Search

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the G i and G q heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways., (© 2024. The Author(s).)

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here, we show that a nonnatural retinal analog, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals., Competing Interests: Competing interests statement:The authors declare no competing interest.

Opsins play a key role in the ability to sense light both in image-forming vision and in non-visual photoreception (NVP). These modalities, in most animal phyla, share the photoreceptor protein: an opsin-based protein binding a light-sensitive chromophore by a lysine (Lys) residue. So far, visual and non-visual opsins have been discovered throughout the Metazoa phyla, including the photoresponsive Hydra , an eyeless cnidarian considered the evolutionary sister species to bilaterians. To verify whether light influences and modulates opsin gene expression in Hydra , we utilized four expression sequence tags, similar to two classic opsins (SW rhodopsin and SW blue-sensitive opsin) and two non-visual opsins (melanopsin and peropsin), in investigating the expression patterns during both diurnal and circadian time, by means of a quantitative RT-PCR. The expression levels of all four genes fluctuated along the light hours of diurnal cycle with respect to the darkness one and, in constant dark condition of the circadian cycle, they increased. The monophasic behavior in the L12:D12 cycle turned into a triphasic expression profile during the continuous darkness condition. Consequently, while the diurnal opsin-like expression revealed a close dependence on light hours, the highest transcript levels were found in darkness, leading us to novel hypothesis that in Hydra , an "internal" biological rhythm autonomously supplies the opsins expression during the circadian time. In conclusion, in Hydra , both diurnal and circadian rhythms apparently regulate the expression of the so-called visual and non-visual opsins, as already demonstrated in higher invertebrate and vertebrate species. Our data confirm that Hydra is a suitable model for studying ancestral precursor of both visual and NVP, providing useful hints on the evolution of visual and photosensory systems., (© 2024 the author(s), published by De Gruyter.)

Animal opsin is a G-protein coupled receptor (GPCR) and binds retinal as a chromophore to form a photopigment. The Opsin 5 (Opn5) group within the animal opsin family comprises a diverse array of related proteins, such as Opn5m, a protein conserved across all vertebrate lineages including mammals, and other members like Opn5L1 and Opn5L2 found in non-mammalian vertebrate genomes, and Opn6 found in non-therian vertebrate genomes, along with Opn5 homologs present in invertebrates. Although these proteins collectively constitute a single clade within the molecular phylogenetic tree of animal opsins, they exhibit markedly distinct molecular characteristics in areas such as retinal binding properties, photoreaction, and G-protein coupling specificity. Based on their molecular features, they are believed to play a significant role in physiological functions. However, our understanding of their precise physiological functions and molecular characteristics is still developing and only partially realized. Furthermore, their unique molecular characteristics of Opn5-related proteins suggest a high potential for their use as optogenetic tools through more specialized manipulations. This review intends to encapsulate our current understanding of Opn5, discuss potential manipulations of its molecular attributes, and delve into its prospective utility in the burgeoning field of animal opsin optogenetics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Rhodopsin (Rho) and cone opsins are essential for detection of light. They respond via photoisomerization, converting their Schiff-base-adducted 11-cis-retinylidene chromophores to the all-trans configuration, eliciting conformational changes to activate opsin signaling. Subsequent Schiff-base hydrolysis releases all-trans-retinal, initiating two important cycles that maintain continuous vision-the Rho photocycle and visual cycle pathway. Schiff-base hydrolysis has been thoroughly studied with photoactivated Rho but not with cone opsins. Using established methodology, we directly measured the formation of Schiff-base between retinal chromophores with mammalian visual and nonvisual opsins of the eye. Next, we determined the rate of light-induced chromophore hydrolysis. We found that retinal hydrolysis from photoactivated cone opsins was markedly faster than from photoactivated Rho. Bovine retinal G protein-coupled receptor (bRGR) displayed rapid hydrolysis of its 11-cis-retinylidene photoproduct to quickly supply 11-cis-retinal and re-bind all-trans-retinal. Hydrolysis within bRGR in native retinal pigment epithelium microsomal membranes was >6-times faster than that of bRGR purified in detergent micelles. N-terminal-targeted antibodies significantly slowed bRGR hydrolysis, while C-terminal antibodies had no effect. Our study highlights the much faster photocycle of cone opsins relative to Rho and the crucial role of RGR in chromophore recycling in daylight. By contrast, in our experimental conditions, bovine peropsin did not form pigment in the presence of all-trans-retinal nor with any mono-cis retinal isomers, leaving uncertain the role of this opsin as a light sensor., Competing Interests: Conflict of interest K. P. is a consultant for Polgenix Inc. and AbbVie Inc. and serves on the Scientific Advisory Board of Hyperion Eye Ltd. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Insect vision starts with light absorption by visual pigments based on opsins that drive Gq-type G protein-mediated phototransduction. Since Drosophila , the most studied insect in vision research, has only Gq-coupled opsins, the Gq-mediated phototransduction has been solely focused on insect vision for decades. However, genome projects on mosquitos uncovered non-canonical insect opsin genes, members of the Opn3 or c-opsin group composed of vertebrate and invertebrate non-visual opsins. Here, we report that a homologue of Opn3, MosOpn3 (Asop12) is expressed in eyes of a mosquito Anopheles stephensi . In situ hybridization analysis revealed that MosOpn3 is expressed in dorsal and ventral ommatidia, in which only R7 photoreceptor cells express MosOpn3. We also found that Asop9, a Gq-coupled visual opsin, exhibited co-localization with MosOpn3. Spectroscopic analysis revealed that Asop9 forms a blue-sensitive opsin-based pigment. Thus, the Gi/Go-coupled opsin MosOpn3, which forms a green-sensitive pigment, is co-localized with Asop9, a Gq-coupled opsin that forms a blue-sensitive visual pigment. Since these two opsin-based pigments trigger different phototransduction cascades, the R7 photoreceptors could generate complex photoresponses to blue to green light. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior., Competing Interests: Declaration of interests H. Adesnik has a patent related to this work: 3D Sparse Holographic Temporal focusing, 2016, L. Waller, N. Pegard, and H. Adesnik, Provisional Patent Application #62-429,017., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups., (© 2022. The Author(s).)

