Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.

Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue-light sensor, and a light-driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes.

Midnight/midday-synchronized expression of cryptochrome genes in the eyes of three teleost species, zebrafish, goldfish, and medaka.

Photoperiodic responses are observed in many organisms living in the temperate zones. The circadian clock is involved in photoperiodic time measurement; however, the underlying molecular mechanism for detection of the day length remains unknown. We previously compared the expression profiles of the Cryptochrome(Cry) genes in the zebrafish eye and reported that Cry1ab has a double peak with variable expression duration depending on the photoperiod.

Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle.

Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADH) upon blue light absorption.

Adaptive light: a lighting control method aligned with dark adaptation of human vision.

Light exposure before sleep causes a reduction in the quality and duration of sleep. In order to reduce these detrimental effects of light exposure, it is important to dim the light. However, dimming the light often causes inconvenience and can lower the quality of life (QOL).

Clock Gene Expression in the Eye Exhibits Circadian Oscillation and Light Responsiveness but is Not Necessary for Nocturnal Locomotor Activity of Japanese Loach, .

There are few model fish that are both edible and suitable for use in the laboratory. The Japanese loach () is a traditional food in Japan, but is highly neglected despite its great nutritional value. To understand its circadian system and photic input pathway for synchronization of physiological activities to environmental light-dark cycles, we measured locomotor activity under light-dark and constant dark (DD) conditions.

A photoperiodic time measurement served by the biphasic expression of Cryptochrome1ab in the zebrafish eye.

The zebrafish (Danio rerio) is a model species that is used to study the circadian clock. It possesses light-entrainable circadian clocks in both central and peripheral tissues, and its core circadian factor cryptochromes (CRYs) have diverged significantly during evolution. In order to elucidate the functional diversity and involvement of CRYs in photoperiodic mechanisms, we investigated the daily expression profiles of six Cry transcripts in central (brain and eye) and peripheral (fin, skin and muscle) tissues.

THETA system allows one-step isolation of tagged proteins through temperature-dependent protein-peptide interaction.

Tools to control protein-protein interactions by external stimuli have been extensively developed. For this purpose, thermal stimulation can be utilized in addition to light. In this study, we identify a monoclonal antibody termed C13 mAb, which shows an approximately 480-fold decrease in the affinity constant at 37 °C compared to that at 4 °C.

Moonlight controls lunar-phase-dependency and regular oscillation of clock gene expressions in a lunar-synchronized spawner fish, Goldlined spinefoot.

Goldlined spinefoot, Siganus guttatus, inhabits tropical and subtropical waters and synchronizes its spawning around the first quarter moon likely using an hourglass-like lunar timer. In previous studies, we have found that clock genes (Cryptochrome3 and Period1) could play the role of state variable in the diencephalon when determining the lunar phase for spawning. Here, we identified three Cry, two Per, two Clock, and two Bmal genes in S.

Light- and circadian-controlled genes respond to a broad light spectrum in Puffer Fish-derived Fugu eye cells.

Some cell lines retain intrinsic phototransduction pathways to control the expression of light-regulated genes such as the circadian clock gene. Here we investigated the photosensitivity of a Fugu eye, a cell line established from the eye of Takifugu rubripes, to examine whether such a photosensitive nature is present. Microarray analysis identified 15 genes that showed blue light-dependent change at the transcript level.

Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish, Careproctus rhodomelas, Living near the Deep-Sea Hydrothermal Vent.

Visual systems in deep-sea fishes have been previously studied from a photobiological aspect; however, those of deep-sea fish inhabiting the hydrothermal vents are far less understood due to sampling difficulties. In this study, we analyzed the visual pigment of a deep-sea snailfish, Careproctus rhodomelas, discovered and collected only near the hydrothermal vents of oceans around Japan. Proteins were solubilized from the C.

Overexpression in yeast, photocycle, and in vitro structural change of an avian putative magnetoreceptor cryptochrome4.

Cryptochromes (CRYs) have been found in a wide variety of living organisms and can function as blue light photoreceptors, circadian clock molecules, or magnetoreceptors. Non-mammalian vertebrates have CRY4 in addition to the CRY1 and CRY2 circadian clock components. Though the function of CRY4 is not well understood, chicken CRY4 (cCRY4) may be a magnetoreceptor because of its high level of expression in the retina and light-dependent structural changes in retinal homogenates.

Hypothalamic expression and moonlight-independent changes of Cry3 and Per4 implicate their roles in lunar clock oscillators of the lunar-responsive Goldlined spinefoot.

Lunar cycle-associated physiology has been found in a wide variety of organisms. Studies suggest the presence of a circalunar clock in some animals, but the location of the lunar clock is unclear. We previously found lunar-associated expression of transcripts for Cryptochrome3 gene (SgCry3) in the brain of a lunar phase-responsive fish, the Goldlined spinefoot (Siganus guttatus).

Identification and characterization of cryptochrome4 in the ovary of western clawed frog Xenopus tropicalis.

