InteractiveFly: GeneBrief

neither inactivation nor afterpotential E: Biological Overview | References

Many animals, including the fruit fly, are sensitive to small differences in ambient temperature. The ability of Drosophila larvae to choose their ideal temperature (18°C) over other comfortable temperatures (19° to 24°C) depends on a thermosensory signaling pathway that includes a heterotrimeric guanine nucleotide-binding protein (G protein), a phospholipase C, and the transient receptor potential TRPA1 channel. Mutation of the gene (ninaE) encoding a classical G protein-coupled receptor (GPCR), Drosophila rhodopsin, eliminates thermotactic discrimination in the comfortable temperature range. This role for rhodopsin in thermotaxis toward 18°C was light-independent. Introduction of mouse melanopsin restored normal thermotactic behavior in ninaE mutant larvae. It is proposed that rhodopsins represent a class of evolutionarily conserved GPCRs that are required for initiating thermosensory signaling cascades (Shen, 2011).

Temperature sensation in animals is mediated largely by direct activation of transient receptor potential (TRP) ion channels. An exception is a TRP channel in Drosophila larvae that functions indirectly in the selection of their optimal temperature (18°C) over other comfortable temperatures (19° to 24°C) and does so through a signaling cascade that includes a heterotrimeric guanine nucleotide-binding protein (G protein) G_q, phospholipase C (PLC), and the TRPA1 channel (Kwon, 2008). A thermosensory signaling cascade is also required in Caenorhabditis elegans, which includes guanylate cyclases and a guanosine 3',5'-monophosphate (cGMP)-gated channel. Thermosensory signaling cascades may contribute to amplification of small temperature differences and to adaptation to temperatures that are less than optimal, but still permissive for survival (Shen, 2011).

G protein-coupled receptors (GPCRs) are candidates to initiate thermosensory cascades because they couple to pathways that include G_q, PLC, and TRP channels, as well as to cascades that engage guanylate cyclases and cGMP-gated channels. However, there are up to 200 hundred GPCRs encoded in flies and over one thousand in worms, and there is no precedent for a GPCR that functions in thermosensation (Shen, 2011).

It was of interest to find out whether the canonical GPCR (rhodopsin) might be required for thermosensation, even though it is thought to function exclusively in light sensation. The basis for this proposal is that the same G_q (Gα49B) and PLC [No Receptor Potential A (NORPA)] that function in light sensation and link rhodopsin to activation of TRP channels are required for larvae to move preferentially toward the 18°C region when the alternative zone is held at another temperature in the 19° to 24°C range (Kwon, 2008). If this behavior requires rhodopsin, it would be a light-independent function, because thermotaxis takes place effectively in the dark (Kwon, 2008; Shen, 2011 and references therein).

To test temperature selection, larvae were placed on a plate between two temperature zones, one of which was kept at 18°C and the other at an alternative temperature. After 10 min, the larvae in each zone were counted and the preference index (PI) was calculated. A lack of temperature bias results in a PI of 0, whereas a complete preference for 18°C or the alternative temperature results in a PI of 1.0 or -1.0, respectively. Wild-type larvae select 18°C over any other temperature, including other temperatures in their comfortable range (20° to 24°C) (Shen, 2011).

To address whether the major opsin (Rh1) encoded by the ninaE gene was required for thermotaxis in their comfortable temperature range, flies were tested with a deletion that removed the ninaE coding region (ninaE^I17). The ability to distinguish 18° from 24°C was impaired in ninaE^I17 larvae and in animals containing the ninaE^I17 mutation in trans with another deletion (Df) that removed ninaE on the homologous chromosome. This phenotype was indistinguishable from the thermotaxis deficits resulting from mutations disrupting PLC (norpA^P24) or the TRPA1 channel (trpA1¹). Flies with any of five of six additional ninaE alleles showed deficits in discrimination between 18° and 24°C, but not between 18°C and cooler or very warm temperatures. Larvae with one missense allele, ninaE^P332, strongly preferred 18°C over 24°C, although the bias for 18°C was eliminated when the alternative temperature was either 20° or 22°C (Shen, 2011).

To confirm that the thermotaxis defect was due to mutation of ninaE, tests were performed for rescue of the phenotype with a wild-type transgene, using the GAL4-UAS system. This approach employs the yeast GAL4 transcription factor that binds to the upstream activation sequence (UAS) to promote transcription. Only ninaE¹⁷ larvae containing both the ninaE-GAL4 and UAS-ninaE transgenes effectively chose 18°C over 24°C. Another GPCR (serotonin receptor; UAS-5-HT₂), which is most similar to mammalian G_q-coupled serotonin receptors, does not rescue the ninaE^I17 deficit. Similar to the norpA^P24 and trpA1¹ phenotypes, loss of ninaE impaired discrimination between 18°C and other temperatures in the comfortable range, 20° or 22°C, but not selection of 18°C over cooler (14° or 16°C) or warmer temperatures (26° to 32°C) (Shen, 2011).

In Drosophila, the vitamin A-derived chromophore stably binds to the opsin and is required for Rh1 to exit the endoplasmic reticulum. Wild-type larvae grown on food depleted of vitamin A, or mutant larvae (santa maria¹) missing a scavenger receptor required for chromophore generation (Wang, 2007), showed impaired temperature discrimination in the 18°C to 24°C range. The defect in santa maria¹ was reversed by adding all trans-retinal to the food (Shen, 2011). To address whether Rh1 might function in the same cells as other components involved in 18° to 24°C thermotaxis, UAS-RNAi transgenes were expressed under the transcriptional control of the ninaE-GAL4 or the trpA1-GAL4. Expression of Gα49B, norpA, or trpA1 RNA interference (RNAi) transgenes using the ninaE-GAL4 reduced the biases toward 18°C over 22° or 23°C. Similarly, the preference for 18°C was diminished in larvae expressing the ninaE RNAi under control of the trpA1-GAL4. Expression of UAS-ninaE⁺ under control of the trpA1-GAL4 restored 18° versus 24°C temperature discrimination in ninaE^I17 larvae (Shen, 2011).

Because rhodopsin is a light sensor, whether thermotactic behavior is altered by light was tested. Wild-type larvae chose 18° over 24°C equally well in the light or dark. Moreover, ninaE^I17 displayed similar thermotactic impairments in the presence or absence of light. Thus, selection of 18° over 24°C was light-independent (Shen, 2011).

Larvae that were unresponsive to light were also tested. Wild-type early third instar larvae avoid white or blue, but not orange, light. For larvae given a choice between 18° and 23°C, the aversion to light overcame the preference for 18°C. Bolwig’s organs, which consist of larval photoreceptor cells that function in the avoidance of moderate light intensities, do not express the trpA1-GAL4. norpA^P24 animals are not negatively phototactic, and expression of UAS-norpA, under the control the trpA1-GAL4 does not restore negative phototaxis. These larvae discriminated temperatures in the 18° to 23°C range, and this behavior was not affected by light (Shen, 2011).

The ninaE gene appeared to be expressed at an exceptionally low level because no signal was detected in larvae with Rh1 antibodies or using the ninaE-GAL4 to drive UAS-GFP. Low amounts of Rh1 might prevent efficient light activation of Rh1 in thermosensory neurons, which might impair thermotactic discrimination. To provide additional evidence that ninaE was coexpressed with trpA1, neurons were dissected from the body wall and the anterior region that expressed the trpA1-reporter (trpA1-GAL4 and UAS-mCD8-GFP; mCD8 is the mouse CD8 receptor), and reverse transcription polymerase chain reaction (RT-PCR) was performed. ninaE RT-PCR products were detected in 5 out of 15 green fluorescent protein (GFP)-positive neurons (3 out of 8 from the body wall; 2 out of 7, anterior region), but not in any dissected GFP negative neurons (Shen, 2011).

Selection of 17.5° to 18°C over cooler temperatures occurs through avoidance that results from increased turning at slightly lower temperatures. To test whether the preference for 18° over slightly higher temperatures occurred through a similar mechanism, larvae were tracked. Wild-type larvae appeared to progress only a short distance into the 24°C area before they paused, stretched their heads, and initiated their first turns. However, ninaE^I17, norpA^P24, and trpA1¹ mutant larvae did not appear to turn until they traversed far into the 24°C zone (Shen, 2011).

To quantify turning behavior, a simple assay was developed. The 24°C zone was demarcated with 20 lines, the larvae were released on the 18°C side near the 24°C interface, and the last line crossed before the larvae made their first turn was tabulated. Larvae were only counted that moved perpendicular to the lines (~5° deviation). Wild-type larvae turned near line 3. However, the mutant larvae traveled to near line 14 in the 24°C area before turning. The much greater distances traveled by the mutants before turning did not appear to be due to increased movement speeds, because all the larvae moved at similar rates. In a reciprocal experiment, larvae were placed on the 24°C side, and animals were monitored that crossed perpendicular to the lines demarcating the 18°C zone. Wild-type larvae did not turn until line 10, and there were only small variations between wild-type and mutant animals (Shen, 2011).

Tests were performed to see whether the higher rate of larval turning at 24°C was dependent on prior exposure to a lower temperature. Wild-type larvae placed on a plate uniformly held at a single temperature showed similar turning frequencies at all temperatures tested (18° to 24°C). Similar results were obtained with the ninaE^I17, norpA^P24, and trpA1¹ larvae. Thus, turning at 24°C was dependent on prior exposure to 18°C (Shen, 2011).

