BIOLOGICAL OVERVIEW

The fly has a circadian cycle of activity; its activities respond to and are influenced by light (see reviews by Hardin, The Circadian Timekeeping System of Drosophila and Vallone, Start the clock! Circadian rhythms and development). How does a biological system regulate such photoperiod responses? The circadian clock is entrained (reset) by changes in the light regime. In the laboratory, the light cycle is 12 hours on, 12 off. At the beginning of the photoperiod day (lights on), TIM and PER (Period) RNA levels begin to rise. The regulation of transcription and nuclear localization in this two protein system is at the core of the photoperiod response. Regulation of PER nuclear entry is critical. Without Timeless, PER cannot enter the nucleus. Nuclear entry of PER takes place only during photoperiod night (lights off), and only when PER and TIM are combined as a protein heterodimer. When lights are on, both PER and TIM proteins are initially degraded; then the cycle begins anew. PER nuclear entry is also regulated by phosphorylation.

PER is unstable in timeless mutants, but TIM protein is stable in per mutants. Moreover, light induces loss of TIM without requiring PER, and therefore TIM light dependent cycling is independent of PER. This result suggests that TIM is responsible for coupling intracellular circadian cycles to light stimulation. If a light pulse is given in the middle of the night, TIM diminution is immediately followed by reaccumulation. This resets the molecular cycle of TIM to an earlier time point, and results in a phase delay in locomotor activity. The magnitude of the molecular phase shift corresponds with that of the behavioral shift (Myers, 1996).

A model for entrainment of the circadian pacemaker shows TIM protein along with PER declining at dawn, due to the light sensitive response of TIM. The drop in PER and TIM levels kindles the initiation of per and tim transcription during the day, and by dusk, PER and TIM mRNA has reached high enough levels to begin to yield significant TIM and PER protein levels. TIM nuclear localization depends on PER. In the middle of the night, nuclear PER and TIM reach sufficiently high concentration to begin suppressing PER and TIM mRNA accumulation, slowing accumulation of the messenger RNA. At daybreak, TIM light sensitivity results in the destruction of both TIM and its dimerization partner PER (Myers, 1996).

Two papers appearing in 1998, one by Yang et al. and another by Suri, et al., suggest that photic information is transduced in a cell autonomous fashion to the endogenous clock directly through the timeless gene product. This signal transduction takes place directly in the brain and does not involve light reception by the eyes.

In Drosophila, as in mammals, the eyes contain an autonomous oscillator that is thought to mediate an eye-specific function. However, the eyes appear to be generally dispensable for the generation of behavioral rhythms, as well as for their entrainment to light-dark cycles; thus, the existence of extraretinal photoreceptors is generally speculated. Nonetheless, there are data indicating defects in behavioral rhythms in at least one mutant that lacks eyes. The degree to which the visual transduction pathway is involved in circadian photoreception remains a major unanswered question. In Drosophila, visual transduction involves the sequential activation of a rhodopsin molecule, then signal transduction by a heterotrimeric G protein. This leads to the the involvement of phospholipase C, which activates the opening of ion channels resulting in a light-activated conductance composed mostly of calcium ions. norpA flies, containing a mutation in the eye-specific phospholipase C that results in vision blindness, display circadian rhythms that are 1 hr shorter than wild-type but otherwise entrain normally to light-dark cycles (Yang, 1998 and references).

In Drosophila, recent experiments suggest that photic information is transduced to the clock through the timeless gene product, Tim. Genetic and spectral evidence is provided supporting the relevance of Tim light responses to clock resetting. Since flies that lack eyes can, in general, entrain to light-dark cycles, one would expect that the lateral neurons in these mutants can still perceive light. However, an acute effect of light was never assayed in eyeless flies, leaving open the possibility that they might be impaired in this more sensitive test of a circadian light response. To determine whether external photoreceptor cells are required for nonparametric entrainment (entrainment to discrete pulses), the Tim response was assayed in the lateral neurons of sine oculisD (soD) flies, which lack eyes. As lateral neurons account for a very small part of the total Tim signal in the adult fly head, immunofluorescence assays, rather than Western blots, were used to assay soD flies. Flies were exposed to a 1 min light pulse of 2800 lux at ZT21 (nine hours after the beginning of the dark period) and were collected 90 min later. Lateral neurons were localized by costaining sections with antibodies to pigment-dispersing hormone (PDH). Light eliminates Tim expression in the lateral neurons of soD mutants, suggesting that acute circadian responses to light are maintained in eyeless flies (Yang, 1998).

To address the question of whether the circadian rhythm transduction mechanisms are similar to those used in the visual system, the Tim response to light was measured in visual transduction mutants. The trp gene, encoding a Ca2+ channel, is required for a light-activated conductance in the visual transduction cascade. Together with the product of the trpl gene, it mediates calcium conductance in photoreceptor cells. In the transient receptor potentialcm (trpcm) mutant, the TIM response to light is normal in photoreceptor cells but is reduced in lateral neurons: it is eliminated in some lateral neurons but persists in others.Double mutants that lack both trp and trpl gene products were obtained and the Tim response was assayed in photoreceptor cells and lateral neurons. In the double mutants, the Tim response to a 1 min pulse of 2800 lux is reduced in both cell types. A 10 min light pulse elicits an increased response in the double mutant, but it is still weaker than that seen in wild-type flies: staining in photoreceptor cells and lateral neurons is still visible in the mutants after the light treatment. The Tim response is also unaffected in ninaE flies that lack the rhodopsin protein (rh1). These results support the hypothesis that circadian entrainment does not rely on the visual system and likely involves a dedicated pathway for photoreception. (Yang, 1998)

The similar features of Per and Tim cycling are likely related to the fact that both proteins are present in a heterodimeric complex. Stimuli that phase shift the clock might cause rapid changes in one or more clock components, such as Per and Tim. Light is one such stimulus and affects several features of circadian oscillations: phase, period, and amplitude. Tim appears more closely connected to light than Per, because a decrease in Tim levels appears to be the first detectable response of a molecular clock component to acute light exposure. Tim levels respond to illumination even in arrhythmic per01 flies, i.e., in the absence of Per, and it has been proposed that a signal transduction pathway from a light receptor to Tim is an important part of the light-mediated entrainment pathway in Drosophila. In many organisms, the circadian light receptor is unknown. Dose-response curves and action spectra for light effects on circadian rhythms have been determined in plants and animals. During certain times of day, there is little or no response to light, whereas at other times there are pronounced delays or advances (dead zone, delay zone, and advance zone, respectively). Studies of the action spectrum for the eclosion delay zone phase shift of Drosophila pseudoobscura have shown that the action spectra for advance and delay phase shifts are very similar. The most effective wavelengths are between 420 and 480 nm, with a sharp decline above 550 nm; the eclosion clock is virtually insensitive to wavelength above 570 nm. This similarity suggests that both phase advances and phase delays are mediated by the same type of photoreceptor. The spectrum is significantly different from that of the major route of visual photoreception in Drosophila, which involves a rhodopsin species in all of the outer photoreceptors, suggesting that a different type of photopigment may be involved in the circadian gating of eclosion (Suri, 1998 and references).

During the past 25 years, D. melanogaster has generally replaced D. pseudoobscura as the most common experimental fly species for circadian rhythm research. Locomotor activity rhythms have also replaced adult eclosion as the most common Drosophila rhythm assay. This is due in part to the fact that the periods and responses to environmental stimuli of individual flies can be measured in the locomotor activity assays. But no action spectra have been reported for locomotor activity rhythms in D. melanogaster. Moreover, no action spectra data exist for the recently described light-mediated decrease in Tim levels. Since Tim is proposed to be the key clock molecule mediating light reception, the Tim disappearance spectrum should parallel the behavioral action spectrum. This is shown to be the case and the the results support the notion that the visual phototransduction pathway is dispensable for Tim degradation (Suri, 1998).

Per's expression pattern in the fly head has been extensively studied. It is present in photoreceptor cells, putative glia in various ganglia, and neurons in lateral as well as dorsal regions of the central brain. Expression in lateral neurons is probably necessary and may be sufficient for locomotor activity rhythms. Per undergoes robust cycling in all of these tissues as assayed by histochemistry. Although less well studied, the expression pattern of Tim is similar, as expected from the biochemical relationship between the two proteins. Multiple expression sites raise a problem for comparing the behavioral and biochemical action spectra: the former probably reflects changes in Tim levels in brain lateral neurons, whereas the latter largely reflects changes in Tim levels in eye photoreceptor cells (Suri, 1998).

