Fas III becomes delocalized from the lateral plasma membrane and is degraded by this stage. Here we examined the effect of PI3K (III) on the localization of Fas III at 30 h APF and it mostly disappeared from the basal region of the lateral plasma membrane but remained in the SJ (Fig. 5). The dVps15 knockdown greatly increased the amount of Fas III remaining in the lateral plasma membrane region (BLPM, Fig. 5) even at 30 h APF. This phenotype is strikingly distinct from that of the intracellular accumulation of Fmi, DE-cadherin and b-integrin induced by dVps15 knockdown. These results suggest that PI3K (III) might regulate Fas III and these other proteins in a different manner. Rbsn-5, a FYVE domain-containing protein, is selectively required for the proper localization of Fas III and b-integrin The above results indicate that PI3K (III) is required for the degradation and correct localization of Fmi, DE-cadherin, Fas III and b-integrin. To identify the factor under the control of PI(3)P, we selected genes from the Drosophila genome database that contain FYVE and/or PX domains, since these domains are known to bind to PI(3)P. The dsRNAs were then expressed, corresponding to each of the selected genes in the wing discs, and the effect on the localization of these membrane proteins determined. From this screen, we found that a Drosophila Rbsn-5 knockdown influenced the localization of Fas III and b-integrin. In wild type cells, b-integrin was found to localize at or near the basal membrane as large clusters but was absent from the apical region at 27 h APF (Fig. 6A). However, knockdown of Rbsn-5 markedly reduced the amount of b-integrin localized at or near the basal membrane and slightly increased the amount of intracellular vesicles located in the apical region (Fig. 6A and B). Furthermore, at 30 h APF for wild type cells, Fas III was mainly localized in the apical region of the lateral plasma membrane, while only a small fraction of Fas III was localized in the basolateral region (Fig. 6A). By contrast, when Rbsn-5 was knocked down, Fas III was also found at the basal region of the lateral plasma membrane (Fig. 6A and B). These Rbsn-5 knockdown cell phenotypes were reminiscent of those of the PI3K (III) knockdown cells. To further investigate the roles of Rbsn-5 in the regulation of membrane protein localization, mutant cells were analyzed from a allele for Rbsn-5C241 [46]. Compared to the wild type cells (GFP-positive cells), b-integrin accumulated in significant amounts in the intracellular vesicles in the rbsn-5 mutant clone cells (GFP- negative cells) at 30 h APF (Fig. 7C; a and b). In the case of Fas III, the amount that was localized to the basolateral plasma membrane was greatly increased (Fig. 7C: c and d). However, importantly, the Rbsn-5 mutation did not result in an intracellular accumulation of Fmi, DE-cadherin or Arm, under the same conditions where it affected the localization of b-integrin and Fas III (Fig. 7C; e-g). Also the Rbsn-5 mutation had no effect on the actin bundling that could be observed at the apical surface (Fig. 7C: h). Therefore, Rbsn-5 appears to selectively regulate the localization of b-integrin and Fas III in this context. These results strongly suggest that Rbsn-5 mediates only a part of the function of PI3K (III). Rbsn-5 increases the size of PI(3)P-positive endosomes, a phenomena prominent at the basal side of the cell Given that Rbsn-5 regulates the localization of basolateral membrane proteins but not that of apical junctional membrane proteins, it is possible that it preferentially regulates basolateral vesicular trafficking. It has been reported in mammalian cells (WIF-B hepatocytes) that PI(3)P-positive endosomes at the apical regions are larger, relative to those at the basolateral region [15], PI3K & Rbsn-5 in Drosophila however, with Drosophila wing disc cells the opposite was true. When PI(3)P-containing endosomes were visualized by GFP- 2xFYVE, small endosomes (,0.5 mm diameter) were found more frequently at the apical side, whereas large endosomes (1.0–1.5 mm diameter) were found more frequently at the basal side in wild type cells (Fig. 6 and 7). Strikingly, the Rbsn-5 knockdown reduced the size of GFP-2xFYVE-positive endosomes throughout the apical- basal axis of the cell (Fig. 6 and 7). This suggests that Rbsn-5 increases the size of PI(3)P-containing endosomes which appear more prominent in the basolateral regions, likely due to the fusion of small PI(3)P-containing endosomes. The selective roles of Rbsn-5 in the regulation of basolateral membrane proteins prompted us to examine the localization of endogenous Rbsn-5. We found that Rbsn-5 was localized to intracellular endosomes at both the apical and basolateral regions (data not shown). It is therefore unlikely that the selective roles of Rbsn-5 were due to a selective localization of this molecule along the apical-basal axis. In addition to PI(3)P, Rbsn-5 has been shown to associate with Rab5 and Rab4 and to be controlled by these Rab family small GTPases, which govern endosome fusion and recycling processes, respectively [31]. Drosophila Rbsn-5 can bind to the GTP form of Rab5, but not to Rab4 or Rab7 in vitro [35,46]. Although Rbsn-5 has been proposed to function at Rab5 or Rab4-positive endosomes in mammalian cells, we found that it was localized not only at the Rab5-positive endosomes but also the Rab7- positive endosomes, whereas it was absent from Rab11-positive endosomes (Fig. 8A). This supports the notion that Rbsn-5 is involved in the sorting to late endosomes in the degradation pathway. Functional interactions between PI3K (III) and Rbsn-5 Finally, we examined the functional relationship between PI3K (III) and Rbsn-5. Although Rbsn-5 contains a FYVE domain that binds to the PI3K (III) product PI(3)P, and it has been shown to be essential for the localization of Rbsn-5 to early endosomes [33], a functional relationship between the two molecules has remained unknown. Firstly, the effect of dVps15 knockdown on the distribution and abundance of Rbsn-5 was examined. The intensity of Rbsn-5 immunostaining was markedly reduced in the dVps15 knockdown cells (Fig. 8B). This suggests that PI3K (III) regulates the level of Rbsn-5 protein, in addition to its localization. Next we examined whether Rbsn-5 genetically interacts with PI3K (III). To this end, we examined phenotypes produced by knockdown of dVps34, dVps15, Rbsn-5, or a combination of these (Fig. 8C). dsRNA-mediated knockdown of target mRNAs is usually incomplete, and a low expression level of the mRNA is expected to remain (Fig. 1B). The malformation of wing morphology, a detachment of the dorsal and ventral surfaces, induced by the knockdown of dVps15 was strongly enhanced by a simultaneous knockdown of dVps34 or Rbsn-5 (Fig. 8C). By contrast, we did not find any genetic interaction between Rbsn-5 or dVps34 and the PI3K class I/p110 (Fig. 8C). Since the attachment of the dorsal and ventral wing surfaces requires the basal localization of integrins to form basal junctions, we also examined whether the abnormal localization of b-integrin by the single knockdown for dVps15 or Rbsn-5 is enhanced by their double knockdown. To demonstrate the synergistic enhancement, we induced dsRNAs weakly for either dVps15 or Rbsn-5 to induce only marginal defects (Fig. 8D). Under this condition, the double knockdown resulted in obvious delocalization of b-integrin (Fig. 8D), which is consistent with the adult wing phenotype. These results support the notion that Rbsn-5 functions under the control of PI3K (III). PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7306