Several results argue strongly against a developmental defect underlying the thermotaxis impairment in the comfortable range. First, although ninaE^P332 larvae were impaired in selecting 18°C over 20° or 22°C, they were able to choose 18°C over 24°C. Second, multiple ninaE missense mutations, including ninaE^P332 and ninaE^P318, have no apparent effects on morphogenesis and are not associated with retinal degeneration, which suggests that these alleles do not affect development of the thermosensory neurons. Third, indistinguishable numbers and morphological appearances of GFP-positive cells were found in wild-type and ninaE^I17 larvae that expressed UAS-mCD8-GFP under control of the trpA1-GAL4 (Shen, 2011).

Advantage was taken of the slightly higher PI exhibited by ninaE^P332 (18° versus 24°C) to test whether other genes required for thermotaxis functioned subsequent to ninaE. Introduction of the Gα49B¹, norpA^P24 or trpA1¹ mutations into the ninaE^P332 background prevented 18°C selection over 24°C. Another mutation that causes a higher-than-normal PI disrupts the rhodopsin phosphatase (rdgC³⁰⁶). The combination of ninaE^I17 or Gα49B¹ with rdgC³⁰⁶ eliminated the bias for 18° over 24°C. These analyses indicate that G_q, PLC, and TRPA1 function in a pathway downstream of Rh1 (Shen, 2011).

Drosophila encodes additional opsins (Rh2-6). To determine whether other opsins could substitute for Rh1, Rh2-6 were expressed under control of the ninaE promoter in ninaE^I17 flies, and 18° versus 24°C selection was assayed. With the exception of Rh3, other opsins could replace Rh1. However, the transgenic flies showed significant differences from wild type when given a choice between 18° and 20° to 22°C. Another GPCR coupled to G_q [5-hydroxytryptamine (5-HT₂)] did not function in place of Rh1 (Shen, 2011).

The mammalian opsin that is most similar to Drosophila Rh1 is melanopsin (OPN4). Expression of Opn4 under control of the ninaE promoter did not reverse the phototransduction defect in adult ninaE^I17. However, Opn4 enabled the ninaE^I17 larvae to distinguish between 18°C and 24°C (Shen, 2011).

The observations that Rh1 is required for thermosensory discrimination and that OPN4 could substitute for Rh1 suggest that Rh1 and related opsins might be intrinsic thermosensors. However, the intrinsic rate of thermal activation, which is ~1/min in fly photoreceptor cells, is far too low to account for the requirement for Rh1 for thermosensation. It is suggested that an accessory factor might interact with Rh1 and accelerates its intrinsic thermal activity. Finally, because rhodopsin has dual roles, it is interesting to consider the question as to whether the archetypal role for rhodopsin was in light sensation or in thermosensation (Shen, 2011).

Targeting of Drosophila rhodopsin requires helix 8 but not the distal C-terminus

The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs. Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, it is proposed that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres (Kock, 2009; full text of article).

Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila

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 (see Normalized spectral quantum sensitivity of the different rhodopsins in the photoreceptor subtypes R7p, R7y, R8p, R8y, and R1-R6). This study 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 (Yamaguchi, 2010).

Phototaxis consists of at least three behavioral components: (1) detection of an object or a light source in visual space; (2) the attractiveness or aversiveness of the light stimulus at this location; and (3) a motor output (goal-directed walking and turning). Genetic dissection of the visual input to phototaxis relies on the assumption that phototaxis stays intact even if some of these inputs are deleted, i.e., that the mutants can still detect the locations of the two stimuli and move toward or away from them. Although in mutants affecting the R1-R6 subsystem, walking speed, turning, and orientation toward stationary objects are affected, these behaviors are sufficiently intact to support phototaxis. In the experiments reported in this study, light intensities were chosen such that all mutants had at least one photoreceptor type that could detect the light sources and guide walking and turning behavior. This allowed measuring of the second component, the differential attractiveness of the two stimuli, which determined the choice between the two monochromatic lights depending upon the photoreceptor types available in the respective fly strains. Flies could judge the attractiveness of a light source on the basis of its spectral composition and/or spectrally weighted brightness. In the following paragraphs the contributions of the four types of photoreceptors R1-R6, R7, R8p, and R8y to the attractiveness function are discussed (Yamaguchi, 2010).

Blocking or removing any one of the photoreceptor types shifts the preference away from the point of neutrality in at least one of the choice tests. Silencing the R1-R6 cells leaves the flies with a modest blue preference in the UV/B choice, and replacing their broadly sensitive opsin Rh1 by the UV-sensitive opsin Rh3 shifts the preference in the B/G choice to blue. Removing or silencing R7 in the presence of R1-R6 shifts the preference in the UV/B choice to blue and in the B/G choice to green. Inactivating the green-sensitive Rh6 opsin in R8y cells shifts the preference in the B/G choice to blue and in the UV/B choice to UV. Inactivating blue-sensitive Rh5 in the R8p cells causes a green preference in the B/G choice and a UV preference in the UV/B choice (Yamaguchi, 2010).

Each of the three photoreceptor subsystems (R1-R6, R7, R8) alone can drive phototaxis. Flies with only photoreceptors R1-R6 (sev rh5² rh6¹ triple mutants) show a blue preference in the UV/B choice, implying that this subsystem not only mediates optomotor responses and orientation but also can mediate the attractiveness function in phototaxis, i.e., that, with only R1-R6 photoreceptors, flies compare the quantum flux of two light sources 180° apart (Yamaguchi, 2010).

The R8 subsystem alone can also mediate the attractiveness function in phototaxis (sev ninaE¹⁷), as had been shown before for the sev rdgB double mutant. The sev ninaE¹⁷ flies have a pronounced green preference in the B/G choice test, consistent with the absorption spectrum of Rh6 expressed in all R8 photoreceptors (Yamaguchi, 2010).

Flies that have only R7 photoreceptors operating (ninaE¹⁷, rh5², rh6¹) show phototaxis. Surprisingly, they have a very strong preference for blue in both choice tests. This could be explained if R7p inhibit R7y photoreceptors. Otherwise, the flies would show a strong UV preference in the UV/B choice. As an alternative explanation, however, it is possible that phototaxis is mediated by the ocelli in flies with only R7 photoreceptors (Yamaguchi, 2010).

As long as only one of the central photoreceptor subsystems R7 or R8 is inactivated, the results can be explained by a model in which the respective photoreceptors contribute roughly additively to the attractiveness function. In the honeybee, all three subtypes of photoreceptors (UV, blue, and green) also feed into phototaxis. Spectral mixing experiments in phototaxis are consistent with simple summation of quantal fluxes, and the action spectra of phototactic behavior also suggest pooling of all three photoreceptor types. Color information, i.e., comparison between different photoreceptors, does not appear to be used in honeybee phototaxis (Yamaguchi, 2010 and references therein).

A simple summation model thus can no longer explain the results of all of the genetic manipulations of photoreceptor types in Drosophila presented in this study. When all four photoreceptor types are intact, UV light is more attractive than would be expected from the sum of the UV attractiveness values of the two isolated major subsystems, R1-R6 and R7/R8. In the UV/B choice test that is balanced for wild type, both flies with only the R1-R6 subsystem (sev rh5² rh6¹) and flies with only the R7/R8 subsystem (rh1 > shi^ts or ninaE¹⁷) show a blue preference. It is thus necessary to postulate that an interaction between the two major retinal subsystems R1-R6 and R7/R8 enhances UV attractiveness. The interaction cannot be explained by an attenuation of the attractiveness of blue because no increased blue preference of the two mutants is observed in the B/G choice. The interaction can unambiguously be traced to the R7 cells. In fact, the strength of the contribution of R7 to the attractiveness function in the UV/B choice depends upon which of the other photoreceptor types are functional. No effect of R7 on spectral preference can be detected without a functional R1-R6 subsystem (comparing ninaE¹⁷ and sev ninaE¹⁷ in both choice tests. A moderate effect is found in the presence of R1-R6 and R8 (comparing wild type and sev; whereas the effect of R7 is large in the absence of the R8 subsystem (comparing rh5² rh6¹ and sev rh5² rh6¹ in UV/B choice (Yamaguchi, 2010).

The most parsimonious explanation of the data is to assume that, in the presence of a functional R8 subsystem, neither the R1-R6 nor the R7 subsystems on their own have a direct input to the attractiveness function. In the absence of R7 (mutant sev), the R1-R6 subsystem seems not to matter for attractiveness although the R8 subsystem is lacking spectral sensitivity in the UV. Likewise, in the absence of a functional R1-R6 subsystem, the R7 subsystem seems not to contribute. Interestingly, as soon as R8y photoreceptors (or R8p and R8y) are inactivated, the other two subsystems exert their influence on the attractiveness function independently of each other. A full model of these interactions would require a more complete investigation (Yamaguchi, 2010).

R1-R6 rhabdomeres degenerate in ninaE¹⁷ mutants, and this degeneration may have secondary effects on the R7/R8 cells. However, there is no significant difference between ninaE¹⁷ mutants and flies expressing rh1>shi^ts, in which R1-R6 function is disrupted without affecting rhabdomere structures. Moreover, it was recently reported that R8y are functional in ninaE¹⁷ mutants because circadian entrainment to red light, which is still observed in ninaE¹⁷, is abolished in ninaE¹⁷ rh6¹ double mutants (Yamaguchi, 2010).

