RESUMO
Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.
Assuntos
Transdução de Sinal Luminoso , Reabilitação Neurológica , Mudanças Depois da Morte , Retina , Animais , Autopsia , Morte Celular/efeitos da radiação , Sistema Nervoso Central/efeitos da radiação , Humanos , Transdução de Sinal Luminoso/efeitos da radiação , Macaca , Camundongos , Retina/metabolismo , Retina/efeitos da radiação , Fatores de TempoRESUMO
Light is essential for photosynthesis but light levels that exceed an organism's assimilation capacity can cause serious damage or even cell death. Plants and microalgae have developed photoprotective mechanisms collectively referred to as non-photochemical quenching to minimize such potential damage. One such mechanism is energy-dependent quenching (qE), which dissipates excess light energy as heat. Over the last 30 years, much has been learned about the molecular mechanism of qE in green algae and plants. However, the steps between light perception and qE represented a gap in our knowledge until the recent identification of light-signaling pathways that function in these processes in the green alga Chlamydomonas reinhardtii. In this review, we summarize the high light and UV-mediated signaling pathways for qE in Chlamydomonas. We discuss key questions remaining about the pathway from light perception to photoprotective gene expression in Chlamydomonas. We detail possible differences between green algae and plants in light-signaling mechanisms for qE and emphasize the importance of research on light-signaling mechanisms for qE in plants.
Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso , Processos Fotoquímicos , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Modelos BiológicosRESUMO
Although roots are mainly embedded in the soil, recent studies revealed that light regulates mineral nutrient uptake by roots. However, it remains unclear whether the change in root system architecture in response to different rhizosphere nutrient statuses involves light signaling. Here, we report that blue light regulates primary root growth inhibition under phosphate-deficient conditions through the cryptochromes and their downstream signaling factors. We showed that the inhibition of root elongation by low phosphate requires blue light signal perception at the shoot and transduction to the root. In this process, SPA1 and COP1 play a negative role while HY5 plays a positive role. Further experiments revealed that HY5 is able to migrate from the shoot to root and that the shoot-derived HY5 autoactivates root HY5 and regulates primary root growth by directly activating the expression of LPR1, a suppressor of root growth under phosphate starvation. Taken together, our study reveals a regulatory mechanism by which blue light signaling regulates phosphate deficiency-induced primary root growth inhibition, providing new insights into the crosstalk between light and nutrient signaling.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Transdução de Sinal Luminoso/efeitos da radiação , Luz , Oxirredutases/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Criptocromos/metabolismo , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso/genética , Oxirredutases/genética , Fosfatos/deficiência , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Plants have evolved various photoreceptors to adapt to changing light environments, and photoreceptors can inactivate the large CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS) protein complex to release their repression of photoresponsive transcription factors. Here, we tracked the origin and evolution of COP/DET/FUS in Archaeplastida and found that most components of COP/DET/FUS were highly conserved. Intriguingly, the COP1-SUPPRESSOR OF PHYA-105 (SPA) protein originated in Chlorophyta but subsequently underwent a distinct evolutionary history in Viridiplantae. SPA experienced duplication events in the ancestors of specific clades after the colonization of land by plants and was divided into two clades (clades A and B) within euphyllophytes (ferns and seed plants). Our phylogenetic and experimental evidences support a new evolutionary model to clarify the divergence and convergence of light signaling during plant evolution.
Assuntos
Eucariotos/metabolismo , Eucariotos/efeitos da radiação , Evolução Molecular , Transdução de Sinal Luminoso , Eucariotos/classificação , Eucariotos/genética , Transdução de Sinal Luminoso/efeitos da radiação , FilogeniaRESUMO
Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.
