RESUMEN
Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature: light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below â¼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Criptocromos , Proteínas de la Matriz de Golgi , Optogenética , Animales , Membrana Celular/química , Membrana Celular/efectos de la radiación , Análisis por Conglomerados , Citoplasma/química , Citoplasma/efectos de la radiación , Luz , Criptocromos/química , Criptocromos/efectos de la radiación , Proteínas de la Matriz de Golgi/química , Proteínas de la Matriz de Golgi/efectos de la radiación , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/efectos de la radiación , Multimerización de ProteínaRESUMEN
Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.
Asunto(s)
Benzoquinonas/metabolismo , Criptocromos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas del Ojo/metabolismo , Flavinas/metabolismo , Animales , Criptocromos/genética , Criptocromos/efectos de la radiación , Proteínas de Drosophila/genética , Proteínas de Drosophila/efectos de la radiación , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Espectroscopía de Resonancia por Spin del Electrón , Proteínas del Ojo/genética , Proteínas del Ojo/efectos de la radiación , Luz , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Relación Estructura-ActividadRESUMEN
Cryptochromes (CRYs) are evolutionarily conserved photoreceptors that mediate various light-induced responses in bacteria, plants, and animals. Plant cryptochromes govern a variety of critical growth and developmental processes including seed germination, flowering time and entrainment of the circadian clock. CRY's photocycle involves reduction of their flavin adenine dinucleotide (FAD)-bound chromophore, which is completely oxidized in the dark and semi to fully reduced in the light signaling-active state. Despite the progress in characterizing cryptochromes, important aspects of their photochemistry, regulation, and light-induced structural changes remain to be addressed. In this study, we determine the crystal structure of the photosensory domain of Arabidopsis CRY2 in a tetrameric active state. Systematic structure-based analyses of photo-activated and inactive plant CRYs elucidate distinct structural elements and critical residues that dynamically partake in photo-induced oligomerization. Our study offers an updated model of CRYs photoactivation mechanism as well as the mode of its regulation by interacting proteins.
Asunto(s)
Proteínas de Arabidopsis/efectos de la radiación , Criptocromos/efectos de la radiación , Secuencia de Aminoácidos , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Estructura Cuaternaria de ProteínaRESUMEN
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.
Asunto(s)
Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Fototransducción/efectos de la radiación , Luz , Optogenética , Criptocromos/genética , Criptocromos/metabolismo , Criptocromos/efectos de la radiación , Flavoproteínas/genéticaRESUMEN
Infrared (IR) spectroscopy is an undoubtedly valuable tool for analyzing vibrations, conformational changes, and chemical reactions of biological macromolecules. Currently, there is a lack of theoretical methods to create a model successfully and efficiently simulate and interpret the origin of the spectral signatures, which are often complex to analyze. Here, we develop a new method for IR vibrational spectroscopy based on analytic second derivatives of electrostatic embedding QM/MM energy, the computation of electric dipole moments with respect to nuclear perturbations and the localization of normal modes. In addition to the IR spectrum, the method can provide the origin of each peak from clearly identified molecular motions of constituent fragments. As a proof of concept, we analyze the IR spectra of flavin adenine dinucleotides in water and in Arabidopsis thaliana cryptochrome proteins for four redox forms, in addition to the difference IR spectra before and after illumination with blue light. We show that the main peaks in the difference spectrum are due to N-H hydrogen out-of-plane motions and hydrogen bendings.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Criptocromos/química , Proteínas de Arabidopsis/efectos de la radiación , Criptocromos/efectos de la radiación , Flavina-Adenina Dinucleótido/química , Luz , Oxidación-Reducción , Prueba de Estudio Conceptual , Teoría Cuántica , Espectrofotometría InfrarrojaRESUMEN
Photoactivation of cryptochrome-family proteins by blue light is a well-established reaction regulating physiology of plants, fungi, bacteria, insects and birds, while impact of blue light on cryptochrome synthesis and/or activity in human non-visual cells remains unknown. Here, we show that 453 nm blue light induces cryptochrome 1 (CRY1) accumulation in human keratinocytes and the hair follicle. CRY1 is prominently expressed in the human anagen hair follicle, including epithelial stem cells. Specific silencing of CRY1 promotes catagen, while stimulation of CRY1 by KL001 prolongs anagen ex vivo by altering the expression of genes involved in apoptosis and proliferation. Together, our study identifies a role for CRY1 in sustaining human hair growth. Previously, we demonstrated positive effects of 453 nm blue light on hair growth ex vivo. Taken all together, our study suggests that CRY1 might mediate blue light-dependent positive effects on hair growth.
