CRY1 is involved in the take-off behaviour of migratory Cnaphalocrocis medinalis individuals.

BACKGROUND: Numerous insect species undertake long-distance migrations on an enormous scale, with great implications for ecosystems. Given that take-off is the point where it all starts, whether and how the external light and internal circadian rhythm are involved in regulating the take-off behaviour remains largely unknown. Herein, we explore this issue in a migratory pest, Cnaphalocrocis medinalis, via behavioural observations and RNAi experiments. RESULTS: The results showed that C. medinalis moths took off under conditions where the light intensity gradually weakened to 0.1 lx during the afternoon or evening, and the take-off proportions under full spectrum or blue light were significantly higher than that under red and green light. The ultraviolet-A/blue light-sensitive type 1 cryptochrome gene (Cmedcry1) was significantly higher in take-off moths than that of non-take-off moths. In contrast, the expression of the light-insensitive CRY2 (Cmedcry2) and circadian genes (Cmedtim and Cmedper) showed no significant differences. After silencing Cmedcry1, the take-off proportion significantly decreased. Thus, Cmedcry1 is involved in the decrease in light intensity induced take-off behaviour in C. medinalis. CONCLUSIONS: This study can help further explain the molecular mechanisms behind insect migration, especially light perception and signal transmission during take-off phases.

Forkhead box protein FOXK1 disrupts the circadian rhythm to promote breast tumorigenesis in response to insulin resistance.

The dysregulation of circadian rhythm oscillation is a prominent feature of various solid tumors. Thus, clarifying the molecular mechanisms that maintain the circadian clock is important. In the present study, we revealed that the transcription factor forkhead box FOXK1 functions as an oncogene in breast cancer. We showed that FOXK1 recruits multiple transcription corepressor complexes, including NCoR/SMRT, SIN3A, NuRD, and REST/CoREST. Among them, the FOXK1/NCoR/SIN3A complex transcriptionally regulates a cohort of genes, including CLOCK, PER2, and CRY2, that are critically involved in the circadian rhythm. The complex promoted the proliferation of breast cancer cells by disturbing the circadian rhythm oscillation. Notably, the nuclear expression of FOXK1 was positively correlated with tumor grade. Insulin resistance gradually became more severe with tumor progression and was accompanied by the increased expression of OGT, which caused the nuclear translocation and increased expression of FOXK1. Additionally, we found that metformin downregulates FOXK1 and exports it from the nucleus, while HDAC inhibitors (HDACi) inhibit the FOXK1-related enzymatic activity. Combined treatment enhanced the expression of circadian clock genes through the regulation of FOXK1, thereby exerting an antitumor effect, indicating that highly nuclear FOXK1-expressing breast cancers are potential candidates for the combined application of metformin and HDACi.

Time-of-Day Adrenal Modulation of Corticosterone Synthesis is Affected by Sex and Diet but Not by Proanthocyanidins in Rat.

SCOPE: The aim of this study is to investigate the effect of time-of-day on serum hormones and gene expression in adrenal glands, studying the impact of sex, obesogenic diet, and timing of proanthocyanidins administration, with a focus on glucocorticoids synthesis by this gland. METHODS AND RESULTS: Female and male rats, assigned to a standard chow or a cafeteria diet-fed group, receive a daily oral dose of a grape seed proanthocyanidin extract (GSPE), or a vehicle (when light is turned on, or when light is turned off). Corticosterone, estradiol, and testosterone serum levels, and the expression analysis of clock genes and genes related to corticosterone synthesis pathway, are assessed. Serum hormone levels exhibited a marked time-of-day effect also see in the expression of scavenger receptor class B member 1 (Scarb1) and cyp11b genes. The correlation between these two genes and period circadian regulator 2 (Per2) is also extended to other clock genes, although to a lesser extent: cryptochrome (Cry) and nuclear receptor subfamily 1 group D member 1 (Rev-erba). CONCLUSION: The strong correlations found suggest an important role of local Per2 (but also of Cry and Rev-erbA) in regulating the expression of the enzymes involved in the corticosterone synthesis pathway. The expression of clock genes in adrenals is influenced by sex and diet but not by GSPE.

