Localization of multiple opsins in ocular and non-ocular tissues of deep-sea shrimps and the first evidence of co-localization in a rhabdomeric R8 cell (Caridea: Oplophoroidea).

Bioluminescence is a prevalent phenomenon throughout the marine realm and is often the dominant source of light in mesophotic and aphotic depth horizons. Shrimp belonging to the superfamily Oplophoroidea are mesopelagic, perform diel vertical migration, and secrete a bright burst of bioluminescent mucous when threatened. Species in the family Oplophoridae also possess cuticular light-emitting photophores presumably for camouflage via counter-illumination. Many species within the superfamily express a single visual pigment in the retina, consistent with most other large-bodied mesopelagic crustaceans studied to date. Photophore-bearing species have an expanded visual opsin repertoire and dual-sensitivity visual systems, as evidenced by transcriptomes and electroretinograms. In this study, we used immunohistochemistry to describe opsin protein localization in the retinas of four species of Oplophoroidea and non-ocular tissues of Janicella spinicauda. Our results show that Acanthephyra purpurea (Acanthephyridae) retinas possess LWS-only photoreceptors, consistent with the singular peak sensitivity previously reported. Oplophoridae retinas contain two opsin clades (LWS and MWS) consistent with dual-sensitivity. Oplophorus gracilirostris and Systellaspis debilis have LWS in the proximal rhabdom (R1-7 cells) and MWS2 localized in the distal rhabdom (R8 cell). Surprisingly, Janicella spinicauda has LWS in the proximal rhabdom (R1-7) and co-localized MWS1 and MWS2 opsin paralogs in the distal rhabdom, providing the first evidence of co-localization of opsins in a crustacean rhabdomeric R8 cell. Furthermore, opsins were found in multiple non-ocular tissues of J. spinicauda, including nerve, tendon, and photophore. These combined data demonstrate evolutionary novelty and opsin duplication within Oplophoridae, with implications for visual ecology, evolution in mesophotic environments, and a mechanistic understanding of adaptive counter-illumination using photophore bioluminescence.

Membrane lipid modulations by methyl-ß-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating.

Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-ß-cyclodextrin (MßCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cß (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MßCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MßCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MßCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MßCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MßCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MßCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MßCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating.

A single photoreceptor splits perception and entrainment by cotransmission.

Vision enables both image-forming perception, driven by a contrast-based pathway, and unconscious non-image-forming circadian photoentrainment, driven by an irradiance-based pathway1,2. Although two distinct photoreceptor populations are specialized for each visual task3-6, image-forming photoreceptors can additionally contribute to photoentrainment of the circadian clock in different species7-15. However, it is unknown how the image-forming photoreceptor pathway can functionally implement the segregation of irradiance signals required for circadian photoentrainment from contrast signals required for image perception. Here we report that the Drosophila R8 photoreceptor separates image-forming and irradiance signals by co-transmitting two neurotransmitters, histamine and acetylcholine. This segregation is further established postsynaptically by histamine-receptor-expressing unicolumnar retinotopic neurons and acetylcholine-receptor-expressing multicolumnar integration neurons. The acetylcholine transmission from R8 photoreceptors is sustained by an autocrine negative feedback of the cotransmitted histamine during the light phase of light-dark cycles. At the behavioural level, elimination of histamine and acetylcholine transmission impairs R8-driven motion detection and circadian photoentrainment, respectively. Thus, a single type of photoreceptor can achieve the dichotomy of visual perception and circadian photoentrainment as early as the first visual synapses, revealing a simple yet robust mechanism to segregate and translate distinct sensory features into different animal behaviours.

Drosophila Phosphatase of Regenerating Liver Is Critical for Photoreceptor Cell Polarity and Survival during Retinal Development.

Establishing apicobasal polarity, involving intricate interactions among polarity regulators, is key for epithelial cell function. Though phosphatase of regenerating liver (PRL) proteins are implicated in diverse biological processes, including cancer, their developmental role remains unclear. In this study, we explore the role of Drosophila PRL (dPRL) in photoreceptor cell development. We reveal that dPRL, requiring a C-terminal prenylation motif, is highly enriched in the apical membrane of developing photoreceptor cells. Moreover, dPRL knockdown during retinal development results in adult Drosophila retinal degeneration, caused by hid-induced apoptosis. dPRL depletion also mislocalizes cell adhesion and polarity proteins like Armadillo, Crumbs, and DaPKC and relocates the basolateral protein, alpha subunit of Na+/K+-ATPase, to the presumed apical membrane. Importantly, this polarity disruption is not secondary to apoptosis, as suppressing hid expression does not rescue the polarity defect in dPRL-depleted photoreceptor cells. These findings underscore dPRL's crucial role in photoreceptor cell polarity and emphasize PRL's importance in establishing epithelial polarity and maintaining cell survival during retinal development, offering new insights into PRL's role in normal epithelium.

A conventional PKC critical for both the light-dependent and the light-independent regulation of the actin cytoskeleton in Drosophila photoreceptors.

Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, we show Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. We explored the light-dependent regulation of Pkc53E and demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCß4/NorpA as degeneration of norpAP24 photoreceptors was enhanced by a reduced Pkc53E activity. We further show that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton.

Functions of neuronal Synaptobrevin in the post-Golgi transport of Rhodopsin in Drosophila photoreceptors.

Polarized transport is essential for constructing multiple plasma membrane domains in the cell. Drosophila photoreceptors are an excellent model system to study the mechanisms of polarized transport. Rab11 is the key factor regulating the post-Golgi transport of rhodopsin 1 (Rh1; also known as NinaE), a photoreceptive protein, to the rhabdomere, a photoreceptive plasma membrane. Here, we found that neuronal Synaptobrevin (nSyb) colocalizes with Rab11 on the trans-side of Golgi stacks and post-Golgi vesicles at the rhabdomere base, and nSyb deficiency impairs rhabdomeric transport and induces accumulation of Rh1 and vesicles in the cytoplasm; this is similar to the effects of Rab11 loss. These results indicate that nSyb acts as a post-Golgi SNARE toward rhabdomeres. Surprisingly, in Rab11-, Rip11- and nSyb-deficient photoreceptors, illumination enhances cytoplasmic accumulation of Rh1, which colocalizes with Rab11, Rabenosyn5, nSyb and Arrestin 1 (Arr1). Arr1 loss, but not Rab5 dominant negative (Rab5DN) protein expression, inhibits the light-enhanced cytoplasmic Rh1 accumulation. Rab5DN inhibits the generation of Rh1-containing multivesicular bodies rather than Rh1 internalization. Overall, these results indicate that exocytic Rh1 mingles with endocytosed Rh1 and is then transported together to rhabdomeres.

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels.

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gαq and phospholipase Cß (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca2+ imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

Expression of a homologue of a vertebrate non-visual opsin Opn3 in the insect photoreceptors.

Insect vision starts with light absorption by visual pigments based on opsins that drive Gq-type G protein-mediated phototransduction. Since Drosophila, the most studied insect in vision research, has only Gq-coupled opsins, the Gq-mediated phototransduction has been solely focused on insect vision for decades. However, genome projects on mosquitos uncovered non-canonical insect opsin genes, members of the Opn3 or c-opsin group composed of vertebrate and invertebrate non-visual opsins. Here, we report that a homologue of Opn3, MosOpn3 (Asop12) is expressed in eyes of a mosquito Anopheles stephensi. In situ hybridization analysis revealed that MosOpn3 is expressed in dorsal and ventral ommatidia, in which only R7 photoreceptor cells express MosOpn3. We also found that Asop9, a Gq-coupled visual opsin, exhibited co-localization with MosOpn3. Spectroscopic analysis revealed that Asop9 forms a blue-sensitive opsin-based pigment. Thus, the Gi/Go-coupled opsin MosOpn3, which forms a green-sensitive pigment, is co-localized with Asop9, a Gq-coupled opsin that forms a blue-sensitive visual pigment. Since these two opsin-based pigments trigger different phototransduction cascades, the R7 photoreceptors could generate complex photoresponses to blue to green light. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Temporally dynamic antagonism between transcription and chromatin compaction controls stochastic photoreceptor specification in flies.

Stochastic mechanisms diversify cell fates during development. How cells randomly choose between two or more fates remains poorly understood. In the Drosophila eye, the random mosaic of two R7 photoreceptor subtypes is determined by expression of the transcription factor Spineless (Ss). We investigated how cis-regulatory elements and trans factors regulate nascent transcriptional activity and chromatin compaction at the ss gene locus during R7 development. The ss locus is in a compact state in undifferentiated cells. An early enhancer drives transcription in all R7 precursors, and the locus opens. In differentiating cells, transcription ceases and the ss locus stochastically remains open or compacts. In SsON R7s, ss is open and competent for activation by a late enhancer, whereas in SsOFF R7s, ss is compact, and repression prevents expression. Our results suggest that a temporally dynamic antagonism, in which transcription drives large-scale decompaction and then compaction represses transcription, controls stochastic fate specification.

The Role of Membrane Lipids in Light-Activation of Drosophila TRP Channels.