The data reveal a further deviation from a simple summation of photoreceptor inputs to the attractiveness function. In the melt^GOF mutant, green-sensitive R8y cells are transformed into blue-sensitive R8p cells. This should shift their preference in the UV/B choice to blue. Yet, a UV shift is observed. In contrast, in the absence of R7 cells, the transformation of R8y to R8p photoreceptors (comparison between sev and sev melt^GOF) increases the blue preference in the UV/B choice as would be expected from spectral sensitivities. This might indicate that R8p cells enhance the input of the UV channel (R1-R6 × R7) to the attractiveness function. These findings, however, should be treated with caution as unknown developmental defects in the sev and melt^GOF mutants might account for the phenotypes (Yamaguchi, 2010).

Using a different phototaxis paradigm, Jacob (1977) proposed a model to explain the nonadditive effects observed in his phototaxis experiments. An inhibition of R1-R6 by R7/R8 was postulated and it was suggested that R7 cells function only when the R1-R6 system is intact. The current results ask for revision of this model. No general inhibition of the R1-R6 receptor subsystem by R7/R8 was found. The data suggest that neither the R1-R6 nor the R7 subsystems have access to the attractiveness function on their own, except in the absence of functional R8y photoreceptors. Moreover, R8p cells in the current tests had an effect only in the presence of functional R8y cells (Yamaguchi, 2010).

Rh3-expressing R8 cells in the dorsal rim area account for about 10% of all Rh3 expressing cells. These R8 are still present in sev flies, but are switched off in panR7> shi^ts flies. The comparison of these two lines does not reveal an effect of the Rh3-expressing R8 cells in the UV/B choice. Taking these cells into consideration therefore does not change any of the above conclusions (Yamaguchi, 2010).

This study has shown that all four photoreceptor types [R1-R6, R7(p, y), R8p, and R8y] are involved in phototaxis, in contrast to motion detection, which relies exclusively on R1-R6 photoreceptors. The wild-type attractiveness function cannot be described as the sum of the attractiveness functions of flies lacking one or more functional photoreceptor types. A multiplicative interaction is observed between photoreceptors R1-R6 and R7. In the absence of functional R1-R6 or R7, the attractiveness function is governed by R8. Only in the absence of R8 and one of the other two subsystems does the remaining subsystem govern the attractiveness function. A recent study on the neuronal substrate of spectral preference identified postsynaptic interneurons in the medulla (Gao, 2008) that are good candidates for mediating some of these interactions. Their involvement in differential phototaxis can now be tested (Yamaguchi, 2010).

The attractiveness function of differential phototaxis is easy to record, robust, and sensitive (for example, Rh5-expressing blue-sensitive cells account for only ~4% of all photoreceptors, yet yield a significant phenotype when they are not functional). Differential phototaxis may lend itself to mutant screens affecting other chromaticity computations in the brain, including color vision, at the circuit level (Yamaguchi, 2010).

Light-induced translocation of Drosophila visual Arrestin2 depends on Rac2

Photoreceptor cells are remarkable in their ability to adjust their sensitivity to light over a wide range of intensities. Rapid termination of the photoresponse is achieved in part by shuttling proteins in and out of the light-transducing compartment of the photoreceptor cells. One protein that undergoes light-dependent translocation is the rhodopsin regulatory protein arrestin. However, the mechanisms coupling rhodopsin to arrestin movement are poorly understood. This study shows that light-dependent shuttling of the major arrestin in Drosophila photoreceptor cells, Arrestin2 (Arr2), occurs independently of known elements of the phototransduction cascade. Disruptions of the trimeric G protein, phospholipase Cβ, the TRP channel, or the Na⁺/Ca²⁺ exchanger did not influence Arr2 localization. Rather, it was found that loss of the small GTPase Rac2 severely impaired Arr2 movement and prolonged the termination of the photoresponse. These findings demonstrate that light-induced translocation of Arr2 occurs through a noncanonical rhodopsin/Rac2 pathway, which is distinct from the classical phototransduction cascade (Elsaesser, 2010).

Activity-dependent shuttling of signaling proteins between the cell surface and intracellular compartments is a widespread phenomenon which contributes to the magnitude and duration of signaling in neurons and many other cell types. One of the earliest demonstrations of activity-dependent translocation of signaling proteins from one cell compartment to another was the light-induced translocation of visual arrestin from the inner to the outer segments of rod photoreceptor cells over the course of a few minutes (Calvert, 2006). Light-dependent shuttling of signaling proteins is an evolutionarily conserved phenomenon, as photostimulation also triggers the movement of the Drosophila visual arrestins from the cell bodies to the fly counterpart to rod outer segments, the rhabdomeres (Kiselev, 2000; Lee, 2003). The trimeric G proteins that function in mammalian and Drosophila phototransduction undergo light-dependent translocation as well, as does the Drosophila transient receptor potential-like (TRPL) channel. However, in contrast to the arrestins, these latter proteins shuttle out of the outer segments and rhabdomeres in response to light. The movements of these signaling proteins have important physiological consequences, as they contribute to light adaptation and termination of the photoresponse and thus are crucial for the ability of photoreceptor cells to adjust their sensitivity to the surrounding light conditions (Elsaesser, 2010).

The mechanisms and signaling pathways controlling the translocation of the Drosophila arrestins, G protein, and TRPL proteins have been explored but are incompletely understood. The light-dependent movement of the major visual arrestin, referred to as Arrestin2 (Arr2), requires interaction with PIP₃. In addition, the NinaC myosin III has been reported to contribute to the spatial reorganization of G_q, TRPL, and Arr2, although Arr2 depends on NINAC only under blue but not white light. Because light triggers the translocations, they would be expected to require activity of the phototransduction cascade. In flies, light-activated rhodopsin engages a heterotrimeric G protein, G_q, leading to stimulation of a phospholipase C (PLC) and opening of the TRP and TRPL cation channels. Visual arrestin binds to rhodopsin and attenuates signaling by dislodging the heterotrimeric G protein associated with the light-activated rhodopsin. Indeed, movement of TRPL requires G_q and PLC, although the light-dependent shuttling of G_q has been reported to occur independently of PLC, TRP, or TRPL (Elsaesser, 2010).

The current work found that the dynamic spatial redistribution of Arr2 from the cell bodies to the rhabdomeres required rhodopsin, but did not depend on any of the other known components of the phototransduction cascade. These include G_q, PLC, TRP, TRPL, the Na⁺/Ca²⁺ exchanger (CalX), and protein kinase C. Rather, it was found that the small GTPase Rac2 interacts with rhodospsin and is essential for the translocation of Arr2 into the rhabdomeres. As is the case with photoreceptor cells expressing Arr2 derivatives that do not translocate efficiently, mutations in rac2 cause a defect in termination of the photoresponse. These data indicate that the light-dependent movement of Arr2 depends on a parallel phototransduction cascade that is initiated by coupling of rhodopsin to Rac2 (Elsaesser, 2010).

Rhodopsin is the archetypal G-protein-coupled receptor, which defines a family of highly related visual pigments. Before the current work, all light-activated pathways known to be physiologically required in photoreceptor cells functioned through heterotrimeric G proteins. Alternative candidate effectors for rhodopsin were small GTPases, because mammalian rhodopsin interacts with a Rho family member and activates this small GTPase in a light-dependent fashion (Balasubramanian, 2003). However, no role has been ascribed for transducing light activity through a rhodopsin/small GTPase pathway (Elsaesser, 2010).

This study found that the light-induced movement of Arr2 from the cell bodies to the rhabdomeres was strictly dependent on Rac2. Moreover, termination of the photoresponse was severely impaired in rac2^δ flies. Thus, the findings indicate a signaling mechanism underlying one form of light adaptation which entails transduction of the light signal through a small GTPase, rather than engaging Rh1 with the G_q/PLC/TRP cascade (Elsaesser, 2010).

In addition to its role as a light receptor, rhodopsin plays a structural role during photoreceptor cell morphogenesis (Kumar, 1995). This light-independent function of rhodopsin is mediated by Rac1 (Chang, 2000). In contrast, the requirement for Rac2 for Arr2 movement did not appear to be due to a morphological defect because the ultrastructures of wild-type and rac2^δ null mutant photoreceptor cells were indistinguishable. Thus, although the roles of Rac1 and Rac2 are often considered interchangeable, Rac2 is specifically required for light-dependent translocation of Arr2 translocation in adult photoreceptor cells, whereas Rac1 has a structural role during development. Other nonredundant functions of Rac2 have been described in the Drosophila cellular immune response (Elsaesser, 2010).

It appears that two phototransduction pathways contribute to light-dependent movements of signaling proteins in Drosophila photoreceptor cells. The classical pathway is required for shuttling of TRPL because it is dependent on most elements of the phototransduction cascade including the PLC. However, the movement of Arr2 depends on a second noncanonical, Rac2-dependent pathway. Light-dependent shuttling of the Gα_q may also function through this second pathway because it occurs normally in mutants missing PLC, protein kinase C, or TRP (Elsaesser, 2010).

The current work raises questions concerning the nature of the proteins that function in concert with Rh1/Rac2 signaling in photoreceptor cells. Finally, because the mammalian Rac1, which is the GTPase most related to Drosophila Rac2, is activated in a light-dependent manner, it is proposed that rhodopsin/Rac signaling may be an evolutionarily conserved mechanism controlling light-induced arrestin translocation and light adaptation in mammalian rods and cones (Elsaesser, 2010).

