Molecular mechanisms of entrainment in the Neurospora circadian clock

Light and temperature are 2 of the most important environmental influences on all circadian clocks, and Neurospora provides an excellent system for understanding their effects. Progress made in the past decade has led to a basic molecular understanding of how the Neurospora clock works and how environmental factors influence it. The purpose of this review is to summarize what we currently know about the molecular mechanism of light and temperature entrainment in Neurospora.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

The wild-type circadian period of Neurospora is encoded in the residual network of the null frq mutants

We model theoretically the response of the widely studied circadian oscillator of Neurospora crassa to inactivation of the frq gene. The resulting organism has been termed "arrhythmic" under constant conditions. Under entrainment to periodic temperature cycles Roenneberg, Merrow and coworkers have shown that the phase angle at which spore formation occurs depends on the entrainment period, curiously even in the null frq mutants (frq9 and frq10). We show that such a response does not imply the presence of a self-sustained free-running oscillator. We derive a simple candidate model (a damped harmonic oscillator) for the null frq mutants that successfully reproduces the observed phase angle response. An endogenous period of 21 h for the damped harmonic oscillator coincides with the endogenous period of wild-type Neurospora. This suggests that the (noise driven) "residual system" present in the mutants may have a significant timekeeping role in the wild-type organism. Our model (with no change of parameters) was then used to investigate spore formation patterns under constant conditions and reproduces the corresponding experimental data of Aronson et al. (Proc. Natl. Acad. Sci. USA 91 (1994) 7683.)     

The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas).

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are "blind" to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. By contrast, ccg-2(eas) mRNA is photoinduced in clock-null strains such as frequency (bd;frq). The results in the clock mutants show that an intact circadian oscillator is not required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a dual regulation that involves separable regulation by light and circadian rhythm.     

Circadian Nature of a Rhythm Expressed by an Invertaseless Strain of Neurospora crassa

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25 degrees ); B) the period is relatively temperature-independent (Q(10) is between 0.95 and 1.21 for temperature range of 18 to 35 degrees ); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.     

The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.     

Mutually synchronized relaxation oscillators as prototypes of oscillating systems in biology

Summary This paper discusses the analogy between phenomena in populations of coupled biological oscillators and the behaviour of systems of synchronized mathematical oscillators. Frequency entrainment in a set of coupled relaxation oscillators is investigated with perturbation methods. This analysis leads to quantitative results for entrainment and explains phenomena such as travelling waves in systems of spatially distributed oscillators.     

Data-driven modelling captures dynamics of the circadian clock of Neurospora crassa

Eukaryotic circadian clocks are based on self-sustaining, cell-autonomous oscillatory feedback loops that can synchronize with the environment via recurrent stimuli (zeitgebers) such as light. The components of biological clocks and their network interactions are becoming increasingly known, calling for a quantitative understanding of their role for clock function. However, the development of data-driven mathematical clock models has remained limited by the lack of sufficiently accurate data. Here we present a comprehensive model of the circadian clock of Neurospora crassa that describe free-running oscillations in constant darkness and entrainment in light-dark cycles. To parameterize the model, we measured high-resolution time courses of luciferase reporters of morning and evening specific clock genes in WT and a mutant strain. Fitting the model to such comprehensive data allowed estimating parameters governing circadian phase, period length and amplitude, and the response of genes to light cues. Our model suggests that functional maturation of the core clock protein Frequency causes a delay in negative feedback that is critical for generating circadian rhythms.     

Interleukin 1beta enhances non-rapid eye movement sleep and increases c-Fos protein expression in the median preoptic nucleus of the hypothalamus

Both temporary access to a running wheel and temporary exposure to light systematically influence the phase producing entrainment of the circadian activity rhythm in the golden hamster (Mesocricetus auratus). However, precise determination of entrainment limits remains methodologically difficult, because such calculations may be influenced by varying experimental paradigms. In this study, effects on the entrainment of the activity pattern during successive light-dark (LD) cycles of stepwise decreasing periods, as well as wheel running activity, were investigated. In particular, the hamster activity rhythm under LD cycles with a period (T) shorter than 22 h was studied, i.e., when the LD cycle itself had been shown to be an insufficiently strong zeitgeber to synchronize activity rhythms. Indeed, it was confirmed that animals without a wheel do not entrain under 11:11-h LD cycles (T = 22 h). Subsequently providing hamsters continuous access to a running wheel established entrainment to T = 22 h. Moreover, this paradigm underwent further reductions of the T period to T = 19.6 h without loss of entrainment. Furthermore, restricting access to the wheel did not result in loss of entrainment, while even entrainment to T = 19 h was observed. To explain this observed shift in the lower entrainment limit, our speculation centers on changes in pacemaker response facilitated by stepwise changes of T spaced very far apart, thus allowing time for adaptation.     

氧限制下粗糙脉孢菌乙醇发酵的数学模型及过程模拟

粗糙脉孢菌(Mzcrospora crassan)具有直接转化植物纤维性物质生产乙醇的能力。研究了不同氧限制条件对粗糙脉孢菌发酵葡萄糖生产乙醇的影响,构建了该过程的数学模型,并利用数学模型进行了模拟和预测研究。结果表明,数学模型能够很好地预测氧限制条件下乙醇的发酵过程,即使微量氧对乙醇发酵也有较大的负面影响。