Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.     

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY.

BACKGROUND: The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS: Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS: This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.     

Serotonin modulates circadian entrainment in Drosophila

Entrainment of the Drosophila circadian clock to light involves the light-induced degradation of the clock protein timeless (TIM). We show here that this entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (d5-HT1B). d5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of d5-HT1B are synergistic with a mutation in the circadian photoreceptor cryptochrome (CRY) and are mediated by SHAGGY (SGG), Drosophila glycogen synthase kinase 3beta (GSK3beta), which phosphorylates TIM. Levels of serotonin are decreased in flies maintained in extended constant darkness, suggesting that modulation of the clock by serotonin may vary under different environmental conditions. These data identify a molecular connection between serotonin signaling and the central clock component TIM and suggest a homeostatic mechanism for the regulation of circadian photosensitivity in Drosophila.     

USP2a protein deubiquitinates and stabilizes the circadian protein CRY1 in response to inflammatory signals

The mammalian circadian clock coordinates various physiological activities with environmental cues to achieve optimal adaptation. The clock manifests oscillations of key clock proteins, which are under dynamic control at multiple post-translational levels. As a major post-translational regulator, the ubiquitination-dependent proteasome degradation system is counterbalanced by a large group of deubiquitin proteases with distinct substrate preference. Until now, whether deubiquitination by ubiquitin-specific proteases can regulate the clock protein stability and circadian pathways remains largely unclear. The mammalian clock protein, cryptochrome 1 (CRY1), is degraded via the FBXL3-mediated ubiquitination pathway, suggesting that it is also likely to be targeted by the deubiquitination pathway. Here, we identified that USP2a, a circadian-controlled deubiquitinating enzyme, interacts with CRY1 and enhances its protein stability via deubiquitination upon serum shock. Depletion of Usp2a by shRNA greatly enhances the ubiquitination of CRY1 and dampens the oscillation amplitude of the CRY1 protein during a circadian cycle. By stabilizing the CRY1 protein, USP2a represses the Per2 promoter activity as well as the endogenous Per2 gene expression. We also demonstrated that USP2a-dependent deubiquitination and stabilization of the CRY1 protein occur in the mouse liver. Interestingly, the pro-inflammatory cytokine, TNF-α, increases the CRY1 protein level and inhibits circadian gene expression in a USP2a-dependent fashion. Therefore, USP2a potentially mediates circadian disruption by suppressing the CRY1 degradation during inflammation.     

Exquisite Light Sensitivity of Drosophila melanogaster Cryptochrome

Drosophila melanogaster shows exquisite light sensitivity for modulation of circadian functions in vivo, yet the activities of the Drosophila circadian photopigment cryptochrome (CRY) have only been observed at high light levels. We studied intensity/duration parameters for light pulse induced circadian phase shifts under dim light conditions in vivo. Flies show far greater light sensitivity than previously appreciated, and show a surprising sensitivity increase with pulse duration, implying a process of photic integration active up to at least 6 hours. The CRY target timeless (TIM) shows dim light dependent degradation in circadian pacemaker neurons that parallels phase shift amplitude, indicating that integration occurs at this step, with the strongest effect in a single identified pacemaker neuron. Our findings indicate that CRY compensates for limited light sensitivity in vivo by photon integration over extraordinarily long times, and point to select circadian pacemaker neurons as having important roles.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

A fly's eye view of circadian entrainment

The Drosophila circadian clock is an ideal model system for teasing out the molecular mechanisms of circadian behavior and the means by which animals synchronize to day-night cycles. The clock that drives behavioral rhythms, located in the lateral neurons in the central brain, consists of a feedback loop of the circadian genes period (per) and timeless (tim). The molecular cycle, roughly 24 h long, is constantly reset by the environment. This review focuses on the main input pathways of the dominant circadian zeitgeber, light. Light acts directly on the clock primarily through cryptochrome (cry), a deep brain blue-light photoreceptor. CRY activation causes rapid TIM degradation, which is a predicted means of resetting the clock both on a daily basis at dawn and on an acute basis following an entraining light pulse during the night hours. In the absence of cry, the clock can still be driven by photic input through the visual system, though the mechanisms underlying this entrainment are unclear. Temperature can also entrain the clock, although the mechanisms by which this occurs are also unclear.     

