InteractiveFly: GeneBrief

no receptor potential A : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin), which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE). Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration, which is believed to cause closure of the cGMP-gated ion (cyclic nucleotide gated or CNG) channel in the plasma membrane and the generation of an electrical response. In Drosophila, rhodopsin, acting through G proteins, targets the protein NorpA encoded by a phosphoinositide-specific phospholipase C (PLC). NorpA in turn catalyzes the breakdown of phospholipids and generates inositol trisphosphate (IP3) and diacylglycerol. Diacylglycerol is a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both TRP (transient receptor potential) and TRPL (TRP-like) are cation channels that are activated in the visual transduction process. These two proteins share homology with alpha-subunits of voltage-gated calcium and sodium channels in vertebrates. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Since arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

Of special interest is evidence for functioning of the visual cascade in Drosophila testis. DGq is the alpha subunit of the heterotrimeric GTPase (G alpha), which couples rhodopsin to phospholipase C in Drosophila vision. Three duplicated exons were identified in dgq by scanning the GenBank data base for unrecognized coding sequences. These alternative exons encode sites involved in GTPase activity and G beta-binding, NorpA-binding, and rhodopsin-binding. In vivo splicing of dgq was examined in adult flies: in all regions, other than the male gonads, only two isoforms are expressed. One isoform, dgqA, is the original visual isoform and is expressed in eyes, ocelli, brain, and male gonads. The other, dgqB, has the three novel exons and is widely expressed. Remarkably, all three nonvisual B exons are highly similar (82% identity at the amino acid level) to domains of the Gq alpha family consensus, from Caenorhabditis elegans to human, but all three visual A exons are divergent (61% identity). Intriguingly, a third isoform, dgqC, is found that is specifically and abundantly expressed in male gonads, and shares the divergent rhodopsin-binding exon of dgqA. It is suggested that DGqC is a candidate for the light-signal transducer of a testes-autonomous photosensory clock. This proposal is supported by the finding that norpA is expressed in male gonads, as well as the photoreceptor-cell-specific genes rhodopsin 2 and arrestin 1 (Alvarez, 1996).

These observations suggest that the visual DGqA is generated by the duplication of three DGqB exons. This allows the photoreceptor-specific DGqA to be optimized for phototransduction at the Gbeta-interacting/GTPase site, the NorpA-binding site and the Rh binding site. That this specialization occurs is suggested by the existence of eye-specific forms of Gbeta, NorpA, and Rh in Drosophila. NorpA, for example, has two cloned isoforms that vary at one alternatively spliced exon: one (I) is expressed at high levels and appears to be eye-specific, and the other (II) is expressed at low levels throughout development and in various tissues -- head, thorax, abdomen, and legs (Kim, 1995). The visual norpA.I is not expressed in testes, but norpA.II is. Vertebrate sensory systems also show organ specific isoform adaptation: these systems have specialized Galphas (of non-q class) for rod-vision (G_t1alpha), cone-vision (G_t2alpha), olfaction (G_olfalpha), and taste (G_gustalpha). However, squid, the only other invertebrate with a known Rh-linked (Gq_alpha), uses a Galpha more similar to DGqB than to the visual DGqA. This suggests that before the divergence of squid and fly, there was a single DGqB-like G_qalpha that mediated vision and at least one other, even more ancient, signaling pathway (Alvarez, 1996).

A central problem in sensory system biology is the identification of the signal transduction pathways used in different sensory modalities. Odorant response in the maxillary palps olfactory organ of Drosophila (see Odorant Receptors), but not the response of the antennal olfactory cells, depends on the norpA phospholipase C gene, providing evidence for use of the inositol 1,4,5-trisphosphate (IP3) signal transduction pathway. Consistent with the demonstration of a role for norpA in the maxillary palp, but not the antenna, norpA is found to be expressed in the maxillary palp, but not the antenna. Staining is localized along the lateral surface of the maxillary palp, in a region that contains a high density of olfactory hairs. Staining occurs not only on cell bodies but also in axons (the maxillary nerve). Since the norpA gene is also essential to phototransduction, this work demonstrates overlap in the genetic and molecular underpinnings of vision and olfaction. Genetic and molecular data also indicate that some olfactory information flows through a pathway that does not depend on norpA. The maxillary palp response is not abolished in norpA null flies, suggesting the existence of an additional PLC independent pathway for the odorant response (Riesgo-Escovar, 1995).

Regulation of dual oxidase activity by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity

All metazoan guts are in constant contact with diverse food-borne microorganisms. The signaling mechanisms by which the host regulates gut-microbe interactions, however, are not yet clear. This study shows that phospholipase C-β (PLCβ) signaling modulates dual oxidase (DUOX) activity to produce microbicidal reactive oxygen species (ROS) essential for normal host survival. Gut-microbe contact rapidly activates PLCβ through Gαq, which in turn mobilizes intracellular Ca²⁺ through inositol 1,4,5-trisphosphate generation for DUOX-dependent ROS production. PLCβ mutant flies have a short life span due to the uncontrolled propagation of an essential nutritional microbe, Saccharomyces cerevisiae, in the gut. Gut-specific reintroduction of the PLCβ restores efficient DUOX-dependent microbe-eliminating capacity and normal host survival. These results demonstrate that the Gαq-PLCβ-Ca²⁺-DUOX-ROS signaling pathway acts as a bona fide first line of defense that enables gut epithelia to dynamically control yeast during the Drosophila life cycle (Ha, 2009).

All organisms are in constant contact with a large number of different types of microbes. This is especially true in the case of the gut epithelia, which control life-threatening pathogens as well as food-borne microbes. In addition to this microbe-eliminating capacity, gut epithelia also need to protect normal commensal microbes which are in a mutually beneficial relationship. Therefore, gut epithelia must be equipped to differentially operate innate immunity in order to efficiently eliminate life-threatening microbes while protecting beneficial microbes. Studies using Drosophila as a genetic model have greatly enhanced understanding of the microbe-controlling mucosal immune strategy in gut epithelia. Previous studies in a gut infection model using oral ingestion of pathogens revealed that the redox system has an essential role in host survival by generating microbicidal effectors such as reactive oxygen species (ROS) (Ha, 2005a; Ha, 2005b). In this redox system, dual oxidase (DUOX), a member of the nicotinamide adenine dinucleotide phosphate (NADP)H oxidase family, is responsible for the production of ROS in response to gut infection (Ha, 2005a). Following microbe-induced ROS generation, ROS elimination is assured by immune-regulated catalase (IRC), thereby protecting the host from excessive oxidative stress (Ha, 2005b). In addition to the redox system, the mucosal immune deficiency (IMD)/NF-κB signaling pathway, which leads to the de novo synthesis of microbicidal effector molecules such as antimicrobial peptides (AMPs), has an essential complementary role to the redox system when the host encounters ROS-resistant pathogenic microbes. These findings indicate that the different spectra of microbicidal activity encompassed by ROS and AMPs may provide the versatility necessary for Drosophila gut immunity to control microbial infections. Furthermore, in the absence of gut infection, a selective repression of IMD/NF-κB-dependent AMPs is mediated by the homeobox gene Caudal, which is required for protection of the resident commensal community and host health. Therefore, fine-tuning of different gut immune systems appears to be essential for both the elimination of pathogens and the preservation of commensal flora (Ha, 2009).

Most studies evaluating gut immunity have been performed in an oral infection model in which the pathogens are ingested. However, the gut epithelia constitute the interface between the host and the microbial environment; therefore, it is likely that animals in nature have already been subjected to continuous microbial contact, even in the absence of oral infection. Thus, it is essential to determine the mechanism by which this natural and continuous microbial interaction produces ROS at a tightly controlled, yet adequate level that allows for healthy gut-microbe interactions and gut homeostasis, because deregulated generation of ROS is believed to lead to a pathophysiologic condition in the gut epithelia. Although the DUOX system is of central importance in gut immunity, the signaling pathway(s) by which gut epithelia regulate DUOX-dependent microbicidal ROS generation are poorly understood (Ha, 2009).

Drosophila feed on microbes, and one of their most essential microbial food sources is baker's yeast, Saccharomyces cerevisiae. As early as 1930, yeast was discovered to be an essential nutrient source for Drosophila and is now used as a major ingredient in standard laboratory Drosophila food recipes. Further, Drosophila-Saccharomyces interaction occurs in wild-captured Drosophila, which suggests that this interaction is an evolutionarily ancient natural phenomenon. Although many studies have investigated the effect of yeast on Drosophila metabolism and aging, very few works have been reported on the effect of yeast in terms of the host immunity. Specifically, it has previously been shown that dietary yeast contributes to the cellular immune responsiveness of Drosophila against a larval parasitoid, Leptopilina boulardi. However, the relationship between yeast and Drosophila gut immunity during the normal life cycle has never been closely examined. Therefore, in this study, a Drosophila-yeast model was used to investigate the intracellular signaling pathway by which the host mounts mucosal antimicrobial immunity, as well as the in vivo value of this pathway in the host's natural life. Through biochemical and genetic analyses, this study revealed that the Gαq-mediated phospholipase C-β (PLCβ) pathway is involved in the routine control of dietary yeast in the Drosophila gut. PLCβ is dynamically activated in the presence of ingested yeast and subsequently mobilizes the intracellular Ca²⁺ to produce ROS in a DUOX-dependent manner. The presence of all of these signaling components of the Gαq-PLCβ-Ca²⁺-DUOX-ROS pathway in the gut is essential to ensure routine control of dietary yeast and host fitness, highlighting the importance of this immune signaling as a bona fide first line of defense in Drosophila (Ha, 2009).

This study demonstrates that the Gαq-PLCβ-Ca²⁺ signaling pathway controls the mucosal gut epithelial defense system through DUOX-dependent ROS generation, which is responsible for routine microbial interactions in the gut epithelia in the absence of infection. The PLCβ pathway impacts a wide variety of biological processes through the generation of a lipid-derived second messenger. In this process, the hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, by PLCβ generates two intracellular messengers, IP₃ and diacylglycerol. This process is one of the earliest events through which more than 100 extracellular signaling molecules regulate functions in their target cells. It has been shown that Gαq-PLCβ signaling is essential for the activation of the phototransduction cascade in Drosophila. This study revealed a physiological role of PLCβ wherein it is involved in the regulation of DUOX enzymatic activity, which leads to the generation of microbicidal ROS in the mucosal epithelia (Ha, 2009).

PLCβ signaling is very rapid, with only a few seconds necessary to activate Ca²⁺ release and ROS production. This rapid response may be advantageous for the host and may be the mechanism by which dynamic and routine control of microbes in the gut epithelia is achieved. Because the gut is in continuous contact with microbes such as dietary microorganisms, it is conceivable that under normal conditions routine microbial contact dynamically induces a certain level of basal Gαq-PLCβ activity that varies depending on the local microbe concentration. This basal Gαq-PLCβ-DUOX activity seems to be sufficient for host survival. In such conditions of low bacterial burden, NF-κB-dependent AMP expression is known to be largely repressed by Caudal repressor for the preservation of commensal microbiota (Ryu, 2008). However, in the case of high bacterial burden (e.g., gut infection condition), the DUOX-ROS system would be strongly activated for full microbicidal activity. Furthermore, all of the flies that contained impaired signaling potentials for the Gαq-PLCβ-Ca²⁺-DUOX pathway were totally intact following septic injury but short-lived under natural rearing conditions or under gut infection conditions, indicating that the mucosal immune pathway is distinct from the systemic immune pathway (Ha, 2009).

It is not clear how Gαq- and PLCβ-induced Ca²⁺ modulates DUOX enzymatic activity. Because the DUOX lacking Ca²⁺-binding EF hand domains is unable to rescue the DUOX-RNAi flies (Ha, 2005a), it is plausible that Ca²⁺ directly modulates the enzymatic activity of DUOX through binding to the EF hand domains (Ha, 2009).

It is also important to determine what pathogen-associated molecular patterns (PAMPs) are responsible for the activation of PLCβ signaling. In Drosophila, peptidoglycan and β-1,3-glucan are the only two PAMPs known to induce the NF-κB signaling pathway in the systemic immunity. The results showed that neither peptidoglycan nor β-1,3-glucan was able to induce ROS in S2 cells, which suggests that a previously uncharacterized type(s) of PAMP is involved in the mucosal immunity. Because the Gαq protein acts as an upstream signaling component of the PLCβ-Ca²⁺ pathway, a microbe-derived ligand capable of activating G protein coupled receptor(s) and/or Gαq protein may be the best candidate for the Gαq-PLCβ-Ca²⁺-DUOX signaling pathway. Given the broad spectrum of microbes that activate the response, it remains possible that the unknown upstream sensors resemble a stress response more than a PAMP response. Elucidation of the molecular nature of such agonists will greatly enhance understanding of bacteria-modulated redox signaling in the gut epithelia. In conclusion, this study demonstrates that mucosal epithelia have evolved an innate immune strategy, which is functionally distinct from the NF-κB-dependent systemic innate immune system. The rapid Gαq-PLCβ-Ca²⁺-DUOX signaling is adapted to the routine and dynamic control of gut-associated microbes and may impact the long-term physiology of the intestine and host fitness (Ha, 2009).

Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. This study shows the two phases are separable. Combining genetics, pharmacology, and electrophysiology, a signaling system consisting of PLCbeta, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors was shown to be required only for the maintenance of homeostatic plasticity. It was also found that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent (James, 2019).

The Drosophila melanogaster NMJ is an ideal model synapse for studying the basic question of how synapses work to counter destabilizing perturbations. At this NMJ, reduced sensitivity to single vesicles of glutamate initiates a retrograde, muscle-to-nerve signaling cascade that induces increased neurotransmitter vesicle release, or quantal content (QC). As a result, the NMJ maintains a normal postsynaptic response level. Mechanistically, this increase in QC depends upon the successful execution of discrete presynaptic events, such as increases in neuronal Ca2+ influx and an increase in the size of the readily releasable pool (RRP) of synaptic vesicles. The field has termed this compensatory signaling process as presynaptic homeostatic potentiation (PHP) (Delvendahl, 2019). Two factors that govern the expression of PHP are the nature of the NMJ synaptic challenge and the amount of time elapsed after presentation of the challenge. Acute pharmacological inhibition of postsynaptic glutamate receptors initiates a rapid induction of PHP that restores synaptic output in minutes. By contrast, genetic lesions and other long-term reductions of NMJ sensitivity to neurotransmitter induce PHP in a way that is sustained throughout life (James, 2019).

Previously work has identified the Plc21C gene as a factor needed for PHP (Brusich, 2015). Plc21C encodes a Drosophila Phospholipase Cβ (PLCβ) homolog known to be neuronally expressed, but recent ribosomal profiling data also indicates possible muscle expression of Plc21C (Chen, 2017). In canonical signaling pathways, once PLCβ is activated by Gαq, it cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG can affect synaptic function by activating Protein Kinase C (PKC), while IP3 binds its receptor (IP3R) to trigger release of calcium from intracellular stores. It is not understood which aspects of this signaling machinery are mobilized during PHP. Potential downstream consequences of PLCβ activity at the NMJ include phosphorylation of neuronal proteins, modulation of ion channel activity, and changes in localization of neurotransmission machinery (James, 2019).

This study scrutinized PLCβ-directed signaling further. Tests were performed to see whether PLCβ-directed signaling was required solely for the maintenance of PHP or if it could also be required for induction. In addition to PLCβ, the IP3 Receptor (Drosophila Itpr, herein IP3R) and Ryanodine receptor (Drosophila RyR) were identified as being part of the same signaling process. Neither PLCβ, nor IP3R, nor RyR are required for the rapid induction of PHP. Additionally, it was found that the rapid induction of PHP is still possible in synapses already sustaining PHP. Surprisingly, it was found that NMJs are capable of rapidly inducing PHP -- even when the sustained expression of PHP is already blocked by impairments in PLCβ, IP3R, or RyR signaling. Taken together, these data show that the induction and maintenance of PHP are separable. Even though there is compelling evidence that parts of the induction and maintenance signaling mechanisms overlap (Goel, 2017), it is also true that acute PHP is possible in scenarios where long-term PHP is not (James, 2019).

This study divides the acute induction and chronic maintenance stages of presynaptic homeostatic potentiation. The data support two core findings. The first is that the short-term induction and long-term maintenance of PHP are separable by genetic and pharmacological manipulations. The second is that an IP3-mediated signaling system is specifically required for the maintenance of PHP (see Model depicting PLCβ/IP3R/RyR signaling underling the maintenance of PHP in both muscle and neuron.) (James, 2019).

For several years, one assumption has been that both the acute and chronic forms of PHP are executed in a similar way, and possibly by shared mechanisms. The issue has been clouded by the fact that both PhTox and a GluRIIA deletion mutant -- the primary reagents utilized to induce PHP -- have the same molecular target, that is GluRIIA-containing glutamate receptors. The process of combining these acute and chronic forms of plasticity within a single genotypic background was cumbersome due to a lack of reagents available to conduct temporally separate targeting experiments (James, 2019).

Several groups ascertained insights into temporal requirements by targeting potential homeostatic signaling genes. The main finding has been that the majority of molecules identified are essential to both the acute and chronic forms of PHP. Neurons tightly control neurotransmitter release probability, and the core presynaptic machinery directly responsible for increasing quantal content is shared. These shared components include the CaV2-type voltage-gated calcium channel or factors gating influx through the channel. They also include factors that regulate the size of the readily releasable pool (RRP) of presynaptic vesicles or factors that control the baseline excitability or plasticity of the presynaptic motor neuron, and neurotransmitter fusion events themselves. As a result, both the acute and chronic forms of PHP signaling cause increases in readily releasable pool (RRP) size and CaV2-mediated calcium influx; and in turn, these presynaptic mechanisms underlie the increases in QC which constitute PHP (James, 2019).

This study shows that although the acute and chronic processes might overlap, they are functionally separable. The fact that they are separable is not necessarily surprising. This finding mirrors data for discrete molecules required for long-term PHP maintenance, such as Target of Rapamycin (Tor), the Rho-type guanine exchange factor Ephexin, the transcription factor Gooseberry, C-terminal Src Kinase, innate immune signals molecules IMD, IKKβ, Relish and the kinesin adaptor Arl8. Importantly, this list contains molecules implicated both in neuron and muscle. This study has added PLCβ (Brusich, 2015) and its effectors IP3R and RyR to this list (James, 2019).

Recent studies have augmented the idea of overlapping signaling pathways and added a degree of specificity. Both acute and chronic forms of PHP begin as instructive retrograde signals after perturbations are detected in the muscle. These forms of PHP involve a decrease in phosphorylation of muscle CaMKII levels, and converge upon the same signaling components in the presynaptic neuron. These studies suggest that Tor signaling converges on the same molecular targets as acute forms of PHP (Goel, 2017). However, the precise roles for Tor and CaMKII in either form of PHP are as yet unknown (James, 2019).

These data appear to contradict the idea of PHP pathway convergence (Goel, 2017). Yet, the current findings are not incompatible with this idea. Multiple lines of evidence indicate discrete signaling requirements for acute forms of PHP on both sides of the synapse. A convergence point is undefined. Accounting for the separation of acute and chronic forms of PHP -- as well as their discrete signaling requirements -- long-term maintenance of PHP might integrate multiple signals between the muscle and neuron over time. For future studies, it will be important to clearly define roles of signaling systems underlying PHP and how distinct signaling systems might be linked (James, 2019).

This work presents unexpected findings. The first is that even in the face of a chronic impairment or block of homeostatic potentiation, the NMJ is nevertheless capable of a full rapid induction of PHP. Given that most molecules required for PHP identified to date are needed for both phases, significant functional separation between them was not expected. A priori it was expected that a failure of the chronic maintenance of PHP would make core machinery unavailable for its acute induction. The second unexpected finding is how quickly the chronic maintenance of PHP can be nullified by pharmacology (10 min), resulting in a return to baseline neurotransmitter release probability after only minutes of drug exposure. This study showed that homeostatic potentiation in GluRIIA mutant larvae or GluRIIC knock-down larvae was abrogated by four different reagents previously known to block IP3R or RyR. Those findings are reminiscent of prior work showing that acute blockade of DAG/ENaC channels with the drug benzamil abolishes PHP in both a GluRIIA mutant background, as well as in the presence of PhTox (Younger, 2013). A difference between benzamil application and the pharmacological agents used in the current study is that the drugs employed in this study only abolished PHP in a chronically challenged background (James, 2019)?

It is unclear how signaling systems that drive homeostatic plasticity transition from a state of induction to a state of maintenance. It is also not understood how interdependent short-term and long-term HSP implementation mechanisms are. A more complete understanding of the timing and perdurance of these properties could have important implications for neurological conditions where synapse stability is episodically lost (James, 2019).

