Patricia L. Lakin-Thomas

Transcriptional Feedback Oscillators: Maybe, Maybe Not...

The molecular mechanism of circadian rhythmicity is usually modeled by a transcription/translation feedback oscillator in which clock proteins negatively feed back on their own transcription to produce rhythmic levels of clock protein mRNAs, which in turn cause the production of rhythmic levels of clock proteins. This mechanism has been applied to all model organisms for which molecular data are available. This review summarizes the increasing number of anomalous observations that do not fit the standard molecular mechanism for the model organisms Acetabularia, Synechococcus, Drosophila, Neurospora, and mouse. The anomalies fall into 2 classes: observations of rhythmicity in the organism when transcription of clock genes is held constant, and rhythmicity in the organism when clock gene function is missing in knockout mutants. It is concluded that the weight of anomalies is now so large that the standard transcription/translation mechanism is no longer an adequate model for circadian oscillators. Rhythmic transcription may have other functions in the circadian system, such as participating in input and output pathways and providing robustness to the oscillations. It may be most useful to think in terms of a circadian system that uses a noncircadian oscillator consisting of metabolic feedback loops, which acquires its circadian properties from additional regulatory molecules such as the products of canonical clock genes.

Amplitude Model for the Effects of Mutations and Temperature on Period and Phase Resetting of the Neurospora Circadian Oscillator

This paper analyzes published and unpublished data on phase resetting of the circadian oscillator in the fungus Neurospora crassa and demonstrates a correlation between period and resetting behavior in several mutants with altered periods: As the period increases, the apparent sensitivity to resetting by light and by cycloheximide decreases. Sensitivity to resetting by temperature pulses may also decrease. We suggest that these mutations affect the amplitude of the oscillator and that a change in amplitude is responsible for the observed changes in both period and resetting by several stimuli. As a secondary hypothesis, we propose that temperature compensation of period in Neurospora can be explained by changes in amplitude: As temperature increases, the compensation mechanism may increase the amplitude of the oscillator to maintain a constant period. A number of testable predictions arising from these two hypotheses are discussed. To demonstrate these hypotheses, a mathematical model of a time-delay oscillator is presented in which both period and amplitude can be increased by a change in a single parameter. The model exhibits the predicted resetting behavior: With a standard perturbation, a smaller amplitude produces type 0 resetting and a larger amplitude produces type 1 resetting. Correlations between period, amplitude, and resetting can also be demonstrated in other types of oscillators. Examples of correlated changes in period and resetting behavior in Drosophila and hamsters raise the possibility that amplitude changes are a general phenomenon in circadian oscillators.

Choline Depletion, frq Mutations, and Temperature Compensation of the Circadian Rhythm in Neurospora crassa

In the fungus Neurospora crassa, the chol-1 mutation blocks the synthesis of the lipid phosphatidylcholine and also lengthens the period of the circadian rhythm of conidiation under conditions of choline depletion. The frq mutations, which have no known metabolic defect, affect both the period of the rhythm and temperature compensation. In this article, the chol-1 mutant strain has been further characterized with respect to its temperature compensation and its interactions with frq. Choline depletion of chol-1 abolishes good temperature compensation: Low temperatures lengthen the period under choline-depleted conditions, and low choline lengthens the period at any one temperature. Double-mutant strains carrying both chol-1 and one of a series of frq alleles demonstrate interactions between chol-1 and frq: On high choline, the periods of the double mutants are identical to the corresponding chol + strains, whereas on low choline all double mutants display very long periods (greater than 50 h). Short-period frq mutations shorten the long period on low choline, whereas long-period frq mutations have no effect. A null frq mutation in the chol-1 background is arrhythmic on high choline but is robustly rhythmic on low choline and has no effect on the long period. The interactions between frq and chol-1 are similar to the interactions between frq and cel, another lipid-deficient mutant. These results provide support for the hypothesis that membrane lipids may be involved in temperature compensation of the circadian rhythm. The possibility is discussed that the frq gene may not be required for circadian rhythmicity under some conditions and therefore may not be a central component of the circadian oscillator but rather a component of an input pathway.

The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes: Effects of thyroid status and fatty acids

In less than 1 min ouabain maximally inhibits oxygen consumption due to gramicidin-induced ATP turnover by the Na+/K+-ATPase in hepatocytes. Ouabain rapidly inhibits respiration on palmitate or glucose by only 6-10% indicating that the Na+/K+-ATPase plays a minor role in cell ATP turnover. 29% of the extra oxygen consumption of hepatocytes isolated from hyperthyroid rats was inhibited by ouabain showing that the Na+/K+-ATPase is responsible for some but not the majority of the stimulation of respiration induced by thyroid hormone.

