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Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo- ), SK channel (dSK- ), or both (dSK- ;; dSlo- ) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that Drosophila with a single copy of the AOS-like Su(H) allele behave in an opposing manner as flies with a Su(H) null allele due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants decrease DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby a single RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

Copyright © 2020 American Chemical Society. We report the surface functionalization of anionic layer by layer nanoparticles (LbL NPs) with cationic tumor-penetrating peptides (TPPs) via electrostatic adsorption while retaining particle stability and charge characteristics. This strategy eliminates the need for structural modifications of the peptide and enables facile functionalization of surface chemistries difficult to modify or inaccessible via covalent conjugation strategies. We show that both carboxylated and sulfated LbL NPs are able to accommodate linear and cyclic TPPs and used fluorescence-based detection assays to quantify peptide loading per NP. We also demonstrate that TPP activity is retained upon adsorption, implying sufficient numbers of peptides take on the appropriate surface orientation, enabling efficient uptake of functionalized NPs in vitro, as characterized via flow cytometry and deconvolution microscopy. Overall, we believe that this strategy will serve as a broadly applicable approach to impart electrostatically assembled NPs with bioactive peptide motifs.

Ca(2+)-activated K(+) channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo(−)), SK channel (dSK(−)), or both (dSK(−);; dSlo(−)) is shaped in the female fruit fly (Drosophila melanogaster) R1–R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1–R6 Shaker and Shab K(+) conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca(2+)-activated K(+) channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information. SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.

Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt–Jakob disease, kuru and Gerstmann–Straussler–Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein.

Since the accident with a cascade failure of photomultiplier tubes (PMTs) in the Super-Kamiokande experiment in 2001, the mechanical performance of large format semi-hemispherical PMTs has become a critical issue for large water Cherenkov detectors. The subject of this study is the survival of an assembled array of PMTs under significant hydrostatic pressure and subjected to shock waves caused by the failure of a single PMT. This paper details the results of the second stage of a R&D program focused on the design and testing of different PMT assemblies to mitigate the risk of a “chain-reaction” of PMT failures. The initial results show that our PMT assembly design can effectively reduce the magnitude of the shock wave. With the testing results in this paper and the hydrodynamic simulation calculation, we can further improve the design of PMT deployment to mitigate the risk of chain reactions caused by implosion induced shock waves.

Large, deep, well shielded liquid detectors have become an important technology for the detection of neutrinos over a wide dynamic range from few MeV to TeV. The critical component of this technology is the large format semi-hemispherical photo-multiplier tube with diameters in the range of 25–50 cm. The survival of an assembled array of these photo-multiplier tubes under high hydrostatic pressure is the subject of this study. These are the results from an R&D program which is intended to understand the modes of failure when a photo-multiplier tube implodes under hydrostatic pressure. Our tests include detailed measurements of the shock wave which results from the implosion of a photo-multiplier tube and a comparison of the test data to modern hydrodynamic simulation codes. Using these results we can extrapolate to other tube geometries and make recommendation on deployment of the photo-multiplier tubes in deep water detectors with a focus on risk mitigation from a tube implosion shock wave causing a chain reaction loss of multiple tubes.

Large, deep, well shielded liquid detectors have become an important technology for the detection of neutrinos over a wide dynamic range of a few MeV to TeV. The critical component of this technology is the large format semihemispherical photo-multiplier tube with diameters in the range of 25 to 50 cm. The survival of an assembled array of these photo-multiplier tubes under high hydrostatic pressure is the subject of this study. These are the results from an R&D program which is intended to understand the modes of failure when a photo-multiplier tube implodes under hydrostatic pressure. Our tests include detailed measurements of the shock wave which results from the implosion of a photo-multiplier tube and a comparison of the test data to modern hydrodynamic simulation codes. Using these results we can extrapolate to other tube geometries and make recommendation on deployment of the photo-multiplier tubes in deep water detectors with a focus on risk mitigation from a tube implosion shock wave causing a chain reaction loss of multiple tubes.

