Long and short isoforms of Neurospora clock protein FRQ support temperature-compensated circadian rhythms

The large (l) and small (s) isoforms of FREQUENCY (FRQ) are elements of interconnected feedback loops of the Neurospora circadian clock. The expression ratio of l-FRQ vs. s-FRQ is regulated by thermosensitive splicing of an intron containing the initiation codon for l-FRQ. We show that this splicing is dependent on light and temperature and displays a circadian rhythm. Strains expressing only l-FRQ or s-FRQ support short and long temperature-compensated circadian rhythms, respectively. The thermosensitive expression ratio of FRQ isoforms influences period length in wt. Our data indicate that differential expression of FRQ isoforms is not required for temperature compensation but rather provides a means to fine-tune period length in response to ambient temperature.     

How temperature affects the circadian clock of Neurospora crassa

Light and temperature are major environmental cues that influence circadian clocks. The molecular effects of these zeitgebers on the circadian clock of Neurospora crassa have been studied intensively during the last decade. While signal transduction of light into the circadian clock is quite well characterized, we have only recently begun to understand the molecular mechanisms that underlie temperature sensing. Here we summarize briefly the current knowledge about the effects of temperature on the circadian clock of Neurospora crassa.     

How temperature affects the circadian clock of Neurospora crassa

Light and temperature are major environmental cues that influence circadian clocks. The molecular effects of these zeitgebers on the circadian clock of Neurospora crassa have been studied intensively during the last decade. While signal transduction of light into the circadian clock is quite well characterized, we have only recently begun to understand the molecular mechanisms that underlie temperature sensing. Here we summarize briefly the current knowledge about the effects of temperature on the circadian clock of Neurospora crassa.     

Circadian and light-induced expression of luciferase in Neurospora crassa

We have constructed a plasmid vector for expressing firefly luciferase in Neurospora crassa under control of the light- and clock-regulated ccg-2 (eas) promoter. The sequence of the luciferase gene in the vector has been modified to reflect the N. crassa codon bias. Both light-induced activity and circadian activity are demonstrated. Expression of luciferase in strains carrying mutant frequency alleles shows appropriate period length alterations. These data demonstrate that luciferase is a sensitive reporter of gene expression in N. crassa. Our results also show that the modified luciferase is expressed in Aspergillus nidulans.     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

pH homeostasis of the circadian sporulation rhythm in clock mutants of Neurospora crassa

The influence of environmental (extracellular) pH on the sporulation rhythm in Neurospora crassa was investigated for wild-type (frq⁺) and the mutants chr, frq¹, frq⁷, and frq⁸. In all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular pH in the range 4-9. On the other hand, for the same pH range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq¹. A loss of pH homeostasis of the period, however, was observed in the mutants frq⁷ and frq⁸, which also are known to have lost temperature compensation. Concerning the influence of extracellular pH on growth rates, a clear correspondence between growth rates and the concentration of available H₂PO₄^- ion has been found, indicating that the uptake of H₂PO₄^- may be a limiting factor for growth under our experimental conditions. The loss of pH compensation in the frq⁷ and frq⁸ mutants may be related to less easily degradable FRQ^7,8 proteins when compared with wild-type FRQ. Results from recent model considerations and experimental results predict that, with increasing extra-and intracellular pH, the FRQ⁷ protein degradation increases and should lead to shorter period lengths. (Chronobiology International, 17(6), 733-750, 2000)     

Role of nitrogen in the photoinduction of protoperithecia and carotenoids in Neurospora crassa

Nitrogen, as KNO₃ or NH₄NO₃, can inhibit the photoinduction of protoperithecia in Neurospora crassa when present in the medium at a high concentration but does not inhibit the photoinduction of carotenoids. The point at which the presence of high nitrogen levels is no longer inhibitory is 5 h after illumination.Abbreviations al albino mutant - wc white-collar mutant - WM Westergaard and Mitchell (1947) medium Dedicated to Professor Wilhelm Nultsch in honour of his 60th birthday     

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.     

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Energy-dependent phases of the circadian clock and the clock-controlled leaf movement in Phaseolus coccineus L.

