InteractiveFly: GeneBrief

neither inactivation nor afterpotential E: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

Single-base pair differences in a shared motif determine differential Rhodopsin expression

The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, this study compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11-base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli (Rister, 2015).

In the visual system, different photoreceptor neurons express specific light-sensing pigments; however, common downstream factors amplify and convert the response to the visual stimulus into a neuronal signal. For instance, each unit eye (ommatidium) of the Drosophila retina contains eight photoreceptors (R1 to R8) that express different light-sensing Rhodopsins (Rhs) that are restricted to specific photoreceptor subsets. Outer photoreceptors R1 to R6 express Rh1. Inner photoreceptors R7 and R8 express either Rh3 in pR7s coupled with Rh5 in pR8s, or Rh4 in yR7s with Rh6 in yR8s. R1 to R8 all share broadly expressed phototransduction factors that amplify and convert the response to the visual stimulus into a neuronal signal (Rister, 2015).

This study examined the cis-regulatory mechanisms that distinguish restricted from broad expression patterns for Rhodopsins and downstream phototransduction factors, respectively. All Rhs share the conserved Rhodopsin Core Sequence I (RCSI), which resembles the palindromic P3 motif (TAATYNRATTA), an optimal binding site for paired-class homeodomain proteins. Almost all known broadly expressed phototransduction genes contain a P3 motif in their proximal promoter. The presence of a conserved P3/RCSI motif within 100 base pairs (bps) of the Rh transcription start site (TSS) is significantly associated with enrichment in adult eyes. P3/RCSI is required for activation in photoreceptors because its mutation caused either a loss or a strong reduction in expression of 16 broad or restricted reporters, with the exception of Arr1. Moreover, expression of 10 out of 15 reporters was lost in mutants for the photoreceptor-specific transcription factor Pph13, a paired-class homeodomain protein that binds P3 and the Rh6 RCSI in vitro (Rister, 2015).

Because each Rh promoter has a highly conserved RCSI variant, the sufficiency of P3 and RCSI were tested to determine the significance of the specific differences between perfectly palindromic (P3) and imperfect motifs (RCSI). Four copies of the P3 motif (including four neighboring bps for spacing; the contribution of these additional bps was only tested for Rh4) from the broadly expressed ninaC, rdgA, or trpl drove broad expression in all photoreceptors, consistent with previous results. In sharp contrast, multimerized RCSI motifs drove expression in subsets of photoreceptors. The RCSI of Rh3 and Rh6 contains a K₅₀ motif, a binding site for K₅₀ homeodomain proteins such as the Dve repressor or the Otd activator. Expression of [Rh3 RCSI]₄ and [Rh6 RCSI]₄ was biased to inner photoreceptors: [Rh3 RCSI]₄ mediated restricted expression in R8 and R7, with a strong bias toward the pR7 subset, where Rh3 is normally expressed. This pattern is complementary to the expression of Dve, which is indeed responsible for the restricted expression as [Rh3 RCSI]₄ drove a broad, P3-like pattern in dve mutants. [Rh6 RCSI]₄ drove restricted expression in R8s and R7s; expression in R1 to R6 was very weak in comparison to P3 motifs, which was due to dve-dependent repression (Rister, 2015).

[Rh1 RCSI]₄ drove variable expression in R1 to R6, where Rh1 is expressed. This outer photoreceptor-specific pattern is complementary to the inner photoreceptor expression of [Rh3 RCSI]₄. Rh4 has the same RCSI as Rh1. However, adding the synergistic 3' RCSII motif led to expression in yR7s, where Rh4 is expressed. Although [Rh5 RCSI]₄ was not sufficient for reporter expression, adding three K₅₀ motifs to a single Rh5 RCSI ([K₅₀]₃ + [Rh5 RCSI]₁) led to expression in R8 and pR7 photoreceptors (Rister, 2015).

In summary, the RCSI motifs of specific Rhs differ from palindromic P3 motifs in broadly expressed genes: They drive expression that is biased toward the endogenous Rh expression patterns. It is shown below that full subtype specificity and activation often requires the repetition of motifs that are present in the RCSI (Rister, 2015).

As specific RCSI motifs directed restricted expression in different photoreceptor subsets, albeit with incomplete subtype specificity and with some variability in expression levels, it was asked whether the single-bp differences are required for subtype specificity in a wild-type promoter context and which other motifs are required for full restriction. The K₅₀ (Otd/Dve) motifs (TAATCC) were mutated to Q₅₀ (Pph13) motifs (TAATTG/A) to disrupt repression while preserving RCSI-mediated activation. Mutating the Rh3 RCSI resulted in an expansion to yR7s, where Dve is present at low levels. Mutating the Rh6 RCSI caused derepression in R1 to R6 and the ocelli. Rh3 and Rh6 have K₅₀/Dve repressor motifs repeated upstream, and mutation of individual motifs also caused derepression in yR7s and R1 to R6 and the ocelli, respectively. Taken together, single-bp changes create K₅₀ motifs in the Rh3 and Rh6 RCSI, which are required for subtype-specific expression together with their upstream repeats (Rister, 2015).

The importance of the disrupted P3 palindrome-i.e., the imperfect 3' homeodomain binding motif in the RCSIs of Rh1 toRh5 was examined. Creating a palindromic motif in theRh3 RCSI (TAATCCAATTC->TAATCCAATTA) caused derepression in yR7s that depended on Pph13. Therefore, derepression appears to be due to increased activation through the newly created Q₅₀/Pph13 site. The same ATTC→ATTA mutation in the Rh5 RCSI led to partial derepression in yR8s. This single-bp change created abinding site for the activator Otd (AGATTA), and indeed derepression in yR8s was lost in otd mutants, as was activation in pR8s (Rister, 2015).

The 3' ATTC motif in the RCSI of Rh3 and Rh5 is repeated upstream. Mutating the upstream repeat without creating a Q₅₀/Pph13 site (ATTC->CAAA) also caused derepression in yR7s (Rh3) or yR8s (Rh5). Mutating both ATTCs of Rh5 enhanced derepression into almost all yR8s. Therefore, this study has identified repressor motifs in the RCSIs of four Rhs (K₅₀/Dve motifs in Rh3/Rh6 and ATTC motifs in Rh3/Rh5). These motifs are repeated upstream within less than 100 bps and are required for full subtype specificity (Rister, 2015).

A single-bp ATTT->ATTA mutation in the Rh4 RCSI caused derepression in R1 to R6, pR7s, and the ocelli. The correct pattern was restored by crossing the mutant Rh4 reporter in a Pph13 mutant background, indicating that the A -> T change prevents Pph13 from overcoming repression in the 'wrong' photoreceptor subsets, as was the case for Rh3 and Rh6. The same mutation in the Rh1 RCSI caused no detectable derepression. Replacing two bps in the RCSI of the ocelli-specific Rh2 to obtain a Q₅₀/Pph13 site led to derepression in R1 to R6 photoreceptors that depended on Pph13 (Rister, 2015).

In vivo data revealed that a cell-fate decision requires single-bp differences in RCSI motifs. They complement previous findings in cell culture that subtle sequence differences in a glucocorticoid receptor or nuclear factor κB (NF-κB) binding site can specify the mode of transcriptional regulatio and that small differences in binding-site sequences can lead to distinct Hox specificities in vivo and in vitro. (1) Single bps in RCSI prevent binding of dimers of broadly expressed activators such as Pph13, tipping the balance of activator/repressor binding. This weakened activation allows repressors to prevent activation in other photoreceptor subtypes. (2) They generate specific combinations of overlapping activator and repressor motifs, often repeated upstream to provide robust expression and full subtype specificity. Creating overlap of activator and repressor motifs is an efficient way of blocking a key activator site in the 'wrong' cell types that express a repressor, especially because the RCSI motifs are very close to the transcription start site and repression there could block other activators. The precise tuning of RCSI motifs within their respective promoter context leads to incompatibility in other Rh promoters, as revealed by RCSI swap experiments: Replacing a given RCSI with another one resulted in two main outcomes: loss of expression or derepression in specific subsets of photoreceptors (Rister, 2015).

