Akihisa Terakita

A Novel Go-mediated Phototransduction Cascade in Scallop Visual Cells

Scallop retinas contain ciliary photoreceptor cells that respond to light by hyperpolarization like vertebrate rods and cones, but the response is generated by a different phototransduction cascade from those of rods and cones. To elucidate the cascade, we investigated a visual pigment and a G-protein functioning in the hyperpolarizing cell. Sequencing of cDNAs andin situ hybridization experiments showed that the hyperpolarizing cells express a novel subtype of visual pigment, which showed significant differences in amino acid sequence from other visual pigments. Cloning cDNA genes of G-protein and immunohistochemical analysis revealed the presence of an alpha subunit of a Gotype G-protein, 83% identical in amino acid sequence to mammalian Go(α) in the nervous system, in the photoreceptive region of the cells. The results demonstrate that a novel, Go-mediated, phototransduction cascade is present in the hyperpolarizing cells. The phototransduction cascade in the scallop hyperpolarizing cell provides an alternative system to investigate Go-mediated transduction pathways in the nervous system. Molecular phylogenetic analysis strongly suggests that the Go-mediated phototransduction system emerged before the divergence of animals into vertebrate and invertebrate in the course of evolution.

Jellyfish vision starts with cAMP signaling mediated by opsin-Gs cascade

Light sensing starts with phototransduction in photoreceptor cells. The phototransduction cascade has diverged in different species, such as those mediated by transducin in vertebrate rods and cones, by Gq-type G protein in insect and molluscan rhabdomeric-type visual cells and vertebrate photosensitive retinal ganglion cells, and by Go-type G protein in scallop ciliary-type visual cells. Here, we investigated the phototransduction cascade of a prebilaterian box jellyfish, the most basal animal having eyes containing lens and ciliary-type visual cells similar to vertebrate eyes, to examine the similarity at the molecular level and to obtain an implication of the origin of the vertebrate phototransduction cascade. We showed that the opsin-based pigment functions as a green-sensitive visual pigment and triggers the Gs-type G protein-mediated phototransduction cascade in the ciliary-type visual cells of the box jellyfish lens eyes. We also demonstrated the light-dependent cAMP increase in the jellyfish visual cells and HEK293S cells expressing the jellyfish opsin. The first identified prebilaterian cascade was distinct from known phototransduction cascades but exhibited significant partial similarity with those in vertebrate and molluscan ciliary-type visual cells, because all involved cyclic nucleotide signaling. These similarities imply a monophyletic origin of ciliary phototransduction cascades distributed from prebilaterian to vertebrate.

Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores

Because of low contents in the native organs and failure of the expression in cultured cells, the chromophore configurations of the pigments in Go‐coupled opsin and peropsin groups in the opsin family are unknown. Here we have succeeded in expression of the amphioxus homologs of these groups in HEK293s cells and found that they can be regenerated with 11‐cis‐ and all‐trans‐retinals, respectively. Light isomerized the chromophores of these opsins into the all‐trans and 11‐cis forms, respectively. The results strongly suggest that the physiological function of peropsin would be a retinal photoisomerase, while 11‐cis configuration is necessary for the Go‐coupled opsin groups.

Distinct roles of the second and third cytoplasmic loops of bovine rhodopsin in G protein activation.

In contrast to the extensive studies of light-induced conformational changes in rhodopsin, the cytoplasmic architecture of rhodopsin related to the G protein activation and the selective recognition of G protein subtype is still unclear. Here, we prepared a set of bovine rhodopsin mutants whose cytoplasmic loops were replaced by those of other ligand-binding receptors, and we compared their ability for G protein activation in order to obtain a clue to the roles of the second and third cytoplasmic loops of rhodopsin. The mutants bearing the third loop of four other Go-coupled receptors belonging to the rhodopsin superfamily showed significant Go activation, indicating that the third loop of rhodopsin possibly has a putative site(s) related to the interaction of G protein and that it is simply exchangeable with those of other Go-coupled receptors. The mutants bearing the second loop of other receptors, however, had little ability for G protein activation, suggesting that the second loop of rhodopsin contains a specific region essential for rhodopsin to be a G protein-activating form. Systematic chimeric and point mutational studies indicate that three amino acids (Glu134, Val138, and Cys140) in the N-terminal region of the second loop of rhodopsin are crucial for efficient G protein activation. These results suggest that the second and third cytoplasmic loops of bovine rhodopsin have distinct roles in G protein activation and subtype specificity.

Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue

Most opsins selectively bind 11-cis retinal as a chromophore to form a photosensitive pigment, which underlies various physiological functions, such as vision and circadian photoentrainment. Recently, opsin 3 (Opn3), originally called encephalopsin or panopsin, and its homologs were identified in various tissues including brain, eye, and liver in both vertebrates and invertebrates, including human. Because Opn3s are mainly expressed in tissues that are not considered to contain sufficient amounts of 11-cis retinal to form pigments, the photopigment formation ability of Opn3 has been of interest. Here, we report the successful expression of Opn3 homologs, pufferfish teleost multiple tissue opsin (PufTMT) and mosquito Opn3 (MosOpn3) and show that these proteins formed functional photopigments with 11-cis and 9-cis retinals. The PufTMT- and MosOpn3-based pigments have absorption maxima in the blue-to-green region and exhibit a bistable nature. These Opn3 homolog-based pigments activate Gi-type and Go-type G proteins light dependently, indicating that they potentially serve as light-sensitive Gi/Go-coupled receptors. We also demonstrated that mammalian cultured cells transfected with the MosOpn3 or PufTMT became light sensitive without the addition of 11-cis retinal and the photosensitivity retained after the continuous light exposure, showing a reusable pigment formation with retinal endogenously contained in culture medium. Interestingly, we found that the MosOpn3 also acts as a light sensor when constituted with 13-cis retinal, a ubiquitously present retinal isomer. Our findings suggest that homologs of vertebrate Opn3 might function as photoreceptors in various tissues; furthermore, these Opn3s, particularly the mosquito homolog, could provide a promising optogenetic tool for regulating cAMP-related G protein-coupled receptor signalings.

Expression and comparative characterization of Gq-coupled invertebrate visual pigments and melanopsin.

A non‐visual pigment melanopsin, which is localized in photosensitive retinal ganglion cells and is involved in the circadian photoentrainment and pupillary responses in mammals, is phylogenetically close to the visual pigments of invertebrates, such as insects and cephalopods. Recent studies suggested that melanopsin is a bistable pigment and drives a Gq‐mediated signal transduction cascade, like the invertebrate visual pigments. Because detailed electrophysiological properties are somewhat different between the visual cells and the photosensitive ganglion cells, we here expressed and purified the invertebrate visual pigment and melanopsin to comparatively investigate their Gq‐activation abilities. We successfully expressed and purified UV and blue light‐sensitive visual pigments of the honeybee as well as the amphioxus melanopsin. Although the purified UV‐sensitive pigment and the melanopsin lost their bistable nature during purification, reconstitution of the pigments in lipid vesicles resulted in return of the bistable nature. The light‐dependent Gq‐activation abilities among these reconstituted pigments are similar, suggesting that the electrophysiological differences do not depend on the Gq‐activation step but rather on the other signal transduction steps and/or on cell properties. Our findings are also important in that this is the first report describes a heterologous large‐scale expression of the Gq‐coupled invertebrate visual pigments in cultured cells.