Asunto(s)
Criptocromos/metabolismo , Criptocromos/efectos de la radiación , Folículo Piloso/metabolismo , Apoptosis/efectos de los fármacos , Carbazoles/farmacología , Color , Criptocromos/genética , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Folículo Piloso/efectos de la radiación , Humanos , Queratinocitos/metabolismo , Sulfonamidas/farmacologíaRESUMEN
After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes.
Asunto(s)
Arabidopsis/metabolismo , Criptocromos/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/análisis , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Sistema Libre de Células , Cotiledón/anatomía & histología , Cotiledón/efectos de los fármacos , Cotiledón/efectos de la radiación , Criptocromos/metabolismo , Criptocromos/efectos de la radiación , Medios de Cultivo , Hipocótilo/anatomía & histología , Hipocótilo/efectos de los fármacos , Hipocótilo/efectos de la radiación , Procesamiento de Imagen Asistido por Computador , Luz , Fenotipo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/efectos de la radiación , Proteínas Recombinantes/biosíntesis , Plantones/efectos de los fármacos , Plantones/efectos de la radiación , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADHâ) upon blue light absorption. Herein, we used millisecond flash photolysis to investigate the cCRY4 photocycle, in both the presence and absence of dithiothreitol (DTT); detecting the anion radical form of FAD (FADâ-) under both conditions. Using spectral data obtained during flash photolysis and UV-visible photospectroscopy, we estimated the absolute absorbance spectra of the photointermediates, thus allowing us to decompose each spectrum into its individual components. Notably, in the absence of DTT, approximately 37% and 63% of FADâ- was oxidized to FADOX and protonated to form FADHâ, respectively. Singular value decomposition analysis suggested the presence of two FADâ- molecular species, each of which was destined to be oxidized to FADOX or protonated to FADHâ. A tyrosine neutral radical was also detected; however, it likely decayed concomitantly with the oxidation of FADâ-. On the basis of these results, we considered the occurrence of bifurcation prior to FADâ- generation, or during FADâ- oxidization, and discussed the potential role played by the tyrosine radical in the radical pair mechanism.
Asunto(s)
Proteínas Aviares/química , Criptocromos/química , Animales , Proteínas Aviares/efectos de la radiación , Pollos , Criptocromos/efectos de la radiación , Ditiotreitol/química , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/efectos de la radiación , Radicales Libres/química , Luz , Oxidación-Reducción , Fotólisis , Tirosina/químicaRESUMEN
Cryptochromes (CRYs) are flavin-binding proteins that sense blue and near-ultraviolet light and participate in the photoreactions of organisms and the regulation of biological clocks. In this study, the complete open reading frame (ORF) of CiCRY-DASH1 (GenBank ID MK392361), encoding one kind of cryptochrome, was cloned from the Antarctic microalga Chlamydomonas sp. ICE-L. The quantitative real-time PCR study showed that the CiCRY-DASH1 had the highest expression at 5 °C and salinity of 32. The CiCRY-DASH1 was positively regulated by blue, yellow, or red light. Moreover, the CiCRY-DASH1 can positively respond to extreme polar day and night treatment and exhibit a certain circadian rhythm, which indicated that CiCRY-DASH1 participated in the circadian clock and its expression was regulated by circadian rhythms. And the CiCRY-DASH1 was more noticeably affected by ultraviolet-B radiation than ultraviolet-A radiation, indicating ultraviolet-B light does obvious damage to Antarctic microalgae.
