InteractiveFly: GeneBrief

jetlag: Biological Overview | References

BIOLOGICAL OVERVIEW

Organisms ranging from bacteria to humans synchronize their internal clocks to daily cycles of light and dark. Photic entrainment of the Drosophila clock is mediated by proteasomal degradation of the clock protein Timeless. Mutations have been indentified in jetlag (a gene coding for an F-box protein with leucine-rich repeats) that result in reduced light sensitivity of the circadian clock. Mutant flies show rhythmic behavior in constant light, reduced phase shifts in response to light pulses, and reduced light-dependent degradation of Tim. Expression of Jet along with the circadian photoreceptor Cryptochrome (Cry) in cultured S2R cells confers light-dependent degradation onto Tim, thereby reconstituting the acute positive response of the circadian clock to light in a cell culture system. These results suggest that Jet is essential for resetting the clock by transmitting light signals from Cry to Tim (Koh, 2006).

In the course of characterizing rest:activity rhythms of various fly strains, a strain was discovered with anomalous activity patterns in constant light (LL). Whereas wild-type flies became arrhythmic after a day or two in LL, the mutant flies were rhythmic for more than a week. Although the mutants could be entrained to light:dark (LD) cycles, they took longer to be re-entrained to a new schedule than wild-type flies, and so the mutation was named jetlag. The behavior of jet flies in LD and in constant darkness (DD) conditions was normal. These phenotypes are reminiscent of those of cry mutants and suggest a defect in circadian photoreception (Koh, 2006).

Using meiotic recombination and deficiency mapping strategies, the mutation was mapped to a small region containing 18 genes on the left arm of the second chromosome. One of these genes, CG8873, encodes an F-box protein with leucine-rich repeats (LRRs), a putative component of a Skp1/Cullin/F-box (SCF) E3 ubiquitin ligase complex. The coding region of the gene was sequenced in 13 strains, including some wild-type strains, the original mutant strain, and several other strains that did not complement the original mutation for the LL phenotype. In six of the seven mutant strains, a phenylalanine-to-isoleucine substitution was found in a conserved LRR domain. In the remaining mutant strain, there was a serine-to-leucine substitution in an adjacent LRR domain. The two mutations will be referred to as common and rare (c and r), respectively. The two alleles did not complement each other, nor did they complement chromosomal deletions that remove the jet locus (Koh, 2006).

The Jet protein contains an N-terminal F-box domain thought to be involved in binding the Skp1 component of the SCF complex, as well as seven LRRs constituting a protein-protein interaction domain thought to be involved in target recognition. Functions of the mammalian F-box proteins with highest similarity to Jet (F-box and LRR protein 15) have yet to be determined (Koh, 2006).

Almost all (>96%) of the jet^r and jet^c flies had rhythmic behavior in LL, whereas very few of the wild-type control flies did. In contrast, the mutants' behavior was indistinguishable from wild-type behavior in DD, which suggests that the mutants have a largely intact circadian system with a specific defect in the light input pathway. Consistent with its limited role in free-running rhythms, the jet mRNA does not cycle in a circadian fashion. The reduced light sensitivity of jet mutants is similar to that of cry mutants; however, unlike cry mutants, jet mutants showed rhythmic activity of a luciferase reporter for a clock gene, period (per), in DD, which suggests that their peripheral clocks function normally. Because luciferase is assayed in whole flies and therefore reports the activity of multiple peripheral clocks, rhythmic luciferase activity in jet mutants also indicates synchrony among these clocks. Peripheral clocks can be entrained to an LD cycle via Cry-independent pathways, which may account for the synchrony of peripheral clocks in jet mutants. Loss of per-luciferase cycling in cry mutants most likely occurs because Cry, in addition to its role as a circadian photoreceptor, has a role in the regulation of core clock components in the periphery (Koh, 2006).

To characterize the behavioral light sensitivity of jet mutants in more detail, phase shifts were measured in response to brief light pulses at night. jet mutants had significantly reduced phase shifts relative to wild-type control flies. Expression of wild-type Jet from a UAS-jet transgene under the control of a cry- or tim-Gal4 driver partially rescued the mutant phenotype. The increase in phase shifts was greater with tim-Gal4 than with cry-Gal4, probably because the former is a stronger driver. Together with the sequence data described above, the rescue results provide strong evidence that the mutations in the jet locus are responsible for the observed mutant phenotypes (Koh, 2006).

