Clocked cell cycle clocks.

The cell division cycle of both mammalian cells and microorganisms, which apparently has both deterministic and probabilistic features, is a clock of sorts in that the sequence of events that comprise it measures time under a given set of environmental conditions. The cell division cycle may itself be regulated by a programmable clock that, under certain conditions, can generate circadian periodicities by interaction with a circadian pacemaker. These clocks must insert time segments into the cell division cycle in order to generate the observed variability in cellular generation times.

Circadian rhythm of cell division in Euglena: effects of random illumination regimen.

A persisting, "free-running," circadian rhythm of cell division in autotrophically grown Euglena gracilis is obtained upon placing either an exponentially increasing population or a culture that has been synchronized by a 10:14 light-dark cycle in a random illumination regimen that affords a total of 8 hours of light each 24 hours. These results are interpreted as implicating an endogenous biological clock which "gates" the specific event of cell division in the cell developmental cycle.

Persisting circadian rhythm of cell division in a photosynthetic mutant of Euglena.

A persisting, free-running, circadian rhythm of cell division in a heterotrophically grown mutant of Euglena gracilis var. bacillaris having impaired photosynthesis is obtained upon placing a culture that has been previously synchronized by a 10,14 light-dark cycle into continuous darkness at 19 degrees C (but not at 25 degrees C). A similar persisting rhythm is initiated in exponentially increasing cultures (growing in darkness at 19 degrees C) by a single "switch-up" in irradiance to continuous bright illumination. The results implicate an endogenous biological clock which "gates" the specific event of cell division in the cell developmental cycle.

Biochemical modeling of an autonomously oscillatory circadian clock in Euglena.

Eukaryotic microorganisms, as well as higher animals and plants, display many autonomous physiological and biochemical rhythmicities having periods approximating 24 hours. In an attempt to determine the nature of the timing mechanisms that are responsible for these circadian periodicities, two primary operational assumptions were postulated. Both the perturbation of a putative element of a circadian clock within its normal oscillatory range and the direct activation as well as the inhibition of such an element should yield a phase shift of an overt rhythm generated by the underlying oscillator. Results of experiments conducted in the flagellate Euglena suggest that nicotinamide adenine dinucleotide (NAD+), the mitochondrial Ca2+-transport system, Ca2+, calmodulin, NAD+ kinase, and NADP+ phosphatase represent clock "gears" that, in ensemble, might constitute a self-sustained circadian oscillating loop in this and other organisms.

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.

Role of cyclic GMP in the mediation of circadian rhythmicity of the adenylate cyclase-cyclic AMP-phosphodiesterase system in Euglena.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are two second messengers that have been proposed to act as a dualistic system in biological regulation. To determine if cGMP plays a role in the mediation of circadian rhythmicity of the adenylate cyclase (AC)-cAMP-phosphodiesterase (PDE) system in the achlorophyllous ZC mutant of the unicellular flagellate Euglena, the levels of cAMP and cGMP were monitored in synchronized cell populations, and the effects of the cGMP analog 8-bromo-cGMP (8-Br-cGMP) and the cGMP inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583) on the activity of AC and PDE, as well as on the level of cAMP, were measured in vivo. A bimodal, 24-hr rhythm of cGMP content was found in both dividing and nondividing cultures in either a 12-hr:12-hr light-dark cycle or constant darkness. The peaks and troughs of the cGMP rhythm occurred 2 hr in advance of those of the cAMP rhythm that has been reported previously. The addition of 8-Br-cGMP at different circadian times increased the cAMP level in vivo by two to five times, whereas LY 83583 reduced the amplitude of the cAMP rhythm so that it disappeared. The effects of 8-Br-cGMP on the activity of AC and PDE were circadian phase-dependent and consistent with the changes in cAMP content. These findings suggest that cGMP may serve as an upstream effector that mediates the cAMP oscillation by regulation of the AC-cAMP-PDE system.

Cell division cycles and circadian oscillators in Euglena.

