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Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

Fishes often have cone photoreceptors organized in lattice-like mosaic formations. In flatfishes, these lattices undergo dramatic changes during metamorphosis whereby a honeycomb mosaic of single cones in the larva is replaced by a square mosaic of single and double cones in the adult. The spatio-temporal dynamics of this transition are not well understood. Here, we describe the photoreceptors and mosaic formations that occur during the larva to juvenile transition of Atlantic halibut from the beginning of eye migration to its completion. To gauge the possibility of colour vision, visual pigments in juveniles were measured by microspectrophotometry and the opsin repertoire explored using bioinformatics. At the start of eye migration, the larva had a heterogeneous retina with honeycomb mosaic in the dorsonasal and ventrotemporal quadrants and a square mosaic in the ventronasal and dorsotemporal quadrants. By the end of metamorphosis, the square mosaic was present throughout the retina except in a centrodorsotemporal area where single, double and triple cones occurred randomly. Six cone visual pigments were found with maximum absorbance (λmax, in nm) in the short [S(431) and S(457)], middle [M(500), M(514) and M(527)], and long [L(550)] wavelengths, and a rod visual pigment with λmax at 491 nm. These pigments only partially matched the opsin repertoire detected by query of the Atlantic halibut genome. We conclude that the Atlantic halibut undergoes a complex re-organization of photoreceptors at metamorphosis resulting in a multi-mosaic retina adapted for a demersal life style.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Optical stimulation is a paradigm-shifting approach to modulating neural activity that has the potential to overcome the issue of current spread that occurs with electrical stimulation by providing focused stimuli. But optical stimulation either requires high power infrared light or genetic modification of neurons to make them responsive to lower power visible light. This work examines optical activation of auditory neurons following optogenetic modification via AAV injection in two species (mouse and guinea pig). An Anc80 viral vector was used to express the channelrhodopsin variant ChR2-H134R fused to a fluorescent reporter gene under the control of the human synapsin-1 promoter. The AAV was administered directly to the cochlea (n = 33) or posterior semi-circular canal of C57BL/6 mice (n = 4) or to guinea pig cochleae (n = 6). Light (488 nm), electrical stimuli or the combination of these (hybrid stimulation) was delivered to the cochlea via a laser-coupled optical fibre and co-located platinum wire. Activation thresholds, spread of activation and stimulus interactions were obtained from multi-unit recordings from the central nucleus of the inferior colliculus of injected mice, as well as ChR2-H134R transgenic mice (n = 4). Expression of ChR2-H134R was examined by histology. In the mouse, transduction of auditory neurons by the Anc80 viral vector was most successful when injected at a neonatal age with up to 89% of neurons transduced. Auditory neuron transductions were not successful in guinea pigs. Inferior colliculus responses to optical stimuli were detected in a cochleotopic manner in all mice with ChR2-H134R expression. There was a significant correlation between lower activation thresholds in mice and higher proportions of transduced neurons. There was no difference in spread of activation between optical stimulation and electrical stimulation provided by the light/electrical delivery system used here (optical fibre with bonded 25 µm platinum/iridium wire). Hybrid stimulation, comprised of sub-threshold optical stimulation to ‘prime’ or raise the excitability of the neurons, lowered the threshold for electrical activation in most cases, but the impact on excitation width was more variable compared to transgenic mice. This study demonstrates the impact of opsin expression levels and expression pattern on optical and hybrid stimulation when considering optical or hybrid stimulation techniques for neuromodulation.