To determine the molecular correlates of the behavioral defects, the changes in TIM levels were examined in central clock neurons after brief light pulses. Light-dependent degradation of Tim was substantially reduced in jet mutants and was restored in rescued flies expressing the UAS-jet transgene, which suggests that the behavioral defects in the mutants are mediated by defects in Tim degradation. Light-dependent Tim degradation was also reduced in head extracts of mutants, implying that Jet facilitates Tim degradation in the peripheral clock in the eye as well (Koh, 2006).

To further explore the role of Jet in Tim degradation, an S2R+ Drosophila embryonic cell line was used. Unlike in the fly, in S2R+ cells, Tim does not degrade in response to light. To test whether Jet is the crucial component missing in these cells, Jet was expressed with the use of a constitutive promoter. The Jet protein had little effect on Tim levels in the dark, but it rapidly reduced Tim levels upon light exposure. This light-induced degradation of Tim required coexpression of Cry and was blocked by a proteasome inhibitor, MG132. Jet did not promote degradation of another core clock protein, PER, demonstrating specificity for its target selection. In addition, light-dependent degradation of Cry did not require Jet, although it was facilitated by Jet in the presence of Tim. The Jet protein itself was also not affected by light in S2R+ cells (Koh, 2006).

One of the mutant versions of the protein (the r allele) was significantly less effective than wild-type Jet at promoting Tim degradation. The r mutation reduced the stability of the Jet protein, which may explain its mutant phenotype. The other mutant allele (c) was also less effective than the wild-type allele, although the difference was not statistically significant. The r mutation is in a residue conserved among insects and mammals, but the c mutation is in a residue conserved only among insects, which may explain the stronger effects of the r allele in both behavioral and molecular assays (Koh, 2006).

Wild-type Jet protein also promoted ubiquitination of Tim in cultured cells. In the presence of wild-type Jet and Cry, a significant increase in Tim ubiquitination was observed after only 10 min of exposure to light. Mutant proteins, especially the r allele, were less effective at ubiquitination of Tim. Consistent with its role as a component of an SCF complex, Jet interacts with SkpA, one of several Skp1 homologs in Drosophila. In addition, Jet physically associates with Tim, and the association is stronger in light than in dark (Koh, 2006).

Flies share many of the core clock components with mammals, but their mechanism for light-induced phase resetting appears to differ. Circadian photoreception in mammals relies on adenosine 3',5'-monophosphate response element–binding protein (CREB)–mediated induction of mPer-1 transcription and does not appear to involve Cry. Fly circadian photoreception resembles that of plants, where Cry functions as a circadian photoreceptor, although the mechanism is somewhat different from that of Drosophila Cry. Notably, the plant F-box protein ZEITLUPE mediates dark-dependent degradation of the clock protein TimING OF CAB EXPRESSION 1 (Koh, 2006).

jet mutants did not show any detectable defects in the free-running rhythm in constant darkness. It is proposed that the Drosophila circadian system uses two separate mechanisms for controlling Tim levels: a clock-controlled one for maintaining rhythm in the dark, and a light-dependent one for entraining the clock to the photic environment. Both mechanisms use SCF complexes but with distinct F-box proteins: SLIMB for the clock-controlled mechanism and Jet for the light-dependent mechanism (Koh, 2006).

This study has identified a component of the Drosophila light entrainment pathway that is critical for light-induced degradation of Tim. Single amino acid substitutions in Jet lead to molecular and behavioral defects in light entrainment. These results suggest the following model of how light resets the clock in Drosophila. Upon light exposure, Cry undergoes conformational change, allowing it to bind Tim. Tim is then modified by phosphorylation, which allows Jet to target Tim for ubiquitination and rapid degradation by the proteasome pathway (Koh, 2006).

Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock

Organisms use the daily cycles of light and darkness to synchronize their internal circadian clocks with the environment. Because they optimize physiological processes and behavior, properly synchronized circadian clocks are thought to be important for the overall fitness. In Drosophila, the circadian clock is synchronized with the natural environment by light-dependent degradation of the clock protein Timeless, mediated by the blue-light photoreceptor Cryptochrome (Cry). This paper report identification of a genetic variant, Veela, which severely disrupts this process, because these genetically altered flies maintain behavioral and molecular rhythmicity under constant-light conditions that usually stop the clock. The Veela strain carries a natural timeless allele (ls-tim), which encodes a less-light-sensitive form of Timeless in combination with a mutant variant of the F-box protein Jetlag. However, neither the ls-tim nor the jetlag genetic variant alone is sufficient to disrupt light input into the central pacemaker. A strong interaction between Veela and cryptochrome genetic variants, demonstrating that the Jetlag, Timeless, and Cry proteins function in the same pathway. Veela also reveals a function for the two natural variants of timeless, which differ in their sensitivity to light. In combination with the complex array of retinal and extraretinal photoreceptors known to signal light to the pacemaker, this previously undescribed molecular component of photic sensitivity mediated by the two Timeless proteins reveals that an unexpectedly rich complexity underlies modulation of this process (Peschel, 2006).

Veela is abnormally rhythmic in constant light, similar to mutations affecting the blue-light photoreceptor Cry. Veela's phenotype is due to the simultaneous presence of the ls-tim allele (encoding a less-sensitive form of Tim) and the jet^c variant encoding a mutant form of the F-box protein Jet. Veela genetically and molecularly interacts with cry^b, indicating that Tim, Jet, and Cry function in the same circadian light-synchronization pathway. These findings show that additional factors are necessary to elicit the phenotypes previously associated with jet variants. In particular, only when jet^c is linked to the ls-tim allele, which encodes a less-light-sensitive form of Tim, can abnormal behavioral rhythmicity in LL be observed. The importance of the Jet protein per se in the light-entrainment process remains unclear, also when considering certain aspects of the original jet study in conjunction with the findings presented in his study. All control flies used by Koh (2006) came from a y w genetic background, which carries the s-tim allele. Contrarily, all jet^c or jet^r mutant flies carried the ls-tim allele (necessarily; otherwise, they would have behaved like WT). It follows that behavioral and molecular differences between control and mutant flies reported by Koh in fact reflect the combined effects of ls-tim (vs. s-tim) and jet^c (vs. jet ⁺). In conjunction with Western blot data showing an increased jet-independent stability of the larger Tim form compared with the smaller one, it seems that the effects on Tim degradation previously attributed to jet variants are mainly a reflection of the different features of the two Tim proteins. This may also explain why Koh saw only very subtle effects of their mutant Jet proteins on Tim degradation in vitro (Peschel, 2006).

Nevertheless, it is clear that jet influences the light-input pathway of the circadian clock; WT flies behave arrhythmically in LL, even though they carry ls-tim. Moreover, Veela strongly interacts with Cry, a crucial protein for circadian light input in flies. Importantly, these findings reveal that, with the current knowledge, an in vivo function for jet's F-box protein can be demonstrated only when the available jet variants are combined with ls-tim. To ultimately resolve the specific function of the Jet protein in the light-input pathway, loss-of-function jet mutants (Debruyne, 2006) or specific RNAi transgenics need to be generated and analyzed chronobiologically (Peschel, 2006).

Characterization of Veela also led to the assignment of a biological function for the two natural tim variants that were identified many years ago. This study has show that Tim encoded by the ls-tim allele is more stable after light exposure, and that this increased stability has behavioral consequences when flies are exposed to constant light; if the ls-tim allele is linked to jet^c, these flies behave abnormally rhythmically in LL. If jet^c is linked to s-tim, the flies behave like WT and become arrhythmic in LL. Therefore, the less-light-sensitive Tim form encoded by ls-tim is necessary and sufficient to block light input into the circadian clock of jet^c flies. In nature, the natural polymorphism at the tim (and perhaps jet) locus might be used to fine-tune the light sensitivity of Drosophila's circadian clock on a purely molecular level. In conjunction with various anatomical light-input routes that are known to send light to Drosophila's circadian pacemaker, these findings reveal a glimpse of the potential complexity of this process. The frequent and random occurrence of tim and jet variants in currently used laboratory strains also speaks to a more cautious strain selection and genotyping in all studies concerning light-input pathways to the circadian clock (Peschel, 2006).

