FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

The phytochrome (phy) family of photoreceptors is of crucial importance throughout the life cycle of higher plants. Light-induced nuclear import is required for most phytochrome responses. Nuclear accumulation of phyA is dependent on two related proteins called FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like), with FHY1 playing the predominant function. The transcription of FHY1 and FHL are controlled by FHY3 (Far-red elongated HYpocotyl 3) and FAR1 (FAr-red impaired Response 1), a related pair of transcription factors, which thus indirectly control phyA nuclear accumulation. FHY1 and FHL preferentially interact with the light-activated form of phyA, but the mechanism by which they enable photoreceptor accumulation in the nucleus remains unsolved. Sequence comparison of numerous FHY1-related proteins indicates that only the NLS located at the N-terminus and the phyA-interaction domain located at the C-terminus are conserved. We demonstrate that these two parts of FHY1 are sufficient for FHY1 function. phyA nuclear accumulation is inhibited in the presence of high levels of FHY1 variants unable to enter the nucleus. Furthermore, nuclear accumulation of phyA becomes light- and FHY1-independent when an NLS sequence is fused to phyA, strongly suggesting that FHY1 mediates nuclear import of light-activated phyA. In accordance with this idea, FHY1 and FHY3 become functionally dispensable in seedlings expressing a constitutively nuclear version of phyA. Our data suggest that the mechanism uncovered in Arabidopsis is conserved in higher plants. Moreover, this mechanism allows us to propose a model explaining why phyA needs a specific nuclear import pathway.

Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.

OBJECTIVE: To weight the rod-, cone-, and melanopsin-mediated activation of the retinal ganglion cells, which drive the pupil light reflex by varying the light stimulus wavelength, intensity, and duration. DESIGN: Experimental study. PARTICIPANTS: Forty-three subjects with normal eyes and 3 patients with neuroretinal visual loss. METHODS: A novel stimulus paradigm was developed using either a long wavelength (red) or short wavelength (blue) light given as a continuous Ganzfeld stimulus with stepwise increases over a 2 log-unit range. The pupillary movement before, during, and after the light stimulus was recorded in real time with an infrared illuminated video camera. MAIN OUTCOME MEASURES: The percent pupil contraction of the transient and sustained pupil response to a low- (1 cd/m(2)), medium- (10 cd/m(2)), and high-intensity (100 cd/m(2)) red- and blue-light stimulus was calculated for 1 eye of each subject. From the 43 normal eyes, median and 25th, 75th, 5th, and 95th percentile values were obtained for each stimulus condition. RESULTS: In normal eyes at lower intensities, blue light evoked much greater pupil responses compared with red light when matched for photopic luminance. The transient pupil contraction was generally greater than the sustained contraction, and this disparity was greatest at the lowest light intensity and least apparent with bright (100 cd/m(2)) blue light. A patient with primarily rod dysfunction (nonrecordable scotopic electroretinogram) showed significantly reduced pupil responses to blue light at lower intensities. A patient with achromatopsia and an almost normal visual field showed selective reduction of the pupil response to red-light stimulation. A patient with ganglion cell dysfunction owing to anterior ischemic optic neuropathy demonstrated global loss of pupil responses to red and blue light in the affected eye. CONCLUSIONS: Pupil responses that differ as a function of light intensity and wavelength support the hypothesis that selected stimulus conditions can produce pupil responses that reflect phototransduction primarily mediated by rods, cones, or melanopsin. Use of chromatic pupil responses may be a novel way to diagnose and monitor diseases affecting either the outer or inner retina.

Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.

Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice.

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4(-/-)) mice under various light-dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7-10 Hz) and gamma (40-70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4(-/-) mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-hratio1-h schedule revealed that the failure to respond to light in Opn4(-/-) mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4(-/-) mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4(-/-) mice slept 1 h less during the 12-h light period of a LD 12ratio12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4(-/-) mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4(-/-) mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.

A pea plasma membrane protein exhibiting blue light-induced phosphorylation retains photosensitivity following triton solubilization.

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.

Light receptor action is critical for maintaining plant biomass at warm ambient temperatures.

The ability to withstand environmental temperature variation is essential for plant survival. Former studies in Arabidopsis revealed that light signalling pathways had a potentially unique role in shielding plant growth and development from seasonal and daily fluctuations in temperature. In this paper we describe the molecular circuitry through which the light receptors cry1 and phyB buffer the impact of warm ambient temperatures. We show that the light signalling component HFR1 acts to minimise the potentially devastating effects of elevated temperature on plant physiology. Light is known to stabilise levels of HFR1 protein by suppressing proteasome-mediated destruction of HFR1. We demonstrate that light-dependent accumulation and activity of HFR1 are highly temperature dependent. The increased potency of HFR1 at warmer temperatures provides an important restraint on PIF4 that drives elongation growth. We show that warm ambient temperatures promote the accumulation of phosphorylated PIF4. However, repression of PIF4 activity by phyB and cry1 (via HFR1) is critical for controlling growth and maintaining physiology as temperatures rise. Loss of this light-mediated restraint has severe consequences for adult plants which have greatly reduced biomass.

Therapeutic Blockade of LIGHT Interaction With Herpesvirus Entry Mediator and Lymphotoxin β Receptor Attenuates In Vivo Cytotoxic Allogeneic Responses.

BACKGROUND: Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses. METHODS: A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. RESULTS: We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. CONCLUSION: The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.

Le myélome à chaînes légères ou les pièges du diagnostic du myélome [Light chain myeloma or the pitfalls of myeloma diagnosis]

The diagnosis of multiple myeloma is often suggested by disturbances found in routine laboratory tests such as sedimentation rate, electrophoresis of serum proteins and search for proteinuria. In light chain myeloma these tests are nonspecific and therefore misleading. We present 8 cases of light chain myeloma and discuss the diagnosis of multiple myeloma with its associated pitfalls.

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis.

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red-dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein-protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB-yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB(Ser86Asp) Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light-induced nuclear import and interaction of phyB(Ser86Asp)-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB-green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

Clinical evaluation of a method for detecting superficial surgical transitional cell carcinoma of the bladder by light-induced fluorescence of protoporphyrin IX following the topical application of 5-aminolevulinic acid: preliminary results.

BACKGROUND AND OBJECTIVE: In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of "flat" urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5-aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: Several hours after bladder instillation of an aqueous solution of ALA in 34 patients, a Krypton ion laser or a filtered Xenon arc-lamp was used to excite PpIX fluorescence. Tissue samples for histological analysis were taken while observing the bladder wall either by means of a video camera, or by direct endoscopic observation. RESULTS: A good correlation was found between the PpIX fluorescence and the histopathological diagnosis. On a total of 215 biopsies, 143 in fluorescent and 72 in nonfluorescent areas, all visible tumors on white light cytoscopy appeared in a bright red fluorescence with the photodetection technique. In addition, this method permitted to discover 47 unsuspected carcinomatous lesions on white light observation, among which 40% were carcinoma in situ. CONCLUSION: PpIX fluorescence induced by instillation into the bladder of 5-ALA is an efficient method of mapping the mucosa in bladder carcinoma.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.