The modular architecture of signaling proteins is thought to be a means of coupling different inputs with new output functions, allowing for rapid evolution of new signaling functions. This feature is also important for creating signaling proteins that integrate multiple inputs to trigger a specific output. Protein modularity also can create new input/output relationships such as ultrasensitive responses through cooperative interactions between input domains. This analysis of Pins supports this idea but adds that modularity can shape pathway responses without cooperativity, simply by including multiple input domains. In this system it was shown that decoys can build either ultrasensitivity or thresholding depending on the affinities of the decoys relative to the activating site for the input. A high-affinity decoy sets a strong threshold, but lowering the decoy affinity can change thresholding into a more sigmoidal shaped curve, simply by blending the inflection point between thresholding and activation. This type of ultrasensitivity may be a fairly common component of cell signaling pathways, because autoinhibition and domain repeats are common features of cell signaling proteins. Thus, incorporating more domain repeats through genetic recombination events can modulate the response profile. The relative affinities of these sites could then be 'tuned' through point mutations to build thresholding behavior and/or apparent steepness into the signaling pathway (Smith, 2011).

Heterotrimeric G-proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G-protein-coupled receptors (GPCRs). However, Gα subunits may also be activated in a non-canonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. This study used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger acto-myosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. This study describes a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. It was shown that Ric-8 regulates this pathway through a physical interaction with Cta, and that Ric-8 preferentially interacts with inactive Cta and directs its localization within the cell. This system was also used to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility (Peters, 2013).

A novel assay was established for testing potential Fog pathway components, and it was found that in Drosophila tissue culture Ric-8 is required for pathway activation and not only binds the Gα12/13, Cta, but preferentially binds inactive Cta, CtaGA. A role was defined for Ric-8 as an escort/scaffold for CtaGA by using artificially induced localization of Ric-8 to the mitochondria. Upon Ric-8 translocation it was found that CtaGA co-localizes with ectopically localized Ric-8, while the cellular localization of wild-type and constitutively active Cta were unaffected. Additionally, when Ric-8 was mis-targeted to the mitochondria, cells were impaired in their ability to constrict in response to Fog application. Further, evolutionarily conserved residues were identified within Ric-8 potentially important for 1) establishing a Ric-8/Cta binding interface 2) nucleotide specific recognition of Cta, and 3) successful G-protein signaling downstream of Fog (Peters, 2013).

The novel cell-based assay was ideal for examining Fog-induced activation of the Rho pathway, due to the ease in which it was possible to deplete cells of specific proteins using RNAi, the rapidity of screening multiple genes simultaneously, and the ability to visualize pathway activation using simple microscope-based examination. This assay opens numerous possibilities for the identification of other pathway components, including the unidentified GPCR involved in transduction of the Fog signal, as well as investigation of general cellular functions such as mechanochemical force production and regulation of the acto-myosin cytoskeleton. Additionally, although not highlighted in this study, it was possible to view Fog-induced contractility in real-time. This allows for further investigation of pathway components that specifically affect the kinetics of Fog responsiveness, and/or the longevity and persistence of pathway activation. In Drosophila, and other systems, Ric-8 modulates the behavior of Gα subunits during asymmetric cell divisions. Due to its role in establishing asymmetry in dividing cells and subsequently controlling cell proliferation rates, Ric-8 has become of interest to the field of cancer biology. This model cell culture system provides a streamlined approach for further investigation into parsing out the complicated signaling networks involved in establishing these disease states (Peters, 2013).

Previous work has implicated Ric-8 as a chaperone during Gα biosynthesis to stabilize nascent protein production, and in turn as an essential factor in Gα membrane targeting. This function of Ric-8 has been shown to affect the stability of all classes of mammalian Gα subunits. Given the necessity of Ric-8 in mammalian systems for Gα stabilization and membrane localization it is likely that Ric-8 acts similarly in Drosophila, as evidenced by the mis-targeting of Gαi and Cta, in the absence of Ric-8, to the cortex of the epithelium of Drosophila embryos and the mis-localization of Cta in Drosophila tissue culture cells. However, unlike Gαi, the levels of Cta are not dramatically affected in the absence of Ric-8; additionally, some rescue was seen in cells depleted of endogenous Ric-8, overexpressing constitutively active Cta, indicating that at least a small amount of Cta is localized correctly and functional. Therefore, while plasma membrane levels of Cta are affected by Ric-8 overall levels of protein are not. One possibility, given constitutively active Cta was still able to rescue, is that Ric-8 could be important for Gα cycling at the site of receptor activation, which is thought to be important for spatial regulation of Gα signaling (Peters, 2013).