The RCSI/P3 motif resembles 'terminal selector' motifs that allow the coordinated expression of effector genes that define a particular neuron type. Yet, RCSI motifs exhibit additional layers of regulation that are integrated in a single regulatory element, as their sequence is modified for subtype specificity. Mutating a cis-regulatory motif in many cases appears to be the shortest evolutionary path toward a novel phenotype. Although it was found that it is possible in some cases to eliminate ectopic expression by removing the broadly expressed activator Pph13, this simultaneously causes a loss of expression of several broad phototransduction genes, defects in photoreceptor morphology, and a severe loss of light sensitivity (Rister, 2015).

It is proposed that the modification of a P3-type motif into different RCSI-type motifs allowed partitioning Rh expression to different subtypes of photoreceptors. This opened the possibility to discriminate wavelengths and likely conveyed a selective advantage. In this model, P3 motifs represent a positive regulatory element shared by ancestral genes that were expressed in all photoreceptors. This regulation is conserved, as the promoter of the long-wavelength Rh, as well as Gβ76C that are both expressed in all photoreceptors in the beetle Tribolium, contain a palindromic P3-type motif and depend on Pph13 (Rister, 2015).

Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq¹ isoform protein playing a major role in fly phototransduction. However, Gαq¹ null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. This study isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, it was found that mutations in the Gαq gene disrupt light responses, and the Gαq3 isoform protein was demonstrated to be responsible for the residual light response in Gαq¹ null mutants. Moreover, this study reports that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation of stable Rh1-arrestin 2 (Arr2) complexes. These findings clarify essential roles for several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis (Gu, 2020).

Heterotrimeric G proteins and G protein-coupled receptors play pivotal roles in mediating a variety of extracellular signals to intracellular signaling pathways, such as hormones, neurotransmitters, peptides, and sensory stimuli. In the Drosophila visual system, light stimulation activates the major rhodopsin (Rh1) to form metarhodopsin, which in turn activates heterotrimeric G proteins and norpA gene-encoded phospholipase C (PLCβ). Activated PLC catalyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 induces the release of Ca2+ from intracellular Ca2+ stores, whereas both DAG and IP3 may trigger extracellular Ca2+ influx by opening transient receptor potential (Trp) and transient receptor potential-like (TrpL) channels on the cell membrane. The Gαq gene encodes several Gαq splice variants, among which the Gαq-RD variant generates Gαq¹ isoform protein, and other splice variants generate Gαq3 isoform protein. Although both strong alleles of norpA and trpl;trp double mutants show completely abolished photoresponses, the Gαq¹ null mutant allele (Gαq⁹⁶¹) still displays a residual light response. These data indicate that other Gαq splice variants, or the Gq α subunits encoded by additional genes, contribute to the residual light responses in Gαq¹ null mutants (Gu, 2020).

Intracellular Ca2+ homeostasis controlled by Gq signaling is also essential for photoreceptor cell survival. Mutations in phototransduction cascade components, such as those in trp and norpA, prevent normal light-induced Ca2+ influx, resulting in stable Rh1/Arr2 complex formation and severe rapid light-dependent retinal degeneration. Disruption of stable Rh1/Arr2 complexes by genetic removal of Arr2 or suppression of Rh1 endocytosis can suppress the retinal degeneration either in norpA or trp mutant flies. Rh1/Arr2 complex formation is thought to contribute to impaired Ca2+ influx-activated CaM kinase II, which usually phosphorylates Arr2 to release Arr2 from Rh1. However, neither Gαq¹ nor Gαq⁹⁶¹ mutants undergo rapid retinal degeneration, exhibiting only slight retinal degeneration after keeping them in 12-h light/12-h dark cycles for 21 days. The disparate retinal degeneration phenotype between Gαq and norpA mutant is therefore unclear (Gu, 2020).

This study isolated a mutant fly with no detectable light responses and revealed that mutations in the Gαq gene cause the defective light responses. Gαq3 is responsible for the residual light response in Gαq¹ null mutants, and depletion of all Gαq splice variants results in rapid light-dependent retinal degeneration due to formation of stable Rh1/Arr2 complexes. In addition, this study revealed that Gαq3 plays essential roles in Rh1 synthesis. This study clarifies the essential role of different Gαq splice variants in fly phototransduction, retinal degeneration, and rhodopsin synthesis (Gu, 2020).

In Drosophila photoreceptors, G proteins are essential to activate the phototransduction cascade. The Gαq gene encodes several Gαq splice variants, and Gαq¹ has been shown to function as the predominant G protein in fly phototransduction. This study identified a mutation (5501T/A) in the Gαq gene, which specifically mutates Val to Asp at residue 303 in Gαq¹ but not Gαq3 isoforms. Although Val is replaced with Ile at residue 303 in vertebrate Gαq proteins, the hydrophobicity at this position is evolutionally conserved. Structural analyses have shown that the V303 region localizes to the interface between Gα proteins and its downstream effector PLC. The change of a hydrophobic residue to a polar one may affect the interaction between these two proteins. A recent study has shown that Gαq^V303D mutant protein is unable to activate PLC in vivo (Gu, 2020).

Although the 5501T/A Gαq gene mutation largely contributes to abolished light responses, this mutation is not fully responsible for the abolished light responses in no detectable light response (nlr) mutants because both nlr/Gαq¹ and nlr/Gαq⁹⁶¹ flies still exhibited a residual light response similar to Gαq¹ and Gαq⁹⁶¹ mutants. These data also excluded the possibility that Gαq^V303D mutant protein dominantly suppresses the function of Gαq protein. Gαq¹ expression in nlr mutants largely recovers the light response, further excluding the possibility that abolished light responses in nlr mutants are due to the dominant suppression of Gαq^V303D mutant protein (Gu, 2020).

The Gαq gene encodes several Gαq splice variants, and Gαq^221c mutants disrupt the expression of all Gαq splice variants (21). An ERG recording revealed that Gαq^221c null mutant clones showed no light responses. Previous whole-cell voltage-clamp recordings showed that the photoresponse of Gαq¹ homozygous cells is larger than that of Gαq¹ heterozygous cells. These results indicate that other Gαq splice variants might contribute to the residual light response in Gαq¹ null mutants. This study demonstrates that Gαq3 contributes to the residual light response in Gαq¹ null mutants (Gu, 2020).

The Gαq gene encodes several Gαq splice variants. Originally, two cDNAs resulting from different Gαq gene splicing were isolated. These two cDNAs encode Gαq1 and Gαq2 isoform proteins, respectively. Functional studies demonstrated that Gαq1 mediates the light response, whereas Gαq2 has no effect on phototransduction. Subsequently, two additional Gαq splice variants were isolated. To date, seven total Gαq splice variants have been annotated in Flybase, and these splice variants encode three different isoform proteins, including Gαq1, Gαq3, and Gαq4. This study has demonstrated that Gαq3 also mediates phototransduction. Overexpression of Gαq3 in nlr mutants induced detectable light responses but failed to fully restore the light response. Interestingly, the rescue flies exhibited comparable ERG trace amplitude and dynamics as those of Gαq¹ and Gαq⁹⁶¹ flies. These results indicate that different Gαq isoform proteins play different roles in phototransduction. Gαq mediates retinal degeneration (Gu, 2020).