Orientation of birds in radiofrequency fields in the absence of the Earth's magnetic field: a possible test for the radical pair mechanism of magnetoreception.

The magnetic compass sense of migratory songbirds is thought to derive from magnetically sensitive photochemical reactions in cryptochromes located in photoreceptor cells in the birds' retinas. More specifically, transient radical pairs formed by light-activation of these proteins have been proposed to account for the birds' ability to orient themselves using the Earth's magnetic field and for the observation that radiofrequency magnetic fields, superimposed on the Earth's magnetic field, can disrupt this ability. Here, by means of spin dynamics simulations, we show that it may be possible for the birds to orient in a monochromatic radiofrequency field in the absence of the Earth's magnetic field. If such a behavioural test were successful, it would provide powerful additional evidence for a radical pair mechanism of avian magnetoreception.

A majority of circadian clock genes are expressed in estrogen receptor and progesterone receptor status-dependent manner in breast cancer.

Circadian clocks, biochemical oscillators that are regulated by environmental time cues including the day/night cycle, have a central function in the majority of biological processes. The disruption of the circadian clock can alter breast biology negatively and may promote the development of breast tumors. The expression status of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) were used to classify breast cancer into different molecular subtypes such as triple-negative breast cancer (TNBC). Receptor status-dependent expression of circadian clock genes have been previously studied in breast cancer using relatively small sample sizes in a particular population. Here, using TCGA-BRCA data (n=1119), we found that the expressions of CRY1, PER1, PER2, PER3, BMAL1, CLOCK, RORA, RORB, RORC, NR1D1, NR1D2, and FBXL3 were higher in ER+ breast cancer cells compared with those of ER- status. Similarly, we showed that transcript levels of CRY2, PER1, PER2, PER3, BMAL1, RORA, RORB, RORC, NR1D1, NR1D2, and FBXL3 were higher in PR+ breast cancer cells than in PR- breast cancer cells. We report that the expressions of CRY2, PER1, BMAL1, and RORA were lower, and the expression of NR1D1 was higher, in HER2+ breast cancer cells compared with HER2- breast cancer cells. Moreover, we studied these receptor status-dependent changes in the expressions of circadian clock genes also based on the race and age of breast cancer patients. Lastly, we found that the expressions of CRY2, PER1, PER2, PER3, and CLOCK were higher in non-TNBC than in TNBC, which has the worst prognosis among subtypes. We note that our findings are not always parallel to the observations reported in previous studies with smaller sample sizes performed in different populations and organisms. Our study suggests that receptor status in breast cancer (thus, subtype of breast cancer) might be more important than previously shown in terms of its influence on the expression of circadian clock genes and on the disruption of the circadian clock, and that ER or PR might be important regulators of breast cancer chronobiology that should be taken into account in personalized chronotherapies.

Circadian period is compensated for repressor protein turnover rates in single cells.

Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.

A Detailed Re-Examination of the Period Gene Rescue Experiments Shows That Four to Six Cryptochrome-Positive Posterior Dorsal Clock Neurons (DN1p) of Drosophila melanogaster Can Control Morning and Evening Activity.

Animal circadian clocks play a crucial role in regulating behavioral adaptations to daily environmental changes. The fruit fly Drosophila melanogaster exhibits 2 prominent peaks of activity in the morning and evening, known as morning (M) and evening (E) peaks. These peaks are controlled by 2 distinct circadian oscillators located in separate groups of clock neurons in the brain. To investigate the clock neurons responsible for the M and E peaks, a cell-specific gene expression system, the GAL4-UAS system, has been commonly employed. In this study, we re-examined the two-oscillator model for the M and E peaks of Drosophila by utilizing more than 50 Gal4 lines in conjunction with the UAS-period16 line, which enables the restoration of the clock function in specific cells in the period (per) null mutant background. Previous studies have indicated that the group of small ventrolateral neurons (s-LNv) is responsible for controlling the M peak, while the other group, consisting of the 5th ventrolateral neuron (5th LNv) and the three cryptochrome (CRY)-positive dorsolateral neurons (LNd), is responsible for the E peak. Furthermore, the group of posterior dorsal neurons 1 (DN1p) is thought to also contain M and E oscillators. In this study, we found that Gal4 lines directed at the same clock neuron groups can lead to different results, underscoring the fact that activity patterns are influenced by many factors. Nevertheless, we were able to confirm previous findings that the entire network of circadian clock neurons controls M and E peaks, with the lateral neurons playing a dominant role. In addition, we demonstrate that 4 to 6 CRY-positive DN1p cells are sufficient to generate M and E peaks in light-dark cycles and complex free-running rhythms in constant darkness. Ultimately, our detailed screening could serve as a catalog to choose the best Gal4 lines that can be used to rescue per in specific clock neurons.