Though signaling nodes involving GPCRs, Gα subunits, and Ric-8 have been extensively studied there is little known about the structure of Ric-8 and how it interacts with Gα. A predicted model of Ric-8 was used as a conceptual basis to visualize mutants, and key conserved residues important for Cta binding, nucleotide specificity, and execution of productive G-protein pathway activation were identified. Based on these data the structure/function assay of Ric-8 identified four cluster mutations, mutants 1, 9, 10 and 13, that inhibited CtaGA binding, of which three: 1, 9, and 13, also failed to rescue Fog-induced constriction to wild-type levels. Of these four mutants, only mutant 1 (in the N-terminus of Ric-8) was found to have an inhibitory effect on binding to wild-type, constitutively active, and constitutively inactive Cta, while mutants 9, 10 and 13 (in the C-terminus of Ric-8) were only deficient in binding inactive Cta. The Itoh lab found that a truncated version consisting of the N-terminal half (residues 1-301) of Ric-8 was sufficient to bind Gαq. In accordance with these data, it is suggested that residues in mutant 1 are important for non-nucleotide specific Cta interaction, while residues in mutants 9, 10 and 13 confer nucleotide specific recognition of Cta. This study presents the first evidence of specific residues within Ric-8 facilitating interaction with a Gα (Peters, 2013).

Several mutants had effects in only the binding or contractile assay. Mutant 10 inhibited binding, while mutants 6-8 prevented Fog-induced constriction. Mutant 10 was able to modestly rescue cellular constriction but exhibited decreased binding to Cta, implying this mutant is still functional but perhaps folded in a manner unproductive for robust binding to Cta; this may be due to its proximity to mutant 13. Mutants 6-8 are capable of binding Cta, but not rescuing Ric-8 function downstream of pathway activation. While the function of mutant clusters 6-8 is unclear, it is tempting to hypothesize that this region is a potential site for Ric-8 GEF activity (Peters, 2013).

In the early dividing C.elegans embryo, Drosophila melanogaster neuroblasts and epithelium and several mammalian tissue culture cell lines Ric-8 localizes Gα subunits to the plasma membrane. The current data suggest there is an additional level regulating Gα localization that is dependent on the nucleotide-bound state of Gα. This study has identified a cluster of residues that may facilitate this interaction with Cta. Clustered Ric-8 mutants, deficient in binding CtaGA in immunoprecipitation assays, when tagged with a sequence directing them to the mitochondria had varying effects in their ability to ectopically localize CtaGA. Mito-Ric-8 mutant 1 did not recruit CtaGA to its ectopic location at the mitochondria, while Mito-Ric-8 mutants 9, 10, and 13 triggered mitochondrial mis-localization of CtaGA. Interestingly, mutants 9, 10 and 13 exhibited decreased binding to constitutively inactive Cta, CtaGA, but not wild-type nor constitutively active Cta, CtaQL. This implies that these residues may confer temporally regulated nucleotide specific recognition sites for Cta (Peters, 2013).

Based on characterization of Ric-8, and data from the literature, the following model is proposed. Ric-8 acts to initially chaperone the folding of Cta, allowing Cta, Gβ13F, and Gγ1 to form a complex that is then transported to the plasma membrane. Upon Fog/GPCR interaction, GTP-bound Cta is released from the Gβγ heterodimer, and interacts with RhoGEF2 (via its RGS domain), causing hydrolysis of GTP to GDP. Specific, evolutionarily conserved residues regulate the binding of GDP-bound Cta to Ric-8, or alternatively Ric-8 stabilizes a nucleotide-free version of Cta. This allows Cta to bypass destruction and be re-inserted into the Fog pathway to activate downstream targets (Peters, 2013).

Drosophila genome encodes six α-subunits of heterotrimeric G proteins. The α-subunit termed Gαs is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. This study shows that another α-subunit, Gαo, can specifically antagonize the Gαs activities by competing for the Gβ13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. These results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development (Katanayeva, 2010).