Animals have evolved light-sensitive G protein-coupled receptors, known as opsins, to detect coherent and ambient light for visual and nonvisual functions. These opsins have evolved to satisfy the particular lighting niches of the organisms that express them. While many unique patterns of evolution have been identified in mammals for rod and cone opsins, far less is known about the atypical mammalian opsins. Using genomic data from over 400 mammalian species from 22 orders, unique patterns of evolution for each mammalian opsins were identified, including photoisomerases, RGR-opsin (RGR) and peropsin (RRH), as well as atypical opsins, encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5). The results demonstrate that OPN5 and rhodopsin show extreme conservation across all mammalian lineages. The cone opsins, SWS1 and LWS, and the nonvisual opsins, OPN3 and RRH, demonstrate a moderate degree of sequence conservation relative to other opsins, with some instances of lineage-specific gene loss. Finally, the photoisomerase, RGR, and the best-studied atypical opsin, OPN4, have high sequence diversity within mammals. These conservation patterns are maintained in human populations. Importantly, all mammalian opsins retain key amino acid residues important for conjugation to retinal-based chromophores, permitting light sensitivity. These patterns of evolution are discussed along with known functions of each atypical opsin, such as in circadian or metabolic physiology, to provide insight into the observed patterns of evolutionary constraint.

Opsins (also called animal rhodopsins) are universal photoreceptive proteins that provide the molecular basis of visual and nonvisual photoreception in animals, including humans. Opsins consist of seven helical α-transmembrane domains and use retinal, a derivative of vitamin A, as a chromophore. In many opsins, light absorption triggers photo-isomerization from 11-cis retinal to all-trans retinal, resulting in activation via dynamic structural changes in the protein moiety. Activated opsins stimulate cognate trimeric G proteins to induce signal transduction cascades in cells. Recently, molecular and physiological analyses of diverse opsins have progressively advanced. This review introduces the molecular basis and physiological functions of opsins. Based on the functions of opsins, I will discuss the potential of opsins as target molecules to treat and prevent visual and nonvisual diseases such as sleep disorder and depression.

Coupling skin colour with the light/dark cycle helps regulate body temperature in ectotherms. In X. laevis, nocturnal release of melatonin from the pineal complex induces pigment aggregation and skin lightening. This nocturnal blanching is initiated by a sensor (type II opsin) that triggers melatonin release when light intensity falls below a minimum threshold, and an effector (melatonin receptor) in the skin which induces pigment aggregation. The sensor/s and effector/s belong to two families of G-protein coupled receptors that originated from a common ancestor, but diverged with subsequent evolution. The aim of this work was to identify candidate sensor/s and effector/s that regulate melatonin-mediated skin colour variation. In X. laevis, we identified a developmental time (stage 43/44) when skin lightening depends on pineal complex photosensitivity alone. At this stage, the pineal complex comprises the frontal organ and pineal gland. A total of 37 type II opsin (14 duplicated) and 6 melatonin receptor (3 duplicated) genes were identified through a full genome analysis of the allotetraploid, X. laevis. These genes were grouped into subfamilies based on their predicted amino acid sequences and the presence of specific amino acids essential for their function. The pineal complex expresses mainly blue light sensitive opsins [pinopsin, parietopsin, opn3, and melanopsins (opn4 and opn4b)] and UV-light sensitive opsins (opn5 and parapinopsin), while visual opsins and va-ancient opsin are absent, as determined by RT-PCR and in situ hybridization. The photoisomerase retinal G-protein coupled receptor, and an uncharacterized opn6b opsin, are also expressed. The spectral sensitivity that triggers melatonin secretion, and therefore melanophore aggregation, falls in the visible spectrum (470-650 ηm) and peaks in the blue/green range, pointing to the involvement of opsins with sensitivities therein. The effector-melatonin receptors expressed in skin melanophores are mtnr1a and mtnr1c. Our data point to candidate proteins required in the neuroendocrine circuit that underlies the circadian regulation of skin pigmentation, and suggest that multiple initiators and effectors likely participate., (Copyright © 2020 Elsevier B.V. All rights reserved.)

