Interactive Fly, Drosophila

Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggests involvement of the casein kinase I homolog doubletime (dbt) in the Drosophila circadian pacemaker. Two novel dbt mutants have been characterized. Both cause a lengthening of behavioral period and profoundly alter period (per) and timeless (tim) transcript and protein profiles. The Per profile shows a major difference from the wild-type program only during the morning hours, consistent with a prominent role for Dbt during the Per monomer degradation phase. The transcript profiles are delayed, but there is little effect on the protein accumulation profiles, resulting in the elimination of the characteristic lag between the mRNA and protein profiles. These results and others indicate that light and post-transcriptional regulation play major roles in defining the temporal properties of the protein curves and suggest that this lag is unnecessary for the feedback regulation of per and tim protein on per and tim transcription (Suri, 2000).

Both mutations, when presented in the context of the highly similar yeast casein kinase I HRR25, severely reduce kinase activity on peptide substrates. The long-period phenotypes are likely caused by insufficient Dbt activity, so it takes longer to reach some required level of Per phosphorylation. It is also assumed that both mutants are expressed at a level similar to that of wild-type Dbt (Suri, 2000).

Both dbt^h and Dbt^g/+ have ~29 hr periods and are similar in all other respects, suggesting that the phenotypes are not idiosyncratic features of the mutations but reflect the role of Dbt in the pacemaker. Although the mutant flies entrain to imposed 24 hr photoperiods, the LD locomotor activity patterns indicate that there is no anticipation of the morning or evening light/dark transitions, and the evening activity peak is delayed by several hours into the night. The altered LD patterns are probably a consequence of the longer periods. Indeed, flies that carry per^s as well as dbt^h have a period of ~22.5 hr and manifest robust anticipation of both morning and evening transitions as well as an advanced evening activity peak. Both dbt mutant LD profiles resemble that of the 29 hr period per^l mutant strain, consistent with this altered period notion (Suri, 2000).

The molecular features of the per^l circadian program are difficult to compare with those of wild-type flies, because the mutant rhythms are weak and of low amplitude as well as long period even under 12 hr LD entraining conditions. In contrast, Per and Tim cycling in the long-period dbt mutants is robust. Protein levels are comparable with those in wild-type flies during the night, and levels in the two mutant strains appear even higher than wild-type levels during the daytime. Previous work suggests a role for Dbt-catalyzed phosphorylation in targeting Per for degradation: this probably reflects slower protein turnover during the morning in the dbt mutants. The Tim phosphorylation pattern in the mutants did not show any noticeable difference from the wild-type pattern. These observations suggest that the modest mutant effects on the Tim profiles are indirect, perhaps through a primary effect of the dbt mutants on Per (Suri, 2000).

Per phosphorylation is still readily observable in both mutant lines. In fact, there is a hint that Per is even hyperphosporylated in these strains. Although this might reflect phosphorylation events that never take place in a wild-type background, less active Dbt mutants might be expected to depress the magnitude as well as the kinetics of the temporal phosphorylation program. This suggests that Per might not be a direct Dbt substrate in vivo but is only influenced indirectly, through intermediates that are direct Dbt targets. For example, Dbt may phosphorylate and activate a direct Per kinase or a specific protease. In this context, Per has not yet been shown to be a direct Dbt substrate. It is also possible that Dbt is a functionally relevant but minor Per kinase. In this case, the bulk of the Per mobility shift on SDS-PAGE is a consequence of other kinases. Because Per persists for several hours longer in the mutants than in wild-type flies, the other kinases would continue to function and give rise to even more highly phosphorylated species than are usually observed. These would be an indirect consequence of weak dbt activity and delayed degradation. A final possibility is that the enhanced and delayed Per phosphorylation simply reflects some misregulation of Dbt activity (Suri, 2000).

Careful analysis of the Per and Tim protein profiles in the long-period dbt mutants suggests that Dbt acts in the late night and morning phase of the molecular cycle: the mutants leave the early evening protein profile almost unaltered. This indicates that dbt probably targets nuclear, monomeric Per. It has also been suggested that Dbt acts in the early night to destabilize cytoplasmic Per, thus delaying nuclear entry and repression. The dbt mutants reported here do not significantly change this early night, presumptive cytoplasmic phase of accumulation. It is possible that Dbt prefers free Per over Per complexed to Tim. If free Per is a better substrate, then Dbt mutants should show a greater effect in the late night and early morning, after a large fraction of Tim has disappeared. Alternatively, Dbt might influence only marginally the Per accumulation phase for some other reason. But dbt mutant larvae accumulate high levels of hypophosphorylated Per, which suggests that Dbt is the major Per kinase and strongly influences Per accumulation as well as degradation. There is evidence, however, that much of this Per accumulation occurs in cells and tissues where Per is not normally detectable, making the connection with the normal Per-Tim cycle uncertain (Suri, 2000).

To assess the effect of the dbt mutants on transcription, per and tim mRNA cycling was assayed in wild-type and dbt mutant flies. Both mutant profiles are delayed by 4-5 hr. This is presumably because of the delayed disappearance of Per as well as Tim, which has been suggested to repress per and tim transcription. This relationship is very similar to that previously reported for the per^S mutant strain; in this case, the clock proteins disappear more quickly, leading to an advance in the RNA profiles. The per^S effect is more pronounced on Per than on Tim, consistent with the notion that monomeric Per might be the major transcriptional repressor. In any case, comparable results in the three mutants indicate a solid relationship between the timing of the decline in protein levels and the timing of the subsequent increase in per and tim transcription (Suri, 2000).

Based on these observations, a possible model for Dbt function in the Drosophila pacemaker is presented. In the cytoplasm, normal destabilization of Per delays substantial buildup of Per-Tim complexes and the consequent nuclear transport of the dimeric Per-Tim complex. In the nucleus, Per destabilization relieves repression. In Dbt mutants, Per degradation is much slower. This prolongs repression and delays the per and tim mRNA upswing in the next cycle (Suri, 2000).

There is an impressive relationship between the per and tim RNA profiles in comparison to the evening locomotor activity peak. In all cases, these RNA and locomotor activity begin to increase at approximately the same time, i.e., around ZT7 in the middle of the daytime. Mutants or physiological manipulations that affect the timing of the RNA profiles affect the timing of the evening activity peak in parallel. This fits with the emerging view, from mammalian as well as Drosophila work, that cycling transcription plays an important role in circadian output as well as within the central pacemaker oscillator. A further implication of these relationships is that the protein oscillations from one day affect behavior as well as the RNA profiles on the next one: the morning decline and eventual disappearance of Per and Tim terminate a protein cycle from the previous day, which then causes the subsequent increases in both RNA levels and locomotor activity (Suri, 2000).

In contrast, the delayed Per and Tim disappearance in the mutants has little if any effect on the subsequent protein accumulation phase (ZT13-ZT20) under these standard LD conditions; it is hardly affected, and both proteins peak at approximately the same time as they do in the wild-type flies (ZT19-ZT21). Because of the delayed RNA rise in the mutants, the per and tim RNA accumulation profiles almost coincide with those of the proteins, between ZT15 and ZT21. This indicates that the timing of the RNA rise is insufficient to time the protein rise. The increase in protein levels may reflect protein half-life regulation, which is uncoupled from the underlying mRNA levels, at least under some circumstances (Suri, 2000).

The coincidence of the protein and RNA curves also raises doubts about the importance of the 4-6 hr lag between these two accumulation profiles. The data presented in this study indicate that the lag is dispensable for robust behavioral and molecular oscillations. This is especially relevant for the RNA fluctuations. Despite evidence that at least per mRNA fluctuations may not be necessary for core oscillator function, they normally correlate with other molecular and behavioral circadian fluctuations. Moreover, there are substantial data indicating that Per and Tim feedback regulate these transcriptional oscillations. There is also considerable experimental evidence as well as theoretical models, to suggest that the normal 4-6 hr lag between the RNA and protein curves is essential for generating these robust, high-amplitude transcriptional oscillations. The general view is that the protein accumulation delay gives enough time for transcription to increase substantially, before protein levels have increased sufficiently to inhibit transcription. The presence of robust transcriptional oscillations without the delayed protein accumulation makes this scheme less likely. It redirects focus toward some post-transcriptional delay (e.g., the timing of nuclear entry of the Per-Tim dimer), which is predicted to be functional and important for transcriptional feedback regulation. It is important to note that these conclusions are based on biochemical experiments with whole-head extracts. It is still possible that the mRNA-protein lag may be important in the specific pacemaker neurons of Drosophila (Suri, 2000).

All of these experiments were performed under LD conditions. When the light comes on at ZT24, it causes a rapid decline in Tim levels. In DD conditions, therefore, Tim levels are much higher in the early subjective day, as expected. But a major, unanticipated difference was that the Per and Tim profiles in the dbt mutant flies are profoundly delayed in DD, as evidenced by the late appearance of faster-migrating species. This occurs without a comparable change in the RNA profiles, giving rise to a quasi-normal lag between RNA and protein. The light-mediated advance of the protein curves and the absence of a comparable light reset of the RNA profile reinforce the independent regulation of the accumulation phase of the clock RNAs and proteins: only the RNA profiles are influenced by the declining phase of the protein cycle of the previous day, whereas only the protein profiles appear to be reset by the light entrainment stimulus. The data are therefore consistent with a post-translational route of light entrainment, perhaps mediated by some aspect of the normal light effect on Tim. This presumably contributes to the daily advance of the dbt mutant clock under LD conditions, which counteracts the 5 hr period-lengthening effect that would take place under DD conditions (Suri, 2000).

Further understanding of the role of Dbt in the clock will require experiments that directly address Dbt function and regulation. For example, it is possible that temporal regulation of Dbt activity makes a major contribution to the temporal phosphorylation profile and more generally to the normal timing of the circadian program. Additionally, the extent to which Dbt modifies other pacemaker proteins is not clear. It is possible that these other putative Dbt substrates may also be intimately connected to the pacemaker mechanism. Addressing these issues would provide a much deeper understanding of the role of phosphorylation in the pacemaker (Suri, 2000).

Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype has been attributed to premature nuclear translocation of the Period/Timeless heterodimer. Reducing Sgg/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreases Timeless phosphorylation, and GSK-3ß specifically phosphorylates Timeless in vitro. Overexpression of sgg in vivo converts hypophosphorylated Timeless to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated Timeless of wild-type flies. These results indicate a role for Sgg/GSK-3 in Timeless phosphorylation and in the regulated nuclear translocation of the Period/Timeless heterodimer (Martinek, 2001).

Two independent lines of evidence suggest that sgg regulates the period of molecular cycling primarily through effects on nuclear translocation of the Per/Tim heterodimer: (1) the transition point between delays and advances of the phase response curve, an indicator for nuclear entry of Per/Tim complexes, is advanced by 3 hr in flies overexpressing sgg; (2) nuclear Per is detected ~2 hr earlier in the lateral neurons of larvae overexpressing sgg than in wild-type LNs (Martinek, 2001).

sgg-induced shifts in the timing of nuclear translocation are likely to reflect changes in Tim phosphorylation that are in turn connected to altered levels of Per and Tim. Because Per and Tim are overproduced when sgg activity is low, it is suggested that sgg-dependent Tim phosphorylation accelerates Per/Tim heterodimerization or directly promotes nuclear translocation of Per/Tim complexes in wild-type flies. In this view, decreased Tim phosphorylation in sgg mutants would tend to retard nuclear transfer, and so require higher concentrations of the Per and Tim proteins at times of nuclear entry (Martinek, 2001).

Tim can be directly phosphorylated by GSK-3ß in vitro. Such experiments suggest a mechanism involving direct interaction of Sgg/GSK-3 and Tim in vivo, but do not exclude indirect regulation of Tim phosphorylation by this enzyme in the fly. Nor do these results rule out the involvement of additional protein kinases. For example, a tyrosine-linked phosphorylation of Tim has been implicated in the degradation of Tim by the proteasome. Because Sgg would not be expected to promote tyrosine phosphorylation, this kinase should not regulate all aspects of Tim function (Martinek, 2001).

Sgg/GSK-3 is well known for its central role in Wingless/Wnt signaling. Surprisingly, recent work has indicated that the vertebrate ortholog of Double-time, casein kinase Iepsilon, may also participate in this developmental pathway. For example, in Xenopus, inhibition of casein kinase Iepsilon produces developmental abnormalities closely corresponding to a loss of Wnt function. Casein kinase Iepsilon stabilizes ß-catenin and binds and phosphorylates Dishevelled, both established components of the Wnt signal transduction pathway. It is remarkable that two kinases that function together to provide specific developmental regulation may both act as controlling elements in a patently unrelated behavioral process. This could reflect an underlying synergism between Sgg/GSK-3 and casein kinase 1epsilon. Certainly the activities of both kinases must be integrated at some level for coherent transduction of Wnt signals. Because Dbt and Sgg appear to produce opposing effects on Per/Tim nuclear transfer, with Dbt retarding transfer and Sgg accelerating the process, the relative activities of these kinases could establish an important focus for stabilizing the period of Drosophila's circadian rhythms. For example, a control point composed of offsetting kinase activities might contribute to such homeostatic mechanisms as temperature compensation of the clock. In preliminary work, the effects on circadian rhythmicity of two other elements of the wg signal transduction pathway were examined. A temperature-sensitive allele of wg fails to show any effect on rhythmic locomotor activity, and a heat shock-dishevelled-rescued dsh mutant produces no circadian abnormalities. Thus, sgg's participation in the circadian oscillator may be unrelated to its function in wg signaling (Martinek, 2001).

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the Period protein. Whereas the period, timeless, and Clock genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and Dbt protein are constitutively expressed. Robust circadian changes in Dbt subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include Per, Tim, and Dbt, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of Per is strongly enhanced when Tim is removed from Per/Tim/Ddt complexes. The varying associations of Per, Ddt and Tim appear to determine the onset and duration of nuclear Per function within the Drosophila clock (Kloss, 2001).

Dbt RNA levels are constant throughout the day. In this respect, the same is true for Dbt protein levels, since there was no detectable circadian oscillation of Dbt accumulation in timed head extracts. Furthermore, a variety of mutations disrupting the circadian clock and molecular oscillations have no effect on the level of Dbt protein. Thus, production of Dbt protein is not under the control of clock genes. In contrast, the subcellular localization of Dbt in the lateral neurons and photoreceptor cells changes over the course of a daily cycle. Dbt is consistently detected in the nucleus. However, at the end of the day and in the early part of the night, a substantial increase is found in cytoplasmic Dbt, coincident with the cytoplasmic accumulation of Per proteins and Per/Tim complexes. Furthermore, when Per/Tim complexes translocate to the nucleus at ~ZT18, and early during the day when Per remains in the nucleus in absence of Tim, a substantial nuclear accumulation of Dbt is observed. These changes in subcellular location of Dbt appear to be influenced exclusively by the locus of Per accumulation (in the presence or absence of Tim). Tim protein has little or no effect on the localization of Dbt because Dbt is always detected in the nucleus in per⁰¹ flies, which lack Per and have a substantial amount of Tim in the cytoplasm. Consequently, there is circadian regulation of Dbt proteins, in the form of a changing subcellular distribution. The fact that the movement of Per and Tim from the cytoplasm to the nucleus predicts the distribution of Dbt implies a close correspondence between maximum levels of Per/Tim complex and cytoplasmic levels of Dbt. Such a relationship could indicate that Tim associates with cytoplasmic Per once the latter protein has effected cytoplasmic localization of most cellular Dbt (Kloss, 2001).

Because Dbt preferentially accumulates in nuclei in the absence of Per, cytoplasmic Per proteins must affect this default localization at certain times of day in wild-type flies. Although the half-life of Dbt has not been determined, Dbt RNA and proteins are constantly synthesized. Therefore, the subcellular fate of newly translated Dbt may simply depend on whether cytoplasmic Per is available to associate with Dbt and retard its nuclear translocation. Alternatively, accumulation of Dbt may involve mechanisms promoting both nuclear import and export, with the predominant localization of Dbt governed by the presence or absence of cytoplasmic Per. Regardless of the specific mechanism, since Dbt has also been implicated in vital developmental and cellular functions that are not mediated through Per, an important product of any device generating cycling subcellular localization of this kinase could be temporal regulation of its access to alternative substrates (Kloss, 2001).

Dbt has been shown to be a component of the cytoplasmic activity that destabilizes Per. Evidence was also found that Dbt influences the stability of nuclear Per proteins. However, it has been unclear whether Dbt acts in both subcellular compartments, or whether nuclear stability of Per is affected by a Dbt-dependent phosphorylation in the cytoplasm, with delayed effects once Per translocates into the nucleus. This study shows that Dbt proteins are found both in the cytoplasm and in the nucleus. Coupled with the finding that Per proteins are always found associated with Dbt, this suggests that Dbt is required both in the nucleus and in the cytoplasm for Per phosphorylations (Kloss, 2001).

