timeless REGULATION Protein Interactions and Post-transcriptional Regulationtimeless was cloned by chromosomal walking and subsequently used, in yeast, to identify Per as a physical partner. Timeless and Period interact, and both are required for production of circadian rhythms. The tim gene encodes a protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the Per dimerization motif (PAS) indicates that direct interaction between Per andTim would require a heterotypic protein association (Meyers, 1995).Tim was isolated based on its ability to physically interact with the Per protein. A restricted segment of Tim binds directly to PAS, a part of the Per dimerization domain. PerL, a mutation in Per that causes a temperature-sensitive lengthening of circadian period and a temperature-sensitive delay in Per nuclear entry, exhibits a temperature-sensitive defect in binding to Tim (Gekakis, 1995).Tim and Per accumulate in the cytoplasm when independently expressed in cultured (S2) Drosophila cells. If coexpressed, however, the proteins move to the nuclei of these cells. Domains of Per and Tim have been identified that block nuclear localization of the monomeric proteins. These regions of Per and Tim interaction consist of the PAS domain of Per and an adjacent domain also required for cytoplasmic localization (CLD). The sequence of Tim involved in interaction with Per resides between amino acids 505 to 578. Tim and Per both contain domains required for cytoplasmic localization. The site in Per required for nuclear localization is a sequence between amino acids 453 and 511. The sequence of Tim required for cytoplasmic localization (the Tim CLD) is C-terminal. It is thought that the CLD interacts with a cytoplasmic factor that inhibits nuclear localization. The results indicate a mechanism for controlled nuclear localization in which suppression of cytoplasmic localization is accomplished by direct interaction of Per and Tim. No other clock functions are required for nuclear localization. The findings suggest that a checkpoint in the circadian cycle is established by requiring cytoplasmic assembly of a Per/Tim complex as a condition for nuclear transport of either protein (Saez, 1996). To investigate the mechanism of phase shifing of circadian clocks by light stimulation, the effects of light pulses on the protein and messenger RNA products of the Drosophilaclock gene period (per) were measured. Photic stimuli perturb the timing of the Per protein andmessenger RNA cycles in a manner consistent with the direction and magnitude of the phase shift.The recently identified clock protein Timeless interacts with Per in vivo, andthis association is rapidly decreased by light. This disruption of the Per-Tim complex in thecytoplasm is accompanied by a delay in Per phosphorylation and nuclear entry and disruption inthe nucleus by an advance in Per phosphorylation and disappearance. These results suggest amechanism for how a unidirectional environmental signal elicits a bidirectional clock response (Lee, 1996). Many circadian features of the Tim cycle resemble those of the Per cycle. However, Tim israpidly degraded in the early morning or in response to light, releasing Per from the complex. ThePer-Tim complex is a functional unit of the Drosophila circadian clock, and Tim degradation maybe the initial response of the clock to light (Zeng, 1996). Drosophila Clock protein (dClock) is a transcription factor that is required for the expression of the circadianclock genes period (per) and timeless (tim). dClock undergoes circadian fluctuations in abundance, is phosphorylated throughout a daily cycle, andinteracts with Per, Tim, and/or the Per-Tim complex during the night but not during most of the day. Both Per and Tim copurify with dClock in a time-of-day-specific manner: Per and Timare first detected at ZT12 (beginning of the dark period), followed by increases in amounts that reach peak values at ZT23.9 (just before the lights go on). Between ZT16 (a third of the way through lights off) and ZT23.9, the amounts of all three proteins in immune complexes increase, even though the totallevels of Tim and Per in head extracts peak at ZT16 and ZT20, respectively. This suggests that during thenight dClock is present in limiting amounts compared to Per and Tim. Despite the higher levels ofimmunoprecipitated dClock between ZT4 and ZT8 compared to values obtained between ZT12 and ZT16, very little, ifany, Per and Tim are detected. A likely explanation forthis is that between ZT4 and ZT8 the total levels of Per and especially those of Tim are at, or close to, trough values. Thus, the interaction of Per and Tim with dClock is mainly restricted to nighttime hours (Lee, 1998). Analysis of immune complexes derived from a period mutant clearly indicate that in the absence of Per, Tim canstill interact with dClock. Because Tim is apparently located exclusively in thecytoplasm in the absence of Per, this result could suggest that thenuclear localization of dClock also requires Per or a functional oscillator. Alternatively, low levels of Tim might beable to enter the nucleus in the absence of Per. In contrast, several attempts to visualize a specific interaction betweenPer and dClock in the absence of Tim