For many species, vision is one of the most important sensory modalities for mediating essential tasks that include navigation, predation and foraging, predator avoidance, and numerous social behaviors. The vertebrate visual process begins when photons of the light interact with rod and cone photoreceptors that are present in the neural retina. Vertebrate visual photopigments are housed within these photoreceptor cells and are sensitive to a wide range of wavelengths that peak within the light spectrum, the latter of which is a function of the type of chromophore used and how it interacts with specific amino acid residues found within the opsin protein sequence. Minor differences in the amino acid sequences of the opsins are known to lead to large differences in the spectral peak of absorbance (i.e. the λmax value). In our prior studies, we developed a new approach that combined homology modeling and molecular dynamics simulations to gather structural information associated with chromophore conformation, then used it to generate statistical models for the accurate prediction of λmax values for photopigments derived from Rh1 and Rh2 amino acid sequences. In the present study, we test our novel approach to predict the λmax of phylogenetically distant Sws2 cone opsins. To build a model that can predict the λmax using our approach presented in our prior studies, we selected a spectrally-diverse set of 11 teleost Sws2 photopigments for which both amino acid sequence information and experimentally measured λmax values are known. The final first-order regression model, consisting of three terms associated with chromophore conformation, was sufficient to predict the λmax of Sws2 photopigments with high accuracy. This study further highlights the breadth of our approach in reliably predicting λmax values of Sws2 cone photopigments, evolutionary-more distant from template bovine RH1, and provided mechanistic insights into the role of known spectral tuning sites., Competing Interests: The authors have declared that no competing interests exist.

Here, we report genetically encoded AviTag conjugating system for Channelrhodopsin-2(ChR2) in order to attach various nanostructures to the membrane protein in a cell type specific manner. First, AviTag peptide sequence is cloned to N-terminal site of ChR2 construct and expressed at the membrane of primary-cultured hippocampal neurons via lentiviral transduction. Second, with the help of BirA enzyme and ATP, biotin coated quantum dots (Qdots) and streptavidin (SAv) coated Qdots are successfully bound to AviTag sites at the membrane where ChR2 is located and confirmed by fluorescence imaging. Moreover, we synthesize biotinylated Traptavidin-DNA conjugate probes containing a desthio-biotin that has weaker affinity than a regular biotin, and successfully exchange them with pre-conjugated Biotin-AviTag-ChR2 site at the membrane of neuronal cells which can potentially solve the crosslinking issue of Avidin linked probes. Therefore, we expect the AviTag-ChR2 fusion platform to become a great tool for incorporating various nanostructures at the specific sites of a cellular membrane in order to overcome the limits of optogenetic opsins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Photoreceptor cells in the eyes of Bilateria are often classified into microvillar cells with rhabdomeric opsin and ciliary cells with ciliary opsin, each type having specialized molecular components and physiology. First data on the recently discovered xenopsin point towards a more complex situation in protostomes. In this study, we provide clear evidence that xenopsin enters cilia in the eye of the larval bryozoan Tricellaria inopinata and triggers phototaxis. As reported from a mollusc, we find xenopsin coexpressed with rhabdomeric-opsin in eye photoreceptor cells bearing both microvilli and cilia in larva of the annelid Malacoceros fuliginosus . This is the first organism known to have both xenopsin and ciliary opsin, showing that these opsins are not necessarily mutually exclusive. Compiling existing data, we propose that xenopsin may play an important role in many protostome eyes and provides new insights into the function, evolution, and possible plasticity of animal eye photoreceptor cells., Competing Interests: CD, SK, ST, IK, HH No competing interests declared, (© 2020, Döring et al.)

Background: Skin is the organ most extensively exposed to light of a broad range of wavelengths. Several studies have reported that skin expresses photoreceptive molecules called opsins. However, the identity and functional role of opsins in the human skin remain elusive. We aim to summarize current scientific evidence on the types of opsins expressed in the skin and their biological functions., Methods: A primary literature search was conducted using PubMed to identify articles on dermal opsins found in nonhuman animals and humans., Results: Twenty-two articles, representing, however, a non-exhaustive selection of the scientific papers published in this specific field, met the inclusion criteria. In nonhuman animals, opsins and opsin-like structures have been detected in the skin of fruit fly, zebrafish, frog, octopus, sea urchin, hogfish, and mouse, and they mediate skin color change, light avoidance, shadow reflex, and circadian photoentrainment. In humans, opsins are present in various skin cell types, including keratinocytes, melanocytes, dermal fibroblasts, and hair follicle cells. They have been shown to mediate wound healing, melanogenesis, hair growth, and skin photoaging., Conclusion: Dermal opsins have been identified across many nonhuman animals and humans. Current evidence suggests that opsins have biological significance beyond light reception. In nonhuman animals, opsins are involved in behaviors that are critical for survival. In humans, opsins are involved in various functions of the skin although the underlying molecular mechanisms remain unclear. Future investigation on elucidating the mechanism of dermal opsins will be crucial to expand the therapeutic benefits of photobiomodulation for various skin disorders., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å -1 and a valley at 0.6 Å -1 , which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å -1 peak, but no 0.6 Å -1 valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin.

Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC 50 ) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).