Decrease in Tim levels in the eye is shown to be independent of brain pacemaker neurons. A comparison of the Western blot Tim signal from eyeless heads with that from wild-type flies indicates that in head extracts, about two-thirds of Tim comes from the eye (i.e., the photoreceptor cells). The similar action spectra for behavior and Tim level changes might therefore reflect a shared photoreceptor tissue, which detects light and relays the appropriate circadian information to the relevant tissues. Alternatively, these measurements might reflect the fact that the eye and pacemaker neurons in the CNS have independent circadian photoreceptors but share the same photopigment. The extreme version of this second possibility is that the Tim response to light is entirely cell autonomous and reflects a ubiquitous photoreceptor. To begin to address this question, the Tim degradation pattern was examined in disco flies. In this mutant strain, the lateral neurons are largely eliminated, ganglia normally located between the brain and the eyes are disrupted or eliminated, and free-running locomotor activity behavior is largely arrhythmic. This implies that signals that normally emanate from the pacemaker neurons are largely eliminated and that the same or perhaps a slightly greater fraction of Tim in disco extracts is still derived from eyes. To optimize sensitivity, Tim levels were examined as a function of intensity with 450 nm light. It was found that in both disco and wild-type genotypes, the Tim level decrease is a function of light intensity. There was little or no effect of disco, indicating that the pacemaker neurons have little or no effect on light-mediated decrease in Tim levels in the eye. This is consistent with the possibility that the Tim light response in photoreceptors may be cell autonomous; i.e., that the eye may contain all of the components necessary for a light-mediated decrease in TIM levels (Suri, 1998).

Genetic evidence is provided that light-mediated decrease in Tim levels is relevant to the effects of light on behavior. A missense mutant tim, TIM-SL, exhibits greater sensitivity to light in both Tim protein disappearance and locomotor activity phase shifting assays. Analysis of the dose response of Tim disappearance in a variety of mutant genotypes suggests cell-autonomous light responses that are largely independent of the canonical visual transduction pathway (Suri, 1998).

The simplest interpretation of these results is that all cycling tissues contain cell-autonomous oscillators with the same, as yet unidentified, photoreceptor. The data also suggest that the same oscillator tissues contain signal transduction components that impact rapidly on Tim. This interpretation is supported by a report of robust circadian cycling and light-mediated phase shifting in the Malpighian tubules of decapitated flies, where no cross-talk with the brain or eye is possible. It is also consistent with a more recent report demonstrating in vitro light sensitivity of a number of oscillating adult Drosophila tissues. The data indicate that Drosophila has a specialized circadian photoreceptor, and the action spectra suggest that flavin- and/or pterin-based molecules, such as cryptochromes, may be involved. Every oscillator tissue, perhaps every cycling cell, may contain the same machinery for light entrainment as well as for free-running rhythms. The machinery may include novel components for photoreception, signal transduction, protein processing, and gene regulation. This raises the exciting possibility that other components of the light-input pathway may be encoded by novel clock genes. The light effects on Tim levels should aid in the identification of the photoreceptor as well as the unknown signal transduction pathways that link it to the Per-Tim clock cycles (Suri, 1998).

Heat regulates the Drosophila biological clock, and the role of Period and Timeless in this process will be dealt with here. Circadian (approximately 24-h) rhythms are governed by endogenous biochemical oscillators (clocks) that in a wide variety of organisms can be phase shifted (i.e., delayed or advanced) by brief exposure to light and changes in temperature. However, it is not known how changes in temperature reset circadian timekeeping mechanisms. Cyclical change in locomotor activity is one circadian rhythm that is sensitive to temperature shift. For example, flies raised at 25 degreesC were placed at 37 degreesC for 30 minutes at 15 hours (T15) after the last dark to light transition (three hours after the beginning of the dark period). Their behavior was monitored for 7 to 10 days in constant darkness. Heat treatment delays peak locomotion by 2.3 hours from its normal peak (12.7 hours after the dark to light transition). To begin to address the biochemical basis of the behavioral change, the effects of short-duration heat pulses were measured on the protein and mRNA products from the Drosophila circadian clock genes period (per) and timeless (tim) (Sidote, 1998).

Heat treatment at T15 (15 hours after the last dark to light transition, that is 3 hours into the dark period) elicits the rapid disappearance of both Per and Tim proteins. Clear reductions are first observed between 3 to 5 min following the start of the 37 degree incubation, and essentially undetectable levels are reached after 10 to 15 min of heat treatment. Similar heat-induced decreases in the levels of both proteins were observed at all times in a daily cycle. No changes in the levels of either per or tim transcripts are detected during the first 20 min of the heat pulses, strongly suggesting that a posttranscriptional mechanism is solely responsible for mediating the heat-induced decreases in the levels of Per and Tim. The magnitude of the phase shift in the locomotor activity rhythm is also proportional to the temperature of the pulse, consistent with a causal relationship between the heat-induced degradation of Per, Tim, or both and phase resetting. It is thought that the majority of the heat-induced disappearance of Per and Tim is due to protein degradation. It is noteworthy that brief pulses at 37 degreesC elicit a full-blown heat shock response in D. melanogaster, raising the likelyhood that this pathway participates in mediating the enhanced degradation of Per and Tim. For example, thermally denatured proteins are prime targets for proteolysis by the ubiquitin-proteasome system (Sidote, 1998).

Per protein level is sensitive to heat but not light, indicating that individual clock components can markedly differ in sensitivity to environmental stimuli. A similar resetting mechanism involving delays in the per-tim transcriptional-translational feedback loop likely underlies the observation that when heat and light signals are administered in the early night, they both evoke phase delays in behavioral rhythms. Heat induces the degradation of Per and Tim independently, since the heat induced degradation of each protein takes place in flies mutant for the other protein. The results indicate that Per and Tim can be independently regulated by heat and that this degradation does not require a functional clock (Sidote, 1998).

The light-induced degradation of Tim in the late night is accompanied by stable phase advances in the temporal regulation of the Per and Tim biochemical rhythms (Per and Tim protein and phosphorylation levels). A 37 degree heat pulse at T15 evokes stable phase delays of several hours in the biochemical oscillations of Per and Tim, consistent with the magnitude and direction of the phase shift in locomotor activity rhythms produced by identical temperature treatments. The heat-induced delays in the temporal regulation of Per and Tim abundance and phosphorylation are stable for at least 2 days after the environmental perturbation (Sidote, 1998)

The initial heat-induced degradation of Per and Tim in the late night, unlike treatment in the early night, is followed by a transient and rapid increase in the speed of the Per-Tim temporal program. The net effect of these heat-induced changes results in an oscillatory mechanism with a steady-state phase similar to that of the unperturbed control situation; at the same time, there is little effect on locomotion. These findings can account for the lack of apparent steady-state shifts in Drosophila behavioral rhythms by heat pulses applied in the late night (Sidote, 1998).

An intriguing observation is that Per is sensitive to heat but not light, whereas Tim is sensitive to both stimuli. Thus, individual clock components can markedly differ in sensitivity to the two most important environmental entraining cues. Although not well studied, it is highly likely that under natural conditions a wide variety of organisms manifest circadian rhythms that are influenced by multiple temporal cues. In the case of Drosophila, it appears that the photic and heat signal transduction pathways converge at the level of regulating the stability of one or more key clock proteins. The observation that Per and Tim interact to form a functional complex that is involved in an autoregulatory circuit that is central to the timekeeping mechanism might ensure that the effects of light and temperature on individual clock proteins are combined into a coherent temporal cue resulting in daily rhythms that are optimally adapted to the precise local conditions. It will be of interest to determine whether other circadian timekeeping devices are assembled with components that differ in sensitivity to different environmental entraining cues (Sidote, 1998).

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

Antisera against the circadian clock proteins Period (Per) and Timeless (Tim) were used to construct a detailed time course of Per and Tim expression and subcellular localization in a subset of the ventrolateral neurons (vLNs) in the Drosophila accessory medulla (AMe). These neurons, which express pigment-dispersing factor, play a central role in the control of behavioral rhythms. The data reveal several unexpected features of the circadian clock in Drosophila: (1) Tim but not Per is restricted to the cytoplasm of vLNs throughout most of the early night; (2) the timing of Tim and Per nuclear accumulation is substantially different; (3) the two subsets of vLNs, the large and small vLNs, have a similar timing of Per nuclear accumulation but differ by 3-4 hr in the phase of Tim nuclear accumulation. These aspects of Per and Tim expression were not predicted by the current mechanistic model of the circadian clock in Drosophila and are inconsistent with the hypothesis that Per and Tim function as obligate heterodimers. The differing profiles of Tim and Per nuclear accumulation suggest that Per and Tim have distinct functions in the nuclei of vLNs (Shafer, 2002).