The algal flagellate Euglena grown photoautotrophically in L:D 3:3 displays a circadian rhythm of cell division. Oscillatory models for cell cycle (CDC) control (particularly those of the limit cycle variety) include the property of phase perturbation, or resetting. This prediction has been tested in synchronous cultures in which the free-running rhythm has been scanned by 3-hr light signals. A strong (Type 0) phase response curve (PRC), yielding both advances and delays as great as 15 hr, has been derived. A second prediction of the limit cycle model is that there exists a pulse of a critical intensity, which, if given at one specific phase of the rhythm (the singularity point), should result in a phaseless, motionless state in which the rhythmicity disappears. Such a point has been found in Euglena in the late subjective night for light pulses having an intensity ranging from 40 to 700 lx. Finally, circadian oscillators typically display temperature-compensated period lengths within the physiological range of steady-state temperatures, although the length of the CDC is commonly thought to be highly temperature dependent. We have found that over a range of at least 10 degrees C, the period of the division rhythm is only slightly affected, exhibiting a Q10 of about 1.05-1.20. These observations, therefore, collectively implicate a circadian oscillator in the control of the CDC.

The role of ions and second messengers in circadian clock function.

The fact that single cells can exhibit circadian rhythmicity simultaneously in quite different processes, such as those of photosynthesis, bioluminescence, and cell division, suggests that membrane-bound compartmentalization is important for temporal organization. Since these rhythms, as well as others, are known to be affected by changes in the ionic environment and are probably membrane-bound systems, it is not surprising that transmembrane ion transport or flux has been proposed to be a key feature of the underlying circadian oscillator(s). Likewise, signal transduction along the entrainment pathway leading to the clock, among the elements, or "gears," of the timing loop itself, and within the output pathway between the oscillator and its "hands" likely is mediated by ions and second messengers. In this overview, we examine the theoretical and experimental evidence supporting the possible roles of intracellular free calcium and cyclic AMP in these capacities, particularly in view of the fact that oscillations in the concentrations of both species have been proposed to form the basis of pacemaker activity and other biological rhythms.

Circadian variations in the affinities of NAD kinase and NADP phosphatase for their substrates, NAD+ and NADP+, in dividing and nondividing cells of the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z).

We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP+ and NAD+, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CT), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2(+)-calmodulin) or for its substrate, NAD+. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.

Chronobiology at the cellular and molecular levels: models and mechanisms for circadian timekeeping.

This review considers cellular chronobiology and examines, at least in a superficial way, several classes of models and mechanisms that have been proposed for circadian rhythmicity and some of the experimental approaches that have appeared to be most productive. After a brief discussion of temporal organization and the metabolic, epigenetic, and circadian time domains, the general properties of circadian rhythms are enumerated. A survey of independent oscillations in isolated organs, tissues, and cells is followed by a review of selected circadian rhythms in eukaryotic microorganisms, with particular emphasis placed on the rhythm of cell division in the algal flagellate Euglena as a model system illustrating temporal differentiation. In the ensuing section, experimental approaches to circadian clock mechanisms are considered. The dissection of the clock by the use of chemical inhibitors is illustrated for the rhythm of bioluminescence in the marine dinoflagellate Gonyaulax and for the rhythm of photosynthetic capacity in the unicellular green alga Acetabularia. Alternatively, genetic analysis of circadian oscillators is considered in the green alga Chlamydomonas and in the bread mold Neurospora, both of which have yielded clock mutants and mutants having biochemical lesions that exhibit altered clock properties. On the basis of the evidence generated by these experimental approaches, several classes of biochemical and molecular models for circadian clocks have been proposed. These include strictly molecular models, feedback loop (network) models, transcriptional (tape-reading) models, and membrane models; some of their key elements and predictions are discussed. Finally, a number of general unsolved problems at the cellular level are briefly mentioned: cell cycle interfaces, the evolution of circadian rhythmicity, the possibility of multiple cellular oscillators, chronopharmacology and chronotherapy, and cell-cycle clocks in development and aging.

Oscillator control of cell division in Euglena: cyclic AMP oscillations mediate the phasing of the cell division cycle by the circadian clock.