Assuntos
Flavoproteínas/metabolismo , Flavoproteínas/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Luz , Optogenética , Criptocromos/genética , Criptocromos/metabolismo , Criptocromos/efeitos da radiação , Flavoproteínas/genéticaRESUMO
Shading can effectively reduce photoinhibition and improve the quality of tea. Lignin is one of the most important secondary metabolites that play vital functions in plant growth and development. However, little is known about the relationship between shading and xylogenesis in tea plant. To investigate the effects of shading on lignin accumulation in tea plants, 'Longjing 43' was treated with no shading (S0), 40% (S1) and 80% (S2) shading treatments, respectively. The leaf area and lignin content of tea plant leaves decreased under shading treatments (especially S2). The anatomical characteristics showed that lignin is mainly distributed in the xylem of tea leaves. Promoter analysis indicated that the genes involved in lignin pathway contain several light recognition elements. The transcript abundances of 12 lignin-associated genes were altered under shading treatments. Correlation analysis indicated that most genes showed strong positive correlation with lignin content, and CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation under 40% and 80% shading treatments. The results showed that shading may have an important effect on lignin accumulation in tea leaves. This work will potentially helpful to understand the regulation mechanism of lignin pathway under shading treatment, and provide reference for reducing lignin content and improving tea quality through shading treatment in field operation.
Assuntos
Camellia sinensis/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Lignina/biossíntese , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Camellia sinensis/enzimologia , Camellia sinensis/genética , Lignina/antagonistas & inibidores , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Metabolismo Secundário/efeitos da radiação , Luz Solar , Protetores Solares , Xilema/enzimologia , Xilema/genética , Xilema/efeitos da radiaçãoRESUMO
Because of their sessile nature, plants evolved integrated defense and acclimation mechanisms to simultaneously cope with adverse biotic and abiotic conditions. Among these are systemic acquired resistance (SAR) and systemic acquired acclimation (SAA). Growing evidence suggests that SAR and SAA activate similar cellular mechanisms and employ common signaling pathways for the induction of acclimatory and defense responses. It is therefore possible to consider these processes together, rather than separately, as a common systemic acquired acclimation and resistance (SAAR) mechanism. Arabidopsis thaliana flavin-dependent monooxygenase 1 (FMO1) was previously described as a regulator of plant resistance in response to pathogens as an important component of SAR. In the current study, we investigated its role in SAA, induced by a partial exposure of Arabidopsis rosette to local excess light stress. We demonstrate here that FMO1 expression is induced in leaves directly exposed to excess light stress as well as in systemic leaves remaining in low light. We also show that FMO1 is required for the systemic induction of ASCORBATE PEROXIDASE 2 (APX2) and ZINC-FINGER OF ARABIDOPSIS 10 (ZAT10) expression and spread of the reactive oxygen species (ROS) systemic signal in response to a local application of excess light treatment. Additionally, our results demonstrate that FMO1 is involved in the regulation of excess light-triggered systemic cell death, which is under control of LESION SIMULATING DISEASE 1 (LSD1). Our study indicates therefore that FMO1 plays an important role in triggering SAA response, supporting the hypothesis that SAA and SAR are tightly connected and use the same signaling pathways.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/efeitos da radiação , Transdução de Sinal Luminoso , Oxigenases/metabolismo , Estresse Fisiológico , Arabidopsis/enzimologia , Morte Celular/efeitos da radiação , Resistência à Doença , Transdução de Sinal Luminoso/efeitos da radiação , Modelos Biológicos , Folhas de Planta/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos da radiaçãoRESUMO
Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation-of the order of 10 pN-applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods' phototransduction.
