Transduction in photoreceptors with bistable pigments: Intermediate processes

The prolonged depolarizing after potential (PDA) in the R1–6 receptors of the fly was used to isolate intermediate processes in phototransduction which are not manifested directly in the voltage response. It is first demonstrated that a pigment shift by light from metarhodopsin to rhodopsin in four species of the flies: Drosophila, Calliphora, Chrysomya and Musca induces an independent antagonistic process to the PDA, which is manifested in a strong inhibitory effect on PDA induction and is called the anti-PDA.By using mutants of Drosophila the existence of processes underlying the PDA were examined. The norpA ^H52and the trp mutant were used in which the voltage response of the photoreceptors could be reversibly abolished by elavated temperature and long intense light respectively. It is shown that the excitatory process underlying the PDA could be induced and depressed in conditions that block the voltage response of the photoreceptors, thus indicating the existance of intermediate processes which link the pigment activation by light to the PDA voltage response.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Drosophila mosaic screen identifies diehard mutants as norpA P24 suppressors

It is well-known that malfunctioning of the phototransduction mechanism stimulates the cell death machinery, resulting in retinal degeneration both in vertebrates and invertebrates. Genetic screens have often failed to identify modifiers underlying such degenerative syndromes because of the lethality associated with their fundamental function during development. A norpA ^P24 suppressor screen was successful performed with mosaic flies generated by the yeast site-specific recombination FLP-FRT system. Three independent diehard mutants, identified by mutagenizing the 2L FRT chromosome in the norpA ^P24 background, include both essential and non-essential mutations. Their suppressive effects on the norpA ^P24 -triggered retinal degeneration depend on the intensity and duration of light based on a deep pseudopupil analysis and histology. These results suggest that the suppressor screen using mosaic flies provides a valuable tool for recovering both essential and non-essential genes within the fundamental genetic pathways. Further studies of diehard mutants will provide insights into the disease mechanism that underlies the retinal degeneration of phototransduction mutants.     

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

Modulation of the dihydropyridine-sensitive calcium channels in Drosophila by a phospholipase C-mediated pathway

Disruption of phospholipase C-β (PLC) by the norpA mutations of Drosophila renders flies blind by affecting the light-evoked photoreceptor potential. We report here that the norpA-coded PLC modulates the 1,4-dihydropyridine (DHP)-sensitive Ca²⁺ channels in larval muscles. The DHP-sensitive current was reduced in the norpA mutants. Application of 1 μM phorbol 12-myristate 13-acetate (TPA) and 1 μM phorbol 12,13-didecanoate (PDD), activators of protein kinase C (PKC), rescued the current in the mutant fibers without significantly affecting the normal current. 4α-phorbol 12,13-didecanoate (4αPDD), an inactive analog of PDD, did not affect either the normal or the mutant current. One micromolar bisindolylmaleimide (BIM), an inhibitor of PKC, reduced the current in the normal fibers without affecting the mutant current. 300 μM sn-1,2-dioctanoyl-glycerol (DOG), an analog of diacylglycerol (DAG), increased the current in the mutant fibers. These experiments suggest that the DHP-sensitive Ca²⁺ channels in Drosophila may be modulated by the PLC-DAG-PKC pathway, and that the same PLC isozyme which is involved in phototransduction in the adult flies may also modulate muscle Ca²⁺ channels in the larval stage of development. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 265–275, 1997     

Circadian expression of the presynaptic active zone protein bruchpilot in the lamina of Drosophila melanogaster

In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild‐type Canton‐S flies in constant darkness (DD), nor in norpA⁷ phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per⁰ arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton‐S flies in DD, as well as in the norpA⁷ mutant in LD, but is absent in per⁰¹, tim,⁰¹ and cry⁰¹ mutants in LD. In addition both peaks seem to depend on clock gene‐expressing photoreceptors and glial cells of the visual system. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Degeneration of photoreceptors in rhodopsin mutants of Drosophila

Five different, well-characterized mutants of the R1–6 rhodopsin gene (ninaE), which corresponds to the rod opsin gene of vertebrates, have been examined morphologically as a function of age (up to 9 weeks) to determine whether or not the photoreceptors degenerate and to assess the pattern of degeneration. Structural deterioration of R1–6 photoreceptors with age has been found in all five mutants. The structural pattern of degeneration is similar in the five mutants, but the time course of degeneration is allele dependent and varies greatly among the five, with the strongest alleles causing the fastest degeneration. The degeneration appears to be independent of either the illumination cycle to which the animals are exposed or the presence of screening pigments in the eye. Although the degeneration first appears in R1–6 photoreceptors, eventually R7/8 photoreceptors, which correspond to cones of vertebrates, are also affected. In many of these mutants, striking proliferations of membrane processes have been observed in the subrhabdomeric region of R1–6 photoreceptors. It is hypothesized that (1) this accumulation of membranes may be caused by the failure of newly synthesized membranes that are inserted into the base of microvilli to be assembled into R1–6 rhabdomeres and (2) this failure may be caused by the extremely low concentration of normal R1–6 rhodopsin in the nina E mutants. © 1992 John Wiley & Sons, Inc.     

