RESUMO
Proteins exert their function through protein-protein interactions. In Drosophila, G protein-coupled receptors like rhodopsin (Rh1) interact with a G protein to activate visual signal transduction and with arrestins to terminate activation. Also, membrane proteins like Rh1 engage in protein-protein interactions during folding within the endoplasmic reticulum, during their vesicular transport and upon removal from the cell surface and degradation. Here, we expressed a Rh1-TurboID fusion protein (Rh1::TbID) in Drosophila photoreceptors to identify in vivo Rh1 interaction partners by biotin proximity labeling. We show that Rh1::TbID forms a functional rhodopsin that mediates biotinylation of arrestin 2 in conditions where arrestin 2 interacts with rhodopsin. We also observed biotinylation of Rh1::TbID and native Rh1 as well as of most visual signal transduction proteins. These findings indicate that the signaling components in the rhabdomere approach rhodopsin closely, within a range of ca. 10 nm. Furthermore, we have detected proteins engaged in the maturation of rhodopsin and elements responsible for the trafficking of membrane proteins, resembling potential interaction partners of Rh1. Among these are chaperons of the endoplasmic reticulum, proteins involved in Clathrin-mediated endocytosis as well as previously unnoticed contributors to rhodopsin transportation, such as Rab32, Vap33, or PIP82.
Assuntos
Biotina , Rodopsina , Animais , Drosophila , beta-Arrestina 1 , Proteínas de MembranaRESUMO
Guided ultrasonic waves are used for the inspection of multilayered composite aerospace structures. Calculating the corresponding dispersion diagrams is challenging for thick-walled composites with more than 100 layers, such as in modern rocket booster pressure vessels. The Dispersion Calculator (DC) is an open source software for calculating such dispersion diagrams and mode shapes of guided waves. Attenuation caused by viscoelasticity and fluid-loading makes the dispersion curve tracing much more difficult than in the nonattenuated case because the modal solutions are sought in the complex wavenumber plane. The tracing problem is mastered by a reliable algorithm. Whereas leaky Lamb and Scholte waves in coupled and decoupled cases are modeled using the stiffness matrix method, shear horizontal (SH) waves are traced using the transfer matrix method without facing the numerical instability. Through implementation of mode family specific dispersion equations in both matrix techniques for nonattenuated and attenuated cases, symmetric, antisymmetric, and nonsymmetric leaky Lamb, Scholte, and SH waves can be traced separately with better efficiency and robustness. The capabilities of DC are demonstrated by calculating dispersion diagrams and mode shapes for a viscoelastic composite with 400 layers immersed in water. Results are compared against DISPERSE (Imperial College London, London, UK) for selected cases.
RESUMO
Vertebrate and fly rhodopsins are prototypical GPCRs that have served for a long time as model systems for understanding GPCR signaling. Although all rhodopsins seem to become phosphorylated at their C-terminal region following activation by light, the role of this phosphorylation is not uniform. Two major functions of rhodopsin phosphorylation have been described: (1) inactivation of the activated rhodopsin either directly or by facilitating binding of arrestins in order to shut down the visual signaling cascade and thus eventually enabling a high-temporal resolution of the visual system. (2) Facilitating endocytosis of activated receptors via arrestin binding that in turn recruits clathrin to the membrane for clathrin-mediated endocytosis. In vertebrate rhodopsins the shutdown of the signaling cascade may be the main function of rhodopsin phosphorylation, as phosphorylation alone already quenches transducin activation and, in addition, strongly enhances arrestin binding. In the Drosophila visual system rhodopsin phosphorylation is not needed for receptor inactivation. Its role here may rather lie in the recruitment of arrestin 1 and subsequent endocytosis of the activated receptor. In this review, we summarize investigations of fly rhodopsin phosphorylation spanning four decades and contextualize them with regard to the most recent insights from vertebrate phosphorylation barcode theory.