The contribution of the SK (small-conductance calcium-activated potassium) channel to neuronal functions in complex circuits underlying sensory processing and behavior is largely unknown in the absence of suitable animal models. Here, we generated aDrosophilaline that lacks the single highly conservedSKgene in its genome (dSK). In R1–R6 photoreceptors,dSKencodes a slow Ca2+-activated K+current similar to its mammalian counterparts. Compared with wild-type,dSK−photoreceptors and interneurons showed accelerated oscillatory responses and adaptation. These enhanced kinetics were accompanied with more depolarizeddSK−photoreceptors axons, assigning a role for dSK in network gain control during light-to-dark transitions. However, compensatory network adaptation, through increasing activity between synaptic neighbors, overcame many detriments of missing dSK current enablingdSK−photoreceptors to maintain normal information transfer rates to naturalistic stimuli. While demonstrating important functional roles for dSK channel in the visual circuitry, these results also clarify how homeostatically balanced network functions can compensate missing or faulty ion channels.

DNA fragmentation is a critical component of apoptosis but it has not been characterized in non-apoptotic forms of cell death, such as necrosis and autophagic cell death. In mammalian apoptosis, caspase activated DNase (CAD) cleaves DNA into nucleosomal fragments in dying cells, and subsequently DNaseII, an acid nuclease, completes the DNA degradation but acts non-cell-autonomously within lysosomes of engulfing cells. Here we examine the requirement for DNases during two examples of programmed cell death (PCD) that occur in the Drosophila melanogaster ovary, starvation-induced death of mid-stage egg chambers and developmental nurse cell death in late oogenesis. Surprisingly, we found that DNaseII was required cell-autonomously in nurse cells during developmental PCD, indicating that it acts within dying cells. Dying nurse cells contain autophagosomes, indicating that autophagy may contribute to these forms of PCD. Furthermore, we provide evidence that developmental nurse cell PCD in late oogenesis shows hallmarks of necrosis. These findings indicate that DNaseII can act cell-autonomously to degrade DNA during non-apoptotic cell death.

The Precision Reactor Oscillation and Spectrum Experiment, PROSPECT, is designed to make a precise measurement of the antineutrino spectrum from a highly-enriched uranium reactor and probe eV-scale sterile neutrinos by searching for neutrino oscillations over meter-long distances. PROSPECT is conceived as a 2-phase experiment utilizing segmented $^6$Li-doped liquid scintillator detectors for both efficient detection of reactor antineutrinos through the inverse beta decay reaction and excellent background discrimination. PROSPECT Phase I consists of a movable 3-ton antineutrino detector at distances of 7 - 12 m from the reactor core. It will probe the best-fit point of the $\nu_e$ disappearance experiments at 4$\sigma$ in 1 year and the favored region of the sterile neutrino parameter space at $>$3$\sigma$ in 3 years. With a second antineutrino detector at 15 - 19 m from the reactor, Phase II of PROSPECT can probe the entire allowed parameter space below 10 eV$^{2}$ at 5$\sigma$ in 3 additional years. The measurement of the reactor antineutrino spectrum and the search for short-baseline oscillations with PROSPECT will test the origin of the spectral deviations observed in recent $\theta_{13}$ experiments, search for sterile neutrinos, and conclusively address the hypothesis of sterile neutrinos as an explanation of the reactor anomaly., Comment: 21 pages, 21 figures

A meter-long, 23-liter EJ-309 liquid scintillator detector has been constructed to study the light collection and pulse-shape discrimination performance of elongated scintillator cells for the PROSPECT reactor antineutrino experiment. The magnitude and uniformity of light collection and neutron/gamma discrimination power in the energy range of antineutrino inverse beta decay products have been studied using gamma and spontaneous fission calibration sources deployed along the cell long axis. We also study neutron-gamma discrimination and light collection abilities for differing PMT and reflector configurations. Key design features for optimizing MeV-scale response and background rejection capabilities are identified., Comment: 20 pages, 11 figures

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue-specific expression system to express wild-type and disease-associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease-associated insertional PrP mutant. We also found that wild-type PrP molecules are maintained in a detergent-soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent-insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.