Asunto(s)
Chlamydomonas/genética , Criptocromos/metabolismo , Criptocromos/efectos de la radiación , Secuencia de Aminoácidos , Regiones Antárticas , Chlamydomonas/química , Chlamydomonas/crecimiento & desarrollo , Clorofila/química , Clorofila/efectos de la radiación , Ritmo Circadiano , Clonación Molecular , Biología Computacional , Criptocromos/química , Criptocromos/genética , ADN Complementario/genética , Expresión Génica , Luz , Microalgas/metabolismo , Sistemas de Lectura Abierta , Filogenia , Salinidad , Homología de Secuencia de Aminoácido , Temperatura , Rayos UltravioletaRESUMEN
MAIN CONCLUSION: The cryptochrome photoreceptor mutant cry2I404F exhibits hyperactivity in the dark, hypersensitivity in different light conditions, and in contrast to the wild-type protein, its flavin chromophore is reducible even in the absence of light. Plant cryptochromes (cry) are blue-light photoreceptors involved in multiple signaling pathways and various photomorphogenic responses. One biologically hyperactive mutant of a plant cryptochrome that was previously characterized is Arabidopsis cry1L407F (Exner et al. in Plant Physiol 154:1633-1645, 2010). Protein sequence alignments of different cryptochromes revealed that L407 in cry1 corresponds to I404 in cry2. Point mutation of Ile to Phe in cry2 in this position created a novel mutant. The present study provided a baseline data on the elucidation of the properties of cry2I404F. This mutant was still able to bind ATP-triggering conformational changes, as confirmed by partial tryptic digestion and thermo-FAD assays. Surprisingly, the FAD cofactor of cry2I404F was reduced by the addition of reductant even in the absence of light. In vivo, cry2I404F exhibited a cop phenotype in the dark and hypersensitivity to various light conditions compared to cry2 wild type. Overall, these data suggest that the hypersensitivity to red and blue light and hyperactivity of this novel mutant in the dark can be mostly accounted to structural alterations brought forth by the Ile to Phe mutation at position 404 that allows reduction of the flavin chromophore even in the absence of light.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Flavinas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/efectos de la radiación , Criptocromos/química , Criptocromos/efectos de la radiación , Luz , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Conformación Proteica , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Drosophila CRYPTOCHROME (dCRY) mediates electrophysiological depolarization and circadian clock resetting in response to blue or ultraviolet (UV) light. These light-evoked biological responses operate at different timescales and possibly through different mechanisms. Whether electron transfer down a conserved chain of tryptophan residues underlies biological responses following dCRY light activation has been controversial. To examine these issues in in vivo and in ex vivo whole-brain preparations, we generated transgenic flies expressing tryptophan mutant dCRYs in the conserved electron transfer chain and then measured neuronal electrophysiological phototransduction and behavioral responses to light. Electrophysiological-evoked potential analysis shows that dCRY mediates UV and blue-light-evoked depolarizations that are long lasting, persisting for nearly a minute. Surprisingly, dCRY appears to mediate red-light-evoked depolarization in wild-type flies, absent in both cry-null flies, and following acute treatment with the flavin-specific inhibitor diphenyleneiodonium in wild-type flies. This suggests a previously unsuspected functional signaling role for a neutral semiquinone flavin state (FADHâ¢) for dCRY. The W420 tryptophan residue located closest to the FAD-dCRY interaction site is critical for blue- and UV-light-evoked electrophysiological responses, while other tryptophan residues within electron transfer distance to W420 do not appear to be required for light-evoked electrophysiological responses. Mutation of the dCRY tryptophan residue W342, more distant from the FAD interaction site, mimics the cry-null behavioral light response to constant light exposure. These data indicate that light-evoked dCRY electrical depolarization and clock resetting are mediated by distinct mechanisms.
