InteractiveFly: GeneBrief

rugose : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - rugose Synonyms - A kinase anchor protein 550, Akap550, DAKAP550 Cytological map position - 4F4--5 Function - scaffolding protein Keywords - Notch pathway, Egfr pathway

Symbol - rg FlyBase ID: FBgn0003244 Genetic map position - 1-11.0 Classification - protein kinase A anchor protein Cellular location - cytoplasmic and nuclear

BIOLOGICAL OVERVIEW

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose has also been identified in a genetic screen for modifiers of Hairless (H), a Notch pathway antagonist (Schreiber, 2002) and rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Activation of Protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development in Drosophila. Thus, PKA-anchor protein complexes may be involved in controlling these crucial biological processes. Evaluation of this proposition requires knowledge of PKA binding/targeting proteins in the fly. DAKAP550/Rugose is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. Later in development it is found in neuroblasts and is enriched in neurons. It binds regulatory subunits (RII) of both mammalian and Drosophila PKAII isoforms. The anchor protein is expressed in many cells in nearly all tissues throughout the lifespan of the fly. However, DAKAP550 is highly enriched and asymmetrically positioned in subpopulations of neurons and in apical portions of cells in gut and trachea. The combination of RII (PKAII) binding activity with differential expression and polarized localization is consistent with a role for DAKAP550 in creating target loci for the reception of signals carried by cAMP (Han, 1997).

Genetic studies in Drosophila have shown that cAMP and PKA (A kinase or protein kinase A)-mediated signaling is required in a variety of processes, including oogenesis, establishment of tissue polarity in the embryo, imaginal disc morphogenesis, and synaptic function. In the developing eye, PKA plays an important role in the initiation of pattern formation and morphogenesis through its interactions with Hedgehog (Hh), DPP, and Wingless (Wg). Signaling by Sev, Egfr, N, and the cAMP-PKA pathways leads to the activation of a variety of downstream transcription factors. Because these signaling pathways regulate a multitude of cellular functions in different tissues and due to the number of transcription factors involved, it became apparent that a single signaling pathway alone could not specify particular cell fates. Recent studies have demonstrated that the determination of specific cell fates involves the integration of inputs from multiple pathways and a combinatorial code of activated transcription factors generates specific cell types. A kinase anchor proteins (AKAPs) are important components of the cAMP-PKA-mediated signal transduction pathway. AKAPs modulate the specificity of PKA function by targeting and compartmentalization of PKA to specific subcellular structures (Pawson, 1997; Scott, 2000). Studies on mammalian systems have led to the identification and molecular characterization of a large number of AKAPs, which mediate targeting of the PKA to various cellular organelles (Gray, 1998; Eide, 1998; Vo, 1998). Tissue and cell type-specific isoforms of AKAPs have recently been characterized (Dong, 1998). In Drosophila, biochemical characterization and molecular cloning led to the identification of two AKAPs, DAKAP 550 and DAKAP 200; however, no physiological roles have been reported for these proteins (Han, 1997; Li, 1999). Data from the genetic and phenotypic analyses of rugose mutants implicate DAKAP 550 in retinal pattern formation. Data on the genetic interactions of rugose suggest that rugose interacts with components of the signaling pathway mediated by the Egfr and N (Shamloula, 2002 and references therein).

The wild-type Drosophila eye is a hexagonal array of about 800 ommatidia. This arrangement of the ommatidia gives a smooth appearance to the external surface of the eye. Any perturbation of the reiterated pattern of the ommatidia results in a rough eye surface. In the wild-type eye each ommatidium contains eight photoreceptor cells (R cells), which occupy stereotypic positions. The plasma membrane of the R cells is multiply folded to form a tightly compacted array of microvilli known as the rhabdomere. The rhabdomeres of the cells R1–R6 are arranged in an asymmetric trapezoidal manner and extend the length of the retina. The rhabdomeres of the two central cells, R7 and R8, are smaller in profile and shorter in length than the R1–R6 rhabdomeres. They are positioned in the same axial plane as the distal R7 rhabdomere, on top of the more basal R8 rhabdomere. The lens-secreting cone cells are found on top of the apical surface of the R cells. The cone cells extend into thin bag-like structures between the R cells and terminate at the floor of the retina where their feet contribute to the formation of the fenestrated basement membrane. The pigment cells ensheathe the R cells and the cone cells. The anterior and posterior primary pigment cells form a cup-like structure around the cone cells. The secondary and tertiary pigment cells form a hexagonal lattice enclosing the ommatidia within. Each ommatidium shares six secondary pigment cells and three tertiary cells with its neighbors. The feet of the secondary and tertiary pigment cells flatten into the fenestrated membrane and form the floor of the retina. A scanning electron micrograph of the compound eye of a rg null allele (rg^gamma6) shows that the eyes of rg mutants are characterized by a rough external surface. The ommatidial facets of the rg mutants are irregular and not hexagonal as in the wild-type eyes. In addition, the mechano-sensory bristles are irregularly positioned and often multiple bristles are seen in place of a single bristle in each corner of the hexagonal facet seen in the wild type. Frequently in rg mutant eyes, the ommatidia exhibit abnormal numbers of R cells. The precise positions of the R cells are altered and the pigment cell lattice loses its hexagonal arrangement leading to the disorganization of the retina. The missing retinal photoreceptors can be seen in the region below the fenestrated basement membrane as seen in transverse sections of the compound eyes. This mispositioning of the R cells may be due to the defects in the fenestrated basement membrane caused by the abnormal number and organization of the cone cells and the disorganized pigment cell lattice (Shamloula, 2002).

To understand the cellular basis of the rough eye phenotype of the rg mutants the number and organization of the cone cells in wild type and rg mutants was examined. The number of cone cells can be determined by staining pupal stage eyes with cobalt sulfide. The cone cell number and organization in the various rugose mutant alleles has been determined. In the wild type, four cone cells are apical to the R cells and the individual cone cells can be identified by their specific position. On the basis of their positions, the four cone cells can be identified as an anterior cone cell, a posterior cone cell, an equatorial cone cell, and a polar cone cell. The eyes of rg mutants show abnormal numbers of cone cells when compared to the wild type. The number of cone cells varies in the different mutant alleles and is dependent on the level of the residual rg gene function. Partial loss-of-function alleles such as rg^gamma10 show two or three cone cells in the majority of the ommatidia, while strong alleles that behave as genetic nulls have either one or no cone cells in the majority of the ommatidia. Some of the rg mutant alleles also exhibit aberrant numbers of secondary/tertiary pigment cells (Shamloula, 2002).

Cut protein expression was used to identify the differentiating cone cells in the developing eye. Cut protein is a transcription factor and its expression in the developing eye is specific to cells that assume a cone cell fate. To localize cone cells in the developing eye, 60-hr pupal eyes were stained with the anti-Cut antibody. In the wild-type pupal eye, four cone cells are labeled with the anti-Cut antibody in each ommatidium whereas the hypomorphic rugose allele rg^gamma10 shows either one or two Cut positive cells. In the null allele, rg^gamma6, no Cut positive cells are seen showing the lack of differentiating cone cells (Shamloula, 2002).

To ascertain if the aberrant number of cone cells seen in rg mutants is due to failure of cone cell differentiation, developing pupal stage eyes were examined using transmission electron microscopy. In the developing eye, the R7 cell is the last photoreceptor to differentiate, resulting in a mature eight-cell cluster. The cone cells differentiate during the late third larval instar stages of development. Once the R7 cell nucleus completes its apical migration, two cone cell nuclei move up from the basal region and flank the R cell cluster. The cone cells spread over the apical tips of the photoreceptor cells and become the anterior and posterior cone cells. These cone cells contact each other centrally above the R cells. Subsequently, two more cells, the equatorial and polar cone cells, are added resulting in four cone cells. During the early pupal stages, in a wild-type eye a stereotypic arrangement of the R cells and the cone cells can be seen at the four-cone cell stage. In rugose mutants, cone cell specification is abnormal and consequently some of the cone cells fail to differentiate. In the hypomorphic allele, rg^gamma10, a single cone cell is seen, while in the null allele rg^gamma3 no cone cells are seen. In all of the mutant alleles tested, an undifferentiated precursor cell occupies the position of the cone cell and fails to differentiate (Shamloula, 2002).