were unsuccessful. There are atleast two nonmutually exclusive reasons that might account for thr inability to detect Per in dClock-containingimmune complexes prepared from tim mutant flies: (1) the levels of Per are very low in tim mutant flies and as such the amounts of Per that copurify with dClock are below thedetection limit, and (2) the interaction of Per with dClock requires Tim, possibly via formation of the Per-Timcomplex and/or a dependence for nuclear localization (Lee, 1998 and references).Attempts were made to measure the relative amounts of dClock that interact with Per and Tim as a function of time in anLD cycle. Head extracts were incubated with antibodies against either Per or Tim, and immune complexes probed fordClock, Per, and Tim. At ZT20 almost identical levels of dClock copurify with antibodies directedagainst either Per or Tim. Equivalent amounts of Per were also present in both immunepellets, but 1.6-fold more Tim is immunoprecipitated with antibodies toTim, as compared to those directed against Per. These results are almost identicalwith a previous study showing that (1) in head extracts prepared from flies collected at ZT20, 80% of thetotal amount of Per is bound to Tim in a 1:1 stoichiometric relationship, and (2) there is 1.5-1.8 times more Tim, as compared to Per. Thus, the current results suggest that at ZT20 the majority of the Per and Timproteins that interact with dClock are in the form of a heterodimeric Per-Tim complex. During the early day, only lowlevels of dClock are detected in immune complexes obtained using either antibodies to Per or Tim, in agreement with results using anti-dClock antibodies. Furthermore,it is mainly versions of Per and Tim that are essentially free of one another that interact with dClock during the earlyday (Lee, 1998). How might a trimeric complex containing Per, Tim, and dClock be assembled? Presumably the HLH domain ofdClock does not participate in mediating protein-protein interactions in this putative trimeric complex, because neitherPer nor Tim seems to have a similar dimerization region. The only other regions that have been shown to mediateprotein-protein interactions are the PAS domain found in Per and dClock and a not so well characterized region in Tim thatspans 400 amino acids and interacts with the PAS domain of Per. It is tempting tospeculate that one or both of these domains has the capacity to engage in at least trimeric formation. Although these studiesdo not address the nature of the trimeric interaction, they indicate that PAS-containing proteins are not limited to binary interactions (Lee, 1998). These results suggest that Per and Tim participate intranscriptional autoinhibition by physically interacting with dClock or a dClock-containing complex. Nevertheless, in the absence of Per or Tim, thelevels of dClock are constitutively low, indicating that Per and Tim also act as positive elements in the feedback loop by stimulating the production ofdClock. Although Per and Tim inhibit dClock activity, Per and Tim arerequired for the high-level production of dClock protein and mRNA. Thus, Per andTim appear to be the main "motor" of the Drosophila circadian oscillator, driving both positive and negative elements ofthe transcriptional-translational feedback loop. These observations suggest an explanation for the previously unexplainedfinding that the levels of Per mRNA in per mutant flies are approximately half as high as those obtained at peak times inwild-type flies. In contrast, mutations that abolish Neurospora FRQ activity result in high levels of frqRNA, suggesting that the frq-based circadian oscillator in Neurospora is based on a more simple negativetranscriptional feedback loop. How Per and Tim stimulate dClock expression is not clear.They may interact with other transcription factors and act as coactivators. Alternatively, they may block the function ofnegative factors leading to the stimulation of gene expression. In addition to regulating the transcriptional activity of thedClock-CYC complex, Per and Tim might also interact with other transcription factors that are not involved in thecircadian oscillator and as such molecularly couple the timekeeping mechanism to downstream effector pathways (Lee, 1998). The cyclic expression of the Period (Per) and Timeless (Tim)proteins is critical for the molecular circadian feedback loop inDrosophila. The entrainment by light of the circadian clock ismediated by a reduction in Tim levels. To elucidate the mechanism ofthis process, the sensitivity of Tim regulation by light was testedin an in vitro assay with inhibitors of candidate proteolyticpathways. The data suggest that Tim is degraded through aubiquitin-proteasome mechanism. In addition, in cultures fromthird-instar larvae, Tim degradation is blocked specifically byinhibitors of proteasome activity. Degradation appears to bepreceded by tyrosine phosphorylation. Finally, Tim is ubiquitinatedin response to light in cultured cells (Naidoo, 1999).An in vitro assay was developed to investigatethe nature of the Tim light response.Flies were entrained to a 12 hour light/12 hour dark cycle andprepared head extracts from flies collected at either ZT (zeitgebertime) 20 or immediately after a 1-hour