The current findings parallel recent data examining active zone protein intensities in the contexts of induction of PHP and maintenance of PHP at the NMJ. There are multiple results informative to this study. First, the expression of any form of PHP (acute or chronic) appears to correlate with an increased intensity of active zone protein levels, such as CaV2/Cacophony, UNC-13, and the Drosophila CAST/ELKS homolog Bruchpilot. Unexpectedly, however, two of these studies also reported that the rapid induction of PHP does not require this protein increase in order to be functionally executed. These are conundrums for future work. How does rapid active zone remodeling happen in minutes on a mechanistic level? In the absence of such remodeling, how is PHP able to be induced rapidly? Moreover, is the observed short-term active zone remodeling the kernel for the longer-term changes to the active zone and release probability - or is some other compensatory system triggered over long periods of developmental time (James, 2019)?

The current findings add a new dimension to those puzzles with the data that IP3 signaling is continuously required to maintain PHP. If active zone remodeling truly is instructive for PHP maintenance, then it will be interesting to test what roles IP3 signaling and intracellular calcium release play in that process. The screen did include a UAS-RNAi line against unc-13 and an upstream GPCR-encoding gene methuselah. Moreover, a previously study of PHP used a UAS-cac[RNAi] line (Brusich, 2015). Chronic PHP maintenance was intact for all of those manipulations. Those findings are not necessarily contradictory to the recent work from other groups. For instance, knockdown of an active zone protein by RNAi is not a null condition. As such, RNAi-mediated knockdown should leave residual wild-type protein around. In theory, that residual protein could be scaled with homeostatic need (James, 2019).

The data strongly suggest that intracellular calcium channel activation and store release fine tune neurotransmitter release that is implemented by PHP. The exact mechanism by which IP3R and RyR function to maintain PHP at the NMJ is unclear. It appears to be a shared process with IP3. If these store-release channels are acting downstream of IP3 activity, then the data suggest that this would be a coordinated activity involving both the muscle and the neuron, with loss of IP3 signaling in the neuron being more detrimental to evoked release (James, 2019).

It remains unclear what signals are acting upstream. PLCβ is canonically activated by Gαq signaling. From prior work, evidence was garnered that a Drosophila Gq protein plays a role in the long-term maintenance of HSP (Brusich, 2015). Logically, there may exist a G-protein-coupled receptor (GPCR) that functions upstream of PLCβ/IP3 signaling. A screen did not positively identify such a GPCR. Several genes encoding GPCRs were examined, including TkR86C, mAChR-A, GABA-B-R1, PK2-R2, methuselah, AdoR, and mGluR. Genes encoding Gβ subunits or putative scaffolding molecules were also examined, including CG7611 (a WD40-repeat-encoding gene), Gβ13F, and Gβ76C, again with no positive screen hits (James, 2019).

The data are consistent with dual pre- and post-synaptic functions of IP3. This could mean dual pre- and post-synaptic roles for calcium store release, through an undetermined combination of RyR and IP3R activities, again either pre- or post-synaptic. Both RyR and IP3R have been shown to be critical for specific aspects of neuroplasticity and neurotransmission. Activities of both RyR and IP3R can activate molecules that drive plasticity, such as Calcineurin and CaMKII. At rodent hippocampal synapses, electrophysiological measures like paired-pulse facilitation and frequency of spontaneous neurotransmitter release are modulated by RyR and/or IP3R function, as is facilitation of evoked neurotransmitter at the rat neocortex. In addition to vesicle fusion apparatus, activity of presynaptic voltage-gated calcium channels is modulated by intracellular calcium. Work from this lab at the NMJ has shown that impairing factors needed for store-operated calcium release can mollify hyperexcitability phenotypes caused by gain-of-function CaV2 amino-acid substitutions (Brusich, 2018; James, 2019 and references therein).

Within the pre-synaptic neuron, IP3R and RyR could activate any number of calcium-dependent molecules to propagate homeostatic signaling. Some candidates were tested in a screen, but none of those tests blocked PHP. One possibility is that the reagents utilized did not sufficiently diminish the function of target molecules enough to impact PHP in this directed screen. Detection of downstream effectors specific to muscle or neuron might also be ham>pered by the fact that attenuation of IP3 signaling in a single tissue is insufficient to abrogate PHP. Another possibility is that presynaptic store calcium efflux via IP3R and RyR may directly potentiate neurotransmitter release, either by potentiating basal calcium levels or synchronously with CaV2-type voltage-gated calcium channels (James, 2019).

Both pre- and post-synaptic voltage-gated calcium channels are critical for the expression of several forms of homeostatic synaptic plasticity. Much evidence supports the hypothesis that store-operated channels and voltage gated calcium channels interact to facilitate PHP. In various neuronal populations, both RyR and IP3R interact with L-type calcium channels physically and functionally to reciprocally impact the opening of the other channel. In presynaptic boutons, RyR calcium release follows action potential firing. Calcium imaging experiments show that both the acute expression and sustained maintenance of PHP requires an increase in presynaptic calcium following an action potential. Because IP3Rs are activated by both free calcium and IP3, elevated IP3 levels in the case of chronically expressed PHP could allow IP3Rs and RyRs to open in a way that is time-locked with CaV2-mediated calcium influx or in a way to facilitate the results of later CaV2-mediated influx (James, 2019).

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca²⁺-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca²⁺ stores in this important model for Ca²⁺ signaling remains obscure. A low affinity Ca²⁺ indicator (ER-GCaMP6-150) was expressed in the ER, and its fluorescence was measured both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau approximately 0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca²⁺ stores, followed by a slower decay, typically reaching approximately 50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP₃ receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na⁺ and in mutants of the Na⁺/Ca²⁺ exchanger, CalX, which was immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na⁺/Ca²⁺ exchange across the ER membrane induced by Na⁺ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na⁺/Ca²⁺ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca²⁺ in photoreceptors exposed to Ca²⁺-free solutions (Liu, 2020).

Phototransduction in microvillar photoreceptors is mediated by a G-protein-coupled phospholipase C (PLC), which hydrolyzes phosphatidyl inositol (4,5) bisphosphate (PIP₂) to generate diacylglycerol and inositol (1,4,5) trisphosphate (InsP₃). In Drosophila photoreceptors, activation of PLC leads to opening of two related Ca²⁺-permeable nonselective cation channels: TRP (transient receptor potential) and TRP-like (TRPL) in the microvillar membrane. TRP is the founding member of the TRP ion channel superfamily, so named because the light response in trp mutants is transient, decaying rapidly to baseline during maintained illumination. Because the most familiar product of PLC activity is InsP₃, it was initially thought that activation of the TRP/TRPL channels required release of Ca²⁺ from endoplasmic reticulum (ER) stores via InsP₃ receptors (InsP₃Rs) and that in the absence of Ca²⁺ influx via TRP channels the stores depleted leading to the response decay. However, it was subsequently found that phototransduction was intact in InsP₃R mutants, whereas response decay in trp mutants was associated with severe depletion of PIP₂. This suggested an alternative explanation of the trp decay phenotype, namely failure of Ca²⁺-dependent inhibition of PLC and the consequent runaway consumption of its substrate, PIP₂. Nevertheless, a role for InsP₃ and Ca²⁺ stores in Drosophila phototransduction remains debated. For example, a recent study reported that sensitivity to light was attenuated by RNAi knockdown of InsP₃R , although this study was unable to confirm this using either RNAi or null InsP₃R mutants (Bollepalli, 2017; Liu, 2020).

Relevant to this debate, Ca²⁺ imaging reveals a small, but significant light-induced rise in cytosolic Ca²⁺ in photoreceptors bathed in Ca²⁺-free solutions. Although some have attributed this to InsP₃-induced Ca²⁺ release from the ER, it was found that the rise was unaffected in InsP₃R mutants but was dependent on Na+/Ca²⁺ exchange (Hardie, 1996; Asteriti, 2017; Bollepalli, 2017). This suggested that the Ca²⁺ rise was due to Na+/Ca²⁺exchange following Na+ influx associated with the light response. However, it is difficult to understand how such a Ca²⁺ rise could be achieved by Na+/Ca²⁺ exchange across the plasma membrane when extracellular Ca²⁺ was buffered to low nanomolar levels. The source of the Ca²⁺ rise in Ca²⁺-free bath thus remains unresolved, and to date there have been no measurements of ER store Ca²⁺ levels in Drosophila photoreceptors. To address this, flies were generated expressing a low-affinity GCaMP6 variant in the ER lumen. Using this probe, a rapid light-induced depletion of ER Ca²⁺ was demonstrated and characterized, which, like the cytosolic Ca²⁺ signal in Ca²⁺-free bath, was unaffected by InsP₃R mutations, but dependent on Na+ influx and the CalX Na+/Ca²⁺ exchanger. These results indicate that the exchanger is also expressed on the ER membrane, that the Na+ influx associated with the light-induced current leads to Ca²⁺ extrusion from the ER by Na+/Ca²⁺exchange and that this accounts for the rise in cytosolic Ca²⁺ observed in Ca²⁺-free solutions (Liu, 2020).

This study measured ER Ca²⁺ levels using a low affinity GCaMP6 variant targeted to the photoreceptor ER lumen, where it generated bright fluorescence throughout the ER network. The probe (ER-GCaMP6-150), originally developed and expressed in mammalian neurons, has a 45-fold dynamic range, which was confirmed in situ, and allows measurements of ER luminal [Ca²⁺] with excellent signal-to-noise ratio. Not only could ER Ca²⁺ levels be monitored in dissociated ommatidia, it was also straightforward to make in vivo measurements from the eyes of completely intact flies. The results demonstrate rapid light-induced, PLC-dependent depletion of the ER Ca²⁺ stores, which refilled in the dark over a time course of 100-200 s (Liu, 2020).

Strikingly the results indicate that the rapid light-induced store depletion was mediated by Na+/Ca²⁺ exchange. Drosophila CalX belongs to the NCX family of Na+/Ca²⁺ exchangers, which are generally considered to act only at the plasma membrane. Although Drosophila CalX clearly does function at the plasma membrane, the results now provide compelling evidence that it also operates across the ER membrane. NCX activity has not previously been reported on the ER; however, Na+/Ca²⁺ exchange on internal membranes is not without precedent: for example NCX has been reported on the inner nuclear membrane providing a route for Ca²⁺ transfer between nucleoplasm and the nuclear envelope and hence ultimately the ER network with which it is continuous. In addition a dedicated mitochondrial Na+/Ca²⁺ exchanger (NCLX) plays important roles in uptake and release of mitochondrial Ca²⁺ (Liu, 2020).

The time course of the Na+/Ca²⁺-dependent rapid store depletion in Ca²⁺-free solutions appeared very similar to the rise in cytosolic Ca²⁺ reported from dissociated ommatidia in Ca²⁺-free bath, the source of which has been a subject of debate for over 20 years. It had recently been claimed that this 'Ca²⁺-free rise' was due to InsP₃-mediated release from ER Ca²⁺ stores; however, it was found that it was unaffected in null mutants of the InsP₃R. Instead, it was found that the Ca²⁺-free cytosolic rise was dependent on Na+/Ca²⁺ exchange, but it was difficult to understand how this could be mediated by a plasma membrane exchanger when extracellular Ca²⁺ was buffered with EGTA to low nanomolar levels. The demonstration of rapid Na+/Ca²⁺-dependent release of Ca²⁺ from ER with a very similar time course now provides an obvious mechanism for this Ca²⁺-free rise and seems finally to have resolved this long-standing enigma. Interestingly the Na+/Ca²⁺-dependent rapid store depletion signal was most pronounced in very young flies around the time of eclosion. Also of note, it was found that trp mutants were very resistant to depletion, both in vivo and in dissociated ommatidia. This argues strongly and directly against the hypothesis that the trp decay phenotype reflects depletion of the ER Ca²⁺ stores (Liu, 2020).

Although up to ~80% rapid store depletion could be observed in newly eclosed adults, even in 1-d-old flies the rapid store depletion signal in vivo was much reduced (to ~10%). However, a much slower depletion was observed in mature adults in vivo, and in dissociated ommatidia after Na+/Ca²⁺ exchange was blocked. The origin of this slow phase depletion remains uncertain: in dissociated ommatidia from young flies this slower depletion was ~50% attenuated, but not blocked in null InsP₃R mutants (itpr), whereas in vivo measurements of the slow depletion phase in adult itpr mutants appeared similar to wild-type. This suggests that although Ca²⁺ release via InsP₃ receptors may contribute to the slow depletion in young flies, some other mechanism(s), such as Ca²⁺ release via ryanodine receptors, is largely responsible (Liu, 2020).

This evidence strongly suggests a novel role for NCX exchangers in mediating Na+/Ca²⁺ exchange across the ER membrane, but its physiological significance is unclear. Although rapid store depletion was routinely observed under experimental conditions used in this study, the Ca²⁺ released into the cytosol from the ER seems unlikely to play a direct role in phototransduction. First, it has a latency of ~100 ms (cf. ~10 ms for the light-induced current), and second it will in any case be swamped by the much more rapid Ca²⁺ influx via the light-sensitive channels. Thus measurements of cytosolic Ca²⁺ in 0 Ca²⁺ bath indicated a rise to only ~200-300 nm. This compares with much faster rises in the high micromolar range due to direct Ca²⁺ influx via the light-sensitive TRP channels. One possible role for an ER Na+/Ca²⁺ exchanger would be that it normally operates as a Ca²⁺ uptake mechanism and only briefly giving Ca²⁺ extrusion (and store depletion) following the extreme, and unnatural conditions of many of the current experiments. This the sudden onset of bright illumination from a dark-adapted state, which results in a massive transient surge of Na+ influx. Rapid Ca²⁺ uptake (store refilling), presumably via re-equilibration of the exchanger as the initial Na+ level subsided during the peak-to-plateau transition, was in fact routinely observed during maintained blue illumination. Furthermore, it is perhaps significant, that despite lacking the rapid depletion phase, the final level of store Ca²⁺ (i.e., after 30 s illumination) in calx^A mutants was if anything lower than that in wild-type backgrounds, although the cytosolic Ca²⁺ levels experienced in calx^A mutants are much higher because of the failure to extrude Ca²⁺ across the plasma membrane (Liu, 2020).

Although store depletion seems unlikely to contribute to activation of the phototransduction cascade, the possibility cannot be excluded that it may play some role in long-term light adaptation. Maintenance of ER Ca²⁺ levels is also important for many other cellular functions including protein folding and maturation in which Ca²⁺ is a cofactor for optimal chaperone activity. With conspicuously high cytosolic Ca²⁺ levels in the presence of light, photoreceptors face unusual homeostatic challenges and Na+/Ca²⁺ exchange across the ER may provide an important additional mechanism. In principle the balance between forward and reverse Na+/Ca²⁺ exchange (i.e., uptake vs release) by an ER Na+/Ca²⁺ exchanger will depend on the Na+ gradient across the ER membrane and whether this is actively regulated. There is no information on ER Na+ levels, although luminal Na+ in the nuclear envelope (which is continuous with the ER) has been reported to be concentrated (84 mm) in nuclei from hepatocytes by Na/K-ATPase expressed on nuclear membranes. The possibility that Na+/Ca²⁺ exchange across the ER might play only a minor physiological role cannot be excluded, but is an unavoidable consequence of the presence of functional CalX protein in ER membranes during protein synthesis and targeting. At least this may account for the enhanced depletion signal measured around the time of eclosion when there may be a rapid final phase of protein synthesis for the developing rhabdomere (Liu, 2020).

These results provide unique insight into ER Ca²⁺ stores in Drosophila photoreceptors. The ER-GCaMP6-150 probe lights up an extensive ER network and indicates a high luminal Ca²⁺ concentration probably in excess of 0.5 mm. The results reveal a rapid, and uniform light-induced depletion of the ER stores mediated by the CalX Na+/Ca²⁺ exchanger expressed on the ER membrane. The resulting extrusion of Ca²⁺ into the cytosol can readily account for the rise in cytosolic Ca²⁺ observed in dissociated ommatidia in Ca²⁺-free solutions), thus resolving this decades old mystery. In addition to the rapid depletion, a much slower depletion was also resolved that appears to be independent of Na+/Ca²⁺ exchange and also largely independent of InsP₃-induced Ca²⁺ release. The physiological significance of the ER Na+/Ca²⁺ exchange activity remains uncertain. It is perhaps more likely that it serves as a low affinity Ca²⁺ uptake mechanism supplementing the SERCA pump, and that rapid depletion is only seen during unnatural abrupt bright stimulation from dark-adapted backgrounds leading to massive Na+ influx and reverse exchange. Ultimately, to resolve the physiological significance of Na+/Ca²⁺ exchange across the ER membrane it will probably be necessary to selectively disrupt Na+/Ca²⁺ exchange on the ER without affecting the exchanger on the plasma membrane, which is known to play very important roles in Ca²⁺ homeostasis in the photoreceptors with direct consequences for channel activation and adaptation (Liu, 2020).

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca²⁺ influx in response to light, but whether Ca²⁺ is released from internal stores remains controversial. This study generated flies expressing GCaMP6f in their photoreceptors and measured Ca²⁺ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with approximately 1200 effectively absorbed photons and saturated (DeltaF/F0 approximately 10-20) with 10000-30000 photons. In Ca²⁺ free bath, smaller (DeltaF/F0 approximately 4), long latency (~ 200ms) light-induced Ca²⁺ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na⁺ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca²⁺ free rises were also eliminated in Na⁺/Ca²⁺ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca²⁺ free rises are strictly dependent on Na⁺ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na⁺/Ca²⁺ exchange across plasma or intracellular membranes following massive Na⁺ influx. Any tiny Ca²⁺ free rise remaining without exchanger activity was equivalent to <10nM (DeltaF/F0 approximately 0.1), and unlikely to play any role in phototransduction (Asteriti, 2017).

Although Ca²⁺ signals in Drosophila photoreceptors were first studied over 20 years ago using Ca²⁺ indicator dyes, only one, recent study had used genetically encoded Ca²⁺ indicators. That study measured signals from dissociated ommatidia using the Gal4-UAS system, combining UAS-GCaMP6f with GMR-Gal4, which drives expression throughout the retina including all photoreceptor classes as well as accessory cells such as pigment and cone cells. GMR-Gal4 expression also causes significant abnormalities in photoreceptor structure and physiology. In the present study, flies were generated in which GCaMP6f expression was driven directly via the Rh1 (ninaE) promoter ensuring exclusive expression in R1-6 photoreceptors with wild-type morphology and physiology. The excellent signal-to-noise ratio of recordings in ninaE-GCaMP6f flies was distinctly superior to that in GMR-Gal4/UAS-GCaMP6f flies, and in many cases the maximum Δ/F0 ratio approached or exceeded 20 (cf ~3 using GMR-Gal4/UAS-GCaMP6f). This is close to the maximum value (23.5) determined by in situ calibrations or in vitro. Although the blue excitation light used for measuring GCaMP6f fluorescence is a super-saturating stimulus, 2-pulse paradigms allowed sensitive and accurate measurements of intensity and time dependence of signals in response to stimuli in the physiological range. Recordings in vivo from the deep pseudopupil (DPP) of intact flies are simple to perform and can be readily maintained over many hours, making this approach a valuable, and completely non-invasive tool for assessing in vivo photoreceptor performance. Even in the more vulnerable dissociated ommatidia preparation, multiple repeatable measurements could be made for up to at least an hour from the same ommatidium as long as metarhodopsin was reconverted to rhodopsin by long wavelength light after each measurement (Asteriti, 2017).

In vivo (DPP) or in dissociated ommatidia bathed in physiological solutions, the GCaMP6f signal reached 50% Fmax at intensities equivalent to ~1000-2500 effectively absorbed photons. It is believed that the elementary single photon response (quantum bump) is generated by activation of Ca²⁺ permeable channels (TRP and TRPL) within a single microvillus and that the consequent Ca²⁺ rise in the affected microvillus reaches near mM levels. Because such levels inevitably saturate GCaMP6f (Kd 290 nM, saturating at 1-2 μM), to a first approximation the Δ/F0 values under physiological conditions are probably best interpreted as the proportion of microvilli 'flooded' with Ca²⁺. In total, the rhabdomere contains ~30000 microvilli, meaning that 50% Fmax is reached when only ~3-8% of the microvilli have been activated by a photon. This implies that the Ca²⁺ influx into a single microvillus must spread to at least the immediately neighbouring microvilli within the timeframe of the response. In ninaE-calx flies over-expressing the Na+/Ca²⁺ exchanger, or in trp mutants lacking the major Ca²⁺ permeable channel, 50% Fmax was only obtained with flashes containing ~12000-15000 effective photons. This should activate ~50% of the microvilli, suggesting that in these flies Ca²⁺ is largely prevented from spreading to neighbouring microvilli under the same conditions (Asteriti, 2017).