To determine if mutations at the rugose locus affect R cell differentiation, the sequential specification of the R cells was followed in the third instar eye discs. Transmission electron microscopic analysis showed normal differentiation of R cell clusters in both wild-type and rugose mutant eye discs. In addition, immunochemical experiments involving a variety of R cell-specific probes also showed normal specification of the R cells in the developing third instar eye imaginal discs. This further supports the conclusion that rugose mutations do not affect R cell differentiation (Shamloula, 2002).

In addition to phenotypes in the eye, rugose mutants exhibit wing vein defects and embryonic lethality with partial penetrance. The penetrance is variable depending on the particular rugose allele. Loss of rugose function leads to an incomplete wing vein L5. rugose mutants also exhibit an embryonic semilethal phenotype. The extent of lethality varies with the strength of the rugose allele. In the null alleles rg^gamma6 and rg^gamma3, for example, 20%–40% of the fertilized eggs fail to hatch, suggesting defects in the embryonic stages. The cellular and molecular basis for the lethality is not clear at this point. The pleiotropic nature of rugose mutations is consistent with the multiple roles of cAMP-PKA-mediated signaling in Drosophila (Shamloula, 2002).

Thus rg is required for correct retinal pattern formation and mutations at the rugose locus result in rough eyes due to an aberrant ommatidial organization. This phenotype may be the consequence of a malformed retinal basement membrane and a defective pigment cell lattice. Analyses of the cellular basis of this phenotype revealed that, in rugose mutants, cone cell differentiation is abnormal, leading either to the lack of or to reduced numbers of cone cells. Cone cell number is sensitive to the rugose gene dosage, suggesting that rugose function is required for normal differentiation of the cone cells. Developmental studies employing both light and transmission electron microscopy have revealed that, in rugose mutants, cone cell development is defective even at the earliest stages and suggest that rugose is a positive effector of the cone cell determination. Cone cells belong to the R7 equivalence group and begin to differentiate immediately following R7 cell specification. In rugose mutants, the number of secondary/tertiary pigment cells is increased. This may be due to the alternative recruitment of the cone cell precursors to the pigment cell fate. Although the ommatidia are disorganized in rugose mutants, photoreceptor cell specification and differentiation are unaffected (Shamloula, 2002).

Direct evidence for the role of AKAPs in the regulation of signaling comes from studies in mammalian systems and has led to the characterization of a large family of AKAPs. Altered localization of PKA by ectopic expression in tissue culture cells results in the altered pattern of phosphorylation of PKA substrates such as ion channels and CREB (Feliciello, 1997; Gao, 1997; Gray, 1998). The data presented in this study suggest that rugose, a Drosophila AKAP, is important for correct retinal pattern formation in the developing eye, a function likely to be mediated through its effects on the subcellular localization of PKA. If true, mutations in rugose would be expected to result in the mislocalization of PKA, leading to altered signaling. Determining the cellular and subcellular distribution of PKA in rugose mutants will test this. At this point the precise role of the cAMP-PKA signaling pathway in cell fate specification in the Drosophila eye is unclear. The cAMP-PKA signal cascade may play a role in cell fate determination through its interactions with the Notch and Egfr pathways. Recent studies have shown that cell fate determination in the developing eye requires the integration of inputs from multiple signaling pathways and a combinatorial code involving the activation of specific transcription factors. The data from the genetic modifier screen show that rugose interacts with components of the Egfr and Notch signaling pathways in a dosage-dependent manner. Mutations that reduce either Egfr or N signaling behave as dominant enhancers of the rough eye phenotype. Conversely, gain-of-function mutations that increase Egfr or N signaling act as dominant suppressors of the rugose phenotype. The molecular basis of these observed interactions is not clear at the present time. It is proposed that these interactions between PKA signaling and the Egfr and Notch pathways are likely to be indirect (Shamloula, 2002).

In a separate study, Huang (2000) identified AKAP 200, the second Drosophila AKAP (Li, 1999), in a misexpression screen for genes that modulate RAS1 signaling. This suggests interactions between the RAS1 signaling and the cAMP-PKA pathways. Others have also reported interactions between the cAMP-PKA, RAS-MAPK, and the Notch pathways in a variety of systems. However, molecular details of interactions between the various pathways are not yet clear. It is possible that rugose interacts with the Egfr and N pathway components at the protein level. In this model, Rugose, as an AKAP, may be involved in anchoring components of other signaling pathways. Although initially it was thought that AKAPs were specific for PKA, it has been shown subsequently that AKAPs are multivalent and can anchor a wide variety of signaling components (Klauck, 1996; Pawson, 1997). Taken together the results suggest that rugose could exert its effects on cone cell specification and retinal pattern formation through its interactions with the Egfr and Notch pathways by facilitating the cross-talk between the different signaling pathways (Shamloula, 2002).

Temporally and Spatially Localized PKA Activity within Learning and Memory Circuitry Regulated by Network Feedback

Dynamic functional connectivity within brain circuits requires coordination of intercellular signaling and intracellular signal transduction. Critical roles for cAMP-dependent protein kinase A (PKA) signaling are well established in the Drosophila mushroom body (MB) learning and memory circuitry, but local PKA activity within this well-mapped neuronal network is uncharacterized. This study use an in vivo PKA activity sensor (PKA-SPARK) to test spatiotemporal regulatory requirements in the MB axon lobes. Immature animals had little detectable PKA activity, whereas postcritical period adults showed high field-selective activation primarily in just 3/16 defined output regions. In addition to the age-dependent PKA activity in distinct α'/β' lobe nodes, females show sex-dependent elevation compared with males in these same restricted regions. Loss of neural cell body Fragile X mental retardation protein (FMRP) and Rugose [human Neurobeachin (NBEA)] suppresses localized PKA activity, whereas overexpression (OE) of MB lobe PKA-synergist Meng-Po (human SBK1) promotes PKA activity. Elevated Meng-Po subverts the PKA age-dependence, with elevated activity in immature animals, and spatial-restriction, with striking γ lobe activity. Testing circuit signaling requirements with temperature-sensitive shibire (human Dynamin) blockade, broadly expanded PKA activity was found within the MB lobes. Using transgenic tetanus toxin to block MB synaptic output, greatly heightened PKA activity was found in virtually all MB lobe fields, although the age-dependence is maintained. It is concluded spatiotemporally restricted PKA activity signaling within this well-mapped learning/memory circuit is age-dependent and sex-dependent, driven by FMRP-Rugose pathway activation, temporally promoted by Meng-Po kinase function, and restricted by output neurotransmission providing network feedback (Sears, 2022).

This study explored the spatiotemporal regulation of PKA activity within the MB lobes. PKA signaling initiates in early adulthood, with heightened activity in just 3/16 MB lobe output neuron fields (α'1, β'1, and β'2ap). In addition to age-dependence, this regional PKA signaling displays sex-dependence, with elevation in females over males. These findings were made possible with the PKA-SPARK biosensor; this fluorescent reporter uses motifs found in earlier PKA sensors, with pharmacological and genetic approaches promoting and preventing PKA activity verifying this new tool in cell culture and in vivo. While it is possible that the PKA-SPARK reporter is revealing only the strongest PKA activity, at the least α'1, β'1, and β'2ap connectivity regions have much higher PKA activity levels compared with the rest of the MB neuropil. In two disease models of intellectual disability and ASDs, from loss of either FMRP or Rugose/NBEA, PKA activity remains spatiotemporally restricted, but is dramatically reduced. There is surprisingly little change from overactivation of the FMRP→Rg control pathway, but PKA activity is profoundly altered by the PKA pathway Meng-Po kinase, with OE enhancing spatiotemporal PKA signaling and loss suppressing PKA activity. Network feedback downstream of KC neurotransmission strongly suppresses PKA activity, since blocking KC synaptic output with conditional shibire^ts or transgenic TNT induces widespread PKA activity signaling. Thus, localized PKA activity is highly regulated at the circuit level (Sears, 2022).

