RESUMO
Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.
Assuntos
Bombas de Próton/química , Rodopsina/química , Transporte de Íons , Luz , Filogenia , Domínios Proteicos , Rodopsina/fisiologiaRESUMO
The Hippo pathway is an important regulator of organ growth and cell fate. In the R8 photoreceptor cells of the Drosophila melanogaster eye, the Hippo pathway controls the fate choice between one of two subtypes that express either the blue light-sensitive Rhodopsin 5 (Hippo inactive R8 subtype) or the green light-sensitive Rhodopsin 6 (Hippo active R8 subtype). The degree to which the mechanism of Hippo signal transduction and the proteins that mediate it are conserved in organ growth and R8 cell fate choice is currently unclear. Here, we identify Crumbs and the apical spectrin cytoskeleton as regulators of R8 cell fate. By contrast, other proteins that influence Hippo-dependent organ growth, such as the basolateral spectrin cytoskeleton and Ajuba, are dispensable for the R8 cell fate choice. Surprisingly, Crumbs promotes the Rhodopsin 5 cell fate, which is driven by Yorkie, rather than the Rhodopsin 6 cell fate, which is driven by Warts and the Hippo pathway, which contrasts with its impact on Hippo activity in organ growth. Furthermore, neither the apical spectrin cytoskeleton nor Crumbs appear to regulate the Hippo pathway through mechanisms that have been observed in growing organs. Together, these results show that only a subset of Hippo pathway proteins regulate the R8 binary cell fate decision and that aspects of Hippo signalling differ between growing organs and post-mitotic R8 cells.
Assuntos
Linhagem da Célula/fisiologia , Proteínas de Drosophila/fisiologia , Proteínas do Olho/fisiologia , Olho/citologia , Proteínas de Membrana/fisiologia , Rodopsina/fisiologia , Espectrina/fisiologia , Animais , Citoesqueleto/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Olho/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Fotorreceptoras de Invertebrados/fisiologia , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
It is not well understood how the spectral composition (wavelength) of daylight that varies considerably during the day and seasons affects photoperiodic responses in a seasonal species. Here, we investigated the molecular underpinnings of wavelength-dependent photoperiodic induction in migratory redheaded buntings transferred to 13 h long days in neutral (white), 460 nm (blue), 500 nm (green) or 620 nm (red) wavelength that were compared with one another, and to short day controls for indices of the migratory (body fattening and weight gain, and Zugunruhe) and reproductive (testicular maturation) responses. Buntings showed wavelength-dependent photoperiodic response, with delayed Zugunruhe and slower testis maturation under 620 nm red light. Post-mortem comparison of gene expressions further revealed wavelength-dependence of the photoperiodic molecular response. Whereas there were higher retinal expressions of opn2 (rhodopsin) and opn5 (neuropsin) genes in red daylight, and of rhodopsin-like opsin (rh2) gene in green daylight, the hypothalamic opn2 mRNA levels were higher in blue daylight. Similarly, we found in birds under blue daylight an increased hypothalamic expression of genes involved in the photoperiodic induction (thyroid stimulating hormone subunit beta, tshb; eye absent 3, eya3; deiodinase type 2, dio2) and associated neural responses such as the calcium signaling (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, atp2a2), dopamine biosynthesis (tyrosine hydroxylase, th) and neurogenesis (brain-derived neurotrophic factor, bdnf). These results demonstrate transcriptional changes in parallel to responses associated with migration and reproduction in buntings, and suggest a role of daylight spectrum in photoperiodic induction of the vernal response in obligate spring avian migrants.
Assuntos
Migração Animal , Luz , Fotoperíodo , Rodopsina/fisiologia , Estações do Ano , Aves Canoras/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Sinalização do Cálcio , Ritmo Circadiano/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Índia , Masculino , Opsinas , Fenótipo , Retina/metabolismo , Rodopsina/metabolismo , Testículo/crescimento & desenvolvimentoRESUMO
Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival.SIGNIFICANCE STATEMENT Visual arrestin-1 forms dimers and tetramers. The biological role of its oligomerization is unclear. To test the role of arrestin-1 self-association, we expressed oligomerization-deficient mutant in arrestin-1 knock-out mice. The mutant quenches light-induced rhodopsin signaling like wild type, demonstrating that in vivo monomeric arrestin-1 is necessary and sufficient for this function. In rods, arrestin-1 moves from the inner segments and cell bodies in the dark to the outer segments in the light. Nonoligomerizing mutant undergoes the same translocation, demonstrating that the size of the oligomers is not the reason for arrestin-1 exclusion from the outer segments in the dark. High expression of oligomerization-deficient arrestin-1 resulted in rod death. Thus, oligomerization reduces the cytotoxicity of high levels of arrestin-1 monomer.