Light-dependent interactions between the Drosophila circadian clock factors Cryptochrome, Jetlag, and Timeless

Circadian clocks regulate daily fluctuations of many physiological and behavioral aspects in life. They are synchronized with the environment via light or temperature cycles. Natural fluctuations of the day length (photoperiod) and temperature necessitate a daily reset of the circadian clock on the molecular level. In Drosophila, the blue-light photoreceptor Cryptochrome (Cry) mediates a rapid light-dependent degradation of the clock protein Timeless (Tim) via the F box protein Jetlag (Jet) and the proteasome, which initiates the resetting of the molecular clock. Cry is also degraded in the light but whereas the degradation of Tim is well characterized, the mechanism for light-dependent degradation of Cry is mostly unknown. Until now it was believed that these two degradation pathways are distinct. This study revealed that Jetlag also interacts with Cry in a light-dependent manner. After illumination, Jetlag induces massive degradation of Cry, which can be prevented in vitro and in vivo by adding Tim as an antagonist. The affinity of Tim for Cry and Jetlag determines the sequential order of Tim and Cry degradation and thus reveal an intimate connection between the light-dependent degradation of these two proteins by the same proteasomal pathway (Peschel, 2009).

Jetlag is involved in the resetting mechanism of the circadian clock. Jet is a putative component of a Skp1/Cullin/F-Box (SCF) E3 ubiquitin ligase complex that associates with Tim in a light-dependent fashion in an embryonic Drosophila cell line (S2) in the presence of Cry. This interaction promotes the ubiquitination and degradation of Tim in cultured cells. In nature, two Drosophila allelic variants of timeless can be found: one allele produces a 23 amino acid N-terminally shortened and more light-sensitive form of Tim (s-tim), the other allele encodes both forms (ls-tim). At the molecular level, S-Tim's enhanced light sensitivity is correlated with (and likely due to) enhanced binding to the circadian blue-light photoreceptor Cry (Peschel, 2009).

The hypomorphic jet^c mutation carries a single amino acid change in the leucine-rich repeat (LRR) region of Jet, which causes flies to be rhythmic in constant light (LL), but only if they express the less light-sensitive L-Tim protein (encoded by the ls-tim allele) as is the case in Veela flies (Peschel, 2006). The LL-rhythmic Veela phenotype resembles that of cry mutants. Also similar to cry^b mutants, homozygous mutant Veela flies accumulate abnormally high levels of Tim protein in the light. Strikingly, both phenotypes are also observed in transheterozygous Veela/+; cry^b/+ flies (Peschel, 2006). This strong genetic interaction between tim, jet, and cry prompted an investigation of a potential physical association between Jetlag and Cry proteins in the yeast two-hybrid system (Y2H). In addition, the two different Timeless isoforms were also tested for interaction with Jetlag or Cryptochrome. In agreement with an earlier study, light-dependent interaction between both Tim proteins and Cry was observed, whereby S-Tim interacted more strongly with Cry as compared to L-Tim. Surprisingly, a striking light-dependent interaction between Cry and Jet was observed, but not between Tim and Jet. Given that Tim and Jet do interact in S2 cells cotransfected with cry and the finding that Jet interacts with Cry in yeast, an explanation for the lack of Tim:Jet binding could be that Cry is essential for this interaction (Peschel, 2009).

The interaction between Jet^c and Cry is significantly weaker compared with the wild-type protein. Keeping in mind that the LRR is the binding region for the F box proteins' substrate, this weaker association was expected. Additionally, Jet and Jet^c were challenged with different Cry mutations. In Cry^Δ the last 20 residues from the C terminus are missing, resulting in strong, light-independent interactions of Cry^Δ with Tim. A strong light-independent interaction was observed between Jet or Jet^c and Cry^Δ. The Cry^b protein does not interact with Jet or Jet^c, correlating with its inability to bind to Tim in yeast (Peschel, 2009).

The strong biochemical and genetic interaction between cry and jet suggests that the Jet:Cry interaction is important in vivo and perhaps required for efficient light-induced Tim turnover. Given that a direct interaction between Jet and Tim was not detected in yeast, this implies that binding of Cry to Tim could modify Tim in a way that it now can bind Jet to induce degradation. Alternatively, the Jet:Cry complex binds to Tim (via Cry acting as a bridge), thereby inducing Tim degradation (Peschel, 2009).