Mutations in most genes encoding components of the phototransduction cascade result in rapid retinal degeneration, except for Gαq hypomorphic allele Gαq¹ and Gαq¹ isoform null mutant allele Gαq⁹⁶¹. Previous studies have shown that both Gαq¹ and Gαq⁹⁶¹ mutants undergo slow light-dependent retinal degeneration due to slow accumulation of stable Rh1/Arr2 complexes. In these Gαq mutants, the small residual photoresponse may reduce Ca2+ influx, which partially activates CaM kinase II and leads to the slow release of Arr2 from Rh1. This study shows that nlr mutants undergo rapid light-dependent retinal degeneration similar to that observed in norpA mutants. Disruption of stable Rh1/Arr2 complexes formation prevented retinal degeneration in the mutants. Under normal conditions, the interaction between Arr2 and Rh1 is transient, because light-triggered Ca2+ influx may activate CaM kinase II, which subsequently phosphorylates Arr2 to release Arr2 from Rh1. In nlr mutants, photoresponses were completely abolished so that the normal rise in Ca2+ after light stimulation was blocked, causing stable Rh1/Arr2 complex formation and retinal degeneration. These observations and explanations are consistent with mutations such as trp and norpA (Gu, 2020).

This study has shown the first evidence that Gαq3 regulates Rh1 synthesis. Rh1 is transported to the plasma membrane by vesicular transport mechanisms regulated by a large number of trafficking proteins. Previous studies have shown that Gαq homologue CG30054 regulates inositol 1,4,5,-tris-phosphate receptor (IP3R) to mediate calcium mobilization from intracellular stores and promote calcium-regulated secretory vesicle exocytosis. Given that Gαq3 shows high sequence identity to CG30054, they may regulate Rh1 synthesis through promoting calcium-regulated secretory vesicle exocytosis (Gu, 2020).

The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, a portion of activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. This study shows that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. It is proposed that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity (Wang, 2014).

Rhodopsins are G protein-coupled receptors that function as light sensors in photoreceptors (PRs), and defective trafficking of rhodopsins often leads to PR degeneration in humans and flies. Because vision is not required for animal survival, previous studies in Drosophila mostly focused on viable mutations that specifically impair PR function. However, it is likely that numerous additional players encoded by essential genes have remained unidentified. This study performed an eye-specific mosaic genetic screen and found that loss of subunits of the retromer causes light-induced PR degeneration (Wang, 2014).

The retromer, a hetero-multimeric protein complex, retrieves specific proteins from endosomes, thereby preventing the degradation of these proteins in lysosomes. The retromer is composed of Vps26, Vps29, Vps35, and certain sorting nexins (Snx). Most subunits are evolutionarily conserved. Mutations in some subunits (Vps35 or Snx3) of the retromer have been shown to decrease the abundance of Wntless (Wls) and impair the secretion of Wingless (Wg), a ligand of the Wnt signaling pathway. Wls is a transmembrane protein that binds to Wg and is required for Wg secretion. Impaired retromer function leads to excessive degradation of Wls in lysosomes, severely reducing Wg secretion and signaling. The retromer has also been shown to maintain the levels of Crumbs, a transmembrane protein required for maintaining the apicobasal polarity in some tissues. Mutations in human VPS35 have been shown to cause a dominant inherited form of Parkinson's disease (PD). However, the retromer has not been implicated in neurons of the visual system in flies or vertebrates (Wang, 2014).

The Drosophila compound eye comprises ~800 hexagonal units named ommatidia. Each ommatidium contains eight PRs (R1-R8) that express rhodopsin proteins. Rhodopsin 1 (Rh1) is the major rhodopsin that is primarily expressed in R1-R6. It is synthesized and folded in the endoplasmic reticulum (ER) and transported to rhabdomeres, the stacked membranous structures in PRs, via the secretory pathway. The proper transport of Rh1 from ER to rhabdomeres requires molecular chaperones and Rab GTPases. Binding of opsins to chromophores as well as protein glycosylation and deglycosylation are essential for Rh1 folding and maturation. Mutations in genes involved in Rh1 synthesis, folding, or transport can result in defective PR development or PR degeneration (Wang, 2014).

Phototransduction in the PRs relies on the activation of Rh1 by photons. Active Rh1 (metarhodopsin, M*) activates phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG). DAG or its metabolites can activate Transient Receptor Potential (TRP) and TRP-like cation channels that lead to depolarization of the PRs. Similar to fly PRs, the vertebrate intrinsically photosensitive retinal ganglion cells (ipRGCs) use melanopsin (a homolog of fly Rh1) as the light sensor and requires PLC and TRPC channels for activation. ipRGCs project their axons to specific brain areas to control circadian rhythms or pupillary light reflex (Wang, 2014).

Tight regulation of Rh1 activity upon light exposure is required to maintain the integrity of PR cells. M* can be converted into its inactive form upon exposure to orange light. In addition, a significant portion of active Rh1 can be endocytosed and degraded in lysosomes. Mutations that abolish Rh1 deactivation or impair the endolysosomal system can cause PR degeneration due to Rh1 accumulation. However, it is unknown whether Rh1 can be retrieved from the endolysosomal compartments and whether impaired Rh1 recycling leads to PR degeneration (Wang, 2014).

This study shows that loss of the fly Vps26 or Vps35 causes early-onset PR degeneration. Retromer subunits are expressed in PRs in flies and melanopsin-expressing ipRGCs in the mouse retina. In fly mutant PRs, the numbers of late endosomes and lysosomes are significantly elevated. The PR degenerative phenotypes are dependent on exposure to light and the presence of Rh1. The data indicate that the fly retromer recycles Rh1, preventing Rh1 retention in the PR cell bodies and shunting Rh1 from being degraded in lysosomes, thereby promoting Rh1 redelivery to rhabdomeres. In summary, the retromer recycles Rh1, prevents an overload of the endolysosomal pathway, and salvages a substantial fraction of Rh1 from degradation in flies. It may also play a similar role in ipRGCs in the retina of vertebrates (Wang, 2014).

Although the loss of the retromer does not obviously affect eye development, mutations in Vps26 or Vps35 genes lead to strong light-dependent PR degeneration. The demise of Vps²⁶¹ and Vps35^MH20 PRs is associated with a significant increase in the number of late endosomes and lysosomes upon light exposure, showing that the endolysosomal pathway is strongly affected when Rh1 recycling by the retromer is impaired. Indeed, Rh1 accumulates in late endosomes or lysosomes in the mutant PRs. Although Rh1 can be degraded and the function of Vps²⁶¹ mutant PRs is not abolished upon short light exposure, chronic exposure to light is detrimental to Vps²⁶¹ mutants because persistent Rh1 accumulation in the endolysosomal pathway is toxic to PR cells. Hence, reducing Rh1 endocytosis or Rh1 levels in the rhabdomeres suppresses PR degeneration upon prolonged light exposure. Interestingly, increasing Vps35 or Vps26 in mutants that show PR degeneration due to Rh1 accumulation in the endolysosomal compartments suppresses the degenerative phenotypes. In summary, the retromer is required to retrieve Rh1 from endosomes to maintain PR function and integrity (Wang, 2014).

How does Rh1 internalization affect the endolysosomal pathway in retromer mutants? One possibility is that lysosomes in the mutants are unable to cope with the increased levels of internalized Rh1 over time as Rh1 is one of the most abundant proteins in PRs. This in turn triggers an increase in the number of lysosomes, the accumulation of aberrant lysosomes, and the accumulation of endolysosomal intermediates, including late endosomes. Alternatively, loss of the retromer may increase the flux in the endolysosomal pathway, which overpowers the rate of endolysosomal maturation and leads to an adaptive response that eventually leads to the expansion of these compartments. Both pathways can lead to an apparent accumulation of Rh1 in the cell body when stained and analyzed by fluorescence microscopy (Wang, 2014).