The loss of previously adaptive traits is typically linked to relaxation in selection, yet the molecular steps leading to such repeated losses are rarely known. Molecular studies of loss have tended to focus on gene sequences alone, but overlooking other aspects of protein expression might underestimate phenotypic diversity. Insights based almost solely on opsin gene evolution, for instance, have made mammalian color vision a textbook example of phenotypic loss. We address this gap by investigating retention and loss of opsin genes, transcripts, and proteins across ecologically diverse noctilionoid bats. We find multiple, independent losses of short-wave-sensitive opsins. Mismatches between putatively functional DNA sequences, mRNA transcripts, and proteins implicate transcriptional and post-transcriptional processes in the ongoing loss of S-opsins in some noctilionoid bats. Our results provide a snapshot of evolution in progress during phenotypic trait loss, and suggest vertebrate visual phenotypes cannot always be predicted from genotypes alone., Competing Interests: AS, KD, LY, KY, PD, BH, ED, LD, SR, KS No competing interests declared, (© 2018, Sadier et al.)

Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([ 3 H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [ 3 H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [ 3 H]PI hydrolysis increased, indicating that [ 3 H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase., (© 2018 Wang et al.)

Most stomatopod crustaceans have complex retinas in their compound eyes, with up to 16 spectral types of photoreceptors, but members of the superfamily Squilloidea have much simpler retinas, thought to contain a single photoreceptor spectral class. In the Atlantic stomatopod Squilla empusa, microspectrophotometry shows that all photoreceptors absorb light maximally at 517 nm, indicating that a single visual pigment is present in all photoreceptors in the retina. However, six distinct, but partial, long wavelength sensitive (LWS) opsin transcripts, which encode the protein component of the visual pigment, have been previously isolated through RT-PCR. In order to investigate the spectral and functional differences among S. empusa's opsins, we used RT-PCR to complete the 3' end of sequences for five of the six expressed opsins. The extended sequences spanned from the first transmembrane (TM1) helix to the 3' end of the coding region. Using homology-based modeling, we predicted the three-dimensional structure of the amino acid translation of the S. empusa opsins. Based on these analyses, S. empusa LWS opsins share a high sequence identity in TM regions and in amino acids within 15 Å of the chromophore-binding lysine on TM helix 7 (TM7), suggesting that these opsins produce spectrally similar visual pigments in agreement with previous results. However, we propose that these spectrally similar opsins differ functionally, as there are non-conservative amino acid substitutions found in intracellular loop 2 (ICL2) and TM5/ICL3, which are critical regions for G-protein binding, and substitutions in extracellular regions suggest different chromophore attachment affinities. In situ hybridization of two of the opsins (Se5 and Se6) revealed strong co-expression in all photoreceptors in both midband and peripheral regions of the retina as well as in selected ocular and cerebral ganglion neuropils. These data suggest the expression of multiple opsins-likely spectrally identical, but functionally different-in multiple types of neuronal cells in S. empusa. This suggests that the multiple opsins characteristic of other stomatopod species may have similar functional specialization.

Functionally distinct retinal ganglion cells have density and size gradients across the mouse retina, and some degenerative eye diseases follow topographic-specific gradients of cell death. Hence, the anatomical orientation of the retina with respect to the orbit and head is important for understanding the functional anatomy of the retina in both health and disease. However, different research groups use different anatomical landmarks to determine retinal orientation (dorsal, ventral, temporal, nasal poles). Variations in the accuracy and reliability in marking these landmarks during dissection may lead to discrepancies in the identification and reporting of retinal topography. The goal of this study was to compare the accuracy and reliability of the canthus, rectus muscle, and choroid fissure landmarks in reporting retinal orientation. The retinal relieving cut angle made from each landmark during dissection was calculated based on its relationship to the opsin transition zone (OTZ), determined via a custom MATLAB script that aligns retinas from immunostained s-opsin. The choroid fissure and rectus muscle landmarks were the most accurate and reliable, while burn marks using the canthus as a reference were the least. These values were used to build an anatomical map that plots various ocular landmarks in relationship to one another, to the horizontal semicircular canals, to lambda-bregma, and to the earth's horizon. Surprisingly, during normal locomotion, the mouse's opsin gradient and the horizontal semicircular canals make equivalent 6° angles aligning the OTZ near the earth's horizon, a feature which may enhance the mouse's ability to visually navigate through its environment., (© 2018 Wiley Periodicals, Inc.)

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity ( K d = 11 ± 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps.