Research reactors host a wide range of activities that make use of the intense neutron fluxes generated at these facilities. Recent interest in performing measurements with relatively low event rates, e.g. reactor antineutrino detection, at these facilities necessitates a detailed understanding of background radiation fields. Both reactor-correlated and naturally occurring background sources are potentially important, even at levels well below those of importance for typical activities. Here we describe a comprehensive series of background assessments at three high-power research reactors, including $\gamma$-ray, neutron, and muon measurements. For each facility we describe the characteristics and identify the sources of the background fields encountered. The general understanding gained of background production mechanisms and their relationship to facility features will prove valuable for the planning of any sensitive measurement conducted therein., Comment: 26 pages, 28 figures. Accepted for publication in Nuclear Instruments and Methods A. v2 incorporates minor revisions requested by NIMA

G protein-coupled receptors are a large family of signaling molecules that respond to a wide variety of extracellular stimuli. The receptors relay the information encoded by the ligand through the activation of heterotrimeric G proteins and intracellular effector molecules. To ensure the appropriate regulation of the signaling cascade, it is vital to properly inactivate the receptor. This inactivation is achieved, in part, by the binding of a soluble protein, arrestin, which uncouples the receptor from the downstream G protein. In addition to the inactivation of G protein-coupled receptors, arrestins have also been implicated in the endocytosis of receptors and cross talk with other signaling pathways. Due to its central role in cellular signaling, misregulation or misfunction of arrestin can have dramatic affects on cell viability and have direct implications in human disease.

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

Prion diseases are CNS disorders that can occur in sporadic, infectious, and inherited forms. Although all forms of prion disease are associated with the accumulation of pathogenic conformers of the prion protein, collectively termed PrPSc, the mechanisms by which PrPScmolecules form and cause neuronal degeneration are unknown. Using the bipartite galactosidase-4–upstream activating sequence expression system, we generated transgenicDrosophila melanogasterheterologously expressing either wild-type (WT) or mutant, disease-associated (P101L) mouse PrP molecules in cholinergic neurons. Transgenic flies expressing neuronal P101L PrP molecules exhibited severe locomotor dysfunction and premature death as larvae and adults. These striking clinical abnormalities were accompanied by age-dependent accumulation of misfolded PrP molecules, intracellular PrP aggregates, and neuronal vacuoles. In contrast, transgenic flies expressing comparable levels of WT PrP displayed no clinical, pathological, or biochemical abnormalities. These results indicate that transgenicDrosophilaexpressing neuronal P101L PrP specifically exhibit several hallmark features of human Gerstmann–Sträussler–Scheinker (GSS) syndrome. Because the rates of abnormal PrP accumulation and clinical progression are highly accelerated inDrosophilacompared with the rates of these processes in rodents or humans, the P101L mutant may be used for future genetic and pharmacologic studies as a novel invertebrate model of GSS.

Photoreceptor cell death accompanying many retinal degenerative disorders results in irreversible loss of vision in humans. However, the precise molecular pathway that executes cell death is not known. Our results from a Drosophila model of retinal degeneration corroborate previously reported findings that the developmental apoptotic pathway is not involved in photoreceptor cell demise. By undertaking a candidate gene approach, we find that players involved in the immune response against Gram-negative bacteria are involved in retinal degeneration. Here, we report that the NF-κB transcription factor Relish regulates neuronal cell death. Retinal degeneration is prevented in genetic backgrounds that block Relish activation. We also report that the N-terminal domain of Relish encodes unique toxic functions. These data uncover a unique molecular pathway of retinal degeneration in Drosophila and identify a previously unknown function of NF-κB signaling in cell death.

Schedule and Cost Books developed for the Water Cherenkov Detector (WCD) option for the far detector of the Long Baseline Neutrino Experiment (LBNE)

Basis of Estimate (BOE) forms and backup documentation developed for the Water Cherenkov Detector (WCD) option for the far detector of the Long Baseline Neutrino Experiment (LBNE)

Here the authors summarize application of a spatially distributed water balance model that was tested using historical data from the U.S. portion of the Columbia River Basin in the Pacific Northwest for a very dry (1977) and very wet (1972) water year. The model adequately partitions incoming precipitation into evapotranspiration and runoff. Because precipitation in the basin is underestimated from measured data, modeled runoff is less than measured runoff from the basin during both the wet and dry years. The predicted future climate conditions significantly increase potential evapotranspiration, causing a 20% reduction in runoff relative to input precipitation, and a 58% reduction in soil moisture storage. If these changes in regional water balance are realized, the distribution and composition of forests in the Northwest would change markedly, and water resources would become more limited. (Copyright (c) Inter-Research 1993.)

