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1.
Nat Commun ; 12(1): 4478, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294698

RESUMO

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.


Assuntos
Encéfalo/fisiologia , Animais , Comportamento Animal/fisiologia , Comportamento Animal/efeitos da radiação , Encéfalo/efeitos da radiação , Cério , Feminino , Células HEK293 , Humanos , Luminescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Opsinas/metabolismo , Opsinas/efeitos da radiação , Optogenética/instrumentação , Contagem de Cintilação , Tecnologia sem Fio/instrumentação , Raios X
2.
Nat Commun ; 12(1): 4488, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301944

RESUMO

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Optogenética/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Opsinas/genética , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapses/genética , Sinapses/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
3.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
4.
Methods Mol Biol ; 2268: 207-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085271

RESUMO

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gßγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Opsinas/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
5.
Nat Commun ; 12(1): 2730, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980868

RESUMO

Neurostimulant drugs or magnetic/electrical stimulation techniques can overcome attention deficits, but these drugs or techniques are weakly beneficial in boosting the learning capabilities of healthy subjects. Here, we report a stimulation technique, mid-infrared modulation (MIM), that delivers mid-infrared light energy through the opened skull or even non-invasively through a thinned intact skull and can activate brain neurons in vivo without introducing any exogeneous gene. Using c-Fos immunohistochemistry, in vivo single-cell electrophysiology and two-photon Ca2+ imaging in mice, we demonstrate that MIM significantly induces firing activities of neurons in the targeted cortical area. Moreover, mice that receive MIM targeting to the auditory cortex during an auditory associative learning task exhibit a faster learning speed (~50% faster) than control mice. Together, this non-invasive, opsin-free MIM technique is demonstrated with potential for modulating neuronal activity.


Assuntos
Córtex Auditivo/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Animais , Eletrofisiologia , Imuno-Histoquímica , Masculino , Camundongos , Opsinas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo
6.
J Biol Rhythms ; 36(2): 109-126, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33765865

RESUMO

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.


Assuntos
Evolução Molecular , Mamíferos/metabolismo , Opsinas/metabolismo , Opsinas/efeitos da radiação , Animais , Ritmo Circadiano/efeitos da radiação , Sequência Conservada , Humanos , Camundongos , Opsinas/química , Opsinas/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Retina/metabolismo , Retina/efeitos da radiação , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismo , Rodopsina/efeitos da radiação
7.
Dev Biol ; 475: 145-155, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33684435

RESUMO

Vertebrate rod and cone photoreceptors detect light via a specialized organelle called the outer segment. This structure is packed with light-sensitive molecules known as visual pigments that consist of a G-protein-coupled, seven-transmembrane protein known as opsin, and a chromophore prosthetic group, either 11-cis retinal ('A1') or 11-cis 3,4-didehydroretinal ('A2'). The enzyme cyp27c1 converts A1 into A2 in the retinal pigment epithelium. Replacing A1 with A2 in a visual pigment red-shifts its spectral sensitivity and broadens its bandwidth of absorption at the expense of decreased photosensitivity and increased thermal noise. The use of vitamin A2-based visual pigments is strongly associated with the occupation of aquatic habitats in which the ambient light is red-shifted. By modulating the A1/A2 ratio in the retina, an organism can dynamically tune the spectral sensitivity of the visual system to better match the predominant wavelengths of light in its environment. As many as a quarter of all vertebrate species utilize A2, at least during a part of their life cycle or under certain environmental conditions. A2 utilization therefore represents an important and widespread mechanism of sensory plasticity. This review provides an up-to-date account of the A1/A2 chromophore exchange system.


Assuntos
Células Fotorreceptoras de Vertebrados/metabolismo , Vitamina A/análogos & derivados , Vitamina A/metabolismo , Animais , Opsinas/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Opsinas de Bastonetes/metabolismo , Vitamina A/fisiologia
8.
Commun Biol ; 4(1): 177, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33564115

RESUMO

Opsins, combined with a chromophore, are the primary light-sensing molecules in animals and are crucial for color vision. Throughout animal evolution, duplications and losses of opsin proteins are common, but it is unclear what is driving these gains and losses. Light availability is implicated, and dim environments are often associated with low opsin diversity and loss. Correlations between high opsin diversity and bright environments, however, are tenuous. To test if increased light availability is associated with opsin diversification, we examined diel niche and identified opsins using transcriptomes and genomes of 175 butterflies and moths (Lepidoptera). We found 14 independent opsin duplications associated with bright environments. Estimating their rates of evolution revealed that opsins from diurnal taxa evolve faster-at least 13 amino acids were identified with higher dN/dS rates, with a subset close enough to the chromophore to tune the opsin. These results demonstrate that high light availability increases opsin diversity and evolution rate in Lepidoptera.


