79 research outputs found

    DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function

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    The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses

    Mechanisms Underlying Stage-1 TRPL Channel Translocation in Drosophila Photoreceptors

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    Background: TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere), TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels. Methodology/Principal Findings: We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content. Conclusions/Significance: Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protei

    Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

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    Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

    Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

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    Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

    Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila.

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    Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K(+) current (I(A)), encoded by the highly conserved Shal/K(v)4 gene, in neuronal firing patterns and repetitive behaviors. While I(A) is present in nearly all neurons across species, elimination of I(A) has been complicated in mammals because of multiple genes underlying I(A), and/or electrical remodeling that occurs in response to affecting one gene.In Drosophila, the single Shal/K(v)4 gene encodes the predominant I(A) current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v)4 neurons display multiple defects during prolonged stimuli. DNK(v)4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/K(v)4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/K(v)4 function. Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested.Based on our results, Shal/K(v)4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K(v)4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors

    Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors

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    Here, we reveal a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. We show that the multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking

    Increased Dietary Ergosterol Slowed the Rate of TRPL Translocation.

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    <p>(A) Wild-type flies were fed a defined medium containing either wild-type or <i>mot3Δ</i> mutant yeast for 10, 15, 20 or 30 days in the dark. Sterols were extracted from whole fly membranes and then scanned spectrophotometrically. Left, shown are representative spectral profiles (250–300 nm) of sterols from flies raised for 30 days on food made with wild-type yeast (wt-fed) or <i>mot3Δ</i> mutant yeast (<i>mot3Δ</i>-fed). This four-peaked profile is characteristic of ergosterol, the major sterol present in <i>Drosophila</i>. <i>mot3Δ</i>-fed flies consistently displayed an increased absorption spectrum, compared to wt-fed flies. Right, quantification of ergosterol increase in flies fed <i>mot3Δ</i>-food, relative to wt-food, for 10, 15, 20, and 30 days. (B) Left, shown are representative retinal sections immunostained for TRPL from wt-fed or <i>mot3Δ</i>-fed flies. Dark-raised flies showed rhabdomeric localization of TRPL. With <u>17</u> minutes of light-exposure, wt-fed flies displayed a more evident stage-1 TRPL translocation, while flies fed <i>mot3Δ</i>-based food displayed a more rhabdomeric-like TRPL localization. After 30 minutes of light-exposure, both wt-fed and <i>mot3Δ</i>-fed flies appeared to display stage-1 translocation. Right, Box-and-Whisker-Plots showing the relative rhabdomeric TRPL signal between wt-fed and <i>mot3Δ</i>-fed flies when exposed to 17 and 30 min of light. Multiple tissue sections were taken from multiple eyes/flies (across different experiments), as follows. For wild-type, 5 eyes from 4 flies (Dark), <u>10</u> eyes from <u>5</u> flies (LEX, <u>17</u> min), <u>2</u> eyes from <u>2</u> flies (LEX, 30 min); for <i>mot3Δ</i>: <u>5</u> eyes from <u>4</u> flies (dark), <u>7</u> eyes from <u>5</u> flies (LEX, <u>17</u> min), <u>3</u> eyes from <u>3</u> flies (LEX, 30 min). ** t-test analysis indicates a significant difference (P<0.01).</p

    Testing the Requirement of Protein Synthesis for TRPL Channel Translocation.

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    <p>(A) <i>hs-inaD</i> transgenic flies (in an <i>inaD<sup>1</sup></i> null background) express INAD protein only when heat-shocked at 37°C. One hour of heat-shock induces INAD expression that can be easily detected by immunoblot analysis (see -CHX). Flies fed green-colored cycloheximide (CHX) for 30 minutes were selected with for abdomens with various degrees of greenness (degree green), heat-shocked, and analyzed by immunoblot analysis (three heads per lane). Degree greenness had an inverse relationship with inducible expression of INAD, indicating that CHX treatment did indeed block protein synthesis. Anti-syntaxin was used as a loading control. (B) Dark-raised wild-type flies were fed +/− CHX (colored green) in the dark for at least 30 minutes, then light-exposed for either 30 minutes (Stage-1) or 12 hours (Stage-2), while remaining on the +/− CHX food throughout the light-exposure. Only flies with dark-green abdomens were selected for sectioning. Shown are representative retinal sections immunostained for TRPL. TRPL channels translocated to both stage-1 and -2 with CHX treatment. Multiple tissue sections were taken from 11 eyes from 8 flies (Dark), 8 eyes from 7 flies (Stage 1, -CHX), 9 eyes from 7 flies (Stage 1, +CHX). (C) Left, Wild-type flies were fed +/− CHX in the dark for at least 30 minutes to inhibit protein synthesis, light-exposed for 30 minutes to induce stage-1 translocation, followed by 6 hours of dark incubation. Only flies with dark-green abdomens were selected for sectioning and immunostaining. TRPL channels relocalized to the rhabdomeres in –CHX flies, but were unable to translocate back to the rhabdomere in +CHX fed flies. Right, Wild-type flies were light-exposed for 12 hours to induce stage-2 translocation, then transferred to +/− CHX food in continuous light for 1 additional hour, followed by dark incubation for 10 hours. Only flies with dark-green abdomens were selected for sectioning and immunostaining. Representative sections show that TRPL channels relocalized to the rhabdomere from stage-2 in both +/− CHX. Multiple sections were taken from 13 eyes from 9 flies (Stage 1), 7 eyes from 5 flies (Dark 6 hrs, -CHX), 12 eyes from 9 flies (Dark 6 hrs, +CHX), 13 eyes from 11 flies (Stage 2), 11 eyes from 9 flies (Dark 10 hrs, -CHX), 16 eyes from 10 flies (Dark 10 hrs, +CHX).</p
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