Rhythms In Flies Research Articles

FMRP binds Per1 mRNA and downregulates its protein expression in mice

FMRP, an RNA-binding protein, has previously shown to be involved in regulation of circadian rhythms in flies and mice. However, the molecular mechanism remains elusive. Here we demonstrate that core circadian component Per1 mRNA was a target of FMRP and the association leads to reduced PER1 expression. In Fmr1 KO mice, the oscillation of PER1 protein expression was significantly affected in a temporal and tissue-dependent pattern when compared to WT mice. Our work thus identified Per1 mRNA as a novel target of FMRP and suggested a potential role of FMRP in regulation of circadian function.

Gap junction protein Innexin2 modulates the period of free-running rhythms in Drosophila melanogaster

SummaryA neuronal circuit of ∼150 neurons modulates rhythmic activity-rest behavior of Drosophila melanogaster. While it is known that coherent ∼24-hr rhythms in locomotion are brought about when 7 distinct neuronal clusters function as a network due to chemical communication amongst them, there are no reports of communication via electrical synapses made up of gap junctions. Here, we report that gap junction proteins, Innexins play crucial roles in determining the intrinsic period of activity-rest rhythms in flies. We show the presence of Innexin2 in the ventral lateral neurons, wherein RNAi-based knockdown of its expression slows down the speed of activity-rest rhythm along with alterations in the oscillation of a core-clock protein PERIOD and the output molecule pigment dispersing factor. Specifically disrupting the channel-forming ability of Innexin2 causes period lengthening, suggesting that Innexin2 may function as hemichannels or gap junctions in the clock circuit.

Gap Junction Protein INNEXIN2 Modulates the Period of Activity-Rest Rhythms in <i>Drosophila Melanogaster</i>

The circadian neuronal circuit of Drosophila melanogaster is made up of about 150 neurons, distributed bilaterally and distinguished into 7 clusters. Multiple lines of evidence suggest that coherent rhythms in behaviour are brought about when these clusters function as a network. Although chemical modes of communication amongst circadian neurons have been well-studied, there has been no report of communication via electrical synapses made up of gap junctions. Here, we report for the first time that gap junction proteins - Innexins play crucial roles in determining the period of free-running activity rhythms in flies. Our experiments reveal the presence of gap junction protein INNEXIN2 in the ventral lateral neurons. RNA-interference based knockdown of its expression in circadian pacemakers slows down the speed of locomotor activity rhythm. Concomitantly, we find alterations in the oscillation of a core-clock protein PERIOD and in the output molecule Pigment Dispersing Factor in the circadian pacemaker neuron network.

Nipped-A regulates the Drosophila circadian clock via histone deubiquitination.

Psychiatric diseases are often accompanied by circadian disruptions, but the molecular underpinnings remain largely unclear. To address this, we screened genes that have been previously reported to be associated with psychiatric diseases and found that TRRAP, a gene associated with schizophrenia, is involved in circadian rhythm regulation. Knocking down Nipped-A, the Drosophila homolog of human TRRAP, leads to lengthened period of locomotor rhythms in flies. Molecular analysis demonstrates that NIPPED-A sets the pace of the clock by increasing the mRNA and protein levels of core clock genes timeless (tim) and Par domain protein 1ε (Pdp1ε). Furthermore, we found that NIPPED-A promotes the transcription of tim and Pdp1ε possibly by facilitating deubiquitination of histone H2B via the deubiquitination module of the transcription co-activator Spt-Ada-Gcn5 acetyltransferase complex. Taken together, these findings reveal a novel role for NIPPED-A in epigenetic regulation of the clock.

Drosophila PINK1 and parkin loss-of-function mutants display a range of non-motor Parkinson's disease phenotypes

Parkinson's disease (PD) is more commonly associated with its motor symptoms and the related degeneration of dopamine (DA) neurons. However, it is becoming increasingly clear that PD patients also display a wide range of non-motor symptoms, including memory deficits and disruptions of their sleep-wake cycles. These have a large impact on their quality of life, and often precede the onset of motor symptoms, but their etiology is poorly understood. The fruit fly Drosophila has already been successfully used to model PD, and has been used extensively to study relevant non-motor behaviours in other contexts, but little attention has yet been paid to modelling non-motor symptoms of PD in this genetically tractable organism. We examined memory performance and circadian rhythms in flies with loss-of-function mutations in two PD genes: PINK1 and parkin. We found learning and memory abnormalities in both mutant genotypes, as well as a weakening of circadian rhythms that is underpinned by electrophysiological changes in clock neurons. Our study paves the way for further work that may help us understand the mechanisms underlying these neglected aspects of PD, thus identifying new targets for treatments to address these non-motor problems specifically and perhaps even to halt disease progression in its prodromal phase.