One concern of an immunocytochemical study is that differences in staining intensity for two epitopes may reflect a disparity in antibody affinity rather than differences in epitope abundance. Attempts were made to allay this concern by using two to three different Per and Tim antibodies. Similar intensities and patterns of staining are seen for all of the antibodies used in this study, suggesting that differential antibody affinity is not responsible for the differences in staining observed for these two proteins. The best estimate for the relative abundance of Per and Tim has come from the analysis of whole-head extracts; the Tim/Per ratio is ~2:1 at ZT 16 and 1:1 by ZT 23. A variation of this magnitude in the vLNs would be consistent with the relative intensities of staining observed in this study. Given this transient disparity between Tim and Per abundance, the persistence of strong cytoplasmic Tim signals (at times when Per is predominantly nuclear) must not be considered as evidence that these proteins enter the nucleus sequentially. Rather, it is the differing profiles of Tim and Per immunoreactivity in the nuclei of the vLNs on which this conclusion is based (Shafer, 2002).

Several lines of evidence had suggested that Per and Tim are transported into nuclei as a dimeric complex. Biochemical assays in fly-head extracts show that Per and Tim are present as heterodimers throughout much of the night, but the data did not exclude the fact that a fraction of Per and Tim are monomeric or complexed with other proteins. Importantly, it is also uncertain what fraction of the Per/Tim heterodimers are present in vivo and what fraction form in vitro during extract preparation and analysis. Furthermore, these extracts reflect thousands of cells of many different types, including all of the photoreceptors from the compound eye. These contribute the majority of the Per and Tim to the homogenate, effectively swamping out the contribution of the vLNs. There is therefore no reason to expect that the biochemistry of the vLNs will be identical to what has been observed in whole-head extracts. In flies, little or no nuclear Per accumulation is seen without Tim, but this observation is likely attributable to Per instability and consequent low Per levels in mutants that lack Tim. The coexpression of Per and Tim has been shown to be required for the nuclear transport of both proteins in cultured S2 cells. Although these cells have not been shown to express Per and Tim rhythmically, this codependence fits well with the notion that Per and Tim enter the nucleus as a heterodimeric complex. Based on these considerations, it is expected that a time course study would reveal the simultaneous nuclear accumulation of Per and Tim in the vLNs. Instead, the data show that Per appears in nuclei at least 3 hr before Tim, indicating that most, and perhaps all, nuclear Per is not complexed with Tim at these times (Shafer, 2002).

In the large vLNs, Per is already nuclear as well as cytoplasmic when it is first detectable at ZT 16. In the large vLNs, nuclear Per is first detected at times that correlate well with the onset of repression in head extracts. This begins at approximately ZT 15-17 by biochemical criteria. The discrepancy between the timing of Per nuclear accumulation in the small cells and the onset of transcriptional repression in whole-head extracts might reflect differences between the cells and tissues that contribute to the biochemical data (Shafer, 2002).

In contrast to Per, Tim is not nuclear until after ZT 18. This difference with Per is especially prominent in the small cells, where Tim remains cytoplasmic until ZT 22. In both cell types, Tim is degraded in the early morning and is therefore nuclear for only a short time. In other words, Tim becomes nuclear well after transcriptional repression is thought to begin and is degraded well before the next cycle of transcription begins anew (Shafer, 2002).

A central feature of the molecular model of the Drosophila clock is that Per and Tim function as obligate heterodimers in their nuclear transport as well as in the repression of Clock/Cycle-based transcription. However, the different times of nuclear accumulation suggest that Per and Tim are transported independently to nuclei. The lack of high-resolution data for the Doubletime kinase makes it difficult to assess whether the Doubletime nuclear accumulation pattern correlates better with that of Per or of Tim. Importantly, these results do not preclude a role for Tim in the nuclear entry of Per. For example Tim could act catalytically in the cytoplasm to potentiate some required Per phosphorylation event. Moreover, Per and Tim could still enter nuclei as Per/Tim heterodimers, but this would require the subsequent export of Tim to the cytoplasm to explain the apparent absence of nuclear Tim during the middle of the night (Shafer, 2002).

In biochemical assays, Per and Tim are both capable of blocking per and tim transcription in the absence of the other, suggesting that repression does not require the Per/Tim heterodimer. The data indicate that little or no Tim is present in the nuclei of vLNs during times associated with repression in whole-head extracts, suggesting that Per might influence transcription independently of Tim. It has been suggested that Per is still capable of repressing transcription after a mutant form of Tim (TimUL) is degraded by light exposure. It has been concluded that Per can repress transcription in the absence of Tim and it has been suggested that Tim-independent repression by Per normally occurs after dawn. However, there was no reason to challenge the notion that the Per/Tim heterodimer is the agent of nuclear entry and the initiator of repression. The current data indicate that the Tim-independent repression by Per also applies to the onset of repression in the middle of the night. Tim may therefore play no direct role in the feedback repression of Per and Tim transcription (Shafer, 2002).

The persistence of Tim oscillations in the small vLNs but the absence of oscillations in the large vLNs under DD conditions has been noted. This observation has been confirmed, but these data extend this result in several important ways. Per was assayed as well as Tim and these two proteins were followed throughout the first DD cycle. This provides a finer temporal resolution and leads to a surprising conclusion: that the molecular oscillation in the large vLNs appears to arrest during the first subjective day. Based on the patterns of Per and Tim, these cells stop in a state that corresponds to ZT 8-10 under LD conditions (Shafer, 2002).

The individual features of Per and Tim distribution in the large and small vLNs can be added to a growing list of differences between these two classes of PDF-expressing neurons. Cell-specific differences in Per and Tim nuclear accumulation underscore the importance of studying the brain clock in situ and may presage an even more profound heterogeneity among other clock-containing neurons within the brain. This view also reflects the fact that the PDF-positive vLNs represent a minority of clock gene-expressing cells in the central brain. One might therefore expect that the circadian system will vary to an even greater degree among other clock neurons. Such diversity might offer clues as to how different groups of clock neurons interact to create the temporal complexity of behavioral rhythms (Shafer, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A key temporal delay in the circadian cycle of Drosophila is mediated by a nuclear localization signal in the timeless protein

Regulated nuclear entry of the Period (Per) and Timeless (Tim) proteins, two components of the Drosophila circadian clock, is essential for the generation and maintenance of circadian behavior. Per and Tim shift from the cytoplasm to the nucleus daily, and the length of time that Per and Tim reside in the cytoplasm is an important determinant of the period length of the circadian rhythm. This study identified a Tim nuclear localization signal (NLS) that is required for appropriately timed nuclear accumulation of both Per and Tim. Transgenic flies with a mutated Tim NLS produced circadian rhythms with a period of ~30 hr. In pacemaker cells of the brain, Per and Tim proteins rise to abnormally high levels in the cytoplasm of tim^δNLS mutants, but show substantially reduced nuclear accumulation. In cultured S2 cells, the mutant TIM^δNLS protein significantly delays nuclear accumulation of both Tim and wild-type Per proteins. These studies confirm that Tim is required for the nuclear localization of Per and point to a key role for the Tim NLS in the regulated nuclear accumulation of both proteins (Saez, 2011).

This study identified a functional nuclear localization signal in the Tim protein. A role was demonstrated for this NLS in specifying the timing of nuclear accumulation for both Tim and its partner protein, Per. Modification of the Tim NLS impaired the nuclear entry of Per and Tim in vivo and produced an aberrant, but specific, delay in the onset of nuclear accumulation in living cultured cells. The cytoplasmic residence of Per and Tim^δNLS exceeds that of wild-type Per and Tim proteins by ~4 hr in S2 cells. A similar delay in vivo might be expected to extend period length to ~28 hr, which is close to the change in behavioral rhythmicity seen in tim⁰; tim^δNLS mutant flies. Although levels of nuclear accumulation were too low to be assessed directly in neural pacemaker cells (sLNV's) of tim⁰; tim^δNLS flies, the rhythmic behavior of these flies makes it likely that periodic Per/Tim ^δNLS nuclear accumulation also occurs in vivo. Alternatively, the long-period rhythms of tim⁰; tim^δNLS flies could be the result of Per and Tim activity in clock cells other than the sLNV’s (Saez, 2011).

The altered profiles of Per and Tim^δNLS nuclear accumulation do not appear to be due to a loss of physical association between the two proteins. As shown by inmmunoprecipitation and FRET analyses, Per and Tim^δNLS are capable of binding to each other and do so in cultured cells with kinetics that are similar to wild-type Per and Tim. Surprisingly, mutating the identified Tim NLS only delays the time of nuclear translocation in S2 cells. These results suggest that a sequence that poorly resembles a nuclear localization signal can promote nuclear entry in the absence of the known Tim NLS (Saez, 2011).