Assuntos
Arrestinas/metabolismo , Arrestinas/fisiologia , Adaptação Ocular , Animais , Arrestinas/genética , Sobrevivência Celular , Eletrorretinografia , Feminino , Transdução de Sinal Luminoso , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação/genética , Retina/anatomia & histologia , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/fisiologiaRESUMO
We have studied dark-adaptation at three levels in the eyes of the crustacean Mysis relicta over 2-3 weeks after exposing initially dark-adapted animals to strong white light: regeneration of 11-cis retinal through the retinoid cycle (by HPLC), restoration of native rhodopsin in photoreceptor membranes (by MSP), and recovery of eye photosensitivity (by ERG). We compare two model populations ("Sea", Sp, and "Lake", Lp) inhabiting, respectively, a low light and an extremely dark environment. 11-cis retinal reached 60-70% of the pre-exposure levels after 2 weeks in darkness in both populations. The only significant Lp/Sp difference in the retinoid cycle was that Lp had much higher levels of retinol, both basal and light-released. In Sp, rhodopsin restoration and eye photoresponse recovery parallelled 11-cis retinal regeneration. In Lp, however, even after 3 weeks only ca. 25% of the rhabdoms studied had incorporated new rhodopsin, and eye photosensitivity showed only incipient recovery from severe depression. The absorbance spectra of the majority of the Lp rhabdoms stayed constant around 490-500 nm, consistent with metarhodopsin II dominance. We conclude that sensitivity recovery of Sp eyes was rate-limited by the regeneration of 11-cis retinal, whilst that of Lp eyes was limited by inertia in photoreceptor membrane turnover.
Assuntos
Crustáceos/fisiologia , Fotofobia/prevenção & controle , Retinoides/metabolismo , Animais , Adaptação à Escuridão , Lagos , Oceanos e Mares , Regeneração , Rodopsina/fisiologiaRESUMO
Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.
Assuntos
Opsinas/genética , Optogenética , Animais , Animais Geneticamente Modificados , Calibragem , Cloretos/metabolismo , Reação de Fuga , Neurônios Motores/fisiologia , Bombas de Próton/fisiologia , Rodopsina/fisiologia , Gânglio Trigeminal/embriologia , Peixe-Zebra/embriologiaRESUMO
Rhodopsin is the G protein-coupled receptor in rod photoreceptor cells that initiates vision upon photon capture. The light receptor is normally locked in an inactive state in the dark by the covalently bound inverse agonist 11-cis retinal. Mutations can render the receptor active even in the absence of light. This constitutive activity can desensitize rod photoreceptor cells and lead to night blindness. A G90D mutation in rhodopsin causes the receptor to be constitutively active and leads to congenital stationary night blindness, which is generally thought to be devoid of retinal degeneration. The constitutively active species responsible for the night blindness phenotype is unclear. Moreover, the classification as a stationary disease devoid of retinal degeneration is also misleading. A transgenic mouse model for congenital stationary night blindness that expresses the G90D rhodopsin mutant was examined to better understand the origin of constitutive activity and the potential for retinal degeneration. Heterozygous mice for the G90D mutation did not exhibit retinal degeneration whereas homozygous mice exhibited progressive retinal degeneration. Only a modest reversal of retinal degeneration was observed when transducin signaling was eliminated genetically, indicating that some of the retinal degeneration occurred in a transducin-independent manner. Biochemical studies on purified rhodopsin from mice indicated that multiple species can potentially contribute to the constitutive activity causing night blindness.
