Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.

Absorption spectra of opsin-based pigments are tuned from the UV to the red regions by interactions of the chromophore with surrounding amino acid residues. Both vertebrates and invertebrates possess long-wavelengthsensitive (LWS) opsins, which underlie color vision involving "red" sensing. The LWS opsins have independently evolved in each lineage, which suggests the existence of diverse mechanisms in spectral tuning. In vertebrate LWS opsins, the mechanisms underlying spectral tuning have been well characterized by spectroscopic analyses with recombinant pigments of wild type (WT) and mutant opsins. However in invertebrate LWS opsins including insect ones, the mechanisms are largely unknown due to the difficulty in obtaining recombinant pigments. Here we have overcome the problem by analyzing heterologous action spectra based on light-dependent changes in the second messenger in opsin-expressing cultured cells. We found that WTs of two LWS opsins of the butterfly, Papilio xuthus, PxRh3 and PxRh1 have the wavelengths of the absorption maxima at around 570 nm and 540 nm, respectively. Analysis of a series of chimeric mutants showed that helix III is crucial to generating a difference of about 15 nm in the wavelength of absorption maxima of these LWS opsins. Further site-directed mutations in helix III revealed that amino acid residues at position 116 and 120 (bovine rhodopsin numbering system) are involved in the spectral tuning of PxRh1 and PxRh3, suggesting a different spectral tuning mechanism from that of primate LWS opsins.

the velvet belly lanternshark, Etmopterus spinax, uses counterillumination to disappear in the surrounding blue light of its marine environment. this shark displays hormonally controlled bioluminescence in which melatonin (Mt) and prolactin (pRL) trigger light emission, while α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ActH) play an inhibitory role. The extraocular encephalopsin (Es-Opn3) was also hypothesized to act as a luminescence regulator. the majority of these compounds (Mt, α-MSH, ActH, opsin) are members of the rapid physiological colour change that regulates the pigment motion within chromatophores in metazoans. Interestingly, the lanternshark photophore comprises a specific iris-like structure (ILS), partially composed of melanophore-like cells, serving as a photophore shutter. Here, we investigated the role of (i) Es-Opn3 and (ii) actors involved in both Mt and α-MSH/ActH pathways on the shark bioluminescence and ILS cell pigment motions. Our results reveal the implication of Es-Opn3, MT, inositol triphosphate (ip 3), intracellular calcium, calcium-dependent calmodulin and dynein in the iLS cell pigment aggregation. conversely, our results highlighted the implication of the α-MSH/ActH pathway, involving kinesin, in the dispersion of the iLS cell pigment. the lanternshark luminescence then appears to be controlled by the balanced bidirectional motion of iLS cell pigments within the photophore. this suggests a functional link between photoreception and photoemission in the photogenic tissue of lanternsharks and gives precious insights into the bioluminescence control of these organisms. Camouflage is one of the most powerful anti-predatory tools on earth 1. By mimicking the colour of the environment background, many organisms successfully escape predation 1,2. An efficient camouflage strategy needs two essential and interconnected mechanisms: (i) an accurate sensory machinery to evaluate the environment and (ii) the genetic determination for expressing a phenotypic trait mimicking the environment or/and the capability to modulate the skin colouration to match with the background colour. Countershading, a type of camouflage strategy which consists of the gradation of colour from dark on the dorsal side to light on the ventral area, is generally considered as an efficient hiding strategy spread mainly in the marine environment 1-4. The cryptic strategy aims to facilitate the concealment of the projected shadow by the body adding a clear betterment to the organism's survival 5. This mechanism may be passive, with no colour modification during the organism life, or active, with the ability to gradually modify the skin colour to adapt the background colour (i.e. in terms of dark-grey scale or colour). Skin colour modifications need to be under fine-tuned modulation to display an efficient camouflage. Pathways controlling colour modifications involve the motion of pigmented granule (i.e. aggregation and

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G proteins. Thousands of animal opsins have been identifi ed, and molecular phylogenetic and biochemical analyses have revealed that opsin-based pigments have basically diversifi ed in selective activation of G proteins (Gs, Gq, Gi, Go, and transducin). Here, we discuss the optogenetic potentials of diverse animal opsins, particularly Gq-coupled spider opsin, Gs-coupled jellyfi sh opsin, and Gi/Go-coupled mosquito opsin 3 (Opn3). After absorbing light, these purifi ed opsin-based pigments do not release the chromophore retinal, indicating the bleach-resistant nature of their photoproducts. In addition, unlike vertebrate visual opsin-based pigments that have been conventionally used for optogenetic applications, the stable photoproducts of spider opsin-and mosquito Opn3-based pigments revert to their original dark states upon subsequent light absorption, which indicates their photoregeneration ability. Mammalian cultured cells that express spider opsin exhibit light-induced increases in Ca 2+ levels, and jellyfi sh opsin-and mosquito Opn3-expressing cells exhibit light-dependent increases and decreases in cyclic adenosine monophosphate (cAMP) levels, respectively. These fi ndings indicate that these pigments control different second messengers, Ca 2+ and cAMP, in mammalian cultured cells, suggesting that these bleach-resistant opsins have an optogenetic potential.

