BIOLOGICAL OVERVIEW

The G proteins, which have been conserved from yeast to humans, are heterotrimeric GTPases that behave as signal transduction proteins. Their three subunits (alpha, beta, and gamma) form a complex that relay signals arising from a receptor (serpentine receptor) composed of seven transmembrane segments: this complex may be activated by a variety of ligands, including hormones or neuropeptides. In the inactive complex, GDP is bound to the alpha subunit. Activation of the serpentine receptor results in an exchange between GDP and GTP and in the dissociation of the heterotrimer into a monomer (alpha subunit) and a dimer (beta and gamma). Both entities can target the activity of various enzymes and of ionic channels, which in turn control the level of intracellular second messengers.

The gene encoding the alpha subunit of the Drosophila G_o protein is expressed early in embryogenesis in the precursor cells of the heart tube, visceral muscles, and nervous system. This early expression coincides with the onset of a mesenchymal-epithelial transition, which takes place within two cell populations: the cardial cells and the precursor cells of the visceral musculature. A detailed analysis of G-oalpha47A expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm. These cardioblasts depend on the wingless and hedgehog signaling pathways (which originate from the overlying ectoderm) for both their determination and specification. In the nervous system, the expression of G_oalpha shortly precedes the beginning of axonogenesis. Mutants produced in the G-oalpha47A gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed. Furthermore, in G-oalpha47A mutant embryos, in a sporadic manner, the epithelial polarity of cardial cells is not acquired (or maintained) in the cardiac tube. In the nervous system of homozygous Df(2R)47A embryos, which lack the G-oalpha47A gene, longitudinal axons are often missing and important modifications are observed in the guidance and axonal growth of motoneurons. It is thought that G_oalpha might be involved in vesicular traffic of membrane proteins that are responsible for the acquisition of epithelial polarity (Frémion, 1999 and references).

In Drosophila, genes coding for proteins that belong to the three major families of alpha subunits (alpha_s, alpha_i, and alpha_o) have been cloned and characterized. Two G_salpha proteins differing by four amino acids are produced by alternative splicing of a unique gene (Quan, 1990). The unique gene for G_ialpha produces two transcripts that encode the same protein (Provost, 1988). Finally, two G_oalpha proteins have been deduced from cDNA sequence analyses and are issued from a unique gene transcribed in three different transcripts (de Sousa, 1989, Schmidt, 1989, Thambi, 1989 and Yoon, 1989). Two other alpha subunits have been identified as homologs of vertebrate G_falpha (Quan, 1993) and G_qalpha-3 (Talluri, 1995), respectively. All these proteins are expressed throughout embryogenesis in various tissues in a dynamic pattern (Wolfgang, 1991) and, consequently, may play major roles in development although no precise developmental function has been ascribed to any of them. In contrast, it is known that the activity of concertina, which encodes the alpha subunit of a G₁₂ or G₁₃ protein, is necessary for the coordination of cell shape changes in the ventral furrow at gastrulation (Parks, 1991). In addition, concertina is a likely candidate to act downstream of a putative secreted protein, Folded gastrulation, whose receptor is as yet unidentified (Costa, 1994).

Among the different families of G proteins, the G_o protein is perhaps less well characterized in terms of the nature of its associated signal transduction pathway. On the plasma membrane, G_o proteins may regulate Ca²⁺ and K⁺ channels (Van Dongen, 1988; Kleuss, 1991; Lledo, 1992), distinct types of phospholipase C (Moriarty, 1990), and the mitogen-activated protein kinase cascade (Van Biesen, 1996). In addition to interacting with serpentine receptors such as opiate, alpha₂-adrenergic, D₂ dopaminergic, and somatostatin receptors, the G_o protein has been described recently as a regulator of vesicular traffic (Lagriffoul, 1996, Gasman, 1998).