Asunto(s)
Relojes Biológicos/efectos de la radiación , Criptocromos/efectos de la radiación , Proteínas de Drosophila/efectos de la radiación , Proteínas del Ojo/efectos de la radiación , Animales , Animales Modificados Genéticamente , Ritmo Circadiano/efectos de la radiación , Criptocromos/genética , Criptocromos/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Locomoción/efectos de la radiación , Mutación , Triptófano/genéticaRESUMEN
Cryptochromes (CRYs) are an ubiquitously occurring class of photoreceptors, which are important for regulating the circadian rhythm of animals via a time-delayed transcription-translation feedback loop (TTFL). Due to their protein architecture and common FAD chromophore, they belong to the same superfamily as photolyases (PHLs), an enzyme class that repairs UV-induced DNA lesions upon blue light absorption. Apart from their different functions the only prominent structural difference between CRY and PHL is the highly variable C-terminal extension (CTE) of the former. The nature of the CTE is still unclear and highly speculated. In this study, we show by hydrogen/deuterium exchange and subsequent mass-spectrometric analysis that the CTE of the animal-like cryptochrome from the green algae Chlamydomonas reinhardtii (CraCRY) binds to the surface of the photolyase homology region, which flanks the DNA binding site. We also compared the fully oxidized and fully reduced states of the flavoprotein and designed a tool, so called light chamber, for automated HDX-MS measurements of photoreceptors in defined photostates. We could observe some striking differences between the two photostates and propose a model for light-dependent switching of this bifunctional cryptochrome.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Criptocromos/química , Luz , Animales , Sitios de Unión , Criptocromos/metabolismo , Criptocromos/efectos de la radiación , Oxidación-Reducción , Conformación ProteicaRESUMEN
MAIN CONCLUSION: Arabidopsis cryptochrome mediates responses to magnetic fields that have been applied in the absence of light, consistent with flavin reoxidation as the primary detection mechanism. Cryptochromes are highly conserved blue-light-absorbing flavoproteins which have been linked to the perception of electromagnetic stimuli in numerous organisms. These include sensing the direction of the earth's magnetic field in migratory birds and the intensity of magnetic fields in insects and plants. When exposed to light, cryptochromes undergo flavin reduction/reoxidation redox cycles leading to biological activation which generate radical pairs thought to be the basis for magnetic sensitivity. However, the nature of the magnetically sensitive radical pairs and the steps at which they act during the cryptochrome redox cycle are currently a matter of debate. Here, we investigate the response of Arabidopsis cryptochrome-1 in vivo to a static magnetic field of 500 µT (10 × earth's field) using both plant growth and light-dependent phosphorylation as an assay. Cryptochrome responses to light were enhanced by the magnetic field, as indicated by increased inhibition of hypocotyl elongation and increased cryptochrome phosphorylation. However, when light and dark intervals were given intermittently, a plant response to the magnetic field was observed even when the magnetic field was given exclusively during the dark intervals between light exposures. This indicates that the magnetically sensitive reaction step in the cryptochrome photocycle must occur during flavin reoxidation, and likely involves the formation of reactive oxygen species.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Criptocromos/fisiología , Flavinas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Western Blotting , Criptocromos/efectos de la radiación , Oscuridad , Hipocótilo/crecimiento & desarrollo , Campos Magnéticos , Oxidación-Reducción , FosforilaciónRESUMEN
Drosophila melanogaster cryptochrome functions as the primary blue-light receptor that mediates circadian photo entrainment. Absorption of a photon leads to reduction of the protein-bound FAD via consecutive electron transfer along a conserved tryptophan tetrad resembling the signalling state required for conformational changes and induction of subsequent signalling cascades. However, how the initial photochemistry and subsequent dark processes leading to downstream signalling are linked to each other at the molecular level is still poorly understood. Here, we investigated in detail the initial photochemical events in DmCRY by time-resolved and stationary absorption spectroscopy combined with quantum chemical and molecular dynamics calculations. We resolved the early events along the conserved tryptophan tetrad and the final deprotonation of the terminal tryptophanyl radical cation. These initial events lead to conformational changes, such as the known C-terminal tail release, Trp decomposition, and finally FAD release providing evidence that DmCRY does not undergo a photocycle. We propose that light is a negative regulator of DmCRY stability even under in vitro conditions where the proteasomal machinery is missing, that is in line with its biological function, i.e. entrainment of the circadian clock.