Defective regulation of Rh1 can lead to the demise of PRs via apoptosis. Does apoptosis play a critical role in the PR degeneration in retromer mutants? It is argued that this is not the case based on the following observations. First, the retromer mutants exhibit progressive PR degeneration over a 3-wk period, whereas apoptosis typically occurs within hours. Second, mutants that lead to PR loss via apoptosis lose most PRs within ommatidia by the engulfment of surrounding glial cells. However, the degenerating Vps26 and Vps35 mutant PRs are not removed, although their morphology is very severely disrupted. Indeed, they can still be identified in 3-wk-old flies, indicating a lack of engulfment by surrounding cells. Third, overexpressing p35, a pan-caspase inhibitor of apoptosis, fails to suppress the PR degeneration in Vps26 mutants upon light exposure. Indeed, Rh1 accumulation in different subcellular compartments triggers different cellular responses, which leads to PR degeneration of varying severity. Accumulation of Rh1 in the endolysosomal pathway seems particularly toxic to PRs but often does not cause the removal of PRs for many weeks (Wang, 2014).

Loss of Wg affects eye development, whereas loss of Crumbs leads to short and/or fused rhabdomeres. Although the retromer can recycle Wls or Crumbs in some fly tissues, Vps²⁶¹ or Vps35MH20 mutants do not exhibit obvious eye developmental defects. It is therefore very likely that the composition of the eye retromer is different from the wing retromer. Indeed, loss of Snx3 does not cause obvious degenerative phenotypes when compared to loss of Vps26 or Vps35, yet these proteins are all essential for the recycling of Wls and wing development (Wang, 2014).

Many players required for phototransduction in Drosophila are conserved in a phototransduction cascade in vertebrate ipRGCs. These include melanopsin, PLC, and the TRP channels. This phototransduction pathway plays a role in photoentrainment of circadian rhythms and the control of the pupillary light reflex. vps35 is expressed in 92% of the melanopsin-expressing RGCs in the mouse retina. This may be an underestimate due to technical difficulties with β-GAL immunostaining in mouse tissues. In addition, the human Vps26 proteins are able to substitute for the function of the fly homolog in PRs. Because vertebrate melanopsin has very similar photochemical properties to fly rhodopsin, the retromer may play a conserved role in vertebrate ipRGCs. As deletion of vps35 leads to early embryonic lethality, a vps35 conditional KO in ipRGCs will need to be established to address its role in ipRGCs (Wang, 2014).

The retromer has been implicated in human neurodegenerative disease, including Alzheimer's disease (AD) and PD. In AD, a retromer deficiency has been proposed to affect subcellular distribution of β-secretase, which leads to increased amyloid-beta (Aβ) deposits and defective neuronal function. In PD, a missense mutation in ^VPS35 (D620N) has been shown to cause an autosomal dominant late onset form of the disease. The Vps35 D620N mutant protein appears to function as a dominant negative, and Vps35 and LRRK2 (Leucine-Rich Repeat Kinase 2) have been shown to interact. It will therefore be interesting to assess if these mutants affect ipRGCs as PD patients often have sleep issues (Wang, 2014).

Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. This study shows that Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. It is proposed that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport (Xiong, 2012).

This study shows that Crag is a novel GEF for Rab11 and that it is required for the post-Golgi transport of Rh1 to the rhabdomeres during light activation. This regulated transport of Rh1, which is independent of Rh1 transport during the development of the photoreceptors, replenishes the loss of Rh1 induced by light stimulation. Loss of Crag leads to accumulation of secretory vesicles in the cytosol of photoreceptor cells, and eventually leads to a light- and age-dependent photoreceptor degeneration (Xiong, 2012).

During development of photoreceptors, Rh1 and other phototransduction proteins are synthesized in the endoplasmic reticulum and transported to the rhabdomeres to build functional photoreceptors. Some molecular players, including Rab11 and exit protein of rhodopsin and TRP (XPORT), have been shown to play a role in this process. Upon light activation Rh1 is converted to metaRh. MetaRh is then converted back into Rh1 on rhabdomere membranes via absorption of another photon, allowing the maintenance of Rh1 levels in the rhabdomere. In wild-type photoreceptors, a portion of metaRh is phosphorylated and endocytosed, and it has been proposed that internalization of metaRh promotes the clearance of dysfunctional proteins and serves as a proofreading mechanism. Internalized Rh1 is then degraded through an endosomal/lysosomal pathway. Obviously, the gradual loss of Rh1 in wild-type photoreceptors leads to the necessity to constitutively synthesize Rh1 and replenish the rhabdomeric pool. This is nicely illustrated with the loss of retinol dehydrogenase (RDH), which is required for the regeneration of the chromophore of Rh1. Loss of RDH leads to progressive reduction in rhabdomere size and light-dependent photoreceptor degeneration (Xiong, 2012).

The data show that Crag is required to maintain homeostasis of Rh1 upon light stimulation. Loss of Crag leads to Rh1 accumulation in the cytosol and, eventually, retinal degeneration in the presence of light. Mutations in genes that affect metaRh1 turnover, such as Calmodulin and arrestin 2, lead to prolonged deactivation time of the photoresponse. Since both ERGs and single-cell recordings of Crag mutant photoreceptors are normal, it is unlikely that Crag is involved in the recycling of metaRh1 to Rh1. To test whether Crag is required for transport of newly synthesized Rh1 in adult photoreceptors, flies exposed to blue light to trigger massive endocytosis and degradation of Rh1, and then the new synthesis and transport of Rh1 back to the rhabdomeres was measured over time. Crag is not required for the synthesis of Rh1. However, in Crag mutants, the newly synthesized Rh1 accumulates in the cytosol. It is proposed that Crag is required for the delivery of newly synthesized Rh1 to the rhabdomeres and that loss of Crag leads to a gradual reduction in the size of rhabdomeres and to degeneration of the photoreceptor cells. Indeed, the time course and morphological features of degeneration associated with loss of Crag are very similar to the phenotypes observed in RDH mutants, further supporting that Crag is involved in the Rh1 synthesis/delivery pathway (Xiong, 2012).

Rab11 has been implicated in various intracellular membrane trafficking processes. Its diverse functions in different membrane compartments are mediated through its downstream effectors in a context-specific manner; many of these functions have been identified in previous studies. However, GEFs for Rab11 in any context have not yet been identified. In vivo and in vitro data provide compelling evidence that Crag is a GEF for Rab11. First, in Drosophila S2 cells, Crag colocalizes and physically interacts with Rab11. Second, Crag preferably binds to the GDP-bound form of Rab11, and the DENN domains are required for binding. Third, Crag is required for the proper localization of Rab11 in photoreceptors upon light stimulation. Fourth, loss of Crag or Rab11 leads to a similar light-induced photoreceptor degeneration. Fifth, expression of Rab11-CA partially rescues the degeneration caused by Crag mutations. Finally, an in vitro GEF assay shows that Crag facilitates the release of GDP from Rab11. It has been previously established that Rab11 is essential for photoreceptor cell development and Rh1 transport during pupal stages. However both rhabdomere morphology and Rh1 localization are normal in Crag clones in newly eclosed flies. Similarly, initial deposition of TRP is also not affected by Crag mutations, in agreement with previous findings that Rh1 and TRP are co-transported to the rhabdomeres during their development. Interestingly, cytosolic localization of TRP is not observed in Crag mutant photoreceptor cells exposed to light, suggesting that during light stimulation, Rh1 and TRP dynamics are distinct. Indeed, internalization of TRP upon light stimulation has not been reported in previous studies. The current data therefore indicate that other GEFs must exist for Rab11 during photoreceptor development, and that Crag is specifically required for Rab11 GDP/GTP exchange during light activation in adult flies. In addition, Crag may function as a GEF for Rab10 in other processes and cells, such as polarized deposition of basement membrane proteins in follicle cells (Xiong, 2012).