The heterotrimeric G-protein binding site on G-protein coupled receptors remains relatively unexplored regarding its potential as a new target of therapeutic intervention or as a secondary site of action by the existing drugs. Tauroursodeoxycholic acid bears structural resemblance to several compounds that were previously identified to specifically bind to the light-activated form of the visual receptor rhodopsin and to inhibit its activation of transducin. We show that TUDCA stabilizes the active form of rhodopsin, metarhodopsin II, and does not display the detergent-like effects of common amphiphilic compounds that share the cholesterol scaffold structure, such as deoxycholic acid. Computer docking of TUDCA to the model of light-activated rhodopsin revealed that it interacts using similar mode of binding to the C-terminal domain of transducin alpha subunit. The ring regions of TUDCA made hydrophobic contacts with loop 3 region of rhodopsin, while the tail of TUDCA is exposed to solvent. The results show that TUDCA interacts specifically with rhodopsin, which may contribute to its wide-ranging effects on retina physiology and as a potential therapeutic compound for retina degenerative diseases., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Opsins are photoreceptors with important roles in reproductive regulation in birds and fishes. In the present study, we identified an opsin gene from the eyes of the oriental river prawn Macrobrachium nipponense using expressed sequence tag analysis and rapid amplification of cDNA ends. The full-length transcript contained 1382 base pairs, encoding 375 amino acids. It was classified into the long-wavelength opsin group by phylogenetic analysis, and designated Mn-LW. Mn-LW expression demonstrated significant seasonal variation in somatic tissues from both male and female prawns, with the highest expression in the eyes, and expression also shown in the ovary. The expression profiles of Mn-LW in eyes and ovary were positively related to ovarian development. In situ hybridization showed that Mn-LW was present in retinular cells in the eye and oocytes in the ovary. Injection of Mn-LW dsRNA in vivo effectively down-regulated Mn-LW expression levels compared with control levels. Mn-LW dsRNA injection also significantly reduced vitellogenin (Vg) expression, indicating a close relationship between Mn-LW and Vg in ovarian development. These results suggest that Mn-LW may play an important role in Vg synthesis and accumulation during ovarian maturation in M. nipponense., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Most opsins are G protein-coupled receptors that utilize retinal both as a ligand and as a chromophore. Opsins' main established mechanism is light-triggered activation through retinal 11-cis-to-all-trans photoisomerization. Here we report a vertebrate non-visual opsin that functions as a Gi-coupled retinal receptor that is deactivated by light and can thermally self-regenerate. This opsin, Opn5L1, binds exclusively to all-trans-retinal. More interestingly, the light-induced deactivation through retinal trans-to-cis isomerization is followed by formation of a covalent adduct between retinal and a nearby cysteine, which breaks the retinal-conjugated double bond system, probably at the C 11 position, resulting in thermal re-isomerization to all-trans-retinal. Thus, Opn5L1 acts as a reverse photoreceptor. We conclude that, like vertebrate rhodopsin, Opn5L1 is a unidirectional optical switch optimized from an ancestral bidirectional optical switch, such as invertebrate rhodopsin, to increase the S/N ratio of the signal transduction, although the direction of optimization is opposite to that of vertebrate rhodopsin.

Background: Based on evolutionary patterns of the vertebrate eye, Walls (1942) hypothesized that early placental mammals evolved primarily in nocturnal habitats. However, not only Eutheria, but all mammals show photic characteristics (i.e. dichromatic vision, rod-dominated retina) suggestive of a scotopic eye design., Results: Here, we used integrative comparative genomic and phylogenetic methodologies employing the photoreceptive opsin gene family in 154 mammals to test the likelihood of a nocturnal period in the emergence of all mammals. We showed that mammals possess genomic patterns concordant with a nocturnal ancestry. The loss of the RH2, VA, PARA, PARIE and OPN4x opsins in all mammals led us to advance a probable and most-parsimonious hypothesis of a global nocturnal bottleneck that explains the loss of these genes in the emerging lineage (> > 215.5 million years ago). In addition, ancestral character reconstruction analyses provided strong evidence that ancestral mammals possessed a nocturnal lifestyle, ultra-violet-sensitive vision, low visual acuity and low orbit convergence (i.e. panoramic vision)., Conclusions: Overall, this study provides insight into the evolutionary history of the mammalian eye while discussing important ecological aspects of the photic paleo-environments ancestral mammals have occupied.

Organisms exhibit a wide range of seasonal responses as adaptions to predictable annual changes in their environment. These changes are originally caused by the effect of the Earth's cycles around the sun and its axial tilt. Examples of seasonal responses include floration, migration, reproduction and diapause. In temperate climate zones, the most robust variable to predict seasons is the length of the day (i.e. the photoperiod). The first step to trigger photoperiodic driven responses involves measuring the duration of the light-dark phases, but the molecular clockwork performing this task is poorly characterized. Photopigments such as opsins are known to participate in light perception, being part of the machinery in charge of providing information about the luminous state of the surroundings. Aphids (Hemiptera: Aphididae) are paradigmatic photoperiodic insects, exhibiting a strong induction to diapause when the light regime mimics autumn conditions. The availability of the pea aphid (Acyrthosiphon pisum) genome has facilitated molecular approaches to understand the effect of light stimulus in the photoperiodic induction process. We have identified, experimentally validated and characterized the expression of the full opsin gene repertoire in the pea aphid. Among identified opsin genes in A. pisum, arthropsin is absent in most insects sequenced to date (except for dragonflies and two other hemipterans) but also present in a crustacean, an onychophoran and chelicerates. We have quantified the expression of these genes in aphids exposed to different photoperiodic conditions and at different times of the day and localized their transcripts in the aphid brain. Clear differences in expression patterns were found, thus relating opsin expression with the photoperiodic response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The G protein-coupled receptor opsin is a phospholipid scramblase that facilitates rapid transbilayer phospholipid exchange in liposomes. The mechanism by which opsin scrambles lipids is unknown. It has been proposed that lipid translocation may occur at protein-protein interfaces of opsin dimers. To test this possibility, we rationally engineered QUAD opsin by tryptophan substitution of four lipid-facing residues in transmembrane helix 4 (TM4) that is known to be important for dimerization. Atomistic molecular dynamics simulations of wild type and QUAD opsins combined with continuum modeling revealed that the tryptophan substitutions lower the energetically unfavorable residual hydrophobic mismatch between TM4 and the membrane, reducing the drive of QUAD opsin to dimerize. We purified thermostable wild type and QUAD opsins, with or without a SNAP tag for fluorescence labeling. Single molecule fluorescence measurements of purified SNAP-tagged constructs revealed that both proteins are monomers. Fluorescence-based activity assays indicated that QUAD opsin is a fully functional scramblase. However, unlike wild type opsin which dimerizes en route to insertion into phospholipid vesicles, QUAD opsin reconstitutes as a monomer. We conclude that an engineered opsin monomer can scramble phospholipids, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange.