Assuntos
Borboletas/efeitos da radiação , Percepção de Cores/efeitos da radiação , Visão de Cores/efeitos da radiação , Evolução Molecular , Proteínas de Insetos/genética , Luz , Mariposas/efeitos da radiação , Opsinas/genética , Animais , Borboletas/genética , Borboletas/metabolismo , Percepção de Cores/genética , Visão de Cores/genética , Duplicação Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma , Proteínas de Insetos/metabolismo , Mariposas/genética , Mariposas/metabolismo , Opsinas/metabolismo , Filogenia , Transcriptoma
9.
Pharmacol Biochem Behav ; 204: 173147, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33549570

RESUMO

Transgenic neuromodulation tools have transformed the field of neuroscience over the past two decades by enabling targeted manipulation of neuronal populations and circuits with unprecedented specificity. Chemogenetic and optogenetic neuromodulation systems are among the most widely used and allow targeted control of neuronal activity through the administration of a selective compound or light, respectively. Innovative genetic targeting strategies are utilized to transduce specific cells to express transgenic receptors and opsins capable of manipulating neuronal activity. These allow mapping of neuroanatomical projection sites and link cellular manipulations with brain circuit functions and behavior. As these tools continue to expand knowledge of the nervous system in preclinical models, developing translational applications for human therapies is becoming increasingly possible. However, new strategies for implementing and monitoring transgenic tools are needed for safe and effective use in translational research and potential clinical applications. A major challenge for such applications is the need to track the location and function of chemogenetic receptors and opsins in vivo, and new developments in positron emission tomography (PET) imaging techniques offer promising solutions. The goal of this review is to summarize current research combining transgenic tools with PET for in vivo mapping and manipulation of brain circuits and to propose future directions for translational applications.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/diagnóstico por imagem , Optogenética/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Animais Geneticamente Modificados , Encéfalo/fisiologia , Sistema Nervoso Central/fisiologia , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Transgênicos , Vias Neurais/fisiologia , Neurônios/fisiologia , Opsinas/metabolismo , Ratos
10.
Adv Exp Med Biol ; 1293: 407-416, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33398829

RESUMO

Optogenetics, which relies on the use of photons to manipulate cellular and subcellular processes, has emerged as an important tool that has transformed several fields including neuroscience. Improvement of optogenetic topographies, together with integration with complementary tools such as electrophysiology, imaging, anatomical and behavioral analysis, facilitated this transformation. However, an inherent challenge associated with optogenetic manipulation of neurons in living organisms, such as rodents, is the requirement for implanting light-delivering optical fibers. This is partly because the current repertoires of light-sensitive opsins are activated only by visible light, which cannot effectively penetrate biological tissues. Insertion of optical fibers and subsequent photo-stimulation inherently damages brain tissue, and fiber tethering can constrain animal behavior. To overcome these technical limitations, we and other research groups recently developed minimally invasive "fiberless optogenetics," which uses particles that can emit visible light through up-conversion luminescence in response to irradiation with tissue-penetrating near-infrared light. Fiberless optogenetics also offers the opportunity to control neural function over longer time frames in freely behaving animals. In this chapter, we discuss the development of fiberless optogenetics and its application in neuroscience and beyond.


Assuntos
Opsinas , Optogenética , Animais , Encéfalo/metabolismo , Raios Infravermelhos , Neurônios/metabolismo , Opsinas/metabolismo
11.
Exp Eye Res ; 205: 108444, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33516760