The contemporaneous movement of Tim and Per (or of Tim^δNLS and Per) to the nucleus in S2 cells suggests that the Tim NLS has an important role in determining the time of nuclear entry, but not the co-dependent nuclear accumulation of Per and Tim. That is, in the absence of the identified Tim NLS, although nuclear transfer is substantially delayed, the onset of nuclear accumulation is shifted for both the Tim^δNLSprotein and for wild-type Per expressed in the same cell. That wild-type Per does not accumulate in the nucleus with kinetics that are independent of Tim^δNLS indicates that other features of the Tim (and Per) proteins coordinate their timed subcellular movements. Prior studies have repeatedly shown that the presence of Tim significantly enhances the nuclear accumulation of Per. This tight coupling is most likely to reflect the physical association of Per and Tim as they accumulate in the cytoplasm and immediately prior to the time of their nuclear translocation. The NLS of Tim may participate in a mechanism that facilitates nuclear entry of both proteins. One possibility is that a specific importin recognizes the Tim NLS in cytoplasmic Per/Tim complexes and promotes an association of these complexes with the nuclear pore. As earlier studies have indicated that Per and Tim dissociate at the time of nuclear transfer, this model would require recognition of the Tim NLS prior to separation of the proteins, but could account for the similar timing of Per and Tim nuclear accumulation that has been observed in S2 cells. Possibly a Per NLS or alternate segment of the Tim protein can promote a delayed association with the nuclear pore in tim^δNLS mutants (Saez, 2011).

In summary, these studies have confirmed a role for a specific Tim protein sequence in the regulated nuclear accumulation of Tim and Per. Mutation of the Tim NLS suppressed nuclear accumulation of Per and Tim in neural pacemaker cells of Drosophila and lengthened the period of molecular and behavioral rhythms in these flies to ~30 hr. The same tim^δNLS mutation also affected nuclear accumulation of Per and Tim when expressed in cultured Drosophila cells and caused delays in the onset of Per/Tim nuclear accumulation that are well correlated with the long-period circadian rhythms of tim^δNLS flies (Saez, 2011).

Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior

Molecular circadian clocks are interconnected via neural networks. In Drosophila, Pigment-Dispersing Factor (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF⁺ and PDF^- circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. This study has demonstrated that genetic inhibition of protein kinase A (PKA) in PDF^- clock neurons can phenocopy PDF mutants, while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, per was rescued in PDF neurons of arrhythmic per⁰¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component Timeless (Tim) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced Tim levels in non-PDF neurons of per⁰¹ flies. To address how PDF impacts pacemaker neuron output, PDF was focally applied to DN1p neurons and was found to acutely depolarize and increase firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. Evidence is provided for a signaling mechanism (PKA) and a molecular target (Tim) by which PDF resets and synchronizes clocks; PDF exhibits an acute direct excitatory effect on target neurons to control neuronal output. The identification of Tim as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, these results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output (Seluzicki, 2014).

Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, behavior as well as molecular rhythms were assayed within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. CRISPR-mediated neuron-specific circadian clock knockouts were also generated. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light (Schlichting, 2019).

Neuronal networks make myriad contributions to behavior and physiology. By definition, individual neurons within a network interact, and different networks also interact to coordinate specialized functions. For example, the visual cortex and motor output centers must coordinate to react properly to environmental changes. In a less immediate fashion, sleep centers and circadian clocks are intertwined to properly orchestrate animal physiology. The brain clock is of special interest: it not only times and coordinates physiology within neuronal tissues but also sends signals to the body to keep the entire organism in sync with the cycling external environment (Schlichting, 2019).

The small, circumscribed Drosophila clock network is ideal to address circadian communication issues. The comparable region in mammals, the suprachiasmatic nucleus, is composed of thousands of cells depending on the species. There are in contrast only 75 clock neurons per hemisphere in Drosophila. These different clock neurons can be divided into several subgroups according to their location within the fly brain. There are 4 lateral and three dorsal neuron clusters, which have different functions in controlling fly physiology (Schlichting, 2019).

The four small ventro-lateral neurons (sLNvs) are arguably the most important of the 75 clock neurons. This is because ablating or silencing these neurons abolishes rhythms in constant darkness (DD). They reside in the accessory medulla region of the fly brain, an important pacemaker center in many insects, and express the neuropeptide PDF. In addition, they are essential for predicting dawn. A very recent study suggests that the sLNvs are also able to modulate the timing of the evening (E) peak of behavior via PDF. The other ventral-lateral group, the four large-ventro-lateral neurons (lLNvs), also express PDF and send projections to the medulla, the visual center of the fly brain; they are important arousal neurons. Consistent with the ablation experiments mentioned above, the absence of pdf function or reducing PDF levels via RNAi causes substantial arrhythmic behavior in DD (Schlichting, 2019).

Other important clock neurons include the dorso-lateral neurons (LNds), which are essential for the timing of the E peak and adjustment to long photoperiods. Two other clock neuron groups, the lateral-posterior neurons (LPN and a subset of the dorsal neurons (DN1s), were recently shown to connect the clock network to sleep centers in the fly central complex. The DN2 neurons are essential for temperature preference rhythms, whereas no function has so far been assigned to the DN3s (Schlichting, 2019).

Despite these distinct functions, individual clock neuron groups are well-connected to each other. At the anatomical level, all lateral neuron clusters and even DN1 dorsal neurons send some of their projections into the accessory medulla, where they can interact. A second area of common interaction is the dorsal brain; only the lLNvs do not project there (Schlichting, 2019).

Several studies have investigated interactions between different clock neurons. Artificially expressing kinases within specific clock neurons causes their clocks to run fast or slow and also changes the overall free-running period of the fly, indicating that network signaling adjusts behavior. Similarly, speeding up or slowing down individual neurons is able to differentially affect behavioral timing in standard light-dark (LD) cycles. A high level of neuronal plasticity within the network also exists: axons of individual cells undergo daily oscillations in their morphology, and neurons change their targets depending on the environmental condition (Schlichting, 2019).

How neuronal communication influences the fly core feedback loop is not well understood. The latter consists of several interlocked transcriptional-translational feedback loops, which probably underlie rhythms in behavior and physiology. A simplified version of the core feedback loop consists of the transcriptional activators Clock (CLK) and Cycle (CYC) and the transcriptional repressors Period (PER) and Timeless (TIM). CLK and CYC bind to E-boxes within the period (per) and timeless (tim) genes (among other clock-controlled genes) and activate their transcription. After PER and TIM synthesis in the cytoplasm, they form a heterodimer and enter the nucleus toward the end of the night. There they interact with CLK and CYC, release them from their E-box targets and thereby inhibit their own transcription. All 75 pairs of clock neurons contain this canonical circadian machinery, which undergoes daily oscillations in level. Indeed, the immunohistochemical cycling of PER and TIM within these neurons is a classic assay to visualize these molecular oscillations (Schlichting, 2019).

Silencing PDF neurons stops their PER cycling, indicating an important role of neuronal firing in maintaining circadian oscillations. However, only two time points were measured, and the results were possibly confounded by developmental effects. PDF neuron silencing also phase advances PER cycling in downstream neurons, suggesting that PDF normally serves to delay cycling in target neurons. This is consistent with experiments showing that PDF signaling stabilizes PER. In addition, neuronal activation is able to mimic a light pulse and phase shift the clock due to firing-mediated TIM degradation (Schlichting, 2019).

To investigate more general features of clock neuron interactions on the circadian machinery, the majority of the fly brain clock neurons were silenced, and behavior and clock protein cycling within the circadian network was examined in a standard light-dark cycle (LD) as well as in constant darkness (DD). Silencing abolished rhythmic behavior but had no effect on clock protein cycling in LD, indicating that the silencing affects circadian output but not oscillator function in a cycling light environment. Silencing similarly abolished rhythmic behavior in DD but with very different effects on clock protein cycling. Although protein cycling in the LNds was not affected by neuronal silencing in DD, the sLNvs dampened almost immediately. Interestingly, this differential effect is under transcriptional control, suggesting that some Drosophila clock neurons experience activity-regulated clock gene transcription. Cell-specific CRISPR/Cas9 knockouts of the core clock protein Per further suggests that network properties are critical to maintain wild-type activity-rest rhythms. The data taken together show that clock neuron communication and firing-mediated clock gene transcription are essential for high amplitude and synchronized molecular rhythms as well as rhythmic physiology (Schlichting, 2019).

The central clock of animals is essential for dictating the myriad diurnal changes in physiology and behavior. Knocking out core clock components such as period or Clock severely disrupts circadian behavior as well as molecular clock properties in flies and mammals. This study show that similar behavioral effects occur when the central clock neurons are silenced and thereby abolish communication within this network and with downstream targets, that is fly behavior becomes arrhythmic in LD as well as DD conditions and resembles the phenotypes of core clock mutant strains (Schlichting, 2019).