To distinguish between these two possibilities, CoIP experiments were performed in an embryonic Drosophila cell line (S2). A full-length Jetlag protein fused to a HIS-tag (Jet-H) and untagged versions of Cry and Tim proteins were overexpressed in S2 cells and immunoprecipitated with HIS antibody. Cells were grown in darkness and exposed to light for 15 min before performing the assay. As expected from the Y2H results, Cry also interacts with Jet-H in S2 cells. Contrary to the Y2H results, Tim also interacts with Jet-H, without the addition of Cry. When Tim and Cry were simultaneously expressed in the presence of Jet-H, only minimal amounts of Tim protein could be detected in the input or CoIP fractions. It is speculated that the low Tim levels were caused because a fully functional light-sensitive clock-resetting protein complex was reconstituted. To test this, the CoIP experiments were also conducted in the presence of the proteasomal inhibitor MG-132, which led to an overall stabilization of the proteins and a clear demonstration of Tim:Jet interactions in S2 cells. The interaction of Tim with Jet is increased in the presence of Cry, supporting the idea that Cry:Tim or Jet:Cry complexes promote binding of Tim to Jet (Peschel, 2009).

Why could Tim:Jet interactions be detected in S2 cells but not in yeast? The reason for this could be that a crucial phosphorylation step necessary for the detection of Tim by Jet is not performed in yeast, but does occur in Drosophila cells. Alternatively, the low endogenous Cry levels in these cells could promote the Tim:Jet interaction, perhaps contributing to the required posttranslational modification of Tim. Therefore attempts were made to reduce the low endogenous CRY levels even further by cry-dsRNA-mediated interference before conducting the CoIP experiments. dsRNA treatment efficiently reduces transfected Cry levels, indicating that endogenous Cry levels should also be reduced by this treatment. CoIP experiments in the presence of MG-132 show that Jet:Tim interactions are dramatically reduced (but still detectable) after dsRNA treatment. This demonstrates that endogenous Cry levels are supporting Jet:Tim interaction observed in S2 cells (Peschel, 2009).

In cells transfected only with tim, a Jet:Tim interaction was detected, but not a Jet:Cry interaction. Even though the input levels of endogenous Cry and transfected Tim are very low, one would expect to precipitate equal amounts of both proteins bound to Jet, if Cry would indeed form a bridge between Tim and Jet. This was not observed, and only Tim was repeatedly precipitated, indicating the existence of Tim:Jet complexes that are free of Cry. The results therefore support a model in which Cry modifies Tim, allowing Tim to interact with Jet after dissociation of the Cry:Tim complex (Peschel, 2009).

If the Jet:Cry interaction is biologically relevant, an effect on Cry degradation should be detectable in flies with reduced jet function. Indeed, jet^c flies exhibited mildly increased Cry levels after 2 and 11 hr in light. Interestingly, in the light phase, s-tim animals accumulate higher levels of Cry compared to ls-tim flies, both in jet⁺ and jet^c genetic backgrounds. Cry associates stronger with S-Tim compared to L-Tim, and in flies this probably leads to a more efficient light-dependent degradation of S-Tim. This suggests that the affinity of the Cry:Tim interaction dictates the temporal order of Tim and Cry degradation by Jet -- in other words, S-Tim would be preferentially degraded, followed by the turnover of Cry, whereas L-Tim enhances the degradation of Cry because of its lower affinity to this photoreceptor (Peschel, 2009).

Because the differences in Cry degradation caused by jet^c were subtle, it was desirable to confirm this effect by creating a more severe reduction of Jet function. For this, the stronger jet^r allele was combined with jet^c or a deficiency of the jet locus. Both combinations lead to substantially increased Cry levels compared to controls and homozygous jet^c mutants. This unequivocally demonstrates that jet influences Cry stability in flies. It was also noticed that the absence or presence of eye pigments influences the amount of Cry degradation after light exposure, perhaps because the pigments 'protect' Cry from the light (Peschel, 2009).

Although cry is expressed in S2 cells, the endogenous Cry protein is unstable in S2 cells. jet (but not tim) is also expressed in these cells. Endogenous jet expression may explain the previous observation of Tim ubiquitination in S2 cells without cotransfection of Jet. This suggests that the amount of Jet (and) or Cry is limiting for triggering Tim degradation. To test this, S2 cells were first transformed with cry, jet, and s-tim or l-tim. Cells transfected with cry and tim showed little degradation of Tim, regardless of the Tim form present. In contrast, cotransfection of jet led to massive Tim degradation, suggesting that the endogenous Jet amount is limiting. A slight reduction of Tim degradation was also observed after cotransfection of jet^c and the long isoform of Tim, confirming previous results obtained in adult flies (Peschel, 2009).

