Circadian rhythms: biological clocks work in phospho-time

The 24 hour molecular oscillator requires precisely calibrated degradation of core clock proteins, like PERIOD. New studies shed light on a sequential series of PERIOD phosphorylation events that first inhibits, then accelerates PERIOD degradation.     

Circadian clocks are resounding in peripheral tissues

Circadian rhythms are prevalent in most organisms. Even the smallest disturbances in the orchestration of circadian gene expression patterns among different tissues can result in functional asynchrony, at the organism level, and may to contribute to a wide range of physiologic disorders. It has been reported that as many as 5%-10% of transcribed genes in peripheral tissues follow a circadian expression pattern. We have conducted a comprehensive study of circadian gene expression on a large dataset representing three different peripheral tissues. The data have been produced in a large-scale microarray experiment covering replicate daily cycles in murine white and brown adipose tissues as well as in liver. We have applied three alternative algorithmic approaches to identify circadian oscillation in time series expression profiles. Analyses of our own data indicate that the expression of at least 7% to 21% of active genes in mouse liver, and in white and brown adipose tissues follow a daily oscillatory pattern. Indeed, analysis of data from other laboratories suggests that the percentage of genes with an oscillatory pattern may approach 50% in the liver. For the rest of the genes, oscillation appears to be obscured by stochastic noise. Our phase classification and computer simulation studies based on multiple datasets indicate no detectable boundary between oscillating and non-oscillating fractions of genes. We conclude that greater attention should be given to the potential influence of circadian mechanisms on any biological pathway related to metabolism and obesity.     

Circadian rhythms of locomotor activity in Drosophila

Drosophila is by far the most advanced model to understand the complex biochemical interactions upon which circadian clocks rely. Most of the genes that have been characterized so far were isolated through genetic screens using the locomotor activity rhythms of the adults as a circadian output. In addition, new techniques are available to deregulate gene expression in specific cells, allowing to analyze the growing number of developmental genes that also play a role as clock genes. However, one of the major challenges in circadian biology remains to properly interpret complex behavioral data and use them to fuel molecular models. This review tries to describe the problems that clockwatchers have to face when using Drosophila activity rhythms to understand the multiple facets of circadian function.     

Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells

Genes and components of the circadian clock may represent relevant drug targets for diseases involving circadian dysfunctions. By exploiting an established cell line derived from human retinal pigment epithelium (HRPE), the cell constituting the blood-retinal barrier that is essential to maintain the visual functions of the sensorineural retina, we showed serum-shock induction of rhythmic changes in forskolin-evoked adenylyl cyclase (AC) activity. In the presence of Ca2+ and protein kinase A, the forskolin-induced AC activity is significantly, but not completely inhibited, suggesting the involvement of both Ca2+-sensitive and Ca2+-insensitive AC isoforms in the regulation of circadian rhythmicity in these cells. Semi-quantitative RT-PCR showed circadian profile in the expression of three AC isoforms, the Ca2+-inhibitable AC5 and AC6 and the Ca2+-insensitive AC7, and the clock genes hPer1 and hPer2. Our results demonstrate for the first time circadian rhythmicity in a human cell line, identifying the isoforms involved in the circadian profile of AC activity and showing a rhythmicity of the clock gene mRNA expression in these cells. Therefore, the results reported here provide evidence for an intertwine between AC/[Ca2+]i signalling pathways and Per genes in the HRPE circadian clockwork.     

Circadian clocks are modulated by compartmentalized oscillating translation

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黑腹果蝇昼夜活动节律及行为时间分配

采用试验室单管观察记录的方法,对3,4,5日龄野生型黑腹果蝇Drosophila melanogaster Meigenw1118成虫每日活动节律进行研究。试验将果蝇活动划分为强活动(飞行和爬行)、弱活动(梳理、觅食等原地发生的运动)和静息(身体不发生移动的休息)3种类型。强活动和弱活动之和为总运动。研究结果显示,野生型黑腹果蝇w1118的昼夜活动表现为明显的双峰模态,晨峰和晚峰分别处于开、关灯前后;雌、雄果蝇总体活动无差异,关灯(18:30)前后雌蝇活动稍强于雄蝇,开灯(6:30)前后则相反;果蝇强活动的节律与总运动基本一致,而弱活动节律不明显;静息节律为单峰模式,其高峰期位于夜间1:00~5:00;雌蝇的静息活动显著多于雄蝇(P<0·05)。     

Circadian rhythms are suppressed in hyperproliferative mouse epidermis

We examined the chronically hyperproliferative epidermis of the asebia (ab/ab) mouse for circadian rhythms in cell proliferation and in the rate of DNA synthesis, which is related to S phase duration. The curve for the circadian rhythm in cell proliferation for asebia epidermis was suppressed and distorted in comparison to that for BALB/cJ epidermis and in comparison to a composite curve produced by averaging the results from ten other published studies.     

