21 research outputs found
Drosophila tan Encodes a Novel Hydrolase Required in Pigmentation and Vision
Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5′ untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function
A Glial Variant of the Vesicular Monoamine Transporter Is Required To Store Histamine in the Drosophila Visual System
Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems
Activity and coexpression of Drosophila black with ebony in fly optic lobes reveals putative cooperative tasks in vision that evade electroretinographic detection
International audienceDrosophila mutants black and ebony show pigmentation defects in the adult cuticle, which disclose their cooperative activity in β-alanyl-dopamine formation. In visual signal transduction, Ebony conjugates β-alanine to histamine, forming β-alanyl-histamine or carcinine. Mutation of ebony disrupts signal transduction and reveals an electroretinogram (ERG) phenotype. In contrast to the corresponding cuticle phenotype of black and ebony, there is no ERG phenotype observed when black expression is disrupted. This discrepancy calls into question the longstanding assumption of Black and Ebony interaction. The purpose of this study was to investigate the role of Black and Ebony in fly optic lobes. We excluded a presynaptic histamine uptake pathway and confirmed histamine recycling via carcinine formation in glia. β-Alanine supply for this pathway is independent of enzymatic synthesis by Black and β-alanine synthase Pyd3. Two versions of Black are expressed in vivo. Black is a specific aspartate decarboxylase with no activity on glutamate. RNA in situ hybridization and anti-Black antisera localized Black expression in the head. Immunolabeling revealed expression in lamina glia, in large medulla glia, in glia of the ocellar ganglion, and in astrocyte-like glia below the ocellar ganglion. In these glia types, Black expression is strictly accompanied by Ebony expression. Activity, localization, and strict coexpression with Ebony strongly indicate a specific mode of functional interaction that, however, evades ERG detection
Molecular cloning of a putative voltage- and cyclic nucleotide-gated ion channel present in the antennae and eyes of Drosophila melanogaster
Location and functions of Inebriated in the Drosophila eye
Histamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to β-alanine to form β-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and β-alanine. The gene Inebriated (Ine) encodes an Na+/Cl−-dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% β-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and β-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT
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A glial variant of the vesicular monoamine transporter is required to store histamine in the Drosophila visual system.
Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems
Tan Expression and Function in the Eye and Optic Lobes
<div><p>(A) RNA in situ hybridization with an antisense <i>tan</i> cDNA probe to wild-type adult head sections. Horizontal section cut through the eye and the optic lobes.</p><p>(B) Sagittal section through the ommatidial array of the eye.</p><p>(C) Control sense probe does not show significant staining (horizontal section).</p><p>(D–G) ERG phenotypes of wild-type flies (D) and <i>tan</i> mutants (E–G). (D) <i>Canton<sup>S</sup></i> (wild-type) ERG showing normal “on” and “off” transients. Upper trace in each recording indicates duration of light input stimulus (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010063#s4" target="_blank">Materials and Methods</a>). (E) <i>tan<sup>2</sup></i> mutant showing complete absence of “on” and “off” transients. (F) <i>P{d07784}</i> exhibits a normal “on” and “off” transient even though it has a <i>tan</i>-like pigmentation phenotype. (G) <i>P{g1557}</i> lacks “on” and “off” transients.</p><p>(H and I) Normalized “on” (H) and “off” (I) transients for wild-type, <i>tan<sup>2</sup> (t[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010063#pgen-0010063-b002" target="_blank">2</a>])</i>, <i>tan</i>-like <i>P</i> excision lines, and “revertant” <i>P</i> excision lines (see text) expressed as a percentage of the respective sustained negative potential. Insets: Correlation between head histamine content and the normalized magnitude of ERG ‘on’ and ‘off’ transients (see text). Error bars: ± 1 standard error in both dimensions.</p><p>(J) Head histamine contents for <i>tan</i>-like and revertant <i>P</i> excision lines (see text), relative to control <i>w<sup>1118</sup></i> and <i>tan<sup>1</sup>w<sup>1118</sup></i> flies. Head histamine increased in all flies that drank 0.5% carcinine in 4% glucose (black bars), relative to controls that drank only 4% glucose (open bars). Revertant and <i>tan</i>-like excision lines are each arranged in rank order. Note that excision line flies that drank only 4% glucose have histamine contents too small to show above the abscissa. Error bars represent ± 1 standard error.</p><p>La, lamina; Me, medulla; Re, retina. Scale bars = 50 μm.</p></div