The role of Drosophila G_o in heart development will be documented in this overview. In stage 11 embryos, the zygotic G-oalpha47A mRNA is first detected in the precursors of the cardioblasts in a repeated pattern of 11 clusters in the dorsal mesoderm. These clusters, constituted initially of two to four cells, are located in the anterior compartment of each mesodermal parasegment in a position anterior to the domain of Engrailed ectodermal stripes and closely neighboring the Wingless-expressing cells in parasegments 2-12 of the overlying ectoderm. These cardioblast precursors belong to the functional domain of, but are distinct from, Ladybird (Lbd) and Evenskipped (Eve) expressing cells in the mesoderm which are the precursors of a fraction of the pericardial cells and of a dorsal muscle. Within the cluster, the expression of the G-oalpha47A mRNA is initially very intense in only two of the four cells cells and then it becomes detectable in all four cells with an almost uniform intensity. Due to their position relative to the parasegment boundary, these cells are called A (for anterior) cardioblasts. Soon after the onset of G-oalpha47A expression in the first cluster, a second cluster of stained cells appears posterior to the first one and not in continuity with it. This group of cells called P (for posterior) cardioblasts is located in the posterior domain of the next parasegment and it never contains more than two G-oalpha47A-expressing cells. Their position coincides with that of an area posterior to En stripes in the ectoderm (Frémion, 1999).

During germ-band retraction (late stage 11 and stage 12), the cardioblast progenitors, which at this stage are mesenchymal in nature, begin to establish contacts among themselves, to extend filopods, and to reorganize their shapes to form a continuous layer on each side of the dorsal opening. This reorganization process coincides in time with the encounter of the A and P groups of G-oalpha47A-expressing cells in each parasegmental domain. Later in development and due to the shortening of the segment during germ-band retraction, the segmentally repeated six cell clusters spread along the anterior-posterior axis to finally join as a monolayer of polarized epithelial cells. Still further along in the process, the two rows of cardial cells, together with the pericardial cells that are attached to cardial cells' basal membrane, fuse at the dorsal midline to form the heart tube. These observations have led to the conclusion that a mature heart at the end of embryogenesis is made up of 52 cardial cells per hemiembryo, consisting of six cells in every segment from T3 to A7 and in only four cells in the T2 segment. Each segment is made up of two distinct populations of cardial cells: two groups; one composed of four cardial cells and one of two cardial cells (together consitituiting the six cells per hemisegment) that derive from mesodermal cells and that are located in domains corresponding, respectively, to the anterior and posterior compartment of each parasegment. None of the cardioblasts present in parasegments 2 and 3, and only a part of those found in PS4, participate in the mature dorsal vessel: however, it is difficult to follow their fates, since the expression of G-oalpha47A vanishes rapidly from these cells during development. Similarly, by using the EC11 antibody specific for the pericardial cells, 36 such cells have been found distributed 4 cells per segment, from T2 to A7. Eve-expressing cells from the labial segment and the segment T1 do not contribute to the pool of pericardial cells and they probably give rise to other structures that could not be recognized (Frémion, 1999).

The cardial cells, which are also muscle cells, are considered to be epithelial, based on the expression of several markers for polarity. For example, antibodies directed against alpha-spectrin specifically label the basal-lateral membrane of the epithelium; staining by anti-Neurotactin antibodies (Nrt) is restricted to the lateral and apical domains of the cell surface. In contrast, phosphotyrosyl proteins and Armadillo are weakly expressed in the heart cell membranes and have no polarized localization, in good agreement with the lack of authentic Zonulae adherens in Drosophila secondary epithelia. Finally, the basal membrane of the epithelium was visualized with the mAb EC11. This antibody recognizes an antigen that is most probably secreted by the pericardial cells and which is detected on the basal membrane of the cardial cells and around the pericardial cells. In the visceral mesoderm, which also undergoes a mesenchymal-epithelial transition before its differentiation into the visceral musculature, G-oalpha47A is expressed in a repeated pattern of clusters of cells in the same 11 parasegments as the precursors of the cardial cells and anterior to them. These precursor cells in parasegments 4-12 are ultimately transformed into a polarized epithelial ribbon, one cell wide (Frémion, 1999).