Unfolded protein response in a Drosophila model for retinal degeneration

Stress in the endoplasmic reticulum (ER stress) and its cellular response, the unfolded protein response (UPR), are implicated in a wide variety of diseases, but its significance in many disorders remains to be validated in vivo. This study analyzed a branch of the UPR mediated by xbp1 in Drosophila to establish its role in neurodegenerative diseases. The Drosophila xbp1 mRNA undergoes ire-1-mediated unconventional splicing in response to ER stress, and this property was used to develop a specific UPR marker, xbp1-EGFP, in which EGFP is expressed in frame only after ER stress. xbp1-EGFP responds specifically to ER stress, but not to proteins that form cytoplasmic aggregates. The ire-1/xbp1 pathway regulates heat shock cognate protein 3 (hsc3), an ER chaperone. xbp1 splicing and hsc3 induction occur in the retina of ninaE^G69D−/+, a Drosophila model for autosomal dominant retinitis pigmentosa (ADRP), and reduction of xbp1 gene dosage accelerates retinal degeneration of these animals. These results demonstrate the role of the UPR in the Drosophila ADRP model and open new opportunities for examining the UPR in other Drosophila disease models (Ryoo, 2007).

The endoplasmic reticulum (ER) is the cellular organelle where proteins destined for secretion or for diverse subcellular localizations are synthesized and acquire their correct conformation. Perturbations of the environment normally required for protein folding in the ER, or production of large amounts of misfolded proteins exceeding the functional capacity of the organelle, trigger a physiological response in the cell, collectively known as the unfolded protein response (UPR). The UPR serves to cope up with ER stress by transcriptionally regulating ER chaperones and other ER-resident proteins, attenuating the overall translation rate and increasing the degradation of misfolded ER proteins (Ryoo, 2007).

The mechanisms leading to the UPR activation and its short-term response in regulating gene expression are well characterized in various organisms. In Saccharomyces cerevisiae, unfolded proteins in the ER cause oligomerization of the ER transmembrane protein Ire-1. Upon oligomerization, the endoribonuclease activity of Ire-1 is activated, which catalyzes the unconventional splicing of hac-1 mRNA. Splicing of hac-1 mRNA allows its translation and its protein product acts as a transcription factor, by binding to DNA motifs collectively called UPRE. This leads to the induction of proteins that help to alleviate ER stress. Ire-1 in Caenorhabditis elegans and mammals plays a similar role, by splicing the mRNA of xbp1. Other branches of the UPR include transcription factors ATF4 and ATF6, as well as nontranscriptional mechanisms that reduce the overall amount of misfolded proteins in the ER. This occurs in part through the attenuation of protein translation through PERK, an ER transmembrane kinase, and enhanced rate of protein degradation, also known as ERAD (ER-associated degradation). PERK and the components of the ERAD machinery are activated in response to ER stress (Ryoo, 2007 and references therein).

Whereas transient ER stress can be alleviated by the UPR, a prolonged condition of ER stress can trigger apoptosis. In a mouse model of Pelizaeus-Merzbacher disease (PMD), misfolded proteolipid protein in the ER triggers apoptosis of oligodendrocytes, and several components of the UPR have been shown to play a protective role against the progression of the disease. In contrast, certain components of the UPR, including the PERK/ATF4/CHOP branch, are implicated to play a proapoptotic role. Additionally, numerous cell culture studies implicate the UPR in many disorders caused by misfolded proteins in the cytoplasm, such as Huntington's and Parkinson's diseases. However, the links between the UPR and a wide variety of neurodegenerative disorders remain indirect and controversial, in part, owing to limitations of existing animal model systems (Ryoo, 2007).

To better understand the role of the UPR in disease, this study focused on a set of alleles in Drosophila ninaE (or rhodopsin-1 (Rh-1)), which have molecular and phenotypic characteristics identical to those found in class III autosomal dominant retinitis pigmentosa (ADRP). These alleles have mutations either in transmembrane domains or in extracellular loops that are predicted to disrupt Rh-1 folding properties (Colley, 1995; Kurada, 1995). ADRP-afflicted individuals, in both humans and Drosophila, show late-onset retinal degeneration, which occurs through the activation of apoptosis (Davidson, 1998; Galy, 2005). Inhibiting caspases blocks retinal degeneration and blindness in the Drosophila model, demonstrating that apoptosis is the main cause of the disease (Ryoo, 2007).

This study examined whether the UPR contributes to the progression of retinal degeneration in class III ADRP model of Drosophila. xbp1 splicing and the induction of an ER chaperone, heat shock cognate protein 3 (hsc3), are shown to occur in response to ER stress. Using this knowledge, an xbp1-EGFP fusion transgene was devised as an in vivo marker of ER stress, designed to have EGFP expressed in frame with xbp1 only after Ire-1-mediated splicing occurs. xbp1-EGFP splicing occurs after mutant Rh-1 expression, but is not detectable in response to polyglutamine repeats or tau R406W, which cause neurodegenerative disorders in humans by forming cytoplasmic protein aggregates. xbp1-EGFP splicing also occurs in the Drosophila model of ADRP, and xbp1 has a protective role against retinal degeneration. These results demonstrate that ER stress occurs in the Drosophila ADRP model and suggest possible mechanisms by which apoptosis is activated in response to mutant Rh-1 molecules (Ryoo, 2007).

ER stress has been implicated in a wide variety of human diseases, including many neurodegenerative disorders and cancer, but relatively few have been validated in animal models. This study has shown that the basic UPR pathway is conserved from yeast, C. elegans, Drosophila and mammals. Moreover, it was demonstrated that the UPR is activated in the ADRP model of Drosophila, and this plays a protective role against the progression of retinal degeneration (Ryoo, 2007).

xbp1 splicing is a specific indicator of the UPR signaling in Drosophila. Using the xbp1-EGFP fusion construct, this study detected xbp1 splicing in response to ER-stress-causing agents (DTT, thapsigargin and tunicamycin), ectopic expression mutant Rh-1, and in the Drosophila model of ADRP. However, in vivo experiments did not support previous observations that cytoplasmic aggregates can also cause ER stress indirectly, through compromising the proteasome capacity. xbp1 activation in the ADRP model can account for many phenotypes previously reported. One such phenotype is the dramatic enlargement of the ER network. As studies in other organisms have shown that the ire-1/xbp1 branch of UPR promotes ER membrane biogenesis, it is most likely that the enlarged ER-network biogenesis of ninaE^G69D−/+ retina is due to Drosophila xbp1 activation. In contrast to the stressed cells, significant levels of xbp1 (or xbp1-EGFP) splicing were not detected in unstressed developing tissues of embryos or late third instar larvae. This is consistent with previous studies conducted in transgenic mice harboring an xbp1-GFP(venus) construct similar to the one used in this study. In mice, xbp1 splicing was not detected in embryonic or postnatal stages, but only after late postnatal stage (16 days or older) and only in a few tissues. A small number of cells showing xbp1-EGFP splicing was observed in earlier stage larvae, indicating that occasional xbp1 splicing occurs during normal development. As xbp1 mutation is recessive lethal, the low level of natural xbp1 splicing may account for the requirement of this gene in Drosophila development. It cannot be excluded, however, that the unspliced RA form of xbp1 also plays a role during development, accounting for the lethality of xbp1-deficient animals. xbp1 is required during embryonic development in mammals, but not in C. elegans, where the animals can complete their developmental program without xbp1. In addition to xbp1, hsc3 and ire-1, other genes homologous to those implicated in the UPR are found in the Drosophila genome. These include the ER transmembrane kinase perk and the ER tethered transcription factor ATF6 (annotated as CG3136). Although their in vivo function has not been analyzed in Drosophila, their presence and high sequence homology suggest a conserved function in the UPR (Ryoo, 2007).

Previous studies demonstrated that the class III ninaE alleles show caspase-dependent cell death. The underlying mechanism by which caspases become active in these cells remains unclear, but the finding of the UPR activation in these cells provides at least three models. First model is based on the observation that JNK signaling is activated as part of the UPR. In this model, activation of the UPR stimulates Ire-1/TRAF interaction, independent of xbp1 mRNA splicing, leading to JNK activation and apoptosis. In fact, Ire-1/TRAF/JNK signaling is required for apoptosis in response to poly-glutamine repeat accumulation in cultured cells. In addition, enhanced JNK signaling is detected in the retinas of a class III ninaE allele in Drosophila. Finally, activation of JNK signaling in Drosophila results in the induction of the proapoptotic gene hid. The second model is based on the observations that Ca²⁺ release from the ER can activate caspase activation and cell death. It is possible that the disruption of ER homeostasis by the accumulation of misfolded proteins in the ER results in the release of Ca²⁺ into the cytoplasm, activating a proteolytic cascade leading to caspase activation. Third, as CHOP has been implicated to mediate ER-stress-triggered apoptosis in mammals, a similar mechanism may exist in Drosophila. However, there are no obvious homologs of CHOP in the Drosophila genome. Whether these models account for the death of class III ninaE mutant photoreceptors awaits further studies (Ryoo, 2007).

Drosophila provides a unique advantage as a model for studying human diseases associated with ER-stress-triggered cell death, as the mechanisms of stress-provoked caspase activation are largely conserved between the two species. In addition, the short lifespan of Drosophila, combined with a similarly accelerated rate of chronic disease manifestation, may help validate the in vivo significance of the UPR in a growing list of disorders, ranging from neurodegenerative disease involving cytoplasmic protein aggregates, hypoxia during cancer progression and p53-induced cell death. In this regard, the present work on the role of the UPR in Drosophila retinal degeneration may provide a framework for further investigations into the UPR in human disease (Ryoo, 2007).