The energy requirements of the various phases of the circadian clock in the laminar pulvini cells of primary leaves of Phaseolus coccineus L. were investigated using 4-h pulses of NaCN (5 mM) and NaN₃ (1 mM). The induced phase shifts were calculated from the timing of the subjective night position during the third cycle after the treatment. Both inhibitors produce advances during phases which are correlated with the upward movement of the leaf (ca. 0–12 h after the maximum of the subjective night position) and during phases which are correlated with the downward movement of the leaf (ca. 20–28 h after the maximum of the subjective night position). Maximal advances are induced during the phase which is correlated with the maximum of the subjective night position (hour 0), whereas during phases which are correlated with the subjective day position (ca. 12–20 h after the maximum of the subjective night position) the inhibitors have no effect or induce only small advances. These results demonstrate that the part of the circadian cycle which, according to Bünning's tension-relaxation model of the circadian clock, is characterized by features of relaxation, represents a sequence of phases with decreasing energy requirement, whereas the tension part of the circadian cycle represents a sequence of phases with increasing energy requirement. The energy requirement for changing and maintaining the leaf positions was investigated by continuously offering NaCN, NaN₃, and dinitrophenol (DNP) to leaves with intact and half (flexor cut away) pulvini. The substances inhibit in both pulvini the upward movement or induce a downward movement, depending on the leaf position, when the transfer to the inhibitor solution takes place. These results give evidence that the movement of intact pulvini reflects the turgor (volume) state of the extensor cells and that the increase of turgor (volume) and high turgor (volume) state requires more energy than the decrease of turgor (volume) or low turgor (small volume) state. Therefore, the time course of the energy requirements of the circadian clock and the clock-controlled turgor (volume states or leaf movement) is out of phase during a circadian cycle. Consequently the reaction of the clock-controlled leaf movement to the reduced energy supply can mask the clock behavior in pulse and step experiments. The phase response curves towards CN^- and N ₃ ^- reflect the time course of the CN^--induced membrane depolarizations (the energy requirement of the electrogenic pump) in extensor cells of the pulvinus (Freudling et al. (1980), Plant Physiol. 65, 966–968), and both are out of phase with the time course of the energy requirement of the turgor. Consequently it is hypothesized that in Phaseolus advances are due to membrane depolarization and that at least in this organism electric properties of the plasmalemma are essentially involved in the mechanism of the circadian clock.Abbreviations LD light-dark cycle - LL continuous light - DNP dinitrophenol This paper is dedicated to Professor Erwin Bünning on the occasion of his 75th birthdayIn this paper zero corresponds to the second maximum of the subjective night position of the leaves after transfer to constant conditions. Zero to twelve hours corresponds approximately to the upward movement of the leaves, 12–20 h to the elevated (subjective day) position, and 20–28 h to the downward movement of the leaves. In other circadian systems Pittendrigh's CT (circadian time) convention is used. CT 00 is the time of dawn after a 12-h light/12-h dark cycle. Since in Phaseolus the plants are raised in a LD cycle different from 12:12 and since the phases at dawn differ considerably from leaf to leaf and are furthermore not precisely determinable (whereas the subjective night position of the leaves is a well-defined and recognizable phase) this convention is not followed in Phaseolus. Phase zero in Phaseolus corresponds to approximately CT 18 in other systems     

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.     

生物钟体系中色噪音诱导的日夜节律振荡和内信号随机共振

利用脉孢菌生物钟体系,研究了色噪音对其进行诱导所产生的日夜节律振荡信号及其内信号随机共振的行为．结果表明,色噪音的相关时间对该体系内信号随机共振的强弱起较大的影响作用．当无外信号存在时,色噪音的相关时间对体系内信号随机共振强度起抑制的作用,且随相关时间的增大,抑制作用增强．当外信号加到体系中时,由于相关时间和外信号的协同作用,相关时间不仅对其内信号随机共振强度起抑制的作用,而且还影响内信号随机共振峰的数目,即随相关时间的增大,可使单峰随机共振变为随机双共振．存在最佳的外信号频率使体系的内信号随机共振强度得到最大的增强,而其他频率的外信号却起抑制作用．色内噪音和色外噪音相比,前者对该体系进行诱导所得的内信号随机共振强度比后者的更强,而且体系对前者更敏感．另外,存在极限的噪音强度使白噪音和色噪音对该体系内信号随机共振的影响差异得以消失．所得结果可为治疗生物钟紊乱综合症提供理论依据,同时可更好地理解其他节奏机理,如心脏搏动节奏、呼吸节奏以及荷尔蒙水平的波动节奏等．     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Light and serotonin phase-shift the circadian clock in the cricket optic lobe in vitro

