Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

Castro-Longoria, Ernestina; Ferry, Mike; Bartnicki-García, S.; Hasty, Jeff; Brody, Stuart

doi:10.1016/j.fgb.2009.12.013

Cited by 29 publications

(59 citation statements)

References 56 publications

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“…19). The advent of fluorescent protein-tagging experiments on the circadian clock in Neurospora (74,75), as well as reporter experiments in mammalian fibroblasts (76), will allow observation of the effect of temperature changes on multiple subprocesses of the circadian clock in a single experiment. Elucidating the biochemical mechanisms of temperature compensation in the circadian clock remains an open problem.…”

Section: Discussionmentioning

confidence: 99%

Temperature compensation and temperature sensation in the circadian clock

Kidd

Young

Siggia

2015

Proc. Natl. Acad. Sci. U.S.A.

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All known circadian clocks have an endogenous period that is remarkably insensitive to temperature, a property known as temperature compensation, while at the same time being readily entrained by a diurnal temperature oscillation. Although temperature compensation and entrainment are defining features of circadian clocks, their mechanisms remain poorly understood. Most models presume that multiple steps in the circadian cycle are temperature-dependent, thus facilitating temperature entrainment, but then insist that the effect of changes around the cycle sums to zero to enforce temperature compensation. An alternative theory proposes that the circadian oscillator evolved from an adaptive temperature sensor: a gene circuit that responds only to temperature changes. This theory implies that temperature changes should linearly rescale the amplitudes of clock component oscillations but leave phase relationships and shapes unchanged. We show using timeless luciferase reporter measurements and Western blots against TIMELESS protein that this prediction is satisfied by the Drosophila circadian clock. We also review evidence for pathways that couple temperature to the circadian clock, and show previously unidentified evidence for coupling between the Drosophila clock and the heat-shock pathway.circadian clock | temperature compensation | mathematical models C ircadian rhythms are daily oscillations in gene expression and protein concentration that regulate sleep (1, 2), metabolism (3,4), and a host of other biological processes (5-8). Circadian oscillations are known to be present in nearly all animals and plants, as well as some fungi and bacteria (9). In all organisms in which circadian oscillations have been observed, circadian oscillations satisfy three defining properties (10). First, circadian oscillations are self-sustained and spontaneously maintain a period of about 24 h. Second, the circadian rhythm is sensitive to light and temperature and can be synchronized to external oscillations in the quantity of either. Third, the period of the circadian oscillation is "temperature-compensated"; that is, the endogenous period of the oscillation is relatively insensitive to temperature.The genetic basis of circadian clocks has been elucidated in considerable detail over the past two decades, particularly in the fruitfly Drosophila melanogaster, the mouse Mus musculus, and the bread mold Neurospora crassa (11). In Drosophila, a self-sustained circadian oscillation in neurons is generated when a pair of proteins, PERIOD (PER) and TIMELESS (TIM), dimerize and, after some delay, translocate into the nucleus, where the proteins repress their own transcription by inactivating a transcription factor dimer composed of the proteins CLOCK and CYCLE. Light sensitivity is mediated by the blue light photoreceptor CRYPTOCHROME (CRY), which upon activation binds TIMELESS and promotes TIMELESS degradation. A remarkably similar mechanism underlies circadian clocks in mouse and Neurospora. The circadian clock of cyanobacteria has also bee...

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Section: Discussionmentioning

confidence: 99%

Temperature compensation and temperature sensation in the circadian clock

Kidd

Young

Siggia

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The transformation vectors for the targeted insertion of ectopic fragments of r + (r ef ) were created by double-joint PCR (DJ-PCR) (Yu et al 2004). DJ-PCR was also used to construct the vectors for tagging the native sad-6 gene at its 59 end with green fluorescent protein (gfp) (Hammond et al 2011b) and the native spo76 gene at its 59 end with mCherryNC (Castro-Longoria et al 2010). A hygromycin resistance marker was used in all transformation vectors (Carroll et al 1994).…”

Section: Transformations and Transformation Vectorsmentioning

confidence: 99%

Efficient Detection of Unpaired DNA Requires a Member of the Rad54-Like Family of Homologous Recombination Proteins