At a macro level, the α'1 and β'1 regions with the highest localized PKA activity levels have been linked to valence, i.e., whether local activation causes animals to approach or avoid a stimulus. The α'1 and β'2 fields exhibit opposing output valence (positive and negative, respectively), while the β'1 role appears less clear. However, higher PKA signaling can be activated in the γ3 region (via Meng-Po), which in combination with the β'1 region drives positive valence. Moreover, dopaminergic and serotonergic biosensor signals function through the β' region from a variety of external sense stimuli, to which the α'1/β'1 fields display high sensitivity, suggesting broad responsiveness. Very recent work shows high spontaneous activity in α'/β' restricted to young animals , suggesting α'/β' PKA signaling may be controlled by selective developmental activity. Moreover, inhibiting miR-92a in α/β and γ, but not α'/β', was shown to enhance memory, indicating another layer of lobe-selective circuit regulation. Note that the output neurons from these MB lobe regions are different (α'1 cholinergic, β'1 GABAergic, and β'2 glutamatergic), suggesting more complex integrative circuit functions. PKA signaling activation after the early-use critical period is consistent with sensory experience-dependent regulation. Importantly, MB sensory integration functions differ markedly between females and males, correlating with the report in this study of sex-dependent PKA signaling differences in females compared with males (Sears, 2022).

Two learning and memory proteins, FMRP and Rugose, are needed for full PKA activity in the α'1 and β'1 MB lobe output neuron fields. RNA-binding FMRP is a translational regulator known to facilitate PKA signaling, which is lost in the FXS, the commonest heritable cause of intellectual disability and ASD. Rugose/NBEA is a PKA-anchor that facilitates learning and memory, and is also associated with ASDs. Previous work has shown FMRP binds to rugose mRNA to drive KC expression. As predicted, disruption of this FMRP→Rg regulative pathway strongly impairs PKA activity in the MB lobes. In contrast, localized PKA signaling is dramatically strengthened by MB OE of the Meng-Po kinase, which induces early-onset PKA activity before adult sensory experience, spatially expands high PKA activity to the γ3 MBON field, and profoundly elevates PKA activity within all the normal MB lobe regions of heightened PKA signaling. Consistently, Meng-Po kinase OE also greatly improves learning and memory, via PKA phosphorylation, but additionally via signaling feedback synergy. Moreover, meng-po RNAi causes the opposite result of reducing localized PKA-SPARK puncta. Based on both loss and gain of function, it is suggested that Meng-Po enhances localized PKA activity, reflecting circuit level kinase regulation. Determining how Meng-Po-regulated PKA activity determines circuit excitability and regional balance will be a major subject of future research (Sears, 2022).

Two different KC synaptic output blocking methods dramatically expand PKA activity signaling in the MB. Both conditional shibire^ts and transgenic tetanus toxin tools block KC neurotransmission, but through quite different mechanisms. At 33°C, KC-targeted shibire^ts drives PKA activity expansion in the MB γ lobe. This change could indicate cross-compartment network interactions between the γ lobe and other MB regions. Increasing γ1 PKA activity is especially interesting, as γ1 toggles inhibition of other MB regions. The tetanus toxin protease blocks neurotransmission through eliminating SV exocytosis, and therefore provides a stronger and more selective means to silence KC synaptic output. Consistently, TNT animals show a more profound expansion of PKA activity throughout the MB lobes, albeit again affecting only spatial and not temporal patterning. Neither shibire^ts shibire^ts nor TNT blockade alters early PKA activity, suggesting induction of PKA signaling is determined primarily by later experience-driven activity. Localized PKA activity changes with KC output block implies active circuit balance; for example, weighing aversive versus attractive responses to sensory stimuli. The widespread PKA activity upregulation with KC output block leads to a hypothesis that enhancing KC neuron activity should result in elevated PKA signaling (Sears, 2022).

At the MB circuit level, multiple candidate synaptic pathways need to be explored for roles in local PKA activity regulation in different MB lobe output neuron fields. GABAergic inputs to dopaminergic neurons are one likely candidate, since GABA treatment has been shown to correct dfmr1 mutant circuit defects exacerbated by glutamate exposure. Moreover, GABAergic anterior paired lateral (APL) neurons broadly control MB activity through feedback to the KCs, and are most strongly activated by the α'/β' lobes. Recent work shows that treatment with a dopamine transport inhibitor also ameliorates rugose mutant social interaction and memory deficits. Another candidate is the Amnesiac neuropeptide from the serotonergic dorsal paired medial neurons, required for their normal development in the broad innervation of the MB lobes. In the context of these studies, de facto depression may feedback onto KCs to promote PKA activity signaling. The impact of upstream input onto the MB lobes is an important consideration, including how this circuitry combines with spontaneous MB activity and internal lobe circuitry to determine PKA signaling. Future research directions should attempt to dissect how these different layers of neuromodulation control localized PKA activity signaling within the MB lobe circuit, and between females and males, by manipulating input-specific neuronal activity in targeted transgenic studies (Sears, 2022).

In conclusion, this study reports that PKA activity signaling in the Drosophila brain MB learning and memory center is highly induced during early experiential adulthood, with selective upregulation in the α'1, β'1, and β'2ap MB lobe output neuron regions. Age-specific and sex-specific PKA signaling controlled within KCs and downstream of KC output shows that spatiotemporally restricted MB lobe PKA activity is regulated through a combination of both intracellular control and intercellular network-level mechanisms. Importantly, PKA signaling can be precociously promoted and spatial expanded though the activity of the PKA-synergist Meng-Po kinase. Moreover, KC neurotransmission inhibits localized PKA signaling within the MB circuit. Future studies will be aimed toward generating new genetic responder tools to test KC signaling with both neurotransmission output blockade and activity promotion of upstream and downstream MB circuit components, simultaneously and independently of KCs. PKA activity signaling will be tested with the manipulation of specific KC partners, by altering neurotransmission signaling in combination with postsynaptic neurotransmitter receptor mutants to determine network communication cues. Taken together with the current work, these ongoing studies will continue to expand understanding of circuit-level PKA signaling regulation in normal function and in neurologic disease model contexts (Sears, 2022).

During the course of embryogenesis Drosophila expresses several RII-binding proteins with sizes ranging from 70,000 to >200,000 kDa. As the fly matures, the levels of the smaller binding proteins are diminished, thereby suggesting that these polypeptides play specialized roles in early development. In contrast, DAKAP550/Rugose is evident in many cells and nearly all tissues throughout development and becomes the principal anchor protein in adult flies. Although DAKAP550 accumulates in multiple cell types, the content of the anchor protein is regulated. DAKAP550 is significantly enriched in subpopulations of peripheral and central neurons (e.g., photoreceptor neurons) as well as in cells of the gut and trachea. Moreover, immunocytochemical staining revealed that DAKAP550 accumulates asymmetrically (e.g., apical localization) in certain neurons and portions of the gut and trachea. This pattern could potentially facilitate polarized cAMP signaling and is consistent with the previous published observations that targeted PKA activity is essential for anterior/posterior patterning, oocyte development, and differential segregation of mRNAs (Han, 1997).