The biochemical assay shows that the kinetics of Crag GEF activity is slow when compared to the GEF activity of other DENN-domain-containing proteins such as the Rab35 GEF. Crag exhibits GEF activity against Rab10 with much faster kinetics than against Rab11, indicating that the slow kinetics may be due to properties of Rab11. This is further supported by the slow kinetics of EDTA that triggers GDP release of Rab11. It's possible that the GDP/GTP exchange of Rab11 requires other co-factors besides its GEF, as, for example, documented for Rab6 (Xiong, 2012).

CaM is a ubiquitously expressed calcium sensor. In the Drosophila photoreceptor cells, photoactivation leads to influx of Ca2+ and activation of CaM. It has been shown that CaM is required for the termination of the photoresponse in several steps, including TRP inactivation and conformational change of metaRh. Crag contains a CaM binding site and interacts with CaM in a calcium-dependent manner. In an in vitro GEF assay, the presence of CaM and Ca2+ indeed enhances the GEF activity of Crag. Hence, it is possible that a light-induced increase of intracellular Ca2+ level enhances Crag activity via CaM binding. The activation of Crag/Rab11 then may serve to replenish rhabdomeric Rh1, whose loss is also induced by light stimulation (Xiong, 2012).

In vertebrate rod cells, polarized transport of Rh is mediated by post-Golgi vesicles that bud from the TGN and fuse with the base of the outer segment. Rab11 has been detected on rhodopsin-bearing post-Golgi vesicles in photoreceptors; however, it has not yet been shown that Rab11 is required for Rh trafficking. DENND4 proteins are highly similar to Crag. This study showed that expression of the UAS–human DENND4A construct not only rescues the lethality but also rescues the light-induced photoreceptor degeneration caused by loss of Crag, showing that the molecular function of DENND4A is also conserved. Moreover, three different subtypes of Usher syndrome, an inherited condition characterized by hearing loss and progressive vision loss, have been mapped to the vicinity of the DENND4A locus at 15q22.31. Hence, DENND4A may also function through Rab11 in human photoreceptors, and loss of DENND4A may lead to photoreceptor degeneration (Xiong, 2012).

GPI biosynthesis is essential for rhodopsin sorting at the trans-Golgi network in Drosophila photoreceptors

Sorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, mutants were screened in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. Deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes were found to cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na⁺K⁺-ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. These results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency (Satoh, 2013).

In this study, 546 lethal lines were screened for potential defects in Rh1 by examining the localization of Arr2::GFP in FLP/FRT-mediated mosaic retinas using two-color fluorescence imaging. A mutation was found in the Drosophila homolog of human PIG-U (Drosophila PIG-U), which encodes a subunit of GPI transamidase. Mutations in other genes of the GPI synthesis pathway but not in the GPI modification pathway gave rise to the same phenotype. Furthermore, the GPI-linked protein, Chp accumulates in the ER whereas the stalk membrane Crumbs protein and basolaterally localized Na⁺K⁺-ATPase were mis-sorted to the rhabdomere. Rh1 was found to be degraded before entering the post-Golgi vesicles, but Crb and Na⁺K⁺-ATPase are misrouted into vesicles destined for the rhabdomere in PIG mutant cells (Satoh, 2013).

There are two previous reports concerning GPI requirements for the transport of transmembrane proteins. In zebrafish, GPI transamidase has been found to be essential for the surface expression of voltage-gated sodium channels. In yeast, GPI synthesis is required for the surface expression of Tat2p tryptophan permease, which is associated with detergent-resistant membrane (DRM) in wild-type cells. In GPI-deficient yeast, Tat2p and Fur4p fail to associate with DRM and are retained in the ER. Although DRM forms in the ER in yeast, in mammalian cells, it is likely that DRM formation occurs only after Golgi entry. The reason for this is thought to be that GPI lipid remodeling occurs in different places: the ER in yeast and the Golgi body in mammalian cells. In mammalian cells, lipid rafts are postulated to concentrate some fractions of apically destined proteins owing to their affinity for the TGN or recycling endosomes (Satoh, 2013).

Along with the raft model, there are two possible explanations for the sorting phenotype of PIG mutant fly photoreceptors: (1) the polarized sorting of Rh1 depends on its affinity for the raft/DRM and the raft/DRM is deficient in PIG mutants; (2) unidentified GPI-anchored protein(s) play crucial roles in the polarized sorting of Rh1 and Na⁺K⁺-ATPase, and the raft/DRM provides a platform for the GPI-anchored protein(s). The first model predicts raft/DRM deficiency in PIG mutants, Rh1 association with lipid rafts and a stronger phenotype caused by mutations in the genes involved in raft formation. By contrast, the second model predicts that GPI deficiency produces a stronger phenotype than that caused by raft deficiency (Satoh, 2013).

Analysis of lipid raft deficiency does not support the first model in which the loss of polarized sorting of Rh1/Na⁺K⁺-ATPase in PIG mutants is a consequence of raft deficiency; instead, the current results support the second model in which unidentified GPI-anchored protein(s) concentrate Rh1 and exclude Na⁺K⁺-ATPase and Crb from post-Golgi vesicles destined for the rhabdomeres. Thus, loss of the GPI-anchored sorting protein(s) might cause most Rh1 to be directed into the endocytotic pathway and degraded by lysosomes while simultaneously allowing Na⁺K⁺-ATPase and Crb to be loaded into post-Golgi vesicles destined for the rhabdomeres. Chp is the only GPI-anchored protein known to be expressed in fly photoreceptors in the late-pupal stages. However, chp² mutants do not exhibit any mislocalization phenotype of Rh1 or Na⁺K⁺-ATPase. Identifying the GPI-anchored protein(s) responsible for the sorting in the TGN is an important step for understanding this mechanism of polarized transport (Satoh, 2013).

The biosynthetic pathway of GPI-anchored proteins has been well elucidated, but little was known to date about the phenotypic consequences of the loss of GPI synthesis in vivo. The present study demonstrates that GPI synthesis is essential for the sorting of non-GPI-anchored transmembrane proteins, including Rh1 and Na⁺K⁺-ATPase, without obvious defects in adherens junctions. Human PIGM or PIGV deficiency causes seizures or mental retardation. These neurological disorders might be also caused by the mis-sorting of some transmembrane proteins in addition to the defects in the formation of GPI-anchoring proteins. These findings aid the understanding of the pathology of diseases involving deficient GPI-anchoring protein synthesis (Satoh, 2013).

CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila, the major light sensing rhodopsin, Rh1, and the downstream ion channel, Transient Receptor Potential Like (TRPL), are endocytosed in response to light, but the mechanism is unclear. Using an RNA-Sequencing approach, this study discovered CULD (CG17352), a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1- or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1 but not TRPL suppressed retinal degeneration in culd null mutant flies. These data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity (Xu, 2016)

G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors and, therefore, transduce signals from a wide variety of hormones, cytokines, neurotransmitters, as well as sensory stimuli. Each of these interactions triggers distinct intracellular responses through heterotrimeric G proteins. Upon continuous stimulation, GPCRs are deactivated by arrestins, and internalized through dynamin-dependent endocytosis. Many internalized GPCRs undergo lysosomal degradation and/or recycling, leading to downregulation of receptor levels, which is important for reducing the strength and duration of cellular responsiveness following various stimuli (Xu, 2016).

