Role of Rhodopsins as Circadian Photoreceptors in the Drosophila melanogaster.

Light profoundly affects the circadian clock and the activity levels of animals. Along with the systematic changes in intensity and spectral composition, over the 24-h day, light shows considerable irregular fluctuations (noise). Using light as the Zeitgeber for the circadian clock is, therefore, a complex task and this might explain why animals utilize multiple photoreceptors to entrain their circadian clock. The fruit fly Drosophila melanogaster possesses light-sensitive Cryptochrome and seven Rhodopsins that all contribute to light detection. We review the role of Rhodopsins in circadian entrainment, and of direct light-effects on the activity, with a special emphasis on the newly discovered Rhodopsin 7 (Rh7). We present evidence that Rhodopsin 6 in receptor cells 8 of the compound eyes, as well as in the extra retinal Hofbauer-Buchner eyelets, plays a major role in entraining the fly's circadian clock with an appropriate phase-to-light⁻dark cycles. We discuss recent contradictory findings regarding Rhodopsin 7 and report original data that support its role in the compound eyes and in the brain. While Rhodopsin 7 in the brain appears to have a minor role in entrainment, in the compound eyes it seems crucial for fine-tuning light sensitivity to prevent overshooting responses to bright light.

Cryptochrome Interacts With Actin and Enhances Eye-Mediated Light Sensitivity of the Circadian Clock in Drosophila melanogaster.

Cryptochromes (CRYs) are a class of flavoproteins that sense blue light. In animals, CRYs are expressed in the eyes and in the clock neurons that control sleep/wake cycles and are implied in the generation and/or entrainment of circadian rhythmicity. Moreover, CRYs are sensing magnetic fields in insects as well as in humans. Here, we show that in the fruit fly Drosophila melanogaster CRY plays a light-independent role as "assembling" protein in the rhabdomeres of the compound eyes. CRY interacts with actin and appears to increase light sensitivity of the eyes by keeping the "signalplex" of the phototransduction cascade close to the membrane. By this way, CRY also enhances light-responses of the circadian clock.

A New Rhodopsin Influences Light-dependent Daily Activity Patterns of Fruit Flies.

Rhodopsin 7 ( Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000, but its function has remained elusive. We generated an Rh7 null mutant ( Rh70) by P element-mediated mutagenesis and found that an absence of Rh7 had significant effects on fly activity patterns during light-dark (LD) cycles: Rh70 mutants exhibited less morning activity and a longer siesta than wild-type controls. Consistent with these results, we found that Rh7 appears to be expressed in a few dorsal clock neurons that have been previously implicated in the control of the siesta. We also found putative Rh7 expression in R8 photoreceptor cells of the compound eyes and in the Hofbauer-Buchner eyelets, which have been shown to control the precise timing of locomotor activity. The absence of Rh7 alone impaired neither the flies' responses to constant white light nor the ability to follow phase shifts of white LD cycles. However, in blue light (470 nm), Rh70 mutants needed significantly longer to synchronize than wild-type controls, suggesting that Rh7 is a blue light-sensitive photopigment with a minor contribution to circadian clock synchronization. In combination with mutants that lacked additionally cryptochrome-based and/or eye-based light input to the circadian clock, the absence of Rh7 provoked slightly stronger effects.

Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function.

Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells.

Twilight dominates over moonlight in adjusting Drosophila's activity pattern.

Light is the most important zeitgeber for the synchronization of the Drosophila melanogaster circadian clock. In nature, there is twilight, and the nights are rarely completely dark, a fact that is usually disregarded in lab experiments. Recent studies showed contrary effects of simulated twilight and moonlight on fly locomotor activity, with twilight shifting morning and evening activity into the day and moonlight shifting it into the night. A currently unanswered question is, what may happen to locomotor activity when flies are exposed to more natural conditions in which both moonlight and twilight are simulated? Our data demonstrate that flies are able to integrate twilight and moonlight. However, twilight seems to dominate over moonlight as both, morning and evening activity peaks, take place at dawn or at dusk, respectively, and not during the night. Furthermore, nocturnal activity decreases in the presence of twilight. The compound eyes are essential for this behavior, and by investigating different photoreceptor mutants, we unraveled the importance of photoreceptor cells 7 and 8 for wild-type phases of the activity peaks. To adjust nocturnal activity levels to a wild-type manner, all photoreceptor cells work together in a complex way, with rhodopsin 6 having a prominent role.

Moonlight detection by Drosophila's endogenous clock depends on multiple photopigments in the compound eyes.

Many organisms change their activity on moonlit nights. Even the fruit fly Drosophila melanogaster responds to moonlight with a shift of activity into the night, at least under laboratory conditions. The compound eyes have been shown to be essential for the perception of moonlight, but it is unknown which of the 5 rhodopsins in the eyes are responsible for the observed moonlight effects. Here, we show that the outer (R1-R6) and inner (R7 and R8) photoreceptor cells in a fly's ommatidium interact in a complex manner to provoke the moonlight effects on locomotor activity. The shift of the evening activity peak into the night depends on several rhodopsins in the inner and outer photoreceptor cells. The increase in relative nocturnal activity in response to moonlight is mainly mediated by the rhodopsin 6-expressing inner photoreceptor cell R8 together with the rhodopsin 1-expressing outer receptor cells (R1-R6), whereas just rhodopsin 1 of R1 to R6 seems necessary for increasing nocturnal activity in response to increasing daylight intensity.

Fly cryptochrome and the visual system.

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific "regulators" that bind the C terminus of the protein. This C-terminal region harbors several protein-protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visual-signaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY-Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.