The simultaneous changes in subcellular localization of Per, Tim, and Dbt make it likely that direct physical associations among these proteins cause the cycling Dbt localizations. Per and Dbt proteins can associate in vitro and in cultured cells. Per/Dbt complexes can be recovered at all times during the day from head extracts, regardless of whether the majority of these proteins are localized in the cytoplasm or in the nucleus. Thus, Per proteins are associated with Dbt proteins in vivo when Per is in a Per/Tim complex and when Per proteins are free from Tim (Kloss, 2001).

Conversely, while Dbt binds to Per and Per/Tim complexes, no evidence has been found that Tim protein, free from Per, associates with Dbt in vivo. This finding is in line with the conclusion that Dbt's effects on the circadian clock are primarily mediated through Per (Kloss, 2001).

Extensive efforts have failed to obtain a functional assay for bacterially produced, recombinant Dbt in vitro. The putative kinase domains of Dbt and its mammalian ortholog CKIepsilon are very closely related (86% aa identity), so it was surprising to find that recombinant, mammalian CKIepsilon readily phosphorylates Drosophila Per and human Per in vitro. These observations suggest that Dbt function might be tightly regulated in the fly. It has been established that truncation of mammalian CKIepsilon substantially increases its activity in vitro, and truncated forms of the enzyme were used in the above mentioned Per and hPer assays. Although a corresponding truncation of Dbt failed to generate activity, such studies of mammalian CKIepsilon also indicate more complex regulation for this kinase in vivo (Kloss, 2001).

Without direct kinetic measurements of the activity of Dbt at different times of day, it cannot be determine whether Dbt function is under circadian control. However, it can be asked whether Per phosphorylation in vivo is (1) dependent upon the presence of Dbt and (2) influenced by Tim. In tim^UL flies entrained to LD 12:12, where Per remains complexed with Tim^UL for a prolonged interval in the nucleus, Per remains hypophosphorylated during the dark phase. Because wild-type flies begin to phosphorylate their Per proteins during the dark phase of such LD cycles, the results with tim^UL suggest that Tim influences the timing of light-independent Per phosphorylation (Kloss, 2001).

Light-triggered removal of Tim^UL protein is correlated with a rapid and progressive increase in the level of Per phosphorylation. Because a similar, cytoplasmic association of Per and Dbt in tim⁰¹ flies results in cytoplasmic Per degradation, and such Per degradation requires Dbt, the most parsimonious explanation of these results should be that nuclear association of Per with Tim^UL protects Per from phosphorylation and, secondarily, from turnover. It has been shown that light eliminates Tim, but will not promote Per phosphorylation in a hypomorphic mutant of Dbt (dbt^P). Thus, Per phosphorylation appears to be influenced by the formation of Per/Tim complexes, and only when Per is free from Tim is it subject to phosphorylation by a Dbt-dependent mechanism. While this view is favored, it is also possible that light directly activates elements of a Dbt-dependent mechanism to promote some Per phosphorylations, or that additional factors associate with Per (or Dbt) after Tim is removed by light. Such factors would then be essential for Dbt-regulated phosphorylation of Per (Kloss, 2001).

The following is a model for the accumulation, phosphorylation, and degradation of Per: Dbt-dependent phosphorylation of Per in the cytoplasm is thought to delay the accumulation of Per proteins until lights off. Increasing Tim levels result in stable Per/Tim/Dbt complexes containing hypophosphorylated Per. These complexes are translocated to nuclei, where continued physical association of Tim with Per prolongs the cycle. Subsequently, the formation of Per free from Tim allows the clock to advance by Dbt-dependent phosphorylation of nuclear Per. This phosphorylation could be indirectly controlled by Dbt. The cycle restarts after degradation of phosphorylated nuclear Per proteins. According to this model, Dbt would have opposing effects on the cycle at different times of day and in different subcellular compartments. This regulation would determine the onset and duration of Per's activity in the nucleus, and should therefore be required to establish rhythmicity and set the period of Drosophila's circadian clock (Kloss, 2001).

The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila Cryptochrome (Cry), a putative circadian photoreceptor, interacts 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. Behavioral analysis of double mutants of per and cry suggest a genetic interaction between the two loci. To investigate whether this is reflected in a physical interaction, a yeast-two-hybrid system was employed that revealed a dimerization between Per and Cry. This is further supported by a coimmunoprecipitation assay in tissue culture cells. The light-dependent nuclear interactions of Per and Tim with Cry require the C terminus of Cry and may involve a trans-acting repressor. Thus, as in mammals, Drosophila Cry interacts with Per, and, as in plants, the C terminus of Cry is involved in mediating light responses (Rosato, 2001).

The genetic interaction between per and cry prompted an investigatation of the possibility of a physical interaction between Per and Cry using a yeast-two-hybrid system. A full-length Cry protein, directly fused to LexA (bait), was challenged with Per(233-685) as prey. This fragment includes the major protein/protein interaction domains described for Per. A fragment of Tim(377-915) that is known to bind to Per and contains the relevant regions for Per/Tim dimerization as prey was also tested. No interactions were observed between LexA-Cry and both Per(233-685) and Tim(377-915) fragments in the dark. Cry has been shown to interact with full-length Tim, but not Per, under constant light. In light, LexA-Cry binds strongly to Per(233-685), but not to Tim(377-915). LexA-Cry was also challenged with full-length Per and Tim, both in darkness and light. No interactions were observed in the dark. Under constant light, only full-length Tim showed evidence of dimerization with LexA-Cry. Three conclusions are drawn from these results: Per and Tim interactions with LexA-Cry are light dependent; the N and/or the C terminus of Tim are required for the association with LexA-Cry, and there is an inconsistency between the results obtained from full-length Per and the fragment Per(233-685). In regard to the latter, the well-established Per/Tim interaction was retested using LexA-Tim bait with Per and Per(233-685) preys in darkness and light. No interactions were observed using full-length Per. Subsequent Western blot analysis has revealed that, in this system, full-length Per is poorly expressed, thereby explaining the lack of interactions in yeast with this construct. Nevertheless, a strong interaction between LexA-Cry and Per(233-685) could be demonstrated. This discrepancy between the current results and contradictory published results must reside in the different yeast-two-hybrid systems employed. Evidence was also found for a Tim-independent Cry/Per complex using coimmunoprecipitation (Rosato, 2001).

Cryptochromes are believed to interact with a signaling factor after light exposure, and evidence has been found in plants for a role of the C-terminal domain in signaling. Since the coimmunoprecipitation result supports the view that the interaction between LexA-Cry and Per(233-685) in yeast reflects a meaningful association between Per and Cry, the power of yeast genetics was exploited to test the regulatory role of the C terminus of Drosophila Cry. Twenty residues were deleted from the Cry C terminus to create CryDelta and it was challenged with Per(233-685) and full-length Tim in darkness and light. An interaction was evident in both conditions, with no obvious difference between them. It has been suggested that LexA-Cry^b is unable to interact with Tim in yeast cells because it may have lost its photoresponsiveness. Both LexA-Cry^b and LexA-Cry^bDelta, which are strongly expressed in yeast, are nevertheless unable to interact with Per(233-685) or with Tim. Given the light independence of CryDelta, it is suggested that the D[410]N substitution in Cry^b probably confers a gross structural defect to LexA-Cry^b, rather than simply affecting its photoreceptor ability (Rosato, 2001).

To further map the interaction between Cry and Per, LexA-CryDelta was challenged with several overlapping Per fragments. It was confirmed that LexA-Tim (377-915) interacts with the PAS A domain (Per[233-390]) and Per(233-685). LexA-CryDelta does not associate with Per(233-390), nor with the PAS A + B region (Per[233-485]), but interacts with the downstream C domain (Per[524-685], which includes the per^S site. From these results, it is speculated that Tim and Cry may interact with different regions of the Per protein and, since Cry associates with region(s) of Tim external to the (377-915) fragment, it is hypothesized that Per, Tim, and Cry can be found in the same complex (Rosato, 2001).

LexA-Cry requires light in order to interact with Per(233-685) and Tim. However, it cannot be ruled out a priori that it is the temperature increase, caused by the continuous light exposure, rather than light per se, that triggers Cry's interactions. LexA-Cry was therefore challenged with Per(233-685) and Tim at 37°C in the dark, but no interactions were observed. Furthermore, since LexA-CryDelta does not require light, this variant was used to investigate the effect of temperature on Cry interactions (Rosato, 2001).

Yeast patches were grown on X-gal plates at 30°C and 37°C in parallel. It was noted that at 37°C, the LexA-CryDelta interaction with Per(233-685) is considerably weakened, whereas the control LexA-Tim(377-915)/Per(233-685) dimerization does not show any substantial temperature differences. The same temperature dependence is also observed when LexA-CryDelta is challenged with Tim and Per (524-685) (Rosato, 2001).

Oscillations of the period (per) and timeless (tim) gene products are an integral part of the feedback loop that underlies circadian behavioral rhythms in Drosophila melanogaster. Resetting this loop in response to light requires the putative circadian photoreceptor Cryptochrome (Cry). The early events in photic resetting were dissected by determining the mechanisms underlying the Cry response to light and by investigating the relationship between Cry and the light-induced ubiquitination of the Tim protein. In response to light, Cry is degraded by the proteasome through a mechanism that requires electron transport. Various Cry mutant proteins are not degraded, and this suggests that an intramolecular conversion is required for this light response. Light-induced Tim ubiquitination precedes Cry degradation and is increased when electron transport is blocked. Thus, inhibition of electron transport may 'lock' Cry in an active state by preventing signaling required either to degrade Cry or to convert it to an inactive form. High levels of Cry block Tim ubiquitination, suggesting a mechanism by which light-driven changes in Cry could control Tim ubiquitination (Lin, 2001).

The presence of endogenous Cry in S2 cells supports the idea that the light-dependent Tim ubiquitination is mediated by Cry. To determine if the Tim response to light requires Cry, Tim levels were assayed in light-pulsed and unpulsed cry^b flies. While wild-type flies show the characteristic decrease in Tim levels with light treatment, this response is lacking in cry^b flies (Lin, 2001).

The S2 cell system was used to determine the relationship between light-induced Cry degradation and Tim ubiquitination and degradation. One possibility considered was that Cry is required for Tim stability. In this model, light-induced Cry degradation would lead to Tim degradation, perhaps by exposing relevant sites on Tim to phosphorylation and ubiquitination events. Although Tim and Cry do not bind each other in the dark in the yeast two-hybrid system, they can be coimmunoprecipitated from S2 cells, suggesting that they are present in the same complex. Thus, removal of Cry in response to light could affect Tim processing. Alternatively, light exposure may lead to some conformational and/or redox changes in Cry that trigger downstream events, including Tim ubiquitination and Cry degradation. To distinguish between these two possibilities, the time course of Tim ubiquitination and that of Cry were examined for degradation in S2 cells. An increase in Tim ubiquitination is detected within 5 min of light exposure, while Cry levels in the same extracts remain unchanged up to the end of a 30-min light pulse. Thus, overall degradation of Cry does not appear to be required for Tim ubiquitination. The possibility that Cry is removed from a complex with Tim cannot be excluded: it is more likely that in response to light, Cry transmits a signal that leads to Tim ubiquitination (Lin, 2001).

No degradation of Tim in S2 cells could be detected in response to light. This may be due, in part, to the HA tag on the ubiquitin, which could interfere with proteasomal digestion. However, other researchers have also noted that Tim is not turned over upon light exposure in S2 cells. Extended incubation (up to 6 h post-Tim induction) of transfected cells results in Tim degradation in both dark- and light-treated cells (Lin, 2001).

Tim ubiquitination was examined in the presence of electron transport inhibitor DPI. Tim ubiquitination is increased by DPI, although Cry degradation is blocked, which is consistent with the idea that Tim ubiquitination does not require degradation of Cry. In fact, the increased Tim ubiquitination is most likely due to the accumulation of activated Cry, effected through a block either in degradation or in the reconversion of Cry to an inactive form (Lin, 2001).

Light-induced Tim ubiquitination in S2 cells is thought to be mediated by endogenous Cry. To test the effects of increasing Cry levels on light-induced Tim ubiquitination, S2 cells were cotransfected with hs-tim, hs-Ub, and different concentrations of either pIZ-cry or hs-cry and Tim ubiquitination was assayed 2 h after light exposure (Lin, 2001).

High concentrations of both hs-cry and pIZ-cry decrease Tim ubiquitination. However, ubiquitination of Tim is enhanced when hs-cry is transfected. pIZ-cry does not increase Tim ubiquitination when transfected at low concentrations, most likely because this plasmid yields higher levels of Cry expression. Taken together these observations indicate that small increases in Cry promote Tim ubiquitination after 2 h of light exposure but that high levels attenuate it. However, even in the presence of high levels of Cry, Tim ubiquitination increases during the first 10 to 15 min of light treatment. The block at later time points in Cry-overexpressing cells is indicative of a deficit in the maintenance of Tim ubiquitination, which may be due to enhanced deactivation of Cry (Lin, 2001).

The data on the differential effects of low and high Cry concentrations are supported by results of Cry overexpression in transgenic flies. Flies that overexpress Cry under control of the tim promoter show enhanced resetting, while those that express Cry under the actin 5c promoter show a reduction of light-induced phase delays. The difference in the phenotypes of these two overexpression strains may lie in the level of overexpression. To determine whether the reduced resetting in the actin 5c line correlates with reduced Tim degradation in response to light, flies carrying a UAS-cry construct were crossed to others carrying an actin 5c promoter-GAL4 transgene and the resulting progeny was assayed for Tim expression. Tim expression was examined at different times of day by Western blotting of adult fly head extracts. In Cry overexpression flies Tim levels are considerably higher than wild-type levels at time points early in the day but equivalent to wild-type levels at all other time points. Thus, the effect is specific for the early part of the day, when Tim is normally turned over in response to light (Lin, 2001).

Degradation of Cry by light invokes analogies with the plant photoreceptors, phytochrome (PHY) and Cry, both of which are degraded in response to light. Thus, it may be a common mechanism to control levels of the photoreceptor and thereby the strength of the photic response. Moreover, as noted here for Cry, PHY is known to be degraded by the proteasome (Lin, 2001).

The role of the proteasome in degradation of both Cry and Tim also underscores similarities with the cell cycle. The cell cycle is characterized by cycling proteins that undergo phosphorylation and subsequent degradation, in many cases by the proteasome. Both Per and Tim are cyclically phosphorylated and phosphorylation plays a role in turnover of both proteins. For Tim, light-induced degradation is effected through an increase in phosphorylation and ubiquitination. Thus, as for the cell cycle, multiple proteins in the circadian cycle are turned over by the ubiquitin-proteasome pathway. However, Per turnover may utilize a different pathway since ubiquitination of Per has not been observed (Lin, 2001).

Cry has been shown to block Per and Tim autoregulation of their own RNA synthesis in a light-dependent manner in S2 cells. Since Tim degradation is not detectable in S2 cells, it has been suggested that the inhibition of Tim activity by Cry, rather than its degradation, is the primary response to light. This block in Per-Tim activity may be the immediate response of the clock to light. Presumably this block persists as long as the photic signals are present and Cry is not degraded. However, a phase change of several hours, which can be produced with a pulse of <1 min of light, must require an irreversible change in a clock component. Tim is ubiquitinated in S2 cells within 5 min of light treatment. In flies, Tim degradation (which presumably follows ubiquitination) occurs within 30 to 60 min of light treatment and is apparently critical for resetting the clock. A Cry molecule with a functional flavin-binding domain is required for this response (Lin, 2001).

The F-box protein Slimb controls the levels of clock proteins Period and Timeless

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. The product of the slimb (slmb) gene -- a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation -- is shown to be an essential component of the Drosophila circadian clock. slmb mutants are behaviorally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms (Grima, 2002).

To test whether the SCF-mediated ubiquitin proteasome pathway is involved in the control of Per and Tim oscillations, circadian rhythms were examined of flies defective for genes encoding F-box proteins that are known to target phosphorylated substrates for degradation. The slimb (slmb) gene, which encodes an F-box/WD40 protein regulating transcription factors' levels in the wingless and hedgehog signaling pathways was examined. slmb⁸ mutants that normally die as early larvae were brought to adulthood by providing the slmb gene product throughout development under the control of a heat-shock promoter. The rescued HS-slmb slmb⁸ adult flies, hereafter referred to as slmb^m mutants, were then tested for their locomotor activity rhythms in both light:dark (LD) and constant darkness (DD) conditions. slmb^m mutants were completely arrhythmic in DD, whereas the heterozygous genotype displayed wild-type rhythms. The absence of anatomical defects of the PDF-expressing ventral lateral neurons (LNvs), which control the behavioral rhythms, strongly argues against a developmental origin of the mutants' rhythm defect. Furthermore, targeted slmb expression using well characterized LNvs-specific gal4 drivers restores near wild-type activity rhythms, whereas similarly targeted overexpression in a wild-type background lengthens the circadian period, indicating a cell-autonomous role of the slmb gene in circadian rhythmicity. In LD conditions, slmb^m mutants did not display the light-off anticipation of activity that characterizes a functional clock, whereas it was observed in the flies expressing slmb under the LNvs-specific gal1118 driver. These experiments identify the F-box/WD40 protein Slmb as an essential component of the Drosophila brain clock (Grima, 2002).