The Drosophila phototransduction cascade is a model pathway for the dissection of GPCR signaling and associated regulatory processes. Proteins of the visual signal transduction cascade are found within rhabdomeres, which are specialized compartments within photoreceptor cells that contain tightly packed microvilli. Light-induced activation of rhodopsin triggers the phototransduction cascade by stimulating the vision protein phospholipase C, which is encoded by the no receptor potential A (norpA) gene, through the α subunit of the heterotrimeric G protein DGq. This opens the transient receptor potential (TRP) channel and the TRP-like (TRPL) Ca²⁺/cation channel, and depolarizes the photoreceptor neurons. Meanwhile, activated rhodopsin, which is referred to as metarhodopsin, is immediately bound by arrestin and deactivated. After inactivation, metarhodopsin is either photoconverted back into rhodopsin or internalized for degradation. Although the majority of internalized metarhodopsin is degraded, with newly synthesized rhodopsin replenishing the pool, it has recently been reported that internalized rhodopsin (Rh1; encoded by ninaE in Drosophila melanogaster) can be recycled upon stimulation with light. The principle arrestin, Arr2, plays a pivotal role in deactivating rhodopsin, whereas Arr1 binds and internalizes rhodopsin (Xu, 2016).

Long-term adaptation to light stimuli also involves the dynamic activity-dependent translocation of signaling proteins that are not GPCRs. As seen with mammalian Rod photoreceptors, light induces the movement of Arr2 and Arr1 into the rhabdomeres. In Drosophila, TRP and TRPL function as the primary light-activated channels. TRP stably localizes to the rhabdomeres by forming a multiprotein signaling complex, the signalplex with inactivation-no-after-potential D protein (INAD), a protein that contains five PDZ domains. In contrast, illumination results in TRPL translocating from the rhabdomeres to an intracellular storage compartment within the cell body. However, the mechanisms that underlie light-induced translocation and trafficking of rhodopsin and TRPL are not yet fully understood. Furthermore, it is unclear whether this endocytic trafficking of TRPL plays a physiological role in maintaining the integrity of photoreceptor cells (Xu, 2016).

By using an RNA-Sequencing (RNA-Seq) approach, this study identified a so-far-unknown gene that is enriched in photoreceptors, and encodes a transmembrane protein with both a CUB and an LDLa domain. This protein was named CULD (CUB- and LDLa-domain protein). CULD mainly localized to the endocytic TRPL- or Rh1-positive vesicles. Mutations in culd led to endosomal accumulation of Rh1 and TRPL, which disrupted the light sensitivity of photoreceptors; blocking of Arr1-mediated endocytosis eliminated the intracellular accumulation of Rh1. Moreover, culd mutants underwent light-dependent retinal degeneration, and resulted in a phenotype that could be rescued by reducing the levels of Rh1. These data indicate that CULD is essential for the function and survival of photoreceptor cells by promoting the endocytic turnover of Rh1 and TRPL (Xu, 2016).

A microarray analysis has previously been used to compare the genes expressed in wild-type heads with heads from a mutant fly that lacked eyes in order to identify eye-enriched genes, which led to the further identification of some genes functioning in phototransduction. However, owing to multiple cell types in the compound eye, many genes identified in this analysis might not function in photoreceptor cells. This study describes an RNA-Seq screen to identify genes expressed predominantly in photoreceptors. Among the 58 genes identified, 36 genes were known to function in photoreceptor cells, representing most of the genes that play major roles in phototransduction or retinal degeneration. However, 22 genes had not been described as being enriched in photoreceptor cells previously. Among them, cg9935 (Eye-enriched kainate receptor: Ekar) has been recently reported to regulate the retrograde glutamate signal in photoreceptor cells and contribute to light-evoked depolarization during phototransduction (Hu, 2015). This study further characterized the new photoreceptor cell-enriched gene culd as being required for turnover of Rh1 and TRPL. Although culd had also been identified as an eye-enriched gene in the earlier microarray analysis that compared RNA expression in wild-type and eyeless heads, 93 other eye-enriched candidates prevented focusing on CULD. In this RNA-Seq screen, only photoreceptor-cell-enriched genes can be identified, and a reasonable number of candidates might represent new factors functioning in phototransduction. However, some eye-enriched genes important for phototransduction might be missed in this screen. For example, recently identified polyglutamine-binding protein 1 (PQBP1) was not found as a photoreceptor-cell-specific gene in the RNA-Seq screen. This might be because PQBP1 is also expressed in other non-photoreceptor retinal cells. Overall, this screen for photoreceptor-enriched genes sheds a light on further understanding of phototransduction and mechanisms of retinal degeneration (Xu, 2016).

Appropriate signals cause arrestins to translocate to the plasma membrane where they bind to activated GPCRs, thereby inhibiting G-protein-dependent signaling and regulating GPCR endocytosis and trafficking. In Drosophila there are two arrestins within photoreceptors, Arr1 and Arr2. Although Arr2 binds to Rh1, it is Arr1 that primarily colocalizes with Rh1 in internalized vesicles. Therefore, Arr1 might mediate light-dependent endocytosis of Rh1, whereas Arr2 functions to quench activated Rh1. In culd mutant flies, Rh1 was immobilized within endocytic vesicles and Arr1 colocalized with the endocytic Rh1; blocking the Arr1-medicated endocytosis in culd mutant cells eliminated the abnormal intracellular accumulation of Rh1. These data strongly suggest that CULD functions downstream of Arr1-mediated endocytosis of Rh1 (Xu, 2016).

Early endosomes containing Rab5 serve as a focal point of the endocytic pathway. Sorting events initiated in early endosomes determine the subsequent fate of internalized proteins, that is, whether they will be recycled to the plasma membrane or degraded within lysosomes. Rh1 and TRPL share the same internalization pathway, and during light stimulation Rab5 initially mediates this vesicular transport pathway. In wild-type photoreceptors, however, Rh1 and TRPL have different fates from common Rab5-positive early endosomes. The majority of Rh1 is eventually delivered to lysosomes for degradation, whereas most internalized TRPL tends to be stored. In wild-type cells, the photoreceptor-enriched protein CULD colocalized with the endocytic TRPL or Rh1 vesicles, and the majority of CULD-positive vesicles were also Rab5-positive. This spatial pattern indicates that CULD is required for the endocytic trafficking of TRPL and Rh1 after they are internalized (Xu, 2016).

CULD functions during the early steps of endocytosis that immediately follow internalization, which is a pathway involved in rhodopsin and TRPL endocytic turnover. Eliminating CULD had profound effects on the photoreceptor physiology. In both vertebrates and invertebrates, the light sensitivity of photoreceptor cells is primarily determined by functional rhodopsin. The culd mutant flies exhibited a gradual reduction in light sensitivity, which suggests that the amount of functional rhodopsin is reduced in culd mutant flies. As the amount of the monomer form of Rh1 was not affected and a large fraction of Rh1 accumulated within intracellular vesicles in culd mutant photoreceptor cells, the rhabdomeral Rh1 levels might be reduced. It is also likely that the endocytic degradation of Rh1 scavenges damaged Rh1 molecules, and blocking this process might lead to the accumulation of dysfunctional Rh1 in rhabdomeres (Xu, 2016).

TRPL has been reported to translocate from rhabdomeres to intracellular compartments for storage during prolonged light stimulation. However, a recent study suggests that some endocytic TRPL proteins are also delivered to lysosomes for degradation. Mutations in culd impaired TRPL endocytic trafficking upon light stimulation, leading to the retention of TRPL in Rab7-positive vesicles. TRPL protein levels were increased in culd mutants and this is probably due to decreased TRPL degradation (Xu, 2016).