Background Pineal-related organs in cyclostomes, teleosts, amphibians, and reptiles exhibit color opponency, generating antagonistic neural responses to different wavelengths of light and thereby sensory information about its “color”. Our previous studies suggested that in zebrafish and iguana pineal-related organs, a single photoreceptor cell expressing both UV-sensitive parapinopsin and green-sensitive parietopsin generates color opponency in a “one-cell system.” However, it remains unknown to what degree these opsins and the single cell-based mechanism in the pineal color opponency are conserved throughout non-mammalian vertebrates. Results We found that in the lamprey pineal organ, the two opsins are conserved but that, in contrast to the situation in other vertebrate pineal-related organs, they are expressed in separate photoreceptor cells. Intracellular electrophysiological recordings demonstrated that the parietopsin-expressing photoreceptor cells with Go-type G protein evoke a depolarizing response to visible light. Additionally, spectroscopic analyses revealed that parietopsin with 11-cis 3-dehydroretinal has an absorption maximum at ~570 nm, which is in approximate agreement with the wavelength (~560 nm) that produces the maximum rate of neural firing in pineal ganglion cells exposed to visible light. The vesicular glutamate transporter is localized at both the parietopsin- and parapinopsin-expressing photoreceptor terminals, suggesting that both types of photoreceptor cells use glutamate as a transmitter. Retrograde tracing of the pineal ganglion cells revealed that the terminal of the parietopsin-expressing cells is located close enough to form a neural connection with the ganglion cells, which is similar to our previous observation for the parapinopsin-expressing photoreceptor cells and the ganglion cells. In sum, our observations point to a “two-cell system” in which parietopsin and parapinopsin, expressed separately in two different types of photoreceptor cells,  contribute to the generation of color opponency in the pineal ganglion cells. Conclusion Our results indicate that the jawless vertebrate, lamprey, employs a system for color opponency that differes from that described previously in jawed vertebrates. From a physiological viewpoint, we propose an evolutionary insight, the emergence of pineal “one-cell system” from the ancestral “multiple (two)-cell system,” showing the opposite evolutionary direction to that of the ocular color opponency.

In lower vertebrates, brain photoreceptor cells express vertebrate-specific non-visual opsins. We previously revealed that a pineal-related organ-specific opsin, parapinopsin, is UV-sensitive and allows pineal wavelength discrimination in lampreys and teleost. The Australian pouched lamprey was recently reported as having two parapinopsin-related genes. We demonstrate that a parapinopsinlike opsin from the Japanese river lamprey exhibits different molecular properties and distribution than parapinopsin. This opsin activates Gi-type G protein in a mammalian cell culture assay in a light-dependent manner. Heterologous action spectroscopy revealed that the opsin forms a violet to blue-sensitive pigment. Interestingly, the opsin is co-localised with green-sensitive P-opsin in the cells of the M5 nucleus of Schober (M5NS) in the mesencephalon of the river and brook lamprey. Some opsins-containing cells of the river lamprey have cilia and others an axon projecting to the retina. The opsins of the brook lamprey are co-localised in the cilia of centrifugal neurons projecting to the retina, suggesting that cells expressing the parapinopsin-like opsin and P-opsin are sensitive to violet to green light. Moreover, we found neural connections between M5NS cells expressing the opsins and the retina. These findings suggest that the retinal activity might be modulated by brain photoreception.

G protein-coupled receptors (GPCRs) transmit extracellular signals into the cell depending on the type of G protein. To analyze the functions of GPCR signaling, we developed optogenetic tools using animal G protein-coupled bistable rhodopsins that can be controlled into active and inactive states by light irradiation. We expressed Gq- and Gi/o-coupled bistable rhodopsins in hindbrain reticulospinal V2a neurons, which are involved in locomotion, or in cardiomyocytes of zebrafish. Light stimulation of the reticulospinal V2a neurons expressing Gq-coupled spider Rh1 resulted in an increase in the level of cytoplasmic Ca2+ and evoked swimming behavior. Light stimulation of cardiomyocytes expressing the Gi/o-coupled mosquito Opn3, pufferfish TMT opsin, or lamprey parapinopsin induced cardiac arrest, and the effect was suppressed by treatment with pertussis toxin or barium, suggesting that Gi/o-dependent regulation of inwardrectifier K+ channels controls cardiac function. These data indicate that these rhodopsins are useful for optogenetic control of GPCR-mediated signaling in neurons and cardiomyocytes in vivo.

Opsins, light-sensitive G protein-coupled receptors, have been identified in corals but their properties are largely unknown. Here, we identified six opsin genes (acropsins 1–6) from a coral species Acropora millepora, including three novel opsins (acropsins 4–6), and successfully characterized the properties of four out of the six acropsins. Acropsins 1 and 6 exhibited light-dependent cAMP increases in cultured cells, suggesting that the acropsins could light-dependently activate Gs-type G protein like the box jellyfish opsin from the same opsin group. Spectral sensitivity curves having the maximum sensitivities at ~ 472 nm and ~ 476 nm were estimated for acropsins 1 and 6, respectively, based on the light wavelength-dependent cAMP increases in these opsins-expressing cells (heterologous action spectroscopy). Acropsin 2 belonging to the same group as acropsins 1 and 6 did not induce light-dependent cAMP or Ca2+ changes. We then successfully estimated the acropsin 2 spectral sensitivity curve having its maximum value at ~ 471 nm with its chimera mutant which possessed the third cytoplasmic loop of the Gs-coupled jellyfish opsin. Acropsin 4 categorized as another group light-dependently induced intracellular Ca2+ increases but not cAMP changes. Our results uncovered that the Acropora coral possesses multiple opsins coupling two distinct cascades, cyclic nucleotide and Ca2+signaling light-dependently.