To understand how Slmb might affect the circadian oscillator, slmb^m mutants were analyzed for Per and Tim oscillations in the head. In wild-type flies maintained in LD cycles, Per and Tim proteins accumulate and are progressively phosphorylated during night time, with Tim disappearing at the end of the night whereas hyper-phosphorylated Per persists for a few hours in the morning. A similar temporal pattern persists in DD, and is required to sustain behavioral rhythmicity. In contrast, highly phosphorylated Per and Tim are present at all circadian times in slmb^m mutants kept in DD, although low-amplitude oscillations of the hypo-phosphorylated forms indicate a weak residual activity of the molecular clock. In agreement with the persistence of weak protein cycling in slmb^m heads, levels of per and tim transcripts displayed low-amplitude oscillations. Per immunoreactivity was examined in the LNvs that control behavioral rhythms. At circadian time (CT) 0 and CT 12, which correspond to the peak and trough of Per labelling in w flies at 20°C, slmb^m mutants showed low levels of Per immunoreactivity, indicating that the oscillations of the proteins levels are also abolished in the clock cells. To determine whether Slmb acts at the protein level or through a transcriptional control, per was constitutively overexpressed through a transgene. High-molecular-mass Per proteins were observed to accumulate in head extracts of slmb^m but not of wild-type flies carrying GMR-gal4 and UAS-per transgenes that drive strong Per expression in the eye. Altogether, these data indicate that Slmb is involved in the control of phosphorylated Per levels (Grima, 2002).

In LD conditions, Per and Tim degradation in the morning is driven by both the circadian cycle and by light. Light-induced Tim degradation involves ubiquitinylation of the protein, and is blocked by proteasome inhibitors. To test whether Slmb is involved in the light-induced degradation pathway of the clock proteins, Per and Tim levels were assayed in slmb^m flies kept in LD conditions. In contrast to constant darkness, robust oscillations of Per and Tim amounts were observed in LD, with both proteins accumulating during the night and showing a strong day-time decrease. This shows that light-induced Per and Tim degradation does not occur through the same slmb-dependent mechanism as their circadian-cycle-controlled degradation in constant darkness. In addition, the absence of light-off anticipation in the slmb^m activity profiles suggests that the mutants' altered temporal regulation of phosphorylated Per and Tim does not allow rhythmic outputs to be driven, although protein levels clearly cycle (Grima, 2002).

Clock-dependent Per and Tim degradation occurs at the end of the circadian cycle, and relieves the transcriptional repression that the proteins exert on their own genes. Per degradation has also been proposed to take place during the rising phase of the protein levels in the early night, and to be responsible for the shift (of 5 h) between per messenger RNA and Per protein peaks. In order to determine whether Slmb levels vary during a circadian cycle and may therefore affect Per and Tim only during a limited time window, anti-Slmb antibodies were raised and the Slmb protein was followed in head extracts at different circadian times. A strongly reacting protein, as well as a faintly reacting one slightly above, were detected at a relative molecular mass of 45,000 (M_r 45K) in wild-type flies, and did not show any oscillations of their levels over a 24-h time course. Similarly, slmb mRNA did not show any cycling. Slmb therefore appears not to be circadianly regulated, and could therefore act on different steps of the cycle (Grima, 2002).

Both early- and late-night Per degradation steps appear to depend upon Per phosphorylation, which requires the casein kinase I encoded by the double-time (dbt) gene. To find out how Slmb could affect Per and Tim phosphorylation, Tests were performed to see whether Dbt, and Shaggy (Sgg), that has been shown to phosphorylate Tim, are affected in slmb^m mutants. No alterations of the level or the mobility of these kinases were detected in slmb^m head extracts. Next, whether Slmb could associate with the Per protein was examined, by searching for Per-Slmb interactions in co-immunoprecipitation experiments on head extracts. The Slmb protein was found to be co-precipitated by anti-Per antibodies, and anti-Slmb can precipitate Per in wild-type flies collected at CT 0. Similar results were obtained with pooled extracts. In addition, Slmb co-precipitates with Dbt . Because Per, but not Dbt, is profoundly affected in slmb^m mutants, these results support Per rather than Dbt as a Slmb target for ubiquitinylation, and suggest that the three proteins constitute a complex. Slmb was co-immunoprecipitated by anti-Per antibodies in tim⁰ flies, indicating that Per-Slmb complexes can form in the absence of Tim. Although twice as much extract was used for tim⁰ flies to compensate for the low Per levels in this genotype, the amount of immunoprecipitated Slmb suggests that the absence of Tim may favor Per-Slmb complexes. These results fit well with Slmb being involved in the control of unbound Per, either during its cytoplasmic accumulation at the beginning of the protein cycle or during its nuclear degradation at the end. To test whether the formation of Per-Slmb complexes is circadianly controlled, co-immunoprecipitations were performed at the beginning of the night when Per is mostly hypo-phosphorylated, or at the end of the night when Per is highly phosphorylated. All time points showed comparable levels of Per-Slmb complexes, and several forms of Per were immunoprecipitated by the anti-Slmb antibodies (compare CT 1 and CT 13). This indicates that differently phosphorylated Per molecules can be committed to Per-Slmb complexes (Grima, 2002).

Possible explanations for the accumulation of highly phosphorylated Per in slmb^m mutants would be that partially phosphorylated Per is the relevant Slmb substrate for degradation, or that Slmb targets some Per kinase that is bound to Per. The presence of highly phosphorylated Per in slmb^m indicates that Slmb is required for the control of phosphorylated Per accumulation in the early night. Moreover, Slmb overexpression in the LNvs results in a lengthening of the circadian period. In agreement with the behavioral data, Slmb overexpression slows down the oscillations of Per immunoreactivity in these cells, which showed a ~6 hour delay compared to wild-type controls after two days. These data can be explained by high levels of cytoplasmic Slmb inducing too much degradation of cytoplasmic Per, thus further delaying the night accumulation of the protein, whereas high levels of nuclear Slmb would rather precipitate the fall of the Per protein and shorten the circadian period. It is therefore thought that Slmb is, at least, involved in the control of cytoplasmic Per accumulation in the early night (Grima, 2002).

The presence of low-mobility Tim proteins at all circadian times in slmb^m mutants indicates that the accumulation of phosphorylated Tim is also Slmb-dependent. Remarkably, the Tim kinase Sgg controls the Slmb-dependent proteolysis of Cubitus interruptus and degradation of Armadillo. The results suggest that phosphorylated Tim could be a Slmb target or that Tim is phosphorylated by a Slmb-dependent kinase. Because Tim is hypo-phosphorylated in per⁰ flies, it is also possible that the accumulation of hyper-phosphorylated Per in slmb^m influences Tim phosphorylation (Grima, 2002).

Although protein degradation is commonly believed to have a major role in the control of the oscillations of clock proteins, the present work is the first to implicate a characterized component of the ubiquitin proteasome pathway. Because cycling of phosphorylated Per proteins also occurs in the mammalian clock, it would be interesting to determine whether the Slmb mammalian homolog ß-Trcp is involved in the control of phosphorylated Per levels. F-box proteins have been shown to be important at the G1/S transition of the cell cycle, by targeting phosphorylated cyclins and inhibitors of cyclin kinases for degradation by the proteasome. This study therefore suggests that the cell-cycle and the circadian-clock machineries share mechanisms to control the oscillations of phosphorylated proteins (Grima, 2002).

A role for CK2 in the Drosophila circadian oscillator

The posttranslational modification of clock proteins is critical for the function of circadian oscillators. By genetic analysis of a Drosophila melanogaster circadian clock mutant known as Andante, which has abnormally long circadian periods, it has been shown that Casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the ß subunit of CK2 and is predicted to perturb CK2ß subunit dimerization. It is associated with reduced ß subunit levels, indicative of a defect in alpha:ß association and production of the tetrameric alpha2:ß2 holoenzyme. Consistent with a direct action on the clock mechanism, it has been shown that CK2ß is localized within clock neurons and that the clock proteins Period (Per) and Timeless (Tim) accumulate to abnormally high levels in the Andante mutant. Furthermore, the nuclear translocation of Per and Tim is delayed in Andante, and this defect accounts for the long-period phenotype of the mutant. These results suggest a function for CK2-dependent phosphorylation in the molecular oscillator (Akten, 2003).

It is of interest that the Andante mutation affects the nuclear entry of clock proteins in the small LN_v population, but seems to have no effect on the large LN_v neurons. Such a differential effect suggests that CK2 is important for oscillator function in one population but not the other. Indeed, the small LN_v cells have been shown to be critical for the clock regulation of activity rhythms; the small cells send projections to a region of the dorsal brain implicated in clock output, and there is a circadian rhythm in the release of the clock output factor pigment dispersing factor (PDF) in these dorsal projections. Furthermore, it has been reported that the small but not the large LN_v cells show Tim rhythmicity in constant darkness (DD). Finally, there are differences between the large and small LN_v cells, with regard to the timing of Per and Tim nuclear entry. As CK2 deficits seem only to affect nuclear entry in the small LN_v neurons, it is possible that this kinase regulates differences between the two neuronal populations (Akten, 2003).

Although it is likely that CK2 acts within clock cells to help specify period length, these studies indicate neither the cellular compartment in which the kinase acts nor the molecular substrates of the enzyme that are relevant for clock function. A delay in the nuclear accumulation of clock proteins, as seen in Andante, suggests that the kinase functions in the cytoplasm to promote nuclear entry. This function might be mediated by direct phosphorylation of a clock protein (such as Per or Tim) or by activation of a second kinase such as GSK-3/Shaggy, which has been implicated in promoting the nuclear entry of Tim. The elevated levels of Per and Tim observed in Andante suggest a decreased turnover of the clock proteins, and this might arise because of a defect in the targeted degradation of one or both proteins within the nucleus. Similar to the Doubletime kinase, CK2 might act both in the cytoplasm and nucleus of clock cells to determine the timing of nuclear entry and/or stability of clock proteins (Akten, 2003).

These studies of CK2 in Drosophila suggest that this kinase might have an important role in the regulation of circadian period in other animal species. Previous studies in the plant Arabidopsis and the fungus Neurospora have also implicated CK2 activity in circadian oscillator function. The Arabidopsis study showed that overexpression of the CK2ß3 subunit is associated with a shortening of circadian period, a result similar to that obtained in this study of Drosophila CK2ß. The Neurospora study showed abnormal phosphorylation of the Frequency (Frq) protein in a Neurospora CK2alpha mutant, but circadian behavior (such as conidiation rhythms) could not be examined in that study because of reduced viability. Although neither of these previous reports characterize a mutant with decreased CK2 activity, the results are consistent with studies of Andante and indicate an evolutionarily conserved role for CK2 in circadian oscillator function (Akten, 2003).

Sequential nuclear accumulation of the clock proteins Period and Timeless in the pacemaker neurons of Drosophila melanogaster

In Drosophila, two intersecting molecular loops constitute an autoregulatory mechanism that oscillates with a period close to 24 hr. These loops touch when proteins from one loop, Period (Per) and Timeless (Tim), repress the transcription of their parent genes, period and timeless, by blocking positive transcription factors from the other loop. The arrival of Per and Tim into the nucleus of a clock cell marks the timing of this interaction between the two loops; thus, control of Per:Tim nuclear accumulation is a central component of the molecular model of clock function. If a light pulse occurs early in the night as the heterodimer accumulates in the nucleus of clock cells, Tim is degraded, Per is destabilized, and clock time is delayed. Alternatively, if Tim is degraded during the later part of the night, after peak accumulation, clock time advances. Current models state that the effect of a light pulse depends on the state of the Per:Tim oscillation, which turns on the changing levels of Tim. However, previous studies have shown that light:dark (LD) regimes mimicking seasonal changes cause behavioral adjustments while altering clock gene expression. This should be reflected in the adjustment of Per and Tim dynamics. LD cycles were manipulated to assess the effects of altered day length on Per and Tim dynamics in clock cells within the central brain as well as light-induced resetting of locomotor rhythms (Shafer, 2004).

Nuclear accumulation profiles of Per and Tim respond in qualitatively different ways to changes in day length. The pattern of Tim accumulation during the early night is very similar within the range of photoperiods tested here; Tim levels are negligible at lights-off, cytoplasmic levels peaked by about 6 hr later, and obvious nuclear Tim is evident by 8-10 hr after lights-off. During night lengths longer than 10 hr, Tim levels begin to fall by 12 hr after lights-off. Under LD 10:14, 8:16, and 6:18, Tim profiles indicate that expression is not altered by increased night length. Thus, under night lengths longer than 12 hr, nuclear Tim begins to wane in anticipation of dawn. A dramatic exception to this pattern was discovered in very short night conditions. With only 6 hr of darkness, no Tim immunoreactivity is detected in the small LNv's during the night (Shafer, 2004).

Unlike Tim, nuclear accumulation of Per reflects the environmental photoperiod. For instance, whereas Per immunoreactivity reachs peak values by 8 hr after lights-off under LD 8:16, under LD 16:8 peak values are not reached until 10 hr after lights-off, meaning that Per's accumulation extends into the day. Nuclear Per is clearly increased in the small LNv's under short-night conditions. Such compensatory Per accumulation is apparent in all of the photoperiods examined. The net effect of this adjustment is the attainment of similarly low levels of nuclear Per by the end of the day for all the photoperiods tested. Given the suggestion that the decline in Per levels allows the resumption of per and tim transcription, such adjustment of Per levels would ensure that the relief of repression occurs in anticipation of lights-off, under a wide range of LD cycles. Thus, nuclear accumulation profiles of Per and Tim are different both within and between the photoperiodic regimes examined in this study (Shafer, 2004).

The results cast doubt on the requirement of Tim for Per nuclear entry and stability. For example, in LD 16:8 Per levels continue to rise steeply during the 2 hr after lights-on even as Tim levels are falling. This finding differs from another set of observations, that noted the longevity of Per after it had accumulated in a tim mutant background. Moreover, another study has shown that in conditions of constant light, per mRNA and protein continues to be rhythmic, whereas no rhythmicity is evident in the products of timeless. Furthermore, another study suggests that Per is not required for Tim nuclear transport but rather prevents the export of Tim from the nucleus once it has entered. Thus, the current results are inconsistent with the proposal of an obligate heterodimer for nuclear accumulation and with the suggestion of a requirement for stoichiometric amounts of Tim to avoid degradation (Shafer, 2004).

The data suggest that Tim and Per rhythms need not occur in the same cell. Under the 6 hr night condition, small LNv's did not express detectable levels of Tim, yet they show a measurable oscillation in nuclear Per accumulation. The differences observed between cells suggest that the functional role of these molecules depends on their cellular context. Just as timing mechanisms may differ between peripheral and central circadian clocks, so it is possible that data obtained from whole-head homogenates might not apply to the regulation of behavior, especially in light of the fact that most of the relevant proteins assayed in these experiments likely come from the compound eye. In this regard, it is worth noting the recent demonstration that the small and large LNv's could be differentially regulated based on their respective light input pathways (Shafer, 2004 and references therein).

Tim levels are dramatically truncated in the short night lengths, whereas Per is present in the nucleus throughout the day. Tim's sensitivity to night length makes it a better candidate than Per for involvement in photoperiodic time measurement, and it is inferred that Per is the workhorse clock factor for control of day-to-day rhythmicity. Previous studies established that Per function is dispensable for the photoperiodic induction of diapause in Drosophila. The effect of short-night conditions on Tim accumulation is consistent with the hypothesis that this element of the circadian clock is also involved in photoperiodic time measurement (Shafer, 2004).

Studies on the adult eclosion rhythm of Drosophila under a wide range of photoperiod conditions have shown that the timing of the overt rhythm systematically shifts relative to lights-off while the phase of the phase response curve is locked to the lights-off signal. The same relationship is observed for the locomotor rhythm; the evening activity peak shifts markedly relative to lights-off under LD conditions ranging from 16:8 to 6:18. However, throughout this range of photoperiods the setting of the phase response curve is essentially the same. The mechanism by which a change in photoperiod can shift the relationship of the phase response curves and the overt rhythm has not been addressed. The differing responses of Per and Tim to changes in photoperiod represent an explanation for how some aspects of clock function (i.e., responses to phase-shifting light stimuli) remain unchanged in the face of varying day length, whereas others (such as clock gene mRNA levels and cyclical behavior) are adjusted for such environmental changes (Shafer, 2004).

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). This entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (5-HT1B). 5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of 5-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 (Yuan,2005).