light pulse delivered atZT 19 (ZT0 = lights on; ZT12 = lights off).These extracts were incubated with Tim protein immunoprecipitatedfrom fly heads. After a 1-hour incubation at room temperature, Tim levels were assayed by protein immunoblots. Addition of the pulsedextract reduces the Tim signal. Unpulsedhead extract has no effect on the level of Tim, indicating that thereduction is light-specific. This light-induced reduction is alsoobserved in tim0 flies (which lack Tim protein) andis, in fact, routinely higher in these flies, which may suggest somedown-regulation by the clock in wild-type flies. Animmunoprecipitated Per substrate is not degraded by addition of thepulsed extract (Naidoo, 1999).In order to determine the nature of the proteolytic activity, several general classes of protease inhibitors were assayed. Inhibitors ofserine proteases [phenylmethylsulfonyl fluoride (PMSF) and aprotinin]and aspartate proteases (pepstatin) are not very effective inblocking Tim degradation. However,degradation is inhibited by the proteasomal inhibitorsacetyl-leu-leu-norleucinal (ALLN), cbz-leu-leu-norvalinal(ZL2NVaH or MG115) and cbz-leu-leu-leucinal(ZL3H or MG132). These peptidealdehydes strongly inhibit the chymotryptic activity of theeukaryotic 26S proteasome. Timdegradation is also blocked by bestatin, a metalloproteaseinhibitor, and by leupeptin, which inhibits cysteine proteases andhas some effects on other proteolytic systems, including theproteasome. The precise mechanism of actionin this case is not known. Consistent with a role for the proteasome,depletion of ubiquitin from the extract blocks Timdegradation (Naidoo, 1999). Although the in vitroassay indicates a mechanism for Tim's response to light, itsusefulness is limited by its variability.To verify the findings of this assay, an in vivosystem was developed. Thus, a primary culture assay was used to test the effect of two proteasomal inhibitors (lactacystinand MG115) on the Tim light response. Lactacystin, a microbialmetabolite, is the most specific one known; a naturally occuring inhibitorof the proteasome. It spontaneouslyhydrolyzes into clastolactacystin B lactone, which is the activespecies that reacts with the proteasome, inhibiting its chymotrypticand tryptic peptidase-like activity. MG115 is a potent synthetic peptide aldehydeinhibitor. For the assay, the centralnervous system (CNS) of third-instar larvae was dissected and maintained in culture medium for1 hour. Some samples were exposed to a pulse of light for20 min and were fixed at the end of the hour. Dark controlsamples were also incubated for an hour in the dark. Tim expression was then examined in thelateral neurons (clock cells), which were located by costaining withan antibody to pigment-dispersing hormone. Strong Tim staining is seen in lateral neurons ofunpulsed tissue, but little to no Tim in CNS tissue that has receiveda light pulse. The effect of inhibitors was tested by adding them to theculture medium at the start of the incubation. Tissue treated withlactacystin and MG115 before the light pulse revealed robust Timstaining in the lateral neurons. The strong inhibition by MG115 isconsistent with a report that this is a much more effective inhibitorof proteolysis in intact cells than it is of in vitro hydrolysis ofmacromolecular substrates. The 100%block by lactacystin may reflect variable permeability or instabilityof the lactone metabolite (Naidoo, 1999). Proteasomes are multicatalytic, multisubunit proteolytic complexeswith highly conserved structures; they play a key role in avariety of cellular processes, including the cell cycle,transcriptional regulation, removal of abnormal proteins from thecell, antigen presentation, and even in theturnover of a mammalian circadian-regulated protein. The Tim response to light is blockedspecifically, in two different assays, by several inhibitors of theproteasome; this is important, given that lactacystin, which wasthought to affect only the proteasome, has been shown to also act on a second multisubunit enzyme. Because the newly identified enzyme isinsensitive to ALLN, it cannot account for the Tim response. For the ubiquitin-proteasome system, prolineglutamate serine threonine (PEST) regions sometimes serve as putativedegradation/phosphorylation signals in the target molecule. The Tim protein sequence reveals the presenceof seven PEST regions concentrated near the NH2 and COOHtermini (Naidoo, 1999). Most cellular proteins that are degraded by theproteasome are ubiquitinated and then targeted to the proteasome. To determine whether Tim is ubiquitinated,which would also demonstrate that it is a direct target of theproteasome, a cell culture system was used. Tim and ahemagglutinin (HA)-tagged ubiquitin octamer were expressed under heat shock control in Drosophila S2 cells. After a30-min heat shock, cells were either maintained in the dark ortreated with light for 2 hours, after which the cells were lysedand immunoprecipitates of Tim were probed with an antibody to HA. Tim was found to be ubiquitinated in response to light. The effect is specific for Tim, because Peris not ubiquitinated with or without light treatment. Extended light treatment also degrades Tim inthese cells, and this degradation is