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein–coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCβ homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP 2 ), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP 2 and its partitioning into ordered membrane domains. Thus ceramide kinase–mediated maintenance of ceramide level is important for the local regulation of PIP 2 and PLC during phototransduction.

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons., Author Summary Irreversible loss of photoreceptor cells has been attributed as a cause of blindness in many retinal degenerative disorders. One such group of disorders is retinitis pigmentosa, which affects 1 in 3,000 individuals. Over 100 mutations in the light-sensing molecule rhodopsin have been identified in patients with autosomal dominant retinitis pigmentosa. These mutations affect rhodopsin transport to the outer segments of rod photoreceptor cells, rhodopsin folding, and rhodopsin endocytosis. In Drosophila photoreceptors, endocytosis of a large amount of rhodopsin at a rapid rate results in cell death. To further understand the role of endocytosis in triggering cell death, we used previously characterized mutants in which lysosomal degradation is compromised. We show that retinal degeneration can also be induced in these genetic backgrounds after rhodopsin is endocytosed, suggesting that failure to degrade internalized rhodopsin in a timely manner triggers cell death of photoreceptor neurons. We also present direct cellular biological evidence of rhodopsin accumulation in the cell bodies of photoreceptors, only in mutant backgrounds that undergo retinal degeneration. We could rescue the degeneration by preventing rhodopsin endocytosis and accumulation. Thus, our results indicate the vital role of lysosomal turnover of rhodopsin in maintaining photoreceptor viability.

Absorption of a photon of light by rhodopsin triggers mechanisms responsible for excitation as well as regulation of the phototransduction cascade. Arrestins are a family of proteins that appear to be responsible for terminating the active state of G-protein-coupled receptors. One of the major substrates of light-dependent phosphorylation in the visual cascade of Drosophila was purified and partially sequenced. The complete primary structure of the protein was determined by isolating the corresponding gene, which revealed it to be a new isoform of arrestin, Arr2. Arr2 is 401 residues in length, and shares 47% sequence identity with the Drosophila Arr1 protein and 42% with human arrestin. We show that the two Drosophila arrestin genes are differentially regulated, and that Arr2 is a specific substrate for a calcium-dependent protein kinase. This is the first demonstration of in vivo regulation of arrestins in a transduction cascade, and provides a new level of modulation in the function of G-protein-coupled receptors.

The adenovirus tripartite leader is a 200-nucleotide-long 5' noncoding region which facilitates translation of viral mRNAs at late times after infection. The tripartite leader also confers the ability to initiate translation independent of the requirement for cap-binding protein complex or eIF-4F without any requirement for adenovirus gene products. To elucidate the manner by which the tripartite leader functions, the primary determinants of leader activity were investigated in vivo by testing a series of mutations expressed from transfected plasmids. The results of these experiments indicate that the tripartite leader does not promote internal ribosome binding, at least in a manner recently described for picornavirus mRNAs. In addition, despite an unusual arrangement of sequences complementary to the 3' end of 18S rRNA in the tripartite leader, we could find no evidence for involvement in its translation activity. Instead, our results are consistent with a model in which much of the first leader is maintained in an unstructured conformation which determines the ability of the tripartite leader to facilitate translation and bypass a normal requirement for eIF-4F activity. Several possible translation models are discussed, as well as the implications for translation of late viral mRNAs.

The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H3 receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H3 receptor., Author Summary During signaling in the nervous system, individual nerve cells transfer information to one another by a complex process called synaptic transmission. This communication involves the release of a specific neurotransmitter into the synaptic cleft, which then triggers signaling in the downstream neuron by binding to and activating specific cell surface receptors. In order to terminate the neuronal signal, the neurotransmitter must be rapidly removed from the synaptic cleft. This is done by two mechanisms: the neurotransmitter can be degraded or modified, or the transmitter can be taken up by the presynaptic neuron and packaged into vesicles for reuse. In the compound eye of the fruitfly D. melanogaster, the photoreceptor cell responds to light and releases histamine into the synaptic cleft. This signal is terminated by the removal of histamine from the synapse and the enzymatic conversion of histamine to carcinine. We have shown that it is not sufficient just to modify the histamine neurotransmitter, but it is also important to remove carcinine from the photoreceptor synapse. The failure to adequately remove carcinine results in defects in the visual transduction process. Moreover, the work suggests that carcinine itself modulates vision by regulating histamine release into the synapse.

