cPKB during the subjective night. A model for the control of the CDC by the circadian oscillator is proposed, on the basis of these results.