inhibited by the proteasomeinhibitor MG115. Although these data implicate a ubiquitin-proteasomal mechanism,they do not preclude a role for other proteolyic systems (Naidoo, 1999). To investigatea possible role for phosphorylation in the degradation of Tim, the effect of several kinase inhibitors in the in vivoprimary culture assay were examined. The tyrosine kinase inhibitor genisteinblocks the degradation of Tim in the lateral neurons after a pulseof light, whereas the serine-threonine inhibitors staurosporin andcalphostin C and the MEK inhibitor PD98059 do not. These results suggest that tyrosine kinaseactivity precedes degradation of Tim. The concentrations of genisteinthat were effective in this assay suggest ac-src-like kinase activity, althoughthe concentration dependence must be interpreted with caution,because it could be a measure of permeability or drug stability (Naidoo, 1999).To determine whether the tyrosine phosphorylation occurs on Timitself, protein immunoblots of Tim immunoprecipitates were probed withan antibody to phosphotyrosine. After 20 min of light treatmentat ZT19, Tim could be detected with the antibody tophosphotyrosine. Tim in the 'dark'samples is sometimes detected with this antibody but notconsistently, which suggests that tyrosine phosphorylation of Tim isincreased by light. The mobility of the Tim band in the light-treatedsample is also reduced, presumably because of phosphorylation (Naidoo, 1999). Together, these data indicate that the Tim response to lightinvolves tyrosine phosphorylation and ubiquitination, followed byproteasomal degradation. What then is the role of the proteasome pathway infree-running behavioral rhythms? Are the mechanisms that degrade Timin response to light the same as those that degrade it in constantdarkness? If this is the case, light may serve only to further activate aprocess that is already under way. It is proposed that cyclic turnover ofTim under free-running conditions is mediated by phosphorylation,which targets it for degradation, perhaps by the proteasome. Tim isprogressively phosphorylated throughout the night, and maximallyphosphorylated forms are found just before the rapid decline ofprotein levels. From this point on, untilthe middle of the day, Tim levels remain low because of the lowlevels of RNA. As the repression of transcription is released, mostlikely because of the decrease in Per levels, RNA accumulates andprotein also starts to accumulate, albeit slowly, because it is stillsubject to phosphorylation and degradation. When the rate of Timsynthesis exceeds the rate of phosphorylation/degradation, higherlevels of protein are observed, but as the phosphorylation programcontinues and RNA levels are reduced (because of negative feedback),levels of the protein drop off. Light could enhance Tim degradationby increasing Tim phosphorylation and/or by increasing proteolytic activityin some manner. This model would predict that the presenceof light accelerates the falling phase of the protein and delays therising phase, both through the same mechanism (Naidoo, 1999).Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggestsinvolvement 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 morninghours, 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 resultsand 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 isunnecessary for the feedback regulation of per and tim protein on per and tim transcription (Suri, 2000). Bothmutations, when presented in the context of the highly similar yeastcasein kinase I HRR25, severely reduce kinase activity on peptidesubstrates. The long-period phenotypes are likely caused byinsufficient Dbt activity, so it takes longer to reach some requiredlevel of Per phosphorylation. It is also assumed that both mutants areexpressed at a level similar to that of wild-type Dbt (Suri, 2000).Both dbth andDbtg/+ have 29 hr periods and aresimilar in all other respects, suggesting that the phenotypes are notidiosyncratic features of the mutations but reflect the role of Dbt inthe pacemaker. Although the mutant flies entrain to imposed 24 hrphotoperiods, the LD locomotor activity patterns indicate that there isno anticipation of the morning or evening light/dark transitions, andthe evening activity peak is delayed by several hours into the night.The altered LD patterns are probably a consequence of the longerperiods. Indeed, flies that carry pers as well asdbth have a period of 22.5 hr andmanifest robust anticipation of both morning and evening transitions aswell as an advanced evening activity peak. Both dbt mutantLD profiles resemble that of the 29 hr periodperl mutant strain, consistent withthis altered period notion (Suri, 2000).The molecular features of the perlcircadian program are difficult to compare with those of wild-typeflies, because the mutant rhythms are weak and of low amplitude as wellas long period even under 12 hr LD entraining conditions. In contrast, Per and Tim cyclingin the long-period dbt mutants is robust. Protein levels arecomparable with those in wild-type flies during the night, and levelsin the two mutant strains appear even higher than wild-type levelsduring the daytime. Previous work suggests a role forDbt-catalyzed phosphorylation in targeting