Reorganization of circadian activity and the pacemaker circuit under novel light regimes.

Many environmental features are cyclic, with predictable changes across the day, seasons and latitudes. Additionally, anthropogenic, artificial-light-induced changes in photoperiod or shiftwork-driven novel light/dark cycles also occur. Endogenous timekeepers or circadian clocks help organisms cope with such changes. The remarkable plasticity of clocks is evident in the waveforms of behavioural and molecular rhythms they govern. Despite detailed mechanistic insights into the functioning of the circadian clock, practical means to manipulate activity waveform are lacking. Previous studies using a nocturnal rodent model showed that novel light regimes caused locomotor activity to bifurcate such that mice showed two bouts of activity restricted to the dimly lit phases. Here, we explore the generalizability of these findings and leverage the genetic toolkit of Drosophila melanogaster to obtain mechanistic insights into this unique phenomenon. We find that dim scotopic illumination of specific durations induces circadian photoreceptor CRYPTOCHROME-dependent activity bifurcation in male flies. We show circadian reorganization of the pacemaker circuit, wherein the 'evening' neurons regulate the timing of both bouts of activity under novel light regimes. Our findings indicate that such environmental regimes can be exploited to design light cycles, which can ease the circadian waveform into synchronizing with challenging conditions.

CRY2 mediates the cognitive decline induced by sleep deprivation in 5xFAD mice.

BACKGROUND: Cryptochrome-2 (CRY2) is a core rhythm gene that plays a crucial role in DNA damage repair. The present study investigated the potential role of CRY2 in mediating sleep deprivation-induced cognitive decline in 5xFAD mice. METHODS: To assess the effects of SD on different brain regions of the mouse brain, we used 18F FDG PET-CT. Cognitive function was evaluated using the Morris water maze test and Y-maze. Lentivirus was used for the overexpression of CRY2, and small interfering RNA (siRNA) was used for the downregulation of CRY2 to verify the effect of CRY2. We used qRT‒PCR and Western blotting to identify the downstream factors of CRY2 and evaluated the cognitive function of mice to confirm the effects of these factors. RESULTS: The AD mice exhibited cognitive decline after 21 days of SD and had higher expression of CRY2 compared to AD mice with normal sleep. Overexpression of CRY2 led to decreased cognitive function in AD mice, and the downregulation of CRY2 attenuated the SD-induced cognitive decline in AD mice. CRY2 reduced the expression and function of CISH, which reduced the inhibition of STAT1 phosphorylation and led to synaptic dysfunction. CISH overexpression attenuated the impairing effect of sleep deprivation on cognitive function in AD mice. Furthermore, 18F FDG PET-CT revealed that SD significantly reduced glucose metabolism in different brain regions of AD mice. CONCLUSION: Our study demonstrated that sleep deprivation upregulated CRY2 in the hippocampus of AD mice, which resulted in synaptic dysfunction by decreasing CISH-mediated STAT1 phosphorylation.

Knockout of cryptochrome 1 disrupts circadian rhythm and photoperiodic diapause induction in the silkworm, Bombyx mori.

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species, remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on the involvement of cry1 gene in B. mori photoperiodism. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction.

Shaping an evanescent focus of light for high spatial resolution optogenetic activations in live cells.

Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD). We show that, after correcting the optical aberrations, light is confined within a spot of sub-micron lateral size and ∼100 nm axial depth above the coverslip, resulting in a volume of illumination drastically smaller than the one generated by a standard propagative focus. This evanescent focus is sufficient to induce a more intense and localized recruitment compared to a propagative focus on the optogenetic system CRY2-CIBN, improving the resolution of its pattern of activation.

