Abstract Opsins underlie diverse physiological responses to light in animals. In the dark, most opsins bind the chromophore 11- cis retinal, which isomerizes to all- trans form upon light absorption, representing the initial key step in signaling. Maintenance of opsin function therefore requires continuous regeneration of the inactive, 11- cis -retinal-bound state. Here, we report a novel type of opsin, AtAntho2c, from a reef-building coral, whose active form, bound to all -trans retinal, can thermally revert to the initial dark state bound to 11- cis retinal. A cysteine residue in extracellular loop 2 region plays a key role in the self-regeneration ability. Using time-resolved and low-temperature spectroscopies, we identify two spectrally distinct photointermediates prior to the all- trans to 11- cis isomerization in AtAntho2c, whose formation rates and yields are found to vary depending on temperature and pH conditions. The active form of AtAntho2c activates Gi/o G protein, resulting in a transient and repeatable decrease in cellular cAMP levels upon repeated light stimulations, even in the absence of exogenous retinal in cultured cells. Furthermore, we confirm that cells expressing AtAntho2c exhibit membrane hyperpolarization via GIRK channel activation light-dependently. These properties highlight the potential of AtAntho2c as a versatile optogenetic actuator capable of repeatedly modulate Gi/o signaling without retinal supplementation. Significance Statement Light-sensitive proteins, opsins, form active states upon light absorption, leading to intracellular G protein signaling and various cellular outputs. The active states require specific enzymatic machinery or another photon absorption to regenerate inactive opsins ready to respond to repeated light stimuli and maintain continuous responsiveness. In our study, we identify and analyze a coral opsin of which the active state rapidly and autonomously reverts to the inactive state in the dark through thermal isomerization of the retinal chromophore within the opsin. This regeneration mechanism allows the opsin to respond to repeated light stimuli at high temporal resolution and maintain large signal amplitude without the need for exogenous retinal making this opsin potentially useful for developing versatile optogenetic tools.

Today, about 2.8 billion people worldwide suffer from myopia (nearsightedness), and projections place the 2050 burden at 4.8 billion people (the "myopia boom"). Myopia results from abnormal elongation of the eye, which causes images to focus in front of the retina. Building on prior work in mouse models assessing the non-visual opsin OPN5, we test the hypothesis that insufficient exposure to short-wavelength light increases myopia susceptibility. We use the tree shrew, a near-primate model for human myopia. Our findings show that indigo light in the range of 419-446 nm, when used to supplement a warm white light-emitting diode (LED), completely suppresses myopia induced by minus-lens wear. A large body of genetic and epidemiological evidence suggests that the "myopia boom" has an environmental cause. Our findings align with this evidence and suggest that one contributing factor is the relative absence of indigo light in built environments where we spend 86% of our time.

Opsins are universal photoreceptive proteins in animals. Rhodopsin is the best-studied opsin and functions as a visual sensor in rod cells of human and mouse retinas. Rhodopsin produces an active state upon photoreception, which triggers the signal transduction cascade to evoke a hyperpolarizing response of the cells. This active state is a metastable intermediate and cannot convert back to the dark state by either photoreaction or thermal reaction. Thus, vertebrate rhodopsin is categorized as a mono-stable opsin. Recent accumulation of genomic information in animals has expanded the known repertoires of opsin genes, which are responsible for visual and non-visual photoreceptive functions. The analysis of these opsins revealed that many opsins, including non-visual opsins such as Opn4 and Opn5, form a stable active state upon photoreception and this active state can photo-convert back to the dark state. These opsins have the property of photoreversibility between the dark and active states and thus are categorized as bistable opsins. In addition, we previously identified a different type of non-visual opsin, Opn5L1, whose activity is controlled by a photocyclic reaction. This photocyclic reaction is quite similar to that of channelrhodopsin and is achieved by a special mechanism involving a cysteine residue at position 188 that has not been observed in any other opsins so far. This review would like to focus on the unique photocyclic animal opsin in the context of the diversity of visual and non-visual opsins and also discuss the possibility of designing “artificial photocyclic opsins” from natural opsins for potential application in optogenetic gene therapy.

