Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY.

BACKGROUND: The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS: Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS: This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.     

Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.     

Serotonin modulates circadian entrainment in Drosophila

Entrainment of the Drosophila circadian clock to light involves the light-induced degradation of the clock protein timeless (TIM). We show here that this entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (d5-HT1B). d5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of d5-HT1B are synergistic with a mutation in the circadian photoreceptor cryptochrome (CRY) and are mediated by SHAGGY (SGG), Drosophila glycogen synthase kinase 3beta (GSK3beta), which phosphorylates TIM. Levels of serotonin are decreased in flies maintained in extended constant darkness, suggesting that modulation of the clock by serotonin may vary under different environmental conditions. These data identify a molecular connection between serotonin signaling and the central clock component TIM and suggest a homeostatic mechanism for the regulation of circadian photosensitivity in Drosophila.     

Photic signaling by cryptochrome in the Drosophila circadian system

Oscillations of the period (per) and timeless (tim) gene products are an integral part of the feedback loop that underlies circadian behavioral rhythms in Drosophila melanogaster. Resetting this loop in response to light requires the putative circadian photoreceptor cryptochrome (CRY). We dissected the early events in photic resetting by determining the mechanisms underlying the CRY response to light and by investigating the relationship between CRY and the light-induced ubiquitination of the TIM protein. In response to light, CRY is degraded by the proteasome through a mechanism that requires electron transport. Various CRY mutant proteins are not degraded, and this suggests that an intramolecular conversion is required for this light response. Light-induced TIM ubiquitination precedes CRY degradation and is increased when electron transport is blocked. Thus, inhibition of electron transport may "lock" CRY in an active state by preventing signaling required either to degrade CRY or to convert it to an inactive form. High levels of CRY block TIM ubiquitination, suggesting a mechanism by which light-driven changes in CRY could control TIM ubiquitination.     

A fly's eye view of circadian entrainment

The Drosophila circadian clock is an ideal model system for teasing out the molecular mechanisms of circadian behavior and the means by which animals synchronize to day-night cycles. The clock that drives behavioral rhythms, located in the lateral neurons in the central brain, consists of a feedback loop of the circadian genes period (per) and timeless (tim). The molecular cycle, roughly 24 h long, is constantly reset by the environment. This review focuses on the main input pathways of the dominant circadian zeitgeber, light. Light acts directly on the clock primarily through cryptochrome (cry), a deep brain blue-light photoreceptor. CRY activation causes rapid TIM degradation, which is a predicted means of resetting the clock both on a daily basis at dawn and on an acute basis following an entraining light pulse during the night hours. In the absence of cry, the clock can still be driven by photic input through the visual system, though the mechanisms underlying this entrainment are unclear. Temperature can also entrain the clock, although the mechanisms by which this occurs are also unclear.     

Rhodopsin 5- and Rhodopsin 6-mediated clock synchronization in Drosophila melanogaster is independent of retinal phospholipase C-β signaling

Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.     

Membrane electrical excitability is necessary for the free‐running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free‐running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell‐autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K⁺ channel stops the free‐running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free‐running larval pacemaker clock—like that of the adult pacemaker neurons they give rise to—requires membrane electrical activity to oscillate. In contrast to the free‐running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light–dark) conditions. This specific disruption of the free‐running clock caused by targeted K⁺ channel expression likely reflects a specific cell‐autonomous clock‐membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Clock gene expression levels and relationship with clinical and pathological features in colorectal cancer patients

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1? expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p=.002), PER1 (p=.002), PER2 (p=.011), PER3 (p=.003), and CRY2 (p=.012) were lower, whereas the expression level of TIM (p=.044) was higher. No significant difference was observed in the expression levels of CLOCK (p=.778), CRY1 (p=.600), CSNK1 (p=.903), and TIPIN (p=.136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p=.026), and female sex (p=.005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p?=?.015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p= .013) and associated with TNM stages III-IV (p=.005) and microsatellite instability (p=.015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p=.010), PER3 (p= .010), and CSNKIE (p=.024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions.     

Drosophila CRY is a deep brain circadian photoreceptor

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.     

Cryptochrome-positive and -negative clock neurons in Drosophila entrain differentially to light and temperature

The blue-light photoreceptive protein Cryptochrome (CRY) plays an important role in the light synchronization of the Drosophila circadian clock. Previously, we found that among the approximately 150 clock neurons, many but not all neurons express CRY. We speculated that the CRY-positive pacemaker neurons may be especially important for light entrainment, whereas the CRY-negative neurons may be important for other environmental cues, for example, temperature. To investigate this hypothesis, we tested the entrainability of the clock neurons to out-of-phase light and temperature cycles. When light-dark or light-dim light cycles were shifted by 12 h with respect to temperature cycles, behavioral rhythms of wild-type flies were re-entrained by the light cycles. In this condition, we found that TIMELESS (TIM) level was strongly influenced by the temperature cycles in many CRY-negative clock neurons, suggesting that the CRY-negative neurons have higher sensitivity to temperature. Under the same conditions, cry-null mutants entrained to the temperature cycles or very slowly re-entrained to light-dark cycles. Our results suggest that there are 2 types of clock neurons having differential sensitivities to light and temperature, and CRY is a key component for the preferential entrainment to light.