Homozygous Df(2R)47A embryos, lacking G-oalpha47A gene, harbor defects in the three tissues in which G-oalpha47A is expressed. All homozygous Df(2R)47A embryos have defects in the heart, the visceral mesoderm, and the nervous system. When probed with anti-EC11 antibodies, the mutant embryos show interruptions in their dorsal vessel that are also visible when DMef 2 expression is examined. The cardial cells are present and they migrate properly in the dorsal mesodermal position, but, in some places, they are no longer arranged as a continuous layer; rather, they form as unorganized clusters of cells. Fas III expression reveals the same types of defects in the visceral mesoderm. Similar alterations are detected in G-oalpha47A⁰⁰⁷ mutants with the same total penetrance (Frémion, 1999).

The heart and visceral mesoderm phenotypes of Df(2R)47A embryos can be rescued at least partially by expressing the class II G-oalpha47A cDNA under the control of 24B-GAL4 that drives the expression of UAS-cDNAs in the myogenic lineage, including the cardioblasts and the visceral mesoderm. In >65% (n = 250 observed embryos) of homozygous Df(2R)47A embryos, a normal development of these tissues has been restored. In contrast, the phenotype in the nervous system is not abolished as expected, first because the deficiency uncovers the lola gene, and second because of the restricted specificity of expression of the 24B-GAL4 line. The G-oalpha47A⁰⁰⁷ mutation that affects only the G-oalpha47A gene can also be rescued by a G-oalpha47A transgene (Frémion, 1999).

The loss of G-oalpha47A function produces heart and visceral mesoderm phenotypes that suggest a participation of the G_o protein in epithelia formation. During the mesenchymal-epithelial transition, subsets of membrane and cytoskeletal proteins localize to distinct regions of the cell surface to create the apical and basal-lateral membrane domains that confer to the cells their epithelial polarity. The G-oalpha47A mutant phenotype is more accurately described with the aid of markers localized specifically to these different domains. Analysis was focused on regions of the mutant heart in which the cells form a continuous uninterrupted layer but which display abnormal marker expression. The localization of two polarity markers is affected in several places in the heart epithelium of G-oalpha47A mutant embryos. alpha-Spectrin, specific for the basal-lateral membrane of epithelial cells, is abnormally expressed on the entire surface of the mutant cells, however with a somewhat lower intensity than in wild-type cells. In addition, these mutant cells remain round, have no signs of shape remodeling, and fail to express the EC11 antigen in their basal membrane. The pericardial cells that are normally attached to the basal membrane of the cardioblasts are absent from these same regions and are rather often associated in clusters in the domains of high EC11 antigen expression. Similarly, Neurotactin, which is localized to the apical and lateral membranes of wild-type cardial cells, in G-oalpha47A embryos is scattered in several locations on the entire surface of those cells that are round and in which the epithelial array is disorganized. Finally, Fas III expression also reveals phenotypes in the visceral mesoderm is characterized in some regions by a uniform and lower Fas III staining on the surface of still round cells with a partially destroyed epithelial structure (Frémion, 1999).

The myoendothelial heart tube is considered as a secondary epithelium that forms by a mesenchymal-epithelial transition. The mesenchymal cardial precursor cells, after their migration from the ventral site of gastrulation in the direction of their final dorsal position, reorganize their plasma membrane to acquire their cellular polarity and establish cell junctions to build up the cardial epithelium. A signal must be received by the mesenchymal cells to create the first asymmetry on their surface. Interactions with localized extracellular matrix components are believed to be largely responsible for this first event. For example, Drosophila mutants in Laminin A or in an integrin subunit present severe disruptions in their dorsal vessels. The initiating signal is probably emitted in response to inductive interactions between the mesenchymal cells and the overlying dorsal ectoderm. The newly created asymmetry could then trigger a reorganization of the cytoskeleton. Finally, the different membrane domains (apical and basal-lateral) are established by the acquisition of different combinatorials of membrane proteins that have been specifically routed towards them; this sorting-out step relies on an absolute specificity of vesicular traffic. In the absence of G-oalpha47A function, the mesenchymal cardial cells do not remodel their shape and, in the case of the most extreme phenotype, they failed to form a continuous row of cells. In addition, whenever such a layer is eventually formed, in several places some (but not all) cells neither acquire nor maintain a proper polarity (Frémion, 1999 and references).