Biochemical analysis has documented that the content of DAKAP550 is markedly elevated in anterior tissues of Drosophila. A similar pattern of enrichment was reported for Drosophila RII (PKAII) (Foster, 1984). Moreover, the anchor protein was recovered in the cytosolic fraction of tissue homogenates. This result does not preclude a tight association of DAKAP550 with cytoskeleton. Many components of highly-organized cytoskeletal structures appear in cytosol (e.g., microtubule-associated protein-2, actin-binding proteins, and paxillin) when tissues are homogenized in standard, low ionic strength buffers in the absence of specific stabilizing agents. Development of stabilizing conditions for Drosophila cytoskeleton will be essential to accurately monitor the distribution of the anchor protein with various subcellular fractions in future studies. Finally, it is also possible that DAKAP550 serves as a 'cytoplasmic anchor' or 'scaffold' to either (1) focus cAMP signaling to target proteins in cytoplasm, (2) inhibit signaling at certain membranes or cytoskeletal sites, or (3) co-assemble tethered PKAII with substrate/effector proteins directly bound to other domains of the anchor protein (Han, 1997).

REGULATION

Protein Interactions

Activation of protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development in Drosophila. Thus, PKA-anchor protein complexes may be involved in controlling these crucial biological processes. Evaluation of this proposition requires knowledge of PKA binding/targeting proteins in the fly. cDNAs encoding a novel, Drosophila A kinase anchor protein, DAKAP550, have been discovered and characterized. DAKAP550 is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. It binds regulatory subunits (RII) of both mammalian and Drosophila PKAII isoforms. The tethering region of DAKAP550 includes two proximal, but non-contiguous RII-binding sites (B1 and B2). The B1 domain (residues 1406-1425) binds RII ~20-fold more avidly than B2 (amino acids 1350-1369). Affinity-purified anti-DAKAP550 IgGs were exploited to demonstrate that the anchor protein is expressed in many cells in nearly all tissues throughout the lifespan of the fly. However, DAKAP550 is highly enriched and asymmetrically positioned in subpopulations of neurons and in apical portions of cells in gut and trachea. The combination of RII (PKAII) binding activity with differential expression and polarized localization is consistent with a role for DAKAP550 in creating target loci for the reception of signals carried by cAMP. The DAKAP550 gene was mapped to the 4F1.2 region of the X chromosome; flies that carry a deletion for this portion of the X chromosome lack DAKAP550 protein (Han, 1997).

The apparent K_D for the DAKAP550·RIIß complex was estimated to be ~10 nM by overlay binding assays. This value is similar to that observed for the binding of RIIß with AKAPs 75 and 79. The size and sequence of the RII-binding domain of DAKAP550 was characterized by a combination of mutagenesis, expression in E. coli, and RII-binding assays. All DAKAP550 cDNAs isolated from bacteriophage expression libraries by functional screening ( i.e., ³²P-RIIß overlay binding assays) contain a common 2-kbp fragment. Thus, this segment of DAKAP550 cDNA must encode amino acids that constitute an RII tethering site in the anchor protein. The shared 2-kbp cDNA fragment was excised via digestion with EcoRI and subcloned in pBluescript. Subsequently, the location of the RII tethering site was defined more precisely by applying exonuclease III digestion or polymerase chain reaction methodology to systematically delete 5' and/or 3' portions of the target cDNA. The 2-kbp cDNA fragment and its truncated derivatives were subcloned into the isopropyl-1-thio-ß-D-galactopyranoside-inducible E. coli expression plasmid pET14b. Upstream plasmid DNA encodes a 20-residue peptide that is appended at the N termini of the partial DAKAP550 proteins. The fusion peptide contains six consecutive His residues that enables purification of recombinant, chimeric proteins to near-homogeneity by affinity chromatography on a Ni²⁺-chelate resin. The shared 2-kbp cDNA encodes a partial protein (amino acids 1001-1676) in DAKAP550 that avidly binds RIIß. This protein was designated fu-B. (Several proteolytic fragments of fu-B also exhibit RIIß binding activity.) Elimination of 161 N-terminal residues and 240 C-terminal residues from fu-B yielded the D1 protein (residues 1162-1436 in DAKAP550). The RIIß binding activities of D1 and fu-B are very similar. However, further deletion of 26 amino acids from the C terminus of D1 generated a protein (D2) whose RII binding activity was diminished by ~90%. Residual RIIß tethering capacity was evident in the D2-3 (residues 1162-1397) and D3 (residues 1162-1381) recombinant proteins. Elimination of all RII binding activity was observed when the C terminus was further truncated to produce the D4 protein (residues 1162-1355). The recombinant, partial DAKAP550 proteins exhibited the same pattern of binding activity when mouse RIIalpha and Drosophila RII were used as the radiolabeled ligands. The tethering region defined by truncation analysis includes two stretches of 20 amino acids (designated B1 and B2), which contain arrangements of large, aliphatic amino acids (Leu, Ile, and Val) that are similar to each other and to patterns observed for previously characterized RII-binding sites. Residues comprising such binding sites are predicted to be assembled as an amphipathic helix by various algorithms. Moreover, mutagenesis/transfection experiments indicate that the creation of a large hydrophobic surface, rather than conservation of a specific primary sequence, governs high-affinity binding of RII isoforms at tethering sites in AKAPs. Computer-based predictions suggest that both the B1 and B2 regions fold into amphipathic helices that contain extended hydrophobic surfaces. However, the two tethering domains do not contribute equally to overall RII binding activity. A partial DAKAP550 protein that contains B1, but lacks B2, complexes ³²P-RII subunits nearly as avidly as a polypeptide (D1) that includes both B1 and B2. This result and the 90%-95% decrease in RII binding activity associated with selective disruption of B1 in the D2 protein) demonstrate that amino acids 1406-1425 constitute the high-affinity tethering site in the Drosophila anchor protein (Han, 1997).

Thus, the principal RII tethering domain maps to a region of DAKAP550 corresponding to amino acid residues 1406-1425 (B1). The arrangement of amino acids with long, aliphatic side chains (Leu, Ile, and Val) in B1 matches the distribution of critical, Leu, Ile, and Val residues in the RII tethering sites of AKAP75 and other mammalian AKAPs. Substitution of any of these residues with Ala eliminates or sharply reduces the ability of AKAP75 to sequester RII subunits. When the integrity of the B1 tethering site in DAKAP550 is disrupted by mutation, RII binding affinity declines by at least an order of magnitude. In the absence of B1, a reduced, but detectable level of RII binding avidity is contributed by a proximal RII-binding site (residues 1350-1369) designated B2. In contrast, partial DAKAP polypeptides containing either B1 alone or both B1 and B2 bind similar amounts of the RIIß ligand. Thus, under standard assay conditions the B1 tethering region plays a predominant role in sequestering RII subunits. The precise function of B2 remains to be determined. One speculative possibility is that in intact cells (where the PKAII concentration is higher than that used in overlay binding assays) B2 may provide a site for transient, rapidly reversible accumulation of RII subunits (Han, 1997).

Secondary structure predictions suggest that residues 1350-1369 (B2) and 1406-1425 (B1) in DAKAP550 fold as amphipathic helices with large hydrophobic surfaces. Mutagenesis and binding studies on mammalian AKAPs 75 and 79 indicate that both a large hydrophobic surface at the tethering site and a complementary hydrophobic surface on RIIß and RIIalpha dimers are essential for formation of stable AKAP-PKAII complexes. Evidently, binding of DAKAP550 with Drosophila RII (and mammalian RII isoforms) is driven by similar hydrophobic interactions. The preferential binding of RIIß with B1 (versus B2) indicates that the 'core' ligand affinity contributed by the hydrophobic surface is further modulated by additional features in the structure of DAKAP550. Possible factors involved in modulating affinities of B1 and B2 for RII are: (1) substitution of conserved Val or Leu with Thr at the C terminus of B2; (2) the presence of 10 hydrophobic residues on the surface of the predicted B1 amphipathic helix, as compared with 8 hydrophobic amino acids at the corresponding region of B2; (3) differences in non-conserved amino acids in the 20-residue tethering domains, and (4) stabilization/destabilization of RII-tethering site interactions by flanking segments of the DAKAP550 polypeptide. Further systematic studies, employing mutagenesis, protein expression, and RII binding analyses, will be required to identify amino acid residues that enhance or diminish the affinity of the B1 and B2 domains for RII isoforms (Han, 1997).