Despite the loss of all rhythmic behavior, silencing did not impact the molecular machinery in LD conditions: PER and PDP1 protein cycling was normal. These findings suggest that (1) rhythmic behavior requires clock neuron output, which is uncoupled from the circadian molecular machinery by network silencing, and (2) synchronized molecular rhythms of clock neurons do not require neuronal activity. These findings are in agreement with previous work showing that silencing the PDF neurons had no effect on Per cycling within these neurons. The results presumably reflect the strong effect of the external light-dark cycle on these oscillators (Schlichting, 2019).

In DD however, the individual neurons change dramatically: the different neurons desynchronize, and their protein cycling damps to different extents. Interestingly, sLNv cycling relies most strongly on neuronal communication: these neurons cycle robustly in controls but apparently not at all in the silenced state. sLNvs were previously shown to be essential for DD rhythms. Unfortunately, the sensitivity of immunohistochemistry precludes determining whether the molecular clock has actually stopped or whether silencing has only (dramatically) reduced cycling amplitude. However, a simple interpretation of the adult-specific silencing experiment favors a stopped clock: decreasing the temperature to 18 degrees after a week at high temperature failed to rescue rhythmic behavior. A similar experiment in mammals gave rise to the opposite result, suggesting an effect of firing on circadian amplitude in that case. However, different explanations cannot be excluded, for example chronic effects of neuronal silencing or a too large phase difference between the different neuronal subgroups to reverse after a week without communication (Schlichting, 2019).

In either case, a stopped clock or an effect on clock protein oscillation amplitude, these results make another link to the mammalian literature: modeling of the clock network suggests that different neurons resynchronize more easily if the most highly-connected cells are intrinsically weak oscillators. The sLNvs are essential for DD rhythms, known to communicate with other clock neurons and are situated in the accessory medulla; this is an area of extensive neuronal interactions in many insects. These considerations rationalize weak sLNv oscillators (Schlichting, 2019).

An important role of interneuron communication in DD is in agreement with previous work showing that altering the speed of individual neuron groups can change the phase of downstream target neurons. An important signaling molecule is the neuropeptide PDF: its absence changes the phase of downstream target neurons, and silencing PDF neurons causes an essentially identical phenotype to the lack of PDF. However, the effects reported in this study are much stronger and show different levels of autonomy than PDF ablation, suggesting that other signaling molecules and/or the neuronal activity of additional clock neurons are essential to maintain proper rhythmic clock protein expression (Schlichting, 2019).

To address these possibilities, this study took two approaches. First, clock gene RNA levels were investigated after silencing. The goal was to assess whether the damping of silenced neurons is under gene expression control, likely transcriptional control. Indeed, tim mRNA profiles nicely reproduced the protein cycling profiles: robust cycling of all (assayed) clock neurons was maintained in LD even with silencing, but tim-mRNA levels in the sLNvs stopped cycling in DD; in contrast, robust cycling was maintained in the LNds. This suggests that the changes in protein cycling amplitude and also possibly phase are under transcriptional control. Importantly, the tim signal in the sLNvs disappeared upon silencing, suggesting that neuronal activity promotes clock gene transcription at least in this subset of neurons. This recapitulates for the first time in Drosophila the robust positive relationship between neuronal firing and clock gene transcription in mammals. To date, Drosophila neuronal firing had only been connected to post-transcriptional clock protein regulation, namely TIM degradation. Conceivably, these two effects are connected: TIM degradation might be required to relieve transcriptional repression and maintain cycling (Schlichting, 2019).

The second approach was a cell-specific knockout strategy, applied to the clock neuron network. Three guides were generated targeting the CDS of per and also CAS9 was expressed in a cell-specific manner. The guides caused double strand breaks in the per gene, which in turn led to cell-specific per mutations. This adult brain knockout strategy worked reliably and specifically, in glial cells as well as neurons, with high efficiency and with no apparent background effects. This strategy was successfully used to knock out most if not all Drosophila GPCRs and it is believed to be superior to RNAi for most purposes. Importantly, expression of the guides with the clk856-GAL4 driver phenocopied per⁰¹ behavior. To focus on individual clock neurons, cell-specific knockouts were generated in different clock neurons. PERKO in the PDF cells did not increase the level of arrhythmicity, only a PERKO in most lateral neurons, E cells as well as PDF cells, generated high levels of arrhythmic behavior (Schlichting, 2019).

Phosphatase of regenerating liver-1 selectively times circadian behavior in darkness via function in PDF neurons and dephosphorylation of TIMELESS

The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. This study demonstrates in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light and clock-regulated gene timeless (tim). Moreover, this study shows that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, these studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles (Kula-Eversole, 2020).

This study has identified a novel circadian clock gene PRL-1 that mediates the behavioral response to changes in photoperiod. Circadian regulation of this phosphatase provides a mechanism for mediating rhythmic phosphorylation, a widely conserved feature of circadian clocks. Reduction or loss-of-function in PRL-1 can induce potent effects on circadian period length even larger than those caused by the reduction of canonical clock genes. These effects are widely conserved and are evident even in human cells. PRL-1 selectively dephosphorylates and suppresses the accumulation of the light-sensitive clock component TIM. PRL-1 affects behavioral phase in short but not long photoperiods, revealing a novel mechanism for appropriate timing across ecologically relevant photoperiods (Kula-Eversole, 2020).

The data suggest PRL-1 plays an especially important role in setting period length comparable to or exceeding those of the canonical core clock components. RNAi knockdown of PRL-1 with two independent RNAi lines results in among the strongest RNAi-induced period-altering phenotypes seen in Drosophila with period lengths exceeding 28 h. Interestingly, the period effects observed through PRL-1 knockdown in the PDF-positive LNv (pdfGAL4) are larger than those observed with a broader circadian driver (timGAL4) or in the putative null allele of PRL-1, suggesting that PRL-1 may have distinct and even antagonistic period setting roles in different clock neurons. The other clock genes with RNAi effects comparable to PRL-1 are the regulatory beta subunit of the protein kinase CK2 and the E3 ubiquitin ligase circadian trip, further highlighting the role of post-translation modifications in determining period length (Kula-Eversole, 2020).

Several lines of evidence support the model that TIM is an in vivo target of PRL-1. PRL-1 mutants exhibit non-additive effects with a tim allele, tim^S1, suggesting PRL-1 and tim function within the same pathway. Non-additive genetic interactions with specific alleles can reflect functional, even direct biochemical, interactions such as between per and tim78 and between per and Dbt. Simple additive effects on period length with tim^UL flies probably reflect the fact that TIM^UL is stable and exhibits prolonged nuclear localization; therefore, stabilizing effects of tim^UL are not counteracted by the destabilizing effects of PRL-1 mutation during the rising phase of TIM accumulation. Loss of PRL-1 results in substantially reduced and delayed nuclear accumulation of TIM during the first day of DD. These effects are much larger than those simply predicted based on the lengthened period and much larger than effects on PER, suggesting a selective effect on TIM in vivo. Using PRL-1 overexpression and knockdown in clock-less Drosophila S2 cells, it was found that PRL-1 dephosphorylates TIM but not PER. Given that PRL-1 mRNA expression peaks around dusk, it is proposed that PRL-1 accumulation during the early night results in TIM dephosphorylation, stabilization, and nuclear localization (Kula-Eversole, 2020).

Strikingly, period lengthening is evident even after siRNA knockdown of all three highly conserved human orthologs of PRL-1, PTP4a-1, -2, and -3 in human U2OS cells, suggesting a widely conserved role in period determination. Like fly PRL-1, mammalian orthologs of PRL-1 also undergo rhythmic expression in many tissues. PTPs may also function via TIM. Mammalian TIM interacts with CRY1 as well as PER1/2, is involved in CLOCK-BMAL1 repression of CLOCK-BMAL1 activation, and sets period length. Mutation of human TIMELESS also results in familial advanced sleep phase syndrome perhaps via altered light responses. Thus, PTP4 action may similarly function to regulate TIM in mammals (Kula-Eversole, 2020).

The connection between PRL-1 and photosensitive TIM suggested that PRL-1 may function to mediate clock responses to light. PRL-1 mutants exhibit a light-dependent and CRY-independent period phenotype. To examine rhythms in LL, PRL-1 mutant period length was examined in a cry mutant background in which flies exhibit free-running rhythms. These flies retain their long periods in DD. On the other hand, in LL they exhibit period lengths comparable to their PRL-1+ controls. Thus, light can act independently of CRY photoreception to compensate for the PRL-1 mutant period lengthening. It is hypothesized that signaling through the visual system could accomplish this (Kula-Eversole, 2020).

