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The rise of high-throughput RNA sequencing (RNA-seq) and de novo transcriptome assembly has had a transformative impact on how we identify and study genes in the phototransduction cascade of non-model organisms. But the advantage provided by the nearly automated annotation of RNA-seq transcriptomes may at the same time hinder the possibility for gene discovery and the discovery of new gene functions. For example, standard functional annotation based on domain homology to known protein families can only confirm group membership, not identify the emergence of new biochemical function. In this study, we show the importance of developing a strategy that circumvents the limitations of semiautomated annotation and apply this workflow to photosensitivity as a means to discover non-opsin photoreceptors. We hypothesize that non-opsin G-protein-coupled receptor (GPCR) proteins may have chromophore-binding lysines in locations that differ from opsin. Here, we provide the first case study describing non-opsin light-sensitive GPCRs based on tissue-specific RNA-seq data of the common bay scallop Argopecten irradians (Lamarck, 1819). Using a combination of sequence analysis and three-dimensional protein modeling, we identified two candidate proteins. We tested their photochemical properties and provide evidence showing that these two proteins incorporate 11-cis and/or all-trans retinal and react to light photochemically. Based on this case study, we demonstrate that there is potential for the discovery of new light-sensitive GPCRs, and we have developed a workflow that starts from RNA-seq assemblies to the discovery of new non-opsin, GPCR-based photopigments., Introduction An implicit assumption when studying eyes and other photoreceptive structures is that the initial step of light perception is mediated by a single protein subfamily, opsin. This is a [...]

It is rare nowadays to find something that nobody has seen before, especially without using a new methodology or looking in a new place. Using conventional differential interference contrast (DIC) [...]

Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin [B.sub.12] derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide visualizations of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter -35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin [B.sub.12] and provide fundamental insight into a new mode of light-dependent gene regulation., Light allows for photosynthesis and other essential light-dependent chemical reactions. Light also triggers photo-oxidative stress via generation of reactive oxygen species, which rapidly damage the cell (1). Organisms in all [...]

This study tested the hypothesis that sea lampreys (Petromyzon marinus L., 1758) undergo a reduction in the photosensitivity of photoreceptors in the tail after metamorphosing from burrow-dwelling, filter-feeding larval sea lampreys (ammocoetes) into open-water, parasitic juvenile phase animals that attach themselves to and feed on the blood of marine and freshwater fishes. Using a photo-illumination apparatus, ammocoetes and juvenile sea lampreys were exposed to white light at an intensity of 10 lx and the photokinetic response (time to movement) was measured in individual animals. The median latency of the response of juvenile lampreys was 5.4 times longer (i.e., slower response time) than observed in ammocoetes, and only 61% of the juvenile animals responded to the light compared with 95% of ammocoetes. It is concluded that the greater photosensitivity of tail photoreceptors in ammocoetes helps ensure that the animals are totally concealed from potential predators while burrowing. Decreased photosensitivity following metamorphosis is likely related to the transition to an open water, parasitic life style in which tail photoreception would have little selective advantage. Key words: dermal photoreceptors, life history, sensory ecology, light avoidance, Petromyzon marinus, sea lamprey. L'etude a verifie l'hypothese voulant que la photosensibilite des photorecepteurs situes dans la queue des lamproies marines (Petromyzon marinus L., 1758) diminue apres la metamorphose des larves fouisseuses se nourrissant par filtration (ammocetes) en animaux juveniles parasitiques qui vivent dans l'eau et se fixent a des poissons marins et d'eau douce pour se nourrir de leur sang. A l'aide d'un dispositif de photo-illumination, des ammocetes et des lamproies juveniles ont ete exposes a de la lumiere blanche d'une intensite de 10 lx et leur reponse photocinetique (le delai avant un mouvement) a ete mesuree pour differents individus. Le temps de latence median de la reponse des lamproies juveniles etait de 5,4 fois superieur (c.- a-d. reponse moins rapide) a celui des ammocetes, et seulement 61 % des juveniles ont reagi a la lumiere comparativement a 95 % des ammocetes. Nous en concluons que la plus grande photosensibilite des recepteurs de la queue des ammocetes les aide a demeurer completement caches des predateurs potentiels quand ils sont enfouis. La diminution de la photosensibilite suivant la metamorphose est vraisemblablement liee au passage a un mode de vie parasitique en eau libre, dans lequel la photoreception par la queue ne confere pas un grand avantage selectif. [Traduit par la Redaction] Mots-cles: photorecepteurs cutanes, cycle vital, ecologie sensorielle, evitement de la lumiere, Petromyzon marinus, lamproie marine., Introduction The life cycle of lampreys (Petromyzontididae) has three distinct phases that are marked by significant changes in physiology, habitat, and behaviour (for reviews see Youson 2003; Wilkie 2011). For [...]