The dark-adapted 'pedestal' level can be used to gain an estimate of the resting Ca²⁺ concentration in dissociated ommatidia (in physiological solutions) assuming in vitro calibration data. With reference to F0 measured in Ca²⁺ free solution in the same ommatidia, the mean dark-adapted value in normal bath was 0.77 ± 0.14 (mean ± S.E.M. n = 11). This would be equivalent to ~80 nM (assuming Kd = 290 nM and Fmax 23.5). This value was significantly lower in ninaE-calx flies over-expressing the exchanger (0.19 ± 0.04 n = 11 equivalent to ~50 nM) and higher in calx¹ mutants (1.94 ± 0.24 n = 14 equivalent to ~120 nM) (Asteriti, 2017).

The recovery of GCaMP6f fluorescence to baseline is likely to be a reasonably accurate reflection of the falling Ca²⁺ levels during response recovery, although the initial decrease (from initial ~mM levels to low μM levels) will still be subject to saturation effects. With relatively dim flashes (up to ~1000 effectively absorbed photons) the GCaMP6f signal in wild-type backgrounds fell to near baseline within ~2-3 s with a half time (t 1/2) of ~1 s. This is slower than the GCaMP6f off-rate (~200 ms), and thus likely to approximate the true time-course of Ca²⁺ recovery. The recovery was significantly accelerated in ninaE-calx flies (~500 ms), and slowed in calx¹ mutants (~2 s increasing to >10 s following brighter flashes), consistent with a dominant role of the Na+/Ca²⁺ exchanger in Ca²⁺ extrusion. Nevertheless, even after bright flashes, given sufficient dark-adaptation time (~30-60 s), resting [Ca²⁺] in calx¹ mutants fell to levels close to those in dark-adapted wild-type photoreceptors, reflecting either residual function of the exchanger in this hypomorphic mutant and/or alternative extrusion mechanism(s) (Asteriti, 2017).

The smaller signals recorded in Ca²⁺ free bath fall within the dynamic range of GCaMP6f and allow estimates of the absolute Ca²⁺ levels reached under these conditions (e.g., Δ/F0 of 6 corresponds to ~200 nM). These signals were used to investigate the long disputed origin of the light-induced rise in cytosolic Ca²⁺ in Ca²⁺ free solutions. Originally, using INDO-1, it was found that this Ca²⁺ free rise was dependent upon extracellular Na+ and suggested that the rise might be due to re-equilibration of Na+/Ca²⁺ exchange in response to the massive light-induced Na+ influx that persists under these conditions. This was challenged by by a study that confirmed the requirement of external Na+ for a significant Ca²⁺ rise in Ca²⁺ free solutions, Na²⁺, but reported that a rise still occurred in Ca²⁺ free bath in the presence of Na+ when the photoreceptors were voltage clamped at the Na+ equilibrium potential to prevent Na+ influx. It was concluded that a Na+ gradient − but not influx − was required, that the Ca²⁺ free rise reflected release from internal stores, and that the requirement of extracellular Na+ reflected involvement of some other Na+ dependent process, such as Na/H transport. But how this might affect release of Ca²⁺ from intracellular stores is far from clear. A more recent study reported that the Ca²⁺ free rise was attenuated following RNAi knockdown of the IP3R. However, this is difficult to reconcile with an earlier study using INDO-1, where the rise was found to be unaffected in null IP3R mosaic eyes and confirmed again in this study using GCaMP6f (Asteriti, 2017).

This study used a variety of approaches to investigate the source of this Ca²⁺ free signal further. It was first confirmed that it was all but abolished in the absence of external Na+, whether substituted for Li+, Cs+, K+ or NMDG+. Importantly, it was found that the rise was also effectively eliminated in trpl;trp double mutants both in vivo and in dissociated ommatidia despite the presence of normal extracellular solutions containing both Na+ and Ca²⁺. Although it might be argued that, for some reason, PLC activity (and hence InsP3 generation) was compromised in trpl;trp mutants, convincing evidence indicates that net PLC activity is in fact greatly enhanced in trpl;trp due to the lack of Ca²⁺ and PKC dependent inhibition of PLC. Thus the rate and intensity dependence of PIP2 hydrolysis, measured using GFP-tagged PIP2 binding probes are greatly enhanced in trpl;trp mutants, as are the PLC-induced photomechanical contractions, and the acidification due to the protons released by the PLC reaction. Overall, therefore these results strongly suggest that Na+ influx is indeed required for the Ca²⁺ free rise. Crucially, the involvement of the Na+/Ca²⁺ exchanger in this rise was confirmed by finding that it was essentially eliminated in an exchanger mutant (calx¹), but greatly accelerated in ninaE-calx photoreceptors over-expressing the exchanger (Asteriti, 2017).

The question remains, how Na+/Ca²⁺ exchanger activity could generate such a sizeable Ca²⁺ signal (~100-200 nM) in cells perfused with EGTA buffered solutions, when free Ca²⁺ in the bath should be reduced to low nM levels. There is no unequivocal answer to this, and assuming the standard equation for the Na+/Ca²⁺ exchange equilibrium it would seem difficult for reverse Na+/Ca²⁺ exchange to raise Ca²⁺ into the range that was observed. However, at least three, not mutually exclusive factors might result in higher cytosolic Cai levels than predicted. Firstly, external Ca²⁺ might be relatively resistant to buffering in the intra-ommatidial space, and specifically the extremely narrow spaces between the microvilli or their bases, where the exchanger is believed to be localised. For example, with 500 nM Ca_o remaining, it is predicted that 130 nM Cai would be reached with 70 mM Na_i, 110 mM Na_o and the cell depolarised to 0 mV (values that could realistically be reached with the huge inward Na+ currents flowing under these conditions). Although one might also expect Ca²⁺ influx via the light-sensitive channels at such Ca_o concentrations, experiments buffering external Ca²⁺ at different concentrations with EGTA showed that direct Ca²⁺ influx signals could only be detected once external Ca²⁺ was raised above ~400 nM. Secondly, resting cytosolic Ca²⁺ concentration is determined not only by the exchanger, but also by any other Ca²⁺ fluxes, which might include tonic leakage from intracellular compartments such as endoplasmic reticulum (ER) or mitochondria. Massive Na+ influx would compromise the ability of the exchanger to counter any such fluxes. A third possibility is that, contrary to conventional dogma, the exchanger might also be expressed on intracellular membranes of endoplasmic reticulum or other Ca²⁺ containing compartments and that Na+ influx leads to re-equilibration of Na+/Ca²⁺ exchange across these (Asteriti, 2017).

Whatever the exact mechanism, the results indicate that the Ca²⁺ rise in Ca²⁺ free bath is strictly dependent upon both Na+ influx and the activity level of the Na+/Ca²⁺ exchanger, but unaffected in null IP3R mutants. Its time-course, with no detectable rise for ~200 ms, also appears much too slow to play any role in initiating the light response, which has a latency of ~10 ms and peaks within ~100-200 ms in response to bright illumination even under Ca²⁺ free conditions. The residual GCaMP6f signal remaining in the absence of Na+ influx and/or in the absence of Na+/Ca²⁺ exchanger activity − whether achieved by Na+ substitution, trpl;trp or calx mutants − was also still observed in IP3R mutants and was so small that it is questionable whether it reflects a Ca²⁺ signal. Because of the rapid inhibition of PLC by Ca²⁺ influx under physiological conditions any presumptive PLC-mediated Ca²⁺ release under physiological conditions would be even less. Together with a study in which no phototransduction defects were found in null IP3R mutants, these results suggest that InsP3-induced Ca²⁺ release plays no significant role in Drosophila phototransduction (Asteriti, 2017).

REGULATION

Promoter Structure

The 5'-flanking region of the Drosophila melanogaster norpA gene has been sequenced and its promoter characterized. The potential promoter region, which was deduced from the determination of the transcription start point (tsp), lacks a distinct TATA box sequence. Deletion analysis of the promoter region suggests that the minimal promoter necessary for efficient expression of the gene is located between -138 (PstI) and +278 relative to the tsp. Within this minimal promoter region, at least two downstream regulatory elements responsible for the stimulation of gene expression seem to exist in the DNA fragments between +44 and +121 and between +214 and +278. Among these, the DNA fragment between +44 and +121 affects promoter activity more dramatically (about 6-7 fold). This DNA fragment contains the consensus promoter element previously reported to be important for photoreceptor cell-specific expression, and this promoter element seems to be working in the norpA gene expression (Doh, 1997).

Enzymatic activity of NorpA

To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in an extract from normal Drosophila heads, which is absent in an extract from norpA mutant heads, was isolated and purified and its partial amino acid sequences were determined. The purified enzyme was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) are quite different from those of partially purified enzyme. The maximal activity for PIP2 hydrolysis is observed at calcium concentrations between 10(-7) and 10(-5) M for both the crude and partially purified enzymes. In contrast, the activity for PI hydrolysis of the crude enzyme increases with increasing calcium concentrations, while that of the partially purified enzyme reaches a maximum at calcium concentrations between 10(-6) and 10(-4) M, and decreases at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme are similar. The crude enzyme hydrolyzes PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increases with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA, which was suggested to be norpA gene (Toyoshima, 1990).

Fatty acid and InsP3 signaling downstream of NorpA

Light-sensitive channels encoded by the Drosophila transient receptor potential-like gene (trpl) are activated in situ by an unknown mechanism requiring activation of Gq and phospholipase C (PLC). Recent studies have variously concluded that heterologously expressed TRPL channels are activated by direct Gq-protein interaction, InsP3 or Ca2+. In an attempt to resolve this confusion an exploration was carried out of the mechanism for the activation of TRPL channels co-expressed with a PLC-specific muscarinic receptor in a Drosophila cell line (S2 cells). Simultaneous whole-cell recordings and ratiometric Indo-1 Ca2+ measurements indicate that agonist (CCh)-induced activation of TRPL channels is not always associated with a rise in Ca2+. Internal perfusion with BAPTA (10 mM) reduces, but does not block, the response to agonist. In most cases, releasing caged Ca2+ facilitates the level of spontaneous channel activity, but similar concentrations (200-500 nM) can also inhibit TRPL activity. Releasing caged InsP3 invariably releases Ca2+ from internal stores but has only a minor influence on TRPL activity and none at all when Ca2+ release is buffered with BAPTA. Caged InsP3 also fails to activate any light-sensitive channels in situ in Drosophila photoreceptors. Two phospholipase C inhibitors (U-73122 4 microM and bromo-phenacyl bromide 50 microM) reduce both spontaneous and agonist-induced TRPL activity in S2 cells. The results suggest that, as in situ, TRPL activation involves G-protein and PLC; that Ca2+ can both facilitate and in some cases inhibit TRPL channels, but that neither Ca2+ nor InsP3 is the primary activator of the channel (Hardie, 1998).

Phototransduction in invertebrate microvillar photoreceptors is thought to be mediated by the activation of phospholipase C (PLC), but how this leads to gating of the light-sensitive channels is unknown. Most attention has focused on inositol-1,4,5-trisphosphate, a second messenger produced by PLC from phosphatidylinositol-4,5-bisphosphate; however, PLC also generates diacylglycerol, a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both of these fatty acids reversibly activate native light-sensitive channels [transient receptor potential (TRP) and TRP-like (TRPL)] in Drosophila photoreceptors as well as recombinant TRPL channels expressed in Drosophila S2 cells. Recombinant channels are activated rapidly in both whole-cell recordings and inside-out patches, with a half-maximal effector concentration for linolenic acid of approximately 10 microM. Four different lipoxygenase inhibitors, which might be expected to lead to build-up of endogenous fatty acids, also activate native TRP and TRPL channels in intact photoreceptors. Since arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

Drosophila phototransduction is a G protein-coupled, calcium-regulated signaling cascade that serves as a model system for the dissection of phospholipase C (PLC) signaling in vivo. The Drosophila light-activated conductance is constituted in part by the Transient receptor potential (Trp) ion channel, yet trp mutants still display a robust response, which demonstrates the presence of additional channels. The transient receptor potential-like (trpl) gene encodes a protein displaying 40% amino acid identity with TRP. Mammalian homologs of TRP and TRPL recently have been isolated and postulated to encode components of the elusive I(crac) conductance. TRP and TRPL localize to the membrane of the transducing organelle, together with rhodopsin and PLC, consistent with a role in PLC signaling during phototransduction. To determine the function of TRPL in vivo, trpl mutants were isolated and characterized physiologically and genetically. The light-activated conductance is composed of TRP and TRPL ion channels and each can be activated on its own. Genetic and electrophysiological tools were used to study the contribution of each channel type to the light response and show that TRP and TRPL can serve partially overlapping functions (Niemeyer, 1996).

Membrane association of NorpA

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. The predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA. Biochemical assays as well as antisera generated against the major gene product of norpA were used to examine PLC subcellular distribution before and during phototransduction. Both phospholipase C activity and the NorpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as NorpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the NorpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C (McKay, 1994b).

Ca2+ and the phosphoinositide pathway

Photoreceptors of dissociated Drosophila retinae were loaded with the fluorescent Ca2+ indicators, fluo-3 and Calcium Green-5N. In fluo-3-loaded, wild-type photoreceptors, a rapid increase in fluorescence (Ca2+ signal) accompanies the light-evoked inward current. Removal of extracellular Ca2+ greatly reduces the Ca2+ signal, indicating Ca2+ influx as its major cause. In Calcium Green-5N-loaded trp mutants, which lack a large fraction of the Ca2+ permeability underlying the light-evoked inward current, the Ca2+ signal is smaller relative to wild-type photoreceptors. Fluo-3-loaded norpA mutant photoreceptors, which lack a light-activated phospholipase C, generate no light-evoked inward current and no Ca2+ signal. The phosphoinositide pathway therefore appears necessary for both excitation and changes in cytosolic free Ca2+ concentration (Peretz, 1994).

Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. The mechanism of this reaction was investigated by assaying PLC activity in Drosophila head membranes using exogenous phospholipid substrates. PLC activation depends on the photoconversion of rhodopsin to metarhodopsin and is reduced in norpAEE5 PLC and ninaEP332 rhodopsin mutants. NorpA PLC is stimulated by light at free Ca2+ concentrations between 10 nM and 1 microM. This finding is consistent with a Ca(2+)-mediated positive feedback mechanism that contributes to the rapid temporal response of invertebrate photoreceptor cells. The guanyl nucleotide dependence of light-stimulated PLC activity indicates that a G protein regulates NorpA. This was confirmed by the observation that light stimulation of PLC activity is deficient in mutants that lack the eye-specific G protein beta subunit G beta e. These results indicate that G beta e functions as the beta subunit of the G protein coupling rhodopsin to NorpA PLC (Running Deer, 1995).

Drosophila phototransduction is a phosphoinositide-mediated and Ca(2+)-regulated signaling cascade ideal for the dissection of feedback regulatory mechanisms. To study the roles of intracellular Ca2+ ([Ca2+]i) in this process, novel techniques were developed for the measurement of [Ca2+]i in intact photoreceptors. Flies were genetically engineered to express a UV-specific rhodopsin in place of the normal rhodopsin, so that long wavelength light can be used to image [Ca2+]i changes while minimally exciting the photoreceptor cells. Activation with UV generates [Ca2+]i increases that are spatially localized to the rhabdomeres and that are entirely dependent on the influx of extracellular Ca2+. Application of intracellular Ca2+ chelators of varying affinities demonstrates that the Ca2+ influx initially generates a large-amplitude transient that is crucial for negative regulation. Internal Ca2+ stores were revealed by discharging them with thapsigargin. But, in contrast to proposals that IP3-sensitive stores mediate phototransduction, thapsigargin does not mimic or acutely interfere with photoexcitation. Finally, a photoreceptor-specific PKC was identified as essential for normal kinetics of [Ca2+]i recovery (Ranganathan, 1994).

Disruption of phospholipase C-beta (PLC) by the norpA mutations of Drosophila renders flies blind by affecting the light-evoked photoreceptor potential. The norpA-coded PLC modulates the 1,4-dihydropyridine (DHP)-sensitive Ca2+ channels in larval muscles. The DHP-sensitive current is reduced in the norpA mutants. Application of 1 microM phorbol 12-myristate 13-acetate (TPA) and 1 microM phorbol 12,13-didecanoate (PDD), both activators of protein kinase C (PKC), rescues the current in the mutant fibers without significantly affecting the normal current. 4Alpha-phorbol 12,13-didecanoate (4alphaPDD), an inactive analog of PDD, does not affect either the normal or the mutant current. One micromolar bisindolylmaleimide (BIM), an inhibitor of PKC, reduces the current in the normal fibers without affecting the mutant current. 300 microM sn-1,2-dioctanoyl-glycerol (DOG), an analog of diacylglycerol (DAG), increases the current in the mutant fibers. These experiments suggest that the DHP-sensitive Ca2+ channels in Drosophila may be modulated by the PLC-DAG-PKC pathway, and that the same PLC isozyme, which is involved in phototransduction in the adult flies, may also modulate muscle Ca2+ channels in the larval stage of development (Gu, 1997).

The SOCS box protein STOPS is required for phototransduction through its effects on phospholipase C

Phosphoinositide-specific phospholipase C (PLC) isozymes play roles in a diversity of processes including Drosophila phototransduction. In fly photoreceptor cells, the PLCbeta encoded by norpA is critical for activation of TRP channels. This study describes a PLCbeta regulator, STOPS (slow termination of phototransduction), which encodes a SOCS box protein. Mutation of stops resulted in a reduced concentration of NORPA and a defect in stopping signaling following cessation of the light stimulus. NORPA has been proposed to have dual roles as a PLC- and GTPase-activating protein (GAP). The slow termination resulting from expressing low levels of wild-type NORPA was suppressed by addition of normal amounts of an altered NORPA, which had wild-type GAP activity, but no PLC activity. STOPS is the first protein identified that specifically regulates PLCbeta protein concentration. Moreover, this work demonstrates that a PLCbeta derivative that does not promote TRP channel activation, still contributes to signaling in vivo (Wang, 2008).

PLC isozymes play vital roles in signal transduction by cleaving the polar head group of phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). The most intensively studied PLC subtype, PLCβ, functions in signaling cascades initiated by stimulation of seven transmembrane domain G-protein coupling receptors (GPCRs). These GPCRs engage G_q, a member of the heterotrimeric GTP-binding protein family resulting in dissociation of the α and βγ subunits. One of these subunits then binds to and activates PLCβ, increasing its catalytic activity and thereby amplifying signaling (Wang, 2008).

In vitro analyses of mammalian PLCβ indicate that they also serve as GTPase activating proteins (GAPs), in addition to the more classical role in catalyzing the hydrolysis of PIP₂. PLCβ1 increases the steady-state GTPase activity up to 20 fold, resulting in fast deactivation of G_q. Thus, the regulation of G_q through the GAP activity of PLCβs potentially forms a short negative feedback loop that contributes to high signal resolution. An open question is whether PLCβs function as GAPs in vivo (Wang, 2008).

The PLCβ required for Drosophila phototransduction has been suggested to be a dual functional protein, serving as a both a GAP and phospholipase C. Fly visual transduction is initiated by light activation of rhodopsin and engagement of the Gα_q effector, which leads to stimulation of the PLCβ encoded by the norpA (no receptor potential A) locus. The cascade culminates with the opening of the TRP and TRPL cation channels (Wang, 2008).

Null mutations in norpA abolish the light response, while weak alleles display reduced light sensitivity, slow activation and decreased rates in the termination of the photoresponse. Weak norpA alleles express reduced levels of the NORPA protein, leading to the suggestion that the defect in termination results from a reduction in GAP activity. However, an alternative possibility is that the slow termination is a consequence of decreased PLC activity, since the activities of TRP and TRPL are dependent on hydrolysis of PIP₂, and Ca²⁺ influx via the channels is required for rapid termination. Thus the basis for the slow termination associated with expression of low quantities of NORPA remains unresolved. Defects in termination of G protein signaling can lead to many deleterious consequences in mammals, such as cardiac dysfunction, decreased fertility, deficits in the immune system, altered sensory responses, including problems in adapting to abrupt changes in light levels, and cell death. Given the diverse expression patterns and roles of PLCβ, understanding the basis through which PLCβ expression impacts on response termination has broad implications (Wang, 2008).

The expression of different PLCβ isoforms at appropriate levels and in distinct cell types is potentially a mechanism of control that applies to both mammalian and Drosophila PLCβs. For example, the NORPA PLCβ is expressed primarily in the Drosophila retina, and the mammalian PLCβ2 which functions in leukocyte signaling, is highly expressed in cells of the immune system. However, the proteins that regulate the cell-type specific expression of PLC proteins are unknown (Wang, 2008).