Magnetic orientation in juvenile Atlantic herring (Clupea harengus) could involve cryptochrome 4 as a potential magnetoreceptor.

The Earth's magnetic field can provide reliable directional information, allowing migrating animals to orient themselves using a magnetic compass or estimate their position relative to a target using map-based orientation. Here we show for the first time that young, inexperienced herring (Clupea harengus, Ch) have a magnetic compass when they migrate hundreds of kilometres to their feeding grounds. In birds, such as the European robin (Erithacus rubecula), radical pair-based magnetoreception involving cryptochrome 4 (ErCRY4) was demonstrated; the molecular basis of magnetoreception in fish is still elusive. We show that cry4 expression in the eye of herring is upregulated during the migratory season, but not before, indicating a possible use for migration. The amino acid structure of herring ChCRY4 shows four tryptophans and a flavin adenine dinucleotide-binding site, a prerequisite for a magnetic receptor. Using homology modelling, we successfully reconstructed ChCRY4 of herring, DrCRY4 of zebrafish (Danio rerio) and StCRY4 of brown trout (Salmo trutta) and showed that ChCRY4, DrCRY4 and ErCRY4a, but not StCRY4, exhibit very comparable dynamic behaviour. The electron transfer could take place in ChCRY4 in a similar way to ErCRY4a. The combined behavioural, transcriptomic and simulation experiments provide evidence that CRY4 could act as a magnetoreceptor in Atlantic herring.

Importance of Polarizable Embedding for Absorption Spectrum Calculations of Arabidopsis thaliana Cryptochrome 1.

Cryptochromes are essential flavoproteins for circadian rhythms and avian magnetoreception. Flavin adenine dinucleotide (FAD), a chromophore within cryptochromes, absorbs blue light, initiating electron transfer processes that lead to a biological signaling cascade. A key step in this cascade is the formation of the FAD semiquinone radical (FADH•), characterized through a specific red-light absorption. The absorption spectra of FADH• in cryptochromes are, however, significantly different from those recorded for the cofactor in solution, primarily due to protein-induced shifts in the absorption peaks. This study employs a multiscale approach, combining molecular dynamics (MD) simulations with quantum mechanical/molecular mechanical (QM/MM) methodologies, to investigate the influence of protein dynamics on embedded FADH• absorption. We emphasize the role of the protein's polarizable environment in the shaping of the absorption spectrum, crucial for accurate spectral predictions in cryptochromes. Our findings provide valuable insights into the absorption process, advancing our understanding of cryptochrome functioning.

Arabidopsis cryptochromes interact with SOG1 to promote the repair of DNA double-strand breaks.

Cryptochromes (CRYs) are blue light (BL) photoreceptors to regulate a variety of physiological processes including DNA double-strand break (DSB) repair. SUPPRESSOR OF GAMMA RADIATION 1 (SOG1) acts as the central transcription factor of DNA damage response (DDR) to induce the transcription of downstream genes, including DSB repair-related genes BRCA1 and RAD51. Whether CRYs regulate DSB repair by directly modulating SOG1 is unknown. Here, we demonstrate that CRYs physically interact with SOG1. Disruption of CRYs and SOG1 leads to increased sensitivity to DSBs and reduced DSB repair-related genes' expression under BL. Moreover, we found that CRY1 enhances SOG1's transcription activation of DSB repair-related gene BRCA1. These results suggest that the mechanism by which CRYs promote DSB repair involves positive regulation of SOG1's transcription of its target genes, which is likely mediated by CRYs-SOG1 interaction.

Understanding the Red Shift in the Absorption Spectrum of the FAD Cofactor in ClCry4 Protein.