Assuntos
Drosophila , Rodopsina , Animais , Rodopsina/metabolismo , Drosophila/metabolismo , Arrestina/metabolismo , Arrestinas/metabolismo , Fosforilação , Clatrina/metabolismoRESUMO
Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.
Assuntos
Proteínas de Drosophila , Canais de Potencial de Receptor Transitório , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Luz , Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Transporte Proteico/fisiologiaRESUMO
Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20 , PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.
Assuntos
Proteínas de Drosophila , Fosfolipase D , Canais de Potencial de Receptor Transitório , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Luz , Fosfolipase D/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Transporte Proteico/fisiologia , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Luminescentes/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Vias Visuais/metabolismo , Animais , Transporte ProteicoRESUMO
In Drosophila photoreceptor cells, Ca2+ exerts regulatory functions that control the shape, duration, and amplitude of the light response. Ca2+ also orchestrates light adaptation allowing Drosophila to see in light intensity regimes that span several orders of magnitude ranging from single photons to bright sunlight. The prime source for Ca2+ elevation in the cytosol is Ca2+ influx from the extracellular space through light-activated TRP channels. This Ca2+ influx is counterbalanced by constitutive Ca2+ extrusion via the Na+/Ca2+ exchanger, CalX. The light-triggered rise in intracellular Ca2+ exerts its regulatory functions through interaction with about a dozen well-characterized Ca2+ and Ca2+/CaM binding proteins. In this review we will discuss the dynamic changes in Ca2+ concentration upon illumination of photoreceptor cells. We will present the proteins that are known to interact with Ca2+ (/CaM) and elucidate the physiological functions of these interactions.
Assuntos
Cálcio , Drosophila , Células Fotorreceptoras de Invertebrados , Transdução de Sinais , Animais , Antiporters/metabolismo , Cálcio/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/metabolismo , Luz , Células Fotorreceptoras de Invertebrados/fisiologiaRESUMO
Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Olho/crescimento & desenvolvimento , Processamento Alternativo , Animais , Diferenciação Celular , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mutagênese Sítio-Dirigida , Células Fotorreceptoras/metabolismoRESUMO
Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.
Assuntos
Artefatos , Proteínas de Drosophila/análise , Drosophila/metabolismo , Imuno-Histoquímica/métodos , Luz , Células Fotorreceptoras/química , Animais , Drosophila/química , Proteínas de Drosophila/metabolismo , Endocitose , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Transdução de Sinal Luminoso , Microscopia de Fluorescência , Transporte Proteico , Rodopsina/metabolismo , Transdução de Sinais , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
OBJECTIVES: Left ventricular (LV) hypertrophy in resistant hypertensive patients is associated with a reduced intramyocardial perfusion. Renal sympathetic denervation (RDN) has been shown to reduce blood pressure (BP) and sympathetic tone. We aimed to prospectively investigate the effect of RDN on functional myocardial parameters and myocardial perfusion reserve (MPR) using cardiac magnetic resonance imaging (cMRI) in patients with resistant hypertension. METHODS: A total of 15 resistant hypertensive patients (11 male individuals, mean age 62±13 y) were included. Adenosine stress-induced cMRI was performed at baseline, 3, 6, and 12 months after RDN. RDN was performed using a single soft-tip radiofrequency catheter (Symplicity). cMRI semiquantitative perfusion analysis was performed using the upslope of myocardial signal enhancement to derive the myocardial perfusion reserve index. RESULTS: Both systolic-BP and diastolic-BP significantly decreased from 148±14 to 133±14 mm Hg and 87±14 to 80±10 mm Hg, respectively (P<0.05). LV septal wall thickness was significantly reduced (P<0.001). LV ejection fraction and MPR lacked significant trends 12 months after RDN. CONCLUSIONS: In this pilot study, RDN significantly reduced LV mass and LV septal wall thickness, as diagnosed by cMRI, with no significant changes in MPR. cMRI may help in diagnosing clinically relevant changes of functional myocardial parameters after interventional therapy in resistant hypertensive patients.