A beginnner's guide to limit cycles, their uses and abuses

Mathematical models of oscillators fall into two major categories, simple (one‐dimensional) and non‐simple (two‐or‐more dimensional). The type of model used to describe a rhythmic system will influ...

Rhythmic Conidiation in Constant Light in Vivid Mutants of Neurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2–3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).

The Genetics of Circadian Rhythms in Neurospora

This chapter describes our current understanding of the genetics of the Neurospora clock and summarizes the important findings in this area in the past decade. Neurospora is the most intensively studied clock system, and the reasons for this are listed. A discussion of the genetic interactions between clock mutants is included, highlighting the utility of dissecting complex mechanisms by genetic means. The molecular details of the Neurospora circadian clock mechanism are described, as well as the mutations that affect the key clock proteins, FRQ, WC-1, and WC-2, with an emphasis on the roles of protein phosphorylation. Studies on additional genes affecting clock properties are described and place these genes into two categories: those that affect the FRQ/WCC oscillator and those that do not. A discussion of temperature compensation and the mutants affecting this property is included. A section is devoted to the observations pertinent to the existence of other oscillators in this organism with respect to their properties, their effects, and their preliminary characterization. The output of the clock and the control of clock-controlled genes are discussed, emphasizing the phasing of these genes and the layers of control. In conclusion, the authors provide an outlook summarizing their suggestions for areas that would be fruitful for further exploration.

Temperature compensation and membrane composition in Neurospora crassa.

The link between temperature compensation of the circadian rhythm and temperature-induced adjustment of membrane composition in Neurospora crassa is briefly reviewed. In common with most organisms, Neurospora responds to changes in growth temperature by adjusting its lipid composition, primarily by increasing the degree of unsaturation of its fatty acids at low temperature. This may result in maintenance of either membrane fluidity or phase transition behavior over a range of temperatures. In Neurospora, there are three mutations (frq, cel, and chol-1) that affect temperature compensation of the circadian rhythm; cel and chol-1 are defective in lipid synthesis, and frq interacts with the other two in double-mutant strains. This suggests that lipid metabolism may play a role in temperature compensation of the rhythm, and that the FRQ gene product may also be involved in membrane function, either in regulating lipid composition or as a sensor responding to changes in lipid composition.

A new mutation affecting FRQ-less rhythms in the circadian system of Neurospora crassa.

We are using the fungus Neurospora crassa as a model organism to study the circadian system of eukaryotes. Although the FRQ/WCC feedback loop is said to be central to the circadian system in Neurospora, rhythms can still be seen under many conditions in FRQ-less (frq knockout) strains. To try to identify components of the FRQ-less oscillator (FLO), we carried out a mutagenesis screen in a FRQ-less strain and selected colonies with altered conidiation (spore-formation) rhythms. A mutation we named UV90 affects rhythmicity in both FRQ-less and FRQ-sufficient strains. The UV90 mutation affects FRQ-less rhythms in two conditions: the free-running long-period rhythm in choline-depleted chol-1 strains becomes arrhythmic, and the heat-entrained rhythm in the frq10 knockout is severely altered. In a FRQ-sufficient background, the UV90 mutation causes damping of the free-running conidiation rhythm, reduction of the amplitude of the FRQ protein rhythm, and increased phase-resetting responses to both light and heat pulses, consistent with a decreased amplitude of the circadian oscillator. The UV90 mutation also has small but significant effects on the period of the conidiation rhythm and on growth rate. The wild-type UV90 gene product appears to be required for a functional FLO and for sustained, high-amplitude rhythms in FRQ-sufficient conditions. The UV90 gene product may therefore be a good candidate for a component of the FRQ-less oscillator. These results support a model of the Neurospora circadian system in which the FRQ/WCC feedback loop mutually interacts with a single FLO in an integrated circadian system.

Phase Resetting of the Neurospora crassa Circadian Oscillator: Effects of Inositol Depletion on Sensitivity to Light

The input pathway between the blue-light photoreceptor and the circadian oscillator of Neurospora crassa has not yet been identified. To test the hypothesis that an inositol phospholipid signaling system might be involved in blue-light signal transduction, phase resetting by light was assayed in the inositol-requiring inl strain under conditions of inositol depletion. Phase-resetting curves and dose-response curves indicated that cultures maintained on low inositol (25 μM) were several orders of magnitude more sensitive to light than those maintained on high inositol (250 μM). This difference in light sensitivity was a property of inositol auxotrophy and was not seen in the wild type or in an inositol-independent inl + revertant. Phase resetting by temperature was not affected by inositol depletion, indicating that the effect on light resetting is specific to the light input pathway and is not the result of a change in the amplitude of the oscillator itself. The results indicate an indirect role for inositol metabolites in the light input pathway—one that is not likely to involve direct participation of an inositol phospholipid signal transduction mechanism.