As a major light sensor within photoreceptor cells, a small amount of activated Rh1 is internalized and degraded upon light stimulation. This is followed by replenishment of the rhabdomeric Rh1 pool. Therefore, a balance between Rh1 endocytosis and replenishment is required for Rh1 homeostasis under light conditions. Prolonged exposure to blue light triggers massive endocytosis of Rh1 and leads to a gradual loss of Rh1. Mutations in culd blocked Rh1 degradation during prolonged light treatment, indicating that the loss of CULD inhibited the Rh1-degradation pathway. Unlike TRPL, Rh1 levels were not increased, which suggests that Rh1 replenishment is strictly controlled. Given that, in Drosophila, rhodopsin levels are regulated by both the synthesis of the opsin and the chromophore subunits, it might be reasonable that in culd mutant cells, the chromophore is not released from the accumulated rhodopsin, and the reduction of free retinal pool might limit the synthesis of new rhodopsin (Xu, 2016).

Both vertebrates and invertebrates have a family of transmembrane proteins that contain both CUB- and LDLa- domains. However, only a few CUB/LDLa proteins have been functionally characterized. Among these proteins, NETO1 and NETO2 (see Drosophila Neto) have been intensively studied. NETO1 functions as an auxiliary subunit of ionotropic glutamate receptors, N-methyl-D-aspartate receptors and kainate receptors, modulating the channel properties of these glutamate receptors. NETO2 maintains normal levels of the neuron-specific K+-Cl- co-transporter KCC2 (also known as SLC12A5), and loss of NETO2-KCC2 interactions reduces KCC2-mediated Cl- extrusion, and decreases synaptic inhibition in hippocampal neurons. Moreover, in both Drosophila and C. elegans, the CUB/LDLa proteins NETO and SOL-2 are required for the clustering and functioning of glutamine receptors, thereby contributing to neuronal signaling pathways. This study cloned a new gene culd, which encodes a member of the CUB/LDLa family proteins specifically expressed in the Drosophila photoreceptor cell; this protein containing a CUB domain, an LDLa domain and one predicted transmembrane motif. CULD was not directly required for the activity of receptors or channels, but instead mediated the endocytic trafficking of Rh1 and TRPL. Loss of CULD led to the accumulation of Rh1 and TRPL in endocytic vesicles, and subsequent retinal degeneration. Therefore, this study revealed a new function of the CUB/LDLa family proteins, namely the endocytic turnover of receptors and channels (Xu, 2016).

It has been proposed that the accumulation of Rh1-Arr2 complexes in late endosomes triggers cell death of photoreceptor cells. Internalized Rh1-Arr2 complexes are not degraded but instead accumulate in late endosomes of norpA mutant photoreceptor cells. Similar rhodopsin accumulations are seen in mutations that affect the trafficking of late endosomes to lysosomes, which causes light-dependent retinal degeneration. Toxic Rh1-Arr2 complexes also induce retinal degeneration in rdgC, rdgB and fatp mutant flies. The culd mutations caused the accumulation of Rh1-Arr1 complexes and TRPL in endocytic vesicles and light-dependent retinal degeneration, suggesting that endosomal accumulation of either channels or receptors induced cell death. In addition, the evidence that ninaE^P332 but not trpl³⁰² rescued photoreceptor degeneration of the culd¹ mutants suggests that abnormal Rh1-Arr1 accumulation induces cell degeneration, whereas intracellular accumulation of TRPL does not contribute to the neuronal degeneration (Xu, 2016).

Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. This study shows that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. These findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking (Kunduri, 2014).

The Gos28 SNARE mediates intra-golgi transport of Rhodopsin and is required for photoreceptor survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. This study characterize the Golgi SNARE protein, Gos28, and its role in Rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. This study has pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from alpha-mannosidase-II in the medial-Golgi. The necessity of key residues in the Gos28 SNARE motif was confirmed, and it was demonstrated that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, human Gos28 was shown to rescue both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. These results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease (Rosenbaum, 2014).

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).

Proteomic survey reveals altered energetic patterns and metabolic failure prior to retinal degeneration

Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, a comprehensive proteomic profiling of retinas was performed from Drosophila transgenics expressing Rh1^P37H, the equivalent of mammalian Rho^P23H, the most common Rho mutation linked to blindness in humans. Profiling of young Rh1^P37H retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. The relevance of these metabolic changes to PN survival was probed by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1^P37H proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1^P37H-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1^P37H toxicity. It is thus proposed that chronic Rh1^P37H proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1^P37H toxicity in the retina. This study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs (Griciuc, 2014).

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).

Role of rhodopsin and arrestin phosphorylation in retinal degeneration of Drosophila

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. Enhanced green fluorescent protein-tagged arrestin 2 (Arr2) was used to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. It was demonstrated that during degeneration of norpA^P24 photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA^P24 is the failure to activate CaMKII (Ca²⁺/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca²⁺ entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. How reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration was investigated. To uncover the consequence underlying a lack of CaMKII activation, ala¹ flies were characterized in which CaMKII was suppressed by an inhibitory peptide, and it was shown that morphology of rhabdomeres was not affected. In contrast, it was found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA^P24 photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, it is concluded that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration (Kristaponyte, 2012).

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).

Xbp1-independent ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila

The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Inositol-requiring enzyme-1 (Ire1) signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. This study shows that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the Fatty acid transport protein (Fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. These results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm (Coelho, 2013).

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. When the folding capacity of the ER is impaired, the presence of incorrectly folded (misfolded) proteins in the ER causes ER stress and activates the unfolded protein response (UPR), which helps to restore homeostasis in the ER. In higher eukaryotes, the activation of the UPR is accomplished via three signaling pathways induced by ER-resident molecular ER stress sensors: protein kinase (PKR)-like ER kinase (PERK),activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (Ire1). Being conserved in all eukaryotes, Ire1 contains an ER luminal domain, which is involved in the recognition of misfolded proteins, and cytoplasmic endoribonuclease and kinase domains, which are involved in the activation of downstream pathways. Activated Ire1 mediates the nonconventional splicing of an intron from X box binding protein 1 (Xbp1) mRNA (or HAC1 mRNA, the yeast Xbp1 ortholog), causing a frameshift during translation, thereby introducing a different carboxyl domain in the Xbp1 protein. Xbp1^spliced is an effective transcription factor that regulates the expression of ER chaperones and other target genes (Coelho, 2013).

In addition to mediating Xbp1 mRNA splicing, cell culture studies demonstrated that Ire1 promotes the degradation of mRNAs encoding ER-targeted proteins, a process called RIDD (regulated Ire1-dependent decay), to reduce the load of ER client proteins during ER stress. The cytosolic domain of mammalian IRE1 binds Traf2 (tumor necrosis factor receptor-associated factor 2), an upstream activator of the c-Jun N-terminal kinase (JNK) signaling pathway. This IRE1/ Traf2 interaction is also independent of Xbp1 splicing and may lead to the activation of apoptosis after prolonged ER stress (Coelho, 2013 and references therein).

In the Drosophila photoreceptor cells, the rhabdomere is the light-sensing organelle, a stack of photosensitive apical microvilli that is formed during the second half of pupal development. The rhabdomere is formed in the apical domain of each photoreceptor cell, which after a 90
rotation extends its apical domain along the proximal-distal axis of the retina. The growth of the rhabdomere requires the delivery of large amounts of membrane and proteins into this structure, imposing a considerable demand to the cellular mechanisms controlling protein folding and membrane production in the ER (Coelho, 2013).

