Photobiological properties of the inhibition of etiolated Arabidopsis seedling growth by ultraviolet-B irradiation

Alteration of 'normal' levels of ultraviolet-B light (UV-B, 280–320 nm) can affect plant chemical composition as well as growth; however, little is known about how plants perceive UV-B light. We have carried out fluence response curves, and demonstrated that the growth inhibition of etiolated Arabidopsis thaliana seedlings by low fluence UV light is specific to UV-B and not UV-A (320–390 nm). The response shows reciprocity between duration and intensity, at least over a limited range, and thus depends only on photon fluence and not on photon flux. The action spectrum for this response indicates a peak of maximum effectiveness at 290 nm, and response spectra at different fluences indicate that the most effective wavelength at 30 000  µ mol m^–2 is 290 nm, whereas 300 nm light was the most effective at 100 000  µ mol m^–2. This response occurs in mutant seedlings deficient in cryptochrome, phytochrome or phototropin, suggesting that none of the known photoreceptors is the major UV-B photoreceptor. Some null mutants in DNA repair enzymes show hypersensitivity to UV-B, suggesting that even at low fluence rates, direct damage to DNA may be one component of the response to UV-B.     

Identification of tubby and tubby-like protein 1 as eat-me signals by phage display

Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1^−/− mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.     

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

In a previous study, we used differential proteomics to identify retinal proteins whose steady‐state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady‐state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina—retinal recycling and generation of retinoic acid—we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11‐cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 623–635, 2010     

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.     

Development of a rod photoreceptor mosaic revealed in transgenic zebrafish

The number and distribution of neurons within the vertebrate retina are tightly regulated. This is particularly apparent in the highly ordered, crystalline-like arrangement of the cone photoreceptors in the teleost. In this report, using a transgenic line of zebrafish, a novel and developmentally regulated mosaic pattern of the rod photoreceptors is described. The spatial and temporal expression of EGFP, under the control of the Xenopus rhodopsin gene promoter, was nearly identical to the endogenous rhodopsin. EGFP was first detected in the ventral nasal retinal in an area of precocious neurogenesis referred to as the "ventral patch". Subsequent expression of EGFP was observed in isolated cells sporadically distributed across the dorsal and central retina. However, confocal microscopy and spatial analysis of larval eyes or retinal explants from adults revealed a precise arrangement to the rod photoreceptors. The rod terminals were arranged in regularly spaced rows with clearly identifiable telodendria linking neighboring cells. The rod inner segments projected through the cone mosaic in a predictable pattern. In the adult, the rod mosaic originated near the retinal margin where clusters of rods differentiated around the immature short single cone. In the embryo, the sporadic differentiation of the rods led to the gradual formation of the mosaic pattern. With the growing interest in neuronal stem cells, revisiting this model of neurogenesis provides an avenue to uncover mechanisms underlying the precise integration of new neuronal elements into a preexisting neural network.     

Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP Phosphodiesterase: A specific role for a light-sensitive GTPase as a component in the activation sequence

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3′5′-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.