The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by CLOCK (CLK) and CYCLE (CYC) and repressed by PERIOD (PER) and TIMELESS (TIM) 1. The timing of ...CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM 2–4. Although kinases that control PER and TIM levels and subcellular localization have been identified 5–10, additional kinases are predicted to target PER, TIM, and/or CLK to promote time-specific transcriptional repression. We screened for kinases that alter circadian behavior via clock cell-directed RNA interference (RNAi) and identified the proline-directed kinase nemo (nmo) as a novel component of the circadian oscillator. Both nmo RNAi knockdown and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. CLK levels increase when nmo expression is knocked down in clock cells, whereas CLK levels decrease and PER and TIM accumulation are delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering CLK, PER, and TIM expression, thereby contributing to the generation of an ∼24 hr circadian period.
► NMO kinase is a new circadian clock component ► NMO slows the pace of the circadian oscillator ► NMO alters PER, TIM, and CLK expression
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports ...obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.
Significance CRYPTOCHROMES (CRYs) are blue light photoreceptors that mediate phototransduction in brain arousal neurons, as well as circadian light entrainment in Drosophila fruit flies. We describe ...how light-activated Drosophila CRY couples to membrane depolarization and increased action potential firing rate in large ventral lateral arousal neurons. Pharmacological treatments that specifically disrupt the CRY redox-sensitive flavin chromophore or block voltage-gated K ⁺ channels abolish the light response. Correspondingly, we find that the Kvβ channel subunit Hyperkinetic with a well conserved redox sensor domain links light-evoked redox changes in CRY to rapid changes in membrane electrical potential.
Blue light activation of the photoreceptor CRYPTOCHROME (CRY) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster . Here we show that acute arousal behavioral responses to blue light significantly differ in mutants lacking CRY, as well as mutants with disrupted opsin-based phototransduction. Light-activated CRY couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K ⁺) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk ⁻/⁻ mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K ⁺ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go–related gene, are ion conducting channels for CRY/Hk-coupled light response. Light activation of CRY is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor.
Circadian neural circuits generate near 24-hr physiological rhythms that can be entrained by light to coordinate animal physiology with daily solar cycles. To examine how a circadian circuit ...reorganizes its activity in response to light, we imaged period (per) clock gene cycling for up to 6 days at single-neuron resolution in whole-brain explant cultures prepared from per-luciferase transgenic flies. We compared cultures subjected to a phase-advancing light pulse (LP) to cultures maintained in darkness (DD). In DD, individual neuronal oscillators in all circadian subgroups are initially well synchronized but then show monotonic decrease in oscillator rhythm amplitude and synchrony with time. The small ventral lateral neurons (s-LNvs) and dorsal lateral neurons (LNds) exhibit this decrease at a slower relative rate. In contrast, the LP evokes a rapid loss of oscillator synchrony between and within most circadian neuronal subgroups, followed by gradual phase retuning of whole-circuit oscillator synchrony. The LNds maintain high rhythmic amplitude and synchrony following the LP along with the most rapid coherent phase advance. Immunocytochemical analysis of PER shows that these dynamics in DD and LP are recapitulated in vivo. Anatomically distinct circadian neuronal subgroups vary in their response to the LP, showing differences in the degree and kinetics of their loss, recovery and/or strengthening of synchrony, and rhythmicity. Transient desynchrony appears to be an integral feature of light response of the Drosophila multicellular circadian clock. Individual oscillators in different neuronal subgroups of the circadian circuit show distinct kinetic signatures of light response and phase retuning.
•Drosophila whole-brain explants cultured 6 days show circuit-wide response to light•Light induces transient loss of synchrony and individual oscillator rhythmicity•A new state of phase-retuned network synchrony gradually emerges after a light pulse•Neuronal subgroups exhibit distinct kinetic signatures of light response in vivo
Roberts et al. describe how Drosophila whole-brain explants cultured for 6 days show circuit-wide responses to light. Light induces transient desynchrony followed by the gradual emergence of a new state of phase-retuned network synchrony. Neuronal subgroups were found to exhibit distinct kinetic signatures of light response in vivo.
Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS (PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was ...previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.
Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in ...period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. We were surprised to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis.