Based on the timetable of G-oalpha47A expression, its participation in the first step, creating the asymmetry of the cardial cells, is not very likely. Rather, it might be required for the subsequent steps that concern cell shape change and polarization, by addressing membrane proteins to their respective domains (Frémion, 1999).

It has been suggested that heterotrimeric G proteins could contribute to the vesicular protein traffic by regulating early steps in the secretory pathway. This hypothesis stems from the observation that AlF4^-, an activator of heterotrimeric but not of monomeric G proteins, inhibits ER to Golgi and intra-Golgi transport as well as vesicle budding from the trans-Golgi network. In particular, G_o proteins have been implicated in granule exocytosis from chromaffin cells (Vitale, 1993 and Gasman, 1998); insulin secretion (Lang, 1995), and transcytosis (Bomsel, 1992). It has been shown that the secretion of the protease Nexin-1 by glioma cells is under the control of G_o1 (Lagriffoul, 1996). The G_oalpha1 protein has been detected on the membrane of small intracellular vesicles and the secretion of Nexin-1 is stimulated by G_oalpha1 overexpression and by activators of G_o proteins, such as mastoparan. It has been further suggested that the GTPase activity of the G_oalpha1 protein can be stimulated in the absence of a classical serpentine receptor (Lagriffoul, 1996).

Thus, it is tempting to speculate that the Drosophila G_o protein has a function in a particular type of vesicular traffic responsible for the acquisition or maintenance of cell polarity in the cardial and visceral mesoderm cells. Preliminary observations on the subcellular localization of G_o in embryonic cells are consistent with this idea. In the totality of the cells examined, Drosophila G_o is located to the cytoplasm rather than associated to the cell membrane and the staining pattern reveals a typical granular appearance (Frémion, 1999).

Since the exportation and the localization to the plasma membrane of the protein markers that were used were all equally affected in G-oalpha47A mutants, it has been concluded that G-oalpha47A is involved in a general aspect of vesicular traffic rather than in the specific process of the sorting out of membrane proteins. G-oalpha47A might also be required for the reorganization of the cytoskeleton. The protein G encoded by the concertina gene participates in cell shape changes taking place at gastrulation (Parks, 1991), probably via a modulation of the invaginating blastoderm cell cytoskeleton resulting from the activation of RhoA (Hall, 1998). The early role of the G_o protein in the formation of the heart epithelium does not exclude a function in later events leading to the formation of the heart or to the acquisition of heart function. It has been shown recently that knocking-out the G_oalpha gene in the mouse results in heart dysfunction. G_o-deficient mice have lost the muscarinic inhibition of isoproterenol-stimulated cardiac L-type Ca²⁺ currents (Valenzuela, 1997 and Jiang, 1998). It will be interesting to investigate whether the Drosophila G_o protein could also be involved in such a process (Frémion, 1999).

G(o) activation is required for both appetitive and aversive memory acquisition in Drosophila

Heterotrimeric G(o) is an abundant brain protein required for negatively reinforced short-term associative olfactory memory in Drosophila. G(o) is the only known substrate of the S1 subunit of pertussis toxin (PTX) in fly, and acute expression of PTX within the mushroom body neurons (MB) induces a reversible deficit in associative olfactory memory. This study demonstrates that the induction of PTX within the α/β and γ lobe MB neurons leads to impaired memory acquisition without affecting memory stability. The induction of PTX within these MB neurons also leads to a significant defect in an optimized positively reinforced short-term memory paradigm; however, this PTX-induced learning deficit is noticeably less severe than found with the negatively reinforced paradigm. Both negatively and positively reinforced memory phenotypes are rescued by the constitutive expression of G(o)α transgenes bearing the Cys(351)Ile mutation. Since this mutation renders the G(o) molecule insensitive to PTX, the results isolate the effect of PTX on both forms of olfactory associative learning to the inhibition of the G(o) activation (Madalan, 2011).