Circadian clocks in prokaryotes

Prokaryotes have long been thought incapable of expressing circadian (daily) rhythms. Recently, however, such biological 'clocks' have been discovered in several species of cyanobacteria. These endogenous timekeepers control gene expression on a global level in cyanobacteria. Even in cyanobacterial cultures that are growing with average doubling times more rapid than one per 24 h, the circadian clock controls gene expression and cell division. We have isolated mutants of the cyanobacterial circadian pacemaker and are currently characterizing the loci responsible for their altered period phenotypes.     

Closely watched clocks: molecular analysis of circadian rhythms in Neurospora and Drosophila

Circadian rhythms represent a type of cellular regulation common to most eukaryotes. Analysis of the genetic basis of this phenomenon is beginning to provide information about how clocks function at the molecular level. Surprisingly, the first two cloned 'clock genes', one from a fruit fly and one from a fungus, share some common characteristics both genetically and in the nature of the proteins they encode. In related work, the recent identification and molecular analysis of clock-controlled genes is revealing how biological clocks control gene expression, and may pave the way for the isolation of novel 'clock genes' in the future.     

Circadian photoreception in Drosophila: functions of cryptochrome in peripheral and central clocks.

In Drosophila melanogaster, disruption of night by even short light exposures results in degradation of the clock protein TIMELESS (TIM), leading to shifts in the fly molecular and behavioral rhythms. Several lines of evidence indicate that light entrainment of the brain clock involves the blue-light photoreceptor cryptochrome (CRY). In cryptochrome-depleted Drosophila (cry(b)), the entrainment of the brain clock by short light pulses is impaired but the clock is still entrainable by light-dark cycles, probably due to light input from the visual system. Whether cryptochrome and visual transduction pathways play a role in entrainment of noninnervated, directly photosensitive peripheral clocks is not known and the subject of this study. The authors monitored levels of the clock protein TIM in the lateral neurons (LNs) of larval brains and in the renal Malpighian tubules (MTs) of flies mutant for the cryptochrome gene (cry(b)) and in mutants that lack signaling from the visual photopigments (norpA(P41)). In cry(b) flies, light applied during the dark period failed to induce degradation of TIM both in MTs and in LNs, yet attenuated cycling of TIM was observed in both tissues in LD. This cycling was abolished in LNs, but persisted in MTs, of norpA(P41);cry(b) double mutants. Furthermore, the activity of the tim gene in the MTs of cry(b) flies, reported by luciferase, seemed stimulated by lights-on and suppressed by lights-off, suggesting that the absence of functional cryptochrome uncovered an additional light-sensitive pathway synchronizing the expression of TIM in this tissue. In constant darkness, cycling of TIM was abolished in MTs; however, it persisted in LNs of cry(b) flies. The authors conclude that cryptochrome is involved in TIM-mediated entrainment of both central LN and peripheral MT clocks. Cryptochrome is also an indispensable component of the endogenous clock mechanism in the examined peripheral tissue, but not in the brain. Thus, although neural and epithelial cells share the core clock mechanism, some clock components and light-entrainment pathways appear to have tissue-specific roles.     

Circadian clocks and adaptation in Arabidopsis

Endogenous circadian rhythms are almost ubiquitous among organisms from cyanobacteria to mammals and regulate diverse physiological processes. It has been suggested that having an endogenous circadian system enables an organism to anticipate periodic environmental changes and adapt its physiological and developmental states accordingly, thus conferring a fitness advantage. However, it is hard to measure fitness directly and there is, to date, only limited evidence supporting the assumption that having a circadian system can increase fitness and therefore be adaptive. In this article, we report an evolutionary approach to examine the adaptive significance of a circadian system. By crossing Arabidopsis thaliana plants containing mutations that cause changes in circadian rhythms, we have created heterozygous 'Mother' (F1) plants with genetic variance for circadian rhythmicity. The segregating F2 offspring present a range of circadian rhythm periods. We have applied a selection to the F2 plants of short and long T-cycles under different competition strengths and found that the average phenotype of circadian period of the resulting F3 plants show a strong positive correlation with the T-cycle growth conditions for the competing F2 plants. Consistent with their circadian phenotypes, the frequency of long-period alleles was altered in the F3 plants. Our results show that F2 plants with endogenous rhythms that more closely match the environmental T-cycle are fitter, producing relatively more viable offspring in the F3 population. Thus, having a circadian clock that matches with the environment is adaptive in Arabidopsis.     

