Temperature synchronization of the Drosophila circadian clock protein PERIOD is controlled by the TRPA channel PYREXIA.
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ABSTRACT:
SUBMITTER: Roessingh S
PROVIDER: S-EPMC6602953 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the per s phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.
| S-EPMC9201207 | biostudies-literature
Project description:The Drosophila circadian oscillator relies on a negative transcriptional feedback loop, in which the PERIOD (PER) and TIMELESS (TIM) proteins repress the expression of their own gene by inhibiting the activity of the CLOCK (CLK) and CYCLE (CYC) transcription factors. A series of posttranslational modifications contribute to the oscillations of the PER and TIM proteins but few posttranscriptional mechanisms have been described that affect mRNA stability. Here we report that down-regulation of the POP2 deadenylase, a key component of the CCR4-NOT deadenylation complex, alters behavioral rhythms. Down-regulating POP2 specifically increases TIM protein and tim mRNA but not tim pre-mRNA, supporting a posttranscriptional role. Indeed, reduced POP2 levels induce a lengthening of tim mRNA poly(A) tail. Surprisingly, such effects are lost in per 0 mutants, supporting a PER-dependent inhibition of tim mRNA deadenylation by POP2. We report a deadenylation mechanism that controls the oscillations of a core clock gene transcript.
| S-EPMC6431209 | biostudies-literature
Project description:Circadian clocks in eukaryotes rely on transcriptional feedback loops, in which clock genes repress their own transcription resulting in molecular oscillations with a period of approximately 24 h. In Drosophila, the clock proteins Period (PER) and Timeless (TIM) operate in such a feedback loop, whereby they first accumulate in the cytoplasm of clock cells as a heterodimer. Nuclear translocation of the complex or the individual PER and TIM proteins is followed by repression of per and tim transcription, whereby PER seems to act as the prime repressor. We found that in addition to PER:TIM complexes, functional PER:PER homodimers exist in flies. Specific disruption of PER homodimers results in drastically impaired behavioral and molecular rhythmicity, pointing the biological importance of this clock protein complex. Analysis of PER subcellular distribution and repressor competence in the PER dimer mutant revealed defects in PER nuclear translocation and a disruption of rhythmic period transcription. The striking similarity of these phenotypes with that of reduced CKII activity suggests that the formation or function of the PER dimer is closely linked to this kinase. Our results confirm a previous structural model for PER and provide strong evidence that PER homodimers are important for circadian clock function.
| S-EPMC2671555 | biostudies-literature
Project description:There is increasing evidence that aging is affected by biological (circadian) clocks - the internal mechanisms that coordinate daily changes in gene expression, physiological functions and behavior with external day/night cycles. Recent data suggest that disruption of the mammalian circadian clock results in accelerated aging and increased age-related pathologies such as cancer; however, the links between loss of daily rhythms and aging are not understood. We sought to determine whether disruption of the circadian clock affects lifespan and healthspan in the model organism Drosophila melanogaster. We examined effects of a null mutation in the circadian clock gene period (per(01)) on the fly healthspan by challenging aging flies with short-term oxidative stress (24h hyperoxia) and investigating their response in terms of mortality hazard, levels of oxidative damage, and functional senescence. Exposure to 24h hyperoxia during middle age significantly shortened the life expectancy in per(01) but not in control flies. This homeostatic challenge also led to significantly higher accumulation of oxidative damage in per(01) flies compared to controls. In addition, aging per(01) flies showed accelerated functional decline, such as lower climbing ability and increased neuronal degeneration compared to age-matched controls. Together, these data suggest that impaired stress defense pathways may contribute to accelerated aging in the per mutant. In addition, we show that the expression of per gene declines in old wild type flies, suggesting that the circadian regulatory network becomes impaired with age.
| S-EPMC2815745 | biostudies-literature
Project description:Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes. These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, we have shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. Here, we further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced mRNA and protein levels of the core clock gene period (per), likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.
| S-EPMC6864020 | biostudies-literature
Project description:BackgroundIn addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K(+)) channel known to regulate RMP in Drosophila central neurons.Methodology/principal findingsWe show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DN(1), DN(2) and DN(3)), or in subsets of clock neurons (LNd and DNs or DNs alone) increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.Conclusions/significanceBased on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.
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Project description:The daily light-dark cycles represent a key signal for synchronizing circadian clocks. Both insects and mammals possess dedicated "circadian" photoreceptors but also utilize the visual system for clock resetting. In Drosophila, circadian clock resetting is achieved by the blue-light photoreceptor cryptochrome (CRY), which is expressed within subsets of the brain clock neurons. In addition, rhodopsin-expressing photoreceptor cells contribute to light synchronization. Light resets the molecular clock by CRY-dependent degradation of the clock protein Timeless (TIM), although in specific subsets of key circadian pacemaker neurons, including the small ventral lateral neurons (s-LNvs), TIM and Period (PER) oscillations can be synchronized by light independent of CRY and canonical visual Rhodopsin phototransduction. Here, we show that at least three of the seven Drosophila rhodopsins can utilize an alternative transduction mechanism involving the same α-subunit of the heterotrimeric G protein operating in canonical visual phototransduction (Gq). Surprisingly, in mutants lacking the canonical phospholipase C-β (PLC-β) encoded by the no receptor potential A (norpA) gene, we uncovered a novel transduction pathway using a different PLC-β encoded by the Plc21C gene. This novel pathway is important for behavioral clock resetting to semi-natural light-dark cycles and mediates light-dependent molecular synchronization within the s-LNv clock neurons. The same pathway appears to be responsible for norpA-independent light responses in the compound eye. We show that Rhodopsin 5 (Rh5) and Rh6, present in the R8 subset of retinal photoreceptor cells, drive both the long-term circadian and rapid light responses in the eye.
| S-EPMC5988559 | biostudies-literature
Project description:We measured daily gene expression in heads of control and period mutant Drosophila by using oligonucleotide microarrays. In control flies, 72 genes showed diurnal rhythms in light-dark cycles; 22 of these also oscillated in free-running conditions. The period gene significantly influenced the expression levels of over 600 nonoscillating transcripts. Expression levels of several hundred genes also differed significantly between control flies kept in light-dark versus constant darkness but differed minimally between per(01) flies kept in the same two conditions. Thus, the period-dependent circadian clock regulates only a limited set of rhythmically expressed transcripts. Unexpectedly, period regulates basal and light-regulated gene expression to a very broad extent.
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Project description:Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding.
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Project description:The mechanistic basis of eukaryotic circadian oscillators in model systems as diverse as Neurospora, Drosophila, and mammalian cells is thought to be a transcription-and-translation-based negative feedback loop, wherein progressive and controlled phosphorylation of one or more negative elements ultimately elicits their own proteasome-mediated degradation, thereby releasing negative feedback and determining circadian period length. The Neurospora crassa circadian negative element FREQUENCY (FRQ) exemplifies such proteins; it is progressively phosphorylated at more than 100 sites, and strains bearing alleles of frq with anomalous phosphorylation display abnormal stability of FRQ that is well correlated with altered periods or apparent arrhythmicity. Unexpectedly, we unveiled normal circadian oscillations that reflect the allelic state of frq but that persist in the absence of typical degradation of FRQ. This manifest uncoupling of negative element turnover from circadian period length determination is not consistent with the consensus eukaryotic circadian model.
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