Assuntos
Transdução de Sinal Luminoso , Mecanotransdução Celular , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Xenopus laevis/metabolismo , Animais , Cálcio/metabolismo , Fluorescência , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Mecanotransdução Celular/efeitos da radiação , Família Multigênica , Estimulação Luminosa , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Canais de Cátion TRPC/genética , Proteínas de Xenopus/genéticaRESUMO
In neurons, stromal interaction molecule (STIM) proteins regulate store-operated Ca2+ entry (SOCE) and are involved in calcium signaling pathways. However, STIM activity in neurological diseases is unclear and should be clarified by studies that are performed in vivo rather than in cultured cells in vitro. The present study investigated the role of neuronal Stim2b protein in zebrafish. We generated stim2b knockout zebrafish, which were fertile and had a regular lifespan. Using various behavioral tests, we found that stim2b-/- zebrafish larvae were hyperactive compared with wild-type fish. The mutants exhibited increases in mobility and thigmotaxis and disruptions of phototaxis. They were also more sensitive to pentylenetetrazol and glutamate treatments. Using lightsheet microscopy, a higher average oscillation frequency and higher average amplitude of neuronal Ca2+ oscillations were observed in stim2b-/- larvae. RNA sequencing detected upregulation of the annexin 3a and gpr39 genes and downregulation of the rrm2, neuroguidin, and homer2 genes. The latter gene encodes a protein that is involved in several processes that are involved in Ca2+ homeostasis in neurons, including metabotropic glutamate receptors. We propose that Stim2b deficiency in neurons dysregulates SOCE and triggers changes in gene expression, thereby causing abnormal behavior, such as hyperactivity and susceptibility to seizures.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Inativação de Genes , Convulsões/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Ácido Glutâmico/metabolismo , Larva/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Mutação/genética , Neurônios/metabolismo , Fenótipo , Fototaxia/efeitos da radiação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Leaf senescence is tightly regulated by numerous internal cues and external environmental signals. The process of leaf senescence is promoted by a low ratio of red to far-red (R:FR) light, FR light, or extended darkness and is repressed by a high ratio of R:FR light or R light. However, the precise regulatory mechanisms by which plants assess external light signals and their internal cues to initiate and control the process of leaf senescence remain largely unknown. In this study, we discovered that the light-signaling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) negatively regulates age-induced and light-mediated leaf senescence in Arabidopsis (Arabidopsis thaliana). FHY3 directly binds to the promoter region of transcription factor gene WRKY28 to repress its expression, thus negatively regulating salicylic acid biosynthesis and senescence. Both the fhy3 loss-of-function mutant and WRKY28-overexpressing Arabidopsis plants exhibited early senescence under high R:FR light conditions, indicating that the FHY3-WRKY28 transcriptional module specifically prevents leaf senescence under high R:FR light conditions. This study reveals the physiological and molecular functions of FHY3 and WRKY28 in leaf senescence and provides insight into the regulatory mechanism by which plants integrate dynamic environmental light signals and internal cues to initiate and control leaf senescence.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Transdução de Sinal Luminoso , Fitocromo/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/efeitos da radiação , Arabidopsis/efeitos dos fármacos , Sequência de Bases , Luz , Transdução de Sinal Luminoso/efeitos dos fármacos , Transdução de Sinal Luminoso/efeitos da radiação , Mutação/genética , Folhas de Planta/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Ácido Salicílico/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiaçãoRESUMO
UV-B constitutes a critical part of the sunlight reaching the earth surface. The homodimeric plant UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) monomerizes in response to UV-B and induces photomorphogenic responses, including UV-B acclimation and tolerance. REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 are negative feedback regulators that operate by facilitating UVR8 ground state reversion through re-dimerization. Here we show that RUP1 and RUP2 are transcriptionally induced by cryptochrome photoreceptors in response to blue light, which is dependent on the bZIP transcriptional regulator ELONGATED HYPOCOTYL 5 (HY5). Elevated RUP1 and RUP2 levels under blue light enhance UVR8 re-dimerization, thereby negatively regulating UVR8 signalling and providing photoreceptor pathway cross-regulation in a polychromatic light environment, as is the case in nature. We further show that cryptochrome 1, as well as the red-light photoreceptor phytochrome B, contribute to UV-B tolerance redundantly with UVR8. Thus, photoreceptors for both visible light and UV-B regulate UV-B tolerance through an intricate interplay allowing the integration of diverse sunlight signals.
Assuntos
Adaptação Fisiológica , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas Cromossômicas não Histona/metabolismo , Criptocromos/metabolismo , Transdução de Sinal Luminoso , Raios Ultravioleta , Adaptação Fisiológica/efeitos da radiação , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Modelos Biológicos , Multimerização Proteica/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/metabolismo , Plântula/efeitos da radiaçãoRESUMO
Photoreceptors are specialized neurons that rely on Ca2+ to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca2+ homeostasis is disrupted. Ca2+ homeostasis is maintained partly by mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), which can influence cytosolic Ca2+ signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca2+ overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca2+ and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca2+ uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca2+ stress by decreasing MICU3, an enhancer of MCU-mediated Ca2+ uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca2+ can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.
