Biology subjects

Ohuchi, H.

Publications and source records attributed to Ohuchi, H..

5 recordsLinked to original sources

bioRxiv2026

A bistable UV-sensitive opsin from a reef building coral showing a switchable and tunable regulation of Gs-signaling by different wavelengths of light

Cnidarians possess large number of opsins in their genomes for their various photoreceptive functions. In particular, they uniquely possess Gs-coupled opsins that induce intracellular cAMP accumulation in a light-dependent manner. These Gs-coupled opsins, cnidopsins, are powerful optogenetic tools manipulating cAMP-dependent cellular responses. In this study, we characterized a cnidopsin, named as AtCnidop3a, from the coral Acropora tenuis as a Gs-coupled and UV-sensitive bistable pigment. This cnidopsin showed a large spectral shift upon activation from absorption maxima from 395 nm to 560 nm, and the resting and activated states are interconvertible by illumination with UV (or violet) and orange light. The activated state efficiently activated Gs proteins and elevated intracellular cAMP levels in mammalian cultured cells. To engineer the opsin mutant that can be turned on and off upon long wavelength light illumination by utilizing the large spectral separation, negatively charged amino acids were introduced near the retinal Schiff base region. Among tested opsin mutants, the Y1133.28E mutant is capable of being activated by green light unlike the wild-type while retaining the property of being inactivated by orange light like the wild-type, indicating successful conversion of the opsin to a visible light sensitive bistable pigment. The visible light-induced cAMP regulation of the Y1133.28E mutant was enhanced by an additional L942.61G substitution. Our characterization and engineering of the cnidopsin revealed functional diversity of cnidarian opsins and its potential utility as optogenetic tools regulating Gs-dependent physiological responses.

biochemistry↗

bioRxiv2025

How opsins diversified after the teleost whole-genome duplication: Insights from two parietopsins of the red piranha, Pygocentrus nattereri

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.

evolutionary biology↗

bioRxiv2025

A safer fluorescent in situ hybridization protocol for cryosections

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

molecular biology↗

bioRxiv2025

A key spectral tuning site of UV-sensitive vertebrate non-visual opsin 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 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.

biochemistry↗

bioRxiv2023

Direct photoreception of a pituitary endocrine cell, melanotroph, induces a hormone release

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. Ca2+ imaging analysis revealed that a melanotroph, a pituitary endocrine cell secreting melanocyte-stimulating hormone (MSH), robustly increases [Ca2+]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 SummaryAn endocrine cell of the pituitary was proven to be a photoreceptive cell that enables autonomous hormone release.

physiology↗