Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.     

MPer1 and mper2 are essential for normal resetting of the circadian clock

Mammalian Per1 and Per2 genes are involved in the mechanism of the circadian clock and are inducible by light. A light pulse can evoke a change in the onset of wheel-running activity in mice by shifting the onset of activity to earlier times (phase advance) or later times (phase delays) thereby advancing or delaying the clock (clock resetting). To assess the role of mouse Per (mPer) genes in circadian clock resetting, mice carrying mutant mPer1 or mPer2 genes were tested for responses to a light pulse at ZT 14 and ZT 22, respectively. The authors found that mPer1 mutants did not advance and mPer2 mutants did not delay the clock. They conclude that the mammalian Per genes are not only light-responsive components of the circadian oscillator but also are involved in resetting of the circadian clock.     

ELF3 modulates resetting of the circadian clock in Arabidopsis

The Arabidopsis early flowering 3 (elf3) mutation causes arrhythmic circadian output in continuous light, but there is some evidence of clock function in darkness. Here, we show conclusively that normal circadian function occurs with no alteration of period length in elf3 mutants in dark conditions and that the light-dependent arrhythmia observed in elf3 mutants is pleiotropic on multiple outputs normally expressed at different times of day. Plants overexpressing ELF3 have an increased period length in both constant blue and red light; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by using light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and constitutive overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. The phase of ELF3 function correlates with its peak expression levels in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting.     

Temperature effect on entrainment,phase shifting,and amplitude of circadian clocks and its molecular bases

Effects of temperature and temperature changes on circadian clocks in cyanobacteria, unicellular algae, and plants, as well as fungi, arthropods, and vertebrates are reviewed. Periodic temperature with periods around 24 h even in the low range of 1-2 degrees C (strong Zeitgeber effect) can entrain all ectothermic (poikilothermic) organisms. This is also reflected by the phase shifts-recorded by phase response curves (PRCs)-that are elicited by step- or pulsewise changes in the temperature. The amount of phase shift (weak or strong type of PRC) depends on the amplitude of the temperature change and on its duration when applied as a pulse. Form and position of the PRC to temperature pulses are similar to those of the PRC to light pulses. A combined high/low temperature and light/dark cycle leads to a stabile phase and maximal amplitude of the circadian rhythm-when applied in phase (i.e., warm/light and cold/dark). When the two Zeitgeber cycles are phase-shifted against each other the phase of the circadian rhythm is determined by either Zeitgeber or by both, depending on the relative strength (amplitude) of both Zeitgeber signals and the sensitivity of the species/individual toward them. A phase jump of the circadian rhythm has been observed in several organisms at a certain phase relationship of the two Zeitgeber cycles. Ectothermic organisms show inter- and intraspecies plus seasonal variations in the temperature limits for the expression of the clock, either of the basic molecular mechanism, and/or the dependent variables. A step-down from higher temperatures or a step-up from lower temperatures to moderate temperatures often results in initiation of oscillations from phase positions that are about 180 degrees different. This may be explained by holding the clock at different phase positions (maximum or minimum of a clock component) or by significantly different levels of clock components at the higher or lower temperatures. Different permissive temperatures result in different circadian amplitudes, that usually show a species-specific optimum. In endothermic (homeothermic) organisms periodic temperature changes of about 24 h often cause entrainment, although with considerable individual differences, only if they are of rather high amplitudes (weak Zeitgeber effects). The same applies to the phase-shifting effects of temperature pulses. Isolated bird pineals and rat suprachiasmatic nuclei tissues on the other hand, respond to medium high temperature pulses and reveal PRCs similar to that of light signals. Therefore, one may speculate that the self-selected circadian rhythm of body temperature in reptiles or the endogenously controlled body temperature in homeotherms (some of which show temperature differences of more than 2 degrees C) may, in itself, serve as an internal entraining system. The so-called heterothermic mammals (undergoing low body temperature states in a daily or seasonal pattern) may be more sensitive to temperature changes. Effects of temperature elevation on the molecular clock mechanisms have been shown in Neurospora (induction of the frequency (FRQ) protein) and in Drosophila (degradation of the period (PER) and timeless (TIM) protein) and can explain observed phase shifts of rhythms in conidiation and locomotor activity, respectively. Temperature changes probably act directly on all processes of the clock mechanism some being more sensitive than the others. Temperature changes affect membrane properties, ion homeostasis, calcium influx, and other signal cascades (cAMP, cGMP, and the protein kinases A and C) (indirect effects) and may thus influence, in particular, protein phosphorylation processes of the clock mechanism. The temperature effects resemble to some degree those induced by light or by light-transducing neurons and their transmitters. In ectothermic vertebrates temperature changes significantly affect the melatonin rhythm, which in turn exerts entraining (phase shifting) functions.     