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single [...]

Linkage analysis of two occult macular dystrophy (OMD) families was performed by the SNP High Throughput Linkage analysis system (SNP HiTLink) to localize the disease locus to chromosome 8p22-p23. The identified RP1L1 mutations as causative for OMD has potentially broader implications for better understanding the differential cone photoreceptor functions in the fovea and the peripheral retina.

Homozygosity mapping is used to identify homozygous region on chromosome 2p15 in patients with autosomal-recessive retinitis pigmentosa (arRP). The data revealed FAM161A to play significant role in photoreceptors and loss-of-function mutations in FAM161A is the major cause of arRP accounting for ~12% of arRP families in the cohort of patients from Israel and the Palestinian territories.

The study examines autosomal-recessive early-onset retinitis pigmentosa (RP) caused by a mutation in PDE6G, the gene encoding for the inhibitory gamma subunit of rod photoreceptor cyclic GMP (cGMP) phosphodiesterase. Results indicate the positive role of the gamma subunit in maintaining phosphodiesterase activity and confirm the role of PED6G in the etiology of RP in humans.

The time scale of the photoresponse in photoreceptor cells is set by the slowest of the steps that quench the light- induced activity of the phototransduction cascade. In vertebrate photoreceptor cells, this rate-limiting reaction is thought to be either shutoff of catalytic activity in the photopigment or shutoff of the pigment's effector, the transducin-GTP-phosphodiesterase complex. In suction pipette recordings from isolated salamander L-cones, we found that preventing changes in internal [[Ca.sup.2+]] delayed the recovery of the light response and prolonged the dominant time constant for recovery. Evidence that the [Ca.sup.2+]-sensitive step involved the pigment itself was provided by the observation that removal of [Cl.sup.-] from the pigment's anion-binding site accelerated the dominant time constant for response recovery. Collectively, these observations indicate that in L-cones, unlike amphibian rods where the dominant time constant is insensitive to [[Ca.sup.2+]], pigment quenching rate limits recovery and provides an additional mechanism for modulating the cone response during light adaptation. doi/10.1085/jgp.200910394

The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In Drosophila, photoreceptors R1-R6 have the same spectral sensitivity throughout the eye and are responsible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision. We investigated how photoreceptor types contribute to the attractiveness of light by blocking the function of certain subsets and by measuring differential phototaxis between spectrally different lights. In a 'UV vs. blue' choice, flies with only R1-R6, as well as flies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas flies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a 'blue vs. green' choice, flies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1-R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1-R6. differential phototaxis | attractiveness function | R7/R8 sytem | color vision www.pnas.org/cgi/doi/10.1073/pnas.0809398107

We determined the metabolic changes that precede cell death in the dystrophic proline-23-histidine (P23H) line 3 (P23H-3) rat retina compared with the normal Sprague-Dawley (SD) rat retina. Metabolite levels and metabolic enzymes were analyzed early in development and during the early stages of degeneration in the P23H-3 retina. Control and degenerating retinas showed an age-dependent change in metabolite levels and enzymatic activity, particularly around the time when phototransduction was activated. However, lactate dehydrogenase (LDH) activity was significantly higher in P23H-3 than SD retina before the onset of photoreceptor death. The creatine/phosphocreatine system did not contribute to the increase in ATP, because phosphocreatine levels, creatine kinase, and expression of the creatine transporter remained constant. However, [Na.sup.+]-[K.sup.+]-ATPase and [Mg.sup.2+]-[Ca.sup.2+]-ATPase activities were increased in the developing P23H-3 retina. Therefore, photoreceptor apoptosis in the P23H-3 retina occurs in an environment of increased LDH. ATPase activity, and higher-than-normal ATP levels. We tested the effect of metabolic challenge to the retina by inhibiting monocarboxylate transport with [alpha]-cyano-4-hydroxycinnamic acid or systemically administering the phosphodiesterase inhibitor sildenafil. Secondary to monocarboxylate transport inhibition. the P23H-3 retina did not demonstrate alterations in metabolic activity. However, administration of sildenafil significantly reduced LDH activity in the P23H-3 retina and increased the number of terminal deoxynucleotidyl transferase biotin-dUPT nick end-labeled photoreceptor cells. Photoreceptor cells with a rhodopsin mutation display an increase in apoptotic markers secondary to inhibition of a phototransduction enzyme (phosphodiesterase), suggesting increased susceptibility to altered cation entry. phosphocreatine; ATP; [alpha]-cyano-4-hydroxycinnamic acid; sildenafil; lactate dehydrogenase; ATPase doi:10.1152/ajpcell.00253.2009.