Channelrhodopsins are light-gated ion channels that, via regulation of flagellar function, enable single-celled motile algae to seek ambient light conditions suitable for photosynthesis and survival. These plant behavioral responses were initially investigated more than 150 years ago. Recently, major principles of function for light-gated ion channels have been elucidated by creating channelrhodopsins with kinetics that are accelerated or slowed over orders of magnitude, by discovering and designing channelrhodopsins with altered spectral properties, by solving the high-resolution channelrhodopsin crystal structure, and by structural model-guided redesign of channelrhodopsins for altered ion selectivity. Each of these discoveries not only revealed basic principles governing the operation of light-gated ion channels, but also enabled the creation of new proteins for illuminating, via optogenetics, the fundamentals of brain function., (Copyright © 2017, American Association for the Advancement of Science.)

Ciliary opsins were classically thought to function only in vertebrates for vision, but they have also been identified recently in invertebrates for non-visual photoreception. Larvae of the annelid Platynereis dumerilii are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. Platynereis c-opsin is suggested to relay light signals for melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades and that it can directly bind exogenous all- trans -retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require a supply of 11- cis -retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all- trans- retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11- cis -retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the Platynereis brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of Platynereis to enable detection of ambient UV light. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phototransduction is initiated when the absorption of light converts the 11- cis -retinal chromophore to its all- trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11- cis -retinal is continuously regenerated from its all- trans conformation through a series of enzymatic steps comprising the "visual or retinoid" cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11- cis -retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11- cis -6-membered ring (11- cis -6mr)-retinal, which has therapeutic effects against certain degenerative retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11- cis -6mr-retinal and demonstrated that this retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro-205 in green cone opsin could prevent entry and binding of 11- cis -6mr-retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11- cis -6mr-retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11- cis -6mr-retinal. These results and our structural modeling indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11- cis -6mr-retinal., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins., Competing Interests: The authors declare no conflict of interest.

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin., Competing Interests: The authors declare no conflict of interest.

Understanding the link between how proteins function in animals that live in extreme environments and selection on specific properties of amino acids has proved extremely challenging. Here we present the discovery of how the compressibility of opsin proteins in two evolutionarily distinct animal groups, teleosts and cephalopods, appears to be adapted to the high-pressure environment of the deep-sea. We report how in both groups, opsins in deeper living species are calculated to be less compressible. This is largely due to a common set of amino acid sites (bovRH# 159, 196, 213, 275) undergoing positive destabilizing selection in six of the twelve amino acid physiochemical properties that determine protein compressibility. This suggests a common evolutionary mechanism to reduce the adiabatic compressibility of opsin proteins. Intriguingly, the sites under selection are on the proteins' outer faces at locations known to be involved in opsin-opsin dimer interactions., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Background: Opsins are the only class of proteins used for light perception in image-forming eyes. Gene duplication and subsequent functional divergence of opsins have played an important role in expanding photoreceptive capabilities of organisms by altering what wavelengths of light are absorbed by photoreceptors (spectral tuning). However, new opsin copies may also acquire novel function or subdivide ancestral functions through changes to temporal, spatial or the level of gene expression. Here, we test how opsin gene copies diversify in function and evolutionary fate by characterizing four rhabdomeric (G q -protein coupled) opsins in the scallop, Argopecten irradians, identified from tissue-specific transcriptomes., Results: Under a phylogenetic analysis, we recovered a pattern consistent with two rounds of duplication that generated the genetic diversity of scallop G q -opsins. We found strong support for differential expression of paralogous G q -opsins across ocular and extra-ocular photosensitive tissues, suggesting that scallop G q -opsins are used in different biological contexts due to molecular alternations outside and within the protein-coding regions. Finally, we used available protein models to predict which amino acid residues interact with the light-absorbing chromophore. Variation in these residues suggests that the four G q -opsin paralogs absorb different wavelengths of light., Conclusions: Our results uncover novel genetic and functional diversity in the light-sensing structures of the scallop, demonstrating the complicated nature of G q -opsin diversification after gene duplication. Our results highlight a change in the nearly ubiquitous shadow response in molluscs to a narrowed functional specificity for visual processes in the eyed scallop. Our findings provide a starting point to study how gene duplication may coincide with eye evolution, and more specifically, different ways neofunctionalization of G q -opsins may occur.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish., Competing Interests: The authors have declared that no competing interests exist.