The current study identified a mutation in an eye-enriched SOCS box protein, which has been named Slow Termination of Phototransduction (STOPS). Known SOCS box proteins bind to the elongin B/C complex and promote the ubiquitination and proteasomal degradation of target proteins (reviewed in Kile, 2002). In contrast to these other proteins, STOPS functions independently of elongin B and C and is essential for expression of maximal levels of NORPA. STOPS is also required for stopping signaling upon cessation of the light stimulus as a consequence of the reduced NORPA expression. The defective termination in the stops mutant was due to decreased GAP activity of NORPA rather than a reduction in PLC activity, thereby providing strong evidence that NORPA functions as a GAP in vivo. These data demonstrate a novel mode for controlling PLCβ expression in photoreceptor cells (Wang, 2008).

Many PLCβ isoforms, such as the NORPA protein, which functions in fly phototransduction, are expressed in distinct subsets of cells. The current demonstrates that NORPA expression in photoreceptor cells depends on the STOPS protein. Mutations in stops caused a decrease in NORPA protein concentration, resulting in slow termination of the photoresponse (Wang, 2008).

A correlation between low NORPA expression and slow response termination has been proposed previously since multiple norpA alleles as well as inaD² express reduced concentrations of NORPA and display termination defects. However, these mutants have additional alterations, precluding the conclusion that low levels of NORPA prevent normal termination of the photoresponse. For example, hypomophic norpA mutants such as norpA^P57 and norpA^P16 express mutant rather than wild-type NORPA. Moreover, the interaction of NORPA with the INAD scaffold protein is disrupted in inaD² and norpA^C1094S, suggesting that the INAD-NORPA interaction contributes to the termination phenotype in these mutant flies (Wang, 2008).

This study provides two lines of evidence that a normal concentration of NORPA is essential for stopping signaling after cessation of the light stimulus. First, the stops mutant expresses decreased quantities of wild-type NORPA but not other proteins in photoreceptor cells, resulting in slow termination of the light response. Second, the amount of wild-type NORPA was manipulated using the heat shock promoter. The results showed a strong relationship between the termination rate and the concentration of NORPA protein (Wang, 2008).

A question concerns the mechanism through which reduced amounts of NORPA cause a termination defect. In principle, the slow termination could result from a combination of low NORPA levels, and a decrease in intrinsic enzyme activity, due to a requirement for STOPS as a cofactor. However, this does not appear to be the case as the termination defect is indistinguishable between stops⁺ flies that express the same low levels of NORPA (expressed under the control of the hsp70 promoter in a norpA mutant background) as is produced in stops¹ flies. STOPS does not co-immunoprecipitate with the TRP channels, suggesting that it does not cause defects in termination through direct interactions with the TRP channel. Moreover, the ERG response is the same in trp^P343 and trp^P343, stops¹ flies, suggesting that STOPS does not alter the activity of the TRPL channel. Furthermore, STOPS does not co-immunoprecipitate with the scaffold protein, INAD, or alter the concentration of any known member of the signalplex other than NORPA, indicating that the termination phenotype in stops¹ does not result from destabilization of the signalplex (Wang, 2008).

In addition to serving as a phospholipase C, mammalian PLCβs and NORPA have been proposed to be GAPs. Therefore, according to one model, low NORPA levels cause a termination defect by decreasing GAP activity, thereby leading to sustained activity of the Gα_q following light stimulation. However, previous work did not exclude that the slow termination resulting from a reduced NORPA concentration was due to a requirement for PLC activity. Thus, an alternative possibility is that the slow termination is a consequence of decreased light-dependent Ca²⁺ influx, since the NORPA phospholipase C activity leads to opening of the TRP and TRPL channels. A rise in Ca²⁺ is important for termination, as the rate is decreased in flies overexpressing the Na⁺/Ca²⁺ exchanger, CalX, or if the photoreceptor cells are illuminated in a Ca²⁺-free bath (Wang, 2008).

The results indicate that the slow termination due to a low concentration of NORPA is caused by reduced GAP activity rather than diminished intracellular Ca²⁺. In support of this conclusion, the delayed termination was not suppressed by a mutation in calx, which eliminates Ca²⁺ extrusion. Rather, it was found that slow termination of the photoresponse was fully reversed by expression of a PLC-derivative, NORPA^S559A, which contains a mutation in a residue critical for activation of the photoresponse. Since NORPA^S559A fully retained GAP activity, these data indicate that the GAP activity of NORPA is essential for arresting signaling following cessation of the light stimulus. The observation that introduction of normal levels of NORPA^S559A, suppresses the termination defect resulting from expression of a low concentration of wild-type NORPA, indicates that more than one NORPA molecule can interact successively with the same activated Gα_q. This proposal is consistent with the evidence that NORPA forms a homodimer. In addition, it appears that a single activated Gα_q-GTP is capable of interacting sequentially with multiple independent NORPA molecules (Wang, 2008).

Following activation, rapid termination of the light response is essential for high temporal resolution to ensure appropriate responses to subsequent stimuli. Since G-protein deactivation appears to be the rate-limiting step for termination in mammalian phototransduction cascade, timely deactivation of G-protein may be equally important for regulation of Drosophila phototransduction. Given that NORPA is the direct target for the G-protein, stimulation of the GTPase activity of the Gα_q represents a highly efficient and rapid mode of negative feedback regulation (Wang, 2008).

The current work has demonstrated that the SOCS box protein, STOPS, is required specifically for expression of NORPA in photoreceptor cells. SOCS proteins were initially identified as suppressors of cytokine signaling and contain a common C-terminal 40 amino acid SOCS box. Genomic analyses led to the identification of many additional SOCS box-containing proteins, which are subdivided into groups based on their diverse N-terminal domains. Examples include an SH2 domain (SOCS), WD40-repeats (WSB), a SPRY-domain (SSBs), a RAB domain (RAR), a domain conserved in the Neuralized family of proteins (Neuralized-like) and ankyrin-repeats (ASBs). Among the mammalian proteins, STOPS is most similar to the human CRA-b isoform of ASB15, since STOPS and CRA-b share additional homology (STOPS domain) that extends N-terminal to the SOCS box (Wang, 2008).

Current understanding about SOCS box containing proteins is that they function as adaptor molecules for the E3 ubiquitin ligase complex to target signaling proteins to the protein degradation pathway (Kile, 2002). The variable N-terminal domain interacts with target proteins thereby defining the substrate specificity for the E3 ubiquitin ligase complex. The SOCS box binds to a heterodimer composed of ubiquitin-like elongin B and Skp1-like elongin C, and forms an E3 ubiquitin ligase complex with Cullin-2 and Rbx-1. In C. elegans, the ZIF SOCS box protein functions in the exclusion of germ-line proteins from somatic lineages by interacting with the elongin B/C complex and targeting the somatic CCCH finger protein for degradation in a cullin and Rbx-dependent manner (Wang, 2008).

In contrast to all other characterized SOCS box proteins, which function in protein degradation, STOPS has an opposite role in promoting expression of NORPA in photoreceptor cells. Consistent with this observation, the elongin B/C pathway was not required for expression of NORPA. The elongin-B and elongin-C mutants expressed normal amounts of NORPA and displayed wild-type light responses. Thus, as expected the elongin B/C complex does not function as a positive regulator of NORPA. Furthermore, a double elongin-B, stops¹ mutant displayed the same decreased NORPA concentration and termination defect as the single stops¹ mutant, indicating that the elongin B/C complex does not function in a pathway opposing STOPS. Nevertheless, mutations in the SOCS box disrupt STOPS function. Taken together, these data indicate that although STOPS is dependent on the SOCS box, it acts independently of the elongin B/C complex in vivo. Thus, STOPS is distinct from previously characterized SOCS box proteins (Wang, 2008).

The expression patterns of many signaling proteins are restricted to ensure that they function in a cell-type specific manner. In wild-type flies, NORPA is expressed predominately in photoreceptor cells, but is still detected in other tissues. It was found that the STOPS protein is required for expression of NORPA only in photoreceptor cells since in the stops¹ retina, NORPA is reduced by >90% without affecting NORPA in other tissues. In hs-norpA flies, which expressed NORPA under the control of heat-shock promoter, the NORPA protein was expressed primarily in the head but not in the body, although the mRNAs were present in both the head and body. Moreover, in the head of hs-norpA flies, NORPA protein was enriched in the retina (Wang, 2008).

Although STOPS is required for relatively high expression of NORPA, it is not sufficient. In heat-shocked norpA^P24;hs-stops;hs-norpA flies, which expressed both the stops and norpA RNAs broadly, NORPA was still expressed principally in the retina. In HEK293T cells, coexpression of STOPS with NORPA did not result in an elevation of NORPA levels beyond that obtained in cells expressing NORPA alone. Moreover, NORPA and STOPS did not co-immunoprecipitate either in fly heads or after expressing the two proteins in HEK293T cells. Thus, there appear to be one or more additional factors that remain to be identified that function in concert with STOPS to promote NORPA expression. Finally, the current work describes a novel mode for controlling the expression of a PLCβ and raises the possibility that there exist mammalian SOCS box proteins that function independently of the elongin B/C complex to promote the expression of PLCβ and other signaling proteins in distinct subsets of cells (Wang, 2008).

Function of rhodopsin in temperature discrimination in Drosophila

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. 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). If this behavior requires rhodopsin, it would be a light-independent function, because thermotaxis takes place effectively in the dark (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, 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).

Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. Recent studies have shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLC activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. This study shows that in IP3 receptor (IP₃R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP₃R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP₃R knockdown with reduced PLCbeta catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP₃R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP₃R does not participate in light excitation, this study shows that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP₃R found here may represent a common feature of the inositol-lipid signaling (Kohn, 2015).

In this study, in vivo light-response suppression was accompanied by reduced Ca2+ release from IP3-sensitive stores. In addition, the rate of spontaneously produced dark bumps, which is highly sensitive to Gqα-dependent PLCβ catalytic activity and cellular Ca2+ level, was virtually abolished in IP₃R-deficient photoreceptors. This dark-bump elimination indicates that the suppressed Ca2+ release from IP3-sensitive stores underlies the suppressed catalytic activity of PLCβ, leading to suppressed light response in IP₃R-deficient photoreceptors. Further evidence that the suppressed light response arises from inhibition of Ca2+ release from IP3-sensitive stores came from blocking the Ca2+ pump by Tg, which mimicked the phenotype of the IP₃R-deficient photoreceptors in WT flies. The above findings indicate that IP₃R-mediated Ca2+ release has a critical role in light excitation of Drosophila photoreceptors. The combination of the PLCβ mutant norpAH43 with IP₃R-deficient photoreceptors, which synergistically suppressed the light response, strongly suggests that there is functional cooperation between the IP₃R and PLCβ in generation of the light response (Kohn, 2015).

It has been previously shown that an increase in cytosolic Ca2+ participates in light excitation as evidenced by enhancement of the light response following photo release of caged Ca2+ at the rising phase of the light response. The target of Ca2+ action has not been entirely resolved. PLCβ is an important target for Ca2+ action and the regulation of its catalytic activity by Ca2+ has been thoroughly investigated. These studies showed that the positive charge of Ca2+ is used to counterbalance local negative charges formed in the active site during the course of the catalytic reaction. Accordingly, Ca2+ performs electrostatic stabilization of both the substrate and the transition state, thus providing a twofold contribution to lower the activation energy of the enzyme reaction (Kohn, 2015).

The following model explains how functional cooperation between the IP₃R and PLCβ via the released Ca2+ operates and secures quantum-bump production: absorption of a single photon, which induces activation of several PLCβ molecules, is initially insufficient at resting Ca2+ levels to reach the critical level of PLCβ activity required for TRP/TRPL channel activation. Nevertheless, the IP3 molecules produced by the given PLCβ activity are able to activate the nearby IP₃Rs, mobilize Ca2+ from the stores, and elevate PLCβ activity above the threshold required for TRP/TRPL channel activation. In addition, the released Ca2+ may also reduce the threshold of TRP/TRPL channel activation and allow bump generation. According to this model, the following enzymatic reactions may explain the current findings. Each Gqα-activated PLCβ has low catalytic activity due to the relatively low (<160 nM) resting Ca2+ concentration in the cytosol. In addition, each activated PLCβ remains active for only a short (approximately several tens of milliseconds) time due to the GTPase-activating protein activity of PLCβ that causes a rapid hydrolysis of Gqα-GTP followed by inactivation of PLCβ. The initial low catalytic activity of PLCβ is apparently below the threshold required for activation of the TRP and TRPL channels, but this low activity still results in hydrolysis of PIP2 producing IP3. Since there are no IP3 buffers in the microvilli and the IP3 degradation time is relatively slow (~1 s), the produced IP3 molecules diffuse fast along the microvillus at an estimated time of ~1 ms along 1 microm long microvillus and bind to IP₃R located at the nearby submicrovillar cisternae (SMC; the photoreceptors' extensions of smooth ER). IP₃R channels residing at the SMC, which are large channels with high sensitivity for IP3 and thus can be activated at low PLCβ activity, open and release Ca2+ juxtaposed to the base of the microvillus. The released Ca2+ steeply raises the local Ca2+ concentration, probably to the microM range, because of the very small aqueous volume of the microvillus and the relatively large local Ca2+ elevation via the release mechanism (Kohn, 2015).

Accordingly, a single IP₃R channel can release ∼104 Ca2+ ions in 1 ms channel opening and Ca2+-induced Ca2+ release mechanism is a property of the IP₃R channels and of the ryanodine receptors, which reside in the ER. Ca2+ released via IP₃R of the WT SMC diffuse back toward the activated PLCβ and the TRP/TRPL channels in the microvillus. Although Ca2+ diffuses ∼20-fold slower than IP3 due to strong buffering, the diffusion constant strongly depends on Ca2+ concentration. Accordingly, at ~250 μM the Ca2+ diffusion coefficient is as large as that of IP3. Once a single TRP channel is activated, the large Ca2+ influx through this channel is sufficient to facilitate the rest of the active PLCβ molecules or reduce the threshold for TRP/TRPL channel activation in this microvillus and produce a bump that reflects activation of the entire microvillus . When there is abnormally low Ca2+ release via the IP₃R because of low IP₃R expression levels (IP₃R-RNAi), there is not enough Ca2+ to increase PLC activity or to reduce TRP activation threshold, and activated PLC in this microvillus does not produce a bump, leading to abnormally low frequency of dark bumps (Kohn, 2015).

The invasive whole-cell recording technique, which was used in previous studies and avoided Ca2+ buffering of the pipette solution, most likely resulted in abnormally elevated cytosolic Ca2+ concentration, which also allowed the Ca2+ pump to keep the stores full. This artificially elevated cytosolic [Ca2+] together with the constitutive Ca2+ leak from the full stores, bypassed the need to mobilize Ca2+ via functional IP₃R to facilitate PLCβ activity and reach its critical catalytic activity level needed to activate the TRP/TRPL channels. In the present study in the intact eye, a significant reduction in light-response amplitude was observed when the IP₃R level was reduced. Furthermore, when cellular [Ca2+] was reduced by prolonged extracellular EGTA application, the light response of the IP₃R-deficient flies was further suppressed. Moreover, when using invasive patch-clamp whole-cell recordings without Ca2+ buffering of the pipette solution, no significant difference between WT and IP₃R-deficient flies was observed, as found in the previous study. However, when pipette Ca2+ was reduced with EGTA, the phenotype of reduced light excitation was observed in both reduced quantum-bump frequency as well as in macroscopic light-response suppression. Unlike quantum bumps, dark bumps were virtually eliminated even without buffering the pipette Ca2+ in IP₃R-deficient flies, indicating that in the dark the IP₃R-deficiency led to abnormally low cytosolic [Ca2+], possibly due to reduced Ca2+ leak from stores leading to cellular [Ca2+] below the critical level required for PLC activation observed in WT flies. Alternatively, the positive feedback between the released Ca2+ and PLC may function at single PLC molecules. Hence the nominal pipette Ca2+ is not sufficient to allow PLC activity to pass the threshold of channel activation, but the released Ca2+ via IP₃R activation together with pipette Ca2+ allows PLC activity to pass this threshold and generate dark-bump (Kohn, 2015).

There is a striking functional similarity between, the cerebellar Purkinje cell (PC) proteins of the IP signaling and Drosophila photoreceptors, but the link of cerebellar mGluR1 receptor to TRPC3 activation is not clear. Interestingly, in PC neurons, stromal interaction molecule 1 (STIM1) was proved an essential regulator of Ca2+ level in neuronal endoplasmic reticulum Ca2+ stores. Accordingly, STIM1-specific deletion caused impairments in slow synaptic current and cerebellar motor behavior. Strikingly, refilling empty Ca2+ stores through increased Ca2+ level in the cytosol partially rescued the phenotype of the stim1 knock-out mice, reminiscent of the rescue of the phenotype of the IP₃R-deficient fly by artificially elevated cytosolic Ca2+. Thus, the facilitatory role of released Ca2+ on PLC in light excitation of Drosophila photoreceptors represents an essential mechanism that operates in other PI systems (Kohn, 2015).

Protein Interactions

Interaction of NorpA with G protein alpha49B

The roles of the Drosophila Gq alpha proteins (DGq) were examined in the phototransduction pathway. The DGq proteins immunolocalize to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocks the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulates DGq. Dominantly active DGq1 mutants exhibit a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics, as compared with wild-type flies. Dominant DGq2 mutants exhibit a light-independent GTPase activity with normal electrophysiological light responses. Retinas of double mutants of DGq1, but not DGq2, with the light-dependent retinal degeneration mutant rdgB degenerate even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark is norpA-dependent. These results indicate that DGq1 mediates the stimulation by light-activated rhodopsin of the norpA-encoded phospholipase C in the visual transduction cascade (Y. J. Lee, 1994).

Heterotrimeric G proteins mediate a variety of signaling processes by coupling seven-transmembrane receptors to intracellular effector molecules. The Drosophila phototransduction cascade is a G protein-coupled signaling cascade that utilizes a phospholipase C (PLC beta) effector. PLC beta has been shown to be activated by Gq alpha in reconstituted systems. To determine whether a Gq-like protein couples rhodopsin to PLC, and to study its function, a mutant defective in a photoreceptor-specific Gq protein, DGq, was isolated. Gq is demonstrated to be essential for the activation of the phototransduction cascade in vivo. Transgenic flies were generated expressing DGq under an inducible promoter and it is possible to manipulate the sensitivity of a photoreceptor cell by controlled expression of DGq. Characterization of quantum bumps in mutants expressing less that 1% of the levels of DGq reveals that the rhodopsin-G protein interaction does not determine the gain of the single photon responses. Together, these results provide significant insight into the role of Gq in regulating the output of a photoreceptor cell (Scott, 1995).

Interaction of NorpA with Inad

The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, Trp is found associated into a multiprotein complex with the norpA-encoded phospholipase C; an eye-specific protein kinase C (InaC), and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog can be isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel (Huber, 1996b).

Photoreceptors that use a phospholipase C-mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cbeta (PLCbeta), the light-activated Ca2+ channel TRP, and an eye-specific protein kinase C (ePKC) are clustered by the PDZ domain protein InaD. The function of ePKC was investigated in three ways: by cloning the Calliphora homolog of Drosophila ePKC; by precipitating the TRP signaling complex with anti-ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti-ePKC antibodies leads to the coprecipitation of PLCbeta, TRP, InaD and ePKC but not of rhodopsin. Phorbolester- and Ca2+-dependent protein phosphorylation reveals that, apart from the PDZ domain protein InaD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop that regulates Ca2+ influx through the TRP channel (Huber, 1998).

Drosophila InaD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in inaD alleles display mislocalizations of signaling molecules of phototransduction that include the essential effector, phospholipase C-beta (PLC-beta), also known as NORPA. The molecular and biochemical details of this functional link are unknown. InaD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). Altered NORPA proteins lacking binding sites display normal basal PLC activity but can no longer associate with InaD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of InaD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent InaD-NORPA interactions, which are essential for G protein-coupled phototransduction (van Huizen, 1998).

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (InaD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that InaD also binds to eye-specific protein kinase C (INAC) and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis the essential elements of the NORPA-InaD association have been defined and three critical residues in the C-terminal tail of NORPA, required for the interaction, have been identified. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in InaD. To evaluate the functional significance of the InaD-NORPA association in vivo, transgenic flies were derived expressing a modified NORPA that lacks the InaD interaction: NORPAC1094S. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation. Similarly, delayed activation may result from NORPAC1094S being unable to localize in close proximity to the TRP channel. It is concluded that InaD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells (Shieh, 1997).

Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation. eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains. There is a direct association between the third PDZ domain of INAD with TRP, and the carboxyl-terminal half of INAD with the last three residues of NORPA. The molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays. The second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile. The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC. In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive. Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated. These double mutants display a more severe response inactivation than either of the single mutants. Based on these findings, it is concluded that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex (Adamski, 1998).