RESUMO

In recent time, gene therapy has proven to be a promising remedial approach for treating visual disorders either by replacement of nonfunctioning gene(s) or by introduction of light sensitive proteins (opsins) as artificial photoreceptors in retinal cells. Conventional viral vector-based gene delivery method is often confronted with limitations due to immunogenetic reaction, unintended non-targeted delivery, non-feasibility of repeated re-dosing due to immunorejection, and complicated manufacturing process, leading to significant roadblock in translational success. In this regard, non-viral delivery provides a safer, simpler and cost-effective alternative. However, most of the non-viral approaches lack spatial and/or cellular specificity and limited by low transfection efficacy and cytotoxicity. Here, we present a minimally invasive, non-viral and clinically translatable safe targeted gene delivery method utilizing functionalized plasmonic gold nanorods (fGNRs, targeted to attach to specific cell types of the organ of interest) and spatially targeted controlled light irradiation. Targeted in-vivo delivery and expression of opsin-encoding gene in bipolar and ganglion cell layers were achieved by use of cell specific fGNRs concurrent with light irradiation. Evaluation of safety and toxicity associated with the transduction of opsin-encoding genes by use of fGNRs and light irradiation were examined by electrophysiology, Optical coherence tomography, intra-ocular pressure and other analytical methods (confocal microscopy, immunohistochemistry). The non-viral light-based opsin-gene delivery provides a safe and effective alternative to viral-vector based gene delivery and holds promise for corrective cell-specific gene therapies for retinal degenerative diseases.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Ouro/metabolismo , Nanopartículas Metálicas , Opsinas/genética , Degeneração Retiniana/terapia , Animais , Imuno-Histoquímica , Injeções Intravítreas , Camundongos , Microscopia Confocal , Opsinas/metabolismo , Optogenética/métodos , Reação em Cadeia da Polimerase , Ressonância de Plasmônio de Superfície , Tomografia de Coerência Óptica
12.
Am J Respir Cell Mol Biol ; 64(1): 59-68, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33058732

RESUMO

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic ß2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.


Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Relaxamento Muscular/fisiologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Opsinas de Bastonetes/metabolismo , Traqueia/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Opsinas/metabolismo , Transdução de Sinais/fisiologia
13.
Arthropod Struct Dev ; 60: 101007, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33341370

RESUMO

As an obligate ectoparasite of bats, the bat fly Trichobius frequens (Diptera: Streblidae) inhabits the same subterranean environment as their nocturnal bat hosts. In this study, we characterize the macromorphology, optical architecture, rhabdom anatomy, photoreceptor absorbance, and opsin expression of the significantly reduced visual system in T. frequens resulting from evolution in the dark. The eyes develop over a 21-22 day pupal developmental period, with pigmentation appearing on pupal day 11. After eclosion as an adult, T. frequens eyes consist of on average 8 facets, each overlying a fused rhabdom consisting of anywhere from 11 to 18 estimated retinula cells. The dimensions of the facets and fused rhabdoms are similar to those measured in other nocturnal insects. T. frequens eyes are functional as shown by expression of a Rh1 opsin forming a visual pigment with a peak sensitivity to 487 nm, similar to other dipteran Rh1 opsins. Future studies will evaluate how individuals with such reduced capabilities for spatial vision as well as sensitivity still capture enough visual information to use flight to maneuver through dark habitats.


Assuntos
Olho Composto de Artrópodes/anatomia & histologia , Dípteros/anatomia & histologia , Células Fotorreceptoras de Invertebrados/citologia , Animais , Quirópteros/parasitologia , Olho Composto de Artrópodes/ultraestrutura , Dípteros/genética , Dípteros/ultraestrutura , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Microscopia Confocal , Microscopia Eletrônica de Varredura , Opsinas/genética , Opsinas/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestrutura
14.
Cell ; 183(6): 1586-1599.e10, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33159859

RESUMO

The hippocampus is crucial for spatial navigation and episodic memory formation. Hippocampal place cells exhibit spatially selective activity within an environment and have been proposed to form the neural basis of a cognitive map of space that supports these mnemonic functions. However, the direct influence of place cell activity on spatial navigation behavior has not yet been demonstrated. Using an 'all-optical' combination of simultaneous two-photon calcium imaging and two-photon optogenetics, we identified and selectively activated place cells that encoded behaviorally relevant locations in a virtual reality environment. Targeted stimulation of a small number of place cells was sufficient to bias the behavior of animals during a spatial memory task, providing causal evidence that hippocampal place cells actively support spatial navigation and memory.


Assuntos
Hipocampo/citologia , Células de Lugar/citologia , Comportamento Espacial , Memória Espacial , Animais , Comportamento Animal , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Opsinas/metabolismo , Optogenética , Fótons , Recompensa , Corrida , Navegação Espacial
15.
Biomolecules ; 10(9)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32942733

RESUMO

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor. Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Peptídeo Hidrolases/genética , Polyporales/efeitos da radiação , Madeira/microbiologia , Criptocromos/genética , Criptocromos/metabolismo , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Luz , Transdução de Sinal Luminoso , Anotação de Sequência Molecular , Opsinas/genética , Opsinas/metabolismo , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Doenças das Plantas/microbiologia , Polyporales/genética , Polyporales/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos da radiação , Proteólise/efeitos da radiação
16.
Nature ; 585(7825): 420-425, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879486

RESUMO

The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.


