Circadian rhythms: new functions for old clock genes.

The mechanisms of circadian clocks, which time daily events, are being investigated by characterizing 'clock genes' that affect daily rhythms. The core of the clock mechanism in Drosophila, Neurospora, mammals and cyanobacteria is described by a transcription-translation feedback-loop model. However, problems with this model could indicate that it is time to look at the functions of these genes in a different light. Our a priori assumptions about the nature of circadian clocks might have restricted our search for new mutants in ways that prevent us from finding important clock genes.

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.

Circadian rhythms in Neurospora crassa: interactions between clock mutations.

Mutations at four loci in Neurospora crassa that alter the period of the circadian rhythm have been used to construct a series of double mutant strains in order to detect interactions between these mutations. Strains carrying mutations at three of these loci have altered periods on minimal media: prd-1, several alleles at the olir (oligomycin resistance) locus and four alleles at the frq locus. A mutation at the fourth locus, cel, which results in a defect in fatty acid synthesis, also leads to lengthening of the period when the medium is supplemented with linoleic acid (18:2). The cel mutation was crossed into strains carrying the frq, prd-1 and olir mutations, and the periods of the double mutant strains with and without 18:2 supplementation were determined. In addition, data from the literature for other combinations of loci and/or chemical effects on the period have been reanalyzed.--It was found that both prd-1 and olir are epistatic to the effects of 18:2 on cel; in the series of cel frq double mutant strains, the period-lengthening effect of 18:2 is inversely proportional to the period of the frq parent, indicating an interaction between frq and cel; period effects reported in the literature can be described as changes by a fixed ratio or percentage of the period rather than by a fixed number of hours, and the data, therefore, can support a multiplicative as well as an additive model.--Several biochemical interpretations of these interactions are discussed, based on simple chemical kinetics, enzyme inhibition kinetics and the control of flux through metabolic pathways.

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 microM) were several orders of magnitude more sensitive to light than those maintained on high inositol (250 microM). 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.

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 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.

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.

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.

Evidence against a direct role for inositol phosphate metabolism in the circadian oscillator and the blue-light signal transduction pathway in Neurospora crassa.

The inositol-depletion hypothesis proposes that the effects of Li+ on cellular functions are the result of inhibition by Li+ of the inositol monophosphate phosphatase and subsequent depletion of inositol lipids. This mechanism has been proposed to account for the effects of Li+ on the period of the circadian oscillator. Inositol phosphate metabolism has also been proposed as part of the blue-light signal-transduction pathway through which the phase of the circadian oscillator can be reset by light pulses. Four predictions of these two hypotheses have been tested in the fungus Neurospora crassa and all have been found to fail: (1) inositol supplementation does not reverse the effects of Li+ on the period of the circadian rhythm; (2) inositol depletion of an inositol-requiring mutant does not mimic the effects of Li+; (3) depletion of inositol lipids does not inhibit the response to light; and (4) a phase-resetting pulse of light does not increase the levels of inositol phosphates, including Ins(1,4,5)P3.

Effects of inositol starvation on the levels of inositol phosphates and inositol lipids in Neurospora crassa.

An inositol-requiring strain of Neurospora crassa was labelled during growth in liquid medium with [3H]inositol, and the levels of inositol phosphates and phosphoinositides were determined under inositol-sufficient and inositol-starved conditions. Because the mutant has an absolute requirement for inositol, the total mass of inositol-containing compounds could be determined. Inositol-containing lipids were identified by deacylation and co-migration with standards on h.p.l.c.; PtdIns3P, PtdIns4P, and PtdIns(4,5)P2 were found in approximately equal amounts, in addition to large amounts of PtdIns. Inositol starvation decreased the level of PtdIns to 10% of the sufficient level, and decreased the levels of the other phosphoinositides to about 25%. A number of inositol phosphates were found, including several InsP3s, InsP4s and InsP5s and phytic acid. Ins(1,4,5)P3 was identified by co-migration with standards on h.p.l.c. and by digestion with inositol phosphomonoesterase. High concentrations of all inositol phosphates were found in the extracellular medium in inositol-starved cultures. Inositol starvation on both liquid and solid agar media decreased the intracellular levels of some inositol phosphates, but increased the levels of phytic acid and several other inositol phosphates which may be its precursors and/or breakdown products. These results may indicate that inositol starvation induces phytic acid synthesis as a protection against the free-radical production and lipid peroxidation characteristic of inositol-less death.

Circadian rhythms in Neurospora crassa: lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants.

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq(9) and frq(10)) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq(9), chol-1 frq(10), chol-1 wc-1, chol-1 wc-2, cel frq(9), cel frq(10), and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.

sn-1,2-diacylglycerol levels in the fungus Neurospora crassa display circadian rhythmicity.