Assuntos
Mutação , Cegueira Noturna/patologia , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/fisiologia , Transducina/fisiologia , Animais , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Cegueira Noturna/etiologia , Degeneração Retiniana/etiologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Rhodopsin (RH1), the temperature-sensitive visual pigment, attained cold adaptation by functional trade-offs between protein stability and activity. Recent studies suggested convergent selection pressures drove cold adaptation of rhodopsin in high altitude catfishes through nonparallel molecular mechanisms. Here, we tested whether the similar shift occurred in RH1 of Tibetan loaches on the Qinghai-Tibet Plateau (QTP) by investigating the molecular evolution and potential effect on function of RH1. We sequenced RH1 from 27 Triplophysa species, and four lowland loaches and combined these data with published sequences. Tests using a series of models of molecular evolution resulted in strong evidence for accelerated evolution and positive selection in Triplophysa RH1. Three positively selected sites were near key functional domains modulating nonspectral properties of rhodopsin, substitutions of which were likely to compensate for cold-induced decrease in rhodopsin kinetics in cold environments. Moreover, although accelerated evolutionary rates in Tibetan loaches was convergent with those in high altitude catfishes, the sites under positive selection were nonoverlapping. Our findings provide evidence for convergent shift in selection pressures of RH1 in high altitude fish during the ecological transition to cold environment of the QTP.
Assuntos
Cipriniformes/genética , Evolução Molecular , Proteínas de Peixes/genética , Rodopsina/genética , Seleção Genética , Adaptação Biológica , Altitude , Substituição de Aminoácidos , Animais , Temperatura Baixa , Biologia Computacional , Proteínas de Peixes/química , Proteínas de Peixes/fisiologia , Modelos Moleculares , Filogenia , Rodopsina/química , Rodopsina/fisiologia , Alinhamento de Sequência , TibetRESUMO
Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and the underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture at the expense of image detail. Here, we show that retinal optical quality improves 2-fold during terminal development, and that this enhancement is caused by nuclear inversion. We further demonstrate that improved retinal contrast transmission, rather than photon-budget or resolution, enhances scotopic contrast sensitivity by 18-27%, and improves motion detection capabilities up to 10-fold in dim environments. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast transmission as a decisive determinant of mammalian visual perception.
Assuntos
Núcleo Celular/ultraestrutura , Sensibilidades de Contraste/fisiologia , Percepção de Movimento/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Animais , Simulação por Computador , Feminino , Genes Reporter , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Bipolares da Retina/fisiologia , Células Bipolares da Retina/ultraestrutura , Células Ganglionares da Retina/fisiologia , Células Ganglionares da Retina/ultraestrutura , Rodopsina/deficiência , Rodopsina/fisiologia , Espalhamento de RadiaçãoRESUMO
The spectral tuning properties of the whale shark (Rhincodon typus) rod (rhodopsin or Rh1) and long-wavelength-sensitive (LWS) cone visual pigments were examined to determine whether these retinal pigments have adapted to the broadband light spectrum available for surface foraging or to the narrowband blue-shifted light spectrum available at depth. Recently published whale shark genomes have identified orthologous genes for both the whale shark Rh1 and LWS cone opsins suggesting a duplex retina. Here, the whale shark Rh1 and LWS cone opsin sequences were examined to identify amino acid residues critical for spectral tuning. Surprisingly, the predicted absorbance maximum (λmax) for both the whale shark Rh1 and LWS visual pigments is near 500 nm. Although Rh1 λmax values near 500 nm are typical of terrestrial vertebrates, as well as surface foraging fish, it is uncommon for a vertebrate LWS cone pigment to be so greatly blue-shifted. We propose that the spectral tuning properties of both the whale shark Rh1 and LWS cone pigments are most likely adaptations to the broadband light spectrum available at the surface. Whale shark melanopsin (Opn4) deactivation kinetics was examined to better understand the underlying molecular mechanisms of the pupillary light reflex. Results show that the deactivation rate of whale shark Opn4 is similar to the Opn4 deactivation rate from vertebrates possessing duplex retinae and is significantly faster than the Opn4 deactivation rate from an aquatic rod monochromat lacking functional cone photoreceptors. The rapid deactivation rate of whale shark Opn4 is consistent with a functional cone class and would provide the animal with an exponential increase in the number of photons required for photoreceptor signaling when transitioning from photopic to scotopic light conditions, as is the case when diving.
Assuntos
Opsinas dos Cones/fisiologia , Fenômenos Ópticos , Células Fotorreceptoras Retinianas Cones/fisiologia , Rodopsina/fisiologia , Tubarões/fisiologia , AnimaisRESUMO
Retinal degenerations are a major cause of blindness in human patients. The identification of endogenous mechanisms involved in neurodegeneration or neuroprotection helps to understand the response of the retina to stress and provides essential information not only for basic retinal physiology but also for defining molecular targets for neuroprotective strategies. Here we used excessive light exposure as a model system to study mechanisms of photoreceptor degeneration in mice. Using one wild type and four genetically modified mouse strains, we demonstrate that light exposure resulted not only in the degeneration of rods but also in an early but transient repression of several cone-specific genes, in a reversible hyperreflectivity of the outer retina including the outer plexiform layer, and in the loss of horizontal cells. The effects on cones, horizontal cells and the inner retina depended on light absorption by rhodopsin and, at least partially, on leukemia inhibitory factor. This demonstrates the existence of intercellular communication routes that transduce rod stress to other cells, likely to provide support for photoreceptors and increase cell survival in the injured retina.