Assuntos
Cor , Luz , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Neurônios/efeitos da radiação , Opsinas/metabolismo , Área Pré-Óptica/citologia , Termogênese/efeitos da radiação , Tecido Adiposo Marrom/inervação , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/efeitos da radiação , Animais , Temperatura Corporal , Temperatura Baixa , AMP Cíclico/metabolismo , Feminino , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Opsinas/deficiência , Opsinas/genética , Termogênese/genética
17.
Elife ; 92020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32880369

RESUMO

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.


Assuntos
Evolução Molecular , Olho , Opsinas , Peptídeos , Células Fotorreceptoras de Invertebrados , Proteínas de Xenopus , Animais , Briozoários/química , Briozoários/genética , Briozoários/metabolismo , Cílios/química , Cílios/genética , Cílios/metabolismo , Olho/química , Olho/metabolismo , Larva/química , Larva/genética , Larva/metabolismo , Opsinas/química , Opsinas/genética , Opsinas/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Células Fotorreceptoras de Invertebrados/química , Células Fotorreceptoras de Invertebrados/metabolismo , Poliquetos/química , Poliquetos/genética , Poliquetos/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
18.
J Photochem Photobiol B ; 212: 112024, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32957069

RESUMO

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 Î·m) 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.


Assuntos
Meio Ambiente , Luz , Melanóforos/metabolismo , Melanóforos/efeitos da radiação , Opsinas/metabolismo , Receptores de Melatonina/metabolismo , Pigmentação da Pele/efeitos da radiação , Sequência de Aminoácidos , Animais , Opsinas/química , Xenopus laevis
19.
Invest Ophthalmol Vis Sci ; 61(10): 17, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32776140

RESUMO

Purpose: To identify the role of the BBSome protein Bardet-Biedl syndrome 5 (BBS5) in photoreceptor function, protein trafficking, and structure using a congenital mutant mouse model. Methods: Bbs5-/- mice (2 and 9 months old) were used to assess retinal function and morphology. Hematoxylin and eosin staining of retinal sections was performed to visualize histology. Electroretinography was used to analyze rod and cone photoreceptor function. Retinal protein localization was visualized using immunofluorescence (IF) within retinal cryosections. TUNEL staining was used to quantify cell death. Transmission electron microscopy (TEM) was used to examine retinal ultrastructure. Results: In the Bbs5-/- retina, there was a significant loss of nuclei in the outer nuclear layer accompanied by an increase in cell death. Through electroretinography, Bbs5-/- mice showed complete loss of cone photoreceptor function. IF revealed mislocalization of the cone-specific proteins M- and S-opsins, arrestin-4, CNGA3, and GNAT2, as well as a light-dependent arrestin-1 mislocalization, although perpherin-2 was properly localized. TEM revealed abnormal outer segment disk orientation in Bbs5-/-. Conclusions: Collectively, these data suggest that, although BBS5 is a core BBSome component expressed in all ciliated cells, its role within the retina mediates specific photoreceptor protein cargo transport. In the absence of BBS5, cone-specific protein mislocalization and a loss of cone photoreceptor function occur.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Animais , Western Blotting , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Opsinas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Transporte Proteico , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura
20.
Proc Natl Acad Sci U S A ; 117(33): 19629-19638, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759209

RESUMO

The visual phototransduction cascade begins with a cis-trans photoisomerization of a retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. Visual opsins release their all-trans-retinal chromophore following photoactivation, which necessitates the existence of pathways that produce 11-cis-retinal for continued formation of visual pigments and sustained vision. Proteins in the retinal pigment epithelium (RPE), a cell layer adjacent to the photoreceptor outer segments, form the well-established "dark" regeneration pathway known as the classical visual cycle. This pathway is sufficient to maintain continuous rod function and support cone photoreceptors as well although its throughput has to be augmented by additional mechanism(s) to maintain pigment levels in the face of high rates of photon capture. Recent studies indicate that the classical visual cycle works together with light-dependent processes in both the RPE and neural retina to ensure adequate 11-cis-retinal production under natural illuminances that can span ten orders of magnitude. Further elucidation of the interplay between these complementary systems is fundamental to understanding how cone-mediated vision is sustained in vivo. Here, we describe recent advances in understanding how 11-cis-retinal is synthesized via light-dependent mechanisms.


Assuntos
Retinaldeído/biossíntese , Visão Ocular , Animais , Humanos , Luz , Transdução de Sinal Luminoso , Opsinas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/química
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