The velvet belly lanternshark, Etmopterus spinax, uses counterillumination to disappear in the surrounding blue light of its marine environment. This shark displays hormonally controlled bioluminescence in which melatonin (MT) and prolactin (PRL) trigger light emission, while α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) play an inhibitory role. The extraocular encephalopsin (Es-Opn3) was also hypothesized to act as a luminescence regulator. The majority of these compounds (MT, α-MSH, ACTH, opsin) are members of the rapid physiological colour change that regulates the pigment motion within chromatophores in metazoans. Interestingly, the lanternshark photophore comprises a specific iris-like structure (ILS), partially composed of melanophore-like cells, serving as a photophore shutter. Here, we investigated the role of (i) Es-Opn3 and (ii) actors involved in both MT and α-MSH/ACTH pathways on the shark bioluminescence and ILS cell pigment motions. Our results reveal the implication of Es-Opn3, MT, inositol triphosphate (IP3), intracellular calcium, calcium-dependent calmodulin and dynein in the ILS cell pigment aggregation. Conversely, our results highlighted the implication of the α-MSH/ACTH pathway, involving kinesin, in the dispersion of the ILS cell pigment. The lanternshark luminescence then appears to be controlled by the balanced bidirectional motion of ILS cell pigments within the photophore. This suggests a functional link between photoreception and photoemission in the photogenic tissue of lanternsharks and gives precious insights into the bioluminescence control of these organisms.

【概要】下等な脊椎動物は脳内でも光を受容し、脳内の光受容細胞には視覚以外の機能に関わる光受容タンパク質(非視覚オプシン)が発現しています。これまでに、非視覚オプシンの一種であるパラピノプシンは紫外線に感度が高く、ヤツメウナギや硬骨魚類において、松果体と呼ばれる脳器官での波長(色)識別を担うことを見出し、脳内光受容のカギとなる非視覚オプシンであると考えられました。この論文では、ヤツメウナギにおいて、最近見出されたパラピノプシンに類似したオプシンが、パラピノプシンとは異なる分子特性と脳内分布を示すことを明らかにしました。このパラピノプシン類似オプシンは、紫から青に対して高い感度を持ち、効率的に光情報を伝達することが明らかになりました。興味深いことに、2種類の日本産ヤツメウナギでは、このオプシンは、中脳のシェーバーのM5核と呼ばれる脳深部領域に発現し、緑色光に高い感度を持つ別のオプシンと同じ細胞に共局在していました。つまり、この脳領域が紫色から緑色の光の受容に関わると考えられました。さらに、これらのオプシンを発現する脳内光受容細胞と網膜との間に神経接続が認められたことから、網膜の機能が脳の光受容によって制御されている可能性を示唆する初めての発見と言えます。, In lower vertebrates, brain photoreceptor cells express vertebrate-specific non-visual opsins. We previously revealed that a pineal-related organ-specific opsin, parapinopsin, is UV-sensitive and allows pineal wavelength discrimination in lampreys and teleost. The Australian pouched lamprey was recently reported as having two parapinopsin-related genes. We demonstrate that a parapinopsin-like opsin from the Japanese river lamprey exhibits different molecular properties and distribution than parapinopsin. This opsin activates Gi-type G protein in a mammalian cell culture assay in a light-dependent manner. Heterologous action spectroscopy revealed that the opsin forms a violet to blue-sensitive pigment. Interestingly, the opsin is co-localised with green-sensitive P-opsin in the cells of the M5 nucleus of Schober (M5NS) in the mesencephalon of the river and brook lamprey. Some opsins-containing cells of the river lamprey have cilia and others an axon projecting to the retina. The opsins of the brook lamprey are co-localised in the cilia of centrifugal neurons projecting to the retina, suggesting that cells expressing the parapinopsin-like opsin and P-opsin are sensitive to violet to green light. Moreover, we found neural connections between M5NS cells expressing the opsins and the retina. These findings suggest that the retinal activity might be modulated by brain photoreception.

The currently unsurpassed diversity of photoreceptors found in the eyes of stomatopods, or mantis shrimps, is achieved through a variety of opsin-based visual pigments and optical filters. However, the presence of extraocular photoreceptors in these crustaceans is undescribed. Opsins have been found in extraocular tissues across animal taxa, but their functions are often unknown. Here, we show that the mantis shrimp Neogonodactylus oerstedii has functional cerebral photoreceptors, which expands the suite of mechanisms by which mantis shrimp sense light. Illumination of extraocular photoreceptors elicits behaviors akin to common arthropod escape responses, which persist in blinded individuals. The anterior central nervous system, which is illuminated when a mantis shrimp’s cephalothorax protrudes from its burrow to search for predators, prey, or mates, appears to be photosensitive and to feature two types of opsin-based, potentially histaminergic photoreceptors. A pigmented ventral eye that may be capable of color discrimination extends from the cerebral ganglion, or brain, against the transparent outer carapace, and exhibits a rapid electrical response when illuminated. Additionally, opsins and histamine are expressed in several locations of the eyestalks and cerebral ganglion, where any photoresponses could contribute to shelter-seeking behaviors and other functions.

Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.

Transposable elements (TEs) are able to jump to new locations (transposition) in the genome, usually after replication. They constitute the so-called selfish or junk DNA and take over large proportions of some genomes. Due to their ability to move around they can change the DNA landscape of genomes and are therefore a rich source of innovation in genes and gene regulation. Surge of sequence data in the past years has significantly facilitated large scale comparative studies. Cephalochordates have been regarded as a useful proxy to ancestral chordate condition partially due to the comparatively slow evolutionary rate at morphological and genomic level. In this study, we used opsin gene family from three Branchiostoma species as a window into cephalochordate genome evolution. We compared opsin complements in terms of family size, gene structure and sequence allowing us to identify gene duplication and gene loss events. Furthermore, analysis of the opsin containing genomic loci showed that they are populated by TEs. In summary, we provide evidence of the way transposable elements may have contributed to the evolution of opsin gene family and to the shaping of cephalochordate genomes in general.

Chlorophyll derivatives are known to enhance vision in vertebrates. They are thought to bind visual pigments (i.e., opsins apoproteins bound to retinal chromophores) directly within the retina. Consistent with previous findings in vertebrates, here we show that chlorin e6 — a chlorophyll derivative — enhances photophobicity in a flatworm (Dugesia japonica), specifically when exposed to UV radiation (λ = 405 nm) or red light (λ = 660 nm). This is the first report of chlorophyll derivatives acting as modulators of invertebrate phototaxis, and in general the first account demonstrating that they can artificially alter animal response to light at a behavioral level. Our findings show that the interaction between chlorophyll derivatives and opsins virtually concerns the vast majority of bilaterian animals, and also occurs in visual systems based on rhabdomeric (rather than ciliary) opsins.

Optogenetics offers unprecedented possibilities to investigate cortical networks. Yet, the number of successful optogenetic applications in non-human primates is still low, and the consequences of opsin expression in the primate brain are not well documented. We assessed histologically if we can target cerebrocortical networks with three common optogenetic constructs (AAV2/5-CaMKIIα-eNpHR3.0-mCherry, -ChR2-eYFP, -C1V1-mCherry). The frontal eye field or the dorsal premotor area of rhesus macaques were virally injected, and the resulting transduction spread, expression specificity, and opsin trafficking into axons projecting to parietal and visual areas were examined. After variable periods (2–24 months), expression was robust for all constructs at the injection sites. The CaMKIIα promoter driven-expression was predominant, but not exclusive, in excitatory neurons. In the case of eNpHR3.0-mCherry and ChR2-eYFP, opsins were present in axonal projections to target areas, in which sparse, retrogradely transduced neurons could also be found. Finally, the intracellular distribution of opsins differed: ChR2-eYFP had almost exclusive membrane localization, while eNpHR3.0-mCherry and C1V1-mCherry showed additional intracellular accumulations, which might affect neuronal survival in the long-term. Results indicate that all three constructs can be used for local neuronal modulation, but axonal stimulation and long-term use require additional considerations of construct selection and verification.