Yeast two-hybrid and ligand overlay results both indicate that the second PDZ domain of INAD associates predominantly with eye-PKC, whereas no interaction was detected with PDZ4. This result is different from a previous report in which interaction of eye-PKC with the fourth PDZ domain of INAD was detected by affinity chromatography (Tsunoda, 1997). The current study tested a total of five constructs that contain the fourth PDZ domain: no indication of eye-PKC/fourth PDZ interaction was found. The constructs tested included a fusion protein that contained exactly the same region as previously tested. One possible explanation for these conflicting results is that the different assay systems are measuring different types of association between eye-PKC and INAD. INAD may bind and cluster eye-PKC to the signaling complex, and it can also act as a substrate for the kinase activity (Huber, 1996a). Amino acid substitutions made in the second PDZ domain of INAD have been shown to disrupt the eye-PKC binding. None of these amino acid changes are near the serine or threonine residues that are putative PKC phosphorylation sites. Furthermore, mutations in the carboxyl-terminal tail of PKC abolish PKC binding to the second PDZ domain. Thus the interaction described for eye-PKC/INAD is a typical carboxyl-terminal tail/PDZ domain association. The basis of the reported interaction with the fourth PDZ domain remains to be determined. Another provocative explanation could be that eye-PKC may bind different PDZ domains of INAD during different physiological conditions. For example, phosphorylation of INAD may change the relative affinity of the interaction in PDZ2 and PDZ4. Clarification of the role of these two eye-PKC/INAD interactions will require analysis of transgenic flies expressing a modified InaD in which these PDZ domain are mutated (Adamski, 1998).

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. This study shows that the sphingolipid biosynthetic enzyme, Ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in Ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, it was showm that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP₂), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP₂ and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP₂ and PLC during phototransduction (Dasgupta, 2009).

This study shows that DCERK regulates the ceramide level to maintain PLC and membrane organization of PIP₂ during phosphoinositide-mediated GPCR signaling in Drosophila. These results are summarized in a model depicted in a Model showing ceramide kinase regulates PLC activity, function and the local organization of PIP2 in GPCR signaling. Morphometric analysis of PIP₂ clusters suggests that there are still some clusters in dcerk¹; thus, the loss of NORPA in dcerk mutant may not be due only to the loss of PIP2 clustering. Although the effect of ceramide on PIP₂ is central, increased ceramide could affect other membrane properties that can downregulate NORPA or other proteins. Also, INAD is required for localization of NORPA and TRP is required for localization of INAD. Because TRP protein is affected over time in dcerk¹, additional interactions mediated by INAD and TRP could also contribute to NORPA stability. Photoreceptor degeneration seen in cerk mutants is also not simply caused by loss of NORPA protein, as it is more severe and sets in earlier than in norpA null mutants. There could be other effects of ceramide contributing to degeneration (Dasgupta, 2009).

Recent clinical studies have identified mutations in the human ceramide kinase like (CERKL) gene in patients with autosomal recessive retinitis pigmentosa. No CERKL homolog has been identified in the Drosophila genome. Interestingly, DCERK shares 31% identity with human CERKL, and possibly DCERK could perform some CERKL functions also. It would be worthwhile to test whether CERK regulates PLCβ4, the closest homolog of NORPA among mammalian PLCs, thereby participating in the mammalian visual process. Recent analyses of cerk^-/- mice revealed that CERK could function in cerebellar Purkinje cells (which are also enriched in PLC?4) and in neutrophil homeostasis. Because the mechanism by which mammalian CERK regulates these processes is not known, it would be interesting to test whether these functions are also mediated through PLC. Although current understanding limits a direct co-relationship between mammalian CERK and PLCs, it is likely that similar ceramide-regulated microenvironments could operate in other phosphoinositide-dependent signaling such as insulin signaling in adipocytes (Dasgupta, 2009).

In summary, these data show that modulation of the ceramide level by CERK regulates PIP₂ and PLCβ function in Drosophila. Because PIP₂ and PLC are fundamental components of GPCR signaling, uncovering their regulation by ceramide through CERK should lead to a better understanding of lipid regulation in signaling (Dasgupta, 2009).

DEVELOPMENTAL BIOLOGY

Adult

The norpA gene encodes a 7.0-kilobase transcript that can be detected in the head but not in the body of adult flies and a 5.6-kilobase transcript that can be detected throughout development and in both heads and bodies of adults. In situ hybridization of cDNA sequences to tissue sections shows that the gene is expressed in the neuronal cell bodies of the optic lobe, central brain, and thoracic ganglia of adults and the brain of larvae. This tissue distribution of norpA transcripts is identical to the distribution of transcripts from a Drosophila Go alpha-subunit gene (Shortridge, 1991).

Antisera against NorpA recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong NorpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the NorpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction (Schneuwly, 1991).

Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues were all used to examine the tissue-specific expression of the norpA gene. It is expressed in a variety of tissues in addition to the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. The 7.5-kb transcript is missing from eyes absent mutants and norpA mutants. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants. The NorpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays that show that the norpA mutation reduces phospholipase C activity in each of the tissues in which NorpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the NorpA antiserum demonstrates that the NorpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Staining in the brain is strongest in the optic lobes and cerebrum. Since some of the above mentioned tissues that express NorpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, it has been concluded that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction (Zhu, 1993).

EFFECTS OF MUTATION

Eight norpA mutants have been characterized by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued (Pearn, 1996).

Mutations in the norpA gene of Drosophila severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC). A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutants by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with NorpA antiserum show that NorpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutants exhibit very little to none. Immunohistochemical staining of tissue sections using NorpA antiserum confirm that expression of NorpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C, which is utilized in phototransduction (McKay, 1995).

The norpA (no receptor potential) mutant of Drosophila melanogaster has a visual transduction deficit. This study determines whether lack of function leads to structural repercussions in photoreceptor cells of the compound eye and their synapses. For this purpose, thin sections and freeze fracture replicas of norpA were examined using transmission electron microscopy. Ultrastructurally, retinula cells in the compound eye and all aspects of the first optic neuropil (lamina ganglionaris) are essentially normal in newly emerged flies. However, as expected, intraretinular pigment granules fail to show their light elicited aggregation; further, the P face particle density is somewhat lower than in wild type. There are unusual membrane specializations on the plasmalemma of the retinula cell, dubbed 'zippers'. Zippers appear to increase with age and can cause a distorted geometry of ommatidia. Only a few retinula cells ultimately degenerate in norpA, and the proportion may not differ from that of wild type. Despite the absence of the receptor potential in norpA, many aspects of the turnover of rhabdomeric membrane appear to be as in wild type (Stark, 1989).

The electrophysiological characteristics of norpAH52, a temperature sensitive phototransduction mutant of Drosophila melanogaster, were studied in vivo. Upon raising the environmental temperature to 33-37 degrees C, mutant flies exhibit time-dependent changes in photoresponses. Initial observations indicate losses in responsiveness at low light intensities and prolonged receptor potential waveforms. Subsequently, reductions in response amplitudes at higher light intensities occur, until no responses are obtained. On return to lower temperature the electrophysiological properties recover in reverse order. Based on these observations it is concluded that the primary defect of norpA affects the efficiency of the phototransduction process. Enhanced light exposure can offset the receptor potential changes in norpA. With the temperature sensitive mutant, (1) additional light exposure prolongs the time that responses can be observed at the higher temperature; (2) when 1-s illuminations no longer elicit responses at the higher temperature, 1-min illuminations at the same intensity temporarily restores the ability to obtain 1-s-responses, and (3) light accelerates the restoration of responses on return to lower temperatures. Illumination also has an effect on non-temperature sensitive norpA mutants, enabling the production of small photoresponses in norpAH44, a mutant that normally does not exhibit any responses, and improving the low-light-intensity responses of norpAP16. The current study indicates that the PI cycle, which is inhibited in norpA mutants (Yoshioka, 1985), is an important light-sensitive positive step or effector in the production of receptor potential responses (Wilson, 1987).

The norpAH44 phototransduction mutant of Drosophila, an allele that, on eclosion, does not exhibit a receptor potential was found, at later ages, to undergo light and temperature dependent degeneration of its photoreceptors as well as decreases in rhodopsin concentration. Pseudopupil measurements and light and electron microscopy were used to monitor the structure of the photoreceptors. When norpAH44 flies are maintained exclusively in the dark, no changes in structure or rhodopsin concentration are observed. When maintained on a 12 h light-12 h dark cycle, structural changes are first observed at 6 days of age for flies maintained at 24 degrees C or at 12 days of age for flies maintained at 19 degrees C. When the light-dark cycle is initiated after 10 days in the dark there is a more rapid loss of rhodopsin concentration and pseudopupil. The data suggest that even in the dark, although no obvious changes in structure or rhodopsin concentration are observed, certain processes that support these components have been affected. NorpAP12, an allele that exhibits small receptor potential amplitudes, also displays age- and light-dependent photoreceptor degeneration and decreases in rhodopsin concentration, whereas no degeneration or decreases in rhodopsin are observed in norpAP16, an allele that exhibits receptor potential amplitudes similar to those of wild-type. The data suggest that the processes that affect phototransduction, such as the phosphatidylinositol cycle, have a long-term role in the maintenance of rhodopsin concentration and photoreceptor integrity (Meyertholen, 1987).

The formation of stable rhodopsin-arrestin complexes induces apoptosis and photoreceptor cell degeneration

Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. This study demonstrates the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, it is shown that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. It is proposed that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders (Alloway, 2000).

This paper demonstrates the existence of a novel mechanism to explain the light-dependent retinal degeneration that is observed in a subset of Drosophila visual system mutants. Mutations in three distinct genetic loci, norpA, arr2, and rdgB, result in the light-dependent formation of stable rhodopsin-arrestin complexes. Elimination of either member of this complex rescues the retinal degeneration in each of the three genetic backgrounds. In addition, it is shown that the formation of these stable rhodopsin-arrestin complexes triggers apoptotic cell death. Furthermore, endocytosis is essential for inducing cell death in norpA mutants, suggesting that the internalization of the rhodopsin-arrestin complexes is an early step in the initiation of apoptosis of retinal photoreceptors. It is possible that the excessive endocytosis saturates a downstream cellular function (for example, saturation of the early endosome or depletion of an endocytic protein), which signals the cell to undergo programmed cell death. Alternatively, the endocytosis may block a signal that protects the cell from apoptosis (Alloway, 2000)

The inactivation of the Drosophila phototransduction cascade is an extremely rapid event. Drosophila photoreceptors can shut off the light-activated currents in less than a 100 ms following termination of the light stimulus. One way in which photoreceptors have evolved to do this is at the level of the G protein-coupled receptor rhodopsin. Immediately upon activation, rhodopsin is multiply phosphorylated on its C terminus, and this phosphorylation greatly increases its affinity for the abundant soluble protein arrestin. Invertebrate Arr2 is a very basic molecule with a pK_a of ~8.7 and, therefore, has a high affinity for phosphorylated rhodopsin. Thus, this interaction of activated rhodopsin and arrestin occurs very rapidly, and the receptor is quickly inactivated. It is tempting to speculate that the posttranslational modifications of arrestin and rhodopsin are essential to eliminate these complexes. In such a model, it is necessary to phosphorylate Arr2, thereby making it less basic, as well as to dephosphorylate rhodopsin, thereby making it less acidic. Thus, the system is designed such that the influx of calcium activates CamKII, the kinase that phosphorylates arrestin, and rdgC, the phosphatase that dephosphorylates rhodopsin. Both of these steps are crucial, as evidenced by the fact that rdgC mutants and arr2(S366A) mutants both undergo rapid light-dependent retinal degeneration. Evidence suggests that, in the absence of these posttranslational modifications, stable rhodopsin-arrestin complexes persist in the cell and are instrumental in the pathology of retinal degeneration (Alloway, 2000)

The retinal degeneration induced by rhodopsin-arrestin complexes can be partially rescued by a temperature-sensitive allele of dynamin. This observation suggests that the rhodopsin-arrestin complexes are being removed from the photoreceptor rhabdomere by the endocytic machinery. A similar receptor internalization pathway occurs for the vertebrate β-adrenergic receptor. β-arrestin is involved in the inactivation and internalization of the β-adrenergic receptor. A small domain near the carboxyl terminus of β-arrestin directly interacts with clathrin and targets the β-adrenergic receptor-β-arrestin complex for internalization. This clathrin binding site is curiously missing in the visual arrestins. However, in spite of the absence of the clathrin-interacting motif, these complexes seem to have the ability to interact with the endocytic machinery. One attractive model is that visual arrestin serves as an AP2-like adaptor, just like β-arrestin, but recruits clathrin by a different mechanism. The question of whether the endocytic proteins are recognizing motifs in rhodopsin or arrestin awaits further studies. Many models have been proposed to explain retinal degeneration in humans, including constitutive activity of the phototransduction cascade, improper trafficking of photoreceptor cell components, and defects in the recycling of rhodopsin. The results described in this study implicate the endocytic pathway in retinal degeneration and suggest that excessive endocytosis can be a trigger for apoptotic cell death (Alloway, 2000)

The internalization of rhodopsin-arrestin complexes via receptor-mediated endocytosis could partly explain the membrane association of arrestin in certain mutant backgrounds. The enhanced membrane affinity of arrestin could in part be due to the rhodopsin-arrestin complexes rapidly interacting with the endocytic machinery. It is possible that, once the complex is associated with clathrin cages, the arrestin would be locked in the membrane-associated state. In support of this model, it has recently been demonstrated that the unphosphorylated form of arrestin directly interacts with clathrin in vitro. Therefore, it is possible that the role of arrestin phosphorylation is to block interactions with clathrin, and any mutant background that fails to phosphorylate arrestin yields complexes due to clathrin interactions. However, it is still essential that a stable rhodopsin-arrestin complex be formed initially to allow for the assembly of the endocytic proteins (Alloway, 2000)

A large number of mutations in the human rhodopsin gene have been isolated that are responsible for retinal disease. Interestingly, there are several mutations in human rhodopsin that form stable rhodopsin-arrestin complexes. These include a mutation in a highly conserved lysine in the seventh transmembrane domain that has been shown to cause autosomal-dominant retinitis pigmentosa. This mutant form of rhodopsin is found both in vivo and in vitro to be constitutively phosphorylated and tightly bound to arrestin. It had been hypothesized that the complexes between rhodopsin and arrestin may be instrumental in the retinal degeneration process. Possibly, as in Drosophila, these stable human rhodopsin-arrestin complexes are removed from the rod outer segments by the endocytic machinery, and this excessive endocytosis is the direct cause of apoptotic retinal degeneration (Alloway, 2000)

One question yet to be addressed is why photoreceptor cells have a mechanism for eliminating rhodopsin-arrestin complexes if, under nonpathological conditions, these complexes do not persist in the cell. One possible explanation is that arrestin has a secondary function to eliminate defective rhodopsin molecules from the photoreceptor cell. A rhodopsin molecule that becomes photochemically damaged may render the receptor nonfunctional or constitutively active. The presence of a constitutively active rhodopsin molecule could potentially be very detrimental and, therefore, necessitates its removal from the photoreceptor cell. Presumably, any rhodopsin molecule that becomes constitutively active will bind arrestin and generate a stable rhodopsin-arrestin complex. The stabily bound arrestin would target the dysfunctional rhodopsin molecule for endocytosis and degradation. In this manner, arrestin could function as a surveillance protein, eliminating defective rhodopsin molecules from the photoreceptor cell. However, in certain mutant backgrounds, the regulation of rhodopsin-arrestin complex formation is defective, and a large number of complexes are formed. The increased endocytosis of these complexes initiates apoptosis and retinal degeneration (Alloway, 2000).

Photoreceptor cells adapt to bright or continuous light, although the molecular mechanisms underlying this phenomenon are incompletely understood. This paper reports a mechanism of light adaptation in Drosophila, which is regulated by phosphoinositides (PIs). Light-dependent translocation of arrestin is defective in mutants that disrupt PI metabolism or trafficking. Arrestin binds to PIP(3) in vitro, and mutation of this site delays arrestin shuttling and results in defects in the termination of the light response, which is normally accelerated by prior exposure to light. Disruption of the arrestin/PI interaction also suppresses retinal degeneration caused by excessive endocytosis of rhodopsin/arrestin complexes. These findings indicate that light-dependent trafficking of arrestin is regulated by direct interaction with PIs and is required for light adaptation. Since phospholipase C activity is required for activation of Drosophila phototransduction, these data point to a dual role of PIs in phototransduction (Lee, 2003).

The demonstration that translocation of Arr2 is regulated by PIs addresses a lingering question concerning potential roles of PIs in photoreceptor cells. The Drosophila visual transduction cascade is among the most intensively studied GPCR cascades. During the last 30 years, many proteins and mutations have been identified that perturb PI signaling; however, the targets and mechanisms directly regulated by PIs have not been previously described (Lee, 2003).

The regulation of Arr2 shuttling by PIs occurs on the order of a few to many minutes. This is in contrast to the millisecond time scale, which operates in the activation of phototransduction. Although the specific activation mechanism involved in Drosophila phototransduction remains elusive, it is established that it depends on a PLCβ (NORPA). Thus, PLC-mediated hydrolysis of PIP₂ leads to rapid activation of the light-sensitive channels through the millisecond generation of PIP₂ metabolites or reduction in PIP₂ levels. Since adaptation occurs over a much slower timescale, regulation of this latter phenomenon exclusively by direct effects of second messengers on protein activities might be too rapid. Rather, regulation of adaptation by the translocation of signaling proteins provides a mechanism whereby changes in second messengers, such as PIP₃, result in delayed effects on the magnitude and the kinetics of signaling. Therefore, PIs appear to have the capacity to serve a dual role in activation and adaptation by modulating the activities and localization of signaling proteins (Lee, 2003).

Previous reports have shown that stable Arr2/rhodopsin complex formation leads to retinal degeneration in norpA or rdgC flies. Removal of the arr2 gene in a norpA or rdgC background partially suppresses the photoreceptor cell death. This partial suppression could be due to elimination of Arr2/rhodopsin complexes, reduction in endocytosis of rhodopsin, or disruption of some other Arr2 function. In this work, Arr2/rhodopsin binding and PI-regulated trafficking of Arr2 were uncoupled by expressing Arr2^3K/Q, which is defective in movement but not rhodopsin binding. Since the retinal degeneration in norpA is largely rescued in arr2^3K/Q flies, these results suggest that apoptosis in norpA results from endocytosis of Arr2/rhodopsin complexes rather than a defect in Arr2/rhodopsin binding. This conclusion is further supported by the finding that there was even greater suppression of the norpA degeneration in an arr2^3K/Q than in an arr2⁵ null background (Lee, 2003).

Molecular basis of amplification in Drosophila phototransduction: Roles for G Protein, Phospholipase C, and Diacylglycerol kinase

In Drosophila photoreceptors, the amplification responsible for generating quantum bumps in response to photoisomerization of single rhodopsin molecules has been thought to be mediated downstream of phospholipase C (PLC), since bump amplitudes were reportedly unaffected in mutants with greatly reduced levels of either G protein or PLC. It is found that quantum bumps in such mutants are reduced ~3- to 5-fold but are restored to near wild-type values by mutations in the rdgA gene encoding diacylglycerol kinase (DGK) and also by depleting intracellular ATP. The results demonstrate that amplification requires activation of multiple G protein and PLC molecules; they identify DGK as a key enzyme regulating amplification, and implicate diacylglycerol as a messenger of excitation in Drosophila phototransduction (Hardie, 2002).

Many photoreceptors generate discrete responses to effective absorptions of single photons, known as quantum bumps. In Drosophila, these represent simultaneous activation of about 15 light-sensitive channels generating an inward current of ~10 pA. The response is remarkable for its speed, with latencies as short as 20 ms and bump halfwidths of ~20 ms. These kinetics are about 100 times faster than in toad rods recorded at similar temperatures, about 10 times faster than mammalian rods recorded at 37°C, and in general, fly photoreceptors are considered to have the fastest known G protein-coupled signaling cascades. The events leading to quantum bump generation in Drosophila have been inferred from a variety of physiological, biochemical, and genetic evidence. Photoisomerized rhodopsin activates a heterotrimeric G protein (Gq class) releasing the alpha subunit, which in turn activates phospholipase C (PLCß4 isoform) encoded by the norpA gene. By a still unknown mechanism, activation of PLC leads to the opening of at least two classes of Ca²⁺ permeable channels, TRP and TRPL. These are the prototypical members of the TRP ion channel superfamily responsible for a wide variety of Ca²⁺ influx pathways throughout the body. Recent evidence suggests that the Drosophila channels, as well as some vertebrate TRP homologs, may be activated by lipid second messengers rather than by InsP₃. Candidates include diacylglycerol (DAG), polyunsaturated fatty acids (PUFAs), which are DAG metabolites, or a reduction in phosphatidyl inositol 4,5 bisphosphate (PIP₂). Following activation of the first channel(s), Ca²⁺ influx mediates rapid positive and negative feedback, which is required for both amplification and rapid termination of the quantum bump. Most, if not all, elements of the phototransduction cascade are located in the ~30,000 tightly packed microvilli, each only 60 nm in diameter and 1–2 µm long, which together form the light-guiding rhabdomere. Quantitative Western analysis suggests that there are about 25 TRP channels per microvillus, which corresponds closely to the number of channels activated during the quantum bump. It is therefore plausible that quantum bump generation is restricted to a single microvillus, representing activation of all or most of the available channels (Hardie, 2002).

