Regulation of Cell Division Cycles by Circadian Oscillators:Role of the cAMP Pathway

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Among other mechanisms that have been proposed to control microbial cell division cycles, autonomous biological oscillators provide a possible means of integrating the disparate concepts of cell cycle, exit, and transit. The proposed research will be concerned with the means by which this coupling between oscillator and cell division cycle is effected. Cyclic AMP, which exhibits bimodal circadian rhythmicity in the algal flagellate Euglena gracilis, and which is known to have the capacity to control certain rate- limiting steps in the progression of the cell division cycle in many cell types, may play such a coupling role, participating in the gating of cell division cycle events to specific phases of the circadian cycle. Two problems will be addressed: First, how the clock confers rhythmicity upon the adenyl cyclase/cAMP- phosphodiesterase system, and second, how the adenyl cyclase/cAMP- phosphodiesterase interacts with and modulates the cell division cycle. The regulation of the level and rhythmicity of cAMP by adenyl cyclase, phosphodiesterase, and other second messengers (such as cGMP and calcium) will be studied in synchronously dividing and nondividing cultures of the achlorophyllous ZC mutant of Euglena, maintained in constant darkness, by examining the ability of pulses of various drugs and chemicals that are known to activate or inhibit these biochemical species to phase-shift the rhythms of cAMP level, adenyl cyclase and phosphodiesterase activities, and cell division. The cellular receptor for cAMP, which preliminary results suggest is a cAMP-dependent kinase, will be characterized, and a correlation between kinase stimulation and effects of cAMP or cAMP analogs on the cell division cycle will be sought. Finally, a Euglena homolog of p34 (the cdc 2 protein- kinase gene product of fission yeast) will be sought, and if it is identified, its histone H1 kinase activity will be measured at different phases of the cell division cycle in synchronous cultures perturbed by a cAMP pulse. Circadian rhythms have been documented in organisms ranging from unicellular eukaryotes to higher plants and animals and concern activities as varied as leaf movements in plants, locomotor activity in insects and mammals, and photosynthesis in unicellular algae. The cell division cycle of many algae, fungi, and protozoa also exhibit circadian rhythms; cell division occurs only during restricted intervals of the circadian cycle, often at the times corresponding to the dark intervals in a diurnal light-dark cycle. This phenomenon is thought to reflect an interaction between the circadian clock and the cell cycle oscillator. The long range goal of this research is to determine the nature of the circadian clock(s) presumed to couple to the cell division cycle and to generate periodicity of division, as well as to underlie circadian rhythmicity in general.