The recent discovery of nonvisual photoreceptors in various organs has raised expectations for uncovering their roles and underlying mechanisms. In this work, we identified a previously unrecognized hormone-releasing mechanism in the pituitary of the Japanese rice fish (medaka) induced by light. Ca 2+ imaging analysis revealed that melanotrophs, a type of pituitary endocrine cell that secretes melanocyte-stimulating hormone, robustly increase the concentration of intracellular Ca 2+ during short-wavelength light exposure. Moreover, we identified Opn5m as the key molecule that drives this response. Knocking out opn5m attenuated melanogenesis by reducing tyrosinase expression in the skin. Our findings suggest a mechanism in which direct reception of short-wavelength light by pituitary melanotrophs triggers a pathway that might contribute to protection from ultraviolet radiation in medaka.

Centralized nervous systems enable animals to detect environmental cues and coordinate behavior, but their evolutionary origins in deuterostomes remain unclear. Among deuterostomes, echinoderms-such as sea urchins-have long been thought to lack brain-like structures, especially in larval stages. Although recent gene expression and neural activity studies suggest brain-like properties in sea urchin larvae, direct links to behavior are still emerging. Here, we identify a light-sensitive cluster of neurons in the posterior neuroectoderm of sea urchin larvae. These neurons express UV-sensitive Opsin5 and regulatory genes such as rx, otx, six3, and lhx6, which are conserved in the vertebrate diencephalon. We mapped this domain using single-cell RNA sequencing and in situ hybridization. Knockdown of Opn5L impaired light-dependent swimming, indicating an active role in photoreception. While further work is needed to fully establish circuit-to-behavior relationships, our findings add to growing evidence that sea urchin larvae possess a non-visual photoreceptive neural center with molecular features shared by vertebrate brain regions. This suggests that such domains originated in the deuterostome ancestor and contributed to the early evolution of brain function.

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha ( Pygocentrus nattereri ), retain both PT1 and PT2. The Mexican tetra ( Astyanax mexicanus ) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish ( Silurus asotus ) PT2 were located at 517 nm, 528 nm, 517 nm, and ∼535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1 . Based on these findings, functional diversification of opsins after gene duplication is discussed. ### Competing Interest Statement The authors have declared no competing interest.

Opsins underlie visual and non-visual photoreceptions in animals. Vertebrate and arthropod visual opsins belong to different opsin groups and convergently show spectral diversity ranging from the UV to the red region for color vision. Recently, uncharacterized opsins called arthropsin have been identified from various protostome genomes. Arthropsin is clustered with arthropod and mollusk visual opsins and vertebrate blue-sensitive non-visual opsin Opn4. Here, we show that arthropsins have unexpected spectral diversity ranging from the UV to the red region. In particular, water flea (Daphnia magna) expresses red-sensitive arthropsins in the optic lobe and brain. Among the non-visual opsins characterized so far, these arthropsins exhibit the most red-shifted spectral sensitivity. Moreover, the molecular mechanism responsible for the red shift of arthropsins is different from that of red-sensitive vertebrate visual opsins. We characterize arthropsin as a different type of non-visual opsin which acquired the spectral diversity independently of vertebrate and arthropod visual opsins.