The finding of LL-dependent period phenotypes led to an examination of the role of PRL-1 in mediating behavioral adaptations to seasonal changes in photoperiod. PRL-1 mutants display delayed morning and evening activity phases under short (6:18) photoperiods. In contrast, they have no alteration of evening activity phase under long (18:6) photoperiods. These photoperiods approximate those that would be experienced by wild Drosophila melanogaster at more extreme latitudes. It is hypothesized that PRL-1 defines a molecular pathway through which the clock adjusts behavioral phase in response to different photoperiods (Kula-Eversole, 2020).

The phenomenon of photoperiod-dependent network hierarchy suggests that M and E cells differ in their core clock properties, responses to light, and/or their network connectivity. It is hypothesized that PRL-1 represents such a specialization of M cell clocks that enables the appropriate behavioral adjustments to different photoperiods. PRL-1 exhibits robust PDF neuron specific cycling, and PRL-1 knockdown selectively in PDF neurons substantially lengthens period. The finding of period phenotypes in DD and not LL is also consistent with M cell specific function. The findings of photoperiod-dependent effects in PRL-1 mutants could reflect enhanced coupling between LNv and CRY-negative DN1p in short photoperiods, consistent with prior findings of light intensity-dependent sLNv-DN1p coupling. Under this model, selective PRL-1-dependent changes in the M cell phase are propagated to E cells in a photoperiod-dependent manner as observed for other M cell-specific changes. Of note, other core clock components have been identified with differential functions between M and E cells, including those involved in TIM phosphorylation, suggesting a wider role for specialized core clock components in M and E cells. It will be of interest to determine if M or E cell-specific mechanisms also function to control behavioral adaptations to different photoperiods and, if so, how they work with respect to PRL-1 (Kula-Eversole, 2020).

PROTEIN STRUCTURE

Amino Acids - 1389

Structural Domains

TIM protein has a central acidic region followed by a basic region. The basic region has been shown to interact with the PAS domain of PER (Meyers, 1995).

EVOLUTIONARY HOMOLOGS

The complete sequence of a tim gene is reported from Drosophila virilis. Between D. melanogaster and D. virilis, TIM is better conserved than is the PER protein (76 vs. 54% overall amino acid identity); putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis tim gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. Deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene display rhythms with ~24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the conserved segment results in very long activity rhythms with periods ranging from 30.5 to 48 hr (Ousleya, 1998).

The clock genes period and timeless of Drosophila encode cardinal components of the fly's endogenous clock. Their mRNAs and proteins oscillate in abundance with a lag of about 4-6 hours between their respective peaks in numerous cells throughout the Drosophila body and brain. Per and Tim proteins participate in a negative autoregulatory feedback loop, providing a relentless circadian molecular oscillation. A mammalian tim homolog appears to play a minor role in the mammalian clock. In addition, and in contrast to Drosophila, in which a null mutation is viable, a knockout of murine tim (mmTim) is lethal, revealing an essential requirement for mmTim in development. These discrepancies between the tim genes of the two taxa prompted an investigation of the possible existence of paralogs of the tim gene. Southern hybridization of Musca domestica genomic DNA with a D. melanogaster tim probe indicated the possible presence of two tim related sequences. Furthermore, partial sequence data from a Bombyx mori tim-like cDNA clone revealed an unexpectedly higher similarity with mammalian tim compared to Drosophila tim. A database search of the recently completed Drosophila genome was launched, and a similar sequence in the fly was identified using the human TIM (hsTIM) protein for comparison. Subsequent RT-PCR from an Oregon-R strain, followed by DNA sequencing, confirmed the existence of a corresponding 4.3 kb transcript, which encodes a putative protein of 1384 residues. The gene, which has been called timeless2 (tim2; GenBank accession number AF279586), spans 75 kb of genomic DNA and is located on chromosome 3R (87E1-87E3), adjacent to the single-minded (sim) gene. The two proteins show 12% identity and 22% similarity. The corresponding hsTIM and dmTIM2 comparison has 29% identity and 44% similarity. The amino terminus is clearly more conserved than the carboxyl terminus and includes some of the regions of Tim that are proposed to interact with Per proteins. A phylogeny does not indicate when the duplication of tim arose. The absence of more than a single copy of a tim2-like sequence in the Caenorhabditis elegans databases, suggests that tim2 may be the ancestral gene from which tim1 originated after the split between the arthropod and nematode lineages. On the basis of the phylogeny, however, it cannot be ruled out that duplication occurred at an earlier stage, and that the tim1 gene has simply been lost in the worm. If the duplication occurred before the divergence of vertebrates and arthropods, the existence of tim1 genes might be expected in mammals. If it occurred afterwards, then mammals would be predicted to have only tim2 type genes. In mammals, the duplicated mPer and mCry (cryptochrome) genes, have largely taken on the roles that tim1 plays in the fly clock. This in itself could suggest that tim1 genes may not be present in mammals, thereby indirectly favouring the scenario in which the duplication of tim is a relatively recent event, largely confined to the arthropods. Consequently, tim1 may have been freed from its original function and evolved a more central role in the insect clockworks. This could explain the long branch lengths for the insect tim1 genes in the phylogeny, which might reflect a rapid evolutionary rate. Further comparative analysis should clarify the evolutionary history of the tim gene family (Benna, 2000).

Mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene have been cloned and characterized. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals (Sangoram, 1998).

The mouse cDNA of a mammalian homolog of the Drosophila timeless gene has been isolated. The mTim protein shows five homologous regions with Drosophila TIM. The first conserved region (C1) encompasses the amino-terminal regions of both the mouse and fly proteins. Between C1 and C2 of dTIM, there is a stretch of 223 residues not found in mTIM. mTIM appears to lack the 5' half of the first PER interactive domain defined in dTIM. The second PER interactive domain (IAD-2) of dTIM is present in C2-C4 of mTIM. In mTIM, however, this domain is interrupted by two long stretches of amino acids not present in dTIM. Between C4 and C5 of dTIM, there is a stretch of 175 amino acids not found in mTIM. C5 represents a small area in the carboxyl end of mTIM that is highly conserved among dTIM and mTIM and also in silkmoth TIM. Within the nonconserved region between C2 and C3 of mTIM, there is a stretch of 10 basic amino acids and a stretch of 11 acidic amino acids. An acidic region resides in the nonconserved region of dTIM between C1 and C2. No motifs of structural significance are detected in the mTIM protein. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays reveal an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock. Since mTim is expressed in the SCN and eyes, it is still possible that mTIM has a clock-relevant function but that its function is distinct from that described for dTIM. It is also conceivable that an mTIM homolog other that the one characterized here might exist that interacts with the mPER proteins (Zylka, 1998).

The timeless (tim) gene is essential for circadian clock function in Drosophila. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Although mTim is expressed in the SCN, neither its RNA nor protein levels oscillate there under constant conditions. Moreover, mTIM is a nuclear protein, and SCN levels are not altered by light pulses that shift circadian behavior. Mammalian TIM does not interact with the mPER proteins in yeast or in the SCN, but it is reported to interact with mPER1 in COS-7 cells and with Drosophila Per in vitro. Immunofluorescence studies of over-expressed proteins in cell culture fail to show changes in cellular location of mPER proteins when coexpressed with mTIM. mTIM interacts with mCRY1 and mCRY2, both in vivo and in vitro. Here it is shown that mTim is essential for embryonic development, but does not have substantiated circadian function. At embryonic day 7.5, presumptive homozygous embryos lack cellular organization, with necrotic cell debris filling the amniotic cavity, and resorption by maternal tissues has already begun. Developmental abnormalities are observed in embryos as early as embryonic day 5.5, indicating that mTim is essential for development around implantation. The mechanism behind the essential role of mTIM for mouse development is currently not known. At embryonic day 7.5, in situ hybridization shows that mTim RNA is expressed throughout the embryo, particularly in the embryonic germ cell layers and in the ectoplacental cone (Gotter, 2000).

Faithful transmission of the genome requires that a protein complex called cohesin establishes and maintains the regulated linkage between replicated chromosomes before their segregation. This study reports the unforeseen participation of C. elegans TIM-1, a paralog of the Drosophila clock protein Timeless, in the regulation of chromosome cohesion. Biochemical experiments defined the C. elegans cohesin complex and revealed its physical association with TIM-1. Functional relevance of the interaction was demonstrated by aberrant mitotic chromosome behavior, embryonic lethality and defective meiotic chromosome cohesion caused by the disruption of either TIM-1 or cohesin. TIM-1 depletion prevents the assembly of non-SMC (structural maintenance of chromosome) cohesin subunits onto meiotic chromosomes; however, and unexpectedly, a partial cohesin complex composed of SMC components still loads. Further disruption of cohesin activity in meiosis by the simultaneous depletion of TIM-1 and an SMC subunit decreases homologous chromosome pairing before synapsis, revealing a new role for cohesin in metazoans. On the basis of comparisons between Timeless homologs in worms, flies and mice, it is proposed that chromosome cohesion, rather than circadian clock regulation, is the ancient and conserved function for Timeless-like proteins (Chan, 2003).