Alleles of pawn have numerous morphological phenotypes, including dark deposits on the eye, truncated bristles, and semilethality. The gene encoding the pawn locus was cloned. The Pawn protein is predicted to be a large cell adhesion molecule with a single transmembrane domain, a short cytoplasmic tail and two extracellular epidermal growth factor (EGF)-like repeats. The identity of the pawn gene product was confirmed by sequencing the defects in four different pawn alleles. Analysis of cDNAs showed a complex genomic structure with two 5' untranslated exons and developmental-specific RNA splicing. The gene is expressed throughout development with highest expression found in the late embryonic and larval stages.

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue-specific expression system to express wild-type and disease-associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease-associated insertional PrP mutant. We also found that wild-type PrP molecules are maintained in a detergent-soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent-insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.

Heterotrimeric G proteins couple various receptors to intracellular effector molecules. Although the role of the G alpha subunit in effector activation, guanine nucleotide exchange and GTP hydrolysis has been well studied, the cellular functions of the G beta subunits are less well understood. G beta gamma dimers bind G alpha subunits and anchor them to the membrane for presentation to the receptor. In specific systems, the G beta subunits have also been implicated in direct coupling to ion channels and to effector molecules. We have isolated Drosophila melanogaster mutants defective in an eye-specific G-protein beta-subunit (G beta e), and show here that the beta-subunit is essential for G-protein-receptor coupling in vivo. Remarkably, G beta mutants are also severely defective in the deactivation of the light response, demonstrating an essential role for the G beta subunit in terminating the active state of this signalling cascade.

To determine the fate of rhodopsin during endocytosis-mediated retinal degeneration.Drosophila stocks were raised in complete darkness and shifted to light for 24 h prior to dissection and fixation of retinas. 1 microm frozen sections were cut on an ultracryomicrotome, then stained with antibodies specific for rhodopsin or arrestin. Localization of photoreceptor cell-specific proteins was determined by confocal microscopy.Flies that are in the process of undergoing endocytosis-mediated retinal degeneration exhibit an apparent loss of rhabdomeric rhodopsin at early times during the degenerative process. Using different immunological agents, genetic backgrounds, and light treatments, we have found that the binding of arrestin to rhodopsin masked the C-terminal monoclonal antibody epitope and resulted in the loss of rhodopsin immunoreactivity. The loss of immunoreactive rhabdomeric rhodopsin only occurred when rhodopsin was depleted from the plasma membrane such that it was found within the rhabdomere at stoichiometric levels with arrestin.When rhodopsin and arrestin are found at equal levels, binding of arrestin to rhodopsin results in the masking of the antibody epitope on the C-terminus of rhodopsin. Since masking can only occur after most of the rhodopsin has been depleted from the rhabdomere, it can be concluded that during endocytosis-induced retinal degeneration, much of the rhodopsin is localized to the cell body in small puncta. These data suggest that rhodopsin is at extremely high local concentrations in the cytoplasm. The data are discussed in the context of a model for photoreceptor cell apoptosis in retinal degenerative disorders.

G protein-coupled receptors are a large family of signaling molecules that respond to a wide variety of extracellular stimuli. The receptors relay the information encoded by the ligand through the activation of heterotrimeric G proteins and intracellular effector molecules. To ensure the appropriate regulation of the signaling cascade, it is vital to properly inactivate the receptor. This inactivation is achieved, in part, by the binding of a soluble protein, arrestin, which uncouples the receptor from the downstream G protein. In addition to the inactivation of G protein-coupled receptors, arrestins have also been implicated in the endocytosis of receptors and cross talk with other signaling pathways. Due to its central role in cellular signaling, misregulation or misfunction of arrestin can have dramatic affects on cell viability and have direct implications in human disease.

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in theDrosophilavisual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascadein vivo. Here, we report the isolation of nine alleles of theDrosophilaphotoreceptor cell-specificarr2gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.

