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Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells.

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

Optical insulation plays a critical role in the fine visual acuity of the Drosophila compound eye. Screening pigments expressed by a number of cell types contribute to this phenomenon. They provide optical insulation that prevents extraneous light rays from inappropriately activating the photoreceptors. This optical insulation can be divided into two categories; the insulation of the individual ommatidia, and the insulation of the compound eye as a whole. The whole-eye insulation is provided by two sources. The sides of the eye are optically insulated by the pigment rim, a band of pigment cells that circumscribes the eye. The base of the eye is insulated by the subretinal pigment layer; a thick layer of pigment that lies directly underneath the retina. How this subretinal pigment layer is generated has not been clearly described. Here, experiments that manipulate pigment expression during eye development suggest that the subretinal pigment layer is directly derived from pigment cells in the overlying retina., (Copyright © 2012 Wiley Periodicals, Inc.)

The crystalline photoreceptor lattice in the Drosophila eye is a paradigm for pattern formation during development. During eye development, activation of proneural genes at a moving front adds new columns to a regular lattice of R8 photoreceptors. We present a mathematical model of the governing activator-inhibitor system, which indicates that the dynamics of positive induction play a central role in the selection of certain cells as R8s. The "switch and template" patterning mechanism we observe is mathematically very different from the well-known Turing instability. Unlike a standard lateral inhibition model, our picture implies that R8s are defined before the appearance of the complete group of proneural cells. The model reproduces the full time course of proneural gene expression and accounts for specific features of the refinement of proneural groups that had resisted explanation. It moreover predicts that perturbing the normal template can lead to eyes containing stripes of R8 cells. We observed these stripes experimentally after manipulation of the Notch and scabrous genes. Our results suggest an alternative to the generally assumed mode of operation for lateral inhibition during development; more generally, they hint at a broader role for bistable switches in the initial establishment of patterns as well as in their maintenance.

Purpose: To take advantage of specialized microscopic methods and transgenic stocks, to understand the properties of each rhodopsin now that Drosophila's six rhodopsins (Rh1-Rh6) have been isolated., Methods: The visual pigment containing organelles, the rhabdomeres, were imaged in live flies with the pseudopupil in standard and confocal microscopes. Five transgenic Drosophila strains in which Rh2-Rh6 replaced the native Rh1 in R1-6 receptors were compared with normal controls (Rh1 in R1-6) for two lines of work: (1) autofluorescence of rhodopsin; and (2) imaging rhodopsin. Other transgenic Drosophila in which the Rh1, Rh3, and Rh4 promoters drive the green fluorescent protein (GFP) reporter were used for other purposes, especially distinguishing the R7/8 types., Results: We show, for the first time, that visual pigment appears pink in white light, especially for Rh1 and Rh6. While showing that rhodopsin-metarhodopsin conversions were understood by their respective wavelengths, we discovered that, for Rh6, rhodopsin and metarhodopsin could not be spectrally separated. Relative fluorescent emission, Rh1=Rh5>Rh6>Rh2>Rh4>Rh3, was of little value in explaining differences between bright and dim autofluorescence in R7. Rather, analysis of GFP driven by Rh3 and Rh4 promoters show that the rhabdomeres with bright autofluorescence are the ones that contain Rh4., Conclusions: Careful imaging provides a useful approach to analyzing Drosophila rhodopsins. Amid a considerable body of microscopic data, we identify the sources of bright and dim R7 rhabdomeres, and we demonstrate the unique properties of Rh6.

CRYPTOCHROME (CRY) is the primary circadian photoreceptor in Drosophila. We show that CRY binding to TIMELESS (TIM) is light-dependent in flies and irreversibly commits TIM to proteasomal degradation. In contrast, CRY degradation is dependent on continuous light exposure, indicating that the CRY-TIM interaction is transient. A novel cry mutation (cry(m)) reveals that CRY's photolyase homology domain is sufficient for light detection and phototransduction, whereas the carboxyl-terminal domain regulates CRY stability, CRY-TIM interaction, and circadian photosensitivity. This contrasts with the function of Arabidopsis CRY domains and demonstrates that insect and plant cryptochromes use different mechanisms.

Rab proteins of the small G-protein superfamily are known to be involved in intracellular vesicle transport. Here, we describe the unique characteristics of a novel Rab protein, RABRP1 (Rab-Related Protein 1). The Drosophila RabRP1 gene is mainly transcribed in the eyes and testes, where the 3-kb and 1.5-kb mRNAs, respectively, are the predominant gene products. The amino-acid sequence deduced from the longer cDNA indicated that the C-terminal 1/3 of the sequence shares homology with Rab proteins, whereas the rest of the peptide shows no significant homology with any other proteins. Immunoblot analysis using antiserum against the Rab-domain indicated that the multiple translates (94 k, 53 k, 30 k, 29 k and 27 k) were expressed in the eyes. In contrast, only smaller peptides (30 k, 29 k and 27 k) were identified in the testes. Molecular phylogenetic analysis revealed that RABRP1 forms a subgroup with Dictiostelium RabE and mammalian Rab29, Rab32, Rab38 proteins, whose functions have not been identified yet. RABRP1 and its relatives were characterized by the amino acid substitution occurring in the conserved GTP-binding motifs. Immunohistochemical studies demonstrated that RABRP1 was localized on the subrhabdomeric cisternae of photoreceptor cells and on the pigment granules in photoreceptor and pigment cells in the retina. The expression of the dominant negative RABRP1 caused the abnormal accumulation of autophagosome-like vesicles. These data suggest that RABRP1 is involved in the lysosomal vesicle transport pathway, including the biogenesis or degradation of pigment granules.

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.