Bipolar cells are the central neurons of the retina that transmit visual signals from rod and cone photoreceptors to third-order neurons in the inner retina and the brain. A dogma set forth by I early anatomical studies is that bipolar cells in mammalian retinas t receive segregated rod/cone synaptic inputs (either from rods or from cones), and here, we present evidence that challenges this traditional view. By analyzing light-evoked cation currents from morphologically identified depolarizing bipolar cells (DBCs) in the wild-type and three pathway-specific knockout mice (rod transducin knockout [Tr[[alpha].sup.-/-]], connexin36 knockout [[Cx36.sup.-/-]], and transcription factor beta4 knockout [[Bhlhb4.sup.-/-]]), we show that a l subpopulation of rod DBCs ([DBC.sub.R2]s) receives substantial input directly from cones and a subpopulation of cone DBCs ([DBC.sub.C1]s) receives substantial input directly from rods. These results provide evidence of the existence of functional rod-[DBC.sub.C] and cone-[DBC.sub.R] synaptic pathways in the mouse retina as well as the previously proposed rod hyperpolarizing bipolar-cells pathway. This is grounds for revising the mammalian rod/cone bipolar cell dogma. light-evoked cation and chloride currents | axon-terminal stratification connexin36 | wild-type and mutant mice | depolarizing bipolar cell www.pnas.org/cgi/doi/10.1073/pnas.0907178107

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a 'flip-and-rotate' model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket. biliverdin | photoconversion | red-light photoreceptor

Phytochrome interacting factors (PIFs) are nuclear basic helix-loop-helix (bHLH) transcription factors that negatively regulate photomorphogenesis both in the dark and in the light in Arabidopsis. The phytochrome (phy) family of photoreceptors induces the rapid phosphorylation and degradation of PIFs in response to both red and far-red light conditions to promote photomorphogenesis. Although phys have been shown to function under blue light conditions, the roles of PIFs under blue light have not been investigated in detail. Here we show that PIF1 negatively regulates photomorphogenesis at the seedling stage under blue light conditions, pill seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12 hr light/12 hr dark) blue light conditions. Double-mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cryl, and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants trader diurnal blue light conditions. Blue light induced the rapid phosphorylation, polyubiquitination, and degradation of PIF1 through the ubi/26S proteasomal pathway. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the blue light-induced degradation of PIF1. phyA played a dominant role under pulses of blue light, while phyA, phyB, and phyD induced the degradation of PIF1 in an additive manner under prolonged continuous blue light conditions. Interestingly, the absence of cryl and cry2 enhanced the degradation of PIF1 under blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis under blue light conditions and that blue light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis. DOI: 10.1534/genetics.108.099887

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond), fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway. cone | Danio rerio | T-box | mosaic

Although age-related macular degeneration is the most prevalent macular disease in the world, numerous discoveries regarding the molecular bases of vision have been made through genetic association studies of rare inherited maculopathies. In this issue of the JCI, Yang et al. present a functional genetics study that identifies a role for prominin 1 (PROM1), best known as a stem cell and/or progenitor cell marker, in the biogenesis of retinal photoreceptor disk arrays (see the related article beginning on page 2908). This study supports an established model in which disk morphogenesis occurs through membrane evagination and extends other recent studies assigning PROM1 important functions outside of the stem cell niche., Essentials of photoreceptor organization More than 50 years ago, the first ultrastructural evidence of photoreceptor disk organization was published by noted electron microscopist Fritiof Sjostrand (1). Subsequent studies provided more [...]