After establishing conditions that recapitulate light-induced degradation of Tim in cell culture, it became possible to study Cry levels after illumination. Transformation of increasing amounts of jet plasmid DNA is correlated with increased degradation of Cry. This effect is indeed caused by extra jet, because transformation with equal amounts of unrelated plasmid DNA did not result in reduced Cry levels. When the jet^c mutation was used, Cry degradation in the light was reduced but still visible, confirming the results obtained with adult flies. Both effects are possibly caused by the poorer ability of Jet^c to physically interact with Cry (Peschel, 2009).

So far, these results suggest that Tim is preferentially degraded, when both Tim and Cry are present. If true, addition of Tim should stabilize Cry in S2 cells. Indeed, after 10 or 120 min of light exposure, a dramatic 'protection' of Cry by Tim was observed. Cotransfection of Jet restored the light-induced degradation of CRY, at least after 2 hr of light exposure. It is concluded that Tim indeed protects Cry from light-induced degradation, most likely because it is the preferred target of Jet (Peschel, 2009).

To further prove that both proteins are a target of Jet and subsequent proteasomal degradation, the proteasome inhibitor MG-132 was added to cells transfected with Cry and Jet. As previously shown for Tim, light- and Jet-dependent degradation of Cry was largely prevented after adding the drug, suggesting that Tim and Cry are degraded via the same pathway. Similar as in flies, a minor Jet-dependent reduction of Cry levels, which seems independent of light and the proteasome, was observed, indicating that Jet also promotes Cry degradation via a different, light-independent pathway (Peschel, 2009).

An assay was developed that allowed examination of the light-induced degradation of Cry with a higher temporal resolution and in a more quantifiable manner. A constitutively expressed firefly-luciferase cDNA was fused to full-length Cry (Luc-dCry) or to a C-terminal truncated version of Cry (Luc-dCry528). In S2 cells, the fusion protein Luc-dCry is degraded in a similar way as Cry alone, whereas the truncated Luc-dCry528 is expressed at a very low level. After transient transfection of the luc-dCry gene, luminescence was measured in an automated luminescence counter. After illumination, the Luc-dCry protein is swiftly degraded and in darkness Luc-dCry levels recover, demonstrating that the system nicely reflects the light-dependent degradation of Cry. When Jet is added to the cells the fusion protein is degraded even faster -- an effect not observed when Jet^c is added. Lower Luc-dCry levels are observed in the dark portion of the day when Jet is present. Cotransfecting luc-dCry with timeless results in a striking stabilization of Cry in S2 cells, confirming the western blot results. The magnitude of this effect depends both on the isoform and on the total amount of Tim. S-Tim inhibits Luc-dCry degradation more strongly as compared to L-Tim, indicating again that the high-affinity S-Tim:Cry interaction stabilizes Cry more efficiently. Adding Jetlag and Tim at the same time leads to decreased Cry turnover, compared to transfection with Jet alone, but Cry is less protected if Tim is added alone. Overall, these luciferase results nicely confirm the S2-cell and whole-fly western blot results and demonstrate that Jet promotes Cry degradation, which is counteracted by Tim, and especially S-Tim (Peschel, 2009).

Next the Luc-dCry protein was expressed in UAS-luc-dCry transgenic flies under the control of a tim-Gal4 driver. Robust Luc-dCry oscillations, which are due to light-dependent degradation in transgenic flies, was observed, because a sharp decrease of luciferase signals coincides exactly with 'lights-on' in every cycle, and the oscillations immediately stop after transfer to DD. This result is in agreement with light- but not clock-regulated oscillation of the Cry protein in flies. Overexpression of Tim with a UAS-tim transgene led to significantly elevated levels of Luc-dCry during the light phase, which is quite remarkable given that these flies contain the endogenous wild-type allele of jet. Because both transgenic genotypes contained the identical and single copy of the UAS-luc-dCry transgene, this difference in the level of Luc-dCry must be due to the overexpression of Tim. Therefore, the increased daytime Cry levels in the transgenic flies are most likely caused by a stabilization of Cry by Tim, similar to that observed in S2 cells. Also similar as in S2 cells, although the Luc-dCry protein is stabilized by Tim, it can still be degraded by light as long as Jet is present. Interestingly, closer inspection of luc-dCry expression in flies reveals that Cry levels in the UAS-tim flies already recover during the light phase, indicating that Tim mainly protects Cry when light is present. A western blot from flies with the same UAS-tim transgene under the control of a tim-Gal4 driver also reveals a dramatic increase in the levels of Cry and confirms the luciferase results. Both the western blot and real-time luminescence data show that Jet supports the light-dependent degradation of Cry in vitro and in flies and that Tim interferes with this process (Peschel, 2009).