The acute expression of PTX within the α/β and γ lobe neurons defined by the P247 driver is sufficient to inhibit both aversive and appetitive short-term olfactory memories. It was further shown through transgenic rescue experiments that the PTX inhibition of both aversive and appetitive short-term memories requires the G(o)α _Cys351 ADP-ribosylation site. PTX will only ribosylate heterotrimers (not individual α subunits), and the consequence of this ribosylation is inhibition of the heterotrimer activation. The inhibition of G(o) signaling by PTX is, therefore, extremely specific; since the ADP-ribosylated G(o) heterotrimers cannot be activated, they do not generate ectopic Gβ/γ subunits, nor do they sequester free Gβ/γ subunits away from other Gα subunits. Hence, the PTX loss-of-learning phenotype and the rescue of this deficit with the expression of G(o)α_Cys351Iso demonstrates that G(o) activation is required within the mushroom body neurons for the formation of short-term olfactory associative memories (Madalan, 2011).

Since anatomically distinct regions of the mushroom bodies have distinct roles in associative memory acquisition, stabilization, and recall, the identification of neurons that require G(o) activation provides important insight into the function of this signaling pathway during memory formation. Previously, it was found that PTX would partially affect negatively reinforced learning when expression was limited to either the α/β or γ mushroom body neurons, but when expressed in both subpopulations of neurons, as defined by the P247 and c772 Gal4 lines, it would almost completely eliminate memory formation. In contrast, the inhibition of G(o) activation within the α/β core neurons or within the DPM neurons had no effect on aversive memories. This studt further delineated the G(o) requirements during memory formation by excluding α'/β' lobe neurons. The DPM and α'/β' neurons are likely involved in a recurrent circuit that during both appetitive and aversive memory acquisition supports the consolidation of memories within the α/β lobe neurons of the mushroom bodies. The activation of G(o) is, therefore, required for aversive memory formation outside of the acquisition and stabilization events that occur within this α'/β'-DPM neuron circuit (Madalan, 2011).

These data highlight potential functions for this G(o) activation in memory formation. The stability of negatively reinforced olfactory memories appeared unaffected by the inhibition of G(o) activation, which suggests that G(o) activation is required during memory formation but not subsequently. In this last experiment, however, possible effects on memory stability may be hidden by the low performance found in flies expressing PTX within their α/β and γ lobe neurons and in the control flies trained with a single CS-US pairing. G(o) activation is also unlikely to be required for the initial encoding of CS strength or identity within the α/β and γ lobe neurons but may be involved in processing the electric shock or in subsequent memory formation processes. This latter conjecture is based partially on the fact that overtraining in the negatively reinforced learning paradigm will not compensate for the learning defect as would be expected if PTX reduced the salience of the odor stimuli. Moreover, the inhibition of G(o) activation differentially affected performance in the appetitive and aversive paradigms, which is again inconsistent with a change in odor strength or identity. The lower performance asymptote found in the acquisition curve after G(o) inhibition could, however, reflect a defect in processing the electric shock, in memory formation, or in reinforcement events acting downstream from this step, as well as in multiple processes. Although transducing the signal for the electric shock within the α/β and γ lobe neurons could account for the severely reduced learning found with PTX expression in both neuron types, it would fail to account for the phenotype found in appetitive memory. G(o) may also be required for a process shared by both appetitive and aversive memory systems that provides more general information about properties of the reinforcement, such as its predictive value, and, as such, could influence memory strength (Madalan, 2011).

