Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock

Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally.Circadian clocks are biological timing systems that allow organisms to anticipate and prepare for daily changes in the environment. A hallmark of a circadian oscillator is its ability to drive self-sustained rhythms in gene expression and behavior with a period close to 24 h, even in the absence of environmental cues (1). A general challenge for the biochemical machinery that generates rhythms is to precisely define the duration of the day in the face of perturbations, including fluctuations in the cellular abundance of the molecular components. The importance of maintaining precise circadian timing is underscored by experiments showing that mismatch between the clock period and the rhythms in the external environment results in health problems and fitness defects (2, 3).Although circadian clocks are found across all kingdoms of life, the Kai oscillator from cyanobacteria presents a uniquely powerful model system to study the design principles inherent in the molecular interactions that generate rhythms. A mixture of the purified proteins KaiA, KaiB, and KaiC results in stable oscillations in the phosphorylation state of KaiC in vitro that persist for many days and share many of the properties of circadian clocks in vivo (4–6). In particular, the oscillator can successfully generate near–24-h rhythms over a range of concentrations of the clock proteins both in vivo and in vitro (7–9), so fine-tuning of gene expression is not needed to support a functional clock. Much has been learned about the behavior of the isolated Kai proteins, including the determination of high-resolution crystal structures of all three components (10–12). A critical challenge that remains is to understand how the properties of the Kai proteins are integrated together in the full system to generate precisely timed rhythms.KaiC appears to be the central hub of timing information in the oscillator. Each KaiC molecule consists of two AAA+ family ATPase domains that consume the free energy of ATP hydrolysis to drive oscillations. Like many other members of this family, KaiC forms hexamers, and the enzymatic active sites are formed at the subunit interfaces where nucleotides are bound. The C-terminal, or CII, domain of KaiC has additional phosphotransferase activities that are unusual for the AAA+ family: it can phosphorylate and dephosphorylate two residues near the subunit interface, Ser431 and Thr432 (13). KaiC autokinase and autophosphatase activities occur at the same active site (14, 15). In isolation, KaiC has high phosphatase activity, but the enzyme is pushed toward kinase activity by the activator protein KaiA, which interacts directly with the KaiC C-terminal tail (16, 17). Roughly speaking, kinase activity predominates during the day, and phosphatase activity predominates during the night (18). Thus, understanding the feedback mechanisms that generate a precise time delay between these modes is crucial to understanding timing in the oscillator (19).Inactivation of KaiA and a transition from kinase to phosphatase mode occur when KaiB•KaiC complexes form, closing a negative feedback loop by sequestering KaiA in a ternary complex and leaving it unable to act on other KaiC molecules (20, 21). By temporarily removing KaiA molecules from their activating role, this molecular titration mechanism may act to synchronize the activity of all KaiC hexamers in the reaction (20, 22, 23). Phosphorylation and dephosphorylation proceed in a strongly ordered fashion so that in response to a change in KaiA activity, Thr432 is (de)phosphorylated first, followed later by Ser431 (18, 20, 21). It is known that phosphorylated Ser431 is important for allowing the formation of KaiB•KaiC complexes. However, recent work has made it clear that the binding of KaiB involves both KaiC domains—in particular, the slow ATPase activity of the N-terminal CI domain, which is not phosphorylated, is required for KaiB interaction (24, 25).Because of the importance of precisely timing negative feedback via KaiB•KaiC complex formation for generating appropriate rhythms (22), we wanted to understand the role of phosphorylation of the KaiC hexamer in controlling this process. The involvement of both KaiC domains suggests that information about phosphorylation in CII is communicated allosterically through changes in hexamer structure to the CI domain, potentially through ring–ring stacking interactions (24, 26). We therefore hypothesized that the KaiC phosphorylation sites on each subunit might act as allosteric regulators in the context of a hexameric ring so that phosphorylation of one subunit would alter the ability of all other subunits in the ring to engage with KaiA and KaiB, providing a cooperative mechanism to control the timing of these interactions.We conducted a series of biochemical experiments and perturbations to study the effect of altering the status of each phosphorylation site on the KaiC hexamer. To interpret these results, we then developed a mathematical model analogous to classical models of allosteric transitions in multimeric proteins. We constrain the kinetic parameters in this model using experimental measurements of rate constants, allowing us to compare the predictions of the model directly with data. We conclude that maintenance of circadian timing over a range of protein concentrations requires an effectively ultrasensitive switch in each KaiC hexamer from an exclusively KaiA-binding state to a state that can bind to KaiB as phosphorylation proceeds. This effect requires that KaiC hexamers consist of mixtures of differentially phosphorylated subunits, as would be produced by stochastic autophosphorylation of a hexamer. Ultrasensitivity results from opposing effects of phosphorylation on Thr432 and Ser431 in controlling a concerted transition within a given KaiC hexamer. Including this mechanism in the model is necessary to explain the experimentally observed tolerance of the system to altered protein concentrations.     