Serotonin regulates the entrainment of circadian behavioral rhythms in Drosophila by affecting the molecular response to light. By modulating the expression of the 5-HT1B receptor in clock neurons, a role of this receptor subtype has been established in the regulation of Drosophila circadian photosensitivity. The data also demonstrate that the molecular connection between 5-HT1B signaling and the clock is GSK3β, which directly phosphorylates the central clock component Tim. It is proposed that serotonin signaling is a part of the homeostatic regulation that prevents dramatic fluctuations in the phase of the circadian clock. In addition, given the altered levels of serotonin in extended DD, it may confer selectivity on the response of the clock to light under different environmental conditions (Yuan, 2005).

The expression pattern of 5-HT1B, as determined by both UAS-Gal4 experiments and by immunostaining, provides some clues to its functions in Drosophila. Besides LNvs and SE5HT-IR neurons, major compartments of the fly brain that express the 5-HT1B receptor include the optic lobes, PI neurons, and mushroom bodies. Interestingly, expression in each of these locations is consistent with functions proposed for serotonin signaling in other organisms. In the housefly, the neuropil of the optic lobes undergoes daily structural changes regulated possibly by serotonin and PDF. PI neurons are neurosecretory cells that may also participate in the ocellar phototransduction pathway. The mushroom body is important for olfactory learning and memory in Drosophila. Therefore, in addition to its postsynaptic function in the LNvs, 5-HT1B may be involved in other aspects of physiology and behavior (Yuan, 2005).

The effect of 5-HT1B on Tim was especially pronounced in the small LNvs. One of the differences between the large and small LNvs is in the timing of nuclear entry, which is delayed in the small subgroup. If delayed nuclear entry accounts for the increased resistance of Tim to light in the small LNvs, it would suggest that 5-HT1B signaling largely affects cytoplasmic Tim (Yuan, 2005).

In addition to its effect on the light response, 5-HT1B overexpression influences free-running behavioral rhythms of cry^b flies. It is speculated that this is due to the loss of synchrony among LNs. The mutual coupling of oscillators within an organism is important for the generation and synchronization of circadian rhythms, and serotonin is implicated in this process in some insects. Decreased synchrony may also result from the reduced photosensitivity produced by 5-HT1B overexpression. Interestingly, a significant number of glass, cry^b double mutants, which lack CRY as well as all visual photoreceptors, are arrhythmic in DD (Yuan, 2005).

5-HT1B not only affects circadian photosensitivity when over- or under-expressed, it also appears to be the major receptor subtype required for the inhibitory effects of serotonin on entrainment. Notably, when 5-HT1B was knocked down with the RNAi transgene driven by tim-Gal4, the effect on photosensitivity was not as pronounced as with the 5-HT1B-Gal4 driver. This might be due to some background differences in flies carrying the tim-Gal4 transgene, or to nonspecific effects produced by expressing the RNAi construct in irrelevant cells. Also, the possibility that cells other than clock neurons participate in the regulation of light sensitivity via 5-HT1B cannot be excluded. However, clock cells clearly have a major role in this effect, in particular since the circadian response to serotonin is eliminated in the tim-Gal4/RNAi flies (Yuan, 2005).

Effects of serotonin on circadian photosensitivity have been demonstrated in other systems, but the underlying mechanisms were not identified. These studies in Drosophila address this issue by demonstrating an effect of 5-HT1B signaling on the posttranslational modification of Tim via Sgg. In 5-HT1B-overexpressing flies, Tim phosphorylation is reduced, and its stability is increased. In contrast, Sgg phosphorylation is increased (i.e., its activity is decreased) in response to elevated levels of 5-HT1B as well as in response to serotonin treatment. Consistent with this effect of 5-HT1B on Sgg, increased Sgg activity abolishes effects of 5-HT1B overexpression on circadian photosensitivity, while 5-HT1B attenuates the period shortening produced by excess Sgg activity. These reciprocal effects in genetic experiments strongly support the regulation of Sgg activity by 5-HT1B. Expression data indicate that Sgg is expressed predominantly in the cytoplasm. The regulation of cytoplasmic Sgg by 5-HT1B is predicted to affect the phosphorylation status of Tim mainly in the cytoplasm; Sgg-phosphorylated Tim is transported to the nucleus more effectively and is also a better substrate for light-induced degradation (Yuan, 2005).

5-HT1B alone does not significantly affect circadian period, suggesting that its effects on Sgg are limited. In this context, it is noted that, while sgg hypomorphs have a period of ~26 hr, flies hemizygous for the locus have wild-type periods. It is inferred that small (up to 50%) changes in Sgg activity do not alter circadian period but can affect circadian photosensitivity. A role for Sgg in circadian photosensitivity was previously suggested by Martinek (2001) who found that forms of Tim phosphorylated by Sgg were selectively degraded in response to light. In fact, phosphorylated Tim is known to be more sensitive to light. While Sgg appears to be the primary kinase that increases photic sensitivity of Tim, the actual process of light-induced Tim degradation involves the activity of a tyrosine kinase (Yuan, 2005).

These results provide a new mechanism for circadian regulation by a G protein-coupled signaling pathway. A role for GSK3β in the mammalian circadian system was recently reported (Iwahana, 2004). In addition, the mammalian 5-HT1A receptor affects phosphorylation of GSK3β in the mouse brain. It is possible that inhibition of GSK3β activity is a conserved mechanism in the regulation of circadian entrainment in mammals and insects (Yuan, 2005).

Slow dark adaptation has been described in Drosophila, whereby circadian sensitivity to light increases more than 10-fold over 3 days in DD. Increased light responsiveness during dark adaptation occurs in rodents, but the mechanism underlying these effects has not been addressed. Elevated responsiveness to light after prolonged exposure to darkness could be due either to a gain in sensitivity in the sensory system or to an increase in sensory output, which may be caused by a reduction in an inhibitory mechanism. In this study, lower serotonin levels were observed in flies maintained in DD. Given that serotonin signaling modulates circadian light sensitivity, it may be the reduction in this inhibitory mechanism that at least partially accounts for the enhanced light response in prolonged DD (Yuan, 2005).

It is proposed that serotonin signaling, which is itself upregulated by light, is a part of a homeostatic mechanism that regulates circadian light sensitivity. A recent study using human subjects also suggested that serotonin levels in the brain reflect the duration of prior light exposure. This change in serotonin levels with light may be relevant to the etiology and treatment of seasonal affective disorder (SAD), a mood disorder related to the reduced hours of sunlight in winter, particularly at northern latitudes. SAD patients respond to antidepression drug treatments, as well as to light therapy, both of which may produce an increase in serotonin. The interplay of serotonin, light, and the circadian system suggests a close relationship between circadian regulation and mental fitness (Yuan, 2005).

Serotonin modulates the entrainment of the circadian system. In contrast, the current results, and studies done in mammalian systems also, suggest circadian effects on serotonin signaling. (1) Based upon the differences seen in LD versus DD in the fly brain, the level of serotonin is affected by the environmental light cycle. (2) Receptor levels are modulated by circadian components, since 5-HT1B levels are altered in fly circadian mutants. In addition, serotonin release and receptor activity are regulated in a circadian fashion in mammals. Mutual regulation of the circadian and serotonin systems may be necessary to maintain the normal physiological functions of both systems (Yuan, 2005).

PER-TIM interactions in living Drosophila cells: An interval timer for the circadian clock

In contrast to current models, fluorescence resonance energy transfer measurements using a single-cell imaging assay with fluorescent forms of Per and Tim showed that these proteins bind rapidly and persist in the cytoplasm while gradually accumulating in discrete foci. After ~6 hours, complexes abruptly dissociated, as Per and Tim independently moved to the nucleus in a narrow time frame. The per^L mutation delays nuclear accumulation in vivo and in a cultured cell system, but without affecting rates of Per/Tim assembly or dissociation. This finding points to a previously unrecognized form of temporal regulation that underlies the periodicity of the circadian clock (Meyer, 2006).

In Drosophila, Per and Tim are two essential proteins of the circadian clock that shift from the cytoplasm of clock cells to the nucleus once a day, promoting ~24-hour oscillations of per and tim transcription. They do this in a regulated manner, and the period length of Drosophila's circadian rhythm is in part determined by how long these proteins are held in the cytoplasm before entering the nucleus (Meyer, 2006).

Formation of Per/Tim heterodimers appears to promote the nuclear accumulation of both proteins. In vivo, a 4- to 6-hour delay in Per nuclear accumulation may be influenced by the slow cytoplasmic assembly of Per/Tim heterodimers, such that once formed, the Per/Tim heterodimer is rapidly transferred from the cytoplasm to the nucleus. It is thought that in the nucleus Per physically interacts with Clock and Cycle, transcriptional activators of per and tim, inhibiting Clock/Cycle activity and hence closing a delayed feedback loop that contributes to oscillating RNA and protein levels (Meyer, 2006).

Recently, the proposal that Per and Tim translocate to the nucleus as obligate heterodimers, and even the necessity of Tim for Per's nuclear accumulation, have been questioned. To follow Per and Tim during their passage from the cytoplasm to the nucleus and to determine the role of Per/Tim interaction in the regulation of nuclear accumulation, a single-cell, fluorescent, live-imaging assay was developed using a Drosophila cell line (Schneider's line 2, S2). Although S2 cells do not express several clock genes and are not rhythmic, this cultured cell system has become an important tool for investigating intracellular mechanisms contributing to Drosophila's circadian clock (Meyer, 2006).

C-terminal fusions of Per and Tim were constructed with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and these were monitored separately or together in S2 cells. Expression of per-cfp (without Tim) was followed in two cell lines. In one line, Per-CFP production was controlled by a heat shock promoter. These cells were constantly monitored for 10 hours after induction (~100 cells in 10 independent experiments). In the second line, an actin promoter drove per-cfp expression, and 40 cells in two experiments were followed for 10 hours after transfection. Per-CFP was detected only in the cytoplasm of live S2 cells in both studies. In a third study, cells were followed in which tim-yfp was driven by a heat shock promoter in the absence of Per (130 cells in 10 experiments). Tim was retained in the cytoplasm in most but not all cells 10 hours after induction (123 cytoplasmic, 7 nuclear). In contrast, when cotransfected, per-cfp and tim-yfp gave predominantly nuclear fluorescence for both proteins in most cells (209 nuclear out of 265 cells monitored in 39 experiments at 8 hours after induction). The behavior of the proteins in the S2 cell system is therefore concordant with in vivo findings and indicates that the fluorescent tags do not detectably interfere with either cytoplasmic retention of the individually expressed proteins or with interactions that promote nuclear accumulation (Meyer, 2006).

To evaluate the validity of an existing model in which rates of Per/Tim interaction affect the timing of their nuclear translocation, Per-CFP/Tim-YFP fluorescence resonance energy transfer (FRET) measurements were compared dynamically by continuously imaging CFP and YFP in live single cells. Maximum levels of FRET were reached during the earliest stages of Per-CFP and Tim-YFP accumulation (within 30 min of Per and Tim production), indicating that physical interaction followed Per-CFP/Tim-YFP synthesis without a measurable delay. Moreover, high levels of FRET were maintained for several hours preceding the onset of nuclear accumulation of Per and Tim. Unexpectedly, FRET declined rapidly as Per and Tim proteins were transferred from the cytoplasm to the nucleus. As Per and Tim became predominantly nuclear, FRET levels remained low in all subcellular compartments, which were typically monitored for a further 100 min (Meyer, 2006).

Immediately following coinduction, Per and Tim were always diffusely present in the cytoplasm. However, this largely uniform distribution was followed by a gradual accumulation in prominent cytoplasmic foci. These foci remained in the cytoplasm until Per and Tim translocated to the nucleus. Notably, the formation of foci was not observed when either Per or Tim was expressed alone. In addition, when Per and Tim were coexpressed, the foci often disappeared earlier in Per-CFP images than in Tim-YFP images. Thus, formation of these foci may be an important step in the temporal control of nuclear entry (Meyer, 2006).

The abrupt decrease in FRET upon nuclear translocation could reflect either dissociation or a change in conformation of the Per-CFP/Tim-YFP complex. To differentiate between these two possibilities, the rates of nuclear accumulation for Per and Tim were independently measured. If Per and Tim undergo a conformational change but remain physically associated as nuclear translocation occurs, individual rates of Per and Tim nuclear accumulation should be equal (Meyer, 2006).

In a survey of 85 cells, it was found that the onset of nuclear accumulation, determined as the inflexion point of the nuclear accumulation profile for Per-CFP, occurred in a narrow time frame, 340 ± 70 min after heat shock in S2 cells. Consistent with the observation that Per and Tim associate rapidly and that these association kinetics have no influence on the onset of nuclear translocation in the S2 cell system, it was found that the time of onset of nuclear accumulation in these experiments was not correlated with the level of Per-CFPor Tim-YFP expressed in the cytoplasm. To determine whether the kinetics of the nuclear accumulations of Per-CFP and of Tim-YFP were similar, the rate of each protein's nuclear accumulation was calculated as the coefficient of a first-order linear regression. The latter was taken from the steepest slope of the profile of nuclear translocation, scaled to the mean fluorescence in each cell. It was found that the rates of nuclear accumulation of Per-CFP and Tim-YFP were independent. Also, although the rate of accumulation of Per-CFP was positively correlated with the level of Per-CFP, this rate was independent of the level of Tim-YFP produced in the same cell. Similarly, Tim-YFP accumulation rates were correlated with the Tim-YFP level, but not with the Per-CFP level in the same cell (Meyer, 2006).

One issue that is not resolved by measuring these accumulation rates is whether the Per/Tim complex dissociates before or after traveling to the nucleus. Comparisons of Per and Tim nuclear translocations within individual cells reveal that onset of Per nuclear accumulation often precedes that of Tim, as has been reported in vivo. Earlier work has shown that, in the absence of Per, Tim shuttles between the nucleus and cytoplasm through the action of both nuclear localization and nuclear export signals. Possibly, Tim transports Per to the nucleus in a complex, after which the proteins separate, allowing Tim to return to the cytoplasm to transport more Per (Meyer, 2006).

To determine whether this property of Tim contributes to the independent rates of Per and Tim nuclear translocation observed in these studies, leptomycin B was used to block Tim-YFP nuclear export. In the presence of this inhibitor of nuclear export, for cells expressing only Tim-YFP, the protein was constitutively localized to the nucleus in most cells (45 cells out of 50 surveyed). In contrast, in cells expressing only Per-CFP, Per remained in the cytoplasm (50 out of 50 cells) in the presence of the drug. Intriguingly, addition of leptomycin B to cells coexpressing Per-CFP and Tim-YFP suppressed the rapid transfer of Tim-YFP to the nucleus. Instead, both proteins were sequestered in the cytoplasm for several hours before nuclear translocation, as previously observed in the absence of drug. Evidently, even in the presence of leptomycin B, Tim is retained by its interaction with Per. Addition of leptomycin B also failed to modify the divergent profiles of Per and Tim nuclear accumulation; rates of Per and Tim nuclear accumulation remained uncorrelated in a study of these cells. The latter finding indicates that although Tim shuttling between the nucleus and cytoplasm has been confirmed, this mechanism cannot explain the independent rates of Per-CFP and Tim-YFP nuclear accumulation that were observed. The measurements hence favor an alternative mechanism for nuclear translocation wherein most of the cytoplasmically derived complexes dissociate in the cytoplasm as the proteins translocate to the nucleus (Meyer, 2006).

The per^L mutation produces a delayed nuclear translocation phenotype in pacemaker cells of the Drosophila brain. This results in long-period behavioral rhythms of ~28 hours. per^L involves a single amino acid substitution, and it also depresses the physical interaction of Per^L and Tim when the proteins are coexpressed in yeast. The tim^UL mutation is associated with a distinct single–amino acid substitution that delays Per and Tim nuclear turnover, resulting in a 33-hour behavioral rhythm. In contrast to per^L, tim^UL has no effect on the timing of nuclear translocation in vivo (Meyer, 2006).

The mean onset of Per-CFP nuclear accumulation in cells coexpressing Per-CFP and Tim^UL-YFP is 299 ± 33 min (20 cells), and it is also independent of Per-CFP and Tim^UL-YFP levels. Furthermore, no persistent FRET was found when Per-CFP and Tim^UL-YFP moved to the nucleus: FRET decay was not delayed when compared to the onset of nuclear accumulation. A loss of FRET was observed with Tim^UL in parallel with nuclear translocation, which suggests that, as for wild-type Tim, Tim^UL/Per heterodimers dissociate as nuclear translocation proceeds in this mutant. Previous studies have shown that, in tim^UL mutants, Per is found in high molecular weight complexes late at night when it is presumably nuclear. The possibility cannot be ruled out that, following translocation, Per and Tim form new associations that do not support FRET in the nucleus in both wild-type and Tim^UL-expressing cells (Meyer, 2006).