The receptor(s) responsible for activating G(o) during memory formation and the downstream effectors are currently unknown. G(o) signaling pathways in vertebrates are better studied than in Drosophila and can offer insight into possible pathways for activation and effectors during Drosophila associative memory formation. G(o) is an extremely abundant membrane protein in the vertebrate brain, comprising between 1%-2% of total membrane protein, suggesting a common role in neural signal transduction. In vertebrate cells, G(o) is typically activated by GPCRs but can also be activated by non-GPCR receptors such as Amyloid Precursor Protein and GAP-43. The GPCRs that activate G(o) are typically of the G(i/o) coupled family and will generally inhibit neural activity; these include opioid receptors, different neuropeptide receptors, subtypes of mGluR receptors, and GABA_B receptors. However, the specificity of GPCR coupling to specific classes of G proteins is not absolute and may strongly depend on the cellular context of the GPCRs. The promiscuous coupling of GPCRs may also be regulated through post-transcriptional modification. For example, the palmitoylation of the Endothelin_B receptor results in a shift in coupling from G(i) to G(q), and the phosphorylation of the β2-adrengenic receptor by PKA leads to a G(s) to G(i) shift in coupling. Hence, it is not possible to a priori eliminate any GPCR from consideration based on receptor type and preferred coupling. Given this stipulation, one possible candidate for G(o) activating GPCRs within the α/β and γ lobe neurons is the dDA1 receptor. The dDA1 dopamine receptor is prominently expressed in the α/β and γ lobe neurons of the mushroom bodies where G(o) activation is required. Moreover, similar to the effects of PTX, dDA1 mutants display severe impairments in aversive memory and less severe impairments in appetitive memory. However, dDA1, which can activate cAMP synthesis, has not been shown capable of coupling to G(o) (Madalan, 2011).

Few G(o) effectors have been demonstrated in vertebrates, and in most cases, it is the βγ subunits that are responsible for actuating signaling. Presynaptic voltage-gated Ca²⁺ channels represent a major effector for G(o). G-protein βγ-subunits inhibit N-type (Ca_v2.2) and P/Q-type (Ca_v2.1) presynaptic Ca²⁺ channels involved in neurotransmitter release, causing a positive shift in their voltage dependence of activation. The inhibition is lifted by high-frequency action potentials. N-type channels also undergo a voltage-independent inhibition that is mediated by the direct binding of G(o)α to the α_1B subunit, resulting in an inhibition of Ca²⁺ current even after strong depolarization. These effects of activated G(o) on the presynaptic voltage-gated Ca²⁺ channels were described in cultured sensory neurons from embryonic chick dorsal root ganglion and set in motion by noradrenaline (NA), γ-aminobutyric acid (GABA), serotonin (5-HT), enkephalin, and somatostatin GPCRs. This voltage-gated Ca²⁺ channels effector pathway for G(o) is also present in central neurons, e.g., in Purkinje cerebellar neurons, elicited through GABA_B receptors, and in sympathetic neurons, e.g., effected through presynaptic β2-adrenergic autoreceptors, adenosine A1 receptors, and E2 (PGE2). The Drosophila cacophony voltage-gated Ca²⁺ channel α subunit may be a target for G(o) subunits during memory formation (Madalan, 2011 and references therein).

Pheromone binding to the VR2 receptors in rodent vomeronasal organs activates G(o), liberating Gβ/γ, which then activates phospholipase Cβ to mediate pheromone signal transduction. In Drosophila, plcβ21 is coexpressed with G(o)α in essentially the entire nervous system. Recently, Dahdal et al. found that Plcβ21 is an effector for G(o) in the Drosophila LN_vs neurons. Hence, the loss of Plcβ21 activation within the α/β or γ lobe neurons may account for the loss of short-term memory found after PTX expression (Madalan, 2011).

Lastly, the activation of adenylyl cyclase is an important G-protein signaling pathway involved in associative memory formation. In vertebrates, G(o) does not appear to signal through adenylyl cyclase. In Drosophila, G(o) signaling within the mushroom body neurons during memory formation is, at least partially, if not wholly, independent of the rut adenylyl cyclase. This conclusion was based on several considerations including the significantly stronger phenotype of PTX inhibition as compared to rut mutants. Additionally, PTX expression within the α/β lobe neurons will inhibit short-term memory, whereas rut activity within these neurons is not necessary for negatively reinforced short-term memory. Moreover, when PTX was lightly induced in rut²⁰⁸⁰ homozygotes or heterozygotes, PTX displayed additivity in the short-term memory phenotype. When these PTX- rut²⁰⁸⁰ 'double-mutant' experiments were performed, rut²⁰⁸⁰ was reported to be an amorph or, at least, a severe-hypomorph; more recent data indicates that rut mRNA levels are at ~25% wild-type levels in the rut²⁰⁸⁰ allele. Hence, it remains possible that the additivity found between PTX expression and the rut²⁰⁸⁰ may be due to residual activity in the rut²⁰⁸⁰ allele. Nevertheless, the learning phenotype for rut²⁰⁸⁰ is as strong as the reported amorphic rut¹ allele, and it even displays haploinsufficiency, arguing that a high level of the enzyme is required to support memory formation. This, together with the strength of the phenotypic differences and the differences in anatomical requirements for G(o) activation and rut, argues against a direct interaction between the cAMP pathway and G(o) activation during negatively reinforced memory formation. In positively reinforced memory, rut is required in the α'/β' neurons and the projection neurons of the antennal lobe. Thus, the role for G(o) activation in positively reinforced memory also maps outside the rut domains and, therefore, is also rut-independent (Madalan, 2011).