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 (IC50) 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)., Scientific Reports, 9, ISSN:2045-2322

Expression of nuptial color is usually energetically costly, and is therefore regarded as an ‘honest signal’ to reflect mate quality. In order to choose a mate with high quality, both sexes may benefit from the ability to precisely evaluate their mates through optimizing visual systems which is in turn partially regulated by opsin gene modification. However, how terrestrial vertebrates regulate their color vision sensitivity is poorly studied. The green-spotted grass lizard Takydromus viridipunctatus is a sexually dimorphic lizard in which males exhibit prominent green lateral colors in the breeding season. In order to clarify relationships among male coloration, female preference, and chromatic visual sensitivity, we conducted testosterone manipulation with mate choice experiments, and evaluated the change of opsin gene expression from different testosterone treatments and different seasons. The results indicated that males with testosterone supplementation showed a significant increase in nuptial color coverage, and were preferred by females in mate choice experiments. By using quantitative PCR (qPCR), we also found that higher levels of testosterone may lead to an increase in rhodopsin-like 2 (rh2) and a decrease in long-wavelength sensitive (lws) gene expression in males, a pattern which was also observed in wild males undergoing maturation as they approached the breeding season. In contrast, females showed the opposite pattern, with increased lws and decreased rh2 expression in the breeding season. We suggest this alteration may facilitate the ability of male lizards to more effectively evaluate color cues, and also may provide females with the ability to more effectively evaluate the brightness of potential mates. Our findings suggest that both sexes of this chromatically dimorphic lizard regulate their opsin expression seasonally, which might play an important role in the evolution of nuptial coloration.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in signal transduction. Cilia are anchored inside the cell through basal bodies (BBs), modified centrioles also acting as microtubule-organization centers. Photoreceptors (PRs) are sensory neurons, whose primary cilium forms a highly specialized compartment called the outer segment (OS) responsible for sensing incoming light. Thus, ciliopathies often present with retinal degeneration. Mutations in KIAA0586/TALPID3 (TA3) cause Joubert syndrome, in which 30% of affected individuals develop retinal involvement. To elucidate the function of TALPID3 in PRs, we studied talpid3 zebrafish mutants and identified a progressive retinal degeneration phenotype. The majority of PRs lack OS development due to defects in BB positioning and docking at the apical cell surface. Intracellular accumulation of the photopigment opsin leads to PR cell death of moderate severity. Electroretinograms demonstrate severe visual impairement. A small subset of PRs display normally docked BBs and extended OSs through rescue by maternally-deposited Talpid3. While localization of the small GTPase Rab8a, which plays an important role in BB docking, appears unaffected in talpid3-/- PRs, overexpression of constitutively active Rab8a rescues OS formation, indicating that the role of Ta3 in early ciliogenesis lies upstream of Rab8a activation in PRs.

Biased mating due to female preferences towards certain traits in males is a major mechanism driving sexual selection, and may constitute an important evolutionary force in organisms with sexual reproduction. In birds, although the role of male ornamentation, plumage coloration, genetic dissimilarity, and body size have on mate selection by females have been examined extensively, few studies have clarified exactly how these characteristics affect female mate preferences. Here, we show that testosterone (T)-dependent male attractiveness enhances female preference for males of a polygamous species, the Japanese quail. A significant positive correlation between female mating preference and circulating T in the male was observed. The cheek feathers of attractive males contained higher levels of melanin and were more brightly colored. The ability of females to distinguish attractive males from other males was negated when the light source was covered with a sharp cut filter (cutoff

Optogenetic tools have opened a rich experimental landscape for understanding neural function and disease. Here, we present the first validation of eight optogenetic constructs driven by recombinant adeno-associated virus (AAV) vectors and a WGA-Cre based dual injection strategy for projection targeting in a widely-used New World primate model, the common squirrel monkey Saimiri sciureus. We observed opsin expression around the local injection site and in axonal projections to downstream regions, as well as transduction to thalamic neurons, resembling expression patterns observed in macaques. Optical stimulation drove strong, reliable excitatory responses in local neural populations for two depolarizing opsins in anesthetized monkeys. Finally, we observed continued, healthy opsin expression for at least one year. These data suggest that optogenetic tools can be readily applied in squirrel monkeys, an important first step in enabling precise, targeted manipulation of neural circuits in these highly trainable, cognitively sophisticated animals. In conjunction with similar approaches in macaques and marmosets, optogenetic manipulation of neural circuits in squirrel monkeys will provide functional, comparative insights into neural circuits which subserve dextrous motor control as well as other adaptive behaviors across the primate lineage. Additionally, development of these tools in squirrel monkeys, a well-established model system for several human neurological diseases, can aid in identifying novel treatment strategies.

