Opsin Family Research Articles

Light sensitivity in Beroidae ctenophores: Insights from laboratory studies and genomics

Light detection underlies a variety of animal behaviors, including those related to spatial orientation, feeding, avoidance of predators, and reproduction. Ctenophores are likely the oldest animal group in which light sensitivity based on opsins evolved, so they may still have the ancestral molecular mechanisms for photoreception. However, knowledge about ctenophore photosensitivity, associated morphological structures, molecular mechanisms involved, and behavioral reactions is limited and fragmented. We present the initial experiments on the responses of adult Beroe ovata to high-intensity light exposure with different spectra and photosensitivity in various parts of the animal's body. Ctenophores have shown a consistent behavioral response when their aboral organ is exposed to a household-grade laser in the violet spectrum. To investigate the genes responsible for the photosensitivity of Beroidae, we have analyzed transcriptome and genome-wide datasets. We identified three opsins in Beroe that are homologous to those found in Mnemiopsis leidyi (Lobata) and Pleurobrachia bachei (Cydippida). These opsins form clades Ctenopsin1, 2, and 3, respectively. Ctenopsin3 is significantly distinct from other ctenophore opsins and clustered outside the main animal opsin groups. The Ctenopsin1 and Ctenopsin2 groups are sister clusters within the canonical animal opsin tree. These two groups could have originated from gene duplication in the common ancestor of the species we studied and then developed independently in different lineages of Ctenophores. So far, there is no evidence of additional expansion of the opsin family in ctenophore evolution. The involvement of ctenophore opsins in photoreception is discussed by analyzing their protein structures.

Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

Eyeless razor clam Sinonovacula constricta discriminates light spectra through opsins to guide Ca2+ and cAMP signaling pathways

Phototransduction is based on opsins that drive distinct types of Gα cascades. Although nonvisual photosensitivity has long been known in marine bivalves, the underlying molecular basis and phototransduction mechanism are poorly understood. Here, we introduced the eyeless razor clam Sinonovacula constricta as a model to clarify this issue. First, we showed that S. constricta was highly diverse in opsin family members, with a significant expansion in xenopsins. Second, the expression of putative S. constricta opsins was highly temporal-spatio specific, indicating their potential roles in S. constricta development and its peripheral photosensitivity. Third, by cloning four S. constricta opsins with relatively higher expression (Sc_opsin1, 5, 7, and 12), we found that they exhibited different expression levels in response to different light environments. Moreover, we demonstrated that these opsins (excluding Sc_opsin7) couple with Gαq and Gαi cascades to mediate the light-dependent Ca2+ (Sc_opsin1 and 5) and cAMP (Sc_opsin12) signaling pathways. The results indicated that Sc_opsin1 and 5 belonged to Gq-opsins, Sc_opsin12 belonged to Gi-opsins, while Sc_opsin7 might act as a photo-isomerase. Furthermore, we found that the phototransduction function of S. constricta Gq-opsins was dependent on the lysine at the seventh transmembrane domain, and greatly influenced by the external light spectra in a complementary way. Thus, a synergistic photosensitive system mediated by opsins might exist in S. constricta to rapidly respond to the transient or subtle changes of the external light environment. Collectively, our findings provide valuable insights into the evolution of opsins in marine bivalves and their potential functions in nonvisual photosensitivity.

Nematostella vectensis exemplifies the exceptional expansion and diversity of opsins in the eyeless Hexacorallia