Asunto(s)
Criptocromos/química , Criptocromos/efectos de la radiación , Drosophila melanogaster/química , Animales , Electrones , Flavina-Adenina Dinucleótido/química , Luz , Modelos Químicos , Simulación de Dinámica Molecular , Oxidación-Reducción , Conformación Proteica , Protones , Teoría Cuántica , Triptófano/químicaRESUMEN
Objective: To investigate the influence of extremely low-frequency magnetic field on periodical expression of cryptochrome (Cry) gene in mouse embryonic fibroblast NIH3T3 cells. Methods: The NIH3T3 cells were divided into magnetic field group and sham-exposure group. The NIH3T3 cells in the magnetic field group were stimulated by horse serum and then exposed to an extremely low-frequency magnetic field (50 Hz and 0.3 mT) for 48 hours, and those in the sham-exposure group were also stimulated by horse serum and then exposed to a coil for 48 hours. The NIH3T3 cells were collected, total RNA was extracted, and cDNA was obtained via reverse transcription. Real-time fluorescent quantitative RT-PCR was used to measure the changes in transcription cycles of Cry and Period genes in both groups. Results: There was no significant difference in the proliferation rate at 0, 12, 24, and 48 hours of exposure between the two groups (P>0.05) . Both sham-exposure group and magnetic field group showed a rhythmic change in the expression of Cry gene, and compared with the sham-exposure group, the magnetic field group had a significantly shortened circadian rhythm of Cry gene in NIH3T3 cells (t=2.57, P<0.05) . Both groups had rhythmic and periodical expression of Period gene and there was no significant difference between the two groups (t=0.70, P>0.05) . Conclusion: Extremely low-frequency magnetic field can significantly shorten the circadian rhythm of Cry gene in mouse embryonic fibroblasts, while there is no significant change in the circadian rhythm of Period gene.
Asunto(s)
Ritmo Circadiano/efectos de la radiación , Criptocromos/efectos de la radiación , Campos Magnéticos , Células 3T3 NIH/efectos de la radiación , Animales , RatonesRESUMEN
Cryptochromes are blue-light receptors that regulate development and the circadian clock in plants and animals. We found that Arabidopsis cryptochrome 2 (CRY2) undergoes blue light-dependent homodimerization to become physiologically active. We identified BIC1 (blue-light inhibitor of cryptochromes 1) as an inhibitor of plant cryptochromes that binds to CRY2 to suppress the blue light-dependent dimerization, photobody formation, phosphorylation, degradation, and physiological activities of CRY2. We hypothesize that regulated dimerization governs homeostasis of the active cryptochromes in plants and other evolutionary lineages.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Criptocromos/química , Criptocromos/efectos de la radiación , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Homeostasis , Luz , Fosforilación , Procesos Fotoquímicos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Unión Proteica , Multimerización de Proteína/efectos de la radiación , Proteolisis/efectos de la radiación , Transcriptoma/efectos de la radiaciónRESUMEN
Many animals are able to sense the Earth's geomagnetic field to enable behaviors such as migration. It is proposed that the magnitude and direction of the geomagnetic field modulates the activity of cryptochrome (CRY) by influencing photochemical radical pair intermediates within the protein. However, this proposal will remain theoretical until a CRY-dependent effect on a receptor neuron is shown to be modified by an external magnetic field (MF). It is established that blue-light (BL) photoactivation of CRY is sufficient to depolarize and activate Drosophila neurons. Here, we show that this CRY-dependent effect is significantly potentiated in the presence of an applied MF (100 mT). We use electrophysiological recordings from larval identified motoneurons, in which CRY is ectopically expressed, to show that BL-dependent depolarization of membrane potential and increased input resistance are markedly potentiated by an MF. Analysis of membrane excitability shows that these effects of MF exposure evoke increased action potential firing. Almost nothing is known about the mechanism by which a magnetically induced change in CRY activity might produce a behavioral response. We further report that specific structural changes to the protein alter the impact of the MF in ways that are strikingly similar to those from recent behavioral studies into the magnetic sense of Drosophila These observations provide the first direct experimental evidence to support the hypothesis that MF modulation of CRY activity is capable of influencing neuron activity to allow animal magnetoreception. SIGNIFICANCE STATEMENT: The biophysical mechanism of animal magnetoreception is still unclear. The photoreceptor protein cryptochrome has risen to prominence as a candidate magnetoreceptor molecule based on multiple reports derived from behavioral studies. However, the role of cryptochrome as a magnetoreceptor remains controversial primarily because of a lack of direct experimental evidence linking magnetic field (MF) exposure to a change in neuronal activity. Here, we show that exposure to an MF (100 mT) is sufficient to potentiate the ability of light-activated cryptochrome to increase neuronal action potential firing. Our results provide critical missing evidence to show that the activity of cryptochrome is sensitive to an external MF that is capable of modifying animal behavior.