The RII-binding domain is inserted between two lengthy blocks of DAKAP550 sequence (residues 892-955 and 1693-1929) that are homologous with portions of both C. elegans F10F2.1 and the human beige-like protein (BLP). F10F2.1 lacks a comparable tethering site for RII subunits. However, this is expected because C. elegans has a single R subunit gene, which encodes a cAMP-binding protein that is a homolog of mammalian RI, not RII. The inclusion of DAKAP550 homology regions in BLP raises the possibility that the previously uncharacterized human protein may (by analogy) be involved in cAMP-mediated signaling. BLP contains a partially conserved, candidate RII binding sequence that should be amenable to characterization by mutagenesis and overlay binding assays (Han, 1997).

AKAPs act in a two-step mechanism of memory acquisition

Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. This study explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) this study identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. It is proposed that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition (Scheunemann, 2013).

Conditioned odor avoidance is subject to a general dichotomy since multiple memory components are engaged in control of behavior. This is usually analyzed at two time points, i.e., 3 min and 3 h after training. At 3 min, basal and dynamic STM are separable by genetic means as revealed by opposing phenotypes of rut¹ and dnc¹ mutants. Moreover, those components are also separable due to characteristic differences in their acquisition dynamics as revealed by different effects of shock number. A similar dichotomy applied to 3 h memory when ASM and ARM were separable by means of amnestic treatment. It was striking that basal STM and consolidated ARM were instantaneously acquired, resulting in a front line of protection by eliciting conditioned avoidance after a singular experience of a CS/US pairing. Interestingly, consolidated ARM (as defined by means of resisting amnestic treatment) was installed quickly after training. However, it remains to be addressed at the genetic and molecular level, whether 3 h ARM linearly results from the functionally similar basal SMT component (Scheunemann, 2013).

In contrast, the two components of dynamic STM and labile ASM acquire in a dynamic fashion but are clearly dissociated from each other by characteristic chronologies of CS/US pairings required for their acquisition. However, either component contributes to behavioral performance in addition to the appropriate instantaneous component, and hence, increases avoidance probability during the test situation. Considering a potential benefit from avoiding aversive situations this overall dichotomy of behavioral control seems plausible and is also reflected at the genetic level since rut-dependent cAMP signals are limited to support of dynamic but not instantaneous memory components. Rut-dependent STM and ASM, however, are dissociated by means of shock impact and discontinuous formation of ASM is limited to situations where animals repeatedly experience high-impact CS/US pairings within a predefined time window. Experience that does not meet this criterion, however, is not discounted but adds to continuously acquired dynamic STM. By functional means these two components are thus clearly separated but commonly dependent on rut-derived cAMP signals within the KC layer, forming ties between genetically and functionally defined memory components (Scheunemann, 2013).

Genetic dissection of memory has a long-standing history in Drosophila and provided a powerful means to define molecular, cellular, and neuronal networks involved in regulation of conditioned odor avoidance. Among others, the cAMP-signaling cascade has been identified as one central tenet of aversive odor memory foremost by means of two single-gene mutants affecting either a Ca²⁺-sensitive type 1 adenylyl cyclase (AC1) and/or a cAMP-specific type 4 phosphodiesterase (PDE4) affected in the Drosophila learning mutants rutabaga (rut-AC1) and dunce (dnc-PDE4). Although originally isolated due to poor performance in the aversive odor learning paradigm, a general dichotomy has been recently revealed that separates memory components by their dependency on either rut-AC1 or dnc-PDE4 function, and the view was established that two different types of cAMP signals are engaged during the single-cycle training procedure (Scheunemann, 2012). A similar dichotomy is observed at level of acquisition dynamics and suggests that rut-dependent cAMP signals are limited to formation of dynamically acquired memory components, i.e., dynamic STM and ASM. Interestingly, rut-dependent cAMP is also required for long-term memory (LTM), which acquires after spaced and repeated training sessions. Downstream the signaling cascade, however, appropriate cAMP signals are differently channeled to either support LTM in a CREB-dependent manner, ASM via tomosyn-dependent plasticity, or basal STM via synapsin-dependent regulation of synaptic efficacy. It appears that the chronology of CS/US pairings is an important determinant of which downstream effect is triggered and hence molecular mechanisms must be installed that are sensitive to the temporal order of training (Scheunemann, 2013).

At the level of molecular scaffolds, literature suggests that AKAPs serve the integration of cAMP with other signaling processes and are crucially involved in the control of a plethora of cellular functions in any organ. For example, AKAP79 coordinates cAMP and Ca²⁺ signaling in neurons to control ion channel activities. The recognized design principle of AKAPs to serve as molecular switch is well in line with the recognized two-step register mechanism involved in ASM formation. An increasing body of evidence shows that AKAPs are involved in memory processing across phyla and accordantly those studies revealed a contribution for support of matured, but not immediate memories. Communality among all those AKAP-dependent memories is the need for repeated and temporally organized training sessions, i.e., only spaced training sessions are effective to induce protein synthesis-dependent LTM in flies and mammals. Similarly, ASM requires the precise timing of two training sessions and mechanistically acts via an 'activated' state generated by the initial CS/US pairing that persists within the brain for ~5 min. Such temporal integration might well take place at level of AKAPs within the KC layer to operate rut-AC1-dependent cAMP signals finally onto phosphorylation of tomosyn. Identification of the particular AKAPs involved in two-step ASM formation will require further analysis of appropriate mutants. To date, only four Drosophila AKAPs are characterized, i.e., rugose, a 550 kDa protein that impacts on STM performance probably via molecular domains other than its AKAP function; yu/spoonbill that supports LTM; and Nervy and AKAP²⁰⁰ have not been tested for their impact on aversive odor memory (Scheunemann, 2013).

Together, the benchmarking of Drosophila aversive odor memory performance by means of acquisition dynamics that were demonstrated in this study will provide a valuable tool since dynamic aspects of acquisition are obviously informative and add to the steady-state condition determined by the single-cycle training procedure (Scheunemann, 2013).

DEVELOPMENTAL BIOLOGY

Antibodies directed against epitopes located between residues 1472 and 1673 in DAKAP550/Rugose were produced in rabbits and purified by affinity chromatography. In adult Drosophila, DAKAP550 accumulates preferentially in anterior tissues. The concentration of DAKAP550 in cytosol derived from fly heads is ~10-fold higher than the level of anchor protein in extracts of fly bodies. Binding assays also detected a 550-kDa protein, which accounts for a very high proportion of total RII tethering activity in adult Drosophila. Like the immunoreactive DAKAP550 polypeptide, the 550-kDa RIIß/RIIalpha-binding protein is isolated in cytosol and enriched ~8-12-fold in anterior tissues (head). Thus, DAKAP550 appears to be the principal PKA anchoring protein in mature flies (Han, 1997).