G protein-coupled receptors (GPCRs) represent the most populous receptor family in metazoans. Approximately 380 non-olfactory GPCRs are encoded by the human genome, corroborated by ca. 250 GPCRs in insect genomes, making 1%-1.5% of the total gene number dedicated to this receptor superfamily in invertebrates and mammals. GPCRs transmit their signals by activating heterotrimeric G protein complexes inside the cell. A heterotrimeric G protein consists of a GDP-bound α-subunit and a βα-heterodimer. Ligand-stimulated GPCR serves as a guanine nucleotide-exchange factor, activating the GDP-to-GTP exchange on the Gα-subunit. This leads to dissociation of the heterotrimeric complex into Gα-GTP and flγ, which transmit the signal further inside the cell (Katanayeva, 2010).

The β- and γ-subunit repertoire of the Drosophila genome is reduced as compared with that of mammals: only two Gγ and three Gβ genes are present in flies. Gγ30A and Gβ76C are components of the fly phototransduction cascade and are mostly expressed in the visual system. Gγ1 and Gβ13F have been implicated in the asymmetric cell divisions and gastrulation, while the function of Gβ5 is as yet unknown (Katanayeva, 2010).

Despite the fact that βγ can activate signal effectors, the main selectivity in GPCR coupling and effector activation is provided by the Gα-subunits. Sixteen genes for the α-subunits are present in the human genome, and six in Drosophila. All human Gαsubunit subgroups are represented in Drosophila: Gαi and Gαo belonging to the Gαi/o subgroup; Gαq belonging to the Gαq/11 subgroup; Gαs belonging to the Gαs subgroup, and concertina (cta) belonging to the Gα12/13 subgroup. Additionally, Drosophila genome encodes for Gαf which probably represents an insect-specific subfamily of Gαsubunits (Katanayeva, 2010).

Multiple functions have been allocated to different heterotrimeric G proteins in humans and flies. For example, in Drosophila development cta is a crucial gastrulation regulator, Gαo is important for the transduction of the Wnt/Frizzled signaling cascade, and Gαi controls asymmetric cell divisions during generation of the central and peripheral nervous system (the later in cooperation with Gαo. Gαq is the Drosophila phototransduction Gαsubunit, but probably has additional functions. Pleotropic effects arise from defects in Gαs function, while the function of Gαf has not yet been characterized (Katanayeva, 2010).

Among the developmental processes ascribed to the control by Gαs are the latest stages of Drosophila wing development. Newly hatched flies have soft and folded wings, which during the 1-2 hours post-eclosion expand and harden through intensive synthesis of components of the extracellular matrix. These processes are accompanied by epithelial-mesenchymal transition and apoptosis of the wing epithelial cells, producing a strong but mostly dead adult wing structure. Expression of the constitutively active form of Gαs leads to precocious cell death in the wing epidermis, which results in failure of the closure of the dorsal and ventral wing sheets and accumulation of the hemolymph inside the wing, producing wing blistering. Conversely, clonal elimination of Gαs leads to autonomous prevention of the cell death. Kimura (2004) has performed an extensive analysis of the signaling pathway controlling apoptosis at late stages of wing development. That study provided evidence suggesting that the hormone bursicon, synthesized in the head of post-eclosion Drosophila and secreted in the hemolymph, activates a GPCR Rickets on wing epithelial cells, which signals through Gαs to activate the cAMP-PKA pathway, culminating at the induction of apoptosis. However, the identity and importance of the &βγ subunits in bursicon signaling, as well as possible involvement of other Ga proteins remained outside of their investigation. There also remain some uncertainties as to the phenotypic consequences of elimination of the bursicon-Gαs-PKA pathway in wings (Katanayeva, 2010).

This study describes a comprehensive functional analysis of the Drosophila heterotrimeric G protein proteome using loss-of-function and overexpression experiments. Loss of Gαs but not any other Gαsubunit leads to the failure of wing expansion after fly hatching. Gαo, but not another Gα, can compete with Gαs and thus antagonize its function. Finally, the Gβ13F and Gγ1 as the βγ subunits of the heterotrimeric Gs complex responding to the epithelial-mesenchymal transition and cell death-promoting signal (Katanayeva, 2010).

