processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains [29,30]. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells [31–33]. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion [34,35], although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena. To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 we analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis [36]. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA) [36,37], whereas the cell adhesion molecules FasIII and b- integrin are localized in lateral and basal membranes, respectively [38,39]. In this study, it was found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, we found that dVps15 knockdown resulted in the accumulation of FasIII at the lateral membrane, whereas it resulted in intracellular accumulation of DE- cadherin, Fmi and b-integrin. Importantly, inactivation of Rbsn-5 showed accumulation of FasIII and b-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization. These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains. PI3K & Rbsn-5 in Drosophila Results Gal4/UAS -mediated knockdown of PI3K (III) in Drosophila Drosophila PI3K (III) consists of two essential components, the catalytic subunit dVps34/PI3K59F (CG5373) and the anchoring subunit dVps15/p150 (CG9746 (see Fig. 1A). To analyze the function of PI3K (III), transgenic fly strains were generated harboring ,500 bp of inverted cDNA fragment repeats, corre- sponding to the PI3K (III) subunits, under the control of the GAL4-responsive UAS. Crossing these strains with appropriate Gal4 driver strains induces a hairpin type double strand RNA (dsRNA) in a Gal4-dependent manner, both temporally and spatially. Accordingly, dsRNAs in whole or restricted regions of the wing primordia were induced using scalloped-Gal4 (sd-Gal4) or decapentaplegic-Gal4 (dpp-Gal4) driver strains, respectively. The knockdown of either dVps34 or dVps15 by sd-Gal4 resulted in a severe malformation of the wings (Fig. 1C), with similar phenotypes being induced by a knockdown of either dVps34 or dVps15. To test whether PI3K (III) is responsible for this phenotype, we performed a rescue experiment by examining whether the expression of the wild-type cDNA would suppress the phenotype induced by the dsRNA-mediated knockdown. It was found that the defects induced by knockdown of dVps34 were completely suppressed when the wild-type cDNA of dVps34 was simultaneously expressed (Fig. 1C). To examine further the dsRNA-mediated knockdown reduction of in vivo activity of the PI3K (III), the localization of tandem- repeated FYVE domains fused to GFP (GFP-2xFYVE) was investigated [40] (Fig. 1D). GFP-2xFYVE is localized with PI(3)P-rich membrane domains in endosomes, since the FYVE domain binds to PI(3)P generated by PI3K (III) (Fig. 1D). When we reduced dVps15 expression in the wing cells by inducing dVps15 dsRNA, GFP-2xFYVE was no longer localized to the endosomes, Figure 1. Gal4/UAS mediated RNA interference in Drosophila. (A) Schematic representation of Drosophila phosphatidylinosytol 3 kinase class III (PI3K(III)) composed of dVps34 (a catalytic subunit, CG5373) and dVps15 (an adaptor subunit, CG9746). PI3K C2 domain, PI kinase conserved domain (PIK domain), PI3K catalytic domain, serine threonine protein kinase domain, WD 40 domain and N-terminal myristoylation are indicated. Underlining indicates regions used to construct dsRNA inducible vectors. (B) dVps34 and dVps15 mRNA abundance were reduced by transfection of the respective dsRNA (+) in S2 cells. RNAs was quantified by RT-PCR. c-tubulin was used as a control. (C) Adult wings in which dsRNAs for either dVps34 (dVps34 RNAi) or dVps15 (dVps15 RNAi) were expressed by the sd-Gal4 driver. The severe malformation was completely rescued by co- expression of wild-type cDNA with dsRNA for dVps34 (dVps34 Rescue), compared with the wild-type wing (Control). (D) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 32 h APF. Double-headed arrows indicate the regions where only GFP-2xFYVE, or both GFP-2xFYVE and dsRNA for dVps15, were expressed using the dpp-Gal4 driver. GFP-2xFYVE (green) was localized to dot-like structures in the wild type but dispersed in the cytoplasm in the dVps15-knockdown cells. Wing hairs stained by rhodamine phalloidin (red) were pointed and orderly in the wild type but deformed and irregularly pointed in the dVps15-knockdown cells. The scale bars represent 10 mm. doi:10.1371/journal.pone.0007306.g001 PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7306