In contrast to the situation in vertebrate rods, recent evidence has led to the proposal that all amplification in this system is mediated downstream of PLC. The main evidence for this view is that quantum bump amplitude is reported to be unaffected in hypomorphic mutants of G protein (Gaq) and PLC (norpA) where protein levels are reduced to levels (<1%) such that there may often be no more than a single G protein or PLC molecule in each microvillus. This led to the conclusion that levels of G protein do not actively contribute to the gain of the single photon response and that the G protein must act as a 'molecular switch,' triggering bump generation (Hardie, 2002).

In the present study, this view has been questioned because of the observation of spontaneous events in the dark in WT flies. Although the data suggested they were due to spontaneous activation of G proteins, they are much smaller than quantum bumps, seemingly inconsistent with single G proteins triggering full-sized bumps. Quantum bumps were therefore systematically reinvestigated in both Gaq and norpA hypomorphs; contrary to previous reports, bump amplitudes were much reduced, suggesting that there is substantial amplification upstream of PLC. The discrepancy with earlier studies is resolved by showing that bump amplitude in both Gaq and norpA mutants could be increased to near WT levels by omitting ATP from the whole-cell recording pipette. Finally, DAG kinase has been identified as the critical ATP-dependent factor, strongly supporting the proposal that DAG (or its downstream metabolites) is the excitatory messenger responsible for channel activation (Hardie, 2002).

The current view that amplification in the Drosophila phototransduction cascade is mediated downstream of PLC is based largely on reports that bump amplitude and timecourse are unaffected in mutants of G protein or PLC. However, after testing an extensive range of mutants of both G protein and PLC, it was found that in all cases quantum bump amplitude was greatly reduced. Although these results seemed to directly contradict earlier studies, it was found that when ATP was omitted from the electrode, bump amplitude approached WT values. This almost certainly accounts for the apparently conflicting results (Hardie, 2002).

Cook (2000) analyzed bump in detail only from norpA^C1094S flies, which this study has found to have among the largest bumps of the norpA alleles tested (~3 pA with occasional bumps as large as 10 pA). Since Cook (2000) excluded events less than 3 pA in amplitude from their analysis, their results are not necessarily in conflict with the results of this study: small bumps are indeed found, e.g., with norpA^P57 under similar recording conditions. Most significantly, the effect of removing ATP could be closely mimicked by mutations in the rdgA gene encoding DGK. These results indicate that amplification in Drosophila is critically dependent on activation of multiple G protein and PLC molecules and identify DGK as a key enzyme regulating bump amplitude and inactivation (Hardie, 2002).

It is now concluded that amplification in Drosophila phototransduction is critically dependent upon activation of multiple G proteins and PLC molecules. Assuming linear summation, the difference in bump current integral between WT and the most severe Gq and norpA hypomorphs suggests that at least five PLCs need be activated in order to generate a typical WT bump. Since quantum bumps in Drosophila correspond to the simultaneous opening of only about 15 channels at the peak of the bump, amplification at the level of the G protein may in fact represent the major component of amplification in Drosophila. Interestingly, a similar number of G proteins (about eight) are believed to be activated in Limulus ventral photoreceptors, although amplification downstream of PLC dominates in these cells -- probably by very distinct mechanisms (Hardie, 2002).

Significantly, lowering ATP did not further increase bump amplitude in WT photoreceptors, suggesting that the bump-generating machinery is saturated in dark-adapted photoreceptors. This can be understood if quantum bumps represent activation of all available channels within the microvillus. The suggestion that the unit of signaling underlying the quantum bump is the microvillus is also consistent with the finding that the number of channels activated during the quantum bump corresponds closely to the number predicted per microvillus from quantitative Western analysis (Hardie, 2002).

Is DAG the excitatory messenger? The essential role of PLC in Drosophila phototransduction is well established, but the downstream mechanisms responsible for gating the light-sensitive channels remain controversial. Accumulating evidence, including the lack of phenotype in mutants of the only InsP₃ receptor gene known in Drosophila, suggests that InsP₃ may not be involved in excitation. Additional consequences of PLC activation include generation of DAG and a reduction in PIP₂, both of which are also currently under discussion as excitatory messengers for some vertebrate TRP homologs. Both TRP and TRPL channels can be activated by polyunsaturated fatty acids (PUFAs), which might be released from DAG by a DAG lipase. It has been found that heterologously expressed TRPL channels can also be activated by DAG, raising the possibility that DAG may be the endogenous transmitter, with PUFAs mimicking their action. However, at least some of the actions of DAG and PUFAs may be indirect via activation of endogenous PLC; activity of TRPL channels in patches is suppressed by application of PIP₂, suggesting PIP₂ depletion as a potential contributory factor to channel gating (Hardie, 2002).

The analysis of the DGK mutant rdgA in this study provides strong independent evidence for an excitatory role for DAG. TRP and TRPL channels in rdgA mutants are constitutively active and degeneration is largely prevented in rdgA;trp double mutants. The response to light is also rescued in rdgA;trp, revealing a deactivation defect, suggesting a role for DGK in response termination, at least with respect to TRPL channels. These results would be consistent with a role for DAG in excitation; however, DGK is also the first enzyme in the PIP₂ recycling pathway, so that PIP₂ levels may also be affected in the rdgA mutant. Furthermore, the remaining TRPL channels were still constitutively active in the rdgA;trp double mutant, and light responses could only be compared to controls in pupae during a very narrow developmental time window (Hardie, 2002).

In the present study, rdgA double mutants were generated with both Galphaq¹and norpA, allowing analysis in adult flies with intact TRP and TRPL channel function. Strikingly, bump amplitudes in both norpA and Galphaq¹ are restored to WT levels by rdgA mutations, also showing defects in inactivation. In addition, quantum efficiency (Q.E. -- the percentage of absorbed photons eliciting a bump) is enhanced, resulting in some cases in massive (~100-fold) overall increases in sensitivity. Since the evidence indicates that these rdgA phenotypes are not due to reduced PIP₂ levels, this is interpreted as compelling evidence for the role of DAG as messenger of excitation. DAG levels are dynamically determined by the balance between PLC activity (generating DAG from PIP₂) and DGK activity (converting DAG to PA). It seems that when only one PLC molecule is activated, DAG is metabolized too quickly for threshold levels to be reached, except perhaps in the immediate vicinity of the activated PLC. However, if DGK is inactivated by the rdgA mutation or by depleting ATP, then DAG can reach threshold more readily and also diffuse to activate more distant channels so that quantum efficiency and quantum bump amplitudes approach WT values. (Note that the reduction in Q.E. in Gaq¹ has been attributed to most activated rhodopsins being inactivated before they have a chance to encounter a rare G protein; the results of this study suggest that a major factor in the reduction of Q.E. is that, despite activating a PLC, most single activated G proteins result in insufficient DAG generation to overcome bump threshold). Despite these arguments, a contributory role of PIP₂ to channel regulation cannot be excluded. For example, it is conceivable that channels are bound to PIP₂ in the closed state, that channel activation involves exchange of PIP₂ for DAG, and that channel closure following excitation may involve rebinding of PIP₂ (Hardie, 2002).

Multiple, sequential G protein activation is well established as a mechanism of amplification in vertebrate phototransduction. Although it has been concluded that multiple G protein activation is also required for amplification in Drosophila, the molecular strategies remain qualitatively and quantitatively distinct. In rods, each activated G protein rapidly encounters a PDE, which immediately begins to hydrolyze cGMP. The cGMP concentration is sensed continuously by the light-sensitive channels, which progressively close as upward of 100 G protein and PDE molecules are recruited by random diffusional encounters with rhodopsin. This gives rise to quantum bumps with very short latencies, but which rise gradually over a time course of ~1 s in toad or ~100 ms in mammalian rods. In Drosophila and other microvillar photoreceptors, there is a finite and variable latency (~20–100 ms in Drosophila) followed by an abrupt (~10 ms) rising phase, indicative of a threshold and positive feedback—features not found in vertebrate photoreceptors. At rest in the dark, the channels are closed, and the latency presumably represents the time taken for diffusional encounters of, perhaps, five to ten G proteins with rhodopsin and PLC and then for sufficient second messenger (DAG or its PUFA metabolites) to accumulate to activate the first channel. Because of the restricted volume of the microvillus, Ca²⁺ influx via the first channel raises Ca²⁺ rapidly throughout the microvillus. If other channels in the microvillus are already exposed to subthreshold concentrations of DAG generated by other PLC molecules, it is proposed that the raised Ca²⁺ sensitizes these channels (e.g., by increasing the affinity of the channel for DAG/PUFA), resulting in an explosive positive feedback which activates all or most of the channels in the microvillus over a time course of ~10 ms. Ca²⁺, which reaches concentrations of at least 200 µM, then mediates negative feedback terminating the bump. Finally, there is believed to be a refractory period lasting ~100 ms, while the Ca²⁺ is cleared by diffusion and/or Na⁺/Ca²⁺ exchange and necessary biochemical steps of inactivation (e.g., Rh-arrestin binding, GTPase activity, clearance of DAG, resynthesis of PIP₂) run their course (Hardie, 2002).

In summary, the results indicate that amplification in Drosophila phototransduction is critically dependent upon activation of multiple G proteins and PLC molecules, identify DGK as a key enzyme regulating amplification, and strongly support the identification of DAG as messenger of excitation in Drosophila phototransduction. PLC has long been recognized as the effector enzyme in invertebrate phototransduction, playing an analogous role to PDE in vertebrate rods (although PLC generates the active transmitter, while PDE degrades it). The results now suggest that DGK is the key enzyme controlling the supply of second messenger in Drosophila and thus plays an analogous role to guanylate cyclase. Ca²⁺-dependent regulation of guanylate cyclase is a key mechanism of light adaptation and response termination in vertebrate rods. It will be interesting to see whether similar regulation of DGK is involved in regulating sensitivity and kinetics during light adaptation in Drosophila (Hardie, 2002).

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C

Drosophila locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. The control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, it was observed that a mutation in the no-receptor-potential-A(P41) [norpA(P41)] gene, which encodes phospholipase-C, generates an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpA(P41) is directly involved in thermosensitivity (Collins, 2004).

The proportion of per transcripts that were spliced at 18°C and 29°C, averaged over several LD 12:12 cycles was examined in Canton-S WT and per⁰¹, tim⁰¹, cry^b, and per⁰¹; cry^b mutant backgrounds. In all backgrounds splicing levels fall as the temperature rises, with 40%-60% of transcripts spliced at 18°C and 20%-45% at 29°C. However, not all genotypes react in the same way to temperature changes (Collins, 2004).

The smallest but nevertheless significant effect of temperature on splicing levels is observed in per⁰¹; cry^b, suggesting that the temperature-sensing system for splicing may be compromised in the double mutant. A significant temperature x time effect reveals that the temporal patterns of cycling differ among temperatures, and the absence of any other significant interactions suggests that all genotypes respond similarly. There is very little evidence for a significant day/night cycle in the proportion of per transcripts that are spliced at 18°C, but at 29°C, all genotypes reveal a higher level of splicing post lights off (ZT12) compared to the trough at ZT8. At 18°C, the per⁰¹ and tim⁰¹ mutations have no significant effect on the level of splicing of per mRNA compared to WT. However in cry^b flies, splicing levels are significantly elevated, particularly after lights off. This is also the case when per⁰¹; cry^b is compared to WT. At 29°C, splicing levels are generally 5%-10% higher in per⁰¹, tim⁰¹, and cry^b mutants compared to WT in the light, but 15%-20% higher after lights off at ZT12. This suggests that in the presence of light, splicing levels are reduced due largely to a clock-independent mechanism. In darkness, the clock and Cry become critical for maintaining this low splicing level at high temperatures (Collins, 2004).

The double mutant per⁰¹; cry^b shows a highly significant increase in splicing of ~20% throughout the day/night cycle compared to WT. Thus, at high temperature, either the presence of the circadian photoreceptor Cry or a functional circadian clock is sufficient to largely repress daytime splicing. With both eliminated, daytime splicing levels are elevated. In contrast, repression of splicing in the absence of light requires the circadian clock plus Cry. It seems somewhat counterintuitive that Cry, which is activated by light, plays a more prominent role in repressing splicing at night than it does during the day (Collins, 2004).

Cry is likely to be a dedicated circadian photoreceptor yet at 29°C, splicing is repressed during the light phase even in cry^b. This suggests that the light input to the splicing machinery cannot be primarily mediated by Cry. To confirm that light represses splicing, the effects that short photoperiods and constant darkness (DD) have on splicing levels in WT was investigated. There is a significant effect of reducing photoperiod on the splicing level with an elevated level of splicing in DD compared to LD 12:12, and similarly in LD 6:18, splicing levels are enhanced. Because the repression of splicing by light in LD 12:12 at 29°C does not require the presence of Cry, whether the visual system plays a role in setting the splicing level was investigated by examining the splicing of per transcripts in the mutants gl^60j and norpA^P41 (Collins, 2004).

The proportion of per mRNA transcripts that are spliced at both temperatures is increased in both the norpA^P41 and gl^60j backgrounds compared to WT. At 18°C, ~65% of per transcripts are spliced in norpA^P41 and ~60% in gl^60j, whereas at 29°C, these levels fall to ~55% and ~40%, respectively. Apart from a marginal difference between norpA^P41; cry^b, and norpA^P41 at 18°C, there are no significant effects for either norpA^P41 or gl^60j when combined with cry^b. These results indicate that the visual system rather than Cry is primarily responsible for the light-dependent repression of splicing. Unlike WT, per splicing levels do not rise after lights off at 29°C in either norpA^P41 or gl^60j (Collins, 2004).

Interestingly, in gl^60j, there is a 20% difference between the splicing levels at different temperatures (60%-40%), whereas in norpA^P41, this difference is reduced to 10% (65%-55%). The difference in gl^60j is similar to that seen in WT (45%-25%). Thus per splicing in norpA^P41 is relatively insensitive to temperature changes. It is also clear that the level of splicing in norpA^P41 is significantly higher at all times and temperatures than gl^60j. Therefore, the effect of norpA^P41 on splicing is greater than that of gl^60j_, despite gl^60j being the more severe visual mutant (Collins, 2004).

Locomotor activity profiles of all genotypes were also monitored at 18°C and 29°C. Because each genotype shows a higher level of splicing at 18°C than at 29°C, it would be predicted that this would generate an earlier evening activity peak at 18°C. This is the case for WT, cry^b and norpA^P41, but not for norpA^P41; cry^b or gl^60j, where despite elevated splicing levels at higher temperatures, there is no difference in the phase of activity. gl^60j cry^b does not entrain to LD cycles at 25°C, so was not included in this analysis (Collins, 2004).

The average proportion of per transcripts that are spliced at 18°C rises from WT (45%) to cry^b (50%) to gl^60j (60%) to norpA^P41 (65%), and at 29°C from WT (25%) to gl^60j and cry^b (~35%) to norpA^P41 (55%). If the per splicing level is the only determinant of evening locomotor peak position, then a similar progression in the timing of this peak would be expected. The evening activity peaks of these different genotypes at 18°C and 29°C were compared. For norpA^P41, cry^b, and WT, there is an inverse relationship between average splicing levels and the position of the activity peak at 18°C, with norpA^P41 and cry^b having similarly advanced activity peaks compared to WT. At 29°C, the same inverse relationship holds, with norpA^P41 advanced compared to cry^b, which is in turn earlier than WT. Thus, those genotypes that show temperature-dependent changes in their evening activity generally display a correlation between average per splicing levels and the timing of the evening activity peak of the following day. Conversely, norpA^P41; cry^b and gl^60j,, which show no significant differences in the phase of evening activity at different temperatures, have high splicing levels but relatively delayed evening activity peaks (Collins, 2004).

These observations raise the question of why the splicing level does not always relate to the timing of the evening locomotor activity peak, as in gl^60j and norpA^P41; cry^b. Thus the per RNA profiles of gl^60j and norpA^P41 were compared to WT. Because per does not cycle in cry^b whole head homogenates, the underlying cycle in this background was not examined. WT and norpA^P41 show similar profiles, with an earlier per mRNA peak and higher overall level of per at the lower temperature. In contrast, there is no cycle in gl^60j at either temperature, and levels of per are significantly different from WT and norpA^P41 (Collins, 2004).

Therefore, to entrain locomotor behavior to different seasons, the fly's clock must respond to changes in both light and temperature. This is mediated through a molecular switch, whereby increases in temperature repress the splicing of an intron within the 3' UTR of per, delaying the onset of evening locomotor activity. Light also represses splicing, with higher splicing levels seen in shorter photoperiods, allowing locomotor activity to be fine-tuned to any given set of photoperiodic and temperature conditions. During the first day of DD, the level of splicing rises continuously. This is presumably because at the beginning of DD, the level of splicing is set low from the previous day's light input. Normally the light from the next day maintains this repressed level of splicing, but because this light input is absent, the repression of splicing is lifted, leading to a gradual rise in splicing levels (Collins, 2004).

The most obvious source for light input into the splicing machinery is the circadian photoreceptor Cry. However, analysis of the splicing levels in cry^b shows that, although this mutation has an effect on splicing levels at 18°C, this effect is marginal and is seen only after lights off. This implies (1) that any function of Cry in the repression of splicing is not via the activation of this molecule by light; (2) because Cry is relatively dispensable for circadian locomotor rhythmicity per se, it also suggests that any minor role in splicing at low temperature is unrelated to the functioning of the clock. As the temperature rises, Per, Tim, and Cry all become involved in the regulation of per mRNA splicing. At 29°C, all three mutants show the same splicing phenotype, with ~30% of transcripts spliced during the day, but at night splicing is enhanced to ~45%. Although Per, Tim, and Cry are known to associate in light conditions, Cry and Tim can also associate in darkness, so it is not unexpected that the elimination of any one of the three proteins has a similar effect. Night time is also when the levels of these proteins are at their highest, and therefore any effects would be maximal (Collins, 2004).

At 29°C and in the presence of light, the levels of splicing in per⁰¹; cry^b are elevated above those of either single mutant, which are themselves similar to WT. This suggests that the presence of either Per or Cry is required for light to repress splicing at 29°C. After lights off, the elevated levels of splicing of per are very similar in per⁰¹, tim⁰¹, cry^b, and per⁰¹; cry^b. Therefore Per, Tim, and Cry probably work together to repress splicing in the dark at 29°C. An alternative view for the virtually identical per⁰¹, tim⁰¹, and cry^b splicing levels at 29°C is that this reflects a masking effect of light, so that exogenous LD cycles have a greater effect on splicing at night compared to WT, which shows a modest but significant day-night rhythm. Such stronger masking effects on locomotor behavior have also been observed in cry^b mutants, but any mechanism that might relate or explain these observations remains obscure (Collins, 2004).

The examination of whole head homogenates means that the majority of biological material is derived from the eyes so may not represent exactly what occurs in the pacemaker neurons. The eyes are peripheral clocks, and the cry^b mutation stops the cycling of the clock in whole head homogenates, although cycling continues in the pacemaker cells. One possibility is that the splicing observed in cry^b does not truly reflect the role of Cry in setting splicing levels but is instead a consequence of the clock having stopped in the eyes, thus explaining why per⁰¹, tim⁰¹, and cry^b all show the same splicing phenotype. However, if this splicing phenotype is simply what happens when the clock stops, then per⁰¹; cry^b should show the same splicing phenotype as either single mutant. This is not the case, because the daytime splicing in per⁰¹; cry^b at 29°C is dramatically elevated compared to either single mutant. Thus the splicing phenotypes of per⁰¹, tim⁰¹, and cry^b cannot simply be a result of the clock having stopped. This means that it is the presence of these proteins, rather than their clock-dependent cycling, that is important to the regulation of per splicing levels (Collins, 2004).

In gl^60j, there is no per mRNA cycle in whole head homogenates. This means that in the majority of cells in the gl^60j head, the clock has either stopped or cells have become desynchronized. If the former is true, then splicing levels of gl^60j should resemble those of per⁰¹ or tim⁰¹, and this is clearly not the case. If the latter is true, this could prevent the observation of any splicing rhythm, but the level of splicing observed should still represent the average level of splicing in this mutant background, which is clearly significantly different from WT. In any case, splicing levels observed in all visual mutants are likely to represent the effect of removing visual photoreception, because these elevated levels are similar to those observed in WT in DD (Collins, 2004).

norpA^P41 and gl^60j have considerably higher splicing levels than WT and cry^b mutants at both temperatures, indicating that information received via the visual system rather than Cry drives this repression of splicing, which is borne out by analysis of gl^60j cry^b and norpA^P41; cry^b double mutants. The splicing levels of gl^60j and gl^60j cry^b are similar at both temperatures, which is also true of norpA and norpA^P41; cry^b at 29°C. At 18°C, there is slightly more spliced per RNA in norpA^P41; cry^b than in the norpA^P41 single mutant, reflecting the earlier result where cry^b showed a marginal enhancement of splicing at cooler temperatures. These results also demonstrate that unspecific genetic background effects are not responsible for this marginal effect of cry^b, because the double mutant background should make any interacting loci heterozygous. This lack of significant background effects in determining overall splicing levels has been confirmed by examining several natural European D. melanogaster lines. All mutants studied here show the same significantly enhanced splicing patterns when compared to any of the wild-caught isolates (Collins, 2004).