Depth Perception from Image Defocus in a Jumping Spider

A Good Judge of Distance Jumping spiders actively pursue their prey, often jumping relatively long distances in order to catch them. Such feats require accurate depth perception. Nagata et al. (p. 469; see the Perspective by Herberstein and Kemp) show that jumping spiders use a process called image defocus, which allows depth perception to be obtained through the comparison of a nonfocused image to a focused image within the same eye. A single layer within the spiders eye that could not focus green light nevertheless contained a green sensitive pigment. Thus, this layer always receives an unfocused image, while other layers receive images in focus. Confirming this eye arrangements role in depth perception, spiders unlucky enough to be bathed in green light nearly always jumped short of their target. To jump exact distances to capture prey, spiders, like computers, use defocus as a major cue for depth perception. The principal eyes of jumping spiders have a unique retina with four tiered photoreceptor layers, on each of which light of different wavelengths is focused by a lens with appreciable chromatic aberration. We found that all photoreceptors in both the deepest and second-deepest layers contain a green-sensitive visual pigment, although green light is only focused on the deepest layer. This mismatch indicates that the second-deepest layer always receives defocused images, which contain depth information of the scene in optical theory. Behavioral experiments revealed that depth perception in the spider was affected by the wavelength of the illuminating light, which affects the amount of defocus in the images resulting from chromatic aberration. Therefore, we propose a depth perception mechanism based on how much the retinal image is defocused.

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 476–484 nm. These values are consistent with maximal sensitivities of action spectra determined from the physiological and behavioral studies on mammalian melanopsins. The melanopsin isoforms found in this study may function as pineal circadian photoreceptors.

Gq-coupled Rhodopsin Subfamily Composed of Invertebrate Visual Pigment and Melanopsin†

Rhodopsins (rhodopsins and their related photopigments) are phylogenetically classified into at least seven subfamilies, which are also roughly discriminated by molecular function. The Gq‐coupled rhodopsin subfamily, members of which activate the Gq type G protein upon light absorption, contains pigments which underlie both visual and nonvisual physiologic functions. Gq‐coupled visual pigments have been found in the rhabdomeric photoreceptor cells of varied protostomes, and those of molluskans and arthropods have been extensively investigated. Recently, a novel photopigment, melanopsin, and its homologs have been identified in varied vertebrates. In mammals, melanopsin is localized in retinal ganglion cells and is involved in nonvisual systems, including circadian entrainment and pupillary light responses. More recently, we discovered a melanopsin homolog in amphioxus, the closest living invertebrate to vertebrates. Amphioxus melanopsin is localized in putative nonvisual photoreceptor cells with rhabdomeric morphology and exhibits molecular properties almost identical to those of invertebrate Gq‐coupled visual pigments. The localization and properties of amphioxus melanopsin bridged the functional and evolutionary gap between invertebrate Gq‐coupled visual pigments and vertebrate circadian photopigment melanopsins. Research into the Gq‐coupled rhodopsin subfamily, especially invertebrate melanopsins, will provide an opportunity to investigate the evolution of various physiologic functions, based on orthologous genes, during animal evolution.

Diversity of animal opsin-based pigments and their optogenetic potential.

Most animal opsin-based pigments are typical G protein-coupled receptors (GPCR) and consist of a protein moiety, opsin, and 11-cis retinal as a chromophore. More than several thousand opsins have been identified from a wide variety of animals, which have multiple opsin genes. Accumulated evidence reveals the molecular property of opsin-based pigments, particularly non-conventional visual pigments including non-visual pigments. Opsin-based pigments are generally a bistable pigment having two stable and photointerconvertible states and therefore are bleach-resistant and reusable, unlike vertebrate visual pigments which become bleached. The opsin family contains Gt-coupled, Gq-coupled, Go-coupled, Gs-coupled, Gi-coupled, and Gi/Go-coupled opsins, indicating the existence of a large diversity of light-driven GPCR-signaling cascades. It is suggested that these molecular properties might contribute to different physiologies. In addition, various opsin based-pigments, especially nonconventional visual pigments having different molecular characteristics would facilitate the design and development of promising optogenetic tools for modulating GPCR-signaling, which is involved in a wide variety of physiological responses. We here introduce molecular and functional properties of various kinds of opsins and discuss their physiological function and also their potentials for optogenetic applications. This article is part of a Special Issue entitled: Retinal proteins - you can teach an old dog new tricks.