pH 9.4) and slides viewed using a Zeiss 510 META confocal microscope. Measurements of intracellular Ca2+ using aequorin Drosophila S2 cells, cultured under standard conditions [21] were transiently transfected with the apoaequorin ORF [21] and a receptor ORF construct, and expression induced using CuSO4. Transfected S2 cells were harvested and incubated with 2.5 mM coelenterazine in the dark at RT for 1–2 h as previously described [21]. 25,000 cells were then placed in 135 ml Schneider’s medium supplemented with 10% FCS in a well of a white polystyrene 96- well plate (Berthold Technologies). Bioluminescence recordings were carried out using a Mithras LB940 automated 96-well plate reader (Berthold Technologies) and MikroWin software. 15 ml of each of different peptides were applied to final concentrations as required. At the end of each recording samples were disrupted by the addition of 100 ml lysis solution, and the [Ca2+] concentrations calculated as previously described [18]. For assays in Ca2+-free medium, transfected cells were collected and pelleted after incubation with coelenterazine. Cell pellets were re-suspended in Ca2+-free Schneider’s medium (Invitrogen Inc.) without FCS and collected by centrifugation. The washing and pelleting procedure was repeated once more. Experiments were then carried out on 25,000 cells per sample as above. To verify whether the observed calcium signal was linked to phospholipase C (PLC) activation, S2- capaR cells were preincubated with the PLC inhibitor U73122 at concentration of 1023 M to 1028 M for 10 min. Luminometry experiments were carried out on live, intact tubules expressing a targeted transgene for cytosolic-targeted apoaequorin in either principal or stellate cells as previously described [21,22]. For in vivo tubule experiments, flies of the following genotypes were used: c42aeq, capaR-GAL4, UAS-capaR, UAS-capaR RNAi, UAS- NMUR 2 and the resulting progeny from GAL4.UAS crosses. Capa Receptor desensitization via b-arrestin To assess the internalization and resensitization of capaR, Drosophila S2 cells were transiently transfected with the capaR- eYFP construct and either left untreated or treated with 1027 M capa-1 for different times. Cells were subjected to immunocyto- chemistry with anti-GFP antibody, with assessment of fluorescence by confocal microscopy. For analysis of b-arrestin localization in response to capaR activation, S2 cells were transfected with b- arrestin-eYFP construct and real-time localization of b-arrestin- eYFP established by confocal microscopy in response to capa-1 stimulation. Cell Surface biotinylation and immunoblotting For biotinylation experiments, S2 cells were left untreated or treated with 1027 M capa-1 for 0, 5 10, 20 and 30 minutes to induce receptor internalization. An additional sample was incubated for 30 minutes with 1027 M capa-1, washed three times with culture medium without capa-1 followed by 30 minutes incubation in culture medium to allow resensitization. Samples were rapidly cooled on ice followed by two washes with ice-cold PBS-CM. Cells were then subjected to cell surface biotinylation to label plasma membrane proteins [23] using EZ-Link Sulfo-NHS- SS-Biotin (Pierce). Total lysates and biotinylated samples (biotin- labelled protein was captured using streptavidin resin) were analyzed by SDS-PAGE (10% gel) followed by immunoblotting using affinity purified rabbit anti-capaR (1:1000) as primary antibody according to standard techniques for S2 cells [24]. Capa GPCR and NMUR in Fluid Homeostasis Bioluminescence Resonance Energy Transfer (BRET) assay S2 cells were co-transfected with capa receptor tagged with Renilla luciferase and b-arrestin-2 tagged with eYFP (ratio 1:4), using calcium phosphate transfection method (Invitrogen). An additional transfection was performed with only the Renilla luciferase construct and empty expression vector pMT/V5-His TOPO vector. Cells were seeded at 200 000 cells per well into poly-D-lysine coated 96 well plates and coelentrazine-h (Promega, Southampton, UK) was added to a final concentration of 5 mM. Cells were incubated in darkness for 10 min at 37uC before addition of different concentrations of capa-1 peptide. Cells were incubated for a further 15 min at 37uC and subsequent BRET measurements were carried out using a PHERAstar FS reader (BMG-Labtech) that allows simultaneous reading of emission signals detected at 485 nm and 530 nm. The BRET ratio was then calculated as emission at 530 nm?emission21 at 485 nm. Net BRET was defined as the 530 nm/485 nm ratio of cells co- expressing Rluc and eYFP minus the BRET ratio of cells expressing only the Renilla luciferase construct in the same experiment. This value was multiplied by 1000 to obtain mBRET units. Fluid transport assay by intact Malpighian tubules Fluid transport assays were carried out as previously described [25] using live, intact tubules dissected from 7-day-old adults with the following genotypes: wild-type Canton-S, c42-GAL4.UAS- capaR RNAi, actin-GAL4.UAS-NMUR 2. Fluid droplets were collected every 10 min, and the volumes of fluid were calculated. Basal rates of fluid secretion were monitored for 30 min, whereupon peptide was added, and the secretion rate was then recorded for a further 30 min. Survival assays 5–7 day old flies of specified genotype were subjected to a starvation/desiccation stress in empty vials [26] in groups of approximately 30, with three biological replicates of each line. The tubule principal cell GAL4 driver (UO-GAL4) [16] was used to either over-express or knock-down capaR, with outcrossed (Canton- S) GAL4 and UAS lines. While the c42-GAL4 driver unequivo- cally drives expression in tubule principal cells in the adult and so is suitable for studies on acutely dissected adult tubules, it also directs expression in a few other tissues in the adult fly [27]. In order to assess the impact of tubule-targeted capaR transgenes on the survival of whole flies under starvation/desiccation stress, we used our Urate Oxidase-GAL4 driver (which directs expression only in the principal cells of both larval and adult tubule main segment), described in [17]. Before embarking on the desiccation survival assays, possible effects of the genetic background of these transgenic flies were avoided. Therefore, the UO-GAL4 and UAS-capaR/capaR RNAi lines used in this study were outcrossed for five generations in a White Canton-S background. In addition, the UAS-capaR/ capaR RNAi and UO-GAL4 transgenic parental lines were crossed to White Canton-S (WhCS) and heterozygote progeny utilized to avoid the effect of 2 copies of the transgene. Flies were counted until 100% mortality was reached and data expressed as % survival 6 SEM (N = 3). Data were assessed for significance by the LogRank (Mantel-Cox) test using Graph Pad Prism 5.0 software. The number of flies used (N) was sufficiently high to allow for significant differences in survival; and the data were consistent between each of the 3 individual assays. PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e29897