Assuntos
Luz/efeitos adversos , Células Fotorreceptoras Retinianas Cones/fisiologia , Degeneração Retiniana/etiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/fisiologia , Estresse Fisiológico/fisiologia , Animais , Eletrorretinografia , Camundongos , Degeneração Retiniana/fisiopatologiaRESUMO
Bleaching adaptation in rod photoreceptors is mediated by apo-opsin, which activates phototransduction with effective activity 105- to 106-fold lower than that of photoactivated rhodopsin (meta II). However, the mechanism that produces such low opsin activity is unknown. To address this question, we sought to record single opsin responses in mouse rods. We used mutant mice lacking efficient calcium feedback to boosts rod responses and generated a small fraction of opsin by photobleaching â¼1% of rhodopsin. The bleach produced a dramatic increase in the frequency of discrete photoresponse-like events. This activity persisted for hours, was quenched by 11-cis-retinal, and was blocked by uncoupling opsin from phototransduction, all indicating opsin as its source. Opsin-driven discrete activity was also observed in rods containing non-activatable rhodopsin, ruling out transactivation of rhodopsin by opsin. We conclude that bleaching adaptation is mediated by opsin that exists in equilibrium between a predominant inactive and a rare meta II-like state.SIGNIFICANCE STATEMENT Electrophysiological analysis is used to show that the G-protein-coupled receptor opsin exists in equilibrium between a predominant inactive and a rare highly active state that mediates bleaching adaptation in photoreceptors.
Assuntos
Opsinas de Bastonetes/fisiologia , Animais , Sinalização do Cálcio/genética , Feminino , Transdução de Sinal Luminoso/genética , Transdução de Sinal Luminoso/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mutação , Fotodegradação , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinaldeído/química , Rodopsina/química , Rodopsina/genética , Rodopsina/fisiologia , Opsinas de Bastonetes/química , Opsinas de Bastonetes/genética , cis-trans-Isomerases/genética , cis-trans-Isomerases/fisiologiaRESUMO
Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.
Assuntos
Adaptação Biológica , Quirópteros/fisiologia , Ecolocação , Evolução Molecular , Rodopsina/fisiologia , Animais , Adaptação à Escuridão , Cinética , Visão OcularRESUMO
To integrate changing environmental cues with high spatial and temporal resolution is critical for animals to orient themselves. Drosophila larvae show an effective motor program to navigate away from light sources. How the larval visual circuit processes light stimuli to control navigational decision remains unknown. The larval visual system is composed of two sensory input channels, Rhodopsin5 (Rh5) and Rhodopsin6 (Rh6) expressing photoreceptors (PRs). We here characterize how spatial and temporal information are used to control navigation. Rh6-PRs are required to perceive temporal changes of light intensity during head casts, while Rh5-PRs are required to control behaviors that allow navigation in response to spatial cues. We characterize how distinct behaviors are modulated and identify parallel acting and converging features of the visual circuit. Functional features of the larval visual circuit highlight the principle of how early in a sensory circuit distinct behaviors may be computed by partly overlapping sensory pathways.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células Fotorreceptoras de Invertebrados/fisiologia , Rodopsina/fisiologia , Navegação Espacial , Animais , Comportamento Animal , Sinais (Psicologia) , Drosophila/embriologia , Larva/fisiologia , Lasers , Luz , Fototaxia , Probabilidade , Fatores de Tempo , Visão OcularRESUMO
Network oscillations across and within brain areas are critical for learning and performance of memory tasks. While a large amount of work has focused on the generation of neural oscillations, their effect on neuronal populations' spiking activity and information encoding is less known. Here, we use computational modeling to demonstrate that a shift in resonance responses can interact with oscillating input to ensure that networks of neurons properly encode new information represented in external inputs to the weights of recurrent synaptic connections. Using a neuronal network model, we find that due to an input current-dependent shift in their resonance response, individual neurons in a network will arrange their phases of firing to represent varying strengths of their respective inputs. As networks encode information, neurons fire more synchronously, and this effect limits the extent to which further "learning" (in the form of changes in synaptic strength) can occur. We also demonstrate that sequential patterns of neuronal firing can be accurately stored in the network; these sequences are later reproduced without external input (in the context of subthreshold oscillations) in both the forward and reverse directions (as has been observed following learning in vivo). To test whether a similar mechanism could act in vivo, we show that periodic stimulation of hippocampal neurons coordinates network activity and functional connectivity in a frequency-dependent manner. We conclude that resonance with subthreshold oscillations provides a plausible network-level mechanism to accurately encode and retrieve information without overstrengthening connections between neurons.