The circadian clock ensures successful DNA replication in cyanobacteria

Disruption of circadian rhythms causes decreased health and fitness, and evidence from multiple organisms links clock disruption to dysregulation of the cell cycle. However, the function of circadian regulation for the essential process of DNA replication remains elusive. Here, we demonstrate that in the cyanobacterium Synechococcus elongatus, a model organism with the simplest known circadian oscillator, the clock generates rhythms in DNA replication to minimize the number of open replication forks near dusk that would have to complete after sunset. Metabolic rhythms generated by the clock ensure that resources are available early at night to support any remaining replication forks. Combining mathematical modeling and experiments, we show that metabolic defects caused by clock–environment misalignment result in premature replisome disassembly and replicative abortion in the dark, leaving cells with incomplete chromosomes that persist through the night. Our study thus demonstrates that a major function of this ancient clock in cyanobacteria is to ensure successful completion of genome replication in a cycling environment.

Circadian clocks, internally generated rhythms in physiology with a ∼24 h period, are found in all domains of life. These clocks allow organisms to coordinate their physiological activities in anticipation of the daily cycle in the external environmental (1–3). Disruption of clocks caused either by mutation or clock–environment mismatch leads to decreased health and reproductive fitness in multiple organisms (4–6). In mammals, risk for age-related diseases such as cancer and cardiometabolic dysfunction is enhanced by circadian disruption (7, 8).Although much is now understood about the molecular mechanisms that generate rhythms, the origin of these health defects is still incompletely understood. A common target of circadian control shared across many species is the progression of the cell cycle (9–12). In animals, disrupted circadian rhythms are often linked to aberrant cell proliferation and tumorigenesis (13). Successful duplication of the genome is essential for the production of viable progeny. Replicating a bacterial genome can take up to several hours, a timescale over which external illumination from sunlight can change substantially. We therefore speculated that initiation of DNA replication could be a key point of circadian control. The cyanobacterium Synechococcus elongatus, which has the simplest known circadian system, is a powerful model system to address these issues, both because its clock is intimately coupled to cell cycle (9, 14–16) and because clock–environment misalignment has profound effects on reproductive fitness (17). Here, we analyze whether replication is clock-regulated in S. elongatus and the consequences of clock–environment mismatch on DNA replication.     

A model of the cell-autonomous mammalian circadian clock

Circadian timekeeping by intracellular molecular clocks is evident widely in prokaryotes and eukaryotes. The clockworks are driven by autoregulatory feedback loops that lead to oscillating levels of components whose maxima are in fixed phase relationships with one another. These phase relationships are the key metric characterizing the operation of the clocks. In this study, we built a mathematical model from the regulatory structure of the intracellular circadian clock in mice and identified its parameters using an iterative evolutionary strategy, with minimum cost achieved through conformance to phase separations seen in cell-autonomous oscillators. The model was evaluated against the experimentally observed cell-autonomous circadian phenotypes of gene knockouts, particularly retention of rhythmicity and changes in expression level of molecular clock components. These tests reveal excellent de novo predictive ability of the model. Furthermore, sensitivity analysis shows that these knockout phenotypes are robust to parameter perturbation.     