Assuntos
Adaptação Fisiológica , Cálcio/metabolismo , Luz , Metaboloma , Mitocôndrias/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Estresse Fisiológico , Adaptação Fisiológica/efeitos da radiação , Animais , Canais de Cálcio/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Isocitrato Desidrogenase/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Cinética , Transdução de Sinal Luminoso/efeitos da radiação , Mitocôndrias/efeitos da radiação , Mitocôndrias/ultraestrutura , Modelos Biológicos , Fenótipo , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Estresse Fisiológico/efeitos da radiação , Peixe-ZebraRESUMO
The cyanobacterium Synechocystis sp. PCC 6803 can move directionally on a moist surface toward or away from a light source to reach optimal light conditions for its photosynthetic lifestyle. This behavior, called phototaxis, is mediated by type IV pili (T4P), which can pull a single cell into a certain direction. Several photoreceptors and their downstream signal transduction elements are involved in the control of phototaxis. However, the critical steps of local pilus assembly in positive and negative phototaxis remain elusive. One of the photoreceptors controlling negative phototaxis in Synechocystis is the blue-light sensor PixD. PixD forms a complex with the CheY-like response regulator PixE that dissociates upon illumination with blue light. In this study, we investigate the phototactic behavior of pixE deletion and overexpression mutants in response to unidirectional red light with or without additional blue-light irradiation. Furthermore, we show that PixD and PixE partly localize in spots close to the cytoplasmic membrane. Interaction studies of PixE with the motor ATPase PilB1, demonstrated by in vivo colocalization, yeast two-hybrid and coimmunoprecipitation analysis, suggest that the PixD-PixE signal transduction system targets the T4P directly, thereby controlling blue-light-dependent negative phototaxis. An intriguing feature of PixE is its distinctive structure with a PATAN (PatA N-terminus) domain. This domain is found in several other regulators, which are known to control directional phototaxis. As our PilB1 coimmunoprecipitation analysis revealed an enrichment of PATAN domain response regulators in the eluate, we suggest that multiple environmental signals can be integrated via these regulators to control pilus function.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Oxirredutases/metabolismo , Fototaxia/fisiologia , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Oxirredutases/genética , Fotorreceptores Microbianos/metabolismo , Synechocystis/genética , Synechocystis/efeitos da radiaçãoRESUMO
Photostimulation has been widely used in neuromodulation. However, existing optogenetics techniques require genetic alternation of the targeted cell or tissue. Here, we report that neural stem cells (NSCs) constitutionally express blue/red light-sensitive photoreceptors. The proliferation and regulation of NSCs to neuronal or glial cells are wavelength-specific. Our results showed a 4.3-fold increase in proliferation and 2.7-fold increase in astrocyte differentiation for cells under low-power blue monochromatic light exposure (455â¯nm, 300⯵W/cm2). The melanopsin (Opn4)/transient receptor potential channel 6 (TRPC6) non-visual opsin serves as a key photoreceptor response to blue light irradiation. Two-dimensional gel electrophoresis coupled with mass spectrometry further highlighted the Jun activation domain-binding protein 1 (Jab1) as a novel and specific modulator in phototransduction pathways induced by blue light exposure. Quiescent adult NSCs reside in specific regions of the mammalian brain. Therefore, we showed that melanopsin/TRPC6 expressed in these regions and blue light stimulation through optical fibers could directly stimulate the NSCs in vivo. Upconversion nanoparticles (UCNPs) converted deep-penetrating near-infrared (NIR) light into specific wavelengths of visible light. Accordingly, we demonstrated that UCNP-mediated NIR light could be used to modulate in vivo NSC differentiation in a less invasive manner. In the future, this light-triggered system of NSCs will enable nongenetic and noninvasive neuromodulation with therapeutic potential for central nervous system diseases.
Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/citologia , Optogenética , Animais , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Autorrenovação Celular/efeitos da radiação , Raios Infravermelhos , Transdução de Sinal Luminoso/efeitos da radiação , Camundongos Endogâmicos C57BL , Nanopartículas/química , Células-Tronco Neurais/efeitos da radiação , Neuroglia/efeitos da radiação , Neurônios/efeitos da radiaçãoRESUMO
Purpose: Accumulating evidence suggests that dopamine, the major catecholamine in the vertebrate retina, may modulate cAMP-mediated signaling in photoreceptors to optimize vision in the light/dark cycle. The main putative mechanism of dopamine-induced adaptation changes in photoreceptors is activation of D2-like receptors (D2R), which leads to a decrease of the intracellular cAMP level and reduction of protein kinase A (PKA) activity. However, the mechanisms by which dopamine exerts its regulating effect on the phototransduction cascade remain largely unknown. The aim of the present study was to investigate the effects of dopamine and dopamine receptor agonists on rod photoresponses. Methods: The experiments were performed on solitary rods of the Rana ridibunda frog. Photoreceptor currents were recorded using a suction pipette technique. The effects of dopamine (0.1-50 µM) and selective dopamine receptor agonists-D1R agonist SKF-38393 (0.1-50 µM), D2R agonist quinpirole (2.5-50 µM), and D1-D2 receptor heterodimer agonist SKF-83959 (50 µM)-were examined. Results: We found that, when applied to the rod inner segments (RISs), dopamine and dopamine receptor agonists had no effect on photoresponses. In contrast, the rods responded to dopamine and all agonists applied to their outer segments by decreasing sensitivity to light. At the highest tested concentration (50 µM), the most prominent effect on light sensitivity was induced by D1R agonist SKF-38393, while dopamine, D2R agonist quinpirole, and D1-D2 receptor heterodimer agonist SKF-83959 produced somewhat lower and approximately equal effects. Moreover, SKF-38393 reduced sensitivity at all tested concentrations starting from the smallest one (0.1 µM), whereas dopamine and quinpirole started their action from the higher concentrations of 2.5 µM and 50 µM, respectively. In addition, dopamine, SKF-38393, and quinpirole, on average, did not change the intracellular calcium level as judged from the "exchange current", while SKF-83959 increased it by ~1.3 times. Conclusions: Dopamine induces a decrease in rod sensitivity, mostly by reducing the activation rate of the cascade, and to a much lesser extent, speeding up the turning off of the cascade. The sign of the reaction to all tested drugs, lack of selectivity of dopamine and dopamine receptor agonist action, and analysis of factors that determine sensitivity of photoreceptors suggest that, in rod outer segments (ROSs), dopamine action is mediated by D1-D2 receptor heterodimers but not D1R or D2R alone. This work supports the assumption made earlier by other authors that dopamine exercises its regulatory effect via at least two independent mechanisms, which are cAMP and Ca2+ mediated.
Assuntos
Agonistas de Dopamina/farmacologia , Dopamina/farmacologia , Transdução de Sinal Luminoso/efeitos dos fármacos , Ranidae/fisiologia , Receptores de Dopamina D1/agonistas , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Cálcio/metabolismo , Cinética , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Receptores de Dopamina D1/metabolismo , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiação , Fatores de TempoRESUMO
Bryophytes, which comprise liverworts, mosses, and hornworts, are one of the earliest diverging lineages of extant land plants and a key plant group for understanding evolutionary aspects of land plant adaptation. Marchantia polymorpha, a liverwort, has recently been established as a model plant species having molecular genetic tractability. In M. polymorpha, phytochrome is encoded by a single-copy gene, MpPHY, with Mpphy regulating various physiological responses through PHYTOCHROME INTERACTING FACTOR (PIF)-mediated transcriptional regulation. The phytochrome signaling system of M. polymorpha, with its single Mpphy and single PIF (MpPIF), is relatively simple compared with other model plants carrying multiple phytochromes and PIFs. Consequently, investigation of phytochrome signaling using M. polymorpha may provide novel insights into fundamental mechanisms and roles of phytochrome during the course of land plant evolution. This chapter provides a number of basic procedures, along with some tips, for designing and performing experiments with M. polymorpha to study phytochrome signaling.
Assuntos
Marchantia/metabolismo , Fitocromo/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Marchantia/efeitos da radiação , Transdução de Sinais/efeitos da radiaçãoRESUMO
We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHOP23H), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHOP23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction. Abbreviations: CYCS: cytochrome c; bRHOP23H: bovine RHOP23H; Cas9: CRISPR associated protein 9; dpf: days post-fertilization; eGFP: enhanced green fluorescent protein; GNAT1: guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin; HSPA1A/hsp70: heat shock protein of 70 kilodaltons; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1A/1B light chain 3; mRFP: monomeric red fluorescent protein; RHO: rhodopsin; RP: retinitis pigmentosa; RPE65: retinal pigment epithelium-specific 65 kDa protein: sfGFP: superfolding GFP; sgRNA: single guide RNA; WGA: wheat germ agglutinin; RHOp: the Xenopus laevis RHO.2.L promoter.