Unlike terrestrial environments, where humans reside, there is no sunlight in the deep sea. Instead, dim visible light from black-body radiation and bioluminescence illuminates hydrothermal vent areas in the deep sea. A deep-sea hydrothermal vent shrimp, Rimicaris hybisae, is thought to detect this dim light using its enlarged dorsal eye; however, the molecular basis of its photoreception remains unexplored. Here, we characterized the molecular properties of opsins, universal photoreceptive proteins in animals, found in R. hybisae. Transcriptomic analysis identified six opsins: three Gq-coupled opsins, one Opn3, one Opn5, and one peropsin. Functional analysis revealed that five of these opsins exhibited light-dependent G protein activity, whereas peropsin exhibited the ability to convert all-trans-retinal to 11-cis-retinal like photoisomerases. Notably, all the R. hybisae opsins, including Opn5, convergently show visible light sensitivity (around 457-517 nm), whereas most opsins categorized as Opn5 have been demonstrated to be UV sensitive. Mutational analysis revealed that the unique visible light sensitivity of R. hybisae Opn5 is achieved through the stabilization of a protonated Schiff base by a counterion residue at position 83 (Asp83), which differs from the position identified in other opsins. These findings suggest that the vent shrimp R. hybisae has adapted its photoreceptive devices to dim deep-sea hydrothermal light by selectively maintaining a repertoire of visible light-sensitive opsins, including the uniquely tuned Opn5.

Opsins are photoreceptive proteins responsible for visual and non-visual photoreceptions in animals. In general, vertebrates have multiple visual and non-visual opsins whose spectral sensitivities range from the UV to the red region. Among these opsins, Opn5 has been widely identified in vertebrates from fishes to primates and functions as a non-visual opsin in various tissues, including the retina and brain. Vertebrate Opn5 has been characterized as a UV-sensitive bistable opsin. Thus, Opn5 provides one of the molecular mechanisms determining the short wavelength limit that vertebrates can detect. In this study, we searched for the amino acid residue responsible for the UV light sensitivity of Opn5. Our mutational analysis revealed that Opn5 acquired visible light sensitivity by the substitution of Lys91 with an amino acid other than an arginine or tyrosine residue. In addition, the mutations at Lys91 altered the preferential binding of the retinal isomers in Opn5. Therefore, the conservation of Lys91 among vertebrate Opn5 proteins would be necessary to enable Opn5 to work as the shortest wavelength sensor in various tissues.

A group of nonvisual opsins specific to vertebrates is essential to understand evolution of lateral eyes, one of the most prominent innovations in this lineage. Nevertheless, our knowledge of their evolutionary history remains limited. To develop an integrated view of their evolution, we surveyed these non-visual opsins (VA opsin, pinopsin, parapinopsin, parietopsin, and parapinopsin-like) in 451 vertebrate genomes. Through extensive manual curation, we completed a high-quality catalog. We could not find them in 202 mammals, supporting previous reports of their loss. VA opsins are highly conserved among nonmammals. In contrast, other opsin subfamilies experienced more dynamic molecular evolution with many secondary losses. In addition, we found a previously unreported opsin subfamily that we named Q113-Bistable (QB) opsin. We found its orthologs only in several lizards and the tuatara. Nevertheless, QB opsin pseudogenes were discovered in diverse taxa, including ray-finned fishes, indicating its ancient origin. QB opsin, parapinopsin, and parietopsin are extremely prone to be lost in the course of evolution, and loss events involving these opsins seem to occur concomitantly. Furthermore, we demonstrated the spectral properties of QB opsin as a UV-sensitive, bistable photo-pigment. This study provides the first integrated view of the entire evolutionary history of this group of opsins.

Most vertebrates have a rhodopsin gene with a five-exon structure for visual photoreception. By contrast, teleost fishes have an intron-less rhodopsin gene for visual photoreception and an intron-containing rhodopsin (exo-rhodopsin) gene for pineal photoreception. Here, our analysis of non-teleost and teleost fishes in various lineages of the Actinopterygii reveals that retroduplication after branching of the Polypteriformes produced the intron-less rhodopsin gene for visual photoreception, which converted the parental intron-containing rhodopsin gene into a pineal opsin in the common ancestor of the Teleostei. Additional analysis of a pineal opsin, pinopsin, shows that the pinopsin gene functions as a green-sensitive opsin together with the intron-containing rhodopsin gene for pineal photoreception in tarpon as an evolutionary intermediate state but is missing in other teleost fishes, probably because of the redundancy with the intron-containing rhodopsin gene. We propose an evolutionary scenario where unique retroduplication caused a “domino effect” on the functional diversification of teleost visual and pineal opsin genes.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods’ low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