The fact that Tim stabilizes Cry can most easily be explained if Tim is the preferred target for Jet. If true, one would predict that in flies light-induced degradation of both Tim and Cry occurs in sequential order; Tim being degraded ahead of Cry. Therefore Tim and Cry levels were simultaneously measured in head extracts of wild-type flies (y w; s-tim) during the first 10 hr of light in a LD cycle. Although levels of both proteins start to decrease after the lights are turned on, and trough levels are reached at the same time (ZT4), Tim degradation appears to occur more rapid in the early day. This result is in agreement with the idea that Tim is preferentially degraded after initial light exposure. Interestingly, a similar result was reported for Cry and Tim degradation kinetics in adult clock neurons (Peschel, 2009).

Recently, a genome-wide cell-culture-based RNAi screen has been performed in order to identify genes involved in the light-dependent degradation of Cry. Interestingly, Jet was not among the identified candidates. Instead, two other ubiquitin ligases encoded by Bruce and CG17735 were reported to affect light-dependent degradation in flies. The effects reported in this study were caused by eye-color differences between mutants and controls. Therefore, Bruce and CG17735 likely do not contribute to light-dependent Cry degradation in flies, which is also the case for two other ubiquitin ligases that were shown to affect Cry degradation in vitro (Peschel, 2009).

In conclusion, in Drosophila, the clock factor Timeless is degraded after illumination, resulting in a daily reset and adaptation of the circadian clock to its environment. This study has demonstrated that the blue-light photoreceptor Cryptochrome directly interacts with the F box protein Jetlag in a light-dependent manner. This interaction leads to the degradation of Cry by the proteasome and it was unequivocally shown that Jet regulates Cry turnover in vitro and in flies. This is an important and surprising observation, given that so far it was assumed that Cry and Tim are degraded via different pathways. In agreement with previous studies, it was also found that Tim also associates with Jet, but the results suggest that a posttranslational modification of Tim, induced by its binding to Cry, is a prerequisite for the Jet:Tim association. Cry is dramatically stabilized in the presence of Tim, which can be explained by an increased binding affinity of Jet toward light-activated Tim compared to Cry. Based on the results, a more complex model for light resetting is proposed: light induces a conformational change in Cry, allowing it to bind to Tim. S-Tim binds to Cry with higher affinity compared to L-Tim, which leads to more efficient S-Tim degradation by Jet and stabilization of Cry. L-Tim interacts weaker with Cry, presumably resulting in a weaker Jet-L-Tim interaction (or fewer Jet-L-Tim complexes) and less efficient L-Tim degradation. As a result, Cry is less stable in L-Tim flies, because it becomes a better substrate for Jet. Consequently, even less Cry is available to bind to L-Tim, which could further contribute to the reduced light-resetting responses observed in ls-tim flies compared to s-tim flies (Peschel, 2009).

What could be the advantage of such an interdependent binding and degradation of light-regulated clock proteins? The results suggest that Tim and Cry may be degraded in a sequential order. As long as Jet triggers the degradation of Tim, Cry would be spared, presumably because Jet's affinity to light-activated Tim is much higher than to Cry. After Tim levels have decreased to a critical amount, Cry is no longer needed and is now the prime target of Jet. Possibly the degradation of Cry then allows a reaccumulation of Tim in the next circadian cycle, which would also explain why Tim levels start to increase already during the late day (Peschel, 2009).

Search PubMed for articles about Drosophila Jetlag

Debruyne, J. P., et al. (2006). A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron 50: 465-477. PubMed ID: 16675400

Koh, K., Zheng, X. and Sehgal, A. (2006). JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312(5781): 1809-12. 16794082

Peschel, N., Veleri, S. and Stanewsky, R. (2006). Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock. Proc. Natl. Acad. Sci. 103: 17313-17318. PubMed ID: 17068124

Peschel, N., Chen, K. F., Szabo, G. and Stanewsky, R. (2009). Light-dependent interactions between the Drosophila circadian clock factors Cryptochrome, Jetlag, and Timeless. Curr. Biol. 19(3): 241-7. PubMed ID: 19185492

date revised: 25 August 2009

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