Assuntos
Coração/diagnóstico por imagem , Hipertensão/complicações , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/cirurgia , Imageamento por Ressonância Magnética/métodos , Simpatectomia/métodos , Feminino , Seguimentos , Coração/fisiopatologia , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Ventrículos do Coração/cirurgia , Humanos , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/patologia , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Projetos Piloto , Estudos Prospectivos , Artéria Renal , Resultado do TratamentoRESUMO
Drosophila retinal degeneration C (RDGC) is the founding member of the PPEF family of protein phosphatases. RDGC mediates dephosphorylation of the visual pigment rhodopsin and the TRP ion channel. From the rdgC locus, three protein isoforms, termed RDGC-S, -M, and -L, with different N-termini are generated. Due to fatty acylation, RDGC-M and -L are attached to the plasma membrane while RDGC-S is soluble. To assign physiological roles to these RDGC isoforms, we constructed flies that express various combinations of RDGC protein isoforms. Expression of the RDGC-L isoform alone did not fully prevent rhodopsin hyperphosphorylation and resulted in impaired photoreceptor physiology and in decelerated TRP dephosphorylation at Ser936. However, expression of RDGC-L alone as well as RDGC-S/M was sufficient to prevent degeneration of photoreceptor cells which is a hallmark of the rdgC null mutant. Membrane-attached RDGC-M displayed higher activity of TRP dephosphorylation than the soluble isoform RDGC-S. Taken together, in vivo activities of RDGC splice variants are controlled by their N-termini.
Assuntos
Processamento Alternativo , Proteínas de Ligação ao Cálcio , Proteínas de Drosophila , Proteínas de Membrana , Fosfoproteínas Fosfatases , Células Fotorreceptoras de Invertebrados/enzimologia , Acilação , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Domínios Proteicos , Rodopsina/genética , Rodopsina/metabolismoRESUMO
BACKGROUND: Blood flow through the cavities of the heart and great vessels is pulsatile and is subject to time and multidirectional variations. To date, the recording of blood flow in multiple directions and phases has been limited. 4D-flow MRI offers advantages for the recording, visualization and analysis of blood flow. METHOD: The status quo of the method was summarized through analysis with the PubMed database using the keywords "4D-flow MRI, phase-contrast magnetic resonance imaging, MR flow imaging/visualization, MR flow quantification, 3âD cine (time-resolved) phase-contrast CMR, three-directional velocity-encoding MRI". RESULTS/CONCLUSION: This review summarizes the current status of the technical development of 4D-flow MRI, discusses its advantages and disadvantages and describes clinical applications. Finally, the most important principles and parameters are explained to give the reader relevant information about clinical indications, postprocessing methods and limitations of the method. KEY POINTS: · 4D-Fluss-MRT. · 3-dimensionale zeitaufgelöste Phasenkontrast-MRT. · Flussanalyse-MRT (Wall-Shear-Stress/Druckgradienten-Messung/Vortex-Fluss/turbulente kinetische Energie/Flussgeschwindigkeit/Flussrate). CITATION FORMAT: · Sträter A, Huber A, Rudolph J etâal. 4D-Flow MRI: Technique and Applications. Fortschr Röntgenstr 2018; 190: 1025â-â1035.
Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Neoplasias da Próstata/diagnóstico por imagem , Humanos , Masculino , Próstata/diagnóstico por imagem , Sensibilidade e EspecificidadeRESUMO
Protein phosphorylation is an abundant molecular switch that regulates a multitude of cellular processes. In contrast to other subfamilies of phosphoprotein phosphatases, the PPEF subfamily is only poorly investigated. Drosophila retinal degeneration C (RDGC) constitutes the founding member of the PPEF subfamily. RDGC dephosphorylates the visual pigment rhodopsin and the ion channel TRP.However, rdgC null mutant flies exhibit rhodopsin and TRP hyperphosphorylation, altered photoreceptor physiology, and retinal degeneration. Here, we report the identification of a third RDGC protein variant and show that the three RDGC isoforms harbor different N-termini that determine solubility and subcellular targeting due to fatty acylation. Taken together, solubility and subcellular targeting of RDGC splice variants are determined by their N-termini.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acilação/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação ao Cálcio/química , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Espaço Intracelular/metabolismo , Mutação/fisiologia , Fosfoproteínas Fosfatases/química , Domínios Proteicos/genética , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/genética , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , SolubilidadeAssuntos
Implante de Prótese Vascular , Circulação Colateral , Procedimentos Endovasculares , Oclusão de Enxerto Vascular/cirurgia , Obstrução da Artéria Renal/cirurgia , Artéria Renal/cirurgia , Circulação Renal , Prótese Vascular , Implante de Prótese Vascular/efeitos adversos , Implante de Prótese Vascular/instrumentação , Angiografia por Tomografia Computadorizada , Stents Farmacológicos , Procedimentos Endovasculares/instrumentação , Feminino , Oclusão de Enxerto Vascular/diagnóstico por imagem , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Pessoa de Meia-Idade , Artéria Renal/diagnóstico por imagem , Artéria Renal/fisiopatologia , Obstrução da Artéria Renal/diagnóstico por imagem , Obstrução da Artéria Renal/fisiopatologia , Reoperação , Fatores de Tempo , Resultado do Tratamento , Grau de Desobstrução VascularRESUMO
PURPOSE: To examine CTP of the brain in real patient data after reducing tube current down to 80â¯mAs to decrease radiation dose. METHODS: CTP was acquired in 60 suspected stroke patients with 80 (n: 30) or 160 (n: 30) mAs. Data were analyzed retrospectively by two independent readers. SNR, perfusion maps and image quality were compared in hypoperfused and non-affected areas. RESULTS: SNR was significantly higher in CTP with 160â¯mAs compared to 80â¯mAs (pâ¯<â¯0.001) in non-affected regions, but there was no significant difference in hypoperfused regions. Overall, images with 80â¯mAs were rated worse than the ones with 160â¯mAs (3.0⯱â¯0.7 versus 4.0⯱â¯0.7), however, still as sufficient to detect proximal vessel occlusions. CONCLUSION: Tube current of 80â¯mAs is still sufficient for the detection of perfusion deficits of proximal vessel occlusions.
Assuntos
Encéfalo/diagnóstico por imagem , Doses de Radiação , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Acidente Vascular Cerebral/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Vasos Sanguíneos/diagnóstico por imagem , Encéfalo/irrigação sanguínea , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Perfusão , Estudos RetrospectivosRESUMO
Guided waves are used for the non-destructive evaluation in automotive and aerospace industries. There is a trend leaning away from isotropic materials to the manufacturing based on composites. However, the elastic wave dynamics in such materials is considerably more complicated. Much effort has been committed to the calculation of guided waves' dispersion curves in composites. Lots of methods and tools are available, but it becomes difficult when there are more than one hundred layers. In this paper the calculation of dispersion diagrams and mode shapes using the stiffness matrix method is demonstrated. Boundary conditions are implemented into the stiffness matrix method that allow for the separate tracing of the various mode families. Shear horizontal modes are modeled with the transfer matrix method without facing any numerical instability. It is elucidated just how the occurrence of the mode families depends on the system's symmetry and wave propagation direction. As a result, the robustness and reliability of guided wave modeling by using the stiffness method is improved, and more information about the modes is yielded. This is demonstrated on exemplary layups of the fiber reinforced polymer T800/913, with up to 400 layers. Referencing is made against results from DISPERSE® (Imperial College London, London, UK) for selected cases.
RESUMO
Drosophila photoreceptors respond to oscillating light of high frequency (â¼100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca2+-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trpS936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trpS936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trpS936A flies reveal light-induced current (LIC) with short latency relative to trpWT or trpS936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.