The development of a continuous digestive tract, or through-gut, represents a key milestone in bilaterian evolution. However, the regulatory mechanisms in ancient bilaterians (urbilaterians) are not well understood. Our study, using larval sea urchins as a model, reveals a sophisticated system that prevents the simultaneous opening of the pylorus and anus, entry and exit points of the gut. This regulation is influenced by external light, with blue light affecting the pylorus via serotonergic neurons and both blue and longer wavelengths controlling the anus through cholinergic and dopaminergic neurons. These findings provide new insights into the neural orchestration of sphincter control in a simplified through-gut, which includes the esophagus, stomach, and intestine. Here, we propose that the emergence of the earliest urbilaterian through-gut was accompanied by the evolution of neural systems regulating sphincters in response to light, shedding light on the functional regulation of primordial digestive systems. The formation of the continuous digestive tract was a key feature during bilaterian evolution, but how the function of the early through-gut is regulated is not well known. Here they uncover light-modulated neural control of sphincters in sea urchin larvae, providing insights into the evolution of through-gut regulation in ancient bilaterians.

Rhodopsin is a general term for photoreceptive proteins that bind the retinal as a chromophore.Rhodopsins are classically classified into two types, animal-type opsins and microbial-type opsins [1].Although these two types share common structural elements including the seven transmembrane helical domains and a chromophore retinal, they show no sequence similarities with each other, which leads to the diversity of their molecular functions.However, recent accumulation of the molecular properties of rhodopsins has crossed the border between animal-type and microbial-type.Optogenetics is a cutting-edge technology that involves the use of light to control and manipulate the activity of genetically modified cells.The application of various rhodopsins as optogenetics tools has crossed borders between the scientific fields, and greatly contributed to the understanding of the molecular mechanisms underlying the physiological functions in animals [2].Furthermore, rhodopsin opens a new field in the gene therapy of diseases [3].To highlight recent progress in this "borderless" research, we organized a symposium during the 61st Annual Meeting of the Biophysical Society of Japan held in Nagoya in November 2023.The symposium was co-organized by the CREST Program of Japan Science and Technology Agency (JST) named "OptoBio", directed by Prof. Ryoichiro Kageyama (Director, Center for Brain Science, RIKEN).Six researchers covering biophysics, molecular cell biology, neurobiology, and ophthalmology were invited to the talk about their recent achievements.The topics of their talks are categorized into 3 groups and summarized as follows.Recent metagenomic analysis has expanded the number of rhodopsin genes.Dr. Masae Konno (University of Tokyo, Japan) introduced a new microbial rhodopsin subfamily called "bestrhodopsin" from marine unicellular algae [6].One of the surprising features is that bestrhodopsin consists of 1-2 rhodopsin domains fused with the following bestrophin domain.Cryo-EM analysis revealed a megacomplex of the bestrodopsin (~700 kDa) forming a pentameric structure in which rhodopsin domains surround the central bestrophin channel.Bestrodopsin shows a markedly red-shifted absorption maximum wavelength (λmax=661nm), and changes its structure through photoconversion between two metastable states, and works as a light-gated anion channel.Bestrhodopsin showed a unique photoconversion of the retinal chromophore from the all-trans to 11-cis form which is unusual for microbial rhodopsins and common for animal rhodopsins.

Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential activation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types.

In addition to canonical photoreception by the eye, many other organs express non-visual photoreceptors although their biological significance is mostly unknown. Here, we discovered a novel phenomenon in which the pituitary of medaka directly receives light, which induces hormone release. Ca 2+ imaging analysis revealed that a melanotroph, a pituitary endocrine cell secreting melanocyte-stimulating hormone (MSH), robustly increases [Ca 2+ ] i during short-wavelength light irradiation. Moreover, we identified Opn5m as the key molecule of this mechanism. The significance of this phenomenon was suggested to be involved in UV protection because knockout of opn5m significantly reduced the expression of tyrosinase , the rate-limiting enzyme for melanogenesis, in the skin. These results suggest a novel mechanism in which direct reception of short-wavelength light by pituitary endocrine cells triggers the pathway to enhance UV protection. One-Sentence Summary An endocrine cell of the pituitary was proven to be a photoreceptive cell that enables autonomous hormone release.

Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes that are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on the Opn4 (melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have five Opn4 genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history of Opn4 genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon, and gar) fishes. Our synteny analysis suggests that the intron-less Opn4 gene emerged by retroduplication after the branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-less Opn4 gene became abundantly used without substantial changes in the molecular properties of the Opn4 protein. This stepwise evolutionary model of Opn4 genes is quite similar to that of rhodopsin genes in the Actinopterygii. The unique acquisition of rhodopsin and Opn4 retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments.

ヒトを含む多くの脊椎動物は，明所では色を識別できるものの，暗がりでは色を識別できない。これは，明所で働く視細胞・錐体を複数種類持つ一方で，暗がりで働く視細胞・桿体を１種類しか持たないことに起因する。しかし，多くの種が夜行性であるカエルやヤモリは，「暗がりで色を識別できる」特殊な能力を持つことが古くより知られていた。これは，カエルには通常の桿体に加えて特殊なもう１つの桿体（緑桿体）が存在し，また，夜行性ヤモリには３種類の桿体が存在することに起因すると考えられていた。そして通常，桿体には光受容タンパク質・ロドプシンが含まれるが，カエルの緑桿体や夜行性ヤモリの桿体にはロドプシンは含まれず，本来は錐体の中で明所での視覚を担う錐体視物質が含まれる。しかし，錐体視物質に比べてロドプシンは，光がない時の熱活性化頻度を低下させることで暗がりでの視覚に貢献しているため，「桿体に含まれる錐体視物質は暗がりでの視覚に利用できるのか？」という課題があった。そこで私たちの研究グループは，独自に開発した生化学的解析法を駆使することで，カエルと夜行性ヤモリの錐体視物質が，ロドプシンのように熱活性化頻度を低下させていることを明らかにした。カエルと夜行性ヤモリは，収斂進化によってロドプシン様の性質を持つ特別な錐体視物質を生み出したことで，夜にカラーで周囲の状況を認識することが可能となり，自身の生活に役立てていると考えられる。

Eyes shut homolog (EYS) encodes a proteoglycan and the human mutation causes retinitis pigmentosa type 25 (RP25) with progressive retinal degeneration. RP25 most frequently affects autosomal recessive RP patients with many ethnic backgrounds. Although studies using RP models have facilitated the development of therapeutic medications, Eys has been lost in rodent model animals. Here we examined the roles for Eys in the maintenance of photoreceptor structure and function by generating eys-null medaka fish using the CRISPR-Cas9 system. Medaka EYS protein was present near the connecting cilium of wild-type photoreceptors, while it was absent from the eys−/− retina. The mutant larvae exhibited a reduced visual motor response compared with wild-type. In contrast to reported eys-deficient zebrafish at the similar stage, no retinal cell death was detected in the 8-month post-hatching (8-mph) medaka eys mutant. Immunohistochemistry showed a significant reduction in the length of cone outer segments (OSs), retention of OS proteins in the inner segments of photoreceptors, and abnormal filamentous actin network at the base of cone OSs in the mutant retina by 8 mph. Electron microscopy revealed aberrant structure of calyceal processes, numerous vesiculation and lamellar interruptions, and autophagosomes in the eys-mutant cone photoreceptors. In situ hybridization showed an autophagy component gene, gabarap, was ectopically expressed in the eys-null retina. These results suggest eys is required for regeneration of OS, especially of cone photoreceptors, and transport of OS proteins by regulating actin filaments. Enhanced autophagy may delay the progression of retinal degeneration when lacking EYS in the medaka retina.