Unlike the clock and cry^b mutants, there is no day-night difference in splicing levels at 29°C in either gl^60j or norpA^P41. One possibility is that visual system structures are required for the repression of splicing even in the dark, hence the overall elevated splicing levels in norpA^P41 and gl^60j at all times. This would be surprising, because such a role would obviously have to be light independent. More likely, the light input received through the eyes sets the splicing level during the day, and the clock maintains this repression at night. Thus, if the visual input is removed or reduced, as in DD, gl^60j, or norpA^P41 mutants, or in shorter photoperiods, then the subsequent splicing level is set higher. The difference in roles between cry and the visual system on per splicing levels may also partly explain recent observations that cry^b mutants are able to adapt the timing of locomotor activity to long and short photoperiods, whereas flies with defective visual photoreception, including gl^60j, are not (Collins, 2004).

Interestingly, although gl^60j is the more severe visual mutant, norpA^P41 has significantly higher per splicing levels than gl^60j at both 18°C and 29°C. Additionally, whereas the difference between splicing levels at 18°C and 29°C is maintained in gl mutants (~65% and ~45% of transcripts spliced vs. ~45% and 25% in WT at 18°C and 29°C, respectively), this is greatly reduced in norpA^P41 (65% and 55%). One possible explanation for this is that norpA may be a signaling molecule in the temperature-sensing pathway for the clock. The patterns of locomotor activity support a role for norpA in temperature sensing, with the norpA^P41 fly's locomotor patterns seemingly more sensitive to high temperatures than WT. Additionally, norpA^P41 evening locomotor activity peaks early at both 18°C and 29°C, and per mRNA splicing shows a corresponding elevation compared to WT. These are responses associated with low temperatures in WT D. melanogaster, and therefore norpA^P41 mutants behave as if they have an impaired ability to detect high temperatures. norpA^P41 flies still detect temperature changes (witness the altered evening peaks and splicing levels); they just react as if the temperature is colder than it actually is (Collins, 2004).

Thus, the enhanced per splicing seen in norpA^P41 may reflect a direct link between norpA-encoded PLC signaling and the temperature sensitivity of the splicing mechanism, independent of norpA visual function. In the phototransduction cascade, rhodopsin activates a G-protein isoform that in turn activates the PLC encoded by norpA. As a result of this activation, Ca²⁺ permeable light-sensitive channels are opened, including members of the transient receptor potential (TRP) class. Recently it has been demonstrated that dANKTM1, a D. melanogaster TRP channel, is activated by temperatures from 24°C to 29°C. In addition, D. melanogaster painless mutant larvae have a disrupted TRP channel and display defective responses to thermal stimuli. Because several TRP family members act as thermal sensors in mammals, TRP channels appear to have an ancient heat-sensing function that is retained in both vertebrates and invertebrates. Given that this study has identified a heat-sensing role for norpA, and norpA is known to activate TRP channels in photoreception, it is not unreasonable to suppose that norpA plays a general role in responses to temperature stimuli (Collins, 2004).

per splicing levels may also impact on aspects of behavior other than the timing of evening locomotor activity. For instance, the free-running period of norpA^P41 is ~1 h shorter than WT. The splicing levels of per mRNA are greatly elevated in this background, and elevated splicing is predicted to advance the Per protein cycle and thus speed up the clock. In fact, the splicing mechanism should have the effect of speeding up the clock at colder temperatures and slowing it down at high temperatures, thereby providing a potential basis for temperature compensation (Collins, 2004).

The position of the evening activity peak at different temperatures moves in different mutant backgrounds. For WT, norpA^P41, and cry^b, the level of splicing appears to correlate with the position of the evening activity peak at different temperatures. At 18°C, there is a small but significantly greater relative amount of spliced per RNA in cry^b than in WT, resulting in the earlier evening activity peak seen in cry^b flies. This difference in per splicing is greatest after lights off at both temperatures. This is when Per levels will be rising, because Tim is present for Per stabilization, so enhancement of Per accumulation by elevated per splicing is likely to have its most noticeable effect around dusk or early evening. A similarly consistent situation is seen in norpA^P41: there is more spliced per mRNA present at 18°C (65%) than 29°C (55%), accounting for the earlier peak of evening activity at 18°C. Additionally these levels are higher than those seen in either WT (45% and 25% per transcripts spliced at each temperature) or cry^b (55% and 40%) and relates to the earlier phases of locomotor activity seen in norpA^P41 compared to the other genotypes. However, at 18°C there is more spliced per in norpA^P41 than in cry^b, but the evening activity peak occurs at the same time. The simplest explanation is that there is a limit to how early the evening activity peak can occur, no matter what the per splicing level, because splicing alters the accumulation of Per protein; this is limited by the light-dependent degradation of Tim. Therefore, in general, the level of splicing determines when the peak level of locomotor activity will occur (Collins, 2004).

The level of splicing of the per intron cannot be the only determinant of evening peak position, because the relationship between the per splicing level and evening activity peak position breaks down in norpA^P41; cry^b and gl^60j, where there are different levels of splicing at the two temperatures but no corresponding difference in the evening peak position. When the underlying per mRNA cycles of gl^60j, norpA^P41, and WT flies were analyzed at 18°C and 29°C, it was found that whereas per levels cycle in norpA^p41 and WT, this cycle is lost in gl^60j. If there is no underlying per RNA cycle, then there is no mRNA peak to be advanced or delayed by splicing (Collins, 2004).

At the cellular level, although gl is not a clock component, when mutated, it eliminates a number of clock-expressing cells within the head, including the eyes, ocelli, Hofbauer-Buchner (H-B) eyelet, and the dorsal neuron 1 (DN1) cells. Despite this, the primary effect on the clock is to remove most of the visual entrainment pathway, but the clock in the key pacemaker cells of gl^60j mutants must still be functional, because behavior still entrains to LD cycles and remains rhythmic in DD. It is significant that the crosstalk between different classes of clock cells is essential for the generation of robust behavioral rhythms. Thus loss of the overall per mRNA rhythm may be a consequence of disrupting this network in gl^60j, and, while leaving the basic system intact, this affects the more subtle temperature-sensitive aspects of entrainment. A similar argument based on an interruption of the entrainment network can also be proposed to explain the corresponding results with norpA^P41; cry^b double mutants, because in this case per mRNA is assumed to be noncycling because of the cry^b background. However, the locomotor behavior of cry^b single mutants remains thermosensitive even though overall per mRNA is noncycling. Thus, only when the photoreceptive pathway and mRNA cycle are both compromised (as in gl and norpA^P41; cry^b) is locomotor behavior insensitive to temperature-dependent changes in per splicing levels (Collins, 2004).

A model is presented of how light and temperature may set the splicing level of the clock. How temperature is detected by the splicing machinery is not yet clear, but there is compelling evidence that norpA plays a role. At low temperatures, the splicing level is primarily set by light via the visual system rather than Cry, which is then remembered during the night. In longer periods of darkness such as in DD, this memory decays, and splicing levels begin to rise. Thus the visual system represses splicing by enhancing the effects of an unknown repressor molecule(s) that is sensitive to temperature change and the norpA PLC. At high temperatures, the regulation of splicing is more stringent and complex and recruits the circadian clock. Again, the light input received through the visual system sets the low splicing level during the day. This appears to also depend on the presence of at least two of the three molecules, Per, Tim, or Cry, because elimination of any one of these gives a barely detectable daytime rise in splicing, reflecting the very low levels of Per, Tim, and Cry at this time. However, elimination of both Per and Cry in the per⁰¹; cry^b double mutant lifts all light-dependent repression during the day (Collins, 2004).

At night, the level of splicing set during the day by the visual system is again remembered and maintained by the clock at night. If per, tim, or cry is eliminated, then this repression of splicing is lost at night, generating the day/night difference in splicing levels. In gl^60j cry^b or norpA^P41; cry^b, because there is no visual light input during the day, there is no splicing level for the clock to remember, and therefore there is no day/night difference in splicing levels. Thus at high temperature, the visual system activates the repressor molecule during the day, and the clock maintains this activation at night. It is assumed that recruiting the clock at high temperature to inhibit per splicing is required to ensure that the fly's locomotor/foraging behavior is adaptive and does not encroach on those times of the day when there would be a significant risk of desiccation (Collins, 2004).

Temperature synchronization of the Drosophila circadian clock

Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. This study shows that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins Period and Timeless in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. Expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. The phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. The further genetic dissection of temperature-entrainment was initiated, and the novel Drosophila mutation nocte was isolated. nocte mutants are defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. It is concluded that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. These data suggest that temperature synchronization involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte (Glaser, 2005).

Although it has been known for a long time that temperature can serve as a potent Zeitgeber to entrain circadian rhythms in animals, practically nothing was known about thermal-entrainment mechanisms and, thus, about the genes and molecules involved. This study has revealed that temperature entrainment of clock-protein expression can function at the level of isolated tissues, independent of the antennal thermosensors studied with regard to Drosophila's acute thermal responsiveness described earlier. The situation is therefore similar to that of circadian photoreception in flies: Clock-gene-expressing tissues can be synchronized in the absence of external, image-forming photoreceptors, and this synchronization is probably mediated by the blue-light photoreceptor Cryptochrome. Similarly, these findings suggest the existence of a cell-autonomous thermoreceptor dedicated to temperature entrainment of the circadian clock. Among the candidates for such a receptor are the transient receptor potential vanilloid (TRPV) channels that have been shown to function in thermoreception in mammals and fly larvae (Glaser, 2005).

The nocte mutant was isolated in a novel screen for temperature-entrainment variants; nocte specifically affects synchronization of the circadian clock to this Zeitgeber (namely, temperature cycles). Such mutant flies are drastically impaired in molecular entrainment of Per-LUC reporter-gene rhythms as well as those of native Per and Tim expression. Behavioral rhythms can be entrained by light:dark, but not by temperature cycles, in nocte flies. Moreover, nocte flies do not affect circadian clock function as such because mutant flies are robustly rhythmic in constant darkness. The circadian clock of nocte flies is also properly temperature compensated; their free running period does not change as a function of increasing or decreasing constant ambient temperatures. These findings show that the assumed product of this gene plays a central role in, and is specific for, temperature entrainment (Glaser, 2005).

It has been shown that the norpA gene is involved in the light-entrainment pathway that ends at the brain's clock. The phospholipase C (PLC) encoded by this gene is an essential factor of the canonical photo-transduction cascade within Drosophila's external photoreceptors. Loss-of-function norpA mutations (such as norpA^P24 and norpA^P41, as applied in this study) disrupt this pathway and cause visual blindness -- but they also blind the eyes' contribution to circadian entrainment by light (Glaser, 2005).

In addition, norpA contributes to a temperature-sensitive splicing event at the 3' end of the per gene. Splicing of a per intron is enhanced by relatively cold temperatures, and this enhancement leads to an earlier increase of per mRNA during the daily cycle of per RNA accumulation and decline (Majercak, 1999; Majercak, 2004). Correlated with this early upswing is an advanced behavioral activity peak in cold conditions. Because both phenomena are enhanced by shortened photoperiods and suppressed by long photoperiods, it was suggested that the 3' alternative splicing event serves as a mechanism to adjust the fly's behavior to seasonal changes: More locomotion during the day in the winter, with its short photoperiods, and more behavior in the evening during the summer, to avoid the desiccation effects of midday heat (Glaser, 2005).

But are norpA and the 3'splicing mechanism also important for the more basic features of day-by-day temperature entrainment? The answer, from this analysis, is yes and no: PLC is clearly involved because norpA mutations affect both molecular and behavioral entrainment to temperature cycles. This is not true for the alternative splicing event at per's 3' end: Two types of controls (wild-type and y w) robustly synchronized their behavioral rhythms to temperature cycles in constant light and constitutively expressed roughly equal amounts of the spliced and unspliced versions of per RNA in the same LL and temperature-cycling conditions. Therefore, temporal regulation of the 3' splicing event is not necessary for entrainment to temperature cycles (Glaser, 2005).

The idea is favored that the PLC encoded by norpA has an additional role to its known functions in photo transduction and thermal regulation of splicing. It is possible that a signal-transduction cascade, similar to the visual one operating in the compound eye, is used to transduce the temperature signal to the clock. It is not known whether all clock-gene-expressing tissues also express norpA (although it is notable that norpA is expressed in adult tissues way beyond the external eyes. If not, this could explain why the temperature-entrainment defects in norpA mutants in behavior seem less severe than those of nocte. norpA's function in certain tissues could be replaced by other PLC enzymes encoded by different genes. In this respect, one of the more salient norpA mutant defects that was uncovered, that such flies cannot entrain Per-LUC rhythms to temperature cycles, suggests that the temperature-entrainment pathway involves a receptor-coupled signal-transduction cascade that includes a crucial function for PLC (Glaser, 2005).

Robust temperature-entrained reporter-gene rhythms were observed only in transgenic situations in which two-thirds or the entirety of the Per protein was fused to luciferase. These results suggest that the temperature-entrainment mechanism targets clock proteins (at least Per) and does not rely on transcriptional mechanisms. Interestingly, in Neurospora, temperature entrainment is also mainly regulated at the protein level. This supposition is also supported by the surprisingly robust Per and Tim cyclings observed in fly heads under constant light and temperature cycling conditions. These protein oscillations were severely damped in the temperature-entrainment-defective nocte mutant (Glaser, 2005).

Previous work showed that heat pulses of 37°C can induce stable molecular and behavioral phase shifts when applied during the early night but not during the late night. These heat pulses function at the posttranscriptional level because they result in rapid disappearance of Per and Tim. Nevertheless, responsiveness of the clock to heat pulses seems to be mediated by a different mechanism, when compared with daily entrainment analyzed in the current study, because heat pulses involving elevated temperatures below 37°C (i.e., 31°C and 28°C) did not lead to significant clock-protein degradation, whereas temperature cycles applied in the current study (which were in a physiological range, 17°C to 25°C) did cause fluctuations in protein concentrations (Glaser, 2005).

A surprising finding in this study was that temperature-entrained molecular oscillations of Per-LUC and of endogenous Per and Tim proteins are robust in constant light. It was reported earlier that upon transfer to constant light and temperature, Tim protein is expressed at constitutively low levels -- probably by CRY-mediated light absorption followed by Tim:CRY interaction. Per protein continues to oscillate for about 2.5 days after transfer to LL, after which its expression levels also becomes low and constitutive. In the current experiments, Per and Tim still oscillated after 4 days in LL when temperatures were cycling, and Per-LUC luminescence oscillations continued with robust amplitude for more than 5 days. These results clearly show that temperature cycles override the arrhythmia-inducing effects of constant light and explain why circadian entrainment of behavioral and physiological rhythmicity is observed under these conditions (Glaser, 2005).

Future work will illuminate which molecules mediate temperature entrainment in addition to phospholipase C. Given the drastic and specific effects of the nocte mutation on temperature entrainment, the factor encoded by this gene will almost certainly be revealed to play a central role in this process (Glaser, 2005).

This study has shown that temperature cycles induce molecular rhythms of clock-gene expression, even in the presence of constant light, which normally results in complete molecular and behavioral arrhythmia. Synchronization was observed in isolated peripheral clock tissues and in the brain, demonstrating that the process is tissue autonomous and is responsible for the synchronized locomotor behavior under constant-light and temperature-cycling conditions. The data suggest that the mechanism functions at a posttranscriptional level involving at least the clock protein Per. Phospholipase C is likely to be involved in the signaling mechanism from the thermal receptor to the clock. The novel mutation nocte specifies a factor that is specific for thermal synchronization of the circadian clock in flies, demonstrating that this input pathway can be genetically dissected, as has been similarly shown for the light input into the circadian clock (Glaser, 2005).

DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses (Leung, 2008).

Visual transduction in Drosophila utilizes a G protein-coupled, phospholipase C-mediated signaling cascade. Phospholipase C, upon activation via rhodopsin and G protein, Gq, catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) into two potential second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP₃). A body of evidence suggests that IP₃ is not involved in Drosophila phototransduction, leaving the DAG branch as a likely source of messenger(s) of activation for the phototransduction channels, transient receptor potential (TRP) and TRP-like (TRPL). The mechanism by which the diacylglycerol (DAG) branch might activate the TRP/TRPL channels is still unresolved. The first indication that a lipid messenger might be involved was provided by Chyb (1999), who showed that polyunsaturated fatty acids (PUFAs) could activate both TRP and TRPL channels either in intact photoreceptors or heterologous expression systems. Later, evidence was provided that DAG is required for photoreceptor excitation using DAG kinase mutants, rdgA. Because the conversion of DAG to phosphatidic acid is blocked in these mutants, they should have an elevated DAG basal level. TRP/TRPL channels are constitutively active in rdgA, and diminished responses of hypomorphic PLC (norpA) mutants can be greatly enhanced by rdgA mutations, in support of the contention that DAG might be excitatory to the channels. However, rdgA mutations are expected to raise the basal levels of not only DAG but also its metabolites. In addition to these two molecules, phosphatidylinositol 4,5-bisphosphate (PIP₂) has also been suggested to play a role in channel excitation. Currently, no consensus exists as to which, if any, of these might be the excitatory agent for TRP/TRPL channels (Leung, 2008).

Drosophila TRP is the founding member of a superfamily of TRP channel proteins. There are now nearly 30 mammalian members of this superfamily comprising seven subfamilies. Although these channels are heterogeneous in their modes of activation, at least four mammalian TRP channels have been reported to be activated by DAG: TRPC2, -3, -6, and -7. While there may be variations in the mechanisms of activation of these channels, elucidation of Drosophila TRP/TRPL channel activation could provide insight into activation of these channels as well (Leung, 2008).

Because both DAG and its potential metabolite, PUFA, have been implicated in the activation of TRP/TRPL channels, a key enzyme in this process is likely to be DAG lipase, which catalyzes the hydrolysis of DAG. Little is known about DAG lipases. Two mammalian DAG lipase genes, DAGLα and -β, have been identified by a bioinformatics approach and characterized both biochemically and molecularly), and many proteins homologous to DAGα and -β have been identified across species. In the case of Drosophila, , rolling blackout (rbo) has been suggested to be in a DAG lipase gene. The protein encoded by the rbo gene, however, shows little homology to the known mammalian DAG lipases. Moreover, conditional loss of the RBO protein leads to rapid depletion of DAG, the opposite of what one would expect if RBO catalyzes the hydrolysis of DAG. Furthermore, in the absence of previous activity, the receptor potential is normal in rbo mutants, making it unlikely that RBO has any direct involvement in the activation of TRP/TRPL channels. Other than rbo, no candidate DAG lipase that might function in phototransduction has been reported in any species (Leung, 2008).

This work reports on a Drosophila DAG lipase (DAGL) gene, inaE, identified from mutants that are defective in photoreceptor responses to light. The protein isoforms encoded by this gene show high sequence similarity to the two known mammalian DAGLs, exhibit DAGL activity in vitro, are highly expressed in photoreceptors, and have access to rhabdomeres. Genetic evidence suggests that the inaE-encoded DAGLs interact in vivo with the DAG generated in the phototransduction cascade. Analysis of mutants generated by imprecise excision of P element insertion in inaE show that no physiologically meaningful photoreceptor responses can be generated if inaE gene is severely impaired (Leung, 2008).

The inaE gene was identified by two ethylmethane sulfonate (EMS)-induced allelic mutants: N125 and P19. These mutants are characterized by their 'ina' (inactivation, no afterpotential) electroretinogram (ERG) phenotype. Wild-type flies, when placed on a white-eye (w) background, respond to a bright blue stimulus with a large response during light stimulus followed by a prolonged depolarizing afterpotential (PDA) after the light is turned off. A second blue stimulus elicits only a small response, originating from R7/8 photoreceptors, superposed on the PDA. By contrast, in ina mutants, the response begins to decay during stimulus (inactivation), and the decay continues after the stimulus. As a result, the PDA is greatly diminished in amplitude (no afterpotential). This phenotype can also be viewed as a mild form of the 'trp' phenotype displayed by strong mutants of the TRP channel gene. In trp mutants, the response to the first blue stimulus decays nearly to baseline during stimulus, and there is no PDA (Leung, 2008).