Effect of melatonin and diazepam on the dissociated circadian rhythm in rats

The main structures involved in the circadian system in mammals are the suprachiasmatic nuclei (SCN) of the hypothalamus. The SCN contain multiple autonomous single-cell circadian oscillators that are coupled among themselves, generating a single rhythm. However, under determined circumstances, the oscillators may uncouple and generate several rhythmic patterns. Rats exposed to an artificially established 22-h light-dark cycle (T22) express two stable circadian rhythms in their motor activity that reflect the separate activities of two groups of oscillators in the morphologically well-defined ventrolateral and dorsomedial SCN subdivisions. In the experiments described in this paper, we studied the effect of melatonin and diazepam (DZP) administration in drinking water on the dissociated components of rat motor activity exposed to T22, to deduce the possible mechanism of these drugs on the circadian system. In order to suppress the endogenous circadian rhythm of melatonin, in some of the rats the pineal gland or the superior cervical ganglia were removed. The results show that melatonin or DZP treatment increased the manifestation of the light-dependent component to the detriment of the manifestation of the non-light-dependent component and that melatonin, but not DZP, shortens the period of the non-light-dependent component. These findings suggest that both DZP and melatonin favor entrainment to external light, and that melatonin could also act on the SCN, producing changes in the period of the circadian cycle.     

Neuropsin (OPN5)-mediated photoentrainment of local circadian oscillators in mammalian retina and cornea

The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength–sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.Most mammalian tissues contain autonomous circadian clocks that are synchronized by the suprachiasmatic nuclei (SCN) in the brain (1). The SCN clock itself is entrained by external light cycles through retinal rods, cones, and melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) (2, 3). The retina also manifests a local circadian clock, which regulates many important functions, such as photoreceptor disk shedding, photoreceptor gap-junction coupling, and neurotransmitter release (4–6). Surprisingly, local retinal photoentrainment does not require the SCN, and it also does not require rods, cones, or ipRGCs (7, 8). Thus, the rd1/rd1;Opn4^−/− mouse retina, which has lost essentially all rods and cones due to degeneration and also has an ablated Opn4 gene (3), remains synchronized to light/dark cycles both in vivo and ex vivo (7).To determine the photopigment(s) responsible for local circadian entrainment in the retina, we took a candidate gene approach. Because some cone nuclei may persist in degenerate rd1/rd1 retinas (9), and murine short-wavelength–sensitive cone opsin (OPN1SW) has been reported to be present in the ganglion cell layer (10), we tested the necessity of this pigment for local retinal circadian photoentrainment. We also tested for the involvement of two orphan pigments, encephalopsin (OPN3) (11) and neuropsin (OPN5) (12), both of which are expressed in mammalian retina and, when expressed heterologously, form light-sensitive pigments that activate G proteins (13–17). The function of OPN3 in mammals is unknown despite its widespread expression in neural tissues (18). OPN5 appears to be a deep-brain photopigment in the hypothalamus of birds and is thought to contribute to seasonal reproduction (19–22); it has been immunolocalized to the mammalian inner retina (13, 16) (SI Text); however, to date, no retinal function for this mammalian pigment has been identified. We did not examine two other pigments, retinal G protein-coupled receptor (RGR) opsin (23) and peropsin (RRH) (24). RGR opsin participates in retinoid turnover (25, 26), whereas RRH is expressed principally in the retinal pigment epithelium (24), a cell layer absent in the photoentrainable ex vivo retina preparation (7).     