CRY proteins can be classified into several groups based on their phylogenetic relationships, and they function as a photoreceptor, a photolyase, and/or a transcriptional repressor of the circadian clock. In order to elucidate the expression profile and functional diversity of CRYs in vertebrates, we focused on XtCRY4, a member of the uncharacterized cryptochrome family CRY4 in Xenopus tropicalis. XtCRY4 cDNA was isolated by RT-PCR, and a phylogenetic analysis of deduced sequence of XtCRY4 suggested that the vertebrate Cry4 genes evolved at much higher evolutionary rates than mammalian-type Cry genes, such as the CRY1 and CRY2 circadian clock molecules.

Light-dependent structural change of chicken retinal Cryptochrome4.

Animals have several classes of cryptochromes (CRYs), some of which function as core elements of circadian clockwork, circadian photoreceptors, and/or light-dependent magnetoreceptors. In addition to the circadian clock genes Cry1 and Cry2, nonmammalian vertebrates have the Cry4 gene, the molecular function of which remains unknown. Here we analyzed chicken CRY4 (cCRY4) expression in the retina with in situ hybridization and found that cCRY4 was likely transcribed in the visual pigment cells, cells in the inner nuclear layer, and retinal ganglion cells.

Zebrafish respond to the geomagnetic field by bimodal and group-dependent orientation.

A variety of animals use Earth's magnetic field as a reference for their orientation behaviour. Although distinctive magnetoreception mechanisms have been postulated for many migrating or homing animals, the molecular mechanisms are still undefined. In this study, we found that zebrafish, a model organism suitable for genetic manipulation, responded to a magnetic field as weak as the geomagnetic field.

Lunar phase-dependent expression of cryptochrome and a photoperiodic mechanism for lunar phase-recognition in a reef fish, goldlined spinefoot.

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal.

Cryptochrome genes are highly expressed in the ovary of the African clawed frog, Xenopus tropicalis.

Cryptochromes (CRYs) are flavoproteins sharing high homology with photolyases. Some of them have function(s) including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates.

Preferential inhibition of BMAL2-CLOCK activity by PER2 reemphasizes its negative role and a positive role of BMAL2 in the circadian transcription.

In the molecular oscillatory mechanism governing circadian rhythms, positive regulators, including CLOCK and BMAL1, transactivate Per and Cry genes through E-box elements, and translated PER and CRY proteins negatively regulate their own transactivation. Like BMAL1, its paralog BMAL2 dimerizes with CLOCK to activate the E-box-dependent transcription, but the role of BMAL2 in the circadian clockwork is still elusive. Here we characterized BMAL2 function in NIH3T3 cells and found that the cellular rhythms monitored by Bmal1 promoter-driven bioluminescence signals were blunted by RNA interference-mediated suppression of Bmal2 as well as that of Bmal1.

Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.

In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase.

Macrophage inhibitory cytokine MIC-1 is upregulated by short-wavelength light in cultured normal human dermal fibroblasts.

To better understand dermal response to visible light, we used DNA microarray analysis to search genes induced by blue or near-UV light in normal human dermal fibroblasts. Of about 12800 transcripts analyzed, near-UV light most prominently upregulated the transcript level of Mic-1, a gene encoding a TGF-beta superfamily protein. Quantitative RT-PCR and immunoblot analyses revealed that mRNA and protein levels of Mic-1 were upregulated by both short-wavelength light but not by green or red light.

Two isoforms of chicken melanopsins show blue light sensitivity.

Melanopsin is a vertebrate non-visual opsin and functions as a circadian photoreceptor in mammalian retinas. Here we found the expression of two kinds of melanopsin genes in the chicken pineal gland and identified the presence of five isoforms derived from these two genes. Reconstitution of the recombinant proteins with 11-cis-retinal revealed that at least two of these melanopsin protein isoforms can function as blue-sensitive photopigments with absorption maxima at 476-484nm.

Circadian clock gene regulation of steroidogenic acute regulatory protein gene expression in preovulatory ovarian follicles.

It is now known that circadian clocks are localized not only in the central pacemaker but also in peripheral organs. An example of a clock-dependent peripheral organ is the ovary of domestic poultry in which ovulation is induced by the positive feedback action of ovarian progesterone on the neuroendocrine system to generate a preovulatory release of LH during a daily 6-10 h "open period" of the ovulatory cycle. It has been assumed previously that the timing of ovulation in poultry is controlled solely by a clock-dependent mechanism within the neuroendocrine system.

GRK1 and GRK7: unique cellular distribution and widely different activities of opsin phosphorylation in the zebrafish rods and cones.

Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis.

Molecular cloning, mRNA expression, and immunocytochemical localization of a putative blue-light photoreceptor CRY4 in the chicken pineal gland.

In non-mammalian vertebrates, the pineal gland contains an endogenous circadian oscillator and serves as a photosensitive neuroendocrinal organ. To better understand the pineal phototransduction mechanism, we focused on the chicken putative blue-light photoreceptive molecule, Cryptochrome4 (cCRY4). Here we report the molecular cloning of pineal cCry4 cDNA, the in vivo expression of cCry4 mRNA, and the detection of cCRY4 protein.