Asunto(s)
Criptocromos/efectos de la radiación , Luz , Campos Magnéticos , Neuronas/efectos de los fármacos , Potenciales de Acción/efectos de la radiación , Animales , Drosophila melanogaster , Larva , Potenciales de la Membrana/efectos de la radiación , Neuronas Motoras/efectos de la radiaciónRESUMEN
Migratory birds have a light-dependent magnetic compass, the mechanism of which is thought to involve radical pairs formed photochemically in cryptochrome proteins in the retina. Theoretical descriptions of this compass have thus far been unable to account for the high precision with which birds are able to detect the direction of the Earth's magnetic field. Here we use coherent spin dynamics simulations to explore the behavior of realistic models of cryptochrome-based radical pairs. We show that when the spin coherence persists for longer than a few microseconds, the output of the sensor contains a sharp feature, referred to as a spike. The spike arises from avoided crossings of the quantum mechanical spin energy-levels of radicals formed in cryptochromes. Such a feature could deliver a heading precision sufficient to explain the navigational behavior of migratory birds in the wild. Our results (i) afford new insights into radical pair magnetoreception, (ii) suggest ways in which the performance of the compass could have been optimized by evolution, (iii) may provide the beginnings of an explanation for the magnetic disorientation of migratory birds exposed to anthropogenic electromagnetic noise, and (iv) suggest that radical pair magnetoreception may be more of a quantum biology phenomenon than previously realized.
Asunto(s)
Migración Animal/efectos de la radiación , Aves/fisiología , Criptocromos/química , Campos Magnéticos , Modelos Biológicos , Teoría Cuántica , Animales , Simulación por Computador , Criptocromos/fisiología , Criptocromos/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Fenómenos de Retorno al Lugar Habitual/efectos de la radiación , Magnetometría/métodos , Modelos Químicos , Dosis de Radiación , Transducción de Señal/efectos de la radiaciónRESUMEN
MAIN CONCLUSION: In this review we focus on the role of SPA proteins in light signalling and discuss different aspects, including molecular mechanisms, specificity, and evolution. The ability of plants to perceive and respond to their environment is key to their survival under ever-changing conditions. The abiotic factor light is of particular importance for plants. Light provides plants energy for carbon fixation through photosynthesis, but also is a source of information for the adaptation of growth and development to the environment. Cryptochromes and phytochromes are major photoreceptors involved in control of developmental decisions in response to light cues, including seed germination, seedling de-etiolation, and induction of flowering. The SPA protein family acts in complex with the E3 ubiquitin ligase COP1 to target positive regulators of light responses for degradation by the 26S proteasome to suppress photomorphogenic development in darkness. Light-activated cryptochromes and phytochromes both repress the function of COP1, allowing accumulation of positive photomorphogenic factors in light. In this review, we highlight the role of the SPA proteins in this process and discuss recent advances in understanding how SPAs link light-activation of photoreceptors and downstream signaling.