It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin(+)Lrat(-/-)) expressing, transgenically, short-wavelength-sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor's outer segment. In R-opsin's absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin's presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptide-competition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling.

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

Background: Arthropods have received much attention as a model for studying opsin evolution in invertebrates. Yet, relatively few studies have investigated the diversity of opsin proteins that underlie spectral sensitivity of the visual pigments within the diverse beetles (Insecta: Coleoptera). Previous work has demonstrated that beetles appear to lack the short-wavelength-sensitive (SWS) opsin class that typically confers sensitivity to the "blue" region of the light spectrum. However, this is contrary to established physiological data in a number of Coleoptera. To explore potential adaptations at the molecular level that may compensate for the loss of the SWS opsin, we carried out an exploration of the opsin proteins within a group of beetles (Buprestidae) where short-wave sensitivity has been demonstrated. RNA-seq data were generated to identify opsin proteins from nine taxa comprising six buprestid species (including three male/female pairs) across four subfamilies. Structural analyses of recovered opsins were conducted and compared to opsin sequences in other insects across the main opsin classes-ultraviolet, short-wavelength, and long-wavelength., Results: All nine buprestids were found to express two opsin copies in each of the ultraviolet and long-wavelength classes, contrary to the single copies recovered in all other molecular studies of adult beetle opsin expression. No SWS opsin class was recovered. Furthermore, the male Agrilus planipennis (emerald ash borer-EAB) expressed a third LWS opsin at low levels that is presumed to be a larval copy. Subsequent homology and structural analyses identified multiple amino acid substitutions in the UVS and LWS copies that could confer short-wavelength sensitivity., Conclusions: This work is the first to compare expressed opsin genes against known electrophysiological data that demonstrate multiple peak sensitivities in Coleoptera. We report the first instance of opsin duplication in adult beetles, which occurs in both the UVS and LWS opsin classes. Through structural comparisons of known insect opsins, we suggest that opsin duplication and amino acid variation within the chromophore binding pocket explains sensitivity in the short-wavelength portion of the visible light spectrum in these species. These findings are the first to reveal molecular complexity of the color vision system within beetles.

The biophysical mechanism of the sense of smell, or olfaction, is still highly debated. The mainstream explanation argues for a shape-based recognition of odorant molecules by olfactory receptors, while recent investigations suggest the primary olfactory event to be triggered by a vibrationally-assisted electron transfer reaction. We consider this controversy by studying the influence of a receptor on the vibrational properties of an odorant in atomistic details as the coupling between electronic degrees of freedom of the receptor and the vibrations of the odorant is the key parameter of the vibrationally-assisted electron transfer. Through molecular dynamics simulations we elucidate the binding specificity of a receptor towards acetophenone odorant. The vibrational properties of acetophenone inside the receptor are then studied by the polarizable embedding density functional theory approach, allowing to quantify protein-odorant interactions. Finally, we judge whether the effects of the protein provide any indications towards the existing theories of olfaction.

A series of vitamin-A aldehydes (retinals) with modified alkyl group substituents (9-demethyl-, 9-ethyl-, 9-isopropyl-, 10-methyl, 10-methyl-13-demethyl-, and 13-demethyl retinal) was synthesized and their 11-cis isomers were used as chromophores to reconstitute the visual pigment rhodopsin. Structural changes were selectively introduced around the photoisomerizing C11=C12 bond. The effect of these structural changes on rhodopsin formation and bleaching was determined. Global fit of assembly kinetics yielded lifetimes and spectral features of the assembly intermediates. Rhodopsin formation proceeds stepwise with prolonged lifetimes especially for 9-demethyl retinal (longest lifetime τ3 = 7500 s, cf., 3500 s for retinal), and for 10-methyl retinal (τ3 = 7850 s). These slowed-down processes are interpreted as either a loss of fixation (9dm) or an increased steric hindrance (10me) during the conformational adjustment within the protein. Combined quantum mechanics and molecular mechanics (QM/MM) simulations provided structural insight into the retinal analogues-assembled, full-length rhodopsins. Extinction coefficients, quantum yields and kinetics of the bleaching process (μs-to-ms time range) were determined. Global fit analysis yielded lifetimes and spectral features of bleaching intermediates, revealing remarkably altered kinetics: whereas the slowest process of wild-type rhodopsin and of bleached and 11-cis retinal assembled rhodopsin takes place with lifetimes of 7 and 3.8 s, respectively, this process for 10-methyl-13-demethyl retinal was nearly 10 h (34670 s), coming to completion only after ca. 50 h. The structural changes in retinal derivatives clearly identify the precise interactions between chromophore and protein during the light-induced changes that yield the outstanding efficiency of rhodopsin.