S2 cells reproduce the delay in nuclear translocation onset when Per^L is expressed in place of Per. In Per^L-expressing cells, the mean onset of nuclear accumulation was at 492 ± 97 min after induction, as compared with 340 ± 70 min in Per-expressing cells (25 cells. The onset of Per and Tim nuclear accumulation remained independent of Per^L-CFP and Tim-YFP levels. The profiles of nuclear accumulation of these proteins also indicated significant independence in their rates of translocation. FRET decayed as Per^L-CFP and Tim-YFP were transferred to the nucleus, and has been seen from Per/Tim combinations, maximum levels of FRET arose without a measurable delay in cells expressing Per^L.This result was not predicted by earlier models, which assumed that an altered rate of Per^L and Tim physical association chiefly determines the temporal delay found in nuclear accumulation. Because nuclear translocation instead followed a protracted interval of maximum FRET in Per^L-expressing cells, a step distinct from Per/Tim assembly appears to trigger nuclear translocation in S2 cells and is likely also responsible for delayed nuclear translocation in vivo (Meyer, 2006).

These studies indicate that cytoplasmically formed Per/Tim complexes are not translocated to the nucleus: FRET disappears in parallel with Per and Tim nuclear accumulation, suggesting a dissociation of the complex, and measurements of Per and Tim nuclear accumulation rates show that, for a given cell, these are different and independent for each protein. Because Per/Tim associations are not sufficient to initiate nuclear accumulation, these results point to a mechanism in which physical interaction precedes an activity that precisely times nuclear translocation of both proteins. In this respect, Per and Tim appear to act as constituents of an intracellular interval timer. A better understanding of this timer might be sought in the discrete cytoplasmic foci observed to routinely precede nuclear translocation. These foci may reflect condensations of cytoplasmic Per/Tim complexes together with additional factors responsible for their posttranslational modifications. Such factors could include the kinases SGG, DBT, and CK2 or the phosphatase PP2A, each known to affect the phosphorylation of Per and Tim and to influence the timing of nuclear translocation in vivo (Meyer, 2006).

JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of Timeless

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).

PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription

Transcriptional activation by Clock-Cycle (Clk-Cyc) heterodimers and repression by Period-Timeless (Per-Tim) heterodimers are essential for circadian oscillator function in Drosophila. Per-Tim has been found to interact with Clk-Cyc to repress transcription, and this interaction is shown to inhibit binding of Clk-Cyc to E-box regulatory elements in vivo. Coincident with the interaction between Per-Tim and Clk-Cyc is the hyperphosphorylation of Clk. This hyperphosphorylation occurs in parallel with the Per-dependent entry of Double-time (Dbt) kinase into a complex with Clk-Cyc, where Dbt destabilizes both Clk and Per. Once Per and Clk are degraded, a novel hypophosphorylated form of Clk accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that Per-dependent rhythms in Clk phosphorylation control rhythms in E-box-dependent transcription and Clk stability, thus linking Per and Clk function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals (Yu, 2006).

ChIP studies demonstrate that Clk-Cyc is only bound to E-boxes when target genes are being actively transcribed. Since Per-Dbt/Per-Tim-Dbt complexes interact with Clk-Cyc to inhibit transcription (Darlington, 1998; Lee, 1998), these data imply that these Per-containing complexes inhibit transcription by removing Clk-Cyc from E-boxes. It is also possible that binding of these Per-containing complexes to Clk-Cyc effectively blocks Clk and Cyc antibody access, in which case Per complexes would inhibit transcription while Clk-Cyc is bound to E-boxes. Given that the polyclonal Clk and Cyc antibodies used in this study were raised against full-length proteins and have been used to immunoprecipitate Per-containing complexes (Lee, 1998), it is highly unlikely that all Clk and Cyc epitopes are fully blocked by Per complex binding. Thus, it is concluded that Clk-Cyc rhythmically binds E-boxes in concert with target gene activation (Yu, 2006).

Per complex binding could remove Clk-Cyc from E-boxes by directly altering their conformation or by promoting Clk phosphorylation. The region of Per that inhibits Clk-Cyc transcription, called the Clk-Cyc inhibitory domain or CCID, is near the C terminus. The CCID can act independently of the N terminus of Per, where the Dbt-binding domain resides. This observation argues that Per does not inhibit Clk-Cyc binding to E-boxes by promoting Dbt-dependent Clk phosphorylation. Dbt- and CK2-dependent phosphorylation nevertheless enhances transcriptional repression in S2 cells by potentiating Per inhibition or by inhibiting Clk activity directly. Unfortunately, these disparate results from S2 cells do not allow distinguishing between the different effects of Per complex binding to inhibit transcription outlined above (Yu, 2006).

In mammals, mCry complexes bind to CLOCK-BMAL1 and repress transcription without removing CLOCK-BMAL1 from E-boxes. This contrasts with the situation in flies, where Per complexes inhibit transcription by inhibiting Clk-Cyc E-box binding, and suggests that these Per and mCry complexes repress transcription via different mechanisms. Although mCry complexes do not remove CLOCK-BMAL1 from E-boxes, they repress transcription by inhibiting the CLOCK-BMAL1-induced acetylation of histones by blocking p300 histone acetyl transferase function or introducing a histone deacetylase. Even though Per complexes repress transcription by inhibiting Clk-Cyc binding to E-boxes, this does not exclude the possibility that rhythms in histone acetylation are also involved in regulating rhythmic transcription in flies. Since chromatin remodeling is generally accepted as a prerequisite for transcription initiation, it would be surprising if rhythms in transcription were not accompanied by rhythms in histone acetylation or some other form of chromatin remodeling (Yu, 2006).

A rhythm in Clk phosphorylation has been defined in which hyperphosphorylated Clk predominates during times of transcriptional repression and hypophosphorylated Clk predominates during times of transcriptional activation. This rhythm occurs in parallel to the rhythm in Per phosphorylation; hyperphosphorylated Per and Clk accumulate in nuclei during the late night and early morning, then these forms are degraded and hypophosphorylated forms of Per and Clk accumulate in the cytoplasm and nucleus, respectively, during the late day and early evening. The rhythm in Clk and Per phosphorylation are not merely coincidental; the accumulation of hyperphosphorylated Clk is Per dependent. Although Per is not itself a kinase, it is bound by Dbt kinase. Per brings Dbt into the nucleus, where Per-Dbt or Per-Tim-Dbt complexes bind Clk-Cyc to inhibit transcription (Yu, 2006 and references therein).

Since Dbt enters a complex containing Clk-Cyc at times when Clk becomes hyperphosphorylated, Dbt may also act to phosphorylate Clk. However, an in vitro assay for Dbt phosphorylation is not available, thus it iw not known whether Dbt directly phosphorylates Clk. Dbt acts to reduce Clk levels in S2 cells even though Per levels are very low. It is therefore possible that Dbt can act to destabilize Clk in a Per-independent manner, although it is believed this is unlikely to be the case since Clk hyperphosphorylation and complex formation with Dbt are both Per dependent (Yu, 2006).

Clk is phosphorylated to some extent in the absence of Per and is hyperphosphorylated in the absence of functional Dbt, indicating that other kinases act to phosphorylate Clk. The accumulation of hyperphosphorylated Clk in dbt^AR/dbt^P flies suggests that Dbt triggers Clk degradation subsequent to Clk hyperphosphorylation. A similar situation is seen for Per, where hyperphosphorylated Per accumulates in the absence of functional Dbt, and phosphorylation by CK2 precedes Dbt-dependent phosphorylation and Per destabilization. In addition, rhythmically expressed phosphatases may also contribute to Clk phosphorylation rhythms (Yu, 2006 and references therein).

Rhythms in Clk phosphorylation may function to modulate Clk stability, subcellular localization, and/or activity. Clk levels do not change appreciably throughout the daily cycle despite approximately fivefold higher levels of Clk mRNA at dawn than at dusk. If a less stable hyperphosphorylated form of Clk accumulates when Clk mRNA is high and a more stable hypophosphorylated form of Clk accumulates when Clk mRNA is low, they would tend to equalize total Clk levels over the daily cycle. This possibility is supported by results in ARK flies, which express Clk mRNA in the opposite circadian phase (i.e., Clk mRNA peak at dusk rather than dawn). The overall level of Clk cycles in ARK flies with a peak in (hypophosphorylated) Clk around dusk, consistent with hypophosphorylated Clk being more stable than hyperphosphorylated Clk. This possibility is also supported by Dbt-dependent destabilization of Clk in S2 cells since Dbt associates with Clk as hyperphosphorylated Clk accumulates in wild-type flies. If hypophosphorylated Clk is relatively stable, higher levels of Clk might be expected to accumulate in per⁰¹ flies. However, constant low levels of Clk mRNA likely limit Clk accumulation in per⁰¹ flies. Clock phosphorylation is coupled to its degradation in cultured mammalian cells, yet degradation of phosphorylated Clock does not lead to a rhythm in Clock abundance even though Clock mRNA levels are constant (Yu, 2006).

Studies in cultured mammalian cells also demonstrate that Clock phosphorylation promotes Clock-BMAL1 nuclear localization, although the significance of this nuclear localization is not clear given that Clock-BMAL1 binding to E-boxes is either constant or more robust during transcriptional repression in vivo. In contrast, Clk is nuclear throughout the daily cycle in flies (Yu, 2006).

The coincidence between Clock phosphorylation and transcriptional repression in mice supports the possibility that phosphorylation inhibits Clock-BMAL1 activity, perhaps by promoting HDAC binding or inhibiting HAT binding. Likewise, hypophosphorylated and hyperphosphorylated Clk accumulate in parallel with target gene activation and repression, respectively, in flies. This relationship suggests that the state of Clk phosphorylation may alter its ability to activate target genes. Given that target gene activation occurs when Clk-Cyc is bound to E-boxes and that E-box binding coincides with the accumulation of hypophosphorylated Clk, it is possible that Clk hyperphosphorylation compromises Clk-Cyc binding to E-boxes and, consequently, target gene transcription is repressed. Precedent for such a regulatory mechanism is seen in the Neurospora clock, where limiting levels of FREQUENCY (FRQ) promote phosphorylation of WHITE COLLAR 1 (WC1) and WHITE COLLAR 2 (WC2), thereby inhibiting WC1-WC2 binding to C-box regulatory elements and repressing transcription. In contrast to FRQ in Neurospora, Per is considerably more abundant than Clk in Drosophila and forms stable complexes with Clk-Cyc. In addition, Per/Per-Tim can release Clk-Cyc from E-boxes in vitro, thus demonstrating that Per/Per-Tim binding is sufficient to release Clk-Cyc from E-boxes independent of Clk phosphorylation. Taken together with the in vitro E-box binding results, the high levels of Per relative to Clk and the formation of stable Per-Tim-Clk-Cyc complexes in flies argue that Per/Per-Tim binding may also be sufficient to inhibit E-box binding by Clk-Cyc in vivo, although they do not rule out a role for Clk hyperphosphorylation in inhibiting Clk-Cyc E-box binding. For instance, Per/Per-Tim binding could function to initially remove Clk-Cyc from E-boxes, and subsequent Clk phosphorylation could maintain Clk-Cyc in a form that is incapable of binding E-boxes (Yu, 2006).

The constant levels and rhythmic phosphorylation of Clk defined in this study are similar to those previously characterized for mammalian Clock. This similarity extends beyond metazoans to fungi, where positive elements of the Neurospora circadian feedback loop; i.e., WC1 and WC2, are also rhythmically phosphorylated. In each of these organisms, phosphorylation of positive factors increases when they interact with their respective negative feedback regulators, and decreases when they activate target gene transcription in the absence of these feedback inhibitors. This remarkable similarity suggests that phosphorylation controls one or more critical aspects of positive element function, and consequently, the rhythm in the positive element phosphorylation has become a conserved feature of circadian feedback loops in eukaryotes (Yu, 2006 and references therein).

Per-dependent regulation of Clk-Cyc binding to E-boxes, Per-dependent formation of Per-Dbt and/or Per-Dbt-Tim complexes with Clk-Cyc, and Per-dependent rhythms in Clk phosphorylation suggest a model for the regulation of rhythmic transcription. During the late day and early evening, hypophosphorylated Clk-Cyc binds E-boxes to activate transcription of per, tim, and other genes within and downstream of the transcriptional feedback loop. Accumulating levels of per mRNA peak during the early evening, but Per accumulation is delayed due to Dbt-dependent (and possibly CK2-dependent) phosphorylation, which destabilizes Per. Per is subsequently stabilized via Tim binding, which inhibits further phosphorylation of Per by Dbt. Phosphorylation of Tim by SGG then promotes the translocation of Tim-Per-Dbt complexes into the nucleus, where they bind (hypophosphorylated) Clk-Cyc and repress transcription by inhibiting E-box binding and promoting Clk hyperphosphorylation and degradation. These transcriptional repression mechanisms are not mutually exclusive; Clk hyperphosphorylation may inhibit E-box binding as well as promote Clk degradation. Dbt is able to enter the nucleus in per⁰¹ flies, but does not associate with Clk in the absence of Per. This suggests that Per is required to either bring Dbt into a complex with Clk-Cyc, enable phosphorylation of Clk after Dbt enters the complex, or both. Once the Tim-Per-Dbt-Clk-Cyc complex has formed, hyperphosphorylated Per and Clk levels decline in a coordinated fashion by mid-day. Tim is eliminated prior to hyperphosphorylated Per and Clk via separate light-dependent and light-independent mechanisms. As hyperphosphorylated Clk and Clk mRNA decline during the day, hypophosphorylated Clk accumulates. This hypophosphorylated Clk forms complexes with Cyc and binds E-boxes in the absence of nuclear Tim-Per-Dbt complexes, thus initiating the next cycle of transcription (Yu, 2006 and references therein).

Post-translational regulation of the Drosophila circadian clock requires protein phosphatase 1 (PP1); PP1 directly dephosphorylates and stabilizes Tim, which promotes accumulation of Per

Phosphorylation is an important timekeeping mechanism in the circadian clock that has been closely studied at the level of the kinases involved but may also be tightly controlled by phosphatase action. This study demonstrates a role for protein phosphatase 1 (PP1) in the regulation of the major timekeeping molecules in the Drosophila clock, Timeless (Tim) and PERIOD (PER). Flies with reduced PP1 activity exhibit a lengthened circadian period, reduced amplitude of behavioral rhythms, and an altered response to light that suggests a defect in the rising phase of clock protein expression. On a molecular level, PP1 directly dephosphorylates Tim and stabilizes it in both S2R+ cells and clock neurons. However, PP1 does not act in a simple antagonistic manner to Shaggy (Sgg), the kinase that phosphorylates Tim, because the behavioral phenotypes produced by inhibiting PP1 in flies are different from those achieved by overexpressing Sgg. PP1 also acts on Per, and Tim regulates the control of Per by PP1, although it does not affect PP2A action on Per. A modified model is proposed for post-translational regulation of the Drosophila clock, in which PP1 is critical for the rhythmic abundance of Tim/Per while PP2A also regulates the nuclear translocation of Tim/Per (Fang, 2007).

Little is known about the potential action of protein phosphatases in the clock. Unlike the large number of protein kinases (~400) in eukaryotes, there are only ~25 protein phosphatases. Of these, protein phosphatase 2A (PP2A) and PP1 together contribute ~90% of the total serine/threonine phosphatase activity in mammalian cells. PP2A dephosphorylates Per, thereby stabilizing Per and promoting its nuclear translocation. This study examined whether PP1 also has a circadian function. PP1 is a ubiquitous eukaryotic enzyme and plays an important role in many cellular processes, including metabolism, cell cycle, muscle relaxation, and synaptic plasticity. In Drosophila, four genes encode a catalytic subunit of PP1 (PP1c) and are named according to their chromosomal loci: 9C (also called flapwing, flw), 13C, 87B, and 96A. PP1c is highly conserved across species, and the four Drosophila PP1c isoforms are ~90% identical to each other at the amino acid level with indistinguishable activities in vitro. Most PP1 targets and associated proteins contain a conserved PP1c-binding motif, [R/K]-X_0-1-[V/I]-{P}-[F/W] (where X denotes any residue and {P} any residue except proline, so-called RVxF motif), which is also found in Tim (RAIGF, amino acids 77-81). This prompted a test of Tim as a possible PP1 target. This study shows that PP1 dephosphorylates and stabilizes Tim, which is a prerequisite for the rhythmic abundance of Tim/Per, and thus plays an essential role in the post-translational regulation of the Drosophila clock (Fang, 2007).

PP1 directly dephosphorylates and stabilizes Tim, which promotes accumulation of Per. The reduction in Tim/Per abundance caused by PP1 inhibition, somewhat resembling a response to continuous dim light, generates a long period coupled with a reduced circadian rhythmicity phenotype (Fang, 2007).