Variable expression of visual pigment proteins (opsins) in cone photoreceptors of the vertebrate retina is a primary determinant of vision plasticity. Switches in opsin expression or variable co-expression of opsins within differentiated cones have been documented for a few rodents and fishes, but the extent of photoreceptor types affected and potential functional significance are largely unknown. Here, we show that both single and double cones in the retina of a flatfish, the starry flounder (Platichthys stellatus), undergo visual pigment changes through opsin switches or variable opsin co-expression. As the post-metamorphic juvenile (i.e., the young asymmetric flatfish with both eyes on one side of the body) grows from ~5 g to ~196 g, some single cones and one member of unequal double cones switched from a visual pigment with maximum wavelength of absorbance, λmax, at shorter wavelengths (437 nm and 527 nm) to one with longer λmax (456 nm and 545 nm, respectively) whereas other cones had intermediate visual pigments (λmax at 445 nm or 536 nm) suggesting co-expression of two opsins. The shift toward longer wavelength absorbing visual pigments was in line with maximizing sensitivity to the restricted light spectrum at greater depths and achromatic detection of overhead targets.

Cones are responsible for daylight, central, high acuity and color vision. Three proteins found in human cones, i.e. long-wavelength (L)-, middle-wavelength (M)-, and short-wavelength sensitive (S)-opsins, are responsible for red, green and blue color recognition, respectively. Human blue cone monochromacy (BCM) is characterized by functional loss of both L- and M-cone opsins due to mutations in the OPN1LW/OPN1MW gene cluster on the X chromosome. BCM patients, who rely on their vision from only S-cones and rods, suffer severely reduced visual acuity and impaired color vision. Recent studies show that there is sufficient cone structure remaining in the central fovea of BCM patients to consider AAV-mediated gene augmentation therapy. In contrast, mouse retina has only two opsins, S-opsin and M-opsin, but no L-opsin. We generated an M-opsin knockout mouse (Opn1mw

Dissecting neural circuitry in non-human primates (NHP) is crucial to identify potential neuromodulation anatomical targets for the treatment of pharmacoresistant neuropsychiatric diseases by electrical neuromodulation. How targets of deep brain stimulation (DBS) and cortical targets of transcranial magnetic stimulation (TMS) compare and might complement one another is an important question. Combining optogenetics and tractography may enable anatomo-functional characterization of large brain cortico-subcortical neural pathways. For the proof-of-concept this approach was used in the NHP brain to characterize the motor cortico-subthalamic pathway (m_CSP) which might be involved in DBS action mechanism in Parkinson’s disease (PD). Rabies-G-pseudotyped and Rabies-G-VSVg-pseudotyped EIAV lentiviral vectors encoding the opsin ChR2 gene were stereotaxically injected into the subthalamic nucleus (STN) and were retrogradely transported to the layer of the motor cortex projecting to STN. A precise anatomical mapping of this pathway was then performed using histology-guided high angular resolution MRI tractography guiding accurately cortical photostimulation of m_CSP origins. Photoexcitation of m_CSP axon terminals or m_CSP cortical origins modified the spikes distribution for photosensitive STN neurons firing rate in non-equivalent ways. Optogenetic tractography might help design preclinical neuromodulation studies in NHP models of neuropsychiatric disease choosing the most appropriate target for the tested hypothesis.