BackgroundOpsins are the primary proteins responsible for light detection in animals. Cnidarians (jellyfish, sea anemones, corals) have diverse visual systems that have evolved in parallel with bilaterians (squid, flies, fish) for hundreds of millions of years. Medusozoans (e.g., jellyfish, hydroids) have evolved eyes multiple times, each time independently incorporating distinct opsin orthologs. Anthozoans (e.g., corals, sea anemones,) have diverse light-mediated behaviors and, despite being eyeless, exhibit more extensive opsin duplications than medusozoans. To better understand the evolution of photosensitivity in animals without eyes, we increased anthozoan representation in the phylogeny of animal opsins and investigated the large but poorly characterized opsin family in the sea anemone Nematostella vectensis.ResultsWe analyzed genomic and transcriptomic data from 16 species of cnidarians to generate a large opsin phylogeny (708 sequences) with the largest sampling of anthozoan sequences to date. We identified 29 opsins from N. vectensis (NvOpsins) with high confidence, using transcriptomic and genomic datasets. We found that lineage-specific opsin duplications are common across Cnidaria, with anthozoan lineages exhibiting among the highest numbers of opsins in animals. To establish putative photosensory function of NvOpsins, we identified canonically conserved protein domains and amino acid sequences essential for opsin function in other animal species. We show high sequence diversity among NvOpsins at sites important for photoreception and transduction, suggesting potentially diverse functions. We further examined the spatiotemporal expression of NvOpsins and found both dynamic expression of opsins during embryonic development and sexually dimorphic opsin expression in adults.ConclusionsThese data show that lineage-specific duplication and divergence has led to expansive diversity of opsins in eyeless cnidarians, suggesting opsins from these animals may exhibit novel biochemical functions. The variable expression patterns of opsins in N. vectensis suggest opsin gene duplications allowed for a radiation of unique sensory cell types with tissue- and stage-specific functions. This diffuse network of distinct sensory cell types could be an adaptive solution for varied sensory tasks experienced in distinct life history stages in Anthozoans.

Taxonomic distribution of opsin families inferred from UniProt Reference Proteomes and a suite of opsin-specific hidden Markov models

IntroductionOpsins are a large and sequence-diverse family of light-responsive G-protein coupled receptors involved in vision, circadian rhythm, and other processes. Numerous subfamilies have been defined based on sequence similarity, cell-type localization, signal transduction mechanism, or biological function, but there is no consensus classification system.MethodsWe used multiple hidden Markov models (HMMs) to identify opsins in the UniProt Reference Proteomes database. Opsin-specific HMMs were also used in an annotation procedure that represents sequences as a vector of HMM scores and assess the similarity of these vectors to those of annotated sequences. UniProt Reference Proteomes are built from genome sequences, allowing us to make meaningful comparisons of the number of opsins in each of the 260 species available at the time of the survey in absolute terms and relative to a larger superfamily of which opsins are a member.ResultsMore than 2,000 opsins were retrieved from 262 species (all metazoans).DiscussionMerging opsin counts into higher order taxa paints a broad view of the taxonomic distribution of opsins, and of opsin subfamilies, annotated according to three different schemes.

The effects of missense OPN3 mutations in melanocytic lesions on protein structure and light-sensitive function.

Opsin 3 (OPN3), a member of the light-sensitive, retinal-dependent opsin family, is widely expressed in a variety of human tissues and plays a multitude of light-dependent and light-independent roles. We recently identified five missense variants of OPN3, including p. I51T, p. V134A, p. V183I, p. M256I and p. C331Y, in human melanocytic tumours. However, it remains unclear how these OPN3 variants affect OPN3 protein structure and function. Herein, we conducted structural and functional studies of these variant proteins in OPN3 by molecular docking and molecular dynamics simulations. Moreover, we performed in vitro fluorescence calcium imaging to assess the functional properties of five single-nucleotide variant (SNV) proteins using a site-directed mutagenesis method. Notably, the p. I51T variant was not able to effectively dock with 11-cis-retinal. Additionally, in vitro, the p. I51T SNVs failed to induce any detectable changes in intracellular Ca2+ concentration at room temperature. Taken together, these results reveal that five SNVs in the OPN3 gene have deleterious effects on protein structure and function, suggesting that these mutations, especially the p. I51T variant, significantly disrupt the canonical function of the OPN3 protein. Our findings provide new insight into the role of OPN3 variants in the loss of protein function.

OPN3 Regulates Melanogenesis in Human Congenital Melanocytic Nevus Cells through Functional Interaction with BRAFV600E

OPN3, as a member of the opsin family, has various nonlight-dependent functions. Congenital melanocytic nevus (CMN) is a skin lesion with dark pigmentation that appears at birth and can be initiated by the BRAFV600E mutation in melanocytes. However, the role of OPN3 in BRAFV600E CMN cell melanogenesis has never been reported. In this study, we show that OPN3 acts as a negative regulator of melanin production by modulating BRAFV600E signaling in BRAFV600E CMN cells. Knocking down OPN3 expression can inhibit the BRAFV600E/extracellular signal‒regulated kinase signaling pathway and upregulate the expression of microcephaly-related transcription factors, tyrosinase, and TRP1 and TRP2, thus increasing melanin levels in BRAFV600E CMN cells. More remarkably, OPN3 and BRAFV600E were found to form a physical complex. Furthermore, a three-dimensional nevus model was used to further prove the negative regulatory effect of OPN3 on BRAFV600E CMN cell melanogenesis. Our study reveals a mechanism for OPN3-mediated melanogenesis in BRAFV600E CMN cells, and these results may lead to a more personalized and economically viable approach to treating BRAFV600E CMN.