In summary, the activation of G(o) is an essential signaling event for associative memory formation. The G(o) pathway is required for the formation of both appetitive and aversive memories within the α/β and γ lobe neurons. Further elucidation of this pathway within these neurons will likely provide fundamental information on the molecular events underlying memory formation (Madalan, 2011).

Golgi-resident Galphao promotes protrusive membrane dynamics

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. This study found that the Galphao-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Galphao induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Galphao, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Galphao interactors, this study pinpoints several basic cellular activities, including vesicular trafficking, as being regulated by Galphao. It was further found small Golgi-residing GTPases Rab1 and Rab3 act as direct effectors of Galphao. This KDELR --> Galphao --> Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues (Solis, 2017).

Intracellular signaling pathways currently emerge more as dynamic networks of protein interactions rather than linear cascades of activation/inactivation reactions. In this regard, thorough elucidation of the interaction targets of heterotrimeric G proteins (the immediate transducers of GPCRs) is of crucial importance to advance the understanding of this type of signal transduction. It is especially true for Gαo. Being the most abundant G protein in the nervous system and controlling multiple evolutionary conserved developmental, physiologic, and pathologic programs, it has been remarkably shy in revealing its signaling partners. This study discloses results of multiple overlapping screens, identifying > 250 interaction partners of Gαo. Each of the screens performed has its inherent advantages and limitations, and by complementation, it is thought that a near complete coverage was obtained of the Gαo interactome -- an endeavor rarely performed for a signaling protein. Cherry-picking of individual proteins from this network resulted in detailed descriptions of mechanisms of Gαo-controlled regulation of Wnt/Fz signaling, synapse formation, PCP, asymmetric cell divisions, endocytic regulation, etc., validating the interactome findings (Solis, 2017).

As opposed to characterizations of selected individual Gαo partners, this study aimed at identifying functional modules within the interactome. For this, bioinformatics analysis clustering was performed the individual components by their functions. This resulted in appearance of several major cellular activities, which now emerge to be regulated by Gαo-dependent GPCR signaling. One of them, vesicular trafficking, was selected for detailed investigation. Many important components of this cellular function, both endocytic and exocytic, are found among Gαo targets. A study previously characterized interaction of Gαo and the endocytic master regulator Rab5, important for GPCR internalization and signaling. This study now focuses more on the exocytic function of Gαo. In various cell types (neuronal, epithelial, mesenchymal) of different animal groups (insect and mammalian) this study now finds a dual localization of Gαo to Golgi and PM, and the coordinated action of the two pools is found in exocytosis and formation of various types of cellular protrusions. This study further uncovered the evolutionary conserved KDELR --> Gαo --> Rab1/Rab3 pathway at Golgi, required for stimulated material delivery to PM and the growing protrusions (Solis, 2017).