We studied the evolution of opsin genes in 59 ray-finned fish genomes. We identified the opsin genes and adjacent genes (syntenies) in each genome. Then we inferred the changes in gene copy number (N), syntenies, and tuning sites along each phylogenetic branch during evolution. The Exorh (rod opsin) gene has been retained in 56 genomes. Rh1, the intronless rod opsin gene, first emerged in ancestral Actinopterygii, and N increased to 2 by the teleost-specific whole genome duplication, but then decreased to 1 in the ancestor of Neoteleostei fishes. For cone opsin genes, the rhodopsin-like (Rh2) and long-wave-sensitive (LWS) genes showed great variation in N among species, ranging from 0 to 5 and from 0 to 4, respectively. The two short-wave-sensitive genes, SWS1 and SWS2, were lost in 23 and 6 species, respectively. The syntenies involving LWS, SWS2 and Rh2 underwent complex changes, while the evolution of the other opsin gene syntenies was much simpler. Evolutionary adaptation in tuning sites under different living environments was discussed. Our study provides a detailed view of opsin gene gains and losses, synteny changes and tuning site changes during ray-finned fish evolution.

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.

Phosphatidylserine (PS) is asymmetrically distributed between the outer and inner leaflets of the plasma membrane in eukaryotic cells. PS asymmetry on the plasma membrane depends on the activities of P4-ATPases, and disruption of PS distribution can lead to various disease conditions. Folding and transporting of P4-ATPases to their cellular destination requires the β subunit TMEM30A proteins. However, the in vivo functions of Tmem30a remain unknown. To this end, we generated retinal-specific Tmem30a-knockout mice to investigate its roles in vivo for the first time. Our data demonstrated that loss of Tmem30a in mouse cone cells leads to mislocalization of cone opsin, loss of photopic electroretinogram (ERG) responses and loss of cone cells. Mechanistically, Tmem30a-mutant mouse embryonic fibroblasts (MEFs) exhibited diminished PS flippase activity and increased exposure of PS on the cell surface. The broad loss of Tmem30a in adult mice led to a reduced scotopic photoresponse, mislocalization of ATP8A2 to the inner segment and cell body, and increased apoptosis in the retina. Our data demonstrated novel essential roles of Tmem30a in the retina.

Genetic mutations in aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) cause photoreceptor degeneration associated with Leber congenital amaurosis 4 (LCA4) in human patients. Here we report retinal phenotypes of a zebrafish aipl1 mutant, gold rush (gosh). In zebrafish, there are two aipl1 genes, aipl1a and aipl1b, which are expressed mainly in rods and cones, respectively. The gosh mutant gene encodes cone-specific aipl1, aipl1b. Cone photoreceptors undergo progressive degeneration in the gosh mutant, indicating that aipl1b is required for cone survival. Furthermore, the cone-specific subunit of cGMP phosphodiesterase 6 (Pde6c) is markedly decreased in the gosh mutant, and the gosh mutation genetically interacts with zebrafish pde6c mutation eclipse (els). These data suggest that Aipl1 is required for Pde6c stability and function. In addition to Pde6c, we found that zebrafish cone-specific guanylate cyclase, zGc3, is also decreased in the gosh and els mutants. Furthermore, zGc3 knockdown embryos showed a marked reduction in Pde6c. These observations illustrate the interdependence of cGMP metabolism regulators between Aipl1, Pde6c, and Gc3 in photoreceptors.

Opsin proteins are fundamental components of animal vision whose structure largely determines the sensitivity of visual pigments to different wavelengths of light. Surprisingly little is known about opsin evolution in beetles, even though they are the most species rich animal group on Earth and exhibit considerable variation in visual system sensitivities. We reveal the patterns of opsin evolution across 62 beetle species and relatives. Our results show that the major insect opsin class (SW) that typically confers sensitivity to “blue” wavelengths was lost ~300 million years ago, before the origin of modern beetles. We propose that UV and LW opsin gene duplications have restored the potential for trichromacy (three separate channels for colour vision) in beetles up to 12 times and more specifically, duplications within the UV opsin class have likely led to the restoration of “blue” sensitivity up to 10 times. This finding reveals unexpected plasticity within the insect visual system and highlights its remarkable ability to evolve and adapt to the available light and visual cues present in the environment.