From the Sea for the Sight: Marine Derived Products for Human Vision.

Visual impairment, at different degrees, produce a reduction of patient wellness which negatively impact in many aspects of working and social activities. Eye diseases can have common cellular damages or dysfunctions (e.g., inflammation, oxidative stress, neuronal degeneration), and can target several eye compartments, primarily cornea and retina. Marine organisms exhibit high chemical diversity due to the wide range of marine ecosystems where they live; thus, molecules of marine origin are gaining increasing attention for the development of new mutation-independent therapeutic strategies, to reduce the progression of retina pathologies having a multifactorial nature and characterized by high genetic heterogeneity. This review aims to describe marine natural products reported in the recent literature that showed promising therapeutic potential for the development of new drugs to be used to contrast the progression of eye pathologies. These natural compounds exhibited beneficial and protective properties on different in vitro cell systems and on in vivo models, through different mechanisms of action, including anti-inflammatory, antioxidant, antiangiogenic/vasoprotective or cytoprotective effects. We report compounds produced by several marine source (e.g., sponges, algae, shrimps) that can be administrated as food or with target-specific strategies. In addition, we describe and discuss the uses of opsin family proteins from marine organisms for the optimization of new optogenetic therapeutic strategies.

Expression of Retinal G Protein-Coupled Receptor, a Member of the Opsin Family, in Human Skin Cells and Its Mediation of the Cellular Functions of Keratinocytes.

Background: Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported. Objectives: We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes. Methods: The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology. Results: The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis. Conclusions: This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.

Analysis of opsin gene family of Crimson snapper (Lutjanus erythropterus)

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061bp to 86203bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.

528 Opsin3 expression in human nevus and reconstructed nevus model in vitro

Background Opsin3 is a non-visual opsin and is a member of the opsin family. Recent researchers found that opsin3 is highly expressed in human epidermal keratinocytes, melanocytes, hair follicles and fibroblast. However, it is rarely reported in human nevus cells. Objective To investigate whether opsin3 express in human nevus cells, nevus tissues, and 3D model of reconstructed nevus in vitro. Methods The expression of opsin3 in nevus tissues, paired adjacent normal skin tissues were analyzed by quantitative real-time PCR and western blotting.

Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic β2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.

Diversity of Light Sensing Molecules and Their Expression During the Embryogenesis of the Cuttlefish (Sepia officinalis).

Eyes morphologies may differ but those differences are not reflected at the molecular level. Indeed, the ability to perceive light is thought to come from the same conserved gene families: opsins and cryptochromes. Even though cuttlefish (Cephalopoda) are known for their visually guided behaviors, there is a lack of data about the different opsins and cryptochromes orthologs represented in the genome and their expressions. Here we studied the evolutionary history of opsins, cryptochromes but also visual arrestins in molluscs with an emphasis on cephalopods. We identified 6 opsins, 2 cryptochromes and 1 visual arrestin in Sepia officinalis and we showed these families undergo several duplication events in Mollusca: one duplication in the arrestin family and two in the opsin family. In cuttlefish, we studied the temporal expression of these genes in the eyes of embryos from stage 23 to hatching and their expression in two extraocular tissues, skin and central nervous system (CNS = brain + optic lobes). We showed in embryos that some of these genes (Sof_CRY6, Sof_reti-1, Sof_reti-2, Sof_r-opsin1 and Sof_v-arr) are expressed in the eyes and not in the skin or CNS. By looking at a juvenile and an adult S. officinalis, it seems that some of these genes (Sof_r-opsin1 and Sof_reti1) are used for light detection in these extraocular tissues but that they set-up later in development than in the eyes. We also showed that their expression (except for Sof_CRY6) undergoes an increase in the eyes from stage 25 to 28 thus confirming their role in the ability of the cuttlefish embryos to perceive light through the egg capsule. This study raises the question of the role of Sof_CRY6 in the developing eyes in cuttlefish embryos and the role and localization of xenopsins and r-opsin2. Consequently, the diversity of molecular actors involved in light detection both in the eyes and extraocular tissues is higher than previously known. These results open the way for studying new molecules such as those of the signal transduction cascade.

