Neither per, nor tim1, nor cry2 alone are essential components of the molecular circadian clockwork in the Madeira cockroach.
ABSTRACT: Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNAi) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clock´s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels within ~two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rm´perRNAi or Rm´cry2RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´perRNAi and shorter periods for Rm´tim1RNAi and Rm´cry2RNAi.As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.
Project description:An interlocked transcriptional-translational feedback loop (TTFL) is thought to generate the mammalian circadian clockwork in both the central pacemaker residing in the hypothalamic suprachiasmatic nuclei and in peripheral tissues. The core circadian genes, including Period1 and Period2 (Per1 and Per2), Cryptochrome1 and Cryptochrome2 (Cry1 and Cry2), Bmal1, and Clock are indispensable components of this biological clockwork. The cycling of the PER and CRY clock proteins has been thought to be necessary to keep the mammalian clock ticking.We provide a novel cell-permeant protein approach for manipulating cryptochrome protein levels to evaluate the current transcription and translation feedback model of the circadian clockwork. Cell-permeant cryptochrome proteins appear to be functional on the basis of several criteria, including the abilities to (1) rescue circadian properties in Cry1(-/-)Cry2(-/-) mouse fibroblasts, (2) act as transcriptional repressors, and (3) phase shift the circadian oscillator in Rat-1 fibroblasts. By using cell-permeant cryptochrome proteins, we demonstrate that cycling of CRY1, CRY2, and BMAL1 is not necessary for circadian-clock function in fibroblasts.These results are not supportive of the current version of the transcription and translation feedback-loop model of the mammalian clock mechanism, in which cycling of the essential clock proteins CRY1 and CRY2 is thought to be necessary.
Project description:Abnormalities in the circadian clockwork often characterize patients with major depressive and bipolar disorders. Circadian clock genes are targets of interest in these patients. CRY2 is a circadian gene that participates in regulation of the evening oscillator. This is of interest in mood disorders where a lack of switch from evening to morning oscillators has been postulated.We observed a marked diurnal variation in human CRY2 mRNA levels from peripheral blood mononuclear cells and a significant up-regulation (P = 0.020) following one-night total sleep deprivation, a known antidepressant. In depressed bipolar patients, levels of CRY2 mRNA were decreased (P = 0.029) and a complete lack of increase was observed following sleep deprivation. To investigate a possible genetic contribution, we undertook SNP genotyping of the CRY2 gene in two independent population-based samples from Sweden (118 cases and 1011 controls) and Finland (86 cases and 1096 controls). The CRY2 gene was significantly associated with winter depression in both samples (haplotype analysis in Swedish and Finnish samples: OR = 1.8, P = 0.0059 and OR = 1.8, P = 0.00044, respectively).We propose that a CRY2 locus is associated with vulnerability for depression, and that mechanisms of action involve dysregulation of CRY2 expression.
Project description:The circadian clock plays a vital role in monarch butterfly (Danaus plexippus) migration by providing the timing component of time-compensated sun compass orientation, a process that is important for successful navigation. We therefore evaluated the monarch clockwork by focusing on the functions of a Drosophila-like cryptochrome (cry), designated cry1, and a vertebrate-like cry, designated cry2, that are both expressed in the butterfly and by placing these genes in the context of other relevant clock genes in vivo. We found that similar temporal patterns of clock gene expression and protein levels occur in the heads, as occur in DpN1 cells, of a monarch cell line that contains a light-driven clock. CRY1 mediates TIMELESS degradation by light in DpN1 cells, and a light-induced TIMELESS decrease occurs in putative clock cells in the pars lateralis (PL) in the brain. Moreover, monarch cry1 transgenes partially rescue both biochemical and behavioral light-input defects in cry(b) mutant Drosophila. CRY2 is the major transcriptional repressor of CLOCK:CYCLE-mediated transcription in DpN1 cells, and endogenous CRY2 potently inhibits transcription without involvement of PERIOD. CRY2 is co-localized with clock proteins in the PL, and there it translocates to the nucleus at the appropriate time for transcriptional repression. We also discovered CRY2-positive neural projections that oscillate in the central complex. The results define a novel, CRY-centric clock mechanism in the monarch in which CRY1 likely functions as a blue-light photoreceptor for entrainment, whereas CRY2 functions within the clockwork as the transcriptional repressor of a negative transcriptional feedback loop. Our data further suggest that CRY2 may have a dual role in the monarch butterfly's brain-as a core clock element and as an output that regulates circadian activity in the central complex, the likely site of the sun compass.
Project description:In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1(-/-) mice, which are known to be a "short-period mutant," entrained to a shorter period of feeding cycles than did Cry2(-/-) mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1(-/-) mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.
Project description:Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3beta (glycogen synthase kinase 3beta)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein. Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3beta and governs the Ser557 phosphorylation-triggered degradation of CRY2.
Project description:Molecular mechanisms responsible for 24 h circadian oscillations, entrainment to external cues, encoding of day length and the time-of-day effects have been well studied experimentally. However, it is still debated from the molecular network point of view whether each cell in suprachiasmatic nuclei harbors two molecular oscillators, where one tracks dawn and the other tracks dusk activities. A single cell dual morning and evening oscillator was proposed by Daan et al., based on the molecular network that has two sets of similar non-redundant per1/cry1 and per2/cry2 circadian genes and each can independently maintain their endogenous oscillations. Understanding of dual oscillator dynamics in a single cell at molecular level may provide insight about the circadian mechanisms that encodes day length variations and its response to external zeitgebers. We present here a realistic dual oscillator model of circadian rhythms based on the series of hypotheses proposed by Daan et al., in which they conjectured that the circadian genes per1/cry1 track dawn while per2/cry2 tracks dusk and they together constitute the morning and evening oscillators (dual oscillator). Their hypothesis also provides explanations about the encoding of day length in terms of molecular mechanisms of per/cry expression. We frame a minimal mathematical model with the assumption that per1 acts a morning oscillator and per2 acts as an evening oscillator and to support and interpret this assumption we fit the model to the experimental data of per1/per2 circadian temporal dynamics, phase response curves (PRC's), and entrainment phenomena under various light-dark conditions. We also capture different patterns of splitting phenomena by coupling two single cell dual oscillators with neuropeptides vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) as the coupling agents and provide interpretation for the occurrence of splitting in terms of ME oscillators, though they are not required to explain the morning and evening oscillators. The proposed dual oscillator model based on Daan's hypothesis supports per1 and per2 playing the role of morning and evening oscillators respectively and this may be the first step towards the understanding of the core molecular mechanism responsible for encoding the day length.
Project description:The circadian clock generates rhythms of approximately 24?h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwo<sup>RNAi</sup> crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.
Project description:In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.
Project description:In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators that regulate the bouts of dawn and dusk activity. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TOC1 was proposed to activate a subset of morning-expressed oscillator genes. Our systems-biological approach overturns the current view of the Arabidopsis circadian clock showing that TOC1 does not function as an activator but as a timely-controlled general repressor of morning and evening oscillator components. Repression occurs through rhythmic binding to the promoters of all oscillator genes, suggesting a previously unexpected direct connection between the morning and evening loops. Examination of TOC1 genome-wide binding using TOC1 Minigene (TMG) seedlings expressing the genomic fragment of TOC1 fused to the Yellow Fluorescent Protein in a toc1-2 mutant background (TMG-YFP/toc1-2 seedlings) grown under LD cycles (12h light:12h dark).
Project description:Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (PER)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.