Given the vital role of C. elegans tim-1 in chromosome cohesion and the essential function of mouse mTim1 in embryo viability, it is suggested that the evolution of the involvement of timeless in the Drosophila circadian rhythm results from a duplication and subsequent specialization of the fly timeout gene, which more closely resembles the single paralog present in C. elegans, mice and humans. Comparison of predicted protein structures reinforces this view. Drosophila Timeout, C. elegans TIM-1 and mouse mTim1 all have contiguous HEAT/Armadillo (Arm) repeats that reside within a similar region of each protein. In contrast, the HEAT/Arm repeats of Drosophila Timeless are interrupted by one of two Period-interaction domains, indicating that this change could enhance the association between Period and Timeless. Given the unique structure of Drosophila Timeless and the conclusions of the present study, it is proposed that chromosome cohesion, rather than circadian rhythm regulation, is the ancient and conserved function for timeless paralogs in metazoans (Chan, 2003).

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. The human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein (Unsal-Kacmaz, 2005).

An unexpected role for the clock protein timeless in developmental apoptosis

Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work has demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, was identified as a binding partner and substrate for c-Src, and its role in the differentiation of mES cells was probed. To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation. These data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked (O'Reilly, 2011).

CRTC potentiates light-independent timeless transcription to sustain circadian rhythms in Drosophila

Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. This study demonstrates a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, these data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. The study proposes that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species (Kim, 2016).

CREB-dependent transcription has long been implicated in different aspects of circadian gene expression. In mammalian clocks, light exposure triggers intracellular signaling pathways that activate CREB-dependent Per1 transcription, thereby adjusting the circadian phase of master circadian pacemaker neurons in the suprachiasmatic nucleus (SCN). The phase-resetting process involves the specific CREB coactivator CRTC1 and its negative regulator SIK1, constituting a negative feedback in the photic entrainment via a CREB pathway (Sakamoto, 2013; Jagannath, 2013). This report demonstrates a novel role of Drosophila CRTC that serves to coordinate circadian gene expression with 24-hour locomotor rhythms even in the absence of light. CRTC may regulate several clock-relevant genes, including those clock output genes that might be involved in the rhythmic arborizations and PDF cycling of the circadian pacemaker neurons. However, tim transcription was identified as one of the primary targets of Drosophila CRTC to sustain circadian rhythms in the free-running conditions, thus defining its light-independent clock function (Kim, 2016).

CREB could employ another transcriptional coactivator CBP (CREB-binding protein) to activate CRE-dependent transcription. In fact, CBP is a rather general coactivator recruited to gene promoters by other DNA-binding transcription factors. Previous studies have shown that Drosophila CBP associates with CLK, titrating its transcriptional activity. Mammalian CBP and the closely related coactivator p300 also form a complex with CLOCK-BMAL1, a homolog of the Drosophila CLK-CYC heterodimer, to stimulate their transcriptional activity. One possible explanation for CRTC-activated tim transcription is that Drosophila CRTC may analogously target the CLK-CYC heterodimer to stimulate CLK-CYC-tivate CRE-dependent transcription. Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while-induced clock genes. Moreover, CRTC associates with the bZIP domain in CREB protein, whereas CBP/p300 binds CREB through the phosphorylated KID domain, indicating that they might not necessarily target the same transcription factors apart from CREB. Finally, a protein complex of CLK and CRTC could not be detected in Drosophila S2 cells. Thus, it is likely that CRTC and CBP/p300 play unique roles in circadian transcription through their interactions with different DNA-binding transcription factors (Kim, 2016).

If CRTC augments CLK-CYC-dependent tim transcription indirectly, then why do crtc effects require CLK? A recent study suggested that mammalian CLOCK-BMAL1 may regulate the rhythmic access of other DNA-binding transcription factors to their target promoters in the context of chromatin, acting as a pioneer-like transcription factor. Given the structural and functional homology between Drosophila CLK-CYC and mammalian CLOCK-BMAL1, the presence of CLK-CYC in the tim promoter might allow the recruitment of additional transcription factors (e.g., CREB) and their co-activators including CRTC for maximal tim transcription. The transcriptional context of tim promoter might thus define its sensitivity to crtc effects among other clock promoters. In addition, the differential assembly of transcription factors on the tim promoter could explain tissue-specific effects of crtc on TIM oscillations (i.e., circadian pacemaker neurons versus peripheral clock tissues). Interestingly, chromatin immunoprecipitation with V5-tagged CLK protein revealed that CLK-CYC heterodimers associate with both tim and Sik2 gene promoters in fly heads. In LD cycles, however, their rhythmic binding to the Sik2 promoter is phase-delayed by ~4.5 hours compared with that to the tim promoter. These modes of transcriptional regulation may gate crtc effects on tim transcription in a clock-dependent manner, particularly in the increasing phase of tim transcription (Kim, 2016).

Transcription from CREB-responsive reporter genes shows daily oscillations, both in Drosophila and mammals, implicating this transcriptional strategy in the evolution of molecular clocks. In fact, cAMP signaling and CRE-dependent transcription constitute the integral components of core molecular clocks, serving to regulate daily rhythmic transcription of circadian clock genes. For instance, reciprocal regulation of dCREB2 and per at the transcription level has been reported to sustain free-running circadian rhythms in Drosophila. During fasting in mammals, a transcriptional program for hepatic gluconeogenesis is induced by CREB phosphorylation and CRTC2 dephosphorylation. Fasting-activated CREB-CRTC2 then stimulates Bmal1 expression61, whereas CLOCK-BMAL1-induced CRY rhythmically gates CREB activity in this process by modulating G protein-coupled receptor activity and inhibiting cAMP-induced CREB phosphorylation62. This molecular feedback circuit thus mutually links mammalian clocks and energy metabolism in terms of CREB-dependent transcription (Kim, 2016).

On the basis of these observations, a model is proposed for the evolution of CRTC-dependent clocks to explain the distinctive circadian roles of CRTC homologs (see A model for the evolution of CRTC-dependent clocks). CRTC is a transcriptional effector that integrates various cellular signals (Altarejos, 2011). It was reasoned that ancestral clocks may have employed CREB-CRTC-mediated transcription to sense extracellular time cues cell-autonomously and integrate this timing information directly into the earliest transcription-translation feedback loop (TTFL). This strategy would have generated simple but efficient molecular clocks to tune free-running molecular rhythms in direct response to environmental zeitgebers, such as light and the availability of nutrients. A circadian role of CRTC then has differentially evolved along with a selective set of clock targets. In poikilothermic Drosophila, light is accessible directly to circadian pacemaker neurons in the adult fly brain. Therefore, TIM degradation by the blue-light photoreceptor CRY plays a major role in the light entrainment of Drosophila clocks, although the photic induction of CLK/CYC-dependent tim transcription has been reported specifically at lower temperatures. Accordingly, Drosophila CRTC retained a constitutive co-activator function from the ancestral TTFL to support CLK/CYC-activated tim transcription and sustain free-running circadian behaviors. In homeothermic mammals, light input to the SCN is indirectly mediated by neurotransmitter release from presynaptic termini of the retinohypothalamic tract (RHT). Intracellular signaling relays in the SCN converge on the dephosphorylation and nuclear translocation of CRTC1 to activate CRE-dependent transcription. Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while per took over a role in the light-entrainment pathway by retaining CREB-CRTC1-dependent transcriptional regulation from the primitive TTFL. Consequently, mammalian clocks have lost a homolog of the Drosophila-like cry gene family, but instead evolved CRY homologs of the vertebrate-like cry gene family with transcriptional repressor activities in CLOCK-BMAL1-dependent transcription (Kim, 2016).

Regulation of metabolism and stress responses by neuronal CREB-CRTC-SIK pathways has been well documented in Drosophila. Given the demonstration of a circadian role of CRTC in the pacemaker neurons, it is possible that CRTC might sense metabolic cues in the context of circadian neural circuits to entrain molecular clocks cell-autonomously. Alternatively, but not exclusively, CRTC could participate in the regulation of clock-relevant metabolism as clock outputs from pacemaker neurons. These hypotheses remain to be validated in future studies (Kim, 2016).

Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing

The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, this study targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 hits that were identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Psi downregulation shortens the period of circadian rhythms and advances the phase of circadian behavior under temperature cycle. Interestingly, both phenotypes were suppressed in flies that could produce TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, it is concluded that PSI regulates the period of Drosophila circadian rhythms and circadian behavior phase during temperature cycling through its modulation of the tim splicing pattern (Foley, 2019).