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

Arrestins have been implicated in the regulation of many G protein-coupled receptor signaling cascades. Mutations in two Drosophila photoreceptor-specific arrestin genes, arrestin 1 and arrestin 2, were generated. Analysis of the light response in these mutants shows that the Arr1 and Arr2 proteins are mediators of rhodopsin inactivation and are essential for the termination of the phototransduction cascade in vivo. The saturation of arrestin function by an excess of activated rhodopsin is responsible for a continuously activated state of the photoreceptors known as the prolonged depolarized afterpotential. In the absence of arrestins, photoreceptors undergo light-dependent retinal degeneration as a result of the continued activity of the phototransduction cascade. These results demonstrate the fundamental requirement for members of the arrestin protein family in the regulation of G protein-coupled receptors and signaling cascades in vivo.

RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.

A primary determinant of pathogenicity in Erwinia stewartii is the production of extracellular polysaccharide (EPS). A single mutation can abolish both EPS synthesis and pathogenicity; both properties are restored by a single cosmid clone. Subcloning and insertion analysis have defined a single positive regulatory function which shares a number of similarities with the rcsA function of Escherichia coli K-12, a positive regulator for capsular polysaccharide synthesis. In E. stewartii, the gene promotes the transcription of at least two operons (cps) involved in EPS synthesis; we have previously demonstrated a similar function for rcsA in E. coli. Both genes code for proteins of 25 to 27 kilodaltons; both proteins are unstable in E. coli. The E. stewartii RcsA protein was stabilized in E. coli lon mutants, as the RcsA product from E. coli is. The E. stewartii function complemented E. coli rcsA mutants, and the E. coli RcsA function increased cps expression and restored virulence in E. stewartii mutants. Therefore, these two gram-negative organisms share a similar component of their regulatory circuitry for the control of capsular polysaccharide synthesis.

Previously we have shown that a subset of visual transduction mutants in Drosophila melanogaster induce the formation of stable complexes between rhodopsin and arrestin. One such mutant is in a visual system-specific phospholipase C (PLC). The rhodopsin/arrestin complexes generated in PLC mutants induce massive retinal degeneration. Here we demonstrate that both arrestin and rhodopsin undergo light-dependent endocytosis in a PLC mutant background. Interestingly, the internalized rhodopsin is rapidly degraded, but the arrestin is fully stable. The data are discussed with respect to mechanisms of arrestin-mediated endocytosis and human retinal disease.

We have previously cloned the genes for synthesis of capsular polysaccharide (cps) and slime from Erwinia stewartii in cosmid pES2144. In this study, pES2144 was shown to complement 14 spontaneous cps mutants. These mutants were characterized by probing Southern blots of mutant genomic DNA with pES2144; insertions were detected in four mutants and deletions in six mutants. Genetic and physical maps of the pES2144 cps region were constructed by subcloning, restriction analysis, and transposon mutagenesis with Tn5, Tn5lac, and Tn3HoHo1. Mutations affecting the ability of pES2144 to restore mucoidy to cps deletion mutants were located in five regions, designated cpsA to cpsE. None of the cps mutants were able to cause systemic wilting of corn plants, and mutations in cps regions B to E further abolished the ability of the bacterium to cause watersoaked lesions on seedlings. The gene for uridine-5'-diphosphogalactose 4-epimerase (galE) was linked to the cps genes on pES2144. In E. stewartii, galE was constitutively expressed, whereas the genes for galactokinase (galK) and galactose-1-phosphate uridyltransferase (galT) were inducible and not linked to galE. Thus, galE does not appear to be part of the gal operon in this species.

In Erwinia stewartii, three groups of avirulent mutants have been defined by complementation studies using clones from a genomic library constructed in cosmid pVK100. Clone pES2144 (27 kb insert) restored virulence and extracellular polysaccharide synthesis (EPS) to mutants in complementation group III which consists of 14 EPS mutants, two of which are sensitive to galactose (GAL8 and GAL17). A restriction map of pES2144 was constructed. Hybridization of pES2144 probe DNA to Southern blots of genomic DNA from the EPS- mutants revealed deletions of this region in five of the mutants (including GAL8 and GAL17). A subclone, pPD0527, containing a 4 kb HindIII- BamH1 fragment complemented five EPS- mutants and another subclone, pPD011, containing a 6.2 kb HindIII fragment, complemented the galactose-sensitive phenotype of GAL8 and GAL17 but did not restore capsule synthesis. Tn5 lac and Tn3 HoHoI mutagenesis indicated that five separate regions of pES2144 contain EPS genes. Properties of EPS-lac gene fusions will be discussed.

RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.