Per for degradation: thisprobably reflects slower protein turnover during the morning in thedbt mutants. The Tim phosphorylation pattern in the mutantsdid 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 thesestrains. Although this might reflect phosphorylation events that nevertake place in a wild-type background, less active Dbt mutants might beexpected to depress the magnitude as well as the kinetics of thetemporal phosphorylation program. This suggests that Per might not be adirect Dbt substrate in vivo but is only influencedindirectly, through intermediates that are direct Dbt targets. Forexample, Dbt may phosphorylate and activate a direct Per kinase or aspecific protease. In this context, Per has not yet been shown to be adirect Dbt substrate. It is also possible that Dbt is a functionallyrelevant but minor Per kinase. In this case, the bulk of the Permobility shift on SDS-PAGE is a consequence of other kinases. BecausePer persists for several hours longer in the mutants than in wild-typeflies, the other kinases would continue to function and give rise toeven more highly phosphorylated species than are usually observed.These would be an indirect consequence of weak dbt activityand delayed degradation. A final possibility is that the enhanced anddelayed Per phosphorylation simply reflects some misregulation of Dbt activity (Suri, 2000). Careful analysis of the Per and Tim protein profiles in the long-perioddbt mutants suggests that Dbt acts in the late night andmorning phase of the molecular cycle: the mutants leave the earlyevening protein profile almost unaltered. This indicates thatdbt probably targets nuclear, monomeric Per. It hasalso been suggested that Dbt acts in the early night to destabilize cytoplasmic Per, thus delaying nuclear entry and repression. The dbt mutants reportedhere do not significantly change this early night, presumptivecytoplasmic phase of accumulation. It is possible that Dbt prefers freePer over Per complexed to Tim. If free Per is a better substrate, thenDbt mutants should show a greater effect in the late night and earlymorning, after a large fraction of Tim has disappeared. Alternatively,Dbt might influence only marginally the Per accumulation phase for someother reason. But dbt mutant larvae accumulate high levelsof hypophosphorylated Per, which suggests that Dbt is the major Perkinase 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 normalPer-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 anddbt mutant flies. Both mutant profiles are delayed by 4-5hr. This is presumably because of the delayed disappearance of Per aswell as Tim, which has been suggested to repress per andtim transcription. This relationship is very similar to thatpreviously reported for the perSmutant strain; in this case, the clock proteins disappear more quickly,leading to an advance in the RNA profiles. The perSeffect is more pronounced on Per than on Tim, consistent with thenotion that monomeric Per might be the major transcriptional repressor. In any case, comparable results in the threemutants indicate a solid relationship between the timing of the declinein protein levels and the timing of the subsequent increase inper 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 substantialbuildup of Per-Tim complexes and the consequent nuclear transport of the dimeric Per-Tim complex. In the nucleus, Per destabilizationrelieves repression. In Dbt mutants, Per degradation is much slower.This prolongs repression and delays the per andtim mRNA upswing in the next cycle (Suri, 2000). There is an impressive relationship between the per andtim RNA profiles in comparison to the evening locomotoractivity peak. In all cases, these RNA and locomotor activity begin to increase atapproximately the same time, i.e., around ZT7 in the middle of thedaytime. Mutants or physiological manipulations that affect the timing of the RNA profiles affect the timing of the evening activity peak inparallel. This fits withthe emerging view, from mammalian as well as Drosophila work, that cycling transcription plays an important role incircadian output as well as within the central pacemaker oscillator. A further implication of theserelationships is that the protein oscillations from one day affectbehavior as well as the RNA profiles on the next one: the morningdecline and eventual disappearance of Per and Tim terminate a proteincycle from the previous day, which then causes the subsequent increasesin both RNA levels and locomotor activity (Suri, 2000). In contrast, the delayed Per and Tim disappearance in the mutants haslittle 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 asthey do in the wild-type flies (ZT19-ZT21). Because of the delayed RNArise in the mutants, the per and tim RNAaccumulation profiles almost coincide with those of the proteins,between ZT15 and ZT21. This indicates that the timing of the RNA riseis insufficient to time the protein rise. The increase in proteinlevels may reflect protein half-life regulation, which is uncoupledfrom the underlying mRNA levels, at least under some circumstances (Suri, 2000). The coincidence of the protein and RNA curves also raises doubts aboutthe importance of the 4-6 hr lag between these two accumulationprofiles. The