A colorful model of the circadian clock

The migration of the colorful monarch butterfly provides biologists with a unique model system with which to study the cellular and molecular mechanisms underlying a sophisticated circadian clock. The monarch circadian clock is involved in the induction of the migratory state and navigation over long distances, using the sun as a compass.     

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.     

Connecting the navigational clock to sun compass input in monarch butterfly brain

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.     

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.     

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.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

Integration of polarization and chromatic cues in the insect sky compass

Animals relying on a celestial compass for spatial orientation may use the position of the sun, the chromatic or intensity gradient of the sky, the polarization pattern of the sky, or a combination of these cues as compass signals. Behavioral experiments in bees and ants, indeed, showed that direct sunlight and sky polarization play a role in sky compass orientation, but the relative importance of these cues are species-specific. Intracellular recordings from polarization-sensitive interneurons in the desert locust and monarch butterfly suggest that inputs from different eye regions, including polarized-light input through the dorsal rim area of the eye and chromatic/intensity gradient input from the main eye, are combined at the level of the medulla to create a robust compass signal. Conflicting input from the polarization and chromatic/intensity channel, resulting from eccentric receptive fields, is eliminated at the level of the anterior optic tubercle and central complex through internal compensation for changing solar elevations, which requires input from a circadian clock. Across several species, the central complex likely serves as an internal sky compass, combining E-vector information with other celestial cues. Descending neurons, likewise, respond both to zenithal polarization and to unpolarized cues in an azimuth-dependent way.     

Animal type 1 cryptochromes. Analysis of the redox state of the flavin cofactor by site-directed mutagenesis

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD(*-), in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD(ox) when purified, and the flavin is readily reduced to FAD(*-) by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD(*-) in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD(*-) represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.