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 cyclic AMP level. Maximum cyclic AMP 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 in cultures that had been transferred into DD and appeared to be under the control of the circadian oscillator rather than to be cell division cycle (CDC)-dependent, since they continued in cultures that had reached the stationary phase of growth. In the experiments reported in this paper, we tested for the possible role of this periodic cyclic AMP signal in the generation of cell division rhythmicity by examining the effects of exogenous cyclic AMP signals and of forskolin, which permanently increased the cyclic AMP level, on the cell division rhythm. Perturbations of the cyclic AMP oscillation by exogenous cyclic AMP resulted in the temporary uncoupling of the CDC from the circadian timer. The addition of cyclic AMP during the subjective day resulted in delays (up to 9 h) of the next synchronous division step. In contrast, mitosis was stimulated when cyclic AMP was administered in the middle of the subjective night. Measurement of the DNA content of cells by flow cytometry indicated that cyclic AMP injected at CT 06-08 delayed progression through S phase, and perhaps also through mitosis. When added at CT 18-20, cyclic AMP accelerated the G2/M transition. The circadian oscillator was not perturbed by the addition of exogenous cyclic AMP: the division rhythm soon returned to its original phase. On the other hand, the permanent elevation of cyclic AMP levels in the presence of forskolin induced a rapid loss of cell division rhythmicity. These findings are consistent with the hypothesis that cyclic AMP acts downstream from the oscillator and that the cyclic AMP oscillation is an essential component of the signaling pathway for the control of the CDC by the circadian oscillator. The receptors for cyclic AMP in Euglena have been shown to be two cyclic AMP-dependent kinases (cPKA and cPKB). Pharmacological studies using cyclic AMP analogs suggested that cPKA mediates cyclic AMP effects during the subjective day, and cPKB during the subjective night.(ABSTRACT TRUNCATED AT 400 WORDS)

cAMP-dependent kinases in the algal flagellate Euglena gracilis.

Euglena cells grown in diurnal light-dark cycles exhibit circadian variations of their cAMP content, which we believe to be under the control of an endogenous timer because they persist in constant darkness in the absence of any environmental time cue. We think that these cAMP oscillations may play a role in the regulation of some of the numerous cellular activities that are known to display circadian rhythmicities in this organism. The role of cAMP in algal cells is still controversial, however, since the nature of the cAMP "receptor" is unknown. We show that extracts of the achlorophyllous ZC mutant of Euglena gracilis contain two cAMP-binding proteins, which bind cAMP with a high affinity (Kd values of 10 nM and 30 nM) and which can be separated by DEAE-cellulose chromatography. Protein kinase activity was assayed using Kemptide as a substrate. Stimulation of kinase activity by cAMP was observed after partial purification by DEAE-cellulose chromatography. Two peaks of activity were resolved, corresponding to distinct enzymes with different cAMP-analog specificities. Thus, cAMP signaling in plant cells may proceed by the phosphorylation of target proteins by cAMP-dependent kinases, in a manner similar to that of animal cells.

Effects of "skeleton" photoperiods and high frequency light-dark cycles on the rhythm of cell division in synchronized cultures of Euglena.

Two further lines of evidence support the contention (EDMUNDS, 1966) that the cell cycle in autotrophically grown Euglena can be coupled to an endogenous, circadian biological clock under certain conditions. So-called "skeleton" photoperiods (LD: 3,6,3:12 and LD: 4,4,4:12) following a complete photoperiod regime entrain the cell division rhythm in the population to a precise 24 hr period, although the step-sizes of the successive fission bursts are always less than 2.00, indicating that not all cells divide in any one 24 hr interval. These findings imply that the continuous action of light is not required for synchronization and suggest that the putative oscillation underlying the rhythm can be phased by discrete light (or dark) "pulses" or signals.The effects of high frequency LD cycles whose periods were integral submultiples of 24 hr were also investigated. In most regimes (LD:1/4,1/2; LD:1/2,1; LD: 1,2; LD: 1,3; LD: 2,4; LD: 2,6; LD: 4,4) synchronous cell division iccurred in the culture with an average period of 26-27 hr, although only a fraction of the cells divided during any one burst. Similar results were obtained if (i) a synchronized culture was exposed to certain high frequency cycles whose periods were not integral submultiples of 24 hr (e.g., LD: 5,5 or LD: 8,8); (ii) an asynchronous culture (grown in LL) was subsequently exposed to a high frequency cycle; or (iii) a synchronized culture was subjected to a "random" LD cycle. The synchrony does not break down as long as the given LD regime is imposed and shows some indications of persistence in certain ensuing conditions of continuous illumination.A general formula was derived which predicts the time of division, t D , for an individual cell: t D =k+nτ, where k is the initial phase delay, n is an integer, and τ is the free-running period of the rhythm observed in the population. These results are interpreted as indicating that the high frequency cycles employed were unable to entrain the circadian oscillation(s) hypothesized to underly and gate cell division, with the result that the rhythm reverted to its free-running period. Exposure to such cycles, however, apparently either initiates a rhythm or synchronizes the phases of the individual oscillations in the populations of cells. The possible direct interaction between energy supply and the observed somewhat variable period lengths is discussed; also, the relevance of stochastic models for the decay of division synchrony in the absence of a recurrent synchronizing procedure is considered.