PI3K & Rbsn-5 in Drosophila Figure 3. Abnormal MVB-like structures were observed in PI3K (III)-knockdown pupal wing cells. (A–E) Horizontal EM images of the pupal wings of the wild type (A and E), or dVps15 knockdown (B–D), at 30 h APF. A number of electron-dense compartments appeared in the cytoplasm at the apical side (green parentheses in B). These electron-dense compartments appeared to be MVB-like structures (C and D) that were scarcely observed in the wild type cells (A). (D and E) Immuno-EM images of the pupal wings obtained with a 10 nm gold particle-labeled GFP antibody to detect GFP- LAMP1. Signals of anti-GFP antibody in the wild type (E) and dVps15-knockdown wings (D) are shown, respectively. The scale bars represent 1 mm (A–C), 500 nm (D) and 200 nm (E), respectively. doi:10.1371/journal.pone.0007306.g003 These abnormal MVB-like structures were preferentially localized within the apical region but not in the basolateral region of the knockdown cells (data not shown). Also the MVB-like structures were GFP-LAMP1-positive (Fig. 3D), suggesting that they may be immature lysosomes. It can therefore be concluded that PI3K (III) is required for the maturation of MVBs into lysosomes in the apical region of the cells. PI3K (III) regulates the localization of Fmi and DE-cadherin The above results on the regulation of lysosomal degradation processes are largely consistent with the previously reported functions of PI3K (III). We next analyzed the function of PI3K (III) in proteins that localize to distinct membrane domains along the apical-basal axis. In the apical membrane domain, Fmi, a planar cell polarity (PCP) protein, is localized to the apical side of the ZA. Fmi first localizes uniformly to the apical ZA and then accumulates in both the proximal and distal sides of the cell ,30 h after pupal formation (APF) [37] (Fig. 4A). In dVps15 knockdown cells, Fmi accumulated in intracellular small compartments in the apical region (Fig. 4A). The accumulated Fmi was found mostly in LAMP1-positive vesicles (Fig. 4B). Considering that PI3K (III) contributes to the maturation of MVBs into lysosomes, this result suggests that the degradation of Fmi is impaired by the dVps15 knockdown. DE-cadherin is known to localize to the ZA and in wild-type cells was localized mostly to the ZA and to some extent to other intracellular small compartments (Fig. 4A). However, knockdown of dVps15 disrupted the localization of DE-cadherin at the ZA and a significant amount accumulated in the intracellular small compartments in the apical region (Fig. 4A). The accumulated DE-cadherin was partly localized to the LAMP1-positive com- partments (Fig. 4B), suggesting that it was due, at least in part, to a defect in the degradation pathway. Finally, it was found that the compartments where DE-cadherin or Fmi accumulated showed little overlap (Fig. 4B). We also stained Vps15-knockdown discs with antibodies against DE-cadherin together with those against several compartment markers such as Rab5, Rab7 and Rab11. DE-cad co-localized partially with Rab11, but not with Rab5 or Rab7 (data not shown), suggesting that a fraction of DE-cad accumulates in recycling endosomes. The remainder of the DE- cad was localized in unidentified compartments which were negative for both of these markers. DE-cadherin is known to bind to its intracellular partner Arm, accordingly whether the localization of Arm is also affected by the knockdown of PI3K (III) was investigated. In wild type cells, Arm was localized to the ZA (Fig. 4A). However, in the dVps15 knockdown region, Arm accumulated in the apical small compartment (Fig. 4A), similar to DE-cadherin. Together, these results suggest that PI3K (III) is required for the proper localization of Fmi and the DE-cadherin-Arm complex to the ZA. PI3K (III) regulates the localization of Fas III and b-integrin in a distinct manner It was next determined whether PI3K (III) also regulates the localization of basolateral membrane proteins. b-integrin was localized in intracellular vesicles at early pupal stages, but gradually translocated and accumulated to the basal junctions (data not shown). At 30 h APF, b-integrin formed large basal junctions at the basal plasma membrane of the prospective intervein cells (Fig. 5). By contrast, when dVps15 was knocked PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7306