The
Drosophila circadian oscillator consists of interlocked
period (
per)/
timeless (
tim) and
Clock (
Clk) transcriptional/translational feedback loops. Within these feedback loops, CLK and CYCLE ...(CYC) activate
per and
tim transcription at the same time as they repress
Clk transcription, thus controlling the opposite cycling phases of these transcripts. CLK-CYC directly bind E box elements to activate transcription, but the mechanism of CLK-CYC-dependent repression is not known. Here we show that a CLK-CYC-activated gene,
vrille (
vri), encodes a repressor of
Clk transcription, thereby identifying
vri as a key negative component of the
Clk feedback loop in
Drosophila's circadian oscillator. The blue light photoreceptor encoding
cryptochrome (
cry) gene is also a target for VRI repression, suggesting a broader role for VRI in the rhythmic repression of output genes that cycle in phase with
Clk.
Circadian regulation of chemosensory processes is common in animals, but little is known about how circadian clocks control chemosensory systems or the consequences of rhythms in chemosensory system ...function. Taste is a major chemosensory gate used to decide whether or not an animal will eat, and the main taste organ in Drosophila, the proboscis, harbors autonomous circadian oscillators. Here we examine gustatory physiology, tastant-evoked appetitive behavior, and food ingestion to understand clock-dependent regulation of the Drosophila gustatory system.
Here we report that single-unit responses from labellar gustatory receptor neurons (GRNs) to attractive and aversive tastants show diurnal and circadian rhythms in spike amplitude, frequency, and duration across different classes of gustatory sensilla. Rhythms in electrophysiological responses parallel behavioral rhythms in proboscis extension reflex. Molecular oscillators in GRNs are necessary and sufficient for rhythms in gustatory responses and drive rhythms in G protein-coupled receptor kinase 2 (GPRK2) expression that mediate rhythms in taste sensitivity. Eliminating clock function in certain GRNs increases feeding and locomotor activity, mimicking a starvation response.
Circadian clocks in GRNs control neuronal output and drive behavioral rhythms in taste responses that peak at a time of day when feeding is maximal in flies. Our results argue that oscillations in GPRK2 levels drive rhythms in gustatory physiology and behavior and that GRN clocks repress feeding. The similarity in gustatory system organization and feeding behavior in flies and mammals, as well as diurnal changes in taste sensitivity in humans, suggest that our results are relevant to the situation in humans.
► The Drosophila circadian clock controls rhythms in taste sensitivity ► Clocks in gustatory receptor neurons control tastant-evoked physiology and behavior ► G protein receptor kinase controls circadian rhythms in gustatory responses ► Feeding is suppressed by circadian clocks in gustatory receptor neurons
Circadian (∼24 hr) clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor CRYPTOCHROME ...(CRY) synchronizes these feedback loops to light:dark cycles by binding to and degrading TIMELESS (TIM) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, we generated a GFP-tagged-cry transgene that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.
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•A GFP-cry transgene recapitulates CRY function in brain pacemaker neurons•GFP-CRY mediates light entrainment and sustains clock function in body tissues•CRY maintains passive membrane properties in a non-clock body tissue•CRY and K+ channels maintain passive membrane properties independent of light
CRYPTOCHROME (CRY) entrains and/or stimulates firing of Drosophila brain neurons upon light exposure. Agrawal et al. show that CRY both entrains and sustains clocks in body tissues but also plays a novel role together with K+ channels to maintain passive membrane properties in non-clock body tissues independent of light.
In the fruit fly Drosophila melanogaster, CRYPTOCHROME (CRY) functions as a photoreceptor to entrain circadian oscillators to light-dark cycles and as a transcription factor to maintain circadian ...oscillator function in certain peripheral tissues. Given the importance of CRY to circadian clock function, we expected this protein to be expressed in all oscillator cells, yet CRY cellular distribution and subcellular localization has not been firmly established. Here we investigate CRY spatial expression in the brain using a newly developed CRY antibody and a novel set of cry deletion mutants. We find that CRY is expressed in s-LNvs, l-LNvs, and a subset of LNds and DN₁s, but not DN₂s and DN₃s. CRY is present in both the nucleus and the cytoplasm of these neurons, and its subcellular localization does not change over the circadian cycle. Although CRY is absent in DN₂s and DN₃s, cry promoter activity and/or cry mRNA accumulation can be detected in these neurons, suggesting that CRY levels are regulated posttranscriptionally. Oscillators in DN₂s and DN₃s entrain to environmental light-dark cycles, which implies that they are entrained indirectly by retinal photoreceptors, extraretinal photoreceptors, or other CRY-expressing cells.
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