Rhodopsin formation in Drosophila is dependent on the PINTA retinoid-binding protein

Retinoids participate in many essential processes including the initial event in photoreception. 11-cis-retinal binds to opsin and undergoes a light-driven isomerization to all-trans-retinal. In mammals, the all-trans-retinal is converted to vitamin A (all-trans-retinol) and is transported to the retinal pigment epithelium (RPE), where along with dietary vitamin A, it is converted into 11-cis-retinal. Although this cycle has been studied extensively in mammals, many questions remain, including the specific roles of retinoid-binding proteins. This study established the Drosophila visual system as a genetic model for characterizing retinoid-binding proteins. In a genetic screen for mutations that affect the biosynthesis of rhodopsin, a novel CRAL-TRIO domain protein was identified, prolonged depolarization afterpotential is not apparent (PINTA), which binds to all-trans-retinol. PINTA functions subsequent to the production of vitamin A and is expressed and required in the retinal pigment cells. These results represent the first genetic evidence for a role for the retinal pigment cells in the visual response. Moreover, the data implicate Drosophila retinal pigment cells as functioning in the conversion of dietary all-trans-retinol to 11-cis-retinal and suggest that these cells are the closest invertebrate equivalent to the RPE (Wang, 2005; full text of article).

A dominant rhodopsin mutation triggers two mechanisms of retinal degeneration and photoreceptor desensitization

A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, a dominant mutation was identified in the ninaE gene (NinaE^pp100) that encodes RH1, the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration is attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaE^pp100 mutant photoreceptor cells are strongly desensitized, which partially masks their constitutive activity. This desensitization primarily results from both the persistent binding of arrestin (ARR2) to the NINAE^pp100 mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaE^pp100 represents the first rhodopsin mutation that stabilizes this protein complex. Additionally, the NinaE^pp100 mutation leads to elevated levels of G_qalpha in the cytosol, which mediates a novel retinal degeneration pathway. Eliminating both G_qalpha and arrestin completely rescues the NinaE^pp100-dependent photoreceptor cell death, which indicates that the degeneration is entirely dependent on both G_qalpha and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans (Iakhine, 2004).

Ceramidase expression facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors

Transgenic expression of ceramidase suppresses retinal degeneration in Drosophila arrestin and phospholipase C mutants. This study shows that expression of ceramidase facilitates the dissolution of incompletely formed and inappropriately located elements of rhabdomeric membranes in ninaE^I17 mutants lacking the G protein receptor Rh1 in R1-R6 photoreceptor cells. Ceramidase expression facilitates the endocytic turnover of Rh1. Although ceramidase expression aids the removal of internalized rhodopsin, it does not affect the turnover of Rh1 in photoreceptors maintained in dark, where Rh1 is not activated and thus has a slower turnover and a long half-life. Therefore, the phenotypic consequence of ceramidase expression in photoreceptors is caused by facilitation of endocytosis. This study provides mechanistic insight into the sphingolipid biosynthetic pathway-mediated modulation of endocytosis and suppression of retinal degeneration I (Acharya, 2004).

Rh1, a major component of rhabdomeres, is not only the receptor for transducing light signal in R1-R6, but is also required for organization of the rhabdomere terminal web (RTW), an actin-based cytoskeletal scaffold, believed to orchestrate the biogenesis of this organelle. In Rh1 null mutant ninaE^I17, the RTW is not organized during the late pupal stage, and rhabdomere biogenesis is defective. Consequently, photoreceptors examined by transmission electron microscopy 3 days after eclosion show the ill formed rhabdomeres, and membranes of rhabdomeres are seen involuting into the photoreceptors. These involuting curtains of rhabdomere elements are then slowly cleared over two weeks (Acharya, 2004).

Transgenic expression of ceramidase does not affect either the development or function of photoreceptors. However, ceramidase expression suppresses degeneration in arrestin and norpA mutants and in photoreceptors expressing a dominant-negative form of dynamin. This led to the proposal that ceramidase mediates its effect by modulating the endocytic pathway. It was reasoned that the phenotypic consequence of ceramidase expression in Rh1 mutant ninaE^I17 would be different from that observed in arrestin and dynamin mutants. If ceramidase affects endocytosis, then in a ninaE^I17 mutant it should influence the process of involution and clearance of the incompletely formed and inappropriately localized rhabdomeric membranes (Acharya, 2004).

Indeed, expression of ceramidase facilitates the removal of these inappropriately positioned rhabdomeric elements. Three-day-old ninaE^I17 flies have ill-formed rhabdomeres, and remnant rhabdomeres are seen as long contiguous elements of plasma membrane involuting into the cell body in R1-R6 photoreceptors. In contrast, 3-day-old ninaE^I17 flies expressing ceramidase have very little rhabdomeric elements at the apical surface of the photoreceptors. Most of the rhabdomeric membranes have been internalized and are in the process of being cleared. Although R1-R6 cells of almost all ommatidial sections of a ninaE^I17 compound eye have a significant density of remnant rhabdomeric membranes in the apical portion of the cells, more than 95% of the R1-R6 photoreceptors expressing ceramidase have no significant rhabdomeric membranes in the apical region. Instead, the apical regions have plasma membranes that are contiguous with the rest of the photoreceptor cells. The cell- cell adherens junction is intact, indicating the structural integrity of these photoreceptor cells (Acharya, 2004).

Because the development of rhabdomeres is initiated in the pupae and because GMR-Gal4 initiates ceramidase expression during its formation, it can be argued that ceramidase expression could affect the biogenesis of rhabdomeric elements in ninaE^I17, thus resulting in the observed phenotype. To resolve this issue, ceramidase was expressed, after eclosion, in an adult ninaE^I17 mutant. In these experiments, UAS-ceramidase expression was driven by a heat shock Gal4 driver. Newly eclosed ninaE^I17 flies and ninaE^I17 flies with ceramidase transgene were incubated at 37°C for 1 h/day for 3 days. Control ninaE^I17 flies heat shocked for 3 days showed features similar to non-heat-shocked mutant flies. Rhabdomeres were incompletely formed but slightly compact, and membranes were seen involuting into the R1-R6 photoreceptors. In contrast, flies expressing ceramidase cleared most of the rhabdomere from the apical region. Thus, expression of ceramidase specifically accelerates the intracellular dissolution of rhabdomeric membranes in ninaE^I17 mutant photoreceptor cells. The internalized tubular and vesicular elements generated by expression of ceramidase in ninaE^I17 was examined for an antigen specific to rhabdomere. Chaoptin is a photoreceptor specific, leucine-repeat containing, cell adhesion plasma membrane protein that localizes to the outer leaflet and is enriched in rhabdomeres of photoreceptors. ninaE^I17 flies expressing ceramidase were examined by immunoelectron microscopy for chaoptin. Chaoptin immunoprecipitates were localized on internalized membranous tubular and vesicular structures, providing additional evidence of their rhabdomeric origin (Acharya, 2004).

Like other G protein-coupled receptors, rhodopsins undergo a ligand-dependent endocytic turnover. Given the previous data on the consequence of ceramidase expression in endocytic mutants and the current observations with ninaE^I17 mutant, itwas decided to evaluate the effects of ceramidase expression on ligand (light)-induced rhodopsin turnover in wild-type photoreceptors (Acharya, 2004).

The endocytic turnover of Rh1 can be followed using an inducible and uniquely tagged Rh1. Rh1- 1D4 transgenic flies express a heat-shock inducible Drosophila Rh1 carrying a specific tag, 1D4, derived from the C terminus of bovine Rh1. Rh1- 1D4 transgene is functional, because it rescues the phenotypic defects in ninaE^I17. Rh1-1D4 synthesis was initiated in wild-type and ceramidase transgenic flies by a single heat shock at 37°C, and was followed by western analysis for 1D4 tag over a period while being maintained in a normal 12-h light/12-h dark cycle at 25°C. Under these conditions, heat shock induction initiated the synthesis of Rh1, which peaked after 24-48 h, decreased over the next several days in wild-type photoreceptors and was still visible around day 8. Under similar conditions in flies expressing ceramidase, Rh1 levels peaked similar to wild-type flies; however, Rh1 disappeared rapidly thereafter, and none was visible after 4 days. These experiments indicate that ceramidase expression enhances the light-dependent turnover of Rh1 (Acharya, 2004).