Coral reefs are one of the most spectrally diverse environments, both in terms of habitat and animal color. Species identity, sex, and camouflage are drivers of the phenotypic diversity seen in coral reef fishes, but how the phenotypic diversity is reflected in the genotype remains to be answered. The labrids are a large, polyphyletic family of coral reef fishes that display a diverse range of colors, including developmental color morphs and extensive behavioral ecologies. Here, we assess the opsin sequence and expression diversity among labrids from the Great Barrier Reef, Australia. We found that labrids express a diverse palette of visual opsins, with gene duplications in both RH2 and LWS genes. The majority of opsins expressed were within the mid-to-long wavelength sensitive classes (RH2 and LWS). Three of the labrid species expressed SWS1 (ultra-violet sensitive) opsins with the majority expressing the violet-sensitive SWS2B gene and none expressing SWS2A. We used knowledge about spectral tuning sites to calculate approximate spectral sensitivities (λmax) for individual species' visual pigments, which corresponded well with previously published λmax values for closely related species (SWS1: 356-370 nm; SWS2B: 421-451 nm; RH2B: 452-492 nm; RH2A: 516-528 nm; LWS1: 554-555 nm; LWS2: 561-562 nm). In contrast to the phenotypic diversity displayed via color patterns and feeding ecology, there was little amino acid diversity within the known opsin sequence tuning sites. However, gene duplications and differential expression provide alternative mechanisms for tuning visual pigments, resulting in variable visual sensitivities among labrid species., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rhodopsin, the mammalian dim light photoreceptor, is the canonical model for G protein-coupled receptors. Activation of rhodopsin occurs when the covalently bound inverse agonist, retinal, absorbs a photon and undergoes an 11-cis to all-trans isomerization. Two critical components of the visual cycle occur with the (1) hydrolytic release of all-trans retinaldehyde and subsequent (2) uptake of 11-cis retinaldehyde to reform the Schiff base linkage in the apoprotein opsin. Two pores on the surface of opsin are connected via the retinal channel, as discovered upon solution of the X-ray crystal structure (Park et al., Nature, 2008), and could serve as potential entryways for uptake and release. Using molecular dynamics simulations, we examined the behavior of rhodopsin in the Meta-II conformation (active) under Meta-I conditions (inactive), and discovered that the retinal binding pocket is flexible enough to allow a 180° rotation along the long axis of the retinal polyene chain. This result reconciles a discrepancy between the known polyene chain orientation from crystallographic and spectroscopic studies and opens the door for further investigation into the intermolecular interactions between the retinal ligand and the apoprotein opsin. Subsequent docking studies of both isomers of retinal into the opsin channel were then conducted to identify the mechanism for uptake and release. Our results suggest that retinal undergoes unidirectional uptake through Pore A and release through Pore B, and that aromatic sidechain interactions play a key role in stabilizing retinal within the opsin channel. These findings are significant in developing our understanding of the retinoid cycle and how ligand-receptor interactions in rhodopsin relate to G protein-coupled receptor activation.

Opn3/TMT opsins belong to one of the opsin groups with vertebrate visual and non-visual opsins, and are widely distributed in eyes, brains and other internal organs in various vertebrates and invertebrates. Vertebrate Opn3/TMT opsins are further classified into four groups on the basis of their amino acid identities. However, there is limited information about molecular properties of these groups, due to the difficulty in preparing the recombinant proteins. Here, we successfully expressed recombinant proteins of TMT1 and TMT2 opsins of medaka fish (Oryzias latipes) in cultured cells and characterized their molecular properties. Spectroscopic and biochemical studies demonstrated that TMT1 and TMT2 opsins functioned as blue light-sensitive Gi/Go-coupled receptors, but exhibited spectral properties and photo-convertibility of the active state different from each other. TMT1 opsin forms a visible light-absorbing active state containing all-trans-retinal, which can be photo-converted to 7-cis- and 9-cis-retinal states in addition to the original 11-cis-retinal state. In contrast, the active state of TMT2 opsin is a UV light-absorbing state having all-trans-retinal and does not photo-convert to any other state, including the original 11-cis-retinal state. Thus, TMT opsins are diversified so as to form a different type of active state, which may be responsible for their different functions.

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself., (Copyright © 2015 Federation of European Biochemical Societies. All rights reserved.)

Birds time their activities in synchronization with daily and seasonal periodicities in the environment, which is mainly provided by changes in day length (=photoperiod). Photoperiod appears to act at different levels than simply entraining the hypothalamic clock via eyes in birds. Photoreceptor cells that transmit light information to an avian brain are localized in three independent structures, the retina of eyes, pineal gland and hypothalamus, particularly in the paraventricular organ and lateral septal area. These hypothalamic photoreceptors are commonly referred to as encephalic or deep brain photoreceptors, DBPs. Eyes and pineal are known to contribute to the circadian regulation of behavior and physiology via rhythmic melatonin secretion in several birds. DBPs have been implicated in the regulation of seasonal physiology, particularly in photoperiod induced gonadal growth and development. Here, we briefly review limited evidence that is available on the roles of these photoreceptors in the regulation of circadian and seasonal physiology, with particular emphasis placed on the DBPs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Over the past 10 years, the development and convergence of microbial opsin engineering, modular genetic methods for cell-type targeting and optical strategies for guiding light through tissue have enabled versatile optical control of defined cells in living systems, defining modern optogenetics. Despite widespread recognition of the importance of spatiotemporally precise causal control over cellular signaling, for nearly the first half (2005-2009) of this 10-year period, as optogenetics was being created, there were difficulties in implementation, few publications and limited biological findings. In contrast, the ensuing years have witnessed a substantial acceleration in the application domain, with the publication of thousands of discoveries and insights into the function of nervous systems and beyond. This Historical Commentary reflects on the scientific landscape of this decade-long transition.