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

ABSTRACT: 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.

SUBMITTER: Lakin-Thomas PL

PROVIDER: S-EPMC1134186 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa.

Project description:A saturating genetic dissection of 'blind' mutants in Neurospora crassa has identified a total of two non-redundant loci (wc-1 and wc-2) each of which is required for blue-light perception/signal transduction. Previously, we demonstrated that WC1 is a putative zinc finger transcription factor able to bind specifically to a light-regulated promoter. Here, we present the cloning and characterization of the wc-2 gene. We demonstrate using mutation analysis and in vitro DNA-binding assays that WC2, the second partner of this light signal transduction system, encodes a functional zinc finger DNA-binding protein with putative PAS dimerization and transcription activation domains. This molecular genetic dissection of the second of two components of this light signal transduction system has enabled us to devise a model whereby WC1 and WC2 are proposed to interact via homologous PAS domains, bind to promoters of light-regulated genes and activate transcription. As such, this study provides the first insight into two co-operating partners in blue-light signal transduction in any organism and describes the molecular tools with which to dissect this enigmatic process.

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Physical association of the WC-1 photoreceptor and the histone acetyltransferase NGF-1 is required for blue light signal transduction in Neurospora crassa.

Project description:In Neurospora crassa and other filamentous fungi, light-dependent-specific phenomena are regulated by transcription factors WC-1 and WC-2. In addition to its transcriptional activity, WC-1 is able to directly sense light stimuli through a LOV sensor domain. Its location in the nucleus and heterodimerization with WC-2, together with the presence of a zinc-finger DNA-binding domain and an environmental sensor domain, all resemble the functional evolutionary architecture adopted by vertebrate nuclear receptors (NRs). Here we describe a scenario in which WC-1 represents a functional orthologue of NRs and acts through association with the chromatin-modifying coactivator NGF-1, which encodes a homologue of the yeast Gcn5p acetyltransferase. To support this view, we show a direct association between WC-1 and NGF-1 that depends on a WC-1 region containing a conserved functional LXXLL motif, a signature previously described as being an exclusive feature of NR/coactivator interaction. Our data suggest that a WC-1/NGF-1 complex is preassembled in the dark on light-inducible promoters and that, after exposure to light stimulation, NGF-1-associated HAT activity leads to histone H3 acetylation and transcriptional activation. Finally, we provide evidence for a NGF-1-independent acetylated form of WC-1. Overall our data indicate that Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli.

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Circadian rhythms synchronize mitosis in Neurospora crassa.

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Direct transcriptional control of a p38 MAPK pathway by the circadian clock in Neurospora crassa.

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Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors.

Project description:Neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. These circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. N. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. We report a novel method for disrupting circadian gene transcription while maintaining light responsiveness in N. crassa when held in a steady metabolic state using bioreactors. The arrhythmic transcription of core circadian genes and downstream clock-controlled genes was observed in constant darkness (DD) as determined by reverse transcription-quantitative PCR (RT-qPCR). Nearly all core circadian clock genes were up-regulated upon exposure to light during 11hr light/dark cycle experiments under identical conditions. Our results demonstrate that the natural timing of the robust circadian clock in N. crassa can be disrupted in the dark when maintained in a consistent metabolic state. Thus, these data lead to a path for the production of industrial scale enzymes in the model system, N. crassa, by removing the endogenous negative feedback regulation by the circadian oscillator.

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Bright to dim oscillatory response of the Neurospora circadian oscillator.

Project description:The fungus Neurospora crassa constitutes an important model system extensively used in chronobiology. Several studies have addressed how environmental cues, such as light, can reset or synchronize a circadian system. By means of an optimized firefly luciferase reporter gene and a controllable lighting system, we show that Neurospora can display molecular circadian rhythms in dim light when cultures receive bright light prior to entering dim light conditions. We refer to this behavior as the "bright to dim oscillatory response" (BDOR). The bright light treatment can be applied up to 76 h prior to dim exposure, and it can be as short as 15 min in duration. We have characterized this response in respect to the duration of the light pulse, the time of the light pulse before dim, the intensity of dim light, and the oscillation dynamics in dim light. Although the molecular mechanism that drives the BDOR remains obscure, these findings suggest that a long-term memory of bright light exists as part of the circadian molecular components. It is important to consider the ecological significance of such dim light responses in respect to how organisms naturally maintain their timing mechanism in moonlight.

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Project description:In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ?FWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.

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Synchronizing stochastic circadian oscillators in single cells of Neurospora crassa.

Project description:The synchronization of stochastic coupled oscillators is a central problem in physics and an emerging problem in biology, particularly in the context of circadian rhythms. Most measurements on the biological clock are made at the macroscopic level of millions of cells. Here measurements are made on the oscillators in single cells of the model fungal system, Neurospora crassa, with droplet microfluidics and the use of a fluorescent recorder hooked up to a promoter on a clock controlled gene-2 (ccg-2). The oscillators of individual cells are stochastic with a period near 21 hours (h), and using a stochastic clock network ensemble fitted by Markov Chain Monte Carlo implemented on general-purpose graphical processing units (or GPGPUs) we estimated that >94% of the variation in ccg-2 expression was stochastic (as opposed to experimental error). To overcome this stochasticity at the macroscopic level, cells must synchronize their oscillators. Using a classic measure of similarity in cell trajectories within droplets, the intraclass correlation (ICC), the synchronization surface ICC is measured on >25,000 cells as a function of the number of neighboring cells within a droplet and of time. The synchronization surface provides evidence that cells communicate, and synchronization varies with genotype.

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A kinetic study of the effects of light on circadian rhythmicity of the frq promoter of Neurospora crassa.

Project description:The role of the frq gene in the Neurospora crassa circadian rhythm has been widely studied, but technical limitations have hindered a thorough analysis of frq circadian expression waveform. Through our experiments, we have shown an improved precision in defining Neurospora's circadian rhythm kinetics using a codon optimized firefly luciferase gene reporter linked to a frq promoter. In vivo examination of this real-time reporter has allowed for a better understanding of the relationship of the light responsive elements of the frq promoter to its circadian feedback components. We provide a detailed phase response curve showing the phase shifts induced by a light pulse applied at different points of the circadian cycle. Using the frq-luc reporter, we have found that a 12-h light:12-h dark cycle (12L:12D) results in a luciferase expression waveform that is more complex and higher in amplitude than that seen in free-running conditions of constant darkness (DD). When using a lighting regime more consistent with solar timing, rather than a square wave pattern, one observes a circadian waveform that is smoother, lower in amplitude, and different in phasing. Using dim light in place of darkness in these experiments also affects the resulting waveform and phasing. Our experiments illustrate Neurospora's circadian kinetics in greater detail than previous methods, providing further insight into the complex underlying biochemical, genetic, and physiological mechanisms underpinning the circadian oscillator.

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Isoform switching facilitates period control in the Neurospora crassa circadian clock.

Project description:A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q(10) of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild-type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic.

| S-EPMC2267733 | biostudies-literature

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