We used wild-type UTEX481; SF33, a shortened-filament mutant strain that shows normal complementary chromatic adaptation pigmentation responses; and FdBK1l4, an RcaE-deficient strain that lacks light-dependent pigmentation responses, to investigate the molecular basis of the photoregulation of cellular morphology in the cyanobacterium Fremyella diplosiphon. Detailed microscopic and biochemical analyses indicate that RcaE is required for the photoregulation of cell and filament morphologies of F. diplosiphon in response to red and green light.

Byline: Roelof A. Hut (INSERM, Bron, France, r.a.hut@rug.nl, Stem Cell and Brain Research Institute, Department of Chronobiology, Bron, France, University of Lyon, France); Malgorzata Oklejewicz (INSERM, Bron, France, Stem Cell [...]

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light-oxygen-voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BI. irradiation. The adduct decays to the ground state in [approximately equal to] 5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles. blue light receptor | basic-region/leucine-zipper | Vaucheria | light-oxygen-voltage

Highly abundant short-chain alcohol dehydrogenases (RDHs) in the retina were assumed to be involved in the recycling of 11-cis-retinal chromophore in the visual cycle. Mutations in human RDH genes are associated with Fundus albipunctatus, a mild form of night blindness (RDHS) and an autosomal recessive, childhood-onset severe retinal dystrophy (RDH12). Rdh12 knockout mice were found to be susceptible to light-induced photoreceptor apoptosis, whereas Rdh5 and Rdh8 knockout mice displayed only delayed dark adaptation. However, each knockout mouse eventually regenerated normal levels of visual pigments, suggesting that RDHs compensate for each other in the visual cycle. Here, we established RDH double knockout (Rdh[8.sup.-/-]Rdh[12.sup.-/-]) and triple knockout (Rdh[5.sup.-/-]Rdh[8.sup.-/-]Rdh[12.sup.-/-]) mice generated on various genetic backgrounds including a rod [alpha]-transducin knockout to test cone function. RDH activity was severely reduced in Rdh[8.sup.-/-]Rdh[12.sup.-/-] retina extracts, whereas Rdh[8.sup.-/-] RDH activity was intermediate and Rdh[12.sup.-/-] RDH activity was reduced only slightly. Surprisingly, all multiple knockout mice produced sufficient amounts of the chromophore to regenerate rhodopsin and cone pigments in vivo. Three-month-old Rdh[8.sup.-/-]Rdh[12.sup.-/-] mice characteristically displayed a slowly progressing rod-cone dystrophy accompanied by accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic substance known to contribute to retinal degeneration. A2E accumulation and retinal degeneration were prevented by application of retinylamine, a potent retinoid cycle inhibitor. The results suggest that RDH8 and RDH12 are dispensable in support of the visual cycle but appear to be key components in clearance of free all-trans-retinal, thereby preventing A2E accumulation and photoreceptor cell death. cone | photoreceptors | rhodopsin | rod

Classical cadherin-mediated interactions between axons and dendrites are critical to target selection and synapse assembly. However, the molecular mechanisms by which these interactions are controlled are incompletely understood. In the Drosophila visual system, N-cadherin is required in both photoreceptor (R cell) axons and their targets to mediate stabilizing interactions required for R cell target selection. Here we identify the scaffolding protein Liprin-[alpha] as a critical component in this process. We isolated mutations in Liprin-[alpha] in a genetic screen for mutations affecting the pattern of synaptic connections made by R1-R6 photoreceptors. Using eye-specific mosaics, we demonstrate a previously undescribed, axonal function for Liprin-[alpha] in target selection: Liprin-[alpha] is required to be cell-autonomous in all subtypes of R1-R6 cells for their axons to reach their targets. Because Liprin-[alpha], the receptor tyrosine phosphatase LAR, and N-cadherin share qualitatively similar mutant phenotypes in R1-R6 cells and are coexpressed in R cells and their synaptic targets, we infer that these three genes act at the same step in the targeting process. However, unlike N-cadherin, neither Liprin-[alpha] nor LAR is required postsynaptically for R cells to project to their correct targets. Thus, these two proteins, unlike N-cadherin, are functionally asymmetric between axons and dendrites. We propose that the adhesive mechanisms that link pre- and postsynaptic cells before synapse formation may be differentially regulated in these two compartments. axon | synapse formation | cadherin | cell adhesion

