A PER/TIM/DBT Interval Timer for<i>Drosophila</i>'s Circadian Clock

Sáez, Lino; Meyer, Pablo; Young, Michael W.

doi:10.1101/sqb.2007.72.034

Cited by 19 publications

(25 citation statements)

References 46 publications

(71 reference statements)

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“…More recent in vitro experiments using fluorescently labeled clock proteins in individual S2 cells demonstrated that a substantial fraction of DBT localize to the nucleus when over-expressed alone or with TIM (15), consistent with in vivo studies that indicate nuclear localization of DBT in per 01 and per 0 fly heads (8,16). However, when coexpressed with PER, the study showed that DBT is mostly cytoplasmic and the PER-DBT interaction can lead to gradual turnover of PER (15). This preliminary PER-DBT study established that the proteins interact in S2 cells but it did not fully explore the kinetics of the ensuing degradation process.…”

Section: Robust Circadian Oscillations Of the Proteins Period (Per)supporting

confidence: 63%

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Syed

Sáez

Young

2011

Journal of Biological Chemistry

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Robust circadian oscillations of the proteins PERIOD (PER)and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT S ), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT S in cell culture resembles that with wild-type DBT and posits that, in flies DBT S likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation. During a 24-h cycle, levels of PERIOD (PER)2 and TIMELESS (TIM) proteins peak in the late night and begin to diminish thereafter. Genetic and biochemical studies suggest that the kinase Doubletime (DBT), an ortholog of the vertebrate casein kinase 1⑀, modulates phosphorylation and the early morning degradation of PER in core clock neurons (1-4). Degradation of PER occurs through the ubiquitin-proteasome pathway, where the DBT-phosphorylated PER binds the F-box protein SLIMB and is targeted for degradation (5-7). In vivo, DBT physically interacts with PER and is found to be associated with the PER-TIM complex (8) with TIM stabilizing PER by suppressing PER phosphorylation (1, 6).Several mutations of the dbt locus have been isolated and the short-period (dbt S ) and the long-period (dbt L ) alleles are among the best characterized dbt mutants in the literature (1-3, 6, 9). dbt L is a Met-80 3 Ile mutation that causes period lengthening of PER and TIM oscillations and animal behavioral activity, to ϳ27 h. Alterations in PER cyclic profile suggest that the DBT L mutation extends the animal rhythms by reducing the rate of PER phosphorylation. Indeed, in vitro gel shift and phosphorylation experiments indicate DBT L has decreased kinase activity compared with DBT (6, 9, 10), which would likely lead to slower degradation of PER in clock neurons. However, the predicted reduction in PER turnover rate due to the long-mutation on DBT has not been tested. dbt S has a Pro-47 3 Ser mutation that results in shortened PER and TIM oscillation period and fly locomotor activity interval of ϳ18 -20 h. From in vivo studies there is no conclusive evidence as to the mechanism via which the mutation affects period length. In vitro, some studies show a ϳ10% decrease in kinase activity for DBT S when compared with DBT (9, 10), suggesting that the mutant is a hypomorph. In cell culture, gel shift assays report hype...

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Section: Robust Circadian Oscillations Of the Proteins Period (Per)supporting

confidence: 63%

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Syed

Sáez

Young

2011

Journal of Biological Chemistry

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“…Nuclear translocation is a particularly promising avenue for future research. A great deal about the biochemistry of nuclear translocation has been discovered in the last decade (19), and much of this knowledge can be leveraged into experiments on temperature compensation. Additionally, the multifarious network of reversible phosphorylations known to regulate nuclear translocation of the PER-TIM dimer bears an intriguing resemblance to models of temperature compensation in the literature (72,73).…”

Section: Discussionmentioning

confidence: 99%

“…ref. 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.…”

Section: Discussionmentioning

confidence: 99%

“…16 and 17), so generating a 24-h circadian rhythm requires extensive regulation to generate long time delays. In wild-type (WT) Drosophila, the translocation of the PER-TIM dimer into the nucleus takes about 6-8 h (18,19) and relies on a complex set of protein interactions and chemical modifications. The delay between peak expression of per and tim mRNA and protein is also long, about 6 h in constant darkness in WT (see Fig.…”

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confidence: 99%

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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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“…Where the clock proteins are localized within the cell and how this localization may change and contribute to clock function have not been previously addressed. In eukaryotic systems subcellular localization is an important feature of timekeeping and several clock components display rhythms of nuclear localization [25, 26] and even rhythmic formation of sub-nuclear structures [27]. …”

Section: Introductionmentioning

confidence: 99%

Dynamic Localization of the Cyanobacterial Circadian Clock Proteins

et al. 2014

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SUMMARY Background The cyanobacterial circadian clock system has been extensively studied and the structures, interactions and biochemical activities of the central oscillator proteins (KaiA, KaiB and KaiC) have been well elucidated. Despite this rich repository of information, little is known about the distribution of these proteins within the cell. Results Here we report that KaiA and KaiC localize as discrete foci near a single pole of cells in a clock-dependent fashion, with enhanced polar localization observed at night. KaiA localization is dependent on KaiC; consistent with this notion, KaiA and KaiC co-localize with each other as well as with CikA, a key input/output factor previously reported to display unipolar localization. The molecular mechanism that localizes KaiC to the poles is conserved in Escherichia coli, another Gram-negative rod shaped bacterium, suggesting that KaiC localization is not dependent on other clock- or cyanobacterial-specific factors. Moreover, expression of CikA mutant variants that distribute diffusely results in the striking de-localization of KaiC. Conclusions This work shows that the cyanobacterial circadian system undergoes a circadian orchestration of subcellular organization. We propose that the observed spatiotemporal localization pattern represents a novel layer of regulation that contributes to the robustness of the clock by facilitating protein complex formation and synchronizing the clock with environmental stimuli.

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A PER/TIM/DBT Interval Timer forDrosophila's Circadian Clock

Cited by 19 publications

References 46 publications

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Temperature compensation and temperature sensation in the circadian clock

Dynamic Localization of the Cyanobacterial Circadian Clock Proteins

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