Among the proteins targeted to the developing rhabdomeres are the rhodopsins, the light-sensitive proteins, and other proteins involved in the transduction of the light stimuli. Rhodopsin-1 (Rh1) is a seven transmembrane domain protein that starts to be expressed by 78% of pupal life and is delivered to the rhabdomeres of the outer photoreceptors (R1–R6), in a trafficking process that requires the activity of Rab11, MyosinV, and dRip11. The delivery of Rh1 to the rhabdomere is required for rhabdomere morphogenesis because in Rh1-null mutants, the rhabdomere does not form, causing degeneration of the photoreceptors (Coelho, 2013).

In mammalians, the microRNA mir-708 is upregulated by CCAAT enhancer-binding protein homologous protein (CHOP) to control rhodopsin expression levels and prevent an excessive rhodopsin load into the ER . In Drosophila, Ire1 signaling is activated in the photoreceptors upon expression of Rh1 folding mutants or in ninaA mutations that cause the accumulation of misfolded Rh1 in the ER. However, the role of Ire1 signaling during normal photoreceptor differentiation remains unknown. This study shows that Ire1 signaling is activated in the photoreceptors during pupal stages of Drosophila development. Ire1 mutant photoreceptors have defects in the delivery of Rh1 to the rhabdomere and the secretion of Spacemaker/Eyes Shut (Spam/Eys) into the interrhabdomeral space (IRS). Surprisingly, Xbp1-null mutant photoreceptors have a milder phenotype with no defects in Rh1 delivery into the rhabdomere or Spam/Eys secretion. Targets of RIDD are upregulated in Ire1 mutant retinas, including the fatty acid transport protein (fatp), a known regulator of Rh1 protein levels. Finally, it was shown that the regulation of fatp levels by RIDD is critical for normal Rh1 delivery into the rhabdomere (Coelho, 2013).

Studies in mammalian systems revealed that the Ire1/Xbp1 signaling pathway is important during development for the differentiation of secretory cells. For example, Xbp1 'knockout' mice have defects in the differentiation of antibody-secreting plasma cells and secretory cells of the exocrine glands of the pancreas. Presumably, in these cases, activation of Ire1/Xbp1 signaling is required to increase the capacity of the ER to fold and process the high load of secreted proteins (Coelho, 2013).

The present results demonstrate that Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis, a process that also imposes a high demand to the capacity of the ER to fold proteins such as Spam/Eys and Rh1. As shown, Ire1 mutant photoreceptors have defects in the secretion of Spam/Eys to the IRS and in the delivery of Rh1 to the rhabdomere. However, activation is seen of the Xbp1-EGFP reporter starting at 48 hr of pupal development, well before when the Spam/Eys secretion and Rh1 delivery defects are observed. Presumably, the folding of other unidentified proteins during these earlier stages might also require Ire1 signaling. It is noteworthy though, that in mutant B lymphocytes modified to lack antibody production, Ire1 is still activated (and Xbp1 spliced) upon lymphocyte differentiation to plasma cells. Activation of Ire1/Xbp1 signaling in this context seems to be part of the process of plasma cell differentiation, independently of the accumulation of misfolded proteins in the ER lumen (Coelho, 2013).

Ire1 function is also required for the regulation of the membrane lipids. In mammalians, Ire1/Xbp1 signaling regulates the biosynthesis of phospholipids and other lipids. A study in yeast demonstrated that Ire1 is activated by 'membrane aberrancy,' a condition of stress caused by the experimental depletion of inositol. Activation of Ire1 in this case occurs by a mechanism that is distinct from the one involving the recognition of misfolded proteins by the luminal domain of Ire1. Furthermore, Ire1 can be activated by direct binding of flavonoids, such as quercetin, to a pocket present in the cytoplasmic domain of Ire1, in a mechanism that is also independent of the binding of misfolded proteins to Ire1. The present results do not clarify if Ire1 activation in the photoreceptors during pupal stages results from the accumulation of misfolded proteins in the ER lumen or an imbalance in the membrane lipids (Coelho, 2013).

The results demonstrate that Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in an Xbp1-independent manner. Studies using cell culture paradigms demonstrated that, in addition to mediating Xbp1 mRNA splicing, Ire1 also promotes RIDD, the degradation of mRNAs encoding ER-targeted proteins, but the physiological significance of the RIDD mechanism is unknown. Quantitative RT-PCR results show that RIDD targets are upregulated in Ire1 mutant eyes, including fatp, a regulator of Rh1 protein levels. The results show that regulation of fatp mRNA by RIDD is critical for rhabdomere morphogenesis because the experimental downregulation of fatp mRNA by RNAi rescues the Rh1 rhabdomere delivery defect observed in Ire1 mutants (Coelho, 2013).

Rh1 protein levels and Rh1 delivery to the rhabdomere are very sensitive to the levels of sphingolipids and phosphatidic acid. Increased fatp levels may lead to an increase in the levels of fatty acids and, subsequently, phosphatidic acid, which is known to downregulate Rh1 protein levels and cause rhabdomere morphogenesis defects. High levels of phosphatidic acid disrupt the Arf1-dependent transport of membrane to the developing rhabdomere. The results show that phosphatidic acid levels are elevated in Ire1 mutant retinas, and lowering phosphatidic acid levels by expression of LPP rescues the defects observed in Ire1 mutants, demonstrating that Ire1/fatp-dependent regulation of fatty and phosphatidic acids levels is important for rhabdomere morphogenesis in Drosophila. In addition, it is possible that the increase in phosphatidic acid levels in Ire1 mutant photoreceptors is also caused by the activation of PERK because upregulation was observed of the PERK pathway mediator ATF4 in Ire1 mutant photoreceptors, and in cell culture models, it was shown that PERK is able to phosphorylate diacylglycerol and generate phosphatidic acid. In conclusion, the results, using well-characterized genetic tools (Ire1 and Xbp1-null mutations) and a developmental paradigm (photoreceptor differentiation in the Drosophila pupa), demonstrate the physiological relevance of Xbp1-independent mechanisms downstream of Ire1 signaling (Coelho, 2013).

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

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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

Coelho, D. S., Cairrao, F., Zeng, X., Pires, E., Coelho, A. V., Ron, D., Ryoo, H. D. and Domingos, P. M. (2013). Xbp1-independent ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila. Cell Rep 5: 791-801. PubMed ID: 24183663

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

Griciuc, A., Roux, M. J., Merl, J., Giangrande, A., Hauck, S. M., Aron, L. and Ueffing, M. (2014). Proteomic survey reveals altered energetic patterns and metabolic failure prior to retinal degeneration. J Neurosci 34: 2797-2812. PubMed ID: 24553922

Gu, Q., Wu, J., Tian, Y., Cheng, S., Zhang, Z. C. and Han, J. (2020). Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila. J Biol Chem. PubMed ID: 32198182

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

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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

Kristaponyte, I., Hong, Y., Lu, H. and Shieh, B. H. (2012). Role of rhodopsin and arrestin phosphorylation in retinal degeneration of Drosophila. J Neurosci 32: 10758-10766. PubMed ID: 22855823

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

Kunduri, G., Yuan, C., Parthibane, V., Nyswaner, K. M., Kanwar, R., Nagashima, K., Britt, S. G., Mehta, N., Kotu, V., Porterfield, M., Tiemeyer, M., Dolph, P. J., Acharya, U. and Acharya, J. K. (2014). Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors. J Cell Biol 206: 79-95. PubMed ID: 25002678

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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, S., Tan, K. L., Agosto, M. A., Xiong, B., Yamamoto, S., Sandoval, H., Jaiswal, M., Bayat, V., Zhang, K., Charng, W. L., David, G., Duraine, L., Venkatachalam, K., Wensel, T. G. and Bellen, H. J. (2014). The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration. PLoS Biol 12: e1001847. PubMed ID: 24781186

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date revised: 23 June 2023

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