Cryptochrome Mediates Light-Dependent Magnetosensitivity of Drosophila's Circadian Clock

Since 1960, magnetic fields have been discussed as Zeitgebers for circadian clocks, but the mechanism by which clocks perceive and process magnetic information has remained unknown. Recently, the radical-pair model involving light-activated photoreceptors as magnetic field sensors has gained considerable support, and the blue-light photoreceptor cryptochrome (CRY) has been proposed as a suitable molecule to mediate such magnetosensitivity. Since CRY is expressed in the circadian clock neurons and acts as a critical photoreceptor of Drosophila's clock, we aimed to test the role of CRY in magnetosensitivity of the circadian clock. In response to light, CRY causes slowing of the clock, ultimately leading to arrhythmic behavior. We expected that in the presence of applied magnetic fields, the impact of CRY on clock rhythmicity should be altered. Furthermore, according to the radical-pair hypothesis this response should be dependent on wavelength and on the field strength applied. We tested the effect of applied static magnetic fields on the circadian clock and found that flies exposed to these fields indeed showed enhanced slowing of clock rhythms. This effect was maximal at 300 μT, and reduced at both higher and lower field strengths. Clock response to magnetic fields was present in blue light, but absent under red-light illumination, which does not activate CRY. Furthermore, cry^b and cry^OUT mutants did not show any response, and flies overexpressing CRY in the clock neurons exhibited an enhanced response to the field. We conclude that Drosophila's circadian clock is sensitive to magnetic fields and that this sensitivity depends on light activation of CRY and on the applied field strength, consistent with the radical pair mechanism. CRY is widespread throughout biological systems and has been suggested as receptor for magnetic compass orientation in migratory birds. The present data establish the circadian clock of Drosophila as a model system for CRY-dependent magnetic sensitivity. Furthermore, given that CRY occurs in multiple tissues of Drosophila, including those potentially implicated in fly orientation, future studies may yield insights that could be applicable to the magnetic compass of migratory birds and even to potential magnetic field effects in humans.     

A putative flavin electron transport pathway is differentially utilized in Xenopus CRY1 and CRY2.

Xenopus laevis cryptochromes (xCRYs) can suppress xCLOCK/xBMAL1-mediated activation of a period E box-containing promoter. This suppression is a crucial part of the vertebrate circadian oscillator. Similar to CRYs in other species, as well as to the closely related photolyases, xCRYs have a conserved flavin binding domain. We show here that an intact flavin binding domain is required for normal function. However, it appears that each xCRY may utilize the bound flavin differently. Mutation in any of the three conserved tryptophan residues in the putative electron transport chain inhibits xCRY2b function, while only the mutation in the last of the three tryptophans significantly affects xCRY1 function. Although knockout studies in mice have suggested that CRY1 and CRY2 are not totally redundant, this is the first time that molecular/biochemical differences between CRY1 and CRY2 have been demonstrated. Both CRYs seem to require an intact flavin binding domain, suggesting that electron transport is important in their ability to suppress CLOCK/BMAL1 activation. However, only xCRY2b appears to depend on electron transport through the conserved tryptophan pathway.     

Cryptochrome-positive and -negative clock neurons in Drosophila entrain differentially to light and temperature

The blue-light photoreceptive protein Cryptochrome (CRY) plays an important role in the light synchronization of the Drosophila circadian clock. Previously, we found that among the approximately 150 clock neurons, many but not all neurons express CRY. We speculated that the CRY-positive pacemaker neurons may be especially important for light entrainment, whereas the CRY-negative neurons may be important for other environmental cues, for example, temperature. To investigate this hypothesis, we tested the entrainability of the clock neurons to out-of-phase light and temperature cycles. When light-dark or light-dim light cycles were shifted by 12 h with respect to temperature cycles, behavioral rhythms of wild-type flies were re-entrained by the light cycles. In this condition, we found that TIMELESS (TIM) level was strongly influenced by the temperature cycles in many CRY-negative clock neurons, suggesting that the CRY-negative neurons have higher sensitivity to temperature. Under the same conditions, cry-null mutants entrained to the temperature cycles or very slowly re-entrained to light-dark cycles. Our results suggest that there are 2 types of clock neurons having differential sensitivities to light and temperature, and CRY is a key component for the preferential entrainment to light.     

Arabidopsis cryptochrome 2 completes its posttranslational life cycle in the nucleus

CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light-dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light-dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light-dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light-dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light-dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.     

Distinct light and clock modulation of cytosolic free Ca2+ oscillations and rhythmic CHLOROPHYLL A/B BINDING PROTEIN2 promoter activity in Arabidopsis

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca(2+)](cyt)). To dissect the circadian Ca(2+)-signaling network, we monitored circadian [Ca(2+)](cyt) oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca(2+)](cyt). Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca(2+)](cyt), and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca(2+)](cyt) rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca(2+)](cyt). Oscillations of [Ca(2+)](cyt) and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca(2+)](cyt) and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca(2+)](cyt) and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.