KDELR is a Golgi-residing GPCR-like receptor, activated by the cargo delivery from ER and regulating both anterograde and retrograde trafficking from Golgi. This study shows that from Drosophila to mammals, KDELR binds Gαo and activates it, potentiating Gαo-induced cellular responses. Intriguingly, this study shows that it is the βγ-free form of Gαo that is the binding and activation partner of KDELR (in a sharp contrast to the action of typical PM-localized GPCRs, which act on heterotrimeric Gαβγ complexes. It was further found that KDELR and Gαo form a multi-subunit complex, additionally containing Rab1/Rab3 GTPses and αGDI. Activation of KDELR results in the nucleotide exchange on Gαo and its dissociation from KDELR. Although recombinant Rabs interact stronger with the GTP-loaded Gαo in vitro in absence of αGDI, in cells it was found that activation of Gαo leads to dissociation of the Rab1/Rab3-αGDI complexes, ultimately resulting in activation of the small GTPases and stimulated anterograde material delivery, necessary for the growth and stabilization of cellular protrusions. Activation of KDELR is known to induce formation of multicomponent aggregates recruiting a number of additional proteins (Majoul, 2001); recruitment of Rab-GEFs to these complexes to mediate ultimate activation of Rab1/Rab3 is also conceivable but will require further investigation. Importantly, the Golgi pool of Gαo plays key roles in these processes, as the anterograde transport as well as KDELR-mediated Rab1 activation are inhibited upon depletion of Gαo (Solis, 2017).

Based on the data presented in this study, a model emerges whereas specific Gαo pools at PM and Golgi play different but cooperative roles during neuritogenesis and protrusion formation in general. At PM, Gαo initiates neurite formation regulating actin and microtubule cytoskeletons in response to activation by specific GPCRs. At Golgi, the atypical GPCR KDELR induces activation of βγ-free Gαo, which subsequently activates Rab1 and Rab3, and the combined action of these proteins potentiates the PM-directed trafficking required for elongation and stability of membrane protrusions. Being conserved from Drosophila to mammals, this molecular mechanism is of basic importance for the understanding of G protein functions in development, physiology, and disease (Solis, 2017).

Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit

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).

GENE STRUCTURE

A Drosophila gene (G-oalpha47A) encoding a G protein alpha subunit has been isolated by screening genomic and adult head cDNA libraries using bovine transducin alpha subunit cDNA as probe. The gene, which maps to 47A on the second chromosome, encodes two proteins that are both 354 amino acids long but differ in seven amino acids in the amino-terminal region. The deduced amino acid sequences of the two proteins are 81% identical to the sequence of a rat Go alpha subunit. Analysis of genomic clones reveals that there are eight coding exons and that the putative transcripts for the two proteins differ in the 5'-noncoding regions and the first coding exons but share the remaining six coding exons. The arrangement of two different 5'-noncoding regions on the gene suggests that two different promoters regulate the expression of the transcripts encoding the two proteins. RNA blot analysis detected three transcripts: a 3.9-kilobase (kb) transcript found at all stages of development; a 5.4-kb transcript present predominantly in adult heads, and a 3.4-kb transcript present only in adult bodies. In situ hybridizations of a cDNA probe to adult tissue sections shows that the gene is expressed abundantly in neuronal cell bodies in the brain, optic lobe, and thoracic ganglia (Yoon, 1989).

Drosophila cDNA clones coding for the alpha subunit of a Go-like G protein have been isolated. The sequence of two cDNA clones shows there is alternative splicing in the 5'-coding region which, on conceptual translation, would give rise to two proteins with slightly different amino termini. A partial genomic clone indicates there are four introns in the carboxyl-terminal half of the clone. Two transcripts, 3.8 and 5.3 kilobases long, are expressed at a high level in the heads of adult flies and also in larvae, pupae, and embryos. Hybridization of the cDNA probes to sections of adult flies indicates the RNA is present predominantly in nervous tissue (in the cortex of the brain and the thoracic ganglion); it is also expressed in the ovaries. Transcripts corresponding to both cDNAs are present in the central nervous system, but only one of them is found in detectable levels in the ovaries. The gene maps to 47A on the Drosophila second chromosome (de Sousa, 1989).

Exons - Six common and two different 5' exons

PROTEIN STRUCTURE

Amino Acids - 354

Structural Domains

The sequence similarity is particulary striking between Drosophila G protein oalpha 47A proteins and the alpha subunit of rat Go (81% identity). The two Drosophila proteins also show 70% and 61% sequence identity to rat and Drosophila Gi alpha subunits, respectively (Yoon, 1989)

date revised: 1 September 2011

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.