Molecular genetics of retinal degeneration: A Drosophila perspective

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP₂, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

Membrane maintenance and electrical properties of photoreceptors of wild-type andrpa (receptor potential absent) mutant blowflies (Calliphora erythrocephala)

Summary The maintenance of photoreceptor cell membranes in the blowfly was investigated in relation to the diurnal cycle, age, and therpa (receptor potential absent) phototransduction mutation. The effect of disturbed membrane assembly on the electrical membrane properties was examined using single-electrode discontinuous current-clamp techniques. In wild-type flies the cross-sectional dimensions of the rhabdomeres were markedly reduced with age, and the quantity of synthetic organelles decreased concurrently, whereas no correlation was found between the diurnal cycle and membrane turnover. Therpa mutation is thought to block the visual transduction cascade in photoreceptor cells and to lead to degeneration of the photoreceptor cell bodies. The volume of rhabdomeres decreased markedly inrpa mutants and the quantity of synthetic organelles was reduced significantly, indicating an imbalance between photoreceptive membrane renewal and degradation. Also, the plasma membrane underwent degenerative changes. The passive electrical properties of photoreceptor cells — resting membrane voltages and input resistances — were only slightly changed from those of wild-type flies, although the photoreceptive membrane did not depolarize in response to light. This indicates no apparent disturbance in the function of the ionic channels in these membranes. Taken together, these results suggest that the photoreceptor cells need a functional phototransduction cascade with its feedback controls to maintain continuous renewal of rhabdomeres, but that the plasma membrane maintains its normal electrochemical properties despite extreme morphological degeneration of photoreceptor cell.     

Rhodopsin activation causes retinal degeneration in Drosophila rdgC mutant

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

Molecular genetics of retinal degeneration

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP_2, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

Maintenance of Rhodopsin levels in Drosophila photoreceptor and phototransduction requires Protein Kinase D

ABSTRACT

During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. In this study, we report that Protein Kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKD^H), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKD^H. Our results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.     

Characteristics of Drosophila rhodopsin in wild-type and norpA vision transduction mutants

The properties of the major visual pigment of Drosophila melanogaster were evaluated. The visual pigment was isolated from other protein components using acrylamide gel electrophoresis and spectral identification. Sodium dodecyl sulfate (SDS) acrylamide gels of the isolated visual pigment gave a single protein subunit with a mol wt of 37,000 daltons. The rhodopsin480 molar extinction coefficient was 35,000 liter/mol-cm (+/- 2,700 SE). The metarhodopsin580 molar extinction coefficient was approximately 56,000 liter/mol-cm. Microspectrophotometry was used to compare the rhodopsin concentrations in wild-type flies and norpA vision transduction mutants. At 2 days of age (12 h dark-12 h light cycle, 19 degrees C) all of the norpA flies exhibited a similar rhodopsin concentration (75% of the wild-type strain). By 21 days of age some of the norpA alleles showed substantially reduced rhodopsin concentrations (16-43% of normal), whereas others showed no major age-dependent decreases (68-77%). Temperature and light-dark cycle affected the reduction. Alleles with no receptor potential exhibited the largest decreases in rhodopsin concentration. The data indicate that the norpA phototransduction mutant has a defect in the system responsible for maintaining the rhodopsin480 concentration. This defect in the rhodopsin maintenance system does not appear to be the cause of the reduced electroretinogram (ERG) amplitude observed in some of these mutants, but instead is a consequence of the decrease in ERG amplitude, or the flaw(s) responsible for the decrease in ERG amplitude.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

In crayfish photoreceptor cells, Gq-type G-protein plays a central role in the phototransduction pathway, and the translocation of Gqα has been proposed as one of the molecular mechanisms to control photoreceptor sensitivity. We here investigated β subunit of Gq and its localization profiles under various light conditions in the crayfish photoreceptor cells to understand the functional characteristic of visual Gq in the phototransduction pathway. An immunoprecipitation experiment was performed using an anti-Gqα antibody and a thiol-cleavable crosslinker. A 39 kDa protein was co-immunoprecipitated with Gqα, but not by irradiation, in the presence of GTPγS. The partial amino acid sequence of the 39 kDa protein was similar to Gβe in Drosophila photoreceptors, indicating that the crayfish Gβ which combines with Gqα is a Gβe homologue. Immunohistochemical and immunoblot analyses revealed that the amount of the Gβ decreased in the rhabdomeric membranes and increased in the cytoplasm in the light, compared with that in the dark. The profile of the translocation was similar to that reported for Gqα. Since both α and βγ subunits are necessary for G-proteins to be activated by rhodopsin in the rhabdom, the light-modulated translocation of a Gβe homologue possibly controls the amount of Gq which can be activated by light-stimulated rhodopsin. Accepted: 27 June 1998     

Vitamin a deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes

Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5–10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R_1–6 has an absorbance maximum at about 490 nm, but no unusually enhanced β-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives.Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.     

Olfactory physiology in the Drosophila maxillary palp requires the visual system gene rdgB

We describe the kinetics of odorant response in the maxillary palp of Drosophila, and show that the rate of recovery from odorant stimulation is affected by mutation of the rdgB (retinal degeneration B) gene. We use immunocytochemistry to confirm that the rdgB gene product is expressed in the maxillary palp. rdgB has recently been shown to encode a protein with Ca²⁺-binding sites and sequence similarity to rat brain phosphatidylinositol transfer protein; it is located near the rhabdomeric membranes in photoreceptor cells, where it has been suggested to play a role in membrane transport. The delay in recovery kinetics that we observe in olfactory tissue may reflect a defect in membrane restoration at the conclusion of the olfactory transduction cascade. The use of common molecules in the physiology of two olfactory organs, and in both visual and olfactory physiology, is discussed.Abbreviations EAG electroantennogram - EPG electropalpogram - ERG electroretinogram - norpA no receptor potential A - PBS phosphate buffered saline - rdgB retinal degeneration B - PI phosphatidylinositol     

Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.