Affinity-purified IgGs directed against DAKAP550 were employed to monitor the expression and location of the anchor protein during Drosophila development. During early embryogenesis DAKAP550 was evident in most cells. From gastrulation onward the anchor protein seemed to be ubiquitous, but DAKAP550 levels differ markedly in distinct tissues. For example, at gastrulation anchor protein content is elevated in the ventral jacent mesectodermal cells. DAKAP550 immunoreactivity remains high in mesoderm for several hours, and was also elevated in neuroblasts delaminating from the ectoderm during stages 8-9. Some DAKAP550 appeared to be in the nuclei of several neuroblasts. Late in embryogenesis DAKAP550 is reduced in epidermis and skeletal muscle and becomes maximal in ventral nerve cord. Peripheral neurons, subsets of central neurons, hindgut, the tracheal system and salivary gland, also have substantial levels of DAKAP550 at this stage. Postembryonically, DAKAP550 is detected in imaginal discs and primordia for various adult tissues. The anchor protein is especially-enriched in neural cells such as photoreceptor neurons of the developing eye. In embryonic hindgut, trachea, and salivary gland, DAKAP550 is selectively concentrated in the apical portion of highly-polarized cells. In the photoreceptor neurons DAKAP550 is also concentrated apically; in addition, a basal localization is noted for the anchor protein in cytoplasmic granules in the same cells. Granular staining was also seen in cells of the larval brain (Han, 1997).

Synthesized antisense single-strand probes from the RgcD10 cDNA were used to follow the expression of rugose mRNA. The expression of rugose mRNA is dynamic during development and was detected in all the embryonic stages. The early expression at stage 5 showed enrichment anteriorly and expression was also seen ventrally in the posterior regions. The late stage 5 showed strong expression in the region of the cephalic furrow formation. During stage 6, the anterior localization of the message is strong including the cephalic furrow region. At stage 7, rg expression becomes maximized in the region of the anterior midgut and persists through stage 8. During the later stages of development (stages 13–17), the expression is pronounced in the developing nervous system. The RgcD10 expression is also seen in the antenna-maxillary complex. In the developing eye imaginal disc, RgcD10 is expressed throughout the disc and in the region of the morphogenetic furrow. The expression of the RgcD10 transcript is highly reduced or absent in the rg mutant eye discs. The rg transcript expression patterns seen in these experiments are similar to the rg expression pattern reported by Schreiber (2002) and consistent with the DAKAP expression patterns reported by Han in 1997 (Shamloula, 2002).

EFFECTS OF MUTATION

Hairless was identified as antagonist in the Notch signaling pathway based on genetic interactions. Molecularly, Hairless inhibits Notch target gene activation by directly binding to the Notch signal transducer Su(H). Additional functional domains apart from the Su(H) binding domain, however, suggest additional roles for the Hairless protein. To further understanding of Hairless functions, a genetic screen was performed for modifiers of a rough eye phenotype caused by overexpression of Hairless during eye development. A number of enhancers were identified that comprise mutations in components of Notch- and Egfr-signaling pathways, some unknown genes and the gene rugose. Mutant alleles of rugose display manifold genetic interactions with mutants in Notch and Egfr signaling pathway components. Accordingly, the rugose eye phenotype is rescued by Hairless and enhanced by Delta. Molecularly, interactions might occur at the protein level because rugose appears not to be a direct transcriptional target of Notch (Schreiber, 2002).

Although several alleles of the rg mutation have been described in the literature, all but rg¹ are extinct. rg¹ is a hypomorphic allele and the rough eye phenotype is temperature sensitive. At 17° the eyes are almost normal with a very slight rough appearance, whereas at 25° the eyes are moderately rough. When flies are grown at 29° the eyes become severely rough. A number of rugose mutant alleles have been isolated using EMS, gamma-rays and P elements as mutagenic agents. New rg alleles were isolated on the basis of their failure to complement the rg¹ allele. The severity of the eye phenotype varies in the different alleles. On the basis of the degree of roughness of the eye the mutant eyes have been classified into mild, moderate, and severe classes. From mutagenic screens, several alleles have been isolated that have severe eye phenotypes and behave as genetic nulls. Hypomorphic alleles or partial loss-of-function mutations show mild or moderate roughness of the eye. In the gamma-ray and EMS mutagenesis, 60,000 and 35,000 F₁ progeny, respectively, have been screened. Six P-element-induced alleles of rg were isolated from screening 30,000 F₂ progeny (Shamloula, 2002).

P-element- induced mutations can be due to either insertions or deletions caused by imprecise excision of an element. Remobilization of the P element, resulting in the reversion of the mutant phenotype, can identify insertions. Reversion analysis of the two P-induced alleles, rg^P2 and rg^P5, was carried out. The revertants show restoration of the cone cell number as well as the smooth eye characteristic of the wild-type eye. The remobilization of the P element also yielded several partial revertants consistent with imprecise excision of the inserted P element. These results indicate that rg^P2 and rg^P5 are insertion mutants (Shamloula, 2002).

Genetic interactions uncover interrelationships between components of a cellular pathway or interactions between different cellular pathways. Genetic modifier screens offer a simple and powerful method of identifying genetic interactions of the gene of interest. The rough eye phenotype of the rg mutants has been used to identify the genetic interactions of rugose. A dominant modifier F₁ screen was conducted by using autosomal deficiencies from the Bloomington Stock Center deficiency kit. The hemizygous rg males were placed in double mutant combination with a single copy of the autosomal deficiency and scored for either suppression or enhancement of the rough eye phenotype. A total of 118 autosomal deficiency stocks (51 on chromosome II and 68 on chromosome III), covering ~60% of the autosomes, were screened. Genes that mapped within the deficiency breakpoints were identified and mutant alleles were obtained and tested for their interactions with various rugose mutant alleles. Effects of a 50% reduction in the dosage of the interacting locus were sought. For some of the interacting genetic loci, the effect of one copy of the gain-of-function allele was tested on rugose phenotype. To do this, transgenic lines were used carrying heat-shock promoter constructs ectopically expressing the wild-type gene product. The results suggest that rugose interacts with the components of the Drosophila EGFR- and Notch-activated signal transduction cascades (Shamloula, 2002).

Argos (aos) is a secreted protein having an EGF motif. Partial loss-of-function mutations in argos result in rough eyes with supernumerary cone and R cells and extra-wing-vein phenotypes. Complete loss-of-function mutants of argos are embryonic lethals. While loss-of-function argos mutants have increased numbers of cone cells, heat-shock promoter-driven overexpression of Argos leads to a reduction in the number of cone cells. Cell culture experiments have shown that Argos is a negative regulator of the Egfr. argos mutations act as strong suppressors of the rugose mutation. The rough eye phenotype of rg is completely suppressed by a single copy of the argos loss-of-function mutation. rg/Y; argos/+ double mutants have smooth eyes and normal complement of R cells as well as cone cells. Consistent with this result is the finding that heat-shock promoter-driven overexpression of Argos acts as a dominant enhancer of the rg mutant phenotype. In a genetic screen for second-site modifiers of the argos phenotype two interacting genes have been identified and it has been suggested that they may function in the Argos-mediated signaling pathway. Mutations in bulge and soba act as dominant suppressors of the rough eye phenotype of an argos amorphic allele as well as the rough eye phenotype caused by the heat-shock-induced overexpression of the Argos protein. A single copy of bulge and soba dominantly enhance the rough eye phenotype of the rg mutants. These results are consistent with the finding that argos mutations act as strong suppressors of rugose (Shamloula, 2002).