It is still a puzzle that has not been entirely solved how migratory birds utilize the Earth's magnetic field for biannual migration. The most consistent explanation thus far is rooted in the modulation of the biological function of the cryptochrome 4 (Cry4) protein by an external magnetic field. This phenomenon is closely linked with the flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the protein. Cry4 is activated by blue light, which is absorbed by the FAD cofactor. Subsequent electron and proton transfers trigger radical pair formation in the protein, which is sensitive to the external magnetic field. An important long-lasting redox state of the FAD cofactor is the signaling (FADH•) state, which is present after the transient electron transfer steps have been completed. Recent experimental efforts succeeded in crystallizing the Cry4 protein from Columbia livia (ClCry4) with all of the important residues needed for protein photoreduction. This specific crystallization of Cry4 protein so far is the only avian cryptochrome crystal structure available, which, however, has great similarity to the Cry4 proteins of night migratory birds. The previous experimental studies of the ClCry4 protein included the absorption properties of the protein in its different redox states. The absorption spectrum of the FADH• state demonstrated a peculiar red shift compared to the photoabsorption properties of the FAD cofactor in its FADH• state in other Cry proteins from other species. The aim of this study is to understand this red shift by employing the tools of computational microscopy and, in particular, a QM/MM approach that relies on the polarizable embedding approximation.

Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome.

Many biological mechanisms rely on the precise control of conformational changes in proteins. Understanding such dynamic processes requires methods for determining structures and their temporal evolution. In this study, we introduce a novel approach to time-resolved ion mobility mass spectrometry. We validated the method on a simple photoreceptor model and applied it to a more complex system, the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY), to determine the role of specific amino acids affecting the conformational dynamics as reaction to blue light activation. In our setup, using a high-power LED mounted in the source region of an ion mobility mass spectrometer, we allow a time-resolved evaluation of mass and ion mobility spectra. Cryptochromes like CraCRY are a widespread type of blue light photoreceptors and mediate various light-triggered biological functions upon excitation of their inbuilt flavin chromophore. Another hallmark of cryptochromes is their flexible carboxy-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region are not yet fully understood and differ among different cryptochromes types. Here, we addressed the highly conserved C-terminal domain of CraCRY, to study the effects of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. We show that D321, the putative proton acceptor of the terminal proton-coupled electron transfer event from Y373, is essential for triggering the large-scale conformational changes of helix α22 and the CTE in the lit state, while D323 influences the timing.

ADA2b acts to positively regulate blue light-mediated photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) act as blue light photoreceptors to regulate various plant physiological processes including photomorphogenesis and repair of DNA double strand breaks (DSBs). ADA2b is a conserved transcription co-activator that is involved in multiple plant developmental processes. It is known that ADA2b interacts with CRYs to mediate blue light-promoted DSBs repair. Whether ADA2b may participate in CRYs-mediated photomorphogenesis is unknown. Here we show that ADA2b acts to inhibit hypocotyl elongation and hypocotyl cell elongation in blue light. We found that the SWIRM domain-containing C-terminus mediates the blue light-dependent interaction of ADA2b with CRYs in blue light. Moreover, ADA2b and CRYs act to co-regulate the expression of hypocotyl elongation-related genes in blue light. Based on previous studies and these results, we propose that ADA2b plays dual functions in blue light-mediated DNA damage repair and photomorphogenesis.

Structural Rearrangements of Pigeon Cryptochrome 4 Undergoing a Complete Redox Cycle.

Cryptochrome is currently the major contender of a protein to underpin magnetoreception, the ability to sense the Earth's magnetic field. Among various types of cryptochromes, cryptochrome 4 has been identified as the likely magnetoreceptor in migratory birds. All-atom molecular dynamics (MD) studies have offered first insights into the structural dynamics of cryptochrome but are limited to a short time scale due to large computational demands. Here, we employ coarse-grained MD simulations to investigate the emergence of long-lived states and conformational changes in pigeon cryptochrome 4. Our coarse-grained simulations complete the picture by permitting observation on a significantly longer time scale. We observe conformational transitions in the phosphate-binding loop of pigeon cryptochrome 4 upon activation and identify prominent motions in residues 440-460, suggesting a possible role as a signaling state of the protein or as a gated interaction site for forming protein complexes that might facilitate downstream processes. The findings highlight the importance of considering longer time scales in studying cryptochrome dynamics and magnetoreception. Coarse-grained MD simulations offer a valuable tool to unravel the complex behavior of cryptochrome proteins and shed new light on the mechanisms underlying their role in magnetoreception. Further exploration of these conformational changes and their functional implications may contribute to a deeper understanding of the molecular mechanisms of magnetoreception in birds.

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.