The soft folded wings of the young insect freshly hatched from the pupal case within 1-2 hours expand and harden, becoming a robust flight organ. This process is accompanied by epithelial-mesenchymal transition and cell death of the wing epithelial cells. Genetic dissection has revealed the function of the neurohormone bursicon and its wing epithelial receptor rickets in initiation of these processes. The GPCR rickets couples to the heterotrimeric G protein Gs; the Gαs-activated cAMP-PKA pathway culminates at the induction of apoptosis. However, the overall phenotypic consequences of the loss of the Gs signaling pathway in post-eclosion wings were unknown, as well as the nature of the Gβγ subunits of the heterotrimeric Gs complex responding to the bursicon-rickets signaling (Katanayeva, 2010).

This study consisted of an extensive analysis of the heterotrimeric G protein subunits in these post-eclosion stages of wing maturation. The whole-wing down-regulation of Gαs results in the failure of wing expansion, demonstrating that this change in the shape of the wing is the major morphological outcome of the bursicon-rickets-Gs signaling. The Gβ13F and Gγ1 subunits were also identified as the other two constituents of the heterotrimeric Gs complex, as downregulation of Gαs, Gβ13F, or Gγ1, but not any other Ga, Gβ, or Gγ subunits encoded by the Drosophila genome, each leads to the same folded wing phenotype (Katanayeva, 2010).

It was also shown that Gαo, but not any other Gαsubunit, can inhibit the wing expansion program through sequestration of the Gβ13F/Gγ1 heterodimer. The reason for the specificity of Gαo over other Gαsubunits in antagonizing the Gs signaling is unclear. It is unlikely that differences in expression levels of the tested Gαsubunits may account for the selective activity of Gαo. Indeed, most overexpression experiments were done with the X-chromosome-inserted MS1096-Gal4 driver, which results in markedly higher expression levels in males than heterozygous female flies, producing a more penetrant folded wing phenotype in males overexpressing Gαo. However, even in male flies overexpressing other Gαsubunits no instances of the folded wing phenotype could be seen. Furthermore, several independent insertions of the UAS-Ga transgenes were tested; while different Gαo transgenes all produced the folded wing phenotype upon overexpression, other Ga constructs remained ineffective (Katanayeva, 2010).

Similarly, the different Gαsubunits possess a similar affinity towards the interaction with the Gβγ heterodimer, not providing an explanation for a specific ability of Gαo to antagonize the Gs-mediated post-eclosion pathway. It is thus thus tempting to propose that a previously uncharacterized biochemical mechanism may allow for a specific antagonism physiologically existing between the Gs- and Go- mediated signaling pathways. As liberation of high amounts of GDP-loaded Gαo is predicted to be a consequence of activation of multiple Go-coupled GPCRs, and as Go is a heavily expressed G protein representing the major G protein species e.g. in the brain of flies and mammals, this specific ability of Gαo to antagonize the Gs-mediated signaling may have physiological implications in other tissues and organisms than Drosophila wing. However, it is added that these speculations are based on the analysis of the overexpression data and must be treated with caution when translating them into physiological situations (Katanayeva, 2010).

Only the GDP-loaded, but not the activated GTP-loaded form of Gαo is effective in antagonizing Gs. A proteomics analysis was performed of the Drosophila proteins which would discriminate between the two nucleotide forms of Gαo, and surprisingly few targets of this kind were revealed. While the chaperone Hsc70-3 and β1-tubulin preferentially interacted with the GTP-loaded Gαo, Gβ13F was found to specifically interact with Gαo-GDP. These data suggest that many Gαo-interaction partners do not discriminate between the two guanine forms of Gαo. These findings are in agreement with our other experimental findings, as well as mathematical modeling predicting that high concentrations of free (monomeric) signaling-competent Gαo-GDP are produced upon activation of Go-coupled GPCRs (Katanayeva, 2010).

Gαo-mediated sequestration of Gβ13F/Gγ1 depletes the pool of the heterotrimeric Gs complexes. As only heterotrimeric Ga&βγ, but not monomeric Ga proteins can efficiently bind and be activated by their cognate GPCRs, overexpression of Gαo abrogates the rickets-Gs signaling. Phenotypic consequences of this abrogation are the failures of apoptosis and wing expansion. In contrast, expression of the constitutively activated form of Gαs induces premature cell death and wing blistering. This phenotype can be also induced by expression of cholera toxin, revealing that the ability of cholera toxin to specifically overactivate Gαs reported in mammalian systems is reproduced with Drosophila proteins. These data also confirm that not only exogenously overexpressed, but also the endogenous Gαs can induce the precocious cell death upon overactivation (Katanayeva, 2010).