Cycles of circadian illuminance are sufficient to entrain and maintain circadian locomotor rhythms in Drosophila.

Light at night disrupts the circadian clock and causes serious health problems in the modern world. Here, we show that newly developed four-package light-emitting diodes (LEDs) can provide harmless lighting at night. To quantify the effects of light on the circadian clock, we employed the concept of circadian illuminance (CIL). CIL represents the amount of light weighted toward the wavelengths to which the circadian clock is most sensitive, whereas visual illuminance (VIL) represents the total amount of visible light. Exposure to 12 h:12 h cycles of white LED light with high and low CIL values but a constant VIL value (conditions hereafter referred to as CH/CL) can entrain behavioral and molecular circadian rhythms in flies. Moreover, flies re-entrain to phase shift in the CH/CL cycle. Core-clock proteins are required for the rhythmic behaviors seen with this LED lighting scheme. Taken together, this study provides a guide for designing healthful white LED lights for use at night, and proposes the use of the CIL value for estimating the harmful effects of any light source on organismal health.

KPNB1 mediates PER/CRY nuclear translocation and circadian clock function.

Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

Effects of polygamy on the activity/rest rhythm of male fruit flies Drosophila melanogaster.

Although polygamy is common in insects, its extent varies enormously among natural populations. Mating systems influence the evolution of reproductive traits and the difference in extent of polygamy between males and females may be a key factor in determining traits which come under the influence of sexual selection. Fruit flies Drosophila melanogaster are promiscuous as both males and females mate with multiple partners. Mating has severe consequences on the physiology and behaviour of flies, and it affects their activity/rest rhythm in a sex-specific manner. In this study, we attempted to discern the effects of mating with multiple partners as opposed to a single partner, or of remaining unmated, on the activity/rest rhythm of flies under cyclic semi-natural (SN) and constant dark (DD) conditions. The results revealed that while evening activity of mated flies was significantly reduced compared to virgins, polygamous males showed a more severe reduction compared to monogamous males. In contrast, though mated females showed reduction in evening activity compared to virgins, activity levels were not different between polygamous and monogamous females. Although there was no detectable effect of mating on clock period, power of the activity/rest rhythm was significantly reduced in mated females with no difference seen between polygamous and monogamous individuals. These results suggest that courtship motivation, represented by evening activity, is successively reduced in males due to mating with one or more partners, while in females, it does not depend on the number of mating partners. Based on these results we conclude that polygamy affects the activity/rest rhythm of fruit flies D. melanogaster in a sex-dependent manner.

Insights into differential activity patterns of Drosophilids under semi-natural conditions

We showed recently that Drosophila ananassae, a closely related and sympatric species of the commonly studied fruitfly D. melanogaster, shows distinctly deviant patterns in circadian activity/rest rhythm from the latter under a variety of laboratory conditions. To examine whether such differences extend to more natural conditions where a variety of time cues and similar environmental pressures might force different species to adopt similar temporal patterns, we examined these two species under semi-natural conditions over a span of 1.5 years. Furthermore, we asked to what extent features of activity/rest rhythm of flies are conserved across species under changing environmental conditions encountered across seasons, and to do so, we studied two more drosophilid species. We found that while each species exhibits seasonality in activity patterns, this seasonality is marked by interesting inter-specific differences. Similar to laboratory studies, D. ananassae showed activity mostly during the day, while D. melanogaster and D. malerkotliana exhibited almost similar activity patterns across seasons, with predominantly two peaks of activity, one in the morning and another in the evening. Throughout the year, Zaprionus indianus displayed very low levels of activity compared with D. melanogaster, yet, compared with those seen in standard laboratory assays, this species exhibited more robust rhythms under semi-natural conditions. We hypothesise that different ecological factors may have influenced these species to adopt different temporal niches.