Light and serotonin were found to cause phase shifts of the circadian neural activity rhythm in the optic lobe of the cricket Gryllus bimaculatus cultured in vitro. The two phase-shifting agents yielded phase-response curves different in shape. Light induced phase delay and advance in the early and late subjective night, respectively, and almost no shifts in the subjective day, whereas serotonin phase-advances the clock during the subjective day and induced delay shifts during the subjective night. The largest phase advance and delay occurred at circadian time 21 and 12, respectively, for light, and circadian time 3 and 18, respectively, for serotonin. Quipazine, a nonspecific serotonin agonist, induced phase advance and phase delay at circadian time 3 and 18, respectively, like serotonin. (±)8-OH-DPAT, a specific 5-HT_1A agonist, phase delayed by 2 h at the subjective night, but produced no significant phase shifts at the subjective day. When NAN-190, a specific 5-HT_1A antagonist, was applied together with quipazine, it completely blocked the phase delay at circadian time 18, whereas it had no effect on the advance shifts induced by quipazine. The results suggest that the phase dependency of serotonin-induced phase shifts of the clock may be partly attributable to the daily change in receptor type. Accepted: 4 July 1999     

A two variable delay model for the circadian rhythm of Neurospora crassa

A two variable model with delay in both the variables, is proposed for the circadian oscillations of protein concentrations in the fungal species Neurospora crassa. The dynamical variables chosen are the concentrations of FRQ and WC-1 proteins. Our model is a two variable simplification of the detailed model of Smolen et al. (J. Neurosci. 21 (2001) 6644) modeling circadian oscillations with interlocking positive and negative feedback loops, containing 23 variables. In our model, as in the case of Smolen's model, a sustained limit cycle oscillation takes place in both FRQ and WC-1 protein in continuous darkness, and WC-1 is anti-phase to FRQ protein, as observed in experiments. The model accounts for various characteristic features of circadian rhythms such as entrainment to light dark cycles, phase response curves and robustness to parameter variation and molecular fluctuations. Simulations are carried out to study the effect of periodic forcing of circadian oscillations by light-dark cycles. The periodic forcing resulted in a rich bifurcation diagram that includes quasiperiodicity and chaotic oscillations, depending on the magnitude of the periodic changes in the light controlled parameter. When positive feedback is eliminated, our model reduces to the generic one dimensional delay model of Lema et al. (J. Theor. Biol. 204 (2000) 565), delay model of the circadian pace maker with FRQ protein as the dynamical variable which represses its own production. This one-dimensional model also exhibits all characteristic features of circadian oscillations and gives rise to circadian oscillations which are reasonably robust to parameter variations and molecular noise.     

Calcium plays a central role in phase shifting the ocular circadian pacemaker of Aplysia

The eye of the marine mollusk Aplysia californica contains an oscillator that drives a circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate the role of extracellular calcium in both light and serotonin-mediated phase shifts. Low calcium treatments were found to cause phase shifts which resembled those produced by the transmitter serotonin. However, unlike serotonin, low calcium neither increased ocular cAMP levels nor could these phase shifts be prevented by increasing extracellular potassium concentration. Low calcium-induced phase shifts were prevented by the simultaneous application of the translational inhibitor anisomycin and low calcium treatment resulted in changes in [³⁵S]methionine incorporation into several proteins as measured by a two-dimensional electrophoresis gel analysis. Finally, light treatments failed to produce phase shifts in the presence of low calcium or the calcium channel antagonist nickel chloride. These results are consistent with a model in which serotonin phase shifts the ocular pacemaker by decreasing a transmembrane calcium flux through membrane hyperpolarization while light-induced phase shifts are mediated by an increase in calcium flux.Abbreviations ASW artificial seawater - EGTA ethylene glycol-bis(-amino-ethyl ester) N,N,N N-tetraacetic acid - CAP compound action potential - CT circadian time 5-HT serotonin - Ni⁺⁺ nickel