However, prevention of apoptosis is not sufficient to produce the folded wing phenotype. Together with the observation that the constitutively active form of Gαs is ineffective in rescuing the wing expansion defects produced by Gαo overexpression, these data suggest that the Gαs-cAMP-PKA pathway culminating at apoptosis is not the sole signaling branch emanating from the bursicon-rickets GPCR activation. It is proposed that the second signaling branch initiated by the rickets-mediated dissociation of the heterotrimeric Gs complex is represented by the free Gββ subunits, signaling to epithelial-mesenchymal transition. Such a double signaling impact mediated by the two components of the heterotrimeric G protein complex leads to initiation of two cellular programs -- apoptosis and epithelial-mesenchymal transition -- which cumulatively result in wing expansion and solidification, producing the adult flight organ. This two-fold response of the Drosophila wing to the maturation signal, mediated by the two components of the heterotrimeric G protein complex activated by the single hormone-responsive GPCR, provides an elegant paradigm for the coordination of signaling and developmental programs (Katanayeva, 2010).

ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in the larval visual system, focus was placed on investigating the physiological properties and behavioral relevance of larval visual interneurons. The ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Galphao signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, these studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system (Qin, 2019).

ON and OFF selectivity, the differential neuronal responses elicited by signal increments or decrements, is an essential component of visual computation and a fundamental property of visual systems across species. Extensive studies of adult Drosophila optic ganglia and vertebrate retinae suggest that the construction principles of ON and OFF selective pathways are shared among visual systems, albeit with circuit-specific implementations. Anatomically, dedicated neuronal pathways for ON vs. OFF responses are key features in visual circuit construction. Specific synaptic contacts are precisely built and maintained in laminar and columnar structures during development to ensure proper segregation of signals for parallel processing. Molecularly, light stimuli elicit opposite responses in ON and OFF pathways through signaling events mediated by differentially expressed neurotransmitter receptors in target neurons postsynaptic to the photoreceptor cells (PRs). This has been clearly demonstrated in the mammalian retina, where light-induced changes in glutamatergic transmission activate ON-bipolar cells via metabotropic glutamate receptor 6 (mGluR6) signaling and inhibit OFF-bipolar cells through the actions of ionotropic AMPA or kainate receptors. In the adult Drosophila visual system, functional imaging indicates that ON vs. OFF selectivity emerges from visual interneurons in the medulla. However, despite recent efforts in transcriptome profiling and genetic analyses, the molecular machinery mediating signal transformation within the ON and OFF pathways has not yet been clearly identified (Qin, 2019).

Unlike the ~6000 PRs in the adult visual system, larval Drosophila eyes consist of only 12 PRs on each side. Larval PRs make synaptic connections with a pair of visual local interneurons (VLNs) and approximately ten visual projection neurons (VPNs) in the larval optic neuropil (LON). VPNs relay signals to higher brain regions that process multiple sensory modalities. Despite this simple anatomy, larvae rely on vision for negative phototaxis, social clustering, and form associative memories based on visual cues. How the larval visual circuit effectively processes information and supports visually guided behaviors is not understood (Qin, 2019).

Recent connectome studies mapped synaptic interactions within the LON in the first instar larval brain, revealing two separate visual pathways using either blue-tuned Rhodopsin 5 (Rh5-PRs) or green-tuned Rhodopsin 6 (Rh6-PRs). Rh5-PRs project to the proximal layer of the LON (LONp) and form direct synaptic connections with all VPNs, whereas Rh6-PRs project to the distal layer of the LON (LONd) and predominantly target one cholinergic (cha-lOLP) and one glutamatergic (glu-lOLP) local interneurons. The two PR pathways then converge at the level of VPNs (Qin, 2019).