Inhibition of PP1 in flies significantly decreases Tim abundance, especially Tim accumulation in the nucleus, but the onset of Tim nuclear entry appears intact. In cell culture experiments as well it was found that neither PP1c nor nuclear inhibitor of PP1 (NIPP1) affects the subcellular localization of Tim. Thus, although PP1 regulates Tim stability, it does not play a major role in trigging the nuclear entry of Tim, suggesting that additional regulation is required to initiate nuclear translocation. This is consistent with the finding that the onset of nuclear accumulation of Tim and Per is not correlated with their protein levels in the cytoplasm. Moreover, Tim protects Per from inhibition of PP1 but not of PP2A, which allows Tim-stabilized Per to undergo further phosphorylation/dephosphorylation. Since dephosphorylation of Per by PP2A promotes nuclear translocation of Per and nuclear expression of Tim appears to depend on Per, it is concluded that, while PP2A primarily targets Per and controls the timing of Tim/Per nuclear translocation, PP1 plays a central role in stabilizing Tim and Per and regulating their rhythmic abundance (Fang, 2007).

NIPP1-overexpressing flies have a specific early- to mid-night defect, and overexpression of NIPP1 produces an additive effect on period lengthening in a tim^UL background. It is inferred that the destabilizing effect of NIPP1 during the accumulation (rising phase) does not affect the action of the tim^UL mutation, which increases Tim stability specifically in the nucleus during the late night (falling phase). The regulation of Tim stability may involve different mechanisms at different times of the cycle, which is also implied by the different mechanisms used for light-independent and light-triggered Tim degradation. These data also suggest that opposite effects on Tim stability can have the same effect on circadian period if they occur at different times of day; in this case, longer periods are produced either by decreased Tim stability during the rising phase (as produced by inhibition of PP1) or by increased Tim stability during the falling phase (produced by the tim^UL mutation) (Fang, 2007).

Although inhibition of PP1 does not lead to a significant mobility shift, direct dephosphorylation of Tim by PP1 is suggested by the in vitro phosphatase and co-IP assays as well as by the ³²P metabolic labeling in S2R⁺ cells. A mobility change of Tim is observed in Sgg-overexpressing flies, in which Tim nuclear entry is advanced but Tim stability is not significantly decreased. Hence, although PP1 dephosphorylates GSK-3β-phosphorylated Tim in vitro, their target sites may not completely overlap; in addition, the functions of PP1 and Sgg in regulating the clock are not simply antagonistic. It is likely that different phosphorylation sites on Tim mediate different cellular processes and are regulated by different mechanisms. A similar idea has been proposed for the regulation of Drosophila and mammalian Per (Fang, 2007).

Based on these findings, a modified model is proposed for the post-translational regulation of the Drosophila clock by multiple phosphorylation events: Once translated, Tim and Per proteins are subject to modifications including phosphorylation, which targets them for proteasome-mediated degradation. PP1 dephosphorylates Tim at one or a small number of 'stability-critical' phosphorylation sites that enable Tim to accumulate in the cytoplasm. The stabilized Tim binds to and stabilizes Per in the cytoplasm. Per is further stabilized by PP2A, which also promotes Per nuclear translocation. Tim nuclear expression is promoted by Sgg phosphorylation, which does not have a major effect on Tim stability, likely because the 'stability-critical' phosphorylation site is protected by PP1. Tim/Per are continually stabilized by PP1 during their nuclear translocation and accumulation, and they then inhibit their own transcription by repressing Clk and Cyc. However, the data do not exclude additional indirect PP1 regulation of Tim/Per as reported for PP5 in the mammalian clock, nor do they rule out the involvement of additional clock target(s) of PP1 (Fang, 2007).

Although PP1 is no longer viewed as a simple housekeeping gene, a steady state of PP1c levels seems critical for an organism, as PP1c is encoded by multiple genes in most eukaryotic species. In flies, overexpression of NIPP1 using stronger and more widespread drivers such as tim-Gal4, elav-Gal4, and actin-Gal4 causes lethality. In addition, it was found that the expression of the Drosophila PP1c isoforms in S2R⁺ cells is regulated such that the total PP1c transcript level remains stable despite the loss or reduction of one PP1c mRNA. While it is beyond the scope of this study, it would be interesting to explore the mechanism underlying this phenomenon and to determine whether this regulation of PP1c expression exists in other fly cells (Fang, 2007).

The functional diversity of PP1 is exerted via its association with a large variety of regulatory subunits. PP1 regulatory subunits not only confer in vivo substrate specificity by directing PP1c to various subcellular loci for its substrates, but also allow the activity of PP1 to be modulated in response to intracellular signals and extracellular stimuli. It is possible that some adaptor proteins/PP1 regulatory subunits facilitate the interaction between PP1 and Tim documented in this study through co-IP experiments. In addition, although none of the PP1c isoforms is rhythmically expressed in the fly head, the regulatory subunit(s) targeting PP1 to 'clock substrates' may oscillate. The paradigm for the cyclic phosphatase activity concept is PP2A, whose regulatory subunits TWS and WDB are expressed with a robust circadian rhythm and affect Drosophila behavioral rhythms (Fang, 2007).

The mechanisms by which Tim stabilizes Per are not known, but it is possible that they involve phosphorylation. Perhaps most importantly, Per is stable and nuclear in tim⁰¹ flies if the kinase DBT is also knocked down, suggesting that, in the absence of Tim, Per is subject to excessive destabilizing phosphorylation. Thus, Tim may stabilize Per either by decreasing phosphorylation by DBT, or by increasing dephosphorylation by a phosphatase such as PP2A or PP1. Since DBT accumulation is not under circadian control and it is found in complexes with Per at all times in vivo, it is likely that the phosphatase activity is dynamic and limiting, regulating the rhythmic abundance of Per. The data suggest that PP1 is the primary phosphatase involved in the stabilizing effect of Tim on Per, as Tim is not more resistant than Per to PP2A inhibition and does not appear to affect dephosphorylation of Per by PP2A. Given that Per does not contain an RVxF-binding motif as found in Tim, it is tempting to speculate that Tim is a target as well as a regulatory subunit of PP1, which may target PP1c to Per and up-regulate local PP1 activity to antagonize the destabilizing action of clock kinases on Per. It is suggested that identification of the circadian-relevant PP1 regulatory subunit(s) will provide profound insight into the post-translational regulation of the clock (Fang, 2007).

This study demonstrates that PP1 plays an essential role in the regulation of the Drosophila clock. PP1 is one of the most conserved eukaryotic proteins, and it often performs similar essential functions in different species. Indeed, studies in the dinoflagellate and the fungus Neurospora have also implied a clock function for PP1. Consistent with the long period phenotype caused by inhibiting PP1 in flies, short pulses of phosphatase inhibitors in dinoflagellates cause phase delays, and PP1 appears to be the dominant phosphatase mediating this circadian function. In Neurospora, PP1 regulates the stability of the clock component FREQUENCY (FRQ). And recently, PP1 was reported to regulate degradation of the mammalian clock protein Per2 . Together, multiple studies indicate an evolutionarily conserved role for PP1 in the circadian clock (Fang, 2007).

Timeless is an important mediator of CK2 effects on circadian clock function in vivo

Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of Period and Timeless proteins govern period length. Yet little is known about the kinases that phosphorylate Tim in vivo. It has been shown previously that the protein kinase CK2 phosphorylates Tim in vitro. This study identified a role for CK2 in Tim regulation in vivo. Induction of a dominant-negative CK2α, CK2α^Tik (Tik), increases Tim protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic Tim localization. Exposure to light and subsequent Tim degradation results in an increase in the fraction of the transcriptional repressor Per that is nuclear and suppression of per and tim RNA levels. Tim protein, but not tim transcript, levels are elevated in Tik mutants in a per⁰¹ background. In contrast, Tik effects on Per are undetectable in a tim⁰¹ background, suggesting that Tim is required for CK2 effects on Per. To identify potential CK2 target sites, Tim phosphorylation rhythms were assayed in a deletion mutant that removes a conserved serine-rich domain. It was found that Tim protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a tim^UL mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that Tim is an important mediator of CK2 effects on circadian rhythms (Meissner, 2008).

Evidence is presented that CK2 operates through Tim to control circadian clock function in vivo. Expression of the dominant-negative CK2α allele, Tik, elevates trough levels of Tim protein and RNA during constant darkness and alters Tim subcellular localization. Tik effects on Per are undetectable in a tim⁰¹ mutant, whereas Tik effects on Tim are evident in a per⁰¹ background, suggesting direct Tim effects. Behavioral period effects of Tik are reduced in the tim^UL mutant that disrupts a putative CK2 site. The effects on Tim metabolism as well as the genetic requirement of tim for CK2 effects indicate CK2 primarily operates through Tim to regulate the circadian clock (Meissner, 2008).

One potential model consistent with these data is that CK2 regulates Tim abundance and in turn, promotes negative feedback. Elevated trough Tim levels are accompanied by elevated trough tim transcript levels in DD, suggesting impaired turnover, negative feedback, and/or tim transcriptional regulation. Effects on Tim are evident in per⁰¹ flies but are not accompanied by changes in tim transcript levels, suggesting a direct effect on Tim protein, perhaps by regulating Tim stability, although a translational effect cannot be ruled out. Importantly, the per⁰¹ data argue strongly against a direct effect of CK2 on CLK/CYC-driven transcription of tim. The ability of light to degrade Tim in Tik-expressing flies implies that Tim levels can be regulated by two distinct pathways, a light/CRY-dependent pathway and a CK2-dependent pathway. During light/dark entrainment, light robustly degrades Tim in Tik-expressing flies. This degradation is accompanied by a sharp suppression in per and tim transcript levels, suggesting that it is excessive Tim levels, rather than direct Per effects, that abrogate negative feedback in Tik-expressing flies. per and tim RNA oscillations remain phase delayed during LD in homozygous Tik-expressing flies, suggesting that Tik also affects the timing of Per repression (Meissner, 2008).

Some CK2 effects on Per may be mediated by CK2 effects on Tim. CK2 effects on Per levels require Tim. CK2 is localized to the cytoplasm where Per/Tim dimers are likely present. CK2 regulates Per and Tim nuclear entry, a process that likely depends on the Per/Tim dimer. Although effects on Per require Tim, Per is likely a direct in vivo CK2 substrate. CK2 robustly and specifically phosphorylates Per in vitro and in-vitro-defined sites have clear in vivo functions. Tik mutants can also strongly effect Per mobility on Western blots; Per mobility is highly dependent on phosphorylation. These data are most consistent with the idea that CK2 targets the Per/Tim dimer in the cytoplasm and phosphorylates Per to promote nuclear entry. Additional experiments will be required to test the dimer hypothesis. Based on S2 cell experiments, it has been proposed that CK2 phosphorylation of Per promotes its intrinsic repressor activity independent of its effects on nuclear localization. The observation that light-induced Tim degradation results in a suppression of per and tim RNA in Tik-expressing flies suggests that elevated Tim levels block Per repression. Nonetheless, remaining alterations in transcript levels suggest that the freed Per repressor may not be entirely functional, consistent with additional CK2 effects on Per (Meissner, 2008).

Tim may be an in vivo CK2 substrate. CK2 has been shown to phosphorylate Tim in vitro. In vivo, Tik increases Tim levels in the absence of Per, suggesting CK2 effects on Tim may be direct. Importantly, increases in Tim protein are not accompanied by increases in transcript, indicating CK2 regulates Tim posttranscriptionally. The notion is favored that CK2 acts to phosphorylate Tim in vivo, consistent with published in vitro phosphorylation experiments (Meissner, 2008).

One potential argument against the hypothesis that CK2 phosphorylates Tim is the finding that low-mobility Tim accumulates to high levels in timGAL4-62; UAS-Tik homozygotes compared with wild-type controls. Interestingly, this result is similar to the observation that in Dbt^g mutants, low mobility forms of Per accumulate that are even lower in mobility than those in wild-type, although Per is widely established as a DBT substrate. In both cases, phosphorylation-induced mobility changes cannot be attributed solely to a single kinase. It is also possible that elevated Tim levels may render low mobility forms more visible in Tik-expressing flies. Thus, the presence of low-mobility forms does not exclude CK2 as an in vivo kinase for Tim (Meissner, 2008).

Although no phosphorylation sites have been identified in Tim, deletion of a small Tim serine-rich domain (Tim 260-292) reduces or eliminates significant circadian mobility changes. This domain is conserved among insects and may contain multiple phosphorylation sites that are responsible for regulating period length and rhythms in Tim levels and mobility. CK2 may be one of several kinases that phosphorylates Tim serine-rich domain. In addition, although this domain is critical for shifts in Tim mobility, there are likely phosphorylation sites for CK2 outside of this region. When the bacterial expression construct used previously to generate Tim protein for CK2 in vitro phosphorylation assay was sequenced, it was discovered that the construct lacks amino acids 260-292. The possibility cannot be excluded that loss of rhythmic mobility changes may be secondary to reduced Tim levels in serine-rich domain mutants or may not be linked to CK2. Nonetheless, the in vivo data and sequence conservation raise the possibility that the serine-rich domain may be a kinase substrate (Meissner, 2008).

The Tim serine-rich domain contains four predicted CK2 sites. One of these sites, Ser²⁷⁹, is potentially altered in tim^UL mutants by the Glu²⁸³Lys point mutation. CK2 preferentially phosphorylates serine or threonine residues located 2-5 residues N-terminal to acidic amino acid residues such as glutamate or aspartate. The placement of basic residues, such as lysine as in the tim^UL mutant, near CK2 sites inhibits CK2 phosphorylation of the target Ser/Thr residue. tim^UL and Tik show allele-specific genetic interactions such that in tim^UL mutants, Tik period lengthening is partially suppressed. These results are consistent with the hypothesis that tim^UL is a CK2 site mutant. This prediction suggests that Ser²⁷⁹ plays a very important role in regulating circadian period length in flies. It will be of interest to test the hypothesis that CK2 promotes Tim degradation (Meissner, 2008).

It has been claimed that tim^UL shows a late-night specific defect principally based on the phase response curve to light and persistent nuclear Tim levels. CK2, however, is mostly restricted to the cytoplasm and thus would be predicted to act on cytoplasmic Per and/or Tim proteins during the early night. Although it is true that tim^UL shows profound late-night defects, on closer examination, there are alterations in the phase response curve to light during the early night. In addition, Tim protein was not examined in pacemaker neurons during the early night in tim^UL, so the possibility that there is also an early-night defect in tim^UL cannot be ruled out. Therefore it is possible that tim^UL also displays early-night defects, as is proposed for CK2 effects. Second, it is possible that phosphorylation of Tim by CK2 in the cytoplasm could influence the function of phospho-Tim in the nucleus. Third, although CK2 is principally observed in the cytoplasm, the possibility that small but functional levels of CK2 are present in the nucleus cannot be ruled out. Regardless, the allele-specific, nonadditive genetic interaction between Tik and tim^UL argue that tim^UL is important for CK2 effects on circadian behavior (Meissner, 2008).

The data are consistent with the speculation that Tik expression reduces CK2 phosphorylation of Tim. This results in retention of Per in the cytoplasm, thus reducing Per-mediated repression. Light-mediated degradation of Tim can then liberate Per (pending some additional events) to enter the nucleus and repress CLK/CYC. Although additional experiments will be needed to test this model, the data demonstrate that it is likely that CK2 effects through Tim will play an important role in circadian clock function (Meissner, 2008).

NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator

The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by Clock (Clc) and Cycle (Cyc) and repressed by Period (Per) and Timeless (Tim). The timing of Clk-Cyc activation and Per-Tim repression is regulated posttranslationally, in part through rhythmic phosphorylation of Clk, Per, and Tim. Although kinases that control Per and Tim levels and subcellular localization have been identified, additional kinases are predicted to target Per, Tim, and/or Clk to promote time-specific transcriptional repression. A screen was carried out for kinases that alter circadian behavior via clock cell-directed RNA interference (RNAi) and the proline-directed kinase nemo (nmo) was identified as a novel component of the circadian oscillator. Both nmo RNAi knockdown and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. Clk levels increase when nmo expression is knocked down in clock cells, whereas Clk levels decrease and Per and Tim accumulation are delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering Clk, Per, and Tim expression, thereby contributing to the generation of an ~24 hr circadian period (Yu, 2011).

This study has identified nmo as a new component of the Drosophila circadian oscillator. The short-period behavioral rhythms of nmo^P1/Df and nmo RNAi flies indicates that Nmo acts to slow the pace of the circadian oscillator, consistent with the lengthening of circadian period when nmo is overexpressed in clock cells. A nmo P[lacZ] enhancer trap line reveal nmo expression in the sLN_v and other brain clock cells, consistent with the short-period behavioral rhythms that result from expressing nmo RNAi in LN_vs. Nmo is present in complexes with Per, Tim, and Clk when Clk-Cyc transcription is repressed and alters Per, Tim, and Clk levels and/or phosphorylation state. These results suggest that Nmo acts within Per-Tim-Clk regulatory complexes to lengthen circadian period. These results are likely relevant to the mammalian circadian oscillator because mPer and Clock are also highly phosphorylated when transcription is repressed and a single nmo ortholog, Nemo-like kinase (NLK), is present in mice and humans (Yu, 2011).