Star, rhomboid, and spitz belong to the 'spitz' group of genes and encode an essential function necessary for ventral midline development. In addition to the recessive lethal embryonic phenotype, S mutations are haplo-insufficient and show a dominant, rough eye phenotype. During development, S is required in a wide variety of tissues and S mutations show genetic interactions with genes from multiple signaling pathways. S encodes a putative membrane protein that, in combination with Rhomboid (rho), participates in the processing of the EGFR ligand, Spitz. In the modifier screen an S deficiency was identified as a strong enhancer of the rugose rough phenotype. A number of S alleles have been tested for their interactions with multiple alleles of rugose. Mutations at the S locus act as strong enhancers of the rugose eye phenotype and conversely heat-shock promoter-driven overexpression of the wild-type Star protein acts as a dominant suppressor of the rugose mutant phenotype. Consistent with these results, rhomboid (rho) mutations act as dominant enhancers of the rough eye phenotype of rugose mutants. rho encodes a novel intramembrane serine protease and is involved in the proteolytic processing of the EGFR ligand Spitz. A single copy of the rho mutation acts as an enhancer of the rg eye phenotype and a single copy of the hs-rho acts as a weak suppressor (Shamloula, 2002).

The Drosophila homolog of Egfr is an RTK that activates a highly conserved signal transduction cascade in a variety of tissue and cell types during Drosophila development. The activation of the Egfr is dependent on the tissue/cell type-specific ligands at the specific developmental stage. In the developing eye, Egfr function is required for the determination of all retinal cell types. A single copy of the mutations in Egfr acts as a mild enhancer of the rugose eye phenotype. In addition, Ellipse, a dominant mutation in Egfr (Egfr^E), acts as a suppressor suggesting that rugose interacts with the Egfr-mediated signal cascade (Shamloula, 2002).

ras1 is a Drosophila homolog of the human ras genes (H-ras, Ki-ras, and N-ras). Ras1 is a GTPase, which functions as the key transducer in several of the receptor tyrosine kinase-activated cellular signal transduction pathways. In the developing eye, ras1 is required for the specification of photoreceptors as well as cone cells. Reduction or loss of Ras1 activity results in the failure of photoreceptor cell determination. A constitutively active form of Ras1 (Ras^v12) results in the overrecruitment of retinal cells. The effects were tested of the ras1 mutations on the rg eye phenotype. A 50% reduction in ras1 activity acts as a dominant enhancer of the rg rough eye phenotype. In addition, a single copy of the dominant negative mutant form of Ras^N17 acts as a strong enhancer of the rg eye phenotype. In these experiments, a single copy of the constitutively active Ras^v12 was a weak suppressor of the rough eye phenotype of rg. These data suggest that Ras1 and rugose interact in a dose-dependent manner and may function synergistically in retinal pattern formation (Shamloula, 2002).

Rolled is a mitogen-activated protein kinase that functions at the last step in the Ras-MAPK phosphorylation cascade. Activated Rolled activates downstream transcription factors and thus plays a key role in the Egfr-mediated signaling required for cell determination and pattern formation. rll/+; rg/Y double mutants were constructed to test for genetic interactions with rg, and a single copy of the rolled loss-of-function mutation enhances the rough eye phenotype of rg. A single copy of the dominant gain-of-function rolled mutation acts a suppressor of the rough eye phenotype. Taken together these results suggest that rugose interacts with the components of the signal cascade activated by Egfr (Shamloula, 2002).

Sparkling is a Drosophila homolog of the vertebrate pax-2 gene and is involved in cone cell specification. The runt family transcription factor, Lozenge, has been shown to directly regulate spa and Lozenge is a key downstream mediator of the Notch and Egfr pathways. A single copy of the spa mutation acts as dominant enhancer of the rg eye phenotype. These results are consistent with the data showing that rg interacts with the Egfr and Notch signaling pathways (Shamloula, 2002).

The Delta-Notch pathway is involved in a variety of cell fate decisions during development. A single copy of the Delta mutation acts as strong dominant enhancer of the rough eye phenotype of rugose. Similarly, a single copy of the Suppressor of Hairless [Su(H)] mutation also acts as an enhancer of the rugose eye phenotype. Conversely, a single copy of the Notch pathway antagonist, Hairless (H), acts as dominant suppressor of the rg phenotype. The results suggest that rugose interacts with the components of the Notch signaling pathway (Shamloula, 2002).

Dissociation of rugose-dependent short-term memory component from memory consolidation in Drosophila

Extensive investigations show several molecular and neuroanatomical mechanisms underlying short-lived and long-lasting memory in Drosophila. At the molecular level, the genetic pathway of memory formation, which was obtained through mutant research, seems to occur sequentially. So far, studies of Drosophila mutants appear to support the idea that mutants defective in short-term memory (STM) are always associated with long-term memory (LTM) impairment. At the neuroanatomical level, distinct memory traces are partially independently distributed. However, whether memory phase dissociation also exists at the molecular level remains unclear. This study reports on molecular separation of STM and consolidated memory through genetic dissection of rugose mutants. Mutants in the rugose gene, which encodes an evolutionarily conserved A-kinase anchor protein, show immediate memory defects as assayed through aversive olfactory conditioning. Intriguingly, two well-defined consolidated memory components, anesthesia-resistant memory and protein synthesis-dependent LTM, are both normal in spite of the defective immediate memory after 10-session massed and spaced training. Moreover, rugose genetically interacts with cyclic AMP-protein kinase A signaling during STM formation. Considering a previous study that AKAP Yu specifically participates in LTM formation, these results suggest that there exists a molecular level of memory phase dissociation with distinct AKAPs in Drosophila (Zhao, 2013).

Wise, A., Tenezaca, L., Fernandez, R.W., Schatoff, E., Flores, J., Ueda, A., Zhong, X., Wu, C.F., Simon, A.F. and Venkatesh, T. (2015). Drosophila mutants of the autism candidate gene neurobeachin (rugose) exhibit neuro-developmental disorders, aberrant synaptic properties, altered locomotion, and impaired adult social behavior and activity patterns. J Neurogenet 29: 135-143. PubMed ID: 26100104

Drosophila mutants of the autism candidate gene neurobeachin (rugose) exhibit neuro-developmental disorders, aberrant synaptic properties, altered locomotion, and impaired adult social behavior and activity patterns

Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions, and hyperactivity. ASD exhibits a strong genetic component with underlying multigene interactions. Candidate gene studies have shown that the neurobeachin (NBEA) gene is disrupted in human patients with idiopathic autism. The NBEA gene spans the common fragile site FRA 13A and encodes a signal scaffold protein. In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. Rugose (rg) is the Drosophila homolog of the mammalian and human NBEA. Previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the epidermal growth factor receptor or EGFR and Notch-mediated signaling pathways, facilitating cross talk between these and other pathways. This study presents functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion, and hyperactivity. These results demonstrate that Drosophila NBEA (rg) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying ASDs (Wise, 2015).

Data presented in this study implicate Drosophila rg in synaptic development, physiology, and adult behavior. Studies on the larval NMJ show altered morphological features and physiological properties of the synapses. rg mutants show significant decreases in the number of boutons at the NMJ, altered bouton shapes, and no significant changes in the bouton size. Overall, there is a decrease in the number of boutons in all of the alleles tested. Electrophysiological studies on the larval neuromuscular junction show an increase in the size of individual EJPs, and synapses also show pair-pulse depression compared with facilitation in the CS control with repetitive stimuli. In addition, rg mutant larvae display aberrant locomotor behavior with decreases in peristaltic movement and speed, and alterations in posture. These findings are consistent with a functional role for rg at the synapse (Wise, 2015).

Rugose is the Drosophila homolog of the mammalian NBEA, a scaffolding protein implicated in neurotransmitter/endomembrane vesicle trafficking at the synapse. Other studies on NBEA lend further support to its functional role at the synapse. In mice, loss-of-function of NBEA protein completely blocks evoked synaptic transmission at neuromuscular junctions while nerve conduction, synaptic structure, and spontaneous neurotransmitter release remain normal. NBEA has also been implicated in vesicular traffic at the synapse and has been shown to be required for normal development of the synapses. Recent studies have shown that the NBEA gene is disrupted in individuals with ASD and the NBEA gene spans the common fragile site FRA 13A in humans. Individuals with fragile X syndrome and autism have reduced levels of cAMP. This has been shown to lead to a decrease in evoked synaptic potential, dendritic architecture, and actin “clumping” in areas near the post-synaptic membrane (Wise, 2015).