Visual discrimination requires sensory processing followed by a perceptual decision. Despite a growing understanding of visual areas in this behavior, it is unclear what role top-down signals from prefrontal cortex play, in particular as a function of perceptual difficulty. To address this gap, we investigated how neurons in dorso-lateral frontal cortex (dl-FC) of freely-moving ferrets encode task variables in a two-alternative forced choice visual discrimination task with high- and low-contrast visual input. About two-thirds of all recorded neurons in dl-FC were modulated by at least one of the two task variables, task difficulty and target location. More neurons in dl-FC preferred the hard trials; no such preference bias was found for target location. In individual neurons, this preference for specific task types was limited to brief epochs. Finally, optogenetic stimulation confirmed the functional role of the activity in dl-FC before target touch; suppression of activity in pyramidal neurons with the ArchT silencing opsin resulted in a decrease in reaction time to touch the target but not to retrieve reward. In conclusion, dl-FC activity is differentially recruited for high perceptual difficulty in the freely-moving ferret and the resulting signal may provide top-down behavioral inhibition.

Photoreceptors, which initiate the conversion of ambient light to action potentials via retinal circuitry, degenerate in retinal diseases such as retinitis pigmentosa and age related macular degeneration leading to loss of vision. Current prosthetic devices using arrays consisting of electrodes or LEDs (for optogenetic activation of conventional narrow-band opsins) have limited spatial resolution and can cause damage to retinal circuits by mechanical or photochemical (by absorption of intense narrow band light) means. Here, we describe a broad-band light activatable white-opsin for generating significant photocurrent at white light intensity levels close to ambient daylight conditions. White-opsin produced an order of magnitude higher photocurrent in response to white light as compared to narrow-band opsin channelrhodopsin-2, while maintaining the ms-channel kinetics. High fidelity of peak-photocurrent (both amplitude and latency) of white-opsin in response to repetitive white light stimulation of varying pulse width was observed. The significantly lower intensity stimulation required for activating white-opsin sensitized cells may facilitate ambient white light-based restoration of vision for patients with widespread photoreceptor degeneration.

A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of

Optogenetic techniques provide an unprecedented ability to precisely manipulate neural activity in the context of complex neural circuitry. Although the toolbox of optogenetic probes continues to expand at a rapid pace with more efficient and responsive reagents, hardware-based light delivery is still a major hurdle that limits its practical use in vivo. We have bypassed the challenges of external light delivery by directly coupling a bioluminescent light source (a genetically encoded luciferase) to an inhibitory opsin, which we term an inhibitory luminopsin (iLMO). iLMO was shown to suppress action potential firing and synchronous bursting activity in vitro in response to both external light and luciferase substrate. iLMO was further shown to suppress single-unit firing rate and local field potentials in the hippocampus of anesthetized rats. Finally, expression of iLMO was scaled up to multiple structures of the basal ganglia to modulate rotational behavior of freely moving animals in a hardware-independent fashion. This novel class of optogenetic probes demonstrates how non-invasive inhibition of neural activity can be achieved, which adds to the versatility, scalability and practicality of optogenetic applications in freely behaving animals.

Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora, a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans.

Molecular imaging is a powerful tool for investigating intracellular signalling, but it is difficult to acquire conventional fluorescence imaging from photoreceptive cells. Here we demonstrated that human opsin5 (OPN5) photoreceptor mediates light-induced Ca(2+) response in human embryonic kidney (HEK293) and mouse neuroblastoma (Neuro2a) cell lines using a luminescence imaging system with a fluorescent indicator and a newly synthesized bioluminescent indicator. Weak light fluorescence and bioluminescence imaging revealed rapid and transient light-stimulated Ca(2+) release from thapsigargin-sensitive Ca(2+) stores, whereas long-lasting Ca(2+) elevation was observed using a conventional fluorescence imaging system. Bioluminescence imaging also demonstrated that OPN5 activation in HEK293 cells induced a decrease in pertussis toxin-sensitive cAMP, confirming previous reports. In addition, ultraviolet radiation induced the phosphorylation of mitogen-activated protein kinases when OPN5 was stimulated in Neuro2a cells. These findings suggest that the combination of these imaging approaches may provide a new means to investigate the physiological characteristics of photoreceptors.

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.