The Case for the Target of Rapamycin Pathway as a Candidate Circadian Oscillator.

The molecular mechanisms that drive circadian (24 h) rhythmicity have been investigated for many decades, but we still do not have a complete picture of eukaryotic circadian systems. Although the transcription/translation feedback loop (TTFL) model has been the primary focus of research, there are many examples of circadian rhythms that persist when TTFLs are not functioning, and we lack any good candidates for the non-TTFL oscillators driving these rhythms. In this hypothesis-driven review, the author brings together several lines of evidence pointing towards the Target of Rapamycin (TOR) signalling pathway as a good candidate for a non-TTFL oscillator.

Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in .

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of "clock genes." Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus continue even when clock genes (, , and ) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology.

Circadian rhythms, metabolic oscillators, and the target of rapamycin (TOR) pathway: the Neurospora connection.

Circadian (24-h) rhythmicity is a fundamental property of eukaryotic cells, and it is not surprising that it intersects with fundamental metabolic processes. Many links between these two processes have been documented, and speculation has been growing that there may be circadian "metabolic oscillators" that interact with and exist independently of the well-known circadian transcription/translation feedback loops (TTFLs) that have been extensively studied. This review takes a critical look at the evidence for the existence of metabolic oscillators at the cellular level, attempting to answer these questions: does metabolism affect circadian rhythmicity, and vice versa? Is metabolism rhythmic, and if so, is that rhythmicity cell autonomous? Systems displaying "non-canonical rhythmicity" in the absence of functional TTFLs provide opportunities for identifying metabolic oscillators, and this review emphasizes the fungus Neurospora crassa as a model system.

A component of the TOR (Target Of Rapamycin) nutrient-sensing pathway plays a role in circadian rhythmicity in Neurospora crassa.

The TOR (Target of Rapamycin) pathway is a highly-conserved signaling pathway in eukaryotes that regulates cellular growth and stress responses. The cellular response to amino acids or carbon sources such as glucose requires anchoring of the TOR kinase complex to the lysosomal/vacuolar membrane by the Ragulator (mammals) or EGO (yeast) protein complex. Here we report a connection between the TOR pathway and circadian (daily) rhythmicity.

PRD-1, a Component of the Circadian System of Neurospora crassa, Is a Member of the DEAD-box RNA Helicase Family.

The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock.

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.

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.

Activation and localization of protein kinase C in Neurospora crassa.

The Neurosporacrassa protein kinase C (NPKC) is reported to be a regulator of light responsive genes. It phosphorylates the light receptor WC-1 and regulates the levels of the circadian clock protein FRQ and transcription of the light-induced albino-2 gene. In mammals, the conventional and novel isoforms of PKC are activated by diacylglycerol (DAG), which induces PKC translocation from the cytoplasm to membranes.

Effects of prd circadian clock mutations on FRQ-less rhythms in Neurospora.

Rhythmic conidiation (spore formation) in Neurospora crassa provides a model system for investigating the molecular mechanisms of circadian rhythmicity. A feedback loop involving the frq, wc-1, and wc-2 gene products (FRQ/ WCC) is an important component of the mechanism; however, rhythmic conidiation can still be observed when these gene products are absent. The nature of the oscillator(s) that drives this FRQ-less rhythmicity (FLO) is an important question in Neurospora circadian biology.

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.

New models for circadian systems in microorganisms.

Microorganisms provide important model systems for studying circadian rhythms, and they are overturning established ideas about the molecular mechanisms of rhythmicity. The transcription/translation feedback model that has been accepted as the basis of circadian clock mechanisms in eukaryotes does not account for old data from the alga Acetabularia demonstrating that transcription is not required for rhythmicity. Moreover, new results showing in vitro rhythmicity of KaiC protein phosphorylation in the cyanobacterium Synechococcus, and rhythmicity in strains of the fungus Neurospora carrying clock gene null mutations, require new ways of looking at circadian systems.

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.

Circadian clock genes frequency and white collar-1 are not essential for entrainment to temperature cycles in Neurospora crassa.

The fungus Neurospora crassa is a model system for investigating the mechanism of circadian rhythmicity, and the core of its circadian oscillator is thought to be a transcription/translation feedback loop involving the products of the frq (frequency), wc-1 (white-collar-1) and wc-2 (white-collar-2) genes. Several reports of rhythmicity in frq and wc null mutants have raised questions about how central the FRQ/WC loop is to the circadian system of Neurospora. Several research groups have attempted to answer this question by looking for entrainment of the conidiation banding rhythm in frq null mutants.

Time-lapse analysis of the circadian rhythms of conidiation and growth rate in neurospora.

The filamentous fungus Neurospora crassa has frequently served as a model organism for the study of circadian rhythms through its ability to form conidial spores on a daily basis. This phenomenon leaves a spatial pattern of conidiation bands along a solid surface of agar after several days of growth. Using time-lapse video, the authors have quantified the rate of conidiation.

Circadian rhythms in microorganisms: new complexities.

Recent advances in understanding circadian (daily) rhythms in the genera Neurospora, Gonyaulax, and Synechococcus are reviewed and new complexities in their circadian systems are described. The previous model, consisting of a unidirectional flow of information from input to oscillator to output, has now expanded to include multiple input pathways, multiple oscillators, multiple outputs; and feedback from oscillator to input and output to oscillator. New posttranscriptional features of the frq/white-collar oscillator (FWC) of Neurospora are described, including protein phosphorylation and degradation, dimerization, and complex formation.

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 Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity.

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.

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.

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.

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.

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.