Violet-light suppression of thermogenesis by opsin 5 hypothalamic neurons.

The opsin family of G-protein coupled receptors are employed as light detectors in animals. Opsin 5 (neuropsin, OPN5) is a highly conserved, violet light (380 nm λmax) sensitive opsin1,2. In mice, OPN5 is a known photoreceptor in retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which Opn5 expressing POA neurons regulate brown adipose tissue (BAT) thermogenesis. We show Opn5 expression in glutamatergic warm-sensing POA neurons that receive synaptic input from multiple thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5 null mice show overactive BAT, elevated body temperature, and exaggerated thermogenesis when cold challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type, but not in Opn5 null mice. Direct measurements of intracellular cAMP ex vivo reveal that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.

Gene expression patterns of novel visual and non-visual opsin families in immature and mature Japanese eel males.

This study was carried out to identify and estimate physiological function of a new type of opsin subfamily present in the retina and whole brain tissues of Japanese eel using RNA–Seq transcriptome method. A total of 18 opsin subfamilies were identified through RNA–seq. The visual opsin family included Rh2, SWS2, FWO, DSO, and Exo-Rhod. The non-visual opsin family included four types of melanopsin subfamily (Opn4x1, Opn4x2, Opn4m1, and Opn4m2), peropsin, two types of neuropsin subfamily (Opn5-like, Opn5), Opn3, three types of TMT opsin subfamily (TMT1, 2, 3), VA-opsin, and parapinopsin. In terms of changes in photoreceptor gene expression in the retina of sexually mature and immature male eels, DSO mRNA increased in the maturation group. Analysis of expression of opsin family gene in male eel brain before and after maturation revealed that DSO and SWS2 expression in terms of visual opsin mRNA increased in the sexually mature group. In terms of non-visual opsin mRNA, parapinopsin mRNA increased whereas that of TMT2 decreased in the fore-brain of the sexually mature group. The mRNA for parapinopsin increased in the mid-brain of the sexually mature group, whereas those of TMT1 and TMT3 increased in the hind-brain of the sexually mature group. DSO mRNA also increased in the retina after sexual maturation, and DSO and SWS2 mRNA increased in whole brain part, suggesting that DSO and SWS2 are closely related to sexual maturation.

Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor

Opsins form a family of light-activated, retinal-dependent, G protein-coupled receptors (GPCRs) that serve a multitude of visual and nonvisual functions. Opsin 3 (OPN3 or encephalopsin), initially identified in the brain, remains one of the few members of the mammalian opsin family with unknown function and ambiguous light absorption properties. We recently discovered that OPN3 is highly expressed in human epidermal melanocytes (HEMs)-the skin cells that produce melanin. The melanin pigment is a critical defense against ultraviolet radiation (UVR), and its production is mediated by the Gαs-coupled melanocortin 1 receptor (MC1R). The physiological function and light sensitivity of OPN3 in melanocytes are yet to be determined. Here, we show that in HEMs, OPN3 acts as a negative regulator of melanin production by modulating the signaling of MC1R. OPN3 negatively regulates the cyclic adenosine monophosphate (cAMP) response evoked by MC1R via activation of the Gαi subunit of G proteins, thus decreasing cellular melanin levels. In addition to their functional relationship, OPN3 and MC1R colocalize at both the plasma membrane and in intracellular structures, and can form a physical complex. Remarkably, OPN3 can bind retinal, but does not mediate light-induced signaling in melanocytes. Our results identify a function for OPN3 in the regulation of the melanogenic pathway in epidermal melanocytes; we have revealed a light-independent function for the poorly characterized OPN3 and a pathway that greatly expands our understanding of melanocyte and skin physiology.

Molecular palaeontology illuminates the evolution of ecdysozoan vision.