Results from this study are consistent with earlier studies on the effects of altered cAMP metabolism on synaptic plasticity in adult Drosophila and neurotransmission at the larval NMJ. The modulation of the cAMP signaling at the synapse by rg may be through its function as a signal scaffold for protein kinase A or A kinase anchoring protein (AKAP). Mammalian AKAPs have been shown to maintain post-synaptic scaffolds by simultaneously associating with other kinases and phosphatases. For example, AKAP79/150 has been shown to be targeted to dendritic spines by a binding motif in the N-terminus which complexes with phosphatidylinositol-4,5-bisphosphate (PIP2), F-actin, and actin-linked cadherin adhesion molecules. rg may work in a similar manner, which would allow for changes in the appearance of synaptic boutons. AKAPs are key mediators of cAMP as well as other signal transduction pathways. Presynaptically, AKAPs have been shown to regulate ion channel function, particularly that of Ca2+ channels, which are required for vesicle fusion. PKA and AKAPs together can increase the efficiency of these channels by 3-10 fold, allowing for greater current to flow. The molecular mechanism that directly links rg function to evoked synaptic transmission remains unclear. However, AKAPs have also been shown to directly interact with adenylate cyclase in neurons thereby regulating the amount of cAMP that is produced in the cell. This may provide a mechanism by which changes in presynaptic vesicular release lead to observed changes in transmission and plasticity (Wise, 2015).

In humans, disruption of the NBEA gene results in idiopathic autism and the autistic individuals typically display hyperactivity and have difficulty with social interactions. To look for similar adult behavior correlates in flies, in addition to examining rg effects in larvae, the consequences of rg mutation on the behavior of adult flies were studied. It was found that rg mutants are similarly both more active and are socially avoidant. This conclusion is based on outcomes of rg mutants’ behavior in assays of social signals. First, the ability of the flies to avoid the dSO left by agitated flies in the avoidance assay was tested. Next, flies’ response to others in social clustering, the measure of distances to their closest neighbor (their social space), in a stable undisturbed group was tested. Social space and social avoidance probably result from equilibrium between multiple attractive and repulsive cues, in addition to environmental factors. Analyzing both the response to attractive signals, as in individual space, and to repulsive signals, as in social avoidance, can help differentiate between different kinds of social deficits. Individuals who do not perform well in either assay would not recognize or care for social signals, and could be characterized as socially indifferent. However, individuals who would have a bigger individual space, but strong avoidance of stressed individuals would be efficient at recognizing social signals, and decide to avoid interactions; thus could be characterized as socially avoidant. It was found that despite proper olfaction, rg mutants unlike the CS control, tend to not avoid the stress odorant left by stressed flies. Instead, they settle further away from their neighbors. It is worth noting that the speed of flies in motion or their activity levels does not affect the distance at which flies choose to finally settle when they form immobile groups. Thus, the behavior data suggest that rg mutant flies are socially indifferent, since they are less responsive both to stressful social signals in the avoidance assay, and to their neighbor in a stable group (Wise, 2015).

The findings on several aspects of synaptic properties, from formation and development of synaptic structures to synaptic release, post-synaptic response amplitude, and behavioral output, suggest a functional role for rg at the synapse. These results are consistent with the working hypothesis that rg is important for targeting and/or sequestering various proteins of cAMP-PKA signaling pathways to specific areas in the neuron. In addition to this potential role at the synapse, it was found that rg functions in pathways involved in regulating behavior, both at the larval and adult stages, to modulate locomotion, activity levels, and response to social signals. The high degree of structural and functional similarity between rg and NBEA suggests an evolutionarily conserved functional role essential for synapse formation and transmission, in pathways of conserved function, making rg a good candidate gene for studies on autism (Wise, 2015).

REFERENCES

Search PubMed for articles about Drosophila rugose

Dong, F. M., et al. (1998). Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein. J. Biol. Chem. 273: 6533-6541. 9497389

Eide, T. et al. (1998). Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp. Cell Res. 238: 305-316. 9473338

Feliciello, A. Y., et al. (1997). A-kinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus. Curr. Biol. 7: 1011-1014. 9382844

Foster, J. L, Guttman, J. J., Hall, L. M., and Rosen, O. M. (1984) J. Biol. Chem. 259: 13049-13055. 6436241

Gao, T. A., et al. (1997). cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19: 185-196. 9247274

Gray, P. C., Scott, J. D. and Catterall, W. A. (1998). Regulation of ion channels by cAMP-dependent protein kinase and A-kinase anchoring proteins. Curr. Opin. Neurobiol. 8: 330-334. 9687361

Han, J. D., Baker, N. E. and Rubin, C. S. (1997). Molecular characterization of a novel A kinase anchor protein from Drosophila melanogaster. J. Biol. Chem. 272(42): 26611-26619. 9334242

Huang, A. M. and Rubin, G. M. (2000). A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster. Genetics 156: 1219-1230. 11063696

Klauck, T. M., et al. (1996). Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271: 1589-1592. 8599116

Li, Z., et al. (1999). Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene. J. Biol. Chem. 274: 27191-27200. 10480936

Pawson, T. and Scott, J. D. (1997). Signaling through scaffold, anchoring, and adaptor proteins. Science 278: 2075-2080. 9405336

Scheunemann, L., Jost, E., Richlitzki, A., Day, J. P., Sebastian, S., Thum, A. S., Efetova, M., Davies, S. A. and Schwarzel, M. (2012). Consolidated and labile odor memory are separately encoded within the Drosophila brain. J Neurosci 32: 17163-17171. PubMed ID: 23197709

Scheunemann, L., Skroblin, P., Hundsrucker, C., Klussmann, E., Efetova, M. and Schwarzel, M. (2013). AKAPs act in a two-step mechanism of memory acquisition. J Neurosci 33: 17422-17428. PubMed ID: 24174675

Schreiber, S. L., et al. (2002). Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the Notch antagonist Hairless in Drosophila. Genesis 33(3): 141-52. 12124948

Scott, J. D. and Pawson, T. (2000). Cell communication: the inside story. Sci. Am. 282: 72-79. 10862426

Sears, J. C. and Broadie, K. (2022). Temporally and Spatially Localized PKA Activity within Learning and Memory Circuitry Regulated by Network Feedback. eNeuro 9(2). PubMed ID: 35301221

Shamloula, H. K., et al. (2002). rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways. Genetics 161(2): 693-710. 12072466

Vo, N. K., Gettemy, J. M. and Coghlan, V. M. (1998). Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs). Biochem. Biophys. Res. Commun. 246: 831-835. 9618298

Wang, J. W., et al. (2001). Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. J. Immunol. 166(7): 4586-95. 11254716

Wang, X., et al. (2000). Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic. J Neurosci. 20(23): 8551-65. 11102458

Wise, A., Tenezaca, L., Fernandez, R.W., Schatoff, E., Flores, J., Ueda, A., Zhong, X., Wu, C.F., Simon, A.F. and Venkatesh, T. (2015). Drosophila mutants of the autism candidate gene neurobeachin (rugose) exhibit neuro-developmental disorders, aberrant synaptic properties, altered locomotion, and impaired adult social behavior and activity patterns. J Neurogenet 29: 135-143. PubMed ID: 26100104

Zhao, J., Lu, Y., Zhao, X., Yao, X., Shuai, Y., Huang, C., Wang, L., Jeong, S. H. and Zhong, Y. (2013). Dissociation of rugose-dependent short-term memory component from memory consolidation in Drosophila. Genes Brain Behav 12: 626-632. PubMed ID: 23790035

date revised: 25 October 2023

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