Robustness of Circadian Timing Systems Evolves in the Fruit Fly Drosophila melanogaster as a Correlated Response to Selection for Adult Emergence in a Narrow Window of Time

Robustness is a fundamental property of biological timing systems that is likely to ensure their efficient functioning under a wide range of environmental conditions. Here we report the findings of our study aimed at examining robustness of circadian clocks in fruit fly Drosophila melanogaster populations selected to emerge as adults within a narrow window of time. Previously, we have reported that such flies display enhanced synchrony, accuracy, and precision in their adult emergence and activity/rest rhythms. Since it is expected that accurate and precise circadian clocks may confer enhanced stability in circadian time-keeping, we decided to examine robustness in circadian rhythms of flies from the selected populations by subjecting them to a variety of environmental conditions comprising of a range of photoperiods, light intensities, ambient temperatures, and constant darkness. The results revealed that adult emergence and activity/rest rhythms of flies from the selected stocks were more robust than controls, as they displayed enhanced stability under a wide variety of environmental conditions. These results suggest that selection for adult emergence within a narrow window of time results in the evolution of robustness in circadian timing systems of the fruit fly D. melanogaster. (Author correspondence: vsharma@jncasr.ac.in or vksharmas@gmail.com)

NAT1/DAP5/p97 and Atypical Translational Control in the Drosophila Circadian Oscillator

Circadian rhythms are driven by gene expression feedback loops in metazoans. Based on the success of genetic screens for circadian mutants in Drosophila melanogaster, we undertook a targeted RNAi screen to study the impact of translation control genes on circadian locomotor activity rhythms in flies. Knockdown of vital translation factors in timeless protein-positive circadian neurons caused a range of effects including lethality. Knockdown of the atypical translation factor NAT1 had the strongest effect and lengthened circadian period. It also dramatically reduced PER protein levels in pigment dispersing factor (PDF) neurons. BELLE (BEL) protein was also reduced by the NAT1 knockdown, presumably reflecting a role of NAT1 in belle mRNA translation. belle and NAT1 are also targets of the key circadian transcription factor Clock (CLK). Further evidence for a role of NAT1 is that inhibition of the target of rapamycin (TOR) kinase increased oscillator activity in cultured wings, which is absent under conditions of NAT1 knockdown. Moreover, the per 5'- and 3'-UTRs may function together to facilitate cap-independent translation under conditions of TOR inhibition. We suggest that NAT1 and cap-independent translation are important for per mRNA translation, which is also important for the circadian oscillator. A circadian translation program may be especially important in fly pacemaker cells.

Temperature can entrain egg laying rhythm of Drosophila but may not be a stronger zeitgeber than light

In Drosophila multiple circadian oscillators and behavioral rhythms are known to exist, yet most previous studies that attempted to understand circadian entrainment have focused on the activity/rest rhythm and to some extent the adult emergence rhythm. Egg laying behavior of Drosophila females also follows circadian rhythmicity and has been seen to deviate substantially from the better characterized rhythms in a few aspects. Here we report the findings of our study aimed at evaluating how circadian egg laying rhythm in fruit flies Drosophila melanogaster entrains to time cues provided by light and temperature. Previous studies have shown that activity/rest rhythm of flies entrains readily to light/dark (LD) and temperature cycles (TC). Our present study revealed that egg laying rhythm of a greater percentage of females entrains to TC compared to LD cycles. Therefore, in the specific context of our study this result can be taken to suggest that egg laying clocks of D. melanogaster entrains to TC more readily than LD cycles. However, when TC were presented along with out-of-phase LD cycles, the rhythm displayed two peaks, one occurring close to lights-off and the other near the onset of low temperature phase, indicating that upon entrainment by TC, LD cycles may be able to exert a greater influence on the phase of the rhythm. These results suggest that temperature and light associatively entrain circadian egg laying clocks of Drosophila.

Sleep, arousal, and rhythms in flies

Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans the biological, physical, and social sciences.

PERspective on PER phosphorylation

Period (PER) proteins are essential parts of the molecular clocks that control circadian rhythms in flies and mammals. Phosphorylation regulates PER's stability and subcellular localization; however, the physiologically relevant sites have been difficult to identify in spite of knowing the relevant kinase. In this issue of Genes & Development, Chiu and colleagues (1758-1772) identify a key phosphorylation site on PER that recruits the F-box protein Slimb to trigger PER degradation and set clock speed.