In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. Here we identify a null mutation in the Liprin-[alpha] gene based on a similar R7 projection defect. Liprin-[alpha] physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-[alpha] nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-[alpha] proteins have been reported to control the localization of LAR, we find that LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-[alpha]. In addition, Liprin-[alpha] overexpression or loss of function can affect R7 targeting in the complete absence of LAR. We conclude that Liprin-[alpha] does not simply act by regulating LAR localization but also has LAR-independent functions. receptor tyrosine phosphatase | visual system | Drosophila

Abstract The photo-responsiveness of 2 groups of interneurons responding to light in the protocerebrum was investigated at 2 developmental stages, the last instar nymphs and adults, in the cricket Gryllus [...]

Adult dragonflies augment their compound eyes with three simple eyes known as the dorsal ocelli. While the ocellar system is known to mediate stabilizing head reflexes during flight, the ability of the ocellar retina to dynamically resolve the environment is unknown. For the first time, we directly measured the angular sensitivities of the photoreceptors of the dragonfly median (middle) ocellus. We performed a second-order Wiener Kernel analysis of intracellular recordings of light-adapted photoreceptors. These were stimulated with one-dimensional horizontal or vertical patterns of concurrent UV and green light with different contrast levels and at different ambient temperatures. The photoreceptors were found to have anisotropic receptive fields with vertical and horizontal acceptance angles of 15[degrees] and 28[degrees], respectively. The first-order (linear) temporal kernels contained significant undershoots whose amplitudes are invariant under changes in the contrast of the stimulus but significantly reduced at higher temperatures. The second-order kernels showed evidence of two distinct nonlinear components: a fast acting self-facilitation, which is dominant in the UV, followed by delayed self- and cross-inhibition of UV and green light responses. No facilitatory interactions between the UV and green light were found, indicating that facilitation of the green and UV responses occurs in isolated compartments. Inhibition between UV and green stimuli was present, indicating that inhibition occurs at a common point in the UV and green response pathways. We present a nonlinear cascade model (NLN) with initial stages consisting of separate UV and green pathways. Each pathway contains a fast facilitating nonlinearity coupled to a linear response. The linear response is described by an extended log-normal model, accounting for the phasic component. The final nonlinearity is composed of self-inhibition in the UV and green pathways and inhibition between these pathways. The model can largely predict the response of the photoreceptors to UV and green light.

Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

Detection threshold in cone photoreceptors requires the simultaneous absorption of several photons because single photon photocurrent is small in amplitude and does not exceed intrinsic fluctuations in the outer segment dark current (dark noise). To understand the mechanisms that limit light sensitivity, we characterized the molecular origin of dark noise in intact, isolated bass single cones. Dark noise is caused by continuous fluctuations in the cytoplasmic concentrations of both cGMP and [Ca.sup.2+] that arise from the activity in darkness of both guanylate cyclase (GC), the enzyme that synthesizes cGMP, and phosphodiesterase (PDE), the enzyme that hydrolyzes it. In cones loaded with high concentration [Ca.sup.2+] buffering agents, we demonstrate that variation in cGMP levels arise from fluctuations in the mean PDE enzymatic activity. The rates of PDE activation and inactivation determine the quantitative characteristics of the dark noise power density spectrum. We developed a mathematical model based on the dynamics of PDE activity that accurately predicts this power spectrum. Analysis of the experimental data with the theoretical model allows us to determine the rates of PDE activation and deactivation in the intact photoreceptor. In fish cones, the mean lifetime of active PDE at room temperature is ~55 ms. In nonmammalian rods, in contrast, active PDE lifetime is ~555 ms. This remarkable difference helps explain why cones are noisier than rods and why cone photocurrents are smaller in peak amplitude and faster in time course than those in rods. Both these features make cones less light sensitive than rods. KEY WORDS: phototransduction * phosphodiesterase * guanylate cyclase * cGMP * calcium

The [Ca.sup.2+]-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that [Ca.sup.2+]-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster [Ca.sup.2+] dynamics, indicating that recoverin is a significant [Ca.sup.2+] buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that [Ca.sup.2+]-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin. KEY WORDS: calcium-binding proteins * knockout mice * light adaptation * photoreceptors * phototransduction