Drosophila phototransduction is a prototypic G protein-coupled receptor-signaling cascade. Like other G protein signaling cascades, invertebrate rhodopsin undergoes light-dependent turnover. In the blowfly, it has been demonstrated that rhodopsin has an extended half-life in flies maintained in the dark, whereas in flies maintained in light it has a short half-life. Although rhodopsins undergo photochemical interconversion, light-activated rhodopsins are eventually endocytosed and degraded. In Drosophila, visual arrestins act as clathrin adaptors and have been demonstrated to bind and internalize light-activated Rh1. It was hypothesized that if ceramidase facilitates a downstream process of endocytosis, although it can facilitate the turnover of light-activated rhodopsin it should have minimal effect on the half-life of an unactivated rhodopsin in flies maintained in dark. Therefore the effect was examined of ceramidase on rhodopsin turnover in flies maintained in the dark. A pulse of rhodopsin in flies reared in the dark results in longer half-life, and a greater fraction of the pulsed rhodopsin is still visible 8 days after heat shock. Similar levels of rhodopsin were seen in ceramidase expressors maintained in the dark, thus suggesting that ceramidase does not accelerate the process of rhodopsin turnover in flies maintained in the dark. It is therefore concluded that ceramidase enhances turnover of light-activated rhodopsin by facilitating endocytosis. Because ceramidase does not affect rhodopsin that is not light-activated, it is believed that it facilitates an existing normal mechanism for rhodopsin turnover. Although the molecular details of rhabdomere and rhodopsin turnover are yet to be elucidated, it has been known for several years that in flies the photoreceptor membrane is shed into the photoreceptor cell and cleared by endocytosis. Ceramidase expression results in the clearing of ill-formed rhabdomeric elements in ninaE^I17 mutants, whereas its expression in endocytic mutants such as arrestin and dynamin suppresses degeneration. Therefore, the consequence of ceramidase expression is determined by the underlying pathology of the phototransduction mutant (Acharya, 2004).

This study has used a sensitized background to reveal the effects of ceramidase on membrane turnover. By following the phenotypic changes from the long involuting rhabdomeric membranes seen in ninaE^I17 mutant photoreceptor cells to cells almost devoid of rhabdomeres in ninaE^I17 expressing ceramidase, it was shown that ceramidase expression facilitates membrane turnover in these cells. The use of an inducible, tagged Rh1 (hs-Rh1-1D4) has allowed the turnover of rhodopsin to be followed in wild-type photoreceptors. Using the inducible rhodopsin, this study has demonstrated that ceramidase specifically facilitates the turnover of light-activated receptor. Light is the ligand for rhodopsin, and these receptors are not engaged when photoreceptors are maintained in the dark. Ceramidase expression had no effect on the half-life of rhodopsin when maintained in the dark, and it was therefore concluded that ceramidase-facilitated ligand induced endocytic turnover of rhodopsin. The study of rhodopsin turnover in wild-type photoreceptors permitted examination of receptor endocytosis without complications of an underlying mutant phenotype. Ceramidase expression suppresses degeneration in endocytic mutants such as arr2³ and mutant photoreceptors expressing a dominant-negative form of dynamin. Indeed, it would be very interesting to evaluate the turnover of rhodopsin in these mutants. Attempts were made to follow rhodopsin turnover in an inducible dominant-negative dynamin mutant background. However, the mutant photoreceptors degenerated, and the function of these photoreceptors was severely compromised. Because of the degeneration, heat-shock induction of 1D4-tagged Rh1 in these mutants did not result in synthesis of appreciable amounts of protein, and hence turnover could not be followed. A similar degenerative pathology complicates analysis in arrestin mutant background, and the lack of these critical controls makes it difficult to evaluate effects of ceramidase on Rh1 turnover in these mutant backgrounds. Another approach is to investigate the interaction of ceramidase expressors with components of pathways that have been implicated in membrane transport and photoreceptor degeneration. A recent study has addressed the probable dual role of phosphoinositides in activation and adaptation of phototransduction cascade. Arrestin 2 specifically bound phoshphoinositide/inositol phosphates. In this study, flies expressing engineered mutants of arrestin that were defective in phosphoinositide binding but not Rh1 binding were generated. The photoreceptors from these flies show delayed and decreased translocation of arrestin to the rhabdomere upon light activation, and those that were translocated were not internalized efficiently after binding Rh1. In a reverse approach, the this study also showed that light-dependent translocation of arrestin was defective in mutants that disrupt phosphoinositide metabolism. Earlier work on CDP-DAG synthase, an enzyme required for phosphoinositide biosynthesis, and RDGB, a phosphoinositide transfer protein, have implicated phosphoinositides in membrane turnover and signaling in Drosophila photoreceptors. Because phosphoinositides have been implicated in transport of phototransduction components to and from rhabdomeric membranes and sphingolipids are integral membrane components, specific interactions could influence phototransduction at multiple steps. It would thus be worthwhile to examine whether enzymes of the sphingolipid biosynthetic pathway, such as serine palmitoyltransferase and ceramidase, do interact with the phosphoinositide signaling pathway. It is also important to discern whether these interactions, if any, are mediated by specific protein- protein interactions or are caused by effects of changes in metabolite concentrations, such as ceramide and sphingosine, or rather are the result of a combined effect. Use of mass spectrometry and feeding experiments suggested that a decrease in steady-state levels of ceramide contribute to the beneficial effect of ceramidase in suppressing degenerations. It is now believed that facilitation of endocytosis observed in ceramidase expressors is also consequent to its action on ceramide levels in photoreceptors. If so, then mutants such as lace that are defective in ceramide synthesis and upstream of neutral ceramidase in the de novo biosynthetic pathway should have a similar effect as ceramidase expression in ninaE^I17mutant. Formation and breakdown of ceramide can affect the structure of membranes because of its topology, membrane sidedness, and limited flip-flop across membranes. It is believed that these results lend credence to suggestions that many of the actions of ceramide are caused by its role in membrane domain formation, membrane vesiculation, fusion and fission reactions, and trafficking. Sphingolipids are being increasingly implicated in yeast as important regulators of cell growth, heat stress response, and membrane trafficking. In conclusion, whereas earlier studies showed that expression of ceramidase suppresses degeneration in arr2³, norpA, and dynamin mutant backgrounds, the current study has led to the proposal that it does so by facilitating endocytosis and a decrease in ceramide contributes to these processes (Acharya, 2004).

This approach of genetically modulating sphingolipid biosynthetic pathway in Drosophila phototransduction mutants, a prototypic G protein-coupled receptor signaling cascade, will help in integrating signaling, lipid metabolism, membrane turnover, and degenerative pathways (Acharya, 2004).

Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1

Rhodopsin is essential for photoreceptor morphogenesis; photoreceptors lacking rhodopsin degenerate in humans, mice, and Drosophila. Transgenic expression of a dominant-active Drosophila Rho guanosine triphosphatase, Rac1, rescues photoreceptor morphogenesis in rhodopsin-null mutants; expression of dominant-negative Rac1 results in a phenotype similar to that seen in rhodopsin-null mutants. Rac1 is localized in a specialization of the photoreceptor cortical actin cytoskeleton, which is lost in rhodopsin-null mutants. Thus, rhodopsin appears to organize the actin cytoskeleton through Rac1, contributing a structural support essential for photoreceptor morphogenesis (Chang, 2000).

Sensory neurons present a challenge for morphogenesis: to harness the generic mechanisms of the cytoskeleton to shape a cell to the needs of its specific sensory protein. For photoreceptors, it is clear that morphogenesis and maintenance of the photosensitive organelle (in Drosophila, the rhabdomeres; in vetrebrates, the outer segments rods and cones) depends on the organelle's sensory protein: rhodopsin. Rhabdomeres and outer segments are orderly stacks of photosensitive plasma membrane organized from enormously expanded apical cell surfaces. The forces that constrain this expansion and organize it into a dense stack are incompletely understood, but the cortical actin cytoskeleton and its associated proteins are substantial contributors. It has been suggested that in addition to its sensory role, Drosophila rhodopsin organizes the cortical actin cytoskeleton into an essential morphogenetic constraint: the rhabdomere terminal web (RTW). The RTW defines the regular, curving base of the rhabdomere that partitions the rhabdomere from the photoreceptor cytoplasm. In rhodopsin-null mutants, the rhabdomere base fails to organize correctly, and the rhabdomere collapses deep into the photoreceptor cytoplasm in convoluted sheets of apposed membrane (Chang, 2000 and references therein).

A chimeric protein that decorates F-actin with green fluorescent protein (GFP) reveals the RTW as bundled microfilaments extending from the rhabdomere base deep into the photoreceptor. Before rhodopsin expression, the RTW of developing photoreceptors shows less microfilament bundling, resembling a house painter's brush. At about 90% of pupal development (pd), after the onset of rhodopsin expression at 75% pd, RTW microfilaments elongate commensurate with the increasing microvillar length and gathered into bundles (Chang, 2000).

RTW maturation and rhabdomere morphogenesis fail in photoreceptors lacking rhodopsin. Paralleling the normal initiation of microvillar organization observed in rhodopsin-null mutants, the RTW of mutant photoreceptors appears normal before the time when rhodopsin expression would normally commence. The RTW growth and bundling that normally follow rhodopsin expression fail in rhodopsin-null photoreceptors. Unlike wild-type rhabdomeres, the smaller, flattened rhabdomeres formed in the rhodopsin-null mutant collapse into the photoreceptor cytoplasm in convoluted sheets of apposed membrane during the first day after eclosion. The actin cytoskeleton becomes thoroughly disorganized in the absence of rhodopsin (Chang, 2000).

Although rhodopsin contributes about 50% of rhabdomere membrane protein, it is unlikely to support morphogenesis by a simple mass effect. Smaller, but ultrastructurally normal rhabdomeres form in mutants in which Rh1 is reduced by over 99%. Furthermore, a pulse of rhodopsin expression restricted to a narrow window of development is sufficient to rescue rhabdomere morphogenesis in photoreceptors otherwise lacking rhodopsin. It is proposed that an additional role for rhodopsin is to contribute an activity required to organize the RTW into an effective subapical barrier (Chang, 2000).