Modeling Temperature Compensation in Chemical and Biological Oscillators

All physicochemical and biological oscillators maintain a balance between destabilizing reactions (as, for example, intrinsic autocatalytic or amplifying reactions) and stabilizing processes. These two groups of processes tend to influence the period in opposite directions and may lead to temperature compensation whenever their overall influence balances. This principle of “antagonistic balance” has been tested for several chemical and biological oscillators. The Goodwin negative feedback oscillator appears of particular interest for modeling the circadian clocks in Neurospora and Drosophila and their temperature compensation. Remarkably, the Goodwin oscillator not only gives qualitative, correct phase response curves for temperature steps and temperature pulses, but also simulates the temperature behavior of Neurospora frq and Drosophila per mutants almost quantitatively. The Goodwin oscillator predicts that circadian periods are strongly dependent on the turnover of the clock mRNA or clock protein. A more rapid turnover of clock mRNA or clock protein results, in short, a slower turnover in longer period lengths. (Chronobiology International, 14(5), 499–510, 1997)     

Calculating activation energies for temperature compensation in circadian rhythms

Many biological species possess a circadian clock, which helps them anticipate daily variations in the environment. In the absence of external stimuli, the rhythm persists autonomously with a period of approximately 24 h. However, single pulses of light, nutrients, chemicals or temperature can shift the clock phase. In the case of light- and temperature-cycles, this allows entrainment of the clock to cycles of exactly 24 h. Circadian clocks have the remarkable property of temperature compensation, that is, the period of the circadian rhythm remains relatively constant within a physiological range of temperatures. For several organisms, temperature-regulated processes within the circadian clock have been identified in recent years. However, how these processes contribute to temperature compensation is not fully understood. Here, we theoretically investigate temperature compensation in general oscillatory systems. It is known that every oscillator can be locally temperature compensated around a reference temperature, if reactions are appropriately balanced. A balancing is always possible if the control coefficient with respect to the oscillation period of at least one reaction in the oscillator network is positive. However, for global temperature compensation, the whole physiological temperature range is relevant. Here, we use an approach which leads to an optimization problem subject to the local balancing principle. We use this approach to analyse different circadian clock models proposed in the literature and calculate activation energies that lead to temperature compensation.     

A phase response curve for circannual rhythm in the varied carpet beetle <Emphasis Type="Italic">Anthrenus verbasci</Emphasis>

We know that entrainment, a stable phase relationship with an environmental cycle, must be established for a biological clock to function properly. Phase response curves (PRCs), which are plots of phase shifts that result as a function of the phase of a stimulus, have been created to examine the mode of entrainment. In circadian rhythms, single-light pulse PRCs have been obtained by giving a light pulse to various phases of a free-running rhythm under continuous darkness. This successfully explains the entrainment to light-dark cycles. Some organisms show circannual rhythms. In some of these, changes in photoperiod entrain the circannual rhythms. However, no single-pulse PRCs have been created. Here we show the PRC to a long-day pulse superimposed for 4 weeks over constant short days in the circannual pupation rhythm in the varied carpet beetle Anthrenus verbasci. Because the shape of that PRC closely resembles that of the Type 0 PRC with large phase shifts in circadian rhythms, we suggest that an oscillator having a common feature in the phase response with the circadian clock, produces a circannual rhythm.     

The Goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of Neurospora crassa

The Goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of Neurospora and Drosophila. An essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. When protein synthesis in N. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (CHX) the period of the circadian sporulation rhythmicity is only slightly increased. The explanation of this effect may be seen in the inhibition of protein synthesis and protein degradation. In the model, increasing inhibition of both processes led to very similar results with respect to period length. That protein degradation is, in fact, inhibited by CHX is shown by determining protein degradation in N. crassa by means of pulse chase experiments. Phase response curves (PRCs) of the N. crassa sporulation rhythm toward CHX which were reported in the literature and investigated in this paper revealed significant differences between frq+and the long period mutants frq7and csp -1 frq7. These PRCs were also convincingly simulated by the model, if a transient inhibition of protein degradation by CHX is assumed as well as a lower constitutive degradation rate of FRQ-protein in the frq7/ csp -1 frq7mutants. The lower sensitivities of frq7and csp -1 frq7towards CHX may thus be explained by a lower degradation rate of clock protein FRQ7. The phase shifting by moderate temperature pulses (from 25 to 30 degrees C) can also be simulated by the Goodwin model and shows large phase advances at about CT 16-20 as observed in experiments. In case of higher temperature pulses (from 35 to 42 or 45 degrees C=heat shock) the phase position and form of the PRC changes as protein synthesis is increasingly inhibited. It is known from earlier experiments that heat shock not only inhibits the synthesis of many proteins but also inhibits protein degradation. Taking this into account, the Goodwin model also simulates the PRCs of high temperature (heat shock) pulses.     

Robust entrainment of circadian oscillators requires specific phase response curves

The circadian clocks keeping time in many living organisms rely on self-sustained biochemical oscillations entrained by external cues, such as light, to the 24-h cycle induced by Earth's rotation. However, environmental cues are unreliable due to the variability of habitats, weather conditions, or cue-sensing mechanisms among individuals. A tempting hypothesis is that circadian clocks have evolved so as to be robust to fluctuations in the signal that entrains them. To support this hypothesis, we analyze the synchronization behavior of weakly and periodically forced oscillators in terms of their phase response curve (PRC), which measures phase changes induced by a perturbation applied at different times of the cycle. We establish a general relationship between the robustness of key entrainment properties, such as stability and oscillator phase, on the one hand, and the shape of the PRC as characterized by a specific curvature or the existence of a dead zone, on the other hand. The criteria obtained are applied to computational models of circadian clocks and account for the disparate robustness properties of various forcing schemes. Finally, the analysis of PRCs measured experimentally in several organisms strongly suggests a case of convergent evolution toward an optimal strategy for maintaining a clock that is accurate and robust to environmental fluctuations.     

Robust circadian rhythmicity of Per1 and Per2 mutant mice in constant light,and dynamics of Per1 and Per2 gene expression under long and short photoperiods

The Per1 and Per2 genes are components of the mammalian circadian clock. Mutations in these genes alter phase resetting in response to a nocturnal light pulse, and Per2 mutant mice are known to become arrhythmic in constant darkness. We show that under constant light conditions, Per2 mutant mice exhibit robust activity rhythms as well as body temperature rhythms with a period length that is less than 24 h. In Per1 mutants, the period length of both activity and body temperature rhythms is longer than 24 h in constant light. Per1 mutants prolong their period length (tao) when illuminance is increased, whereas Per2 mutants shorten their endogenous period. Additionally, the authors show that the circadian pattern of Per1 and Per2 gene expression in mice is modified under different photoperiods and that there is a mutual influence of these genes on their timing of expression. We propose that, in mice, the phase relationship between Per1 and Per2 gene expression might be critical for transducing day length information to the organism. Per1 could be part of a morning oscillator tracking dawn, and Per2 could be part of an evening oscillator tracking dusk.     