Colour vision is known to have arisen only twice—once in Vertebrata and once within the Ecdysozoa, in Arthropoda. However, the evolutionary history of ecdysozoan vision is unclear. At the molecular level, visual pigments, composed of a chromophore and a protein belonging to the opsin family, have different spectral sensitivities and these mediate colour vision. At the morphological level, ecdysozoan vision is conveyed by eyes of variable levels of complexity; from the simple ocelli observed in the velvet worms (phylum Onychophora) to the marvellously complex eyes of insects, spiders, and crustaceans. Here, we explore the evolution of ecdysozoan vision at both the molecular and morphological level; combining analysis of a large-scale opsin dataset that includes previously unknown ecdysozoan opsins with morphological analyses of key Cambrian fossils with preserved eye structures. We found that while several non-arthropod ecdysozoan lineages have multiple opsins, arthropod multi-opsin vision evolved through a series of gene duplications that were fixed in a period of 35–71 million years (Ma) along the stem arthropod lineage. Our integrative study of the fossil and molecular record of vision indicates that fossils with more complex eyes were likely to have possessed a larger complement of opsin genes.

Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases

BackgroundThe green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two—the channelrhodopsins—were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.ResultsHere we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called “two-component cyclase opsins” (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.ConclusionsCr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.

An all-trans-retinal-binding opsin peropsin as a potential dark-active and light-inactivated G protein-coupled receptor

Peropsin or retinal pigment epithelium-derived rhodopsin homolog, found in many animals, belongs to the opsin family. Most opsins bind to 11-cis-retinal as a chromophore and act as light-activated G protein-coupled receptors. Some peropsins, however, bind all-trans-retinal and isomerise it into 11-cis form by light, and peropsin has been suggested to supply other visual opsins with 11-cis-retinal. Additionally, peropsin has some amino acid sequence motifs that are highly conserved among G protein-coupled opsins. Here, using chimeric mutant peropsins, we found that peropsin potentially generates an “active form” that drives G-protein signalling in the dark by binding to all-trans-retinal and that the active form photo-converts to an inactive form containing 11-cis-retinal. Comparative spectroscopic analysis demonstrated that spider peropsin exhibited catalytic efficiency for retinal photoisomerisation that was much lower than a retinal photoisomerase, squid retinochrome. The chimeric peropsins, constructed by replacing the third intracellular loop region with that of Gs- or Gi-coupled opsin, were active and drove Gs- or Gi-mediated signalling in the dark, respectively, and were inactivated upon illumination in mammalian cultured cells. These results suggest that peropsin acts as a dark-active, light-inactivated G protein-coupled receptor and is useful as a novel optogenetic tool.

Avian central clocking system

In birds many life processes runs in diurnal (e.g. locomotor activity, feeding, melatonin secretion) and seasonal rhythms (e.g. reproduction, song, feathering, migration) depending on the environmental light and the activity of the central clock system (CCS). The structure and mechanisms of the activity of the avian CCS are the most complex among vertebrates. CCS consists of three oscillators (in the retina, SCN and pineal gland) possessing own sensory input system (photopigments) and effective output system (products for direct biological effects). So far in the CCS in birds 14 forms of photopigments (Opn1, Opn2, Opn3, TMT, Opn4x, Opn4m, Opn5, RGR, RRH, VA-opsins, pinopsin, Cry1, Cry2 i Cry4) and 12 clock genes making up oscillators (Bmal1, Bmal2, Clock, NPas2 called also Mop4 and Rorα – positive genes and Cry1, Cry2, Cry4, Per2, Per3, E4bp4 and Rev-erbα – negative genes) have been described. Photopigments are placed in all layers of the retina; in brain - mainly in regions of nuclei: septalis lateralis, premammillaris, habenularis and paraventricularis; in the pineal gland - in all kinds of pinealocytes. Most photopigments belonging to the opsin family are linked with nucleotide phototransduction path, typical for vertebrates, but in avian CCS also phosphoinositol phototransduction path, characteristic for invertebrates, is existing and concerns Opn4x and Opn5. Oscilators are placed in nuclei of cells of all layers of the retina, in mSCN and vSCN (with great species variability) and in pinealocytes. It is supposing, that all nonvisual photopigments have a direct role in the synchronization of the oscillator activity with the environmental light, but molecular mechanisms of the interaction between photopigments and the oscillator remain unknown. The impact of each of the three oscillators of CCS in generating of biological rhythms in birds show great species differentiation. The differences concern both the domination of one of oscillators over the others and the assignation of biological processes which individual oscillator synchronizes rhythmically with the environmental light..