These connectome studies also revealed potential functions for cha- and glu-lOLP. The pair of lOLPs, together with one of the VPNs, the pOLP, are the earliest differentiated neurons in the larval optic lobe and are thus collectively known as optic lobe pioneer neurons (OLPs). Besides relaying visual information from Rh6-PRs to downstream VPNs, the lOLPs also form synaptic connections with each other and receive neuromodulatory inputs from serotonergic and octopaminergic neurons, suggesting that they may act as ON and OFF detectors. This proposal is further supported by recent studies on the role of the Rh6-PR/lOLP pathway in larval movement detection and social clustering behaviors. However, it remains unclear how the lOLPs support differential coding for ON and OFF signals without anatomical separation at either the input or output level (Qin, 2019).

This study investigated the lOLPs' physiological properties and determined the molecular machinery underlying their information processing abilities. Functional imaging studies revealed differential physiological responses towards light increments and decrements in cha-lOLP and glu-lOLP, indicating their functions in detecting ON and OFF signals. Furthermore, it was found that light-induced inhibition on glu-lOLP is mediated by mAchR-B/Gαo signaling, which generates the sign inversion required for the OFF response and encodes temporal information between the cholinergic and glutamatergic transmissions received by downstream VPNs. Lastly, genetic manipulations of glu-lOLP strongly modified the physiological responses of VPNs and eliminated dark-induced pausing behaviors. Together, these studies identify specific cellular and molecular pathways that mediate OFF detection in Drosophila larvae, reveal functional interactions among key components of the larval visual system, and establish a circuit mechanism for ON vs. OFF discrimination in this simple circuit (Qin, 2019).

The Drosophila larval visual circuit, with its small number of components and complete wiring diagram, provides a powerful model to study how specific synaptic interactions support visual computation. Built on knowledge obtained from connectome and behavioral analyses, the current physiological and genetic studies revealed unique computational strategies utilized by this simple circuit for processing complex outputs. Specifically, the results indicate that ON vs. OFF discrimination emerges at the level of the lOLPs, a pair of second-order visual interneurons. In addition, the essential role is demonstrated of glu-lOLP, a single glutamatergic interneuron, in meditating OFF detection at both the cellular and behavior levels and identify mAchR-B/Gαo signaling as the molecular machinery regulating its physiological properties (Qin, 2019).

Functional imaging studies using genetically encoded calcium and voltage indicators provide valuable information regarding the physiological properties of synaptic interactions among larval visual interneurons and projection neurons. However, optical recording approaches have certain technical limitations, including the kinetics and sensitivities of the voltage and calcium sensors, as well as the imaging and visual stimulation protocols. In addition, although glu-lOLP displays a biphasic response towards the light stimulation, calcium reductions and increases for only the initial set of physiological characterizations were quantified. Compared to the delayed calcium rise, the light-induced calcium reductions have low amplitudes and high variabilities, possibly due to the half-wave rectification of the intracellular calcium previously described in adult visual interneurons. For the genetic experiments, focus was placed on evaluating the activation of glu-lOLP, which is reflected by the increase of intracellular calcium signals that lead to neurotransmitter release (Qin, 2019).

To process light and dark information in parallel, both mammalian and adult fly visual systems utilize anatomical segregation to reinforce split ON and OFF pathways. In the larval visual circuit, however, almost all VPNs receive direct inputs from both cha-lOLP and glu-lOLP as well as the Rh5-PRs. Therefore, the response signs of the VPNs cannot be predicted by their anatomical connectivity to ON and OFF detectors. Based on the cumulative evidence obtained through genetic, anatomical, and physiological studies, it is proposed that temporal control of inhibition potentially contributes to ON vs. OFF discrimination in larvae. While cha-lOLP displays clear ON selectivity, the OFF selectivity in glu-lOLP is strengthened by the extended suppression of its light response by mAchR-B/Gαo signaling. This temporal control may also produce a window of heightened responsiveness in cha-lOLP and ON-VPNs towards light signals, similar to the case in mammalian sensory systems where the temporal delay of input-evoked inhibition relative to excitation sharpens the tuning to preferred stimuli. Together, the temporal separation between cholinergic and glutamatergic transmission could reinforce the functional segregation in the VPNs and lead to distinct transmissions of ON and OFF signals. Although further functional validations are needed, temporal control of inhibition provides an elegant solution that may be of general use in similar contexts where parallel processing is achieved without anatomically split pathways (Qin, 2019).