Adjustability of the circadian clock in the cockroaches: a comparative study of two closely related species, Blattella germanica and Blattella bisignata.

A single 2h light pulse (250 lux) was given at various times to phase shift the locomotor circadian rhythm of two species of closely related cockroaches, Blattella bisignata and Blatella germanica. The phase-response curve (PRC) of both species showed a similar pattern. Phase delays and advances were induced by light pulse during the early and late subjective night, respectively, while no clear phase shifting was elicited during the subjective day. However, the magnitude of the phase delay (1.89h +/- 0.66h) and advance (0.69h +/- 0.36h) of B. bisignata was significantly larger than that of B. germanica (0.78h +/- 0.38h and 0.35h +/- 0.18h, respectively). This result indicates the superior adjustability of the circadian clock in B. bisignata. The period-response curve (PdRC) was also constructed for both species. Although both species did not show great flexibility in circadian period changes, the phase shifts were significantly correlated with the period changes in the advance zone of B. bisignata (r = 0.72, P < .1). This allowed the circadian clock of B. bisignata to display better entrainability since the phase advance adjustment was significantly more difficult than that of phase delay. The results indicate the overall adjustability of the circadian clock of B. germanica is inferior to that of B. bisignata. The significance of this finding is discussed from an ecological perspective.     

Arabidopsis response regulators ARR3 and ARR4 play cytokinin-independent roles in the control of circadian period

Light and temperature are potent environmental signals used to synchronize the circadian oscillator with external time and photoperiod. Phytochrome and cryptochrome photoreceptors integrate light quantity and quality to modulate the pace and phase of the clock. PHYTOCHROME B (phyB) controls period length in red light as well as the phase of the clock in white light. phyB interacts with ARABIDOPSIS RESPONSE REGULATOR4 (ARR4) in a light-dependent manner. Accordingly, we tested ARR4 and other members of the type-A ARR family for roles in clock function and show that ARR4 and its closest relative, ARR3, act redundantly in the Arabidopsis thaliana circadian system. Loss of ARR3 and ARR4 lengthens the period of the clock even in the absence of light, demonstrating that they do so independently of active phyB. In addition, in white light, arr3,4 mutants show a leading phase similar to phyB mutants, suggesting that circadian light input is modulated by the interaction of phyB with ARR4. Although type-A ARRs are involved in cytokinin signaling, the circadian defects appear to be independent of cytokinin, as exogenous cytokinin affects the phase but not the period of the clock. Therefore, ARR3 and ARR4 are critical for proper circadian period and define an additional level of regulation of the circadian clock in Arabidopsis.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Can phase response curves of various treatments of circadian rhythms be explained by effects on protein synthesis and degradation?

The phase response curves of circadian rhythms toward pulses of various chemical and physical perturbations are standardized and compared in Gonyaulax, Phaseolus, Kalanchoe, Trifolium, and Aplysia. The time of maximal phase shift, in many of these curves clustered around a given time of day, especially in Gonyaulax and Aplysia. This could be interpreted as being due to a converging effect of these modalities on a single function that is decisive for the mechanism of the circadian clock. Since many of the treatments that result in significant phase response curves (e.g., light pulses, hormones, temperature pulses, changes of ion concentration, etc.) have also been shown in independent studies to be capable of affecting protein synthesis, it is possible that these treatments may all affect circadian rhythmicity by virtue of their direct or indirect effect on protein synthesis. There is also a number of treatments which give phase response curves that are not clustered, especially in Phaseolus. This means either that our original assumption is incorrect or that these treatments impinge on sensitive components of the clock other than protein synthesis. It is emphasized, however, that the phasing of a phase response curve on one hand is subject to variability of the boundary conditions in the different laboratories and on the other hand seems to depend on the strength, direction, and modality of the perturbing pulse.