Auxin redistribution along gravistimulated maize coleoptiles causes differential expression of the auxin-induced [K.sup.+]-channel gene ZMK1 (Zea mays [K.sup.+] channel 1) and precedes the curvature response. To evaluate the role of ZMK1 during phototropism, we here investigated blue light-stimulated coleoptiles. Four hours of blue light stimulation resulted in phototropic bending (23[degrees]). Rotation on a clinostat, at nominally 'zero' gravity, and simultaneous stimulation with unidirectional blue light, however, resulted in up to 51[degrees] bending toward the light. Differential ZMK1 transcription reached a maximum after 90 min of blue light stimulation under gravity, whereas ZMK1 expression remained asymmetric for at least 180 min in photostimulatad coleoptiles on a clinostat. We therefore conclude that the stronger phototropic bending under nominally 'zero' gravity results from prolonged differential expression of ZMKI. Under both conditions, asymmetric expression of ZMK1 could be superimposed on the lateral auxin gradient across the coleoptile tip, whereas the gene for the blue light receptor phototropin 1 (PHOT1), expressed in the tip only, was not differentially regulated in response to blue light. The activation of the two different receptors eliciting the photo- and gravitropic response of the coleoptile thus feeds into a common signaling pathway, resulting in auxin redistribution in the coleoptile tip and finally in differential transcription of ZMK1. In the process of signal integration, gravity transduction restricts the magnitude of the blue light-inducible ZMK1 gradient. The spatial and temporal distribution of ZMK1 transcripts and thus differential [K.sup.+] uptake in both flanks of the coleoptile seem to limit the stimulus-induced bending of this sensory organ.

The experiments reported here were designed to answer the question of whether inositol 1,4,5-trisphosphate (I[P.sub.3])-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. For this purpose the membrane-permeable I[P.sub.3] receptor antagonist 2-aminoethoxydiphenyl borate (2APB) (Maruyama, T., T. Kanaji, S. Nakade, T. Kanno, and K. Mikoshiba. 1997. J. Biochem. (Tokyo). 122:498-505) was used. Previously, 2APB was found to inhibit the light activated current of Limulus ventral photoreceptors and reversibly inhibit both light and I[P.sub.3] induced calcium release as well as the current activated by pressure injection of calcium into the light sensitive lobe of the photoreceptor (Wang, Y., M. Deshpande, and R. Payne. 2002. Cell Calcium. 32:209). In this study 2APB was found to inhibit the response to a flash of light at all light intensities and to inhibit the entire light response to a step of light, that is, both the initial transient and the steady-state components of the response to a step of light were inhibited. The light response in cells injected with the calcium buffer 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) was reversibly inhibited by 2APB, indicating that these light responses result from I[P.sub.3]-mediated calcium release giving rise to an increase in [Ca.sub.i]. The light response obtained from cells after treatment with 100 [micro]M cyclopiazonic acid (CPA), which acts to empty intracellular calcium stores, was reversibly inhibited by 2APB, indicating that the light response alter CPA treatment results from I[P.sub.3]-mediated calcium release and a consequent rise in [Ca.sub.i]. Together these findings imply that I[P.sub.3]-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. KEYWORDS: phototransduction * microvillar photoreceptors * inositol 1,4,5-trisphosphate receptor * 2-aminoethoxydiphenyl borate * calcium buffers

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod [Ca.sup.2+] overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome c release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-[x.sub.L] overexpression completely blocked all apoptotic events, except [Ca.sup.2+] overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an in vivo apoptosis model and shows that Bcl-[x.sub.L] overexpression blocks increased contact sites and apoptosis. These findings extend our in vitro retinal studies with [Pb.sup.2+] and [Ca.sup.2+] and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the [Pb.sup.2+] and [Ca.sup.2+] overloaded rod mitochondria to release cytochrome c. These results have relevance for therapies in where [Ca.sup.2+] overload, lead exposure, and/or mitochondrial dysfunction occur.

The flavin-containing antirepressor AppA functions as a trancription factor and regulates PpsR repressor's binding to DNA by inhibiting the protein binding by either inducing a disulfide bond in PpsR or forming a stable AppA-PpsR(sub)2 antirepressor-repressor complex. Further, the flavin mediated and blue-light induced red shifted photocycle inhibits the complex formation.

Aim: To examine the distribution of melatonin 1 a (MT1) receptors in the human eye. Methods: Seven normal human eyes were examined by immunohistochemical staining of paraffin sections, using an [...]