Drosophila Rac1, localizes to the rhabdomere base beginning with the onset of microvillar organization during midpupal development; it remaines subapical in adult eyes. To explore potential Rac1 functions in rhabdomere morphogenesis, dominant-negative N17Rac1 was expressed at defined stages of eye development. N17Rac1 expression during rhabdomere morphogenesis leads to reduced, disordered rhabdomeres. Fewer microvilli are seen in cross section, and a well-defined rhabdomere base is not formed; apposed sheets of rhabdomere membrane involute into the photoreceptor cytoplasm. Although these defects are reminiscent of those seen in rhodopsin-null mutants, the phenotype is not a consequence of a failure of rhodopsin delivery to the rhabdomeres. The actin cytoskeleton, however, appears diffuse and disordered as a result of transgene expression (Chang, 2000).

The resemblance of the rhabdomere base defects caused by N17Rac1 to those seen in rhodopsin-null mutants suggests that rhodopsin might exert its structural effect through Rac1. If so, it was reasoned that expression of constitutively active V12Rac1 might rescue rhabdomere morphogenesis in photoreceptors lacking rhodopsin. To test this idea, V12Rac1 was expressed during rhabdomere morphogenesis in ninaEI17 mutants that lack rhodopsin in photoreceptors R1 to R6. Substantial rescue of rhabdomere morphogenesis was observed. Occasional loops of rhabdomere membrane intrude into the photoreceptor, but most terminate at a well-defined base. The RTW is more tightly organized in V12Rac1-expressing animals. Similar to rhodopsin-null rhabdomeres rescued by a pulse of rhodopsin expression, V12Rac1-expressing animals show substantial rescue 5 days after eclosion. Thus, V12Rac1 appears to supply a durable organizing activity lost in rhodopsin-null mutants (Chang, 2000).

Similar to the requirement of small amounts of rhodopsin for normal morphogenesis, rescue appears quite sensitive to Rac1V12. About 18% of R1 to R6 rhabdomeres are rescued in non-heat-shocked hsGAL/SM1;UAS-Rac1V12 ninaEI17 eyes, rising to 90% in animals heat-shocked at 80% pd. Substantial rhabdomeres and tighter organization of the RTW are evident. To examine rescue specificity among Rho small GTPases, constitutively active V12Cdc42 and V14Rho were overexpressed in ninaE-null mutants. V12Cdc42 rescues rhodopsin-null morphogenesis, but V14Rho does not. Neither Rho nor Cdc42 immunolocalizes to the RTW of normal flies, but Cdc42 has been found to activate Rac in other systems and may do so here (Chang, 2000).

The observations reported here suggest that Rac1 links rhodopsin to photoreceptor morphogenesis: Targeted delivery of rhodopsin to the developing rhabdomere promotes localized Rac1 activity that, in turn, orchestrates assembly of the RTW. In rhodopsin-null photoreceptors, failure to correctly organize the RTW, likely including a failure of microfilament cross linking, would allow sheets of self-adhesive rhabdomere membrane to intrude unopposed into the photoreceptor cytoplasm. How rhodopsin contributes to Rac1 activity, as well as its downstream effectors, remains to be determined. Two attractive effector candidates are nonmuscle myosin II and moesin, which localize to the base of the developing rhabdomere and which, in other systems, lie downstream of small GTPases (Chang, 2000).

An actin barrier may also shape vertebrate photoreceptors, constraining newly added photosensitive membrane to the outer segment. Actin and actin-associated proteins localize to the site of outer segment disc membrane evagination, and nascent outer segment disc membrane intrudes into the cytoplasm of rabbit photoreceptors exposed to cytochalasin D. Given the several parallels between vertebrate and Drosophila retinal development and the highly conserved mechanisms of the cytoskeleton, it is interesting to speculate that vertebrate rhodopsin may also regulate the photoreceptor cytoskeleton. It is possible that some mutant rhodopsins, including those causing human retinitis pigmentosa, may result in photoreceptor degeneration because of an inability to correctly organize the actin cytoskeleton (Chang, 2000).

Interaction of InaD with the major rhodopsin encoded by the ninaE locus

A suggestion that the major rhodopsin encoded by the ninaE locus may interact with InaD is that it coimmunoprecipitates with the TRP channel from wild-type but not InaDP215 mutant fly heads. To test whether the rhodopsin interacts with InaD in vivo, a coimmunoprecipitation experiment was performed using extracts from Drosophila heads. InaD coimmunoprecipitates with rhodopsin but not in a control reaction carried out with nonimmune serum. In addition, the proteins were co-expressed in vitro using a mammalian tissue culture system, 293T cells, and it was found that ninaE opsin and InaD coimmunoprecipitate. Evidence that the association between the opsin and InaD is direct is that in vitro-translated opsin binds to GST-InaD immobilized on a glutathione-Sepharose column but not to the GST control (Xu, 1998a).

To map the regions in InaD mediating the interactions with the opsin, TRPL, and PKC, a series of InaD constructs were generated and coexpressed with each target protein in 293T cells. Either PDZ3 or PDZ4 is sufficient to interact with the opsin, TRPL, and PKC. However, binding of each target to PDZ3 requires an extra 28 amino acids COOH-terminal to PDZ3 (PDZ3L). A requirement for additional residues for target binding to a PDZ domain is not unique since a similar COOH-terminal extension is necessary for the binding of target peptides to the PDZ domain in neuronal nitric acid synthase. The association of the targets with PDZ3L requires PDZ3 rather than just the extra 28 COOH-terminal amino acids since truncation of the NH2-terminal portion of PDZ3L-obliterates binding. Binding of the opsin, TRPL, and PKC to PDZ3L and PDZ4 appears to be specific since none of the eight other proteins or protein fragments tested bind to PDZ3L or PDZ4. These included TRPC3, Shaker B, Calmodulin, and the PLC encoded by the no receptor potential A (norpA) locus. Furthermore, consistent with a recent report that PLC binds to a GST-PDZ1 fusion protein (van Huizen, 1998), it has been found that the COOH-terminal 123 residues of PLC expressed in 293T cells coimmunoprecipitate with either PDZ1 or PDZ1-PDZ2. These data suggest that the lack of interaction between these latter two PDZ domains and either the opsin, TRPL, or PKC is not due toimproper folding of PDZ1 or PDZ1-PDZ2. Studies in other labs, using bacterial fusion proteins, indicate that PLC binds to either PDZ1 and PDZ5 or PDZ5 only. Although the PLC expressed in 293T cells does not bind to PDZ5, PLC interacts with PDZ5 expressed in E. coli. The lack of interaction of PLC with PDZ5 in 293T cells may be due to interference by post-translational modifications of PDZ5, endogeous proteins that bind to either PLC or PDZ5 (precluding the PLC/PDZ5 interaction), or misfolding of PDZ5 (Xu, 1998).

Rhodopsin plays an essential structural role in Drosophila photoreceptor development

Null mutations of the Drosophila Rh1 rhodopsin gene, ninaE, result in developmental defects in the photosensitive membranes, the rhabdomeres, of compound eye photoreceptors R1-R6. In normal flies, Rh1 expression begins at about 78% of pupal life. At approximately 90% of pupal life, a specialized catacomb-like membrane architecture develops at the base of normal rhabdomeres. In ninaE null mutants, these catacombs do not form and developing rhabdomere membrane involutes into the cell as curtains of apposed plasma membrane. A filamentous cytoskeletal complex that includes F-actin and the unconventional myosin, NINAC, decorates the cytoplasmic surface of these curtains (Kumar, 1995).

The collapse of rhabdomere morphogenesis in rhodopsin null mutants suggests the protein plays a significant structural role in photosensitive membrane development. The onset of rhabdomere dysgenesis is marked by a failure of mutant photoreceptors to organize the catacomb-like membrane architecture that forms the base of normal rhabdomeres; the subsequent intrusion of curtains of rhabdomere membrane into the cytoplasm follows progressively upon this defect. Why an absence of rhodopsin results in this phenotype remains to be determined, but a simple hypothesis is suggested by the failure of microvillar membranes to part at the neck. Strong adhesion between the apposed membranes of the microvillar stack, mediated importantly by the peripheral membrane protein chaoptin, is critical in organizing the rhabdomere. It is possible that rhodopsin helps modulate microvillar membrane adhesivity, allowing apposed membranes to separate at the neck. In the absence of rhodopsin, adhesion between rhabdomere membranes is unchecked, driving the membrane into apposed planar sheets (Kumar, 1995).

Rhodopsin might participate in a direct, steric fashion, for example, by diluting chaoptin density; alternatively, an interaction between rhodopsin and the specialized cytoskeletal web at the base of the rhabdomere might provide a localized force that shapes the base. The latter scenario has the appeal that it includes a cytoskeletal element that could participate in the long-range ordering of the rhabdomere base. The base of the normal rhabdomere is remarkably smooth, and this order does not develop in rhodopsin nulls. It is intriguing that, in vertebrates, disruption of the microfilament cytoskeleton of developing rod outer segments (ROS) results in the failure of nascent discs of ROS membrane to terminate normally, resulting in ROS membrane overgrowth (Kumar, 1995).

Consistent with previous physiological observations, the present study finds that only a small amount of rhodopsin suffices to maintain normal rhabdomere structure. Immunofluorescence of the hypomorphic alleles, ninaEP318, ninaEP332, ninaEP334, ninaEP350 and ninaEUS6275, shows low levels of rhodopsin relative to wild type; particularly notable is that immunostaining is often undetectable in rhabdomeres that are ultrastructurally normal. It is possible that, even if mutant proteins cannot be properly transported into microvillar membranes, their presence in the broad membrane floor of the catacomb might contribute to a required stabilization. The truncated mutant ninaEP352 retains its epitope but evidently lacks a key structural site; rhabdomeres show the null phenotype even though a detectable amount of rhodopsin is present in the cytoplasm (Kumar, 1995).