Entrainment Properties of the Locomotor Activity Rhythm of Drosophila melanogaster Under Different Photoperiodic Regimens

In this paper we report the results of an experiment to assess how closely repeated brief light pulses (LPs) mimic the effects of 12:12 h light/dark (LD) cycles (PPc). The locomotor activity rhythm of individual fruit flies from a laboratory population of Drosophila melanogaster was monitored under four different photoperiodic regimens, created using 12 h of light and 12 h of darkness or brief light pulses (LPs). The phase relationship (Ψ) and the stability (precision) of the locomotor activity rhythm during entrainment were estimated in order to compare the state of the circadian clocks under the four different photoperiodic regimens. The flies (n = 72) were subjected to four different LD cycles: (i) 12 h of light and 12 h of darkness (complete photoperiod, PPc); (ii) a single brief LP of 15 min duration presented close to the onset of activity (SLP-1); (iii) a single brief LP of 15 min duration administered close to the offset of activity (SLP-2); and (iv) two brief LPs administered 12 h apart (skeleton photoperiod, PPs). The locomotor activity rhythm of the flies was first monitored under constant darkness (DD) for about 10 days and then under the four different photoperiodic regimens for about 10 days, and finally in DD for the remainder of the experiment. The Ψ of the locomotor activity rhythm and its precision under PPc and PPs did not differ significantly, but they were significantly different from the SLP-1 and SLP-2 conditions. The results provide interesting insights into photoentrainment mechanisms of circadian clocks in D. melanogaster, and suggest that skeleton photoperiods, but not single brief LPs, mimic the actions of complete photoperiods.

Joseph S. Takahashi, Fred W. Turek and Robert Y.Moore (eds): Circadian Clocks (Handbook of Behavioral Neurobiology, Vol 12)

Circadian Clocks (Handbook of Behavioral Neurobiology, Vol. 12) Takahashi, Joseph S., Turek, Fred W. and Moore, Robert Y. (eds) Kluwer Academic/Plenum Publishers 2001 , $175 , 794 pages, ISBN 0-306-46504-3 Most organisms have been forced to adapt to the once-daily rotation of our planet on its axis. The geophysical day–night cycle is not only reflected as circadian (Latin: circa = about, dies = day) rhythms of physiology and behavior, but is also actually incorporated within organisms as a genetically determined temporal program. This endogenous, temperature-compensated timekeeping mechanism recognizes local time, measures its passage, and contributes to the regulation of homeostasis, seasonal behavior, and migration. Evidence for its existence was still being debated as late as 1960, but by 1981 the field had advanced to such a stage that it was reviewed in Volume 4 of the new Handbook of Behavioral Neurobiology. At that time, single gene mutants affecting rhythms in flies and fungi had been isolated, and the suprachiasmatic nucleus (SCN) was being proposed as a candidate circadian clock in mammals, but the terms ‘genetics’, ‘neurotransmitter’, and ‘shift work’ were not yet listed in the index. In the editor's preface, Jürgen Aschoff wrote, ‘The study of the physiological mechanisms underlying circarhythms is a rapidly developing field, and the question of how discrete is the clock from the rest of the body…may soon be answered in the circadian case…’. Twenty years have now passed and our progress has been almost unimaginable. Among other advances, the body of evidence that has identified the SCN as a master circadian pacemaker is so compelling and multidisciplinary that the strength of this functional localization is unsurpassed by that of any other structure in the vertebrate brain. We have discovered intracellular regulatory molecules and biochemical processes that are likely to constitute the actual oscillatory mechanism of the clock. The practical application of our new knowledge has been recognized, and chronobiologists now advise astronauts. Volume 12 of the Handbook, Circadian Clocks, summarizes how extraordinarily far we have come. It is a comprehensive and authoritative reference work of 26 chapters in 6 parts. Included are chapters on entrainment theory and the ecology and evolution of circadian rhythmicity; on the organization of circadian systems, from unicells to invertebrates and non-mammalian and mammalian vertebrates; on the regulation of mammalian rhythmicity, including photic and non-photic entrainment, development and aging, sleep and wakefulness, and seasonality; on cellular and molecular mechanisms in Synechococcus, Gonyaulax, Neurospora, Drosophila, opisthobranch mollusks, and rodents; and on the human circadian system and its role in sleep, performance, aging, affective disorders, endocrine secretion, and photoperiodic responses. The book is handsomely produced and certain to become a manual for molecular biologists, neuroscientists, physiologists, and physicians working on circadian rhythms. While no existing book matches the scope of Circadian Clocks, a few topics are missing even from this volume. Not considered are the clocks of higher plants, or the role of clocks in tidal rhythms and sun compass orientation. There is little, if any, coverage of how circadian rhythms might impact medical practice, i.e. by influencing the timing of pharmacotherapy or the manifestations of disease. Finally, some chapters on molecular mechanisms were apparently written five years ago and are a bit outdated, although the field is moving so fast anyway that it would be impossible for any book to be completely up to date. So, some remarkable new stories – the mutant clock genes responsible for advanced sleep phase syndrome, the role of peripheral oscillators in body rhythmicity, and the identification of candidate circadian photoreceptors and molecules – will need to wait for the next volume. Circadian Clocks clearly shows why the circadian timekeeping system has become such a unique and powerful model for studying genes, brain and behavior. There are remarkable opportunities to begin to understand a biological system at multiple levels of organization, ranging from the regulation of gene expression, to intercellular networks and multisynaptic pathways, and finally to integrated patterns of physiology and performance. Clock investigators include geneticists cloning clock genes, zoologists examining rhythmic behaviors in the wild, neurophysiologists recording spike trains in the hypothalamus, and psychiatrists treating patients with seasonal affective disorder (to name just a few). By enhancing the dialogue between these researchers and their disciplines, Circadian Clocks should serve as a very valuable resource. William J. Schwartz Department of Neurology University of Massachusetts Medical School 55 Lake Ave. North Worcester MA 01655, USA