The nuclear receptor unfulfilled is required for free-running clocks in Drosophila pacemaker neurons

An intricate neural circuit composed of multiple classes of clock neurons controls circadian locomotor rhythms in Drosophila. Evidence indicates that the small ventral lateral neurons (s-LNvs, M cells) are the dominant pacemaker neurons that synchronize the clocks throughout the circuit and drive free-running locomotor rhythms. Little is known, however, about the molecular underpinning of this unique function of the s-LNvs. Here, we show that the nuclear receptor gene unfulfilled (unf; DHR51) is required for the function of the s-LNvs. UNFULFILLED (UNF) is rhythmically expressed in the s-LNvs, and unf mutant flies are behaviorally arrhythmic. Knockdown of unf in developing LNvs irreversibly destroys the ability of adult s-LNvs to generate free-running rhythms, whereas depletion of UNF from adult LNvs dampens the rhythms of the s-LNvs only in constant darkness. These temporally controlled LNv-targeted unf knockdowns desynchronize circuit-wide molecular rhythms and disrupt behavioral rhythms. Therefore, UNF is a prerequisite for free-running clocks in the s-LNvs and for the function of the entire circadian circuit.     

Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus

Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).     

Separate sets of cerebral clock neurons are responsible for light and temperature entrainment of Drosophila circadian locomotor rhythms

The fruit fly Drosophila melanogaster shows a bimodal circadian locomotor rhythm with peaks at lights-on and before lights-off, which are regulated by multiple clocks in the brain. Even under light-dark cycles, the timing of the evening peak is highly dependent on temperature, starting earlier under lower ambient temperature but terminating almost at the same time. In the present study, using behavioral and immunohistochemical assays, the authors show that separate groups of clock neurons, either light-entrainable or temperature-entrainable, form a functional system driving the locomotor rhythm. When subjected to a light cycle combined with a temperature cycle advanced by 6 h relative to the light cycle, the dorsally located neurons (DNs) and lateral posterior neurons (LPNs) shifted their phase of TIMELESS expression, but the laterally located protocerebral neurons (LNs) basically maintained their original phase. Thus, the LNs seem to be preferentially light-entrainable and the DNs and LPNs to be primarily temperature-entrainable. In pdf(01) mutant flies that lack the neuropeptide PDF in the ventral groups of LNs, the onset of the evening peak was greatly advanced even under synchronized light and temperature cycles and was shifted even more than in wild-type flies in response to a 6-h phase shift of the temperature cycle, suggesting that ventral LNs have a strong impact on the phase of the other cells. It seems likely that the 2 sets of clock cells with different entrainability to light and temperature, and the coupling between them, enable Drosophila to keep a proper phase relationship of circadian activity with respect to the daily light and temperature cycles.     

Decoding olfaction in Drosophila

Recent experiments in Drosophila demonstrate striking stereotypy in the neural architecture of the olfactory system. Functional imaging experiments in mammals and honeybees suggest a mechanism of odor coding that translates discrete patterns of activity in olfactory glomeruli into an odor image. Future experiments in Drosophila may permit a direct test of this odor-coding hypothesis.     

Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent reporter of the circadian clock. In liquid culture, AMC149 expresses a rhythm of bioluminescence that displays the same behavior as circadian rhythms in higher eukaryotes. Improvements in the technique for administering the reporter enzyme's substrate (decanal) and a highly sensitive photon-counting camera allow monitoring the bioluminescence of single colonies. Individual colonies on agar plates displayed a rhythmicity which is essentially the same as that previously reported for liquid cultures.     

Circadian clocks in the mammalian brain

Daily cycles in physiology and behaviour are probably a universal feature of multicellular organisms. These rhythms are predominantly driven by endogenous clocks with a periodicity approximating to one day, i.e. circadian. In mammals, the circadian clock governing activity/ rest, neuroendocrine and autonomic rhythms lies in the hypothalamus, in the suprachiasmatic nuclei (SCN). Intrinsic circadian oscillators are also present in the retina. The SCN "clockwork" is based on a cell autonomous, genetically determined mechanism. Mammalian homologues of a number of Drosophila genes which encode elements of the fly circadian mechanism have recently been identified. In Drosophila, the protein products of these genes interact in a negative feedback loop, establishing a circadian cycle in gene expression. Characterisation of the roles played by putative mammalian clock genes in the SCN, and how the emergent cellular signal imposes order over the entire neuraxis, will provide a fundamental contribution to our understanding of the molecular basis of behaviour. BioEssays 22:23-31, 2000.