Assuntos
Autofagia/genética , Autofagia/efeitos da radiação , Transdução de Sinal Luminoso/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/metabolismo , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Autofagossomos/efeitos da radiação , Ritmo Circadiano/genética , Ritmo Circadiano/efeitos da radiação , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Transdução de Sinal Luminoso/efeitos da radiação , Mutação , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Retinose Pigmentar/genética , Rodopsina/química , Rodopsina/genética , Rodopsina/efeitos da radiação , Fatores de Tempo , Xenopus laevis , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
Stimulus- or context-dependent routing of neural signals through parallel pathways can permit flexible processing of diverse inputs. For example, work in mouse shows that rod photoreceptor signals are routed through several retinal pathways, each specialized for different light levels. This light-level-dependent routing of rod signals has been invoked to explain several human perceptual results, but it has not been tested in primate retina. Here, we show, surprisingly, that rod signals traverse the primate retina almost exclusively through a single pathway - the dedicated rod bipolar pathway. Identical experiments in mouse and primate reveal substantial differences in how rod signals traverse the retina. These results require reevaluating human perceptual results in terms of flexible computation within this single pathway. This includes a prominent speeding of rod signals with light level - which we show is inherited directly from the rod photoreceptors themselves rather than from different pathways with distinct kinetics.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transdução de Sinais , Células Amácrinas/metabolismo , Células Amácrinas/efeitos da radiação , Animais , Cinética , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Macaca , Camundongos Endogâmicos C57BL , Estimulação Luminosa , Células Bipolares da Retina/metabolismo , Células Bipolares da Retina/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Razão Sinal-RuídoRESUMO
In cyanobacteria, high light photoactivates the orange carotenoid protein (OCP) that binds to antennae complexes, dissipating energy and preventing the destruction of the photosynthetic apparatus. At low light, OCP is efficiently deactivated by a poorly understood action of the dimeric fluorescence recovery protein (FRP). Here, we engineer FRP variants with defined oligomeric states and scrutinize their functional interaction with OCP. Complemented by disulfide trapping and chemical crosslinking, structural analysis in solution reveals the topology of metastable complexes of OCP and the FRP scaffold with different stoichiometries. Unable to tightly bind monomeric FRP, photoactivated OCP recruits dimeric FRP, which subsequently monomerizes giving 1:1 complexes. This could be facilitated by a transient OCP-2FRP-OCP complex formed via the two FRP head domains, significantly improving FRP efficiency at elevated OCP levels. By identifying key molecular interfaces, our findings may inspire the design of optically triggered systems transducing light signals into protein-protein interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Luz/efeitos adversos , Synechocystis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Reagentes de Ligações Cruzadas/química , Transdução de Sinal Luminoso/fisiologia , Transdução de Sinal Luminoso/efeitos da radiação , Mutagênese Sítio-Dirigida , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiação , Ligação Proteica/efeitos da radiação , Multimerização Proteica/efeitos da radiação , Espectrometria de Fluorescência , Synechocystis/efeitos da radiaçãoRESUMO
In response to blue light, cryptochromes photoexcite and interact with signal partners to transduce signal almost synchronously in plants. The detailed mechanism of CRY-mediated light signaling remains unclear: the photobiochemical reactions of cryptochrome are transient and synchronous, thus making the monitoring and analysis of each step difficult in plant cells. In this study, we reconstituted the Arabidopsis CRY2 signaling pathway in mammalian cells and investigated the biological role of Arabidopsis CRY2 in this heterologous system, eliminating the interferences of other plant proteins. Our results demonstrated that, besides being the light receptor, Arabidopsis CRY2 binds to DNA directly and acts as a transcriptional activator in a blue-light-enhanced manner. Similar to classic transcription factors, we found that the transcriptional activity of CRY2 is regulated by its dimerization and phosphorylation. In addition, CRY2 cooperates with CIB1 to regulate transcription by enhancing the DNA affinity and transcriptional activity of CIB1 under blue light.