On the Use of the Time Axis for Ecological Separation: Diel Rhythms as an Evolutionary Constraint

On the Use of the Time Axis for Ecological Separation: Diel Rhythms as an Evolutionary Constraint

A quadruple photoreceptor mutant still keeps track of time.

Time measurement and light detection are inextricably linked. Cryptochromes, the blue-light photoreceptors shared between plants and animals, are critical for circadian rhythms in flies and mice [1-3]. WC-1, a putative blue-light photoreceptor, is also essential for the maintenance of circadian rhythms in Neurospora [4]. In contrast, we report here that in Arabidopsis thaliana the double mutant lacking the cryptochromes cry1 and cry2, and even a quadruple mutant lacking the red/ far-red photoreceptor phytochromes phyA and phyB as well as cry1 and cry2, retain robust circadian rhythmicity. Interestingly, the quadruple mutant was nearly blind for developmental responses but perceived a light cue for entraining the circadian clock. These results indicate that cryptochromes and phytochromes are not essential components of the central oscillator in Arabidopsis and suggest that plants could possess specific photosensory mechanisms for temporal orientation, in addition to cryptochromes and phytochromes, which are used for both spatial and temporal adaptation.

Heavyweight Class

Heavyweight Class

Staining in the brain of Pachymorpha sexguttata mediated by an antibody against a Drosophila clock-gene product: labeling of cells with possible importance for the beetle's circadian rhythms.

Central nervous system ganglia within the head of the beetle Pachymorpha sexguttata were labeled using an antibody that recognizes an evolutionarily conserved region of the period (per) gene product of Drosophila melanogaster. per and the protein it encodes (PER) are believed to play a central role in the generation of endogenous circadian rhythms in flies; therefore anti-PER-mediated immunoreactivity may help to uncover cellular components of the circadian clock system in that insect and in others. In the beetle, application of this antibody led to the staining of a distinct set of neurons located in the optic lobes and the central brain, plus small numbers of putative glial cells in the optic lobes. Neuronal perikarya (including their nuclei in a few cases), the axons, and terminal regions of the neurons were stained. The network formed by these labeled cells and processes are candidates for the neuronal basis of the beetle's circadian clock system: the pacemaker region situated next to the medulla neuropil, its connection to the apparent site of Zeitgeber input, and putative efferent pathways projecting to control centers of various effector systems. Anti-PER-mediated labeling and that resulting from application to beetle specimens of an antiserum against pigment-dispersing hormone (PDH) were compared; in the Drosophila brain all "PDH cells" express the per gene as well. In the beetle, however, the set of "PER cells" and PDH ones is at least in part nonoverlapping. The hypothesis that neurons stained by application of anti-PER participate in the control of the beetle's circadian rhythms is discussed in the context of previous electrophysiological and immunohistochemical studies. Also considered are analogies to, and differences from, labeling of the PER protein in fruit flies and PER-like immunoreactivity in other animals.