Assuntos
Potenciais de Ação/fisiologia , Aprendizagem/fisiologia , Modelos Neurológicos , Rede Nervosa/fisiologia , Redes Neurais de Computação , Neurônios/fisiologia , Rodopsina/fisiologia , Animais , Simulação por Computador , Canais Iônicos/fisiologia , CamundongosRESUMO
In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours ('Quint': ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H134R and H134R;D156C enabled increased effects during repetitive stimulation. We also tested new hyperpolarizers (ACR1, ACR2, ACR1(C102A), ZipACR). Particularly ACR1 and ACR2 showed strong effects in behavioral assays and very large currents with fast kinetics. In sum, we introduce highly light-sensitive optogenetic tools, bypassing previous shortcomings, and thus constituting new tools that feature high effectiveness and fast kinetics, allowing better repetitive stimulation or investigating prolonged neuronal activity states in C. elegans and, possibly, other systems.
Assuntos
Caenorhabditis elegans/efeitos da radiação , Luz , Optogenética , Rodopsina/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Mutação PuntualRESUMO
Convergent evolution in response to similar selective pressures is a well-known phenomenon in evolutionary biology. Less well understood is how selection drives convergence in protein function, and the underlying mechanisms by which this can be achieved. Here, we investigate functional convergence in the visual system of two distantly related lineages of high-altitude adapted Andean and Himalayan catfishes. Statistical analyses revealed in the two high-altitude lineages, a parallel acceleration of evolutionary rates in rhodopsin, the dim-light visual pigment. However, the elevated rates were found to be accompanied by substitutions at different sites in the protein. Experiments substituting Andean- or Himalayan-specific residues significantly accelerated the kinetic rates of rhodopsin, destabilizing the ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates, properties of rhodopsin mediating rod sensitivity and visual performance. Our study suggests that molecular convergence in protein function can be driven by parallel shifts in evolutionary rates but via nonparallel molecular mechanisms. Signatures of natural selection may therefore be a powerful guide for identifying complex instances of functional convergence across a wider range of protein systems.
Assuntos
Peixes-Gato/genética , Peixes-Gato/fisiologia , Evolução Molecular , Proteínas de Peixes/genética , Rodopsina/genética , Altitude , Aminoácidos/análise , Animais , Peixes-Gato/classificação , Proteínas de Peixes/química , Proteínas de Peixes/fisiologia , Filogenia , Rodopsina/química , Rodopsina/fisiologia , Seleção GenéticaRESUMO
Retinitis pigmentosa (RP) is a group of inherited retinal degenerative conditions and a leading cause of irreversible blindness. 25%-30% of RP cases are caused by inherited autosomal dominant (ad) mutations in the rhodopsin (Rho) protein of the retina, which impose a barrier for developing therapeutic treatments for this genetically heterogeneous disorder, as simple gene replacement is not sufficient to overcome dominant disease alleles. Previously, we have explored using the genomic short-form of Rho (sgRho) for gene augmentation therapy of RP in a Rho knockout mouse model. We have shown improved gene expression and fewer epigenetic modifications compared with the use of a Rho cDNA expression construct. In the current study, we altered our strategy by delivering a codon-optimized genomic form of Rho (co-sgRho) (for gene replacement) in combination with an RNAi-based inactivation of endogenous Rho alleles (gene suppression of both mutant Rho alleles, but mismatched with the co-sgRho) into a homozygous RhoP23H/P23H knock-in (KI) RP mouse model, which has a severe phenotype of adRP. In addition, we have conjugated a cell penetrating TAT peptide sequence to our previously established CK30PEG10 diblock co-polymer. The DNAs were compacted with CK30PEG10-TAT diblock co-polymer to form DNA nanoparticles (NPs). These NPs were injected into the sub-retinal space of the KI mouse eyes. As a proof of concept, we demonstrated the efficiency of this strategy in the partial improvement of visual function in the RhoP23H/P23H KI mouse model.