data presented in this study indicate that the lag is dispensable forrobust behavioral and molecular oscillations. This is especiallyrelevant for the RNA fluctuations. Despite evidence that at leastper mRNA fluctuations may not be necessary for coreoscillator function, theynormally correlate with other molecular and behavioral circadianfluctuations. Moreover, there are substantial data indicating that Perand Tim feedback regulate these transcriptional oscillations. There is also considerable experimental evidence as well as theoretical models, to suggest that thenormal 4-6 hr lag between the RNA and protein curves is essential forgenerating these robust, high-amplitude transcriptional oscillations. The general view isthat the protein accumulation delay gives enough time for transcriptionto increase substantially, before protein levels have increasedsufficiently to inhibit transcription. The presence ofrobust transcriptional oscillations without the delayed proteinaccumulation makes this scheme less likely. It redirects focus towardsome post-transcriptional delay (e.g., the timing of nuclear entry ofthe Per-Tim dimer), which is predicted to be functional and important fortranscriptional feedback regulation. It is important to note that theseconclusions are based on biochemical experiments with whole-headextracts. It is still possible that the mRNA-protein lag may beimportant in the specific pacemaker neurons of Drosophila (Suri, 2000).All of these experiments were performed under LD conditions. When thelight comes on at ZT24, it causes a rapid decline in Tim levels. In DDconditions, therefore, Tim levels are much higher in the earlysubjective day, as expected. But a major, unanticipated difference wasthat the Per and Tim profiles in the dbt mutant flies areprofoundly delayed in DD, as evidenced by the late appearance offaster-migrating species. This occurs without a comparable change inthe RNA profiles, giving rise to a quasi-normal lag between RNA andprotein. The light-mediated advance of the protein curves and theabsence of a comparable light reset of the RNA profile reinforce theindependent regulation of the accumulation phase of the clockRNAs and proteins: only the RNA profiles are influenced bythe declining phase of the protein cycle of the previous day, whereasonly the protein profiles appear to be reset by the light entrainmentstimulus. The data are therefore consistent with a post-translationalroute of light entrainment, perhaps mediated by some aspect of thenormal light effect on Tim. This presumably contributes tothe daily advance of the dbt mutant clock under LDconditions, which counteracts the 5 hr period-lengthening effect thatwould take place under DD conditions (Suri, 2000). Further understanding of the role of Dbt in the clock will requireexperiments that directly address Dbt function and regulation. Forexample, it is possible that temporal regulation of Dbt activity makesa major contribution to the temporal phosphorylation profile and moregenerally 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 beintimately connected to the pacemaker mechanism. Addressing theseissues would provide a much deeper understanding of the role ofphosphorylation 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).\n\nTim 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 timUL flies entrained to LD 12:12, where Per remains complexed with TimUL 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 timUL suggest that Tim influences the timing of light-independent Per phosphorylation (Kloss, 2001).Light-triggered removal of TimUL 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 TimUL 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 (dbtP). 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-Cryb is unable to interact with Tim in yeast cells because it may have lost its photoresponsiveness. Both LexA-Cryb and LexA-CrybDelta, 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 Cryb probably confers a gross structural defect to LexA-Cryb, 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 perS 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 37C 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 30C and 37C in parallel. It was noted that at 37C, 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 circadianbehavioral rhythms in Drosophila melanogaster. Resetting this loop in response to light requires the putative circadian photoreceptorCryptochrome (Cry). The early events in photic resetting were dissected by determining the mechanisms underlying the Cry response tolight 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 cryb flies. While wild-type flies show the characteristic decrease in Tim levels with light treatment, this response is lacking in cryb 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 Biological Overview Evolutionary Homologs Protein Interactions Developmental Biology Effects of Mutation ReferencesHome page: The Interactive Fly 1997 Thomas B. Brody, Ph.D.\nThe Interactive Fly resides on the\nSociety for Developmental Biology's Web server.\n\nDarkness and Flame: The Dark Side activation code generator\nDownload Zip https://t.co/1LrmoURDf1\n\n\n eebf2c3492\n
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