Increasing evidence indicates that post-transcriptional mechanisms controlling gene expression are also critical for the proper function of circadian clocks in many organisms. In Drosophila, the post-transcriptional regulation of per mRNA has been best studied. per mRNA stability changes as a function of time. In addition, per contains an intron in its 3'UTR (dmpi8) that is alternatively spliced depending on temperature and lighting conditions. On cold days, the spliced variant is favored, causing an advance in the accumulation of per transcript levels as well as an advance of the evening activity peak. This behavioral shift means that the fly is more active during the day when the temperature would be most tolerable in their natural environment. The temperature sensitivity of dmpi8 is due to the presence of weak non-canonical splice sites. However, the efficiency of the underlying baseline splicing is affected by four single nucleotide polymorphisms (SNPs) in the per 3'UTR that vary in natural populations and form two distinct haplotypes. Also, while this splicing is temperature-sensitive in two Drosophila species that followed human migration, two species that remained in Africa lack temperature sensitivity of dmpi8 splicing. Furthermore, it has been recently demonstrated that the the trans-acting splicing factor B52 enhances dmpi8 splicing efficiency, and this effect is stronger with one of the two haplotypes. per is also regulated post-transcriptionally by the TWENTYFOUR-ATAXIN2 translational activation complex. This complex works by binding to per mRNA as well as the cap-binding complex and poly-A binding protein. This may enable more efficient translation by promoting circularization of the transcript. Interestingly, this mechanism appears to be required only in the circadian pacemaker neurons. Non-canonical translation initiation has also been implicated in the control of PER translation. Regulation of PER protein translation has also been studied in mammals, with RBM4 being a critical regulator of mPER1 expression. In flies however, the homolog of RBM4, LARK, regulates the translation of DBT, a PER kinase. miRNAs have emerged as important critical regulators of circadian rhythms in Drosophila and mammals, affecting the circadian pacemaker itself, as well as input and output pathways controlling rhythmic behavioral and physiological processes (Foley, 2019).

RNA-associated proteins (RAPs) include proteins that either bind directly or indirectly to RNAs. They mediate post-transcriptional regulation at every level. Many of these regulated events - including alternative splicing, splicing efficiency, mRNA stability, and translation - have been shown to function in molecular clocks. Thus, to obtain a broad view of the Drosophila circadian RAP landscape and its mechanism of action, an RNAi screen was performed targeting 364 of these proteins. This led to the discovery of a role for the splicing factor P-element somatic inhibitor (PSI) in regulating the pace of the molecular clock through alternative splicing of tim (Foley, 2019).

The results identify a novel post-transcriptional regulator of the circadian clock: PSI. PSI is required for the proper pace of both brain and body clock, and for proper phase-relationship with ambient temperature cycles. When Psi is downregulated, the circadian pacemaker speeds up and behavior phase under temperature cycles is advanced by 3 hr, and these phenotypes appear to be predominantly caused by an abnormal tim splicing pattern. Indeed, the circadian period and behavior phase of flies that can only produce functional TIM protein from a transgene missing most introns is insensitive to Psi downregulation. It is noted however that cwo's splicing pattern is also affected by Psi downregulation, and sgg splicing pattern was not studied, although it might also be controlled by PSI. It is therefore not possible to exclude a small contribution of non-tim splicing events to PSI downregulation phenotypes, or that in specific tissues these other splicing events play a greater role than in the brain (Foley, 2019).

Interestingly, Psi downregulation results in an increase in intron inclusion events that are favored under cold conditions (tim-sc and tim-cold), while an intron inclusion event favored under warm conditions is decreased (tim-M). However, the ability of tim splicing to respond to temperature changes is not abolished when Psi is downregulated. This could imply that an as yet unknown factor specifically promotes or represses tim splicing events in a temperature-dependent manner. Another possibility is that the strength of splice sites or tim's pre-mRNA structure impacts splicing efficiency in a temperature-dependent manner. For example, suboptimal per splicing signals explain the lower efficiency of per's most 3' splicing event at warm temperature (Foley, 2019).

How would the patterns of tim splicing affect the pace of the circadian clock, or advance the phase of circadian behavior under temperature cycles? In all splicing events that were studied, intron retention results in a truncated TIM protein. It is therefore possible that the balance of full length and truncated TIM proteins, which may function as endogenous dominant-negatives, determines circadian period. For example, truncated TIM might be less efficient at protecting PER from degradation, thus accelerating the pacemaker, or affecting its phase. Consistent with this idea, overexpression of the shorter cold-favored tim isoform (tim-sc) shortens period. Strikingly, Psi downregulation increases this isoform's levels and also results in a short phenotype. It has been proposed that production of tim-M transcripts (called tim-tiny in their study) delays the rate of TIM accumulation. Such a mechanism could also contribute to the short period observed when Psi is downregulated, since this reduces tim-M levels, which may accelerate TIM accumulation. Another interesting question is how PSI differentially affects specific splice isoforms of tim. One possibility is that the execution of a specific tim splicing event negatively influences the probability of the occurrence of other splicing events. For example, PSI could downregulate tim-sc and tim-cold by enhancing splicing and removal of the introns whose retention is necessary for production of these isoforms. This could indirectly reduce splicing of the intron that is retained in the warm tim-M isoform and result in tim-M upregulation. Conversely, PSI could directly promote tim-M intron retention and indirectly downregulate production of tim-sc and tim-cold (Foley, 2019).

Other splicing factors have been shown to be involved in the control of circadian rhythms in Drosophila. SRm160 contributes to the amplitude of circadian rhythms by promoting per expression, while B52/SMp55 and PRMT5 regulate per's most 3' splicing, which is temperature sensitive. Loss of PRMT5 results in essentially arrhythmic behavior, but this is unlikely to be explained by its effect on per's thermosensitive splicing. B52/SMp55 knockdown flies show a reduced siesta, which is controlled by the same per splicing. With the identification of Psi, this study has uncover a key regulator of tim alternative splicing pattern and shows that this pattern determines circadian period length, while per alternative splicing regulates the timing and amplitude of the daytime siesta. Interestingly, a recent study identified PRP4 kinase and other members of tri-snRNP complexes as regulators of circadian rhythms. Downregulation of prp4 caused excessive retention of the tim-M intron. PSI and PRP4 might thus have complementary functions in tim mRNA splicing regulation, working together to maintain the proper balance of tim isoform expression (Foley, 2019).

An unexpected finding is the role played by both PDF neurons and other circadian neurons in the short period phenotype observed with circadian locomotor rhythms when Psi was knocked-down. Indeed, it is quite clear from multiple studies that under constant darkness, the PDF-positive sLNvs dictate the pace of circadian behavior. Why, in the case of Psi downregulation, do PDF negative neurons also play a role in period determination? The explanation might be that PSI alters the hierarchy between circadian neurons, promoting the role of PDF negative neurons. This could be achieved by weakening PDF/PDFR signaling, for example (Foley, 2019).

While this study focused on PSI, several other interesting candidates were identified in the screen. The presence of a large number of splicing factors is noted. This adds to the emerging notion that alternative splicing plays a critical role in the control of circadian rhythms. Several per splicing regulators have been mentioned that can impact circadian behavior. In addition, a recent study demonstrated that specific classes of circadian neurons express specific alternative splicing variants, and rhythmic alternative splicing is widespread in these neurons. Interestingly, in this study, the splicing regulator barc, which was identified in the screen and which has been shown to causes intron retention in specific mRNAs, was found to be rhythmically expressed in LNds. Moreover, in mammals, alternative splicing appears to be very sensitive to temperature, and could explain how body temperature rhythms synchronize peripheral clocks. Another intriguing candidate is cg42458, which was found to be enriched in circadian neurons (LNvs and Dorsal Neurons 1). In addition to emphasizing the role of splicing, the screen suggests that regulation of polyA tail length is important for circadian rhythmicity, since several members of the CCR4-NOT complex and deadenylation-dependent decapping enzymes were identified. Future work will be required to determine whether these factors directly target mRNAs encoding for core clock components, or whether their effect on circadian period is indirect. Interestingly, the POP2 deadenylase, which is part of the CCR4-NOT complex, was recently shown to regulate tim mRNA levels post-transcriptionally. It should be noted that while the screen targeted 364 proteins binding or associated with RNA, it did not include all of them. For example, LSM12, which was recently shown to be a part of the ATXN2/TYF complex, was not included in the screen because it had not been annotated as a potential RAP when the screen was initiated (Foley, 2019).

In summary, this work provides an important resource for identifying RNA associated proteins regulating circadian rhythms in Drosophila. It identifies PSI is an important regulator of circadian period and circadian phase in response to thermal cycles, and points at additional candidates and processes that determine the periodicity of circadian rhythms (Foley, 2019).

date revised: 1 March 2024

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