et al. 2014

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Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination. MEIOSIS is fundamental to sexual reproduction. During meiosis, chromosomes are replicated, aligned, recombined, and segregated to nuclei that will develop into gametes. Two of these key processes, alignment and recombination, likely require a search for DNA homology between chromosomes (Barzel and Kupiec 2008;Moore and Shaw 2009). Such homology searching is necessary because sexual organisms inherit a copy of each chromosome from each of its parents. These chromosomes, referred to as homologs, must somehow find each other so that alignment, recombination, and segregation can occur.Although recent research has improved our understanding of homology search mechanisms (Forget and Kowalczykowski 2012;Renkawitz et al. 2013), there are many questions that remain unanswered. The filamentous fungus Neurospora crassa may be useful for investigating the unknowns of homology searching because it possesses a genetically tractable phenomenon called meiotic silencing by unpaired DNA (MSUD) (Aramayo and Selker 2013;Billmyre et al. 2013). MSUD scans pairs of homologs for segments of DNA that are not accurately paired between them. If improper pairing (i.e., unpairing) is identified, the offending sequences are silenced for the duration of sexual development. For example, if a hypothetical gene called "gene A" is on the left arm of one chromosome but on the right arm of its homolog, it will be silenced. The same holds true if gene A has been lost from one of the homologs.A functional MSUD response can be easily detected with alleles that affect ascospore (sexual spore) color or shape. Indeed, MSUD was discovered during studies of ascospore maturation-1 (asm-1), a gene required for the production of pigmented (black) ascospores . A cross between an asm-1 + strain and an asm-1 D strain produces mostly unpigmented (white) ascospores. This is because MSUD silences the unpaired asm-1 + all...

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“…Taken together, we have presented several lines of evidence strongly suggesting that FRQ phosphorylation does not play a major role in regulating FRQ cellular localization. Recently, examination of a fluorescent FRQ-mCherry fusion protein suggested that there are two peaks of FRQ enrichment in the nucleus within a circadian cycle (43). Although it is not clear how the cellular distribution of FRQ-mCherry correlates with its phosphorylation states, this result suggests that regulation of FRQ cellular localization may be complex.…”

Section: (17) As Shown Inmentioning

confidence: 99%

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Cha

Yuan

Liu

2011

Journal of Biological Chemistry

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Eukaryotic circadian clocks employ autoregulatory negative feedback loops to control daily rhythms. In the filamentous fungus Neurospora, FRQ, FRH, WC-1, and WC-2 are the core components of the circadian negative feedback loop. To close the transcription-based negative feedback loop, the FRQ-FRH complex inhibits the activity of the WC complex in the nucleus by promoting the casein kinases-mediated WC phosphorylation. Despite its essential role in the nucleus, most FRQ is found in the cytoplasm. In this study, we mapped the FRQ regions that are important for its cellular localization. We show that the C-terminal part of FRQ, particularly the FRQ-FRH interaction domain, plays a major role in controlling FRQ localization. Both the mutation of the FRQ-FRH interaction domain and the down-regulation of FRH result in the nuclear enrichment of FRQ, suggesting that FRH regulates FRQ localization via a physical interaction. To study the role of FRQ phosphorylation, we examined the FRQ localization in wild-type as well as an array of FRQ kinase, FRQ phosphatase, and FRQ phosphorylation site mutants. Collectively, our results suggest that FRQ phosphorylation does not play a significant role in regulating its cellular localization. Instead, we find that phosphorylation of FRQ inhibits its transcriptional repressor activity in the circadian negative feedback loop. Such an effect is achieved by inhibiting the ability of FRQ to interact with WCC and casein kinase 1a. Our results indicate that the rhythmic FRQ phosphorylation profile observed is an important part of the negative feedback mechanism that drives robust circadian gene expression.The eukaryotic circadian oscillators are comprised of autoregulatory negative feedback loops (1-5). Despite the evolutionary distance between the filamentous fungus Neurospora crassa and higher eukaryotes, their circadian oscillator mechanisms share remarkable similarities (6 -8). In N. crassa, two PAS (PER-ARNT-SIM) domain-containing transcription factors, WC-1 and WC-2, form a complex (WCC) 2 that activates the transcription of the frq gene by binding to its promoter (9 -13). FRQ protein binds FRH to form the FRQ-FRH complex (FFC), which acts as the negative element in the circadian negative feedback loop (14 -17). To close the circadian negative feedback loop, FFC decreases frq mRNA-levels by promoting frq mRNA degradation and by inhibiting frq transcription. To repress frq transcription, FFC inhibits WCC activity by recruiting casein kinase 1a (CK-1a) and CKII to phosphorylate the WC proteins, resulting in a decrease of WCC DNA binding activity and an increase of WCC nuclear export (9, 10, 14 -24). Like the animal PER proteins, FRQ is progressively phosphorylated upon its synthesis and becomes extensively phosphorylated before its disappearance, resulting in robust oscillations of its level and phosphorylation profile (25). Under normal physiological conditions, FRQ is phosphorylated by CK-1a, CKII, and PKA (21,22,(25)(26)(27)(28). On the other hand, FRQ is also dephosphorylated and st...

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Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

Cited by 29 publications

References 56 publications

Temperature compensation and temperature sensation in the circadian clock

Temperature compensation and temperature sensation in the circadian clock

Efficient Detection of Unpaired DNA Requires a Member of the Rad54-Like Family of Homologous Recombination Proteins

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

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