In Drosophila, thorough retinoid deprivation is possible, optimizing investigation of the effects of vitamin A metabolites and retinoic acid on the visual system. Retinoids had been found to control transcription and translation of Drosophila's opsin gene. To follow this line of inquiry, we examined the effect of retinoids on the translation and transcription of a Drosophila Retinoid and Fatty Acid Binding Glycoprotein. Western blots showed that this protein is high in retinoid replete flies and low in deprived flies. Flies grown on media capable of activating the opsin gene's transcription and which contain alternate transcription activators including retinoic acid yielded extracts containing significant amounts of Retinoid and Fatty Acid Binding Glycoprotein. Immunocytochemistry confirmed its absence in deprived flies and its presence in flies reared or replaced on these diverse media containing retinoids or general nutrients. Immunocytochemistry localized Retinoid and Fatty Acid Binding Glycoprotein to the Semper (cone) cells and the intraommatidial matrix (the interphotoreceptor matrix of the ommatidium). Positive staining of Semper cells in mutants of the opsin gene and a mutant lacking receptors suggests that Retinoid and Fatty Acid Binding Glycoprotein does not depend on presence of opsin and that it is not synthesized in receptor cells respectively. Northern blots demonstrated greatly diminished mRNA for Retinoid and Fatty Acid Binding Glycoprotein in flies grown on deprivation food relative to flies grown on normal food. Although the synthesis of Retinoid and Fatty Acid Binding Glycoprotein does not require chromophore precursors as does that of opsin, the control of Retinoid and Fatty Acid Binding Glycoprotein and opsin transcription by retinoids including retinoic acid might very well be the same. Our results suggest that Retinoid and Fatty Acid Binding Glycoprotein may be involved in retinoid transport. Also, Semper cells may be analogous to vertebrate retinal pigment epithelium in retinoid metabolism and/or delivery., (Copyright 1997 Academic Press Limited.)

We have examined the effects of the insect ecdysteroid, 20-hydroxyecdysone, on the differentiation of neuronal and non-neuronal elements in the developing adult visual system, using in vitro methods in Drosophila. We examined the differentiation of early neuronal markers in the presence and absence of 1 microgram/ml 20-hydroxyecdysone. Immunoreactivity to 22C10, a marker of an early neuronal antigen, as well as to the photoreceptor-specific antibody 24B10, suggests that differentiation of neuronal and photoreceptor antigens does not require 20-hydroxyecdysone. In eye-discs cultured from animals 5 hours after the white prepupa (P + 5), ommochrome pigmentation first appeared after 2 days in 1 microgram/ml 20-hydroxyecdysone, but cultures lacked pigmentation without 20-hydroxyecdysone. Our culture conditions failed to support the formation of the second screening pigment, drosopterins, even with 20-hydroxyecdysone. Eye discs from P + 5 also formed lenses and interommatidial bristles in culture when 20-hydroxyecdysone was added but not in cultures devoid of the hormone. The differentiation of synaptotagmin and the elongation of extending photoreceptor neurites from eye disc fragments both occur in the absence of 20-hydroxyecdysone in cultures, but adding the hormone increased average neurite length. The threshold for enhanced neurite length was less than 125 ng/ml 20-hydroxyecdysone. Eye-disc cultures also developed immunoreactivity to histamine, the photoreceptor transmitter, from synthesis not re-uptake, in both the presence and in the absence of 20-hydroxyecdysone. These findings suggest that photoreceptor axons may be able to release transmitter in vivo both when they grow into the optic lobe and during the subsequent events in synapse formation.

Ultraviolet light (UV)-induced DNA damage can be repaired by DNA photolyase in a light-dependent manner. Two types of photolyase are known, one specific for cyclobutane pyrimidine dimers (CPD photolyase) and another specific for pyrimidine (6-4) pyrimidone photoproducts[(6-4)photolyase]. In contrast to the CPD photolyase, which has been detected in a wide variety of organisms, the (6-4)photolyase has been found only in Drosophila melanogaster. In the present study a gene encoding the Drosophila(6-4)photolyase ws cloned, and the deduced amino acid sequence of the product was found to be similar to the CPD photolyase and to the blue-light photoreceptor of plants. A homolog of the Drosophila (6-4)photolyase gene was also cloned from human cells.

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple "smash" technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.

Two classes of phosphorylated homologs of vertebrate arrestins, designated phosrestins I (PRI) and phosrestin II (PRII), are expressed in the photoreceptors of a fruit fly, Drosophila melanogaster. This study presents evidence that the housefly, Musca domestica, also has a protein similar to Drosophila PRI. Our conclusion is based on the following evidence. (1) We identified a Musca photoreceptor protein exhibiting a molecular mass (51 kDa) and an isoelectric point (pI = 8.6) similar to those of Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. (2) Rabbit antibodies raised against Musca PRI, against bovine arrestin, and against a synthetic peptide based on the Drosophila PRI sequence stained the Drosophila and Musca PRIs specifically on 1 and 2-dimensional Western immunoblots. (3) Both Drosophila and Musca PRIs incorporated 32P-radioactivity from gamma-32P-ATP in cell-free homogenates of retinas. Partial peptide digestions of Drosophila and Musca PRIs revealed similarity between these proteins. We observed that Drosophila PRI exists in the random preparation, but it also exists in other subcellular fractions. Immunocytochemistry at the EM level revealed a distribution of both Drosophila and Musca PRI epitopes in membranous vesicular structures in the cytosol as well as in the rhabdomeric microvillar membranes where the visual pigment, rhodopsin, exists. Such distribution of PRI epitopes suggests that PRI and its light-dependent phosphorylation may function in a space remote from the rhabdomere as well as the immediate milieu of photoreception.