Light-mediated TIM degradation within Drosophila pacemaker neurons (s-LNvs) is neither necessary nor sufficient for delay zone phase shifts

Circadian systems are entrained and phase shifted by light. In Drosophila, the model of light-mediated phase shifting begins with photon capture by Cryptochrome (Cry) followed by rapid TIMELESS (Tim) degradation. This study focused on phase delays, and Tim degradation within individual brain clock neurons was assayed in response to light pulses in the early night. Surprisingly, there was no detectable change in Tim staining intensity within the eight pacemaker s-LNvs. This indicates that Tim degradation within s-LNvs is not necessary for phase delays, and similar assays in other genotypes indicate that it is also not sufficient. In contrast, more dorsal circadian neurons appear light-sensitive in the early night. Because Cry is still necessary within the s-LNvs for phase shifting, the results challenge the canonical cell-autonomous molecular model and raise the question of how the pacemaker neuron transcription-translation clock is reset by light in the early night (Tang, 2010; full text of article).

QUASIMODO, a Novel GPI-anchored zona pellucida protein involved in light input to the Drosophila circadian clock

Circadian clocks are synchronized to the solar day via visual and specialized photoreceptors. In Drosophila, Cryptochrome (Cry) is a major photoreceptor that mediates resetting of the circadian clock via light-dependent degradation of the clock protein Timeless (Tim). However, in the absence of Cry, this Tim-mediated resetting still occurs in some pacemaker neurons, resulting in synchronized behavioral rhythms when flies are exposed to light-dark cycles. Even in the additional absence of visual photoreception, partial molecular and behavioral light synchronization persists. Therefore, other important clock-related photoreceptive and synchronization mechanisms must exist. This study identified a novel clock-controlled gene (quasimodo) that encodes a light-responsive and membrane-anchored Zona Pellucida domain protein that supports light-dependent Tim degradation. Whereas wild-type flies become arrhythmic in constant light (LL), quasimodo mutants elicit rhythmic expression of clock proteins and behavior in LL. Quasimodo can function independently of Cry and is predominantly expressed within Cry-negative clock neurons. Interestingly, downregulation of qsm in the clock circuit restores LL clock protein rhythms in qsm-negative neurons, indicating that qsm-mediated light input is not entirely cell autonomous and can be accessed by the clock circuit. These findings indicate that Qsm constitutes part of a novel and Cry-independent light input to the circadian clock. Like Cry, this pathway targets the clock protein Tim. Qsm's light-responsive character in conjunction with the predicted localization at the outer neuronal membrane suggests that its function is linked to a yet unidentified membrane-bound photoreceptor (Chen, 2011).

Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability

The light-input factor Quasimodo (Qsm) regulates rhythmic electrical excitability of clock neurons, presumably via an Na+, K+, Cl- cotransporter (NKCC) and the Shaw K+ channel (dKV3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression (OX) and knockdown (RNAi) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsm^OX led to a constitutive less active, night-like state, and qsm^RNAi led to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of Shaw^OX and NKCC^RNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. It is proposed that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC (Buhl, 2016).

All organisms are subject to predictable but drastic daily environmental changes caused by the earth's rotation around the sun. It is critical for the fitness and well-being of an individual to anticipate these changes, and this anticipation is done by circadian timekeeping systems (clocks). These regulate changes in behavior, physiology, and metabolism to ensure they occur at certain times during the day, thereby adapting the organism to its environment. The circadian system consists of three elements: the circadian clock to keep time, inputs that allow entrainment, and outputs that influence physiology and behavior. Like a normal clock, circadian clocks run at a steady pace (24 h) and can be reset. In nature this environmental synchronization is done via daily light and temperature cycles, food intake, and social interactions (Buhl, 2016).

In Drosophila the central clock comprises 75 neuron pairs grouped into identifiable clusters that subserve different circadian functions. The molecular basis of the circadian clock is remarkably conserved from Drosophila to mammals. This intracellular molecular clock drives clock neurons to express circadian rhythms in electrical excitability, including variation in membrane potential and spike firing. Clock neurons are depolarized and fire more during the day than at night, and circadian changes in the expression of clock-controlled genes encoding membrane proteins such as ion channels and transporters likely contribute to these rhythms. Such cyclical variations in activity play a critical role in synchronizing different clock neurons and conveying circadian signals to other parts of the nervous system and body. Furthermore, they provide positive feedback to the molecular clock, which can dampen rapidly without such feedback (Buhl, 2016).

Light resets the circadian clock every morning to synchronize the clock to the environment via Timeless (Tim) degradation after activation of the blue-light photoreceptor Cryptochrome (Cry), Quasimodo (Qsm), and potentially also visual photoreceptors. Qsm acts either independently or downstream of Cry and also is able to affect clock protein stability in Qsm-negative neurons by an unknown non-cell-autonomous mechanism (Chen, 2011). Recently Cry has been shown to regulate clock neuron excitability via the redox sensor of the Hyperkinetic voltage-gated potassium (KV)-β subunit (Hk) (Fogle, 2015), and this study asked if Qsm affects the clock neurons in a similar way (Buhl, 2016).

Membrane potential is important for control of circadian behavior, and manipulation of Shaw and the Narrow Abdomen (NA) channels, both of which are expressed and function within clock neurons influence neuronal electrical activity, the circadian clock, and clock-controlled behavior in both flies and mice. The firing rate is a key component in mammalian circadian rhythmicity and can be regulated by regional and circadian expression of the sodium potassium chloride cotransporter NKCC, which switches the effects of GABA from inhibitory to excitatory across the day (Buhl, 2016).

This study shows that down-regulation or overexpression of the three membrane proteins encoded by the genes qsm, Shaw, and NKCC leads to rhythmicity in constant illumination (LL) and that these genes interact. All three genes are expressed in the well-characterized pigment-dispersing factor (Pdf)- and Cry-positive large ventral lateral neurons (l-LNv), which are important for arousal and light input to the clock. Whole-cell recordings of l-LNvs were used to characterize their physiological properties and acute light effects across the day, and Qsm was found to help set the circadian state of clock neurons and modify their response to light, possibly by acting via Shaw and NKCC (Buhl, 2016).

Light is the dominant circadian zeitgeber that resets the molecular clock. This study determined how light affects membrane excitability via the membrane proteins Qsm, Shaw, and NKCC. Previously it was shown that Qsm contributes to circadian clock light input with down-regulation in all clock neurons (tim-gal4) resulting in robust rhythmic behavior in LL (Chen, 2011). This study shows that overexpression (qsm^OX) also results in robust LL rhythmicity, but with predominantly ~13-h periods, suggesting a more robust morning oscillator that is normally weakened in DD conditions. Manipulating the expression levels of both the potassium channel Shaw and the ion cotransporter NKCC also resulted in LL rhythmicity, whereas several visual system mutants behaved like wild-type flies and became arrhythmic. These experiments show that the membrane proteins encoded by qsm, Shaw, and NKCC control rhythmic behavior in LL. Furthermore, the rescue of wild-type behavior and neurophysiological properties by reciprocal changes of Qsm and Shaw and by the simultaneous reduction of Qsm and NKCC suggests that Qsm interacts genetically and perhaps directly with Shaw and NKCC (Buhl, 2016).

Clock neurons are more depolarized and fire more during the daytime, and circadian changes in the expression of clock-controlled genes such as ion channels and transporters are likely to play a part. Contributing to this rhythm is a sodium leak current mediated by NA that recently has been shown to depolarize Drosophila clock neurons. This study shows that Shaw, NKCC, and Qsm also contribute to daily electrical activity rhythms: overexpression and RNAi knock-down of qsm and Shaw compared with NKCC resulted in opposing phenotypes. Interestingly, qsm^OX or Shaw^OX and NKCC^RNAi promote the less active nighttime state, whereas qsm^RNAi, Shaw^RNAi, and NKCC^OX push the neurons into the more depolarized daytime state, eliminating the acute day/night differences in all cases. Previous work has shown that Shaw regulates circadian behavior and that, in agreement with the current findings, Shaw regulates membrane potential and firing in Drosophila motoneurons. NKCC activity is electrically neutral but increases the intracellular Cl^- concentration so that the GABA_A receptor opens in response to GABA but, as a consequence, Cl^- presumably exits the cell down its electrochemical gradient, thereby depolarizing the membrane potential so that GABA effectively becomes an excitatory neurotransmitter. The current data show that in Drosophila a similar mechanism occurs, which is consistent with potential NKCC enrichment in l-LNv at dawn. The mechanism setting the neuronal state to either daytime or nighttime via Qsm, Shaw, and NKCC is likely to be predominantly cell-autonomous, because all components have been shown to act or to be expressed in the l-LNv. Although in an earlier study using qsm-gal4 lines qsm expression was not detected in the l-LNv, these lines may not report expression faithfully in all qsm cells. Now, the finding that qsm RNA is enriched in the l-LNv, combined with the strong effects of two qsm-RNAi lines on l-LNv electrical properties presented in this study, indicates that qsm is endogenously expressed in these neurons (Buhl, 2016).

Physiological studies are limited to the Pdf-expressing l-LNv neurons. These neurons are unlikely candidates for driving behavioral rhythms in LL, and previous work has shown that qsm knockdown in Pdf neurons (s-LNv and l-LNv) does not result in robust LL rhythmicity. Therefore the effects of light on the electrical properties of l-LNv reported here do not necessarily explain the LL rhythmicity observed after manipulating qsm, Shaw, and NKCC in all clock neurons. However, the electrophysiological results using tim-gal4 show that Qsm, Shaw, and NKCC could fulfill similar functions in other clock neurons, including those crucial for LL rhythmicity (e.g., LNd and DN₁). Additionally, or alternatively, the manipulated l-LNv could generate signals interfering with normal network function, resulting in the observed rhythmic LL behavior (Buhl, 2016).

Although qsm is a clock-controlled gene, the acute blue-light effects that were observed are too fast to be mediated by transcriptional changes. Therefore, a more direct membrane-localized mechanism is favored in which rapid light-dependent posttranslational changes of Qsm alter the activity of Shaw and NKCC. Because (i) Cry is required for light-dependent Tim degradation in l-LNv, (ii) changing the Qsm level has no effect on Cry levels, and (iii) qsm^OX triggers Tim degradation in the absence of Cry, the most likely explanation for the results reported in this study is that, in addition to activating Hk, Cry acts upstream of Qsm, which in turn regulates the activity of Shaw and NKCC. It is assumed that Qsm is activated by light because a light pulse at night rapidly increases protein levels. Qsm is an extracellular zona-pellucida (ZP) membrane-anchored protein, and it is hypothesized that after light exposure the extracellular ZP domain is cleaved at a conserved furin protease cleavage site, a form of posttranslational processing typical for ZP-domain proteins. It is also possible that Qsm signals to Shaw and NKCC in both membrane-bound and cleaved forms. For example, at night membrane-bound Qsm could block NKCC, whereas light-induced cleavage could release this block, and the freed extracellular part could inactivate Shaw. This mechanism is reminiscent to the mechanism by which the GPI-anchored extracellular protein Sleepless increases Shaker (K_V1 channel) activity for regulating Drosophila sleep (Buhl, 2016).

How Qsm-induced changes in clock neuron activity influence the molecular clock remains an open question. Recent work shows that, in addition to the canonical degradation via Cry and Jetlag, Tim is also degraded via a Cul-3 and neuronal activity-dependent pathway in DD that has been implicated in mediating phase delays in the circadian clock. In contrast to this activity-dependent Cul-3 pathway, the light responses in the current study depend on Cry. Therefore a model is favored in which the combined functions of Qsm, Shaw, and NKCC contribute to the canonical Cry- and Jetlag-dependent Tim-degradation pathway (Buhl, 2016).

In conclusion, this study demonstrates that Qsm affects both daily and acute light responses of l-LNvs, and therefore (Qsm) presumably contributes to light-input to the Drosophila circadian clock. Qsm possibly signals downstream of Cry and acts on Shaw and NKCC to change clock neuronal activity in response to light (Buhl, 2016).

The novel gene twenty-four defines a critical translational step in the Drosophila clock

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. This study reports mutants of a novel Drosophila gene twenty-four (tyf; CG4857) that show weak behavioural rhythms. Weak rhythms are accompanied by marked reductions in the levels of the clock protein Period (Per) as well as more modest effects on Timeless (Tim). Nonetheless, Per induction in pacemaker neurons can rescue tyf mutant rhythms. Tyf associates with a 5'-cap-binding complex, poly(A)-binding protein (PABP), as well as per and tim transcripts. Furthermore, Tyf activates reporter expression when tethered to reporter messenger RNA even in vitro. Taken together, these data indicate that Tyf potently activates Per translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock (Lim, 2011).

It remains unclear how TYF controls translation of its target RNAs. Specific effects on Per and Tim were observed but not on other clock components, and Tyf was found to interact with translation components such as the eIF4E-containing cap-binding complex and PABP. It is proposed that RNA-binding translational repressors associate with newly transcribed per RNA, temporarily postpone translation and thus delay Per feedback repression on its own transcription. Such a delay could contribute to the observed lag between protein and RNA particularly in pacemaker neurons, although post-translational mechanisms may also contribute, at least in the eyes. Tyf, which does not have a known RNA-recognition motif, could then be recruited to target transcripts by these translational repressors, releasing them to stimulate initiation of per translation. It has not been possible to biochemically or genetically link Tyf to RNA-binding proteins FMR, LARK, or the translation regulator Thor/4E-BP, which have been shown to contribute to circadian clock function. Nonetheless, TYF association with eIF4E and their similar polysome profiles implicate TYF as a novel translation initiation factor. In addition, the effects of TYF may be more evident on poorly adenylated transcripts on the basis of in vitro data. Of note, the Drosophila homologue of the clock-regulated deadenylase nocturnin has been shown to be important in dorsal neurons for circadian light responses but neither a LN function nor an RNA target has been described. Nevertheless, unique features of Tyf-regulated transcripts may mediate the highly selective TYF effects on clock components in vivo (Lim, 2011).

Post-transcriptional regulation on per RNA has been considered to be modulatory to clock function. The identification of a critical role for Tyf highlights an important role for Per translation in the Drosophila neural clockwork. It will be of interest to determine if proteins functionally analogous to Tyf serve similarly important and specific functions in the mammalian clock (Lim, 2011).

Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription

Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dClock (dClk)-Cycle (Cyc) transcriptional activity by dPeriod (dPer) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPer function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPer interacts with dClk-Cyc to promote repression is not known. This study identified a small conserved region on dPer required for binding to dClk, termed CBD (for dClk binding domain). In the absence of the CBD, dPer is unable to stably associate with dClk and inhibit the transcriptional activity of dClk-Cyc in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the Doubletime kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPer missing the CBD (dPer^δCBD) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPer^δCBD and dClk is Tim-dependent and modulated by light, revealing a novel and unanticipated in vivo role for Tim in circadian transcription. Finally, dPer^δCBD does not provoke the daily hyperphosphorylation of dClk, indicating that direct interactions between dPer and dClk are necessary for the dClk phosphorylation program but are not required for other aspects of dClk regulation (Sun, 2010).

A shared feature of eukaryotic circadian pacemaker mechanisms is that daily cycles in gene expression involve the phase-specific interaction of one or more repressors with core clock transcription factors. Initial findings identified Per proteins in animals and Frequency (Frq) in Neurospora as key components underlying the main negative feedback loops operating in their respective clocks. Early models, mostly based on work in Drosophila and Neurospora, suggested that the direct binding of Per or Frq to their relevant transcription factors was the biochemical mode-of-action underlying these repressors. More recent work is beginning to refine this view and it is now thought that these 'repressors' function, at least partly, by acting as molecular bridges to ensure the timely assembly and/or delivery of larger repressor complexes that inhibit elements functioning in the positive arms of circadian transcriptional feedback loops. Intriguingly, Per and Frq proteins also share another role in that phosphorylation driven changes in their daily levels are central to setting clock pace. Thus, repressors such as Per and Frq act as a critical nexus in clock mechanisms by connecting phosphorylation-based biochemical timers to the regulation of transcription, yielding appropriately phased daily cycles in gene expression. How the binding of these period-setting repressors to core clock transcription factors leads to inhibition in transactivation potential is not well understood (Sun, 2010).

This study identified the C4 region on dPer as the sole or major dClk binding domain (termed CBD). Despite the inability of dPer^δCBD to bind to dClk, p{dper^δCBD} flies manifest quasi-normal feedback circuitry within the core oscillator mechanism, almost certainly as a result of Tim facilitating the close interaction of dPer with dClk, enabling temporal repression of dClk-Cyc-mediated transcription. Thus, Tim is a bona fide component of the in vivo circadian repressor complex, and presumably plays a role in modulating the interaction between dClk and dPer. Moreover, this study reports that direct binding between dClk and dPer is not necessary for dPer's repressor activity, but is likely necessary for normal hyperphosphorylation of dClk (Sun, 2010).