Melatonin and circadian rhythms in liver diseases: Functional roles and potential therapies

Circadian rhythms and clock gene expressions are regulated by the suprachiasmatic nucleus in the hypothalamus, and melatonin is produced in the pineal gland. Although the brain detects the light through retinas and regulates rhythms and melatonin secretion throughout the body, the liver has independent circadian rhythms and expressions as well as melatonin production. Previous studies indicate the association between circadian rhythms with various liver diseases, and disruption of rhythms or clock gene expression may promote liver steatosis, inflammation, or cancer development. It is well known that melatonin has strong antioxidant effects. Alcohol drinking or excess fatty acid accumulation produces reactive oxygen species and oxidative stress in the liver leading to liver injuries. Melatonin administration protects these oxidative stress-induced liver damage and improves liver conditions. Recent studies have demonstrated that melatonin administration is not limited to antioxidant effects and it has various other effects contributing to the management of liver conditions. Accumulating evidence suggests that restoring circadian rhythms or expressions as well as melatonin supplementation may be promising therapeutic strategies for liver diseases. This review summarizes recent findings for the functional roles and therapeutic potentials of circadian rhythms and melatonin in liver diseases.     

Peripheral circadian clock for the cuticle deposition rhythm in Drosophila melanogaster

Insect endocuticle thickens after adult emergence by daily alternating deposition of two chitin layers with different orientation. Although the cuticle deposition rhythm is known to be controlled by a circadian clock in many insects, the site of the driving clock, the photoreceptor for entrainment, and the oscillatory mechanism remain elusive. Here, we show that the cuticle deposition rhythm is regulated by a peripheral oscillator in the epidermis in Drosophila melanogaster. Free-running and entrainment experiments in vitro reveal that the oscillator for the cuticle deposition rhythm is independent of the central clock in the brain driving the locomotor rhythms. The cuticle deposition rhythm is absent in null and dominant-negative mutants of clock genes (i.e., period, timeless, cycle, and Clock), indicating that this oscillator is composed of the same clock genes as the central clock. Entrainment experiments with monochromatic light-dark cycles and cry(b) flies reveal that a blue light-absorbing photoreceptor, cryptochrome (CRY), acts as a photoreceptor pigment for the entrainment of the cuticle deposition rhythm. Unlike other peripheral rhythms in D. melanogaster, the cuticle deposition rhythm persisted in cry(b) and cry(OUT) mutant flies, indicating that CRY does not play a core role in the rhythm generation in the epidermal oscillator.     

No evidence for a phase delay in human circadian rhythms after a single morning melatonin administration

Although there is good consensus that a single administration of melatonin in the early evening can phase advance human circadian rhythms, the evidence for phase delay shifts to a single melatonin stimulus given in the early morning is sparse. We therefore carried out a double-blind randomized-order placebo-controlled study under modified constant routine (CR) conditions (58 hr bedrest under approximately 8 lux with sleep 23:00-07:00 hr) in nine healthy young men. A single (pharmacological) dose of melatonin (5 mg p.o.) or a placebo was administered at 07:00 hr on the first morning. Core body temperature (CBT) and heart rate (HR) were continuously recorded, and saliva was collected half-hourly for assay of melatonin. Neither the timing of the mid-range crossing times of temperature (MRCT) and HR rhythms, nor dim light melatonin onset (DLMOn) or offset (DLMOff) were phase shifted the day after melatonin administration compared with placebo. The only change was an altered wave form of the CBT rhythm: longer duration of higher-than-average temperature after melatonin administration. Under the same modified CR conditions we have previously demonstrated a clear phase advance of the above circadian rhythms following a single administration of 5 mg melatonin in the evening. This study's failure to find significant delays to a single administration does not negate other positive findings with multiple doses, which may be necessary for a 'weak zeitgeber'.     

At what time are implantable defibrillator shocks delivered?: Evidence for individual circadian variance in sudden cardiac death

Background: As in myocardial infarction and transient ischaemia,out-of-hospital sudden cardiac death has an increased morningincidence. However, sudden death occurring in hospital is evenlydistributed over the 24 h period suggesting that there mightbe subgroups of patients with atypial circadian patterns ofsudden death. Patients who received an implantable defibrillatorconstitute an ideal group for studies of circadian patternsof sudden death since this generation of devices are able tostore the exact time when defibrillation occurred Methods: The distribution of sudden death aborted by the implantabledefibrillator was analysed during the 24 h period for 87 presumedappropriate shocks delivered in a group of 22 patients, 18 menand four women, 58.7 ± 11.9 years old and with a meanleft ventricular ejection fraction of 39.4 ± 17.6%. Results: Each patient received an average of 4.42 ± 3.04shocks during a mean follow-up of 9.4 ± 5.6 months. Apartfrom a clear tendency for shocks to occur during the morninghours (42% of total shocks), five of 16 patients who receivedmultiple shocks also showed a trend to repeat the shocks aroundthe same period during the day. Conclusion: Our results support the accepted view that changesin autonomic tone in the early morning play a role in the circadianvariations of sudden death. Sudden death not only occurs morefrequently in the morning hours, but it also clusters in certainperiods for individual patients.     