The fungus Neurospora crassa is a model organism for investigating the biochemical mechanism of circadian (daily) rhythmicity. When a choline-requiring strain (chol-1) is depleted of choline, the period of the conidiation rhythm lengthens. We have found that the levels of sn-1,2-diacylglycerol (DAG) increase in proportion to the increase in period. Other clock mutations that change the period do not affect the levels of DAG. Membrane-permeant DAGs and inhibitors of DAG kinase were found to further lengthen the period of choline-depleted cultures. The level of DAG oscillates with a period comparable to the rhythm of conidiation in wild-type strains, choline-depleted cultures, and frq mutants, including a null frq strain. The DAG rhythm is present at the growing margin and also persists in older areas that have completed development. The phase of the DAG rhythm can be set by the light-to-dark transition, but the level of DAG is not immediately affected by light. Our results indicate that rhythms in DAG levels in Neurospora are driven by a light-sensitive circadian oscillator that does not require the frq gene product. High levels of DAG may feed back on that oscillator to lengthen its period.

A pantothenate derivative is covalently bound to mitochondrial proteins in Neurospora crassa.

The presence of protein-bound pantothenate in Neurospora crassa was investigated by labelling a pantothenate auxotroph (pan-2) with [14C]pantothenate and examining mycelial homogenates on dodecyl sulfate/polyacrylamide gels. Five peaks of radioactivity were found, with apparent molecular masses of 200, 140, 22, 19, and 9 kDa. The 200-kDa peak was identified as fatty acid synthetase, based on its absence in a fatty acid synthetase mutant. The 22-kDa and 19-kDa peaks co-purified with mitochondrial markers on sucrose gradients. When purified mitochondria were fractionated, the 19-kDa protein was associated with the inner membrane and the 22-kDa protein was enriched in the soluble mitochondrial fraction. The label was quantitatively recovered from the mitochondrial proteins as 4'-phosphopantetheine after mild alkaline hydrolysis. Although the function of this post-translational modification of mitochondrial proteins is not known, several possibilities are discussed: the 4'-phosphopantetheine may act as a carrier group in an enzymatic reaction, or it may perform a regulatory function as part of an enzyme complex.

Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein.

When Neurospora crassa was labeled with [14C]pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of Mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. The 19-kDa band was converted to the 22-kDa band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral pH, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral pH. Mitochondrial subfractionation indicated that the 22-kDa form was preferentially associated with the soluble fraction while the 19-kDa form was found in all fractions. Several properties of the mitochondrial protein were similar to the Escherichia coli acyl carrier protein: Mr on sodium dodecyl sulfate gels, decreased electrophoretic mobility under deacylating conditions, isoelectric point, and covalent attachment of 4'-phosphopantetheine. The 19- and 22-kDa bands may therefore represent acylated and deacylated forms of a mitochondrial acyl carrier protein.

Mitogenic stimulation transiently increases the exchangeable mitochondrial calcium pool in rat thymocytes.

Exchangeable calcium pools were measured in rat thymocytes by 45Ca labelling and selective depletion of intracellular pools with oligomycin in the presence or absence of rotenone. The mitochondrial pool increased by 150% after 3 min of treatment with the mitogen concanavalin A, and decreased to zero 10 min after mitogen addition. No significant change in the ATP-dependent pool could be detected.

Rhythms of differentiation and diacylglycerol in Neurospora.

Although the fungus Neurospora crassa is a relatively simple lower eukaryote, its circadian system may be more complex than previously thought. In this paper we review evidence suggesting that there may be several output pathways coupled in complex ways to a single oscillator, or that there may be more than one oscillator driving independent output pathways. We have described two new rhythms in Neurospora that are not tightly coupled to the rhythm of conidiation bands that is the standard assay for the state of the Neurospora circadian clock. The first is a rhythm in the timing of differentiation, i.e. the production of aerial hyphae and spores. Large regions of the mycelium differentiate synchronously, as if responding to a spatially widespread signal. This rhythm may be distinct from the timer that sets the determination switch controlling the spatial pattern of conidiation bands. The second new rhythm is an oscillation in the levels of the neutral lipid diacylglycerol (DAG). This rhythm is found in all regions of a colony and is not always in phase with the rhythm of conidiation bands. The DAG rhythm shares some characteristics with the differentiation rhythm and has the potential to act as the signal that induces rhythmic differentiation.

Stimulation of respiration by mitogens in rat thymocytes is independent of mitochondrial calcium.

The role of calcium in the control of respiration by the mitogen concanavalin A (ConA) was investigated in rat thymocytes. ConA induced an increase in both mitochondrial respiration and the mitochondrial calcium pool. The stimulation of respiration was shown to be independent of the increase in mitochondrial calcium: the calcium pool declined after 3 min, whereas the respiration increase was persistent, and was not affected by depletion of the calcium pool or by buffering intracellular Ca2+ transients with quin2. The mitogen phytohaemagglutinin stimulated respiration to the same extent as ConA, but did not increase the mitochondrial calcium pool. In addition, respiration was unaffected by changes in the mitochondrial calcium pool induced by increasing or decreasing extracellular calcium. These results indicate that control of respiration is not located in the Ca2+-sensitive mitochondrial dehydrogenases. The ConA-induced increase in respiration could be blocked by oligomycin, suggesting control by cytoplasmic ATP turnover, and was not associated with detectable changes in NAD(P)H fluorescence, indicating a balance between increased electron transfer and increased supply of reduced substrates.