The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network.

Among the several multigene families codified by the genome of T. cruzi, the TcTASV family was the latest discovered. The TcTASV (Trypomastigote, Alanine, Serine, Valine) family is composed of ∼40 members, with conserved carboxi- and amino-termini but with a variable central core. According to the length and sequence of the central region the family is split into 3 subfamilies. The TcTASV family is conserved in the genomes of – at least – lineages TcI and TcVI and has no orthologues in other trypanosomatids. In the present work we focus on the study of the TcTASV-C subfamily, composed by 16 genes in the CL Brener strain. We determined that TcTASV-C is preferentially expressed in trypomastigotes, but it is not a major component of the parasite. Both immunoflourescence and flow cytometry experiments indicated that TcTASV-C has a clonal expression, i.e. it is not expressed by all the parasites of a certain population at the same time. We also determined that TcTASV-C is phosphorylated and glycosylated. TASV-C is attached to the parasite surface by a GPI anchor and is shed spontaneously into the medium. About 30% of sera from infected hosts reacted with TcTASV-C, confirming its exposition to the immune system. Its superficial localization and secretory nature suggest a possible role in host-parasite interactions.

BackgroundThe identification and characterization of antigens expressed in Trypanosoma cruzi stages that parasitize mammals are essential steps for the development of new vaccines and diagnostics. Genes that are preferentially expressed in trypomastigotes may be involved in key processes that define the biology of trypomastigotes, like cell invasion and immune system evasion.Methodology/Principal FindingsWith the initial aim of identifying trypomastigote-specific expressed tags, we constructed and sequenced an epimastigote-subtracted trypomastigote cDNA library (library TcT-E). More than 45% of the sequenced clones of the library could not be mapped to previously annotated mRNAs or proteins. We validated the presence of these transcripts by reverse northern blot and northern blot experiments, therefore providing novel information about the mRNA expression of these genes in trypomastigotes. A 280-bp consensus element (TcT-E element, TcT-Eelem) located at the 3′ untranslated region (3′ UTR) of many different open reading frames (ORFs) was identified after clustering the TcT-E dataset. Using an RT-PCR approach, we were able to amplify different mature mRNAs containing the same TcT-Eelem in the 3′ UTR. The proteins encoded by these ORFs are members of a novel surface protein family in T. cruzi, (which we named TcTASV for T. cruzi Trypomastigote, Alanine, Serine and Valine rich proteins). All members of the TcTASV family have conserved coding amino- and carboxy-termini, and a central variable core that allows partitioning of TcTASV proteins into three subfamilies. Analysis of the T. cruzi genome database resulted in the identification of 38 genes/ORFs for the whole TcTASV family in the reference CL-Brener strain (lineage II). Because this protein family was not found in other trypanosomatids, we also looked for the presence of TcTASV genes in other evolutionary lineages of T. cruzi, sequencing 48 and 28 TcTASVs members from the RA (lineage II) and Dm28 (lineage I) T. cruzi strains respectively. Detailed phylogenetic analyses of TcTASV gene products show that this gene family is different from previously characterized mucin (TcMUCII), mucin-like, and MASP protein families.Conclusions/SignificanceWe identified TcTASV, a new gene family of surface proteins in T. cruzi.

BackgroundMembers of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system.ResultsThe present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light.ConclusionsBased on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.

Background Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. Results Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of β-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. Conclusions These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.