Moreover, responses of inaE mutants resemble those of trp in that they both display refractory properties. Following a response to the first stimulus, only very small responses can be elicited from trp until they recover over a period of minutes, while wild-type responses recover almost immediately. Likewise, inaE mutants exhibit a similar refractory period, although the degree and duration of response suppression are not as pronounced or prolonged as in trp (Leung, 2008).

In addition to the above similarities, inaE^N125 acts as a genetic enhancer of Trp^P365. Trp^P365 is a semidominant allele of trp, which causes constitutive activation of TRP channels and, as a result, massive photoreceptor degeneration from excessive Ca²⁺ influx. In Trp^P365 homozygotes, degeneration is already so advanced in 1- to 2-day-old flies that essentially no ERGs can be elicited. Trp^P365 heterozygotes exhibit a much milder phenotype and elicit ERGs of substantial amplitude at the same age. However, if inaE^N125 is introduced into the Trp^P365/+ background, the resulting phenotype is as severe as that of Trp^P365 homozygotes. The genetic enhancement of Trp^P365/+ by inaE^N125 and the basic similarity of ERG phenotypes between inaE and trp mutants led to the hypothesis that the protein products of these two genes may interact and/or subserve closely related functions (Leung, 2008).

The CG33174 gene had not been characterized previously, and its function was electronically inferred as 'triacylglycerol lipase (TAGL) activity'. However, the possibility is considered that the above annotation could simply reflect a dearth of information on DAGLs. The first two human DAGLs, DAGLα and -β, were cloned and characterized by a bioinformatic approach and shown to be sn-1 type DAGLs. Multiple alignment of INAE-A and INAE-D with DAGLα and -β revealed extensive sequence and domain conservations. All four proteins are predicted to have four transmembrane segments near the N-terminal region, and they all have a lipase_3 domain with a highly conserved serine active site. Overall sequence homology between INAE-D and the two human DAGLs is 39% identity and 56% similarity for DAGLα and 30% identity and 50% similarity for DAGLβ, respectively. In the lipase_3 domain, the sequence homology between INAE-A/D and the mammalian proteins rises to 60% and 45% identity and 73% and 63% similarity for DAGLα and DAGLβ, respectively (Leung, 2008).

To demonstrate that the INAE proteins have DAG lipase activity, the INAE-A and -D protein isoforms were expressed in E. coli and purified to >95% purity to carry out DAG lipase assays using 1-stearoyl-2-arachidonoyl-sn-glycerol as substrate, and the lipase assay products were analyzed by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) for identification of hydrolysis products and kinetic studies (Leung, 2008).

LC-MS detected four products from the analysis of both INAE isoforms: two primary products, stearic acid (18:0) and 2-arachidonoyl glycerol (2-AG), and two minor products, arachidonic acid (20:4) and 1-stearoyl glycerol (1-SG), eluting at 6.3, 9.3, 5.8, and 8.7 min, respectively. Two primary products corresponded to hydrolysis at the sn-1 position of DAG substrate, and the two minor products corresponded to hydrolysis at the sn-2 position. Thus, in vitro, the two recombinant INAE isoforms are both DAG lipases highly preferential for hydrolysis at the sn-1 position, with the D form having much higher activity than the A form (Leung, 2008).

A growing body of evidence suggests that Drosophila phototransduction utilizes the DAG branch of the G protein-coupled, PLC_β-mediated signaling pathway). Although DAG lipase is expected to play a critical role in this pathway, no DAG lipase that could play such a role had been identified previously in Drosophila. This study reports on a DAG lipase identified from the Drosophila mutants, inaE. The inaE gene was found to encode two protein isoforms, INAE-A and INAE-D, by alternative splicing. Both of these proteins are highly homologous to the two previously identified mammalian sn-1 type DAG lipases, and in vitro DAG lipase assays of recombinant INAE-A and INAE-D showed that both are DAG lipases highly preferential for hydrolysis at the sn-1 position. Expression of the INAE protein is not restricted to the eye but occurs throughout the head, consistent with the finding that strong mutations in this gene are homozygous lethal. In photoreceptors, anti-INAE antibody labeling occurs as punctate staining scattered throughout the photoreceptor cytoplasm. Occasionally, some of the puncta are found within the rhabdomeres, indicating that some DAGL enters the rhabdomeres. Results of the norpA inaE double mutant study provide strong functional support for the above observation. In this study, the receptor potential disappears in an inaE allele-dependent manner -- the stronger the inaE allele in the double mutant, the more severe the double mutant phenotype. The allele dependence strongly suggests that the action of inaE-encoded DAGL is responsible for the observed double mutant phenotype. Furthermore, to affect the receptor potential phenotype, DAGL must act on the DAG generated by norpA-encoded PLC_β, and, for that to occur, DAGL must enter the rhabdomeres (Leung, 2008).

Because inaE mutations already available were all relatively mild, severe mutations were generated by imprecise excisions of a P element insertion in the inaE gene. These imprecise excision alleles were homozygous lethal and had to be studied as eye mosaics. Quantitative RT-PCR results showed that even the severest of these imprecise excision mutants, inaE^xl18, is not a null mutant and expresses RNA at ~25% of the normal level. This mutation profoundly affects the photoreceptor responses to light. If xl18 is placed on a norpA^H43 background to reduce the amount of DAG generated, the light stimulus generates no response at all. In xl18 flies themselves, a bright prolonged stimulus generates only a small response of slow kinetics that decays to baseline completely during the stimulus. This response most likely represents the residual DAGL activity in this severely affected mutant. As the severity of mutation progressively decreases in the xl series of mutants, the receptor potential phenotype returns to normal in an allele-dependent manner. Again, the inaE allele dependence strongly argues that the action of inaE-encoded DAGL is responsible for the observed change in the receptor potential phenotype. These results, taken together, suggest (1) that the production of DAG metabolite(s) through the action of the inaE-encoded DAGL is required for the generation of photoreceptor responses to light and (2) that, in the absence of the metabolite, DAG plays little direct role in the activation of channels. However, the identity of the excitatory molecule cannot be specified from this work. It could be one or more of the products generated by INAE, such as monoacylglycerol (2-AG) or stearic acid or even DAGL (INAE) itself (Leung, 2008).

While DAG may not have a direct role in channel activation, evidence was found suggesting that it may be important in regulating the action of the DAG metabolite that acts as an excitatory agent, although the evidence is still largely indirect. The ability of inaE^N125 to act as an enhancer of Trp^P365/+ seems to present a quandary when considered in relation to the results summarized above. If DAG has little or no direct role in channel activation, how does one explain the disappearance of the small response present in P365/+ when N125 is added to this background? A simple explanation for the phenomenon would be that DAG is excitatory to the channels and that adding N125 to the P365/+ background raised the level of DAG to make more channels to become constitutively active in N125;P365/+ than in P365/+. However, results of the experiment replacing N125 with a stronger inaE allele, xl18, in the N125;P365/+ double mutant run counter to this simple explanation. Replacing N125 with xl18 should have sharply raised the basal DAG level further in the double mutant. If DAG were excitatory, the resting potential should have depolarized even more than before the N125 replacement, and no receptor potential at all should have been obtained. Just the opposite results were obtained. A small but distinct receptor potential could be recorded from xl18;P365/+, and the resting potential has returned to the level in P365/+. These results are incompatible with the hypothesis that DAG is excitatory to the channel and instead provide another line of evidence for the conclusions summarized earlier (Leung, 2008).

However, the fact that a much more severe phenotype is obtained in N125;P365/+ than in P365/+ or xl18;P365/+ suggests that DAG may have a role in facilitating, enhancing, and orchestrating the action of the DAG metabolite that serves as the excitatory agent. This action of DAG would be more noticeable under conditions in which a sufficient amount of the excitatory product is produced, as in N125;P365/+ rather than in xl18;P365/+. A similar action of DAG can also be inferred from the norpA inaE double mutant studies. In this series of experiments, hypomorphic norpA mutation, H43, was used to restrict the amount of DAG generated both in the single and double mutants. The response obtained from H43 N125 is short in duration but has nearly the same maximum amplitude as the H43 response and much faster time course of rise than the H43 response. The shortness of response duration may be due to the fact that, under the conditions of this experiment (restricted DAG generation), the response cannot be sustained during a bright prolonged stimulus. This response arises as a result of DAGL activity because further reducing the DAGL activity (xl18 mutation) abolishes the response. However, the fact that adding N125 to the H43 background resulted in a response of faster time course may also be a manifestation of the enhancing and facilitatory effects of DAG on the excitatory agent. Speculating further, the facilitatory action of DAG might be in place to ensure that the excitation of channels is light regulated, because DAGL activity is not light regulated while the generation of DAG is (Leung, 2008).

REFERENCES

Search PubMed for articles about Drosophila no receptor potential A

Adamski, F. M., et al. (1998). Interaction of eye protein kinase C and INAD in Drosophila. Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies. J. Biol. Chem. 273(28): 17713-9. PubMed Citation: 9651370

Alloway, P. G., Howard, L. and Dolph, P. J. (2000). The formation of stable rhodopsin-arrestin complexes induces apoptosis and photoreceptor cell degeneration. Neuron 28: 129-138. 11086989

Alvarez, R. A., et al. (1995). cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C beta 4 (PLCB4). Genomics 29(1): 53-61. PubMed Citation: 8530101

Alvarez, C. E., Robison, K. and Gilbert, W. (1996). Novel Gq alpha isoform is a candidate transducer of rhodopsin signaling in a Drosophila testes-autonomous pacemaker. Proc. Natl. Acad. Sci. 93(22): 12278-82. PubMed Citation: 8901571

Asteriti, S., Liu, C. H. and Hardie, R. C. (2017). Calcium signalling in Drosophila photoreceptors measured with GCaMP6f. Cell Calcium 65: 40-51. PubMed ID: 28238353

Bloomquist, B. T., et al. (1988). Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell 54(5): 723-33. 88311074

Brusich, D. J., Spring, A. M. and Frank, C. A. (2015). A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity. Front Cell Neurosci 9: 107. PubMed ID: 25859184

Brusich, D. J., Spring, A. M., James, T. D., Yeates, C. J., Helms, T. H. and Frank, C. A. (2018). Drosophila CaV2 channels harboring human migraine mutations cause synapse hyperexcitability that can be suppressed by inhibition of a Ca2+ store release pathway. PLoS Genet 14(8): e1007577. PubMed ID: 30080864

Chen, X. and Dickman, D. (2017). Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations. PLoS Genet 13(12): e1007117. PubMed ID: 29194454

Chyb, S., Raghu, P. and Hardie, R. C. (1999). Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397(6716): 255-9. PubMed Citation: 9930700

Collins, B. H., Rosato, E. and Kyriacou, C. P. (2004). Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C. Proc. Natl. Acad. Sci. 101: 1945-1950. 14766972

Cook, B., BarYaacov, M., BenAmi, H. C., Goldstein, R. E., Paroush, Z., Selinger, Z. and Minke, B. (2000). Phospholipase C and termination of G-protein-mediated signalling in vivo. Nature Cell Biol. 2: 296-301. 10806481

Dasgupta, U., et al. (2009). Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction. Proc. Natl. Acad. Sci. 106(47): 20063-8. PubMed Citation: 19892737

Delvendahl, I. and Muller, M. (2019). Homeostatic plasticity-a presynaptic perspective. Curr Opin Neurobiol 54: 155-162. PubMed ID: 30384022

Doh, S., et al. (1997). Promoter region of the Drosophila melanogaster norpA gene. Mol. Cells 7(6): 795-9. PubMed Citation: 9509423

Ferreira, P. A., Shortridge, R. D. and Pak, W. L. (1993). Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila. Proc. Natl. Acad. Sci. 90(13): 6042-6. PubMed Citation: 8327481

Ferreira, P. A. and Pak, W. L. (1994). Bovine phospholipase C highly homologous to the NorpA protein of Drosophila is expressed specifically in cones. J. Biol. Chem. 269(5): 3129-31. PubMed Citation: 8106345

Glaser, F. T. and Stanewsky, R. (2005). Temperature synchronization of the Drosophila circadian clock. Curr. Biol. 15: 1352-1363. PubMed Citation: 16085487

Goel, P., Li, X. and Dickman, D. (2017). Disparate postsynaptic induction mechanisms ultimately converge to drive the retrograde enhancement of presynaptic efficacy. Cell Rep 21(9): 2339-2347. PubMed ID: 29186673

Gu, G. G. and Singh, S. (1997). Modulation of the dihydropyridine-sensitive calcium channels in Drosophila by a phospholipase C-mediated pathway. J. Neurobiol. 33(3): 265-75. PubMed Citation: 9298764

Ha, E. M., et al. (2005a). A direct role for dual oxidase in Drosophila gut immunity. Science 310: 847-850. PubMed Citation: 16272120

Ha, E. M., et al (2005b). An antioxidant system required for host protection against gut infection in Drosophila. Dev. Cell 8: 125-132. PubMed Citation: 15621536

Ha, E. M., et al. (2009). Regulation of DUOX by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity. Dev. Cell 16(3): 386-97. PubMed Citation: 19289084

Hardie, R. C. and Raghu, P. (1998). Activation of heterologously expressed Drosophila TRPL channels: Ca2+ is not required and InsP3 is not sufficient. Cell Calcium 24(3): 153-63. PubMed Citation: 9883270

Hardie, R. C., et al. (2002). Molecular basis of amplification in Drosophila phototransduction: Roles for G Protein, Phospholipase C, and Diacylglycerol kinase. Neuron 36: 689-701. 12441057

Huber, A., Sander, P. and Paulsen, R. (1996a). Phosphorylation of the InaD gene product, a photoreceptor membrane protein required for recovery of visual excitation. J. Biol. Chem. 271(20): 11710-7

Huber, A., et al. (1996b). The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD. EMBO J. 15(24): 7036-45

Huber, A., et al. (1998). The TRP Ca2+ channel assembled in a signaling complex by the PDZ domain protein INAD is phosphorylated through the interaction with protein kinase C (ePKC). FEBS Lett. 425(2): 317-22

James, T. D., Zwiefelhofer, D. J. and Frank, C. A. (2019). Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling. Elife 8: e39643. PubMed ID: 31180325

Jiang, H., Wu, D. and Simon, M. I. (1994). Activation of phospholipase C beta 4 by heterotrimeric GTP-binding proteins. J. Biol. Chem. 269(10): 7593-6

Jiang, H., et al. (1996). Phospholipase C beta 4 is involved in modulating the visual response in mice. Proc. Natl. Acad. Sci. 93(25): 14598-601

Kile, B. T., et al. (2002). The SOCS box: a tale of destruction and degradation. Trends Biochem Sci. 27: 235-241. PubMed Citation: 12076535

Kim, D., et al. (1997). Phospholipase C isozymes selectively couple to specific neurotransmitter receptors. Nature 389(6648): 290-3

Kim, M. J., et al. (1993). Cloning of cDNA encoding rat phospholipase C-beta 4, a new member of the phospholipase C. Biochem. Biophys. Res. Commun. 194(2): 706-12

Kim, M. J., et al. (1998). A cytosolic, galphaq- and betagamma-insensitive splice variant of phospholipase C-beta4. J. Biol. Chem. 273(6): 3618-24

Kim, S., et al. (1995). Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster. J. Biol. Chem. 270(24): 14376-82

Kohn, E., Katz, B., Yasin, B., Peters, M., Rhodes, E., Zaguri, R., Weiss, S. and Minke, B. (2015) Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo. J Neurosci 35: 2530-2546. PubMed ID: 25673847

Koyanagi, M., et al. (1998). Phospholipase C cDNAs from sponge and hydra: antiquity of genes involved in the inositol phospholipid signaling pathway. FEBS Lett. 439(1-2): 66-70.

Lee, C. W., et al. (1993). Purification, molecular cloning, and sequencing of phospholipase C-beta 4. J. Biol. Chem. 268(28): 21318-27

Lee, C. W., et al. (1994). Regulation of phospholipase C-beta 4 by ribonucleotides and the alpha subunit of Gq. J. Biol. Chem. 269(41): 25335-8

Lee, S. J., Xu, H., Kang, L. W., Amzel, L. M. and Montell, C. (2003). Light adaptation through phosphoinositide-regulated translocation of Drosophila visual arrestin. Neuron 39(1):121-32. 12848937

Lee, Y. J., et al. (1994). The Drosophila dgq gene encodes a G alpha protein that mediates phototransduction. Neuron 13(5): 1143-57

Leung, H. T., Tseng-Crank, J., Kim, E., Mahapatra, C., Shino, S., Zhou, Y., An, L., Doerge, R. W. and Pak, W. L. (2008). DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors. Neuron 58(6): 884-96. PubMed Citation: 18579079

McKay, R. R., et al. (1994a). The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye. J. Neurogenet. 9(3):177-87

McKay, R. R., Zhu, L. and Shortridge, R. D. (1994b). Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster. Neuroscience 61(1): 141-8

McKay, R. R., et al. (1995). Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster. J. Biol. Chem. 270(22): 13271-6

Meyertholen, E. P., et al. (1987). Studies of the Drosophila norpA phototransduction mutant. II. Photoreceptor degeneration and rhodopsin maintenance. J. Comp. Physiol. [A]161(6): 793-8

Mitchell, J. and Mayeenuddin, L. H. (1998). Purification, G protein activation, and partial amino acid sequence of a novel phospholipase C from squid photoreceptors. Biochemistry 37(25): 9064-72

Niemeyer, B. A., et al. (1996). The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell 85(5): 651-9

Pearn, M. T., et al. (1996). Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants. J. Biol. Chem. 271(9): 4937-45

Peretz, A., et al. (1994). The light response of Drosophila photoreceptors is accompanied by an increase in cellular calcium: effects of specific mutations. Neuron 12(6): 1257-67

Ranganathan, R., et al. (1994). Cytosolic calcium transients: spatial localization and role in Drosophila photoreceptor cell function. Neuron 13(4): 837-48

Riesgo-Escovar, J., Raha, D. and Carlson, J. R. (1995). Requirement for a phospholipase C in odor response: overlap between olfaction and vision in Drosophila. Proc. Natl. Acad. Sci. 92(7): 2864-8

Roustan, P., et al. (1995). The rat phospholipase C beta 4 gene is expressed at high abundance in cerebellar Purkinje cells. Neuroreport 6(14): 1837-41

Running Deer, J. L., Hurley, J. B. and Yarfitz, S. L. (1995). G protein control of Drosophila photoreceptor phospholipase C. J. Biol. Chem. 270(21): 12623-8

Ryu, J. H., et al. (2008). Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319: 777-782. PubMed Citation: 18218863

Schneuwly, S., et al. (1991). Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster. J. Biol. Chem. 266(36): 24314-9

Scott, K., et al. (1995). Gq alpha protein function in vivo: genetic dissection of its role in photoreceptor cell physiology. Neuron 15(4): 919-27

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

Shieh, B. H., Zhu, M. Y., Lee, J. K., Kelly, I. M. and Bahiraei, F. (1997). Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo. Proc. Natl. Acad. Sci. 94(23): 12682-12687

Shortridge, R. D., et al. (1991). A Drosophila phospholipase C gene that is expressed in the central nervous system. J. Biol. Chem. 266(19): 12474-80

Stark, W. S., Sapp, R. and Carlson, S. D. (1989). Photoreceptor maintenance and degeneration in the norpA (no receptor potential-A) mutant of Drosophila melanogaster. J. Neurogenet. 5(1): 49-59

Toyoshima, S., et al. (1990). Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye. J. Biol. Chem. 265(25): 14842-8

Tsunoda, S., et al. (1997). A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature 388(6639): 243-9

van Huizen, R., et al. (1998). Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for G protein-coupled signaling. EMBO J. 17(8): 2285-97

Wang, T., Wang, X., Xie, Q. and Montell, C. (2008). The SOCS box protein STOPS is required for phototransduction through its effects on phospholipase C. Neuron 57(1): 56-68. PubMed Citation: 18184564

Wilson, M. J. and Ostroy, S. E. (1987). Studies of the Drosophila norpA phototransduction mutant. I. Electrophysiological changes and the offsetting effect of light. J. Comp. Physiol. [A] 161(6): 785-91. 88118417

Yoshioka, T., Inoue, H., Hotta, Y. (1985). Absence of phosphatidylinositol phosphodiesterase in the head of a Drosophila visual mutant, norpA (no receptor potential A). J. Biochem. 97: 1251-1254. 85289108

Younger, M. A., Muller, M., Tong, A., Pym, E. C. and Davis, G. W. (2013). A presynaptic ENaC channel drives homeostatic plasticity. Neuron 79(6): 1183-1196. PubMed ID: 23973209

Zhu, L., McKay, R. R. and Shortridge, R. D. (1993). Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster. J. Biol. Chem. 268(21): 15994-6001

date revised: 13 August 2023

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.