Signaling between periglomerular cells reveals a bimodal role for GABA in modulating glomerular microcircuitry in the olfactory bulb

In the mouse olfactory bulb glomerulus, the GABAergic periglomerular (PG) cells provide a major inhibitory drive within the microcircuit. Here we examine GABAergic synapses between these interneurons. At these synapses, GABA is depolarizing and exerts a bimodal control on excitability. In quiescent cells, activation of GABA_A receptors can induce the cells to fire, thereby providing a means for amplification of GABA release in the glomerular microcircuit via GABA-induced GABA release. In contrast, GABA is inhibitory in neurons that are induced to fire tonically. PG–PG interactions are modulated by nicotinic acetylcholine receptors (nAChRs), and our data suggest that changes in intracellular calcium concentrations triggered by nAChR activation can be amplified by GABA release. Our results suggest that bidirectional control of inhibition in PG neurons can allow for modulatory inputs, like the cholinergic inputs from the basal forebrain, to determine threshold set points for filtering out weak olfactory inputs in the glomerular layer of the olfactory bulb via the activation of nAChRs.The balance of excitation and inhibition is critical for the normal functioning of brain networks. Timed inhibition of principal neurons modulates circuit output and contributes to network synchrony and oscillation. GABAergic interneurons play a key role in regulating these network properties (1, 2). Recent findings (e.g., ref. 3), however, have compelled us to move away from a simple view of transmission in the brain, in which glutamate and GABA represent the major excitatory and inhibitory transmitter systems, to a more nuanced interpretation of their roles.GABAergic neurotransmission has both inhibitory and excitatory effects in the CNS. Whereas the inhibitory actions of GABA on principal neurons in different brain regions have been examined extensively, studies of excitatory GABA have focused mostly on the developmental aspects of neuronal growth and synapse formation (4, 5). Recent evidence suggests that GABA can be excitatory in mature neurons as well (with the term “mature” here referring to neurons that are integral parts of established brain networks) (3, 6).Dynamic GABAergic signaling between inhibitory interneurons is less well understood. The common assumption is that GABAergic signaling between these interneurons would lead to disinhibition of principal neurons in a circuit. Excitatory GABA signaling between these interneurons, on the other hand, could serve as a means for amplification of principal cell inhibition. A combination of the two could effectively buffer interneuron firing rates and possibly normalize circuit output in a given area (7).The modularity in brain circuits allows for application of principles gleaned from the study of one defined circuit to other circuits as well. In the olfactory bulb (OB) glomerulus, the GABAergic periglomerular (PG) cells provide a large fraction of the inhibitory drive for information transfer between the olfactory nerve (ON) and mitral cells (MCs), the principal neurons. In this system, the existence of PG–PG synapses has been demonstrated (8), and GABA has been suggested to be depolarizing, yet inhibitory, on these neurons (9). Whether these synapses participate in glomerular signaling either during odor input or during neuromodulation of glomerular output is not yet known.In this paper, we report that GABAergic connections between PG cells have a bimodal effect on excitation depending on the previous activity state of the neurons. Excitation of PG cells by GABA can lead to amplification of glomerular inhibition via GABA-induced GABA release (GIGR). GABA release from PG cells modulates glomerular output on the activation of nicotinic acetylcholine receptors (nAChRs), wherein weak signals from the ON are filtered out while stronger ones are transmitted (10). Our results suggest that bimodal signaling by GABA could be important in determining set points for inhibition thresholds in the glomerular microcircuit.