Assuntos
DNA/administração & dosagem , Modelos Animais de Doenças , Terapia Genética/métodos , Nanopartículas/administração & dosagem , Retinose Pigmentar/terapia , Rodopsina/fisiologia , Animais , DNA/química , Técnicas de Introdução de Genes/métodos , Genes Dominantes , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Degeneração Retiniana , Retinose Pigmentar/genéticaRESUMO
Peropsin is a non-visual opsin in both vertebrate and invertebrate species. In mammals, peropsin is present in the apical microvilli of retinal pigment epithelial (RPE) cells. These structures interdigitate with the outer segments of rod and cone photoreceptor cells. RPE cells play critical roles in the maintenance of photoreceptors, including the recycling of visual chromophore for the opsin visual pigments. Here, we sought to identify the function of peropsin in the mouse eye. To this end, we generated mice with a null mutation in the peropsin gene (Rrh). These mice exhibited normal retinal histology, normal morphology of outer segments and RPE cells, and no evidence of photoreceptor degeneration. Biochemically, Rrh-/- mice had â¼2-fold higher vitamin A (all-trans-retinol (all-trans-ROL)) in the neural retina following a photobleach and 5-fold lower retinyl esters in the RPE. This phenotype was similar to those reported in mice that lack interphotoreceptor retinoid-binding protein (IRBP) or cellular retinol-binding protein, suggesting that peropsin plays a role in the movement of all-trans-ROL from photoreceptors to the RPE. We compared the phenotypes in mice lacking both peropsin and IRBP with those of mice lacking peropsin or IRBP alone and found that the retinoid phenotype was similarly severe in each of these knock-out mice. We conclude that peropsin controls all-trans-ROL movement from the retina to the RPE or may regulate all-trans-ROL storage within the RPE. We propose that peropsin affects light-dependent regulation of all-trans-ROL uptake from photoreceptors into RPE cells through an as yet undefined mechanism.
Assuntos
Rodopsina/metabolismo , Vitamina A/fisiologia , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Camundongos , Camundongos Knockout , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Retinaldeído/metabolismo , Retinoides/metabolismo , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismo , Rodopsina/genética , Rodopsina/fisiologia , Opsinas de Bastonetes/metabolismo , Vitamina A/metabolismoRESUMO
Two distinct motor areas, the primary and secondary motor cortices (M1 and M2), play crucial roles in voluntary movement in rodents. The aim of this study was to characterize the laterality in motor cortical representations of right and left forelimb movements. To achieve this goal, we developed a novel behavioral task, the Right-Left Pedal task, in which a head-restrained male rat manipulates a right or left pedal with the corresponding forelimb. This task enabled us to monitor independent movements of both forelimbs with high spatiotemporal resolution. We observed phasic movement-related neuronal activity (Go-type) and tonic hold-related activity (Hold-type) in isolated unilateral movements. In both M1 and M2, Go-type neurons exhibited bias toward contralateral preference, whereas Hold-type neurons exhibited no bias. The contralateral bias was weaker in M2 than M1. Moreover, we differentiated between intratelencephalic (IT) and pyramidal tract (PT) neurons using optogenetically evoked spike collision in rats expressing channelrhodopsin-2. Even in identified PT and IT neurons, Hold-type neurons exhibited no lateral bias. Go-type PT neurons exhibited bias toward contralateral preference, whereas IT neurons exhibited no bias. Our findings suggest a different laterality of movement representations of M1 and M2, in each of which IT neurons are involved in cooperation of bilateral movements, whereas PT neurons control contralateral movements.SIGNIFICANCE STATEMENT In rodents, the primary and secondary motor cortices (M1 and M2) are involved in voluntary movements via distinct projection neurons: intratelencephalic (IT) neurons and pyramidal tract (PT) neurons. However, it remains unclear whether the two motor cortices (M1 vs M2) and the two classes of projection neurons (IT vs PT) have different laterality of movement representations. We optogenetically identified these neurons and analyzed their functional activity using a novel behavioral task to monitor movements of the right and left forelimbs separately. We found that contralateral bias was reduced in M2 relative to M1, and in IT relative to PT neurons. Our findings suggest that the motor information processing that controls forelimb movement is coordinated by a distinct cell population.