It might be expected that mutations in other genes that reduce rhodopsin levels would lead to degeneration similar to ninaE nulls. The genes ninaA, ninaB, ninaC and ninaD were isolated, along with ninaE, based on aberrant electroretinograms (Pak, 1979). Using in vivo microspectrophotometry Stephenson (1983) found that rhodopsin levels in these mutants were drastically reduced, from 32% of wild type for ninaC to as low as 3% for ninaD. Of these, only ninaC has smaller, malformed rhabdomeres at eclosion. Rhabdomere degeneration in ninaC is unlike that seen in ninaE nulls; the base of the rhabdomere is normal at eclosion and membrane curtains are not observed. The remaining nina mutants, although having reduced rhodopsin levels, possess intact rhabdomeres. Presumably rhodopsin levels in these mutants are sufficient to maintain normal rhabdomere structure (Kumar, 1995).

Rhabdomere loss in ninaE nulls is also distinct from the phenotypes seen in other retinal degeneration mutants. Defects in the phototransduction cascade (norpA), membrane turnover (rdgB), second messenger pathways (rdgA and rdgC) and cell adhesion molecules (chp) all lead to rhabdomere deterioration but none of these mutants display the deep intrusion of rhabdomere membrane into the photoreceptor cytoplasm. The unique character of retinal degeneration in ninaE nulls supports a specific role for rhodopsin in maintaining rhabdomere structure. Dominant rhodopsin mutants that lead to rhabdomere degeneration have recently been isolated in Drosophila. In contrast to the rapid deterioration of the rhabdomere in null mutants, degeneration in dominant mutants is a slower process (Kurada, 1995). The molecular basis for degeneration in these dominant mutants is thought to result from impaired rhodopsin biogenesis that reduces rhodopsin levels below that needed to maintain normal rhabdomeres. Presumably the small amount of normal protein made in these animals is sufficient to prevent the rhodopsin null phenotype (Kumar, 1995).

The filaments that decorate membrane curtains of null mutants resemble the tooth-like structures in photoreceptors of normal Musca domestica. teeth have been noted in the photoreceptors of normal and mutant (norpA) Drosophila. The disappearance of both teeth and F-actin in ninaC;ninaE double mutants suggests they are multimolecular complexes containing at least NINAC and F-actin and that NINAC is required for their assembly and/or anchorage to the membrane. These results complement the biochemical observations that NINAC directly interacts with F-actin in actin blot overlays (Kumar, 1995).

Interestingly, although axial filaments are reduced in ninaC nulls, actin immunoreactivity and phalloidin staining persists, indicating that microvillar microfilaments can be organized in the absence NINAC. In view of the evidence that unconventional myosins can bind to cell membranes, it possible that, in ninaE nulls, NINAC binds to involuted rhabdomere membrane and in turn organizes F-actin assemblies. The filaments may thus be similar to the rhabdomeral cytoskeleton, but nucleated and stabilized in a different manner (Kumar, 1995).

In contrast with mouse models of retinitis pigmentosa, it is notable that Drosophila photoreceptors, having lost their rhabdomeres, do not undergo cell death. Photoreceptors of transgenic mice containing a dominant RP rhodopsin gene undergo apoptotic cell death shortly after the onset of retinal degeneration. In fly null and hypo4368 morphic alleles, photoreceptors maintain their cell bodies well into late stages of adulthood. A survey of 3- week-old null mutants indicated that, although the rhabdomeres have been lost long ago, the photoreceptors still maintain their cell bodies and no evidence of apoptotic cell death is detectable. This survival should make it possible to test if restoration of rhodopsin expression after rhabdomeres have degenerated suffices to revive microvillar architecture. The transient expression of several cytoskeletal proteins at the base of the developing rhabdomere hints a constellation of proteins may be required for rescue (Kumar, 1995).

Two distinct pathways of rhabdomere degeneration are evident among the mutants studied: nulls show catastrophic ultrastructural failure which commences during development; hypomorphs develop normally and preserve microvillar architecture during a progressive loss of rhabdomere volume. Degeneration in hypomorphs becomes severe in middle aged (4 weeks) and older flies. The latter pattern appears more typical of the vertebrate RP picture and is consistent with a 'metabolic' root for the phenotype, such as an imbalance between membrane addition and turnover or an impairment of normal membrane protein traffic. Fundamental cell biological similarities unite vertebrate and Drosophila photoreceptors. It will be interesting to see if ROS genesis in vertebrate null mutants resembles that seen in flies (Kumar, 1995).

Search PubMed for articles about Drosophila NinaE

Acharya, U., et al. (2004). Ceramidase expression facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors. Proc. Natl. Acad. Sci. 101: 1922-1926. PubMed ID: 14769922

Balasubramanian, N. and Slepak, V. Z. (2003). Light-mediated activation of Rac-1 in photoreceptor outer segments. Curr. Biol. 13: 1306-1310. PubMed ID: 12906790

Calvert, P. D., et al. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends Cell Biol. 16: 560-568. PubMed ID: 16996267

Chang, H. Y. and Ready, D. F. (2000). Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. Science 290(5498): 1978-80. 11110667

Colley, N. J., Cassill, J. A., Baker, E. K. and Zuker, C. S. (1995). Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proc. Natl. Acad. Sci. 92: 3070-3074. PubMed ID: 7708777

Davidson, F. F. and Steller, H. (1998). Blocking apoptosis prevents blindness in Drosophila retinal degeneration mutants. Nature 391: 587-591. PubMed ID: 9468136

Elsaesser, R., Kalra, D., Li, R. and Montell, C. (2010). Light-induced translocation of Drosophila visual Arrestin2 depends on Rac2. Proc. Natl. Acad. Sci. 107(10): 4740-5. PubMed ID: 20176938

Galy, A., et al. (2005). Rhodopsin maturation defects induce photoreceptor death by apoptosis: a fly model for rhodopsinPro23His human retinitis pigmentosa. Hum. Mol. Genet. 14: 2547-2557. PubMed ID: 16049034

Iakhine, R., Chorna-Ornan, I., Zars, T., Elia, N., Cheng, Y., Selinger, Z., Minke, B. and Hyde, D. R. (2004). Novel dominant rhodopsin mutation triggers two mechanisms of retinal degeneration and photoreceptor desensitization. J. Neurosci. 24(10): 2516-26. PubMed ID: 15014127

Kiselev, A., et al. (2000), A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila. Neuron 28: 139-152. PubMed ID: 11086990

Kock, I., et al. (2009). Targeting of Drosophila rhodopsin requires helix 8 but not the distal C-terminus. PLoS One 4(7): e6101. PubMed ID: 19572012

Kumar, J. P. and Ready, D. F. (1995), Rhodopsin plays an essential structural role in Drosophila photoreceptor development. Development 121: 4359-4370. PubMed ID: 8575336

Kurada, P., O'Tousa, J. E. (1995). Retinal degeneration caused by dominant rhodopsin mutations in Drosophila. Neuron 14: 571-579. PubMed ID: 7695903

Kwon, Y., Shim, H. S., Wang, X. and Montell, C. (2008). Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade. Nat. Neurosci. 11: 871-3. PubMed ID: 18660806

Lee, S. J., et al. (2003). Light adaptation through phosphoinositide-regulated translocation of Drosophila visual arrestin. Neuron 39: 121-132. PubMed ID: 12848937

Pak, W. L. (1979). Study of photoreceptor function using Drosophila mutants . In Neurogenetics : Genetic Approaches to the Nervous System. X. Breakefield, editor. Elsevier North-Holland, New York.

Ryoo, H. D., Domingos, P. M., Kang, M.-J. and Steller, H. (2007). Unfolded protein response in a Drosophila model for retinal degeneration. EMBO J. 26: 242-252. PubMed ID: 17170705

Shen, W. L., et al. (2011). Function of rhodopsin in temperature discrimination in Drosophila. Science 331(6022): 1333-6. PubMed ID: 21393546

Stephenson, R. A., O’Tousa, J., Scavarda, N. J., Randall, L. L. and Pak, W. L. (1983). Drosophila mutants with reduced rhodopsin content. In The Biology of Photoreception. (ed. D. J. Cosens and D. Vince-Price). pp. 447- 501. Cambridge England: University Press

Wang, T. and Montell, C. (2005). Rhodopsin formation in Drosophila is dependent on the PINTA retinoid-binding protein. J. Neurosci. 25(21): 5187-5194. PubMed ID: 15917458

Wang, T., Jiao, Y. and Montell, C. (2007). Dissection of the pathway required for generation of vitamin A and for Drosophila phototransduction. J. Cell Biol. 177: 305-16. PubMed ID: 17452532

Yamaguchi, S., Desplan, C. and Heisenberg, M. (2010). Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila. Proc. Natl. Acad. Sci. 107(12): 5634-9. PubMed ID: 20212139

Xu, X. Z., et al. (1998). Coordination of an array of signaling proteins through homo- and heteromeric interactions between PDZ domains and target proteins. J. Cell Biol. 142(2): 545-55. PubMed ID: 9679151

date revised: 20 July 2011

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