Assuntos
Luz , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Drosophila , Cinética , FosforilaçãoRESUMO
Drosophila photoreceptors respond to oscillating light of high frequency (â¼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca2+-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila (trpS936A ) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid (trpS936D ) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions.SIGNIFICANCE STATEMENTDrosophila photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤â¼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and in vivo electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca2+-dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions.
Assuntos
Adaptação Ocular/fisiologia , Proteínas de Drosophila/metabolismo , Estimulação Luminosa/métodos , Células Fotorreceptoras de Invertebrados/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Eletrorretinografia/métodos , Feminino , Masculino , Fosforilação/fisiologiaRESUMO
To prospectively compare image quality and myocardial T1 relaxation times of modified Look-Locker inversion recovery (MOLLI) imaging at 3.0 T (T) acquired with patient-adaptive dual-source (DS) and conventional single-source (SS) radiofrequency (RF) transmission. Pre- and post-contrast MOLLI T1 mapping using SS and DS was acquired in 27 patients. Patient wise and segment wise analysis of T1 times was performed. The correlation of DS MOLLI measurements with a reference spin echo sequence was analysed in phantom experiments. DS MOLLI imaging reduced T1 standard deviation in 14 out of 16 myocardial segments (87.5%). Significant reduction of T1 variance could be obtained in 7 segments (43.8%). DS significantly reduced myocardial T1 variance in 16 out of 25 patients (64.0%). With conventional RF transmission, dielectric shading artefacts occurred in six patients causing diagnostic uncertainty. No according artefacts were found on DS images. DS image findings were in accordance with conventional T1 mapping and late gadolinium enhancement (LGE) imaging. Phantom experiments demonstrated good correlation of myocardial T1 time between DS MOLLI and spin echo imaging. Dual-source RF transmission enhances myocardial T1 homogeneity in MOLLI imaging at 3.0 T. The reduction of signal inhomogeneities and artefacts due to dielectric shading is likely to enhance diagnostic confidence.
Assuntos
Cardiopatias/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Imagem Cinética por Ressonância Magnética/métodos , Miocárdio/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artefatos , Meios de Contraste/administração & dosagem , Feminino , Gadolínio DTPA/administração & dosagem , Cardiopatias/patologia , Humanos , Imagem Cinética por Ressonância Magnética/instrumentação , Masculino , Pessoa de Meia-Idade , Imagens de Fantasmas , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Adulto JovemRESUMO
The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons. Here we used transgenic Drosophila, in which the mouse OPN4 replaced the native Rh1 photopigment of Drosophila R1-6 photoreceptors, resulting in deformed rhabdomeric structure. Immunocytochemistry revealed OPN4 expression at the base of the rhabdomeres, mainly at the rhabdomeral stalk. Measurements of the early receptor current, a linear manifestation of photopigment activation, indicated large expression of OPN4 in the plasma membrane. Comparing the early receptor current amplitude and action spectra between WT and the Opn4-expressing Drosophila further indicated that large quantities of a blue absorbing photopigment were expressed, having a dark stable blue intermediate state. Strikingly, the light-induced current of the Opn4-expressing fly photoreceptors was â¼40-fold faster than that of ipRGC. Furthermore, an intense white flash induced a small amplitude prolonged dark current composed of discrete unitary currents similar to the Drosophila single photon responses. The induction of prolonged dark currents by intense blue light could be suppressed by a following intense green light, suggesting induction and suppression of prolonged depolarizing afterpotential. This is the first demonstration of heterologous functional expression of mammalian OPN4 in the genetically emendable Drosophila photoreceptors. Moreover, the fast OPN4-activated ionic current of Drosophila photoreceptors relative to that of mouse ipRGC, indicates that the slow light response of ipRGC does not arise from an intrinsic property of melanopsin.