Transient Receptor Potential (TRP) channels constitute a large superfamily of polymodal channel proteins with diverse roles in many physiological and sensory systems that function both as ionotropic and metabotropic receptors. From the early days of TRP channel discovery, membrane lipids were suggested to play a fundamental role in channel activation and regulation. A prominent example is the Drosophila TRP and TRP-like (TRPL) channels, which are predominantly expressed in the visual system of Drosophila. Light activation of the TRP and TRPL channels, the founding members of the TRP channel superfamily, requires activation of phospholipase Cß (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into Diacylglycerol (DAG) and Inositol 1, 4,5-trisphosphate (IP3). However, the events required for channel gating downstream of PLC activation are still under debate and led to several hypotheses regarding the mechanisms by which lipids gate the channels. Despite many efforts, compelling evidence of the involvement of DAG accumulation, PIP2 depletion or IP3-mediated Ca2+ release in light activation of the TRP/TRPL channels are still lacking. Exogeneous application of poly unsaturated fatty acids (PUFAs), a product of DAG hydrolysis was demonstrated as an efficient way to activate the Drosophila TRP/TRPL channels. However, compelling evidence for the involvement of PUFAs in physiological light-activation of the TRP/TRPL channels is still lacking. Light-induced mechanical force generation was measured in photoreceptor cells prior to channel opening. This mechanical force depends on PLC activity, suggesting that the enzymatic activity of PLC converting PIP2 into DAG generates membrane tension, leading to mechanical gating of the channels. In this review, we will present the roles of membrane lipids in light activation of Drosophila TRP channels and present the many advantages of this model system in the exploration of TRP channel activation under physiological conditions.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins.

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Activity-dependent circuitry plasticity via the regulation of the histamine receptor level in the Drosophila visual system.

Activity-dependent synaptic plasticity is crucial for responses to the environment. Although the plasticity mechanisms of presynaptic photoreceptor neurons in the Drosophila visual system have been well studied, postsynaptic modifications remain elusive. In addition, further studies on the adaption of the visual system to different light experiences at a circuitry scale are required. Using the modified transcriptional reporter of intracellular Ca2+ method, we describe a way to visualize circuitry changes according to different light experiences. We found enhanced postsynaptic neuronal activity responses in lamina monopolar neuron L2 after prolonged light treatment. Although L1 also has connections with photoreceptors, there were no enhanced activity responses in L1. We also report in this study that activity-dependent transcriptional downregulation of inhibitory histamine receptor (HR), Ort, occurs in postsynaptic neuron L2, but not in L1, during continuous light conditions. We produced exogenous Ort proteins in L2 neurons and found that it attenuated the enhanced activity response caused by constant light exposure. These findings, together with the fact that histamine is the main inhibitory neurotransmitter released by photoreceptors in the Drosophila visual system, confirmed our hypothesis that the activity-dependent transcriptional downregulation of HR is responsible for the constant light exposure-induced circuitry response changes in L2. The results successfully demonstrated the selective circuit change after synaptic remodeling evoked by long-term activation and provided in vivo evidence of circuitry plasticity upon long-term environmental stimulation.

The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress.

The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. Here, we integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When we disrupted the Clock:Cycle complex by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, we observed changes in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, we showed that expression of ClkDN in photoreceptors leads to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, our data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress.

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum.

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20 , PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.

Brain connectivity inversely scales with developmental temperature in Drosophila.

Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. Here, we use developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, we show that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors.

Drosophila fabp is required for light-dependent Rhodopsin-1 clearance and photoreceptor survival.

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.

Spectral responses across a dorsal-ventral array of dermal sensilla in the medicinal leech.

How do animals use visual systems to extract specific features of a visual scene and respond appropriately? The medicinal leech, Hirudo verbana, is a predatory, quasi-amphibious annelid with a rich sensorium that is an excellent system in which to study how sensory cues are encoded, and how key features of visual images are mapped into the CNS. The leech visual system is broadly distributed over its entire body, consisting of five pairs of cephalic eyecups and seven segmentally iterated pairs of dermal sensilla in each mid-body segment. Leeches have been shown to respond behaviorally to both green and near ultraviolet light (UV, 365-375 nm). Here, we used electrophysiological techniques to show that spectral responses by dermal sensilla are mapped across the dorsal-ventral axis, such that the ventral sensilla respond strongly to UV light, while dorsal sensilla respond strongly to visible light, broadly tuned around green. These results establish how key features of visual information are initially encoded by spatial mapping of photo-response profiles of primary photoreceptors and provide insight into how these streams of information are presented to the CNS to inform behavioral responses.

Semper's cells in the insect compound eye: Insights into ocular form and function.

The arthropod compound eye represents one of two major eye types in the animal kingdom and has served as an essential experimental paradigm for defining fundamental mechanisms underlying sensory organ formation, function, and maintenance. One of the most distinguishing features of the compound eye is the highly regular array of lens facets that define individual eye (ommatidial) units. These lens facets are produced by a deeply conserved quartet of cuticle-secreting cells, called Semper cells (SCs). Also widely known as cone cells, SCs were originally identified for their secretion of the dioptric system, i.e. the corneal lens and underlying crystalline cones. Additionally, SCs are now known to execute a diversity of patterning and glial functions in compound eye development and maintenance. Here, we present an integrated account of our current knowledge of SC multifunctionality in the Drosophila compound eye, highlighting emerging gene regulatory modules that may drive the diverse roles for these cells. Drawing comparisons with other deeply conserved retinal glia in the vertebrate single lens eye, this discussion speaks to glial cell origins and opens new avenues for understanding sensory system support programs.

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System.

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.

The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele.

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.