An approach that is providing insights into how these 'phospho-timing repressor' clock proteins function is the identity of regions required for promoting transcriptional inhibition. In Drosophila, early work mapped a region on dPer necessary for strong inhibition of dClk/Cyc activity in an S2 cell transcription assay. This region, termed the CCID domain, contains two highly conserved regions, namely C3 and C4. The C3 region contains the sole or major domain on dPer required for stable interaction with Dbt, termed the dPDBD, and is critical not only for hyperphosphorylation of dPer but also for inhibiting dClk-Cyc-mediated transcription, despite the fact that eliminating this region does not abrogate the ability of dPer to stably interact with dClk in vivo. In contrast, the newly identified CBD is required for the physical interaction of dPer with dClk. Thus, the CCID is comprised of at least two distinct regions with different biochemical modes-of-action; a region required for physical interaction with dClk and another that functions as a scaffold to promote binding of regulatory factors that modulate the activity/metabolism of dClk/Cyc (Sun, 2010).

An unanticipated aspect of this work is that tim can promote the binding of dPer^δCBD to dClk in a simplified cell culture system and in flies, an event that rescues dPer's repressor function. Earlier work suggested that Tim is dispensable for repression of dClk/Cyc transactivity. However, although Tim exhibits little to no repressor activity toward dClk-Cyc-mediated transcription in S2 cells, it enhances that of dPer. This enhancement was largely attributed to the fact that Tim stimulates the nuclear localization of dPer. The current findings suggest a physiological role for Tim in modulating circadian transcription by regulating the interaction between dPer and dClk. Although dPer can bind dClk in the absence of Tim, it is possible that by interacting with both dPer and dClk, Tim influences the properties of the repressor complex. For example, Tim might modulate the conformation of dPer, enhancing the assembly/activity of the repressor complex. In support of this view, brief light stimulation in the night leads to subtle but noticeable effects on dper/tim RNA levels that precede significant changes in the abundance of dPer. Of particular interest, dper/tim RNA levels were induced by light exposure in flies expressing a dPer variant (named dPer-δC2) missing a short stretch of conserved amino acids (515-568). Although the basis for the impaired function of dPer-δC2 was not clear, based on the current results it is possible that its interaction with dClk/Cyc is defective in the absence of Tim (Sun, 2010).

The findings that Tim has a more pivotal role in transcriptional regulation might also be relevant to a recent study suggesting a two-step mechanism for dPer-mediated inhibition of dClk-Cyc activity, whereby during the first phase of inhibition dPer is bound to dClk at the chromatin, followed by a second off-DNA sequestration of dClk by dPer (Menet, 2010). The switch from an on-DNA to an off-DNA mechanism is thought to occur around ZT18, around the time tim begins to accumulate in the nucleus. It is speculated that Tim might regulate progression from an on-DNA to an off-DNA inhibitory mechanism by modulating the interaction between dPer and dClk/Cyc (Sun, 2010).

Although dPer^δCBD in the presence of Tim can suppress dClk-Cyc-mediated transcription, the efficiency is lower compared with that of wild-type dPer. Several different scenarios could account for this, including less favorable spatial alignment between dPer^δCBD and dClk/Cyc and/or effects on the ability of dPer^δCBD to bind and/or deliver regulatory factors to dClk/Cyc. Perhaps a more interesting possibility is suggested by the less extensive phosphorylation of dClk in p{dper^δCBD} flies. Hyperphosphorylated dClk is mainly detected in the late night/early day during times when dClk-Cyc transcriptional activity is inhibited, suggesting that highly phosphorylated dClk is less active. The absence of hyperphosphorylated isoforms of dClk in p{dper^δCBD} flies suggests that direct association between dPer and dClk is required to provoke dPer-dependent dClk phosphorylation. The lack of hyperphosphorylated isoforms of dClk in p{dper^δCBD} flies might also contribute to the higher overall levels of dper/tim transcripts. In this context it is noteworthy that Frq is thought to play a major role in repressing the positive limb of the circadian transcriptional circuits in Neurospora by regulating the phosphorylated state of the WCC complex, the key clock transcription factor driving cyclical gene expression in that system. Future studies will be required to better understand the biochemical function of dClk phosphorylation (Sun, 2010).

In summary, these findings demonstrate that the direct interaction of a key repressor to its target transcription factors is not necessary for its ability to engage in transcriptional inhibition. Moreover, Tim can promote the close association of dPer to dClk in a manner that sustains dPer's repressor capability, revealing a more direct role for tim in functional interactions between the negative and positive limbs of the circadian transcriptional feedback circuits operating in Drosophila. The dClk interaction domain on dPer is situated very close to the dPDBD region that functions, at least partly, by acting as a bridge to promote close interactions between regulatory factors (such as Dbt) and the dClk/Cyc complex. The close spacing on dPer between these two functional regions suggests that complex architectural constraints need to be met to assemble highly efficient repressor complexes. It will be of interest to determine whether other repressors, such as Per proteins in mammals and Frq in Neursopora, also have similar spatial arrangements. Intriguingly, in mammals the C-terminal region of mPer2, which is downstream of the casein kinase binding (CKB) domain, has been reported to be involved in directly binding to BMAL1 (Chen, 2009). Finally, the current findings strongly suggest that direct interactions between dPer and dClk/Cyc are required for dClk hyperphosphorylation. It is possible that some regulatory factors stay tightly bound to key clock repressors (such as Dbt to dPer) and thus require very close contact with central clock transcription factors to modulate them, whereas other factors are 'delivered' and establishing a high local concentration is sufficient to promote efficient transfer from the repressors to circadian-relevant transcription complexes (Sun, 2010).

AKT and TOR signaling set the pace of the circadian pacemaker

The circadian clock coordinates cellular and organismal energy metabolism. The importance of this circadian timing system is underscored by findings that defects in the clock cause deregulation of metabolic physiology and result in metabolic disorders. On the other hand, metabolism also influences the circadian clock, such that circadian gene expression in peripheral tissues is affected in mammalian models of obesity and diabetes. However, to date there is little to no information on the effect of metabolic genes on the central brain pacemaker which drives behavioral rhythms. This study found that the AKT and TOR-S6K pathways, which are major regulators of nutrient metabolism, cell growth, and senescence, impact the brain circadian clock that drives behavioral rhythms in Drosophila. Elevated AKT or TOR activity lengthens circadian period, whereas reduced AKT signaling shortens it. Effects of TOR-S6K appear to be mediated by SGG/GSK3beta, a known kinase involved in clock regulation. Like SGG, TOR signaling affects the timing of nuclear accumulation of the circadian clock protein Timeless. Given that activities of AKT and TOR pathways are affected by nutrient/energy levels and endocrine signaling, these data suggest that metabolic disorders caused by nutrient and energy imbalance are associated with altered rest:activity behavior (Zheng, 2010).

There are several possible mechanisms by which nutrient and energy metabolism could affect peripheral clocks. Local physiological factors dependent on metabolic activity could influence the expression of core clock components and of nuclear receptors that regulate clock gene expression. Indeed, cellular redox state, AMPK activity, NAD+ levels, and SIRT1 activities appear to feed into the circadian clock in peripheral tissues such as the liver. AMPK, which acts upstream of TSC in mammals, directly phosphorylates Cryptochrome in peripheral tissues. However, prior to this work, there was no known mechanism for the modulation of the central pacemaker by nutrient-sensing pathways. This study identifies such a mechanism by demonstrating that metabolic genes such as AKT and TOR-S6K act in the central pacemaker cells in the brain. The lengthened circadian period caused by high-fat diet in mammals is likely mediated by these molecules. This conclusion is further supported by a recent cell-culture-based genome-wide RNAi study that implicated the PI3K-TOR pathway in the regulation of circadian period. In addition, another ribosomal S6 kinase (S6KII) was found to influence the circadian clock through its interaction with casein kinase 2β. Importantly, daily fasting:feeding cycles driven by the central clock regulate circadian gene transcription in the liver, whereas clock function in the liver contributes to energy homeostasis. It is speculated that metabolic stress or energy imbalance affects AKT and TOR-S6K signaling, resulting in general circadian disruption, which in turn exacerbates metabolic deregulation and, consequently, facilitates the development of metabolic syndromes prevalent in modern society (Zheng, 2010).

Cullin-3 controls Timeless oscillations in the Drosophila circadian clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the Period and Timeless proteins accumulate during the night, inhibit the activity of the Clock (Clk)/Cycle (Cyc) transcriptional complex, and are degraded in the early morning. The control of Per and Tim oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. Slmb, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated Per. This study shows that Cullin-3 (Cul-3) is required for the circadian control of Per and Tim oscillations. Expression of either Cul-3 RNAi or dominant negative forms of Cul-3 in the clock neurons alters locomotor behavior and dampens Per and Tim oscillations in light-dark cycles. In constant conditions, Cul-3 deregulation induces behavioral arrhythmicity and rapidly abolishes Tim cycling, with slower effects on Per. Cul-3 affects Tim accumulation more strongly in the absence of Per and forms protein complexes with hypo-phosphorylated Tim. In contrast, Slmb affects Tim more strongly in the presence of Per and preferentially associates with phosphorylated Tim. Cul-3 and Slmb show additive effects on Tim and Per, suggesting different roles for the two ubiquitination complexes on Per and Tim cycling. This work thus shows that Cul-3 is a new component of the Drosophila clock, which plays an important role in the control of Tim oscillations (Grima, 2012).

Flavin reduction activates Drosophila cryptochrome

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation (Vaidya, 2013).

period and timeless mRNA splicing profiles under natural conditions in Drosophila melanogaster

Analysis of Drosophila circadian behavior under natural conditions has revealed a number of novel and unexpected features. This study focused on the oscillations of per and tim mRNAs and their posttranscriptional regulation and observe significant differences in molecular cycling under laboratory and natural conditions. In particular, robust per mRNA cycling from fly heads is limited to the summers, whereas tim RNA cycling is observed throughout the year. When both transcripts do cycle, their phases are similar, except for the very warmest summer months. The natural splicing profiles of per and tim transcripts were also studied and a clear relationship was observed between temperature and splicing. In natural conditions, the relationship between accumulation of the per^spliced variant, low temperature, and the onset of the evening component of locomotor activity, first described in laboratory conditions, was also confirmed. Intriguingly, in the case of tim splicing, the opposite relationship was observed, with tim^spliced expression increasing at higher temperatures. A first characterization of the 4 different TIM protein isoforms (resulting from the combination of the natural N-terminus length polymorphism and the C-terminus alternative splicing) using the 2-hybrid assay showed that the TIM^unspliced isoforms have a stronger affinity for CRY, but not for PER, suggesting that the tim 3' splicing could have physiological significance, possibly in temperature entrainment and/or adaptation to seasonal environments (Montelli, 2015).

Calcium and SOL protease mediate temperature resetting of circadian clocks

Circadian clocks integrate light and temperature input to remain synchronized with the day/night cycle. Although light input to the clock is well studied, the molecular mechanisms by which circadian clocks respond to temperature remain poorly understood. This study found that temperature phase shifts Drosophila circadian clocks through degradation of the pacemaker protein Tim. This degradation is mechanistically distinct from photic Cry-dependent Tim degradation. Thermal Tim degradation is triggered by cytosolic calcium increase and Calmodulin binding to Tim and is mediated by the atypical calpain protease Sol. This thermal input pathway and Cry-dependent light input thus converge on Tim, providing a molecular mechanism for the integration of circadian light and temperature inputs. Mammals use body temperature cycles to keep peripheral clocks synchronized with their brain pacemaker. Interestingly, downregulating the mammalian Sol homolog SOLH blocks thermal mPER2 (see Drosophila Per) degradation and phase shifts. Thus, it is proposed that circadian thermosensation in insects and mammals share common principles (Tataroglu, 2015).

Temperature compensation and temperature sensation in the circadian clock

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. This study shows using timeless luciferase reporter measurements and Western blots against Timeless protein that this prediction is satisfied by the Drosophila circadian clock. Evidence is reviewed for pathways that couple temperature to the circadian clock; previously unidentified evidence is shown for coupling between the Drosophila clock and the heat-shock pathway (Kidd, 2015).

GSK-3 β does not stabilize Cryptochrome in the circadian clock of Drosophila

Cryptochrome (CRY) is the primary photoreceptor of Drosophila's circadian clock. It resets the circadian clock by promoting light-induced degradation of the clock protein Timeless (TIM) in the proteasome. Under constant light, the clock stops because TIM is absent, and the flies become arrhythmic. In addition to TIM degradation, light also induces CRY degradation. This depends on the interaction of CRY with several proteins such as the E3 ubiquitin ligases Jetlag (JET) and Ramshackle (BRWD3). However, CRY can seemingly also be stabilized by interaction with the kinase Shaggy (SGG), the GSK-3 beta fly orthologue. Consequently, flies with SGG overexpression in certain dorsal clock neurons are reported to remain rhythmic under constant light. This study investigated the interaction between CRY, Ramshackle and SGG and started to perform protein interaction studies in S2 cells. Surprisingly, it was not possible to replicate the results that SGG overexpression does stabilize CRY, neither in S2 cells nor in the relevant clock neurons. SGG rather does the contrary. Furthermore, flies with SGG overexpression in the dorsal clock neurons became arrhythmic as did wild-type flies. Nevertheless, the published interaction of SGG with TIM was reproducible, since flies with SGG overexpression in the lateral clock neurons shortened their free-running period. It is concluded that SGG does not directly interact with CRY but rather with TIM. Furthermore, it was demonstrated that an unspecific antibody explains the observed stabilization effects on CRY (Fischer, 2016).

GSK-3 and CK2 kinases converge on Timeless to regulate the master clock

The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, this study identified a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro also delay nuclear accumulation in both tissue culture and in vivo and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity (Top, 2016).

Ubiquitylation dynamics of the clock cell proteome and TIMELESS during a circadian cycle

Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape. An in vivo ubiquitin labeling assay was used to investigate the circadian ubiquitylated proteome of Drosophila melanogaster. Cyclic ubiquitylation affects Megator (Mtor), a chromatin-associated nucleoporin that, in turn, feeds back to regulate the core molecular oscillator. Furthermore, the ubiquitin ligase subunits Cullin-3 (Cul-3) and Supernumerary limbs Slbm) cooperate for ubiquitylating the Timeless protein. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system (Szabo, 2018).

Photosensitive alternative splicing of the circadian clock gene timeless is population specific in a cold-adapted Fly, Drosophila montana

To function properly, organisms must adjust their physiology, behavior and metabolism in response to a suite of varying environmental conditions. One of the central regulators of these changes is organisms' internal circadian clock, and recent evidence has suggested that the clock genes are also important in the regulation of seasonal adjustments. In particular, thermosensitive splicing of the core clock gene timeless in a cosmopolitan fly, Drosophila melanogaster, has implicated this gene to be involved in thermal adaptation. To further investigate this link, the splicing of timeless was examined in a northern malt fly species, Drosophila montana, which can withstand much colder climatic conditions than its southern relative. Northern and southern populations from two different continents (North America and Europe) were examined to find out whether and how the splicing of this gene varies in response to different temperatures and day lengths. Interestingly, it was found that the expression of timeless splice variants was sensitive to differences in light conditions, and while the flies of all study populations showed a change in the usage of splice variants in constant light compared to LD 22:2, the direction of the shift varied between populations. Overall, these findings suggest that the splicing of timeless in northern Drosophila montana flies is photosensitive, rather than thermosensitive and highlights the value of studying multiple species and populations in order to gain perspective on the generality of gene function changes in different kinds of environmental conditions (Tapanainen, 2018).

mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

Circadian rhythms in metazoan eukaryotes are controlled by an endogenous molecular clock. It functions in many locations, including subsets of brain neurons (clock neurons) within the central nervous system. Although the molecular clock relies on transcription/translation feedback loops, posttranscriptional regulation also plays an important role. This study shows that the abundant Drosophila melanogaster microRNA mir-276a regulates molecular and behavioral rhythms by inhibiting expression of the important clock gene timeless (tim). Misregulation of mir-276a in clock neurons alters tim expression and increases arrhythmicity under standard constant darkness (DD) conditions. mir-276a expression itself appears to be light-regulated because its levels oscillate under 24-h light-dark (LD) cycles but not in DD. mir-276a is regulated by the transcription activator Chorion factor 2 in flies and in tissue-culture cells. Evidence from flies mutated using the clustered, regularly interspaced, short palindromic repeats (CRISPR) tool shows that mir-276a inhibits tim expression: Deleting the mir-276a-binding site in the tim 3' UTR causes elevated levels of TIM and approximately 50% arrhythmicity. It is suggested that this pathway contributes to the more robust rhythms observed under light/dark LD conditions than under DD conditions (Chen, 2016).

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