The connectome study identified ten larval VPNs which receive both direct and filtered inputs from two types of PRs and transmit visual information to higher brain regions, including four LNvs (PDF-LaNs), five LaN, nc-LaN1, and two pVL09, VPLN, and pOLP17. Based on these studies on LNvs and pOLP, it is expected the functional diversity in VPNs generated by differential expression of neurotransmitter receptors or molecules involved in electric coupling will be observed. Besides basic ON vs. OFF discrimination, VPNs are also involved in a variety of visually guided behaviors. The temporal regulation of their glutamatergic and cholinergic inputs as well as the local computation within the LON are among potential cellular mechanisms that increase the VPNs' capability to process complex visual information. Further physiological and molecular studies of the VPNs and behavioral experiments targeting specific visual tasks are needed to elucidate their specific functions (Qin, 2019).

Besides the similarities observed between larval lOLPs and the visual interneurons in the adult fly visual ganglia, an analogy can be drawn between lOLPs and interneurons in mammalian retinae based on their roles in visual processing. Cha-lOLP and glu-lOLP carry sign-conserving or sign-inverting functions and activate ON- or OFF-VPNs, respectively, performing similar functions as bipolar cells in mammalian retinae. At the same time, lOLPs also provide inhibitory inputs to either ON- or OFF-VPNs and thus exhibit the characteristics of inhibitory amacrine cells. The dual role of lOLPs is the key feature of larval ON and OFF selectivity, which likely evolved to fulfill the need for parallel processing using limited cellular resources (Qin, 2019).

Lastly, these studies reveal signaling pathways shared between mammalian retinae and the larval visual circuit. Although the two systems are constructed using different neurochemicals, Gαo signaling is responsible for producing sign inversion in both glu-lOLP and the ON-bipolar cell. In mGluR6-expressing ON-bipolar cells, light increments trigger Gαo deactivation, the opening of TrpM1 channels, and depolarization. In larval glu-lOLP, how light induces voltage and calcium responses via mAchR-B signaling has yet to be determined. Gαo is known to have functional interactions with a diverse group of signaling molecules including potassium and calcium channels that could directly link the light-elicited physiological changes in glu-lOLP. Genetic and physiological studies in the larval visual circuit will facilitate the discovery of these target molecules and contribute to the mechanistic understanding of visual computation (Qin, 2019).

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

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

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

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

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

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

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

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

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

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

Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, this study showed Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. The light-dependent regulation of Pkc53E was demonstrated and it was demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCβ4/NorpA as degeneration of norpA(P24) photoreceptors was enhanced by a reduced Pkc53E activity. It was further shown that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton (Shieh, 2023).

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. This work develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, P hoto- i nduced M odulation of G α protein - I nhibition of Gα q (PiGM-Iq), exhibited potent and selective inhibition of Gα q signaling. The behavior of C. elegans and Drosophila was altered with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, it was also shown that this approach is amenable to Gαi signaling (Lockyer, 2023).

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

Katanayeva, N., Kopein, D., Portmann, R., Hess, D. and Katanaev, V. L. (2010). Competing activities of heterotrimeric G proteins in Drosophila wing maturation. PLoS One. 5(8): e12331. PubMed Citation: 20808795

Lockyer, J., Reading, A., Vicenzi, S., Delandre, C., Marshall, O., Gasperini, R., Foa, L. and Lin, J. Y. (2023). Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation. bioRxiv. PubMed ID: 37214843

Peters, K. A. and Rogers, S. L. (2013). Drosophila Ric-8 interacts with the Galpha12/13 subunit, Concertina, during activation of the Folded gastrulation pathway. Mol Biol Cell 24: 3460-3471. PubMed ID: 24006487

Qin, B., Humberg, T. H., Kim, A., Kim, H. S., Short, J., Diao, F., White, B. H., Sprecher, S. G. and Yuan, Q. (2019). Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit. Nat Commun 10(1): 4093. PubMed ID: 31501438

Shieh, B. H., Sun, W. and Ferng, D. (2023). A conventional PKC critical for both the light-dependent and the light-independent regulation of the actin cytoskeleton in Drosophila photoreceptors. J Biol Chem 299(6): 104822. PubMed ID: 37201584

Smith, N. R. and Prehoda, K. E. (2011). Robust spindle alignment in Drosophila neuroblasts by ultrasensitive activation of pins. Mol. Cell 43(4): 540-9. PubMed ID: 21855794

Zygotically transcribed genes

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