but rather was dispersed within the cytoplasm (Fig. 1D), suggesting that the knockdown significantly reduced PI3K (III) activity in Drosophila. The above results taken together led us to conclude that the defects were caused by a reduction in the level of PI3K (III) activity. PI3K (III) is required for efficient endosomal trafficking to the lysosomes The Rab family of GTPases plays pivotal roles in the regulation of vesicular trafficking, the Rab5, Rab7 and Rab11 members contributing to the formation and dynamics of early endosomes, late endosomes and recycling endosomes, respectively [41]. It seems likely that PI3K (III) products might co-localize with these Rab family proteins since they are known to physically associate with Rab5 [10], and possibly with Rab7 [42]. Indeed, GFP- 2xFYVE has been shown to localize with Rab5-positive endo- somes, when expressed in Drosophila S2 and neural cells [40]. We therefore examined which endosomal compartments GFP- 2xFYVE is localized to when expressed in larval wing cells. In larval wing disc cells GFP-2xFYVE was localized mostly to Rab7- positive endosomes, but not to Rab5- or Rab11-positive endosomes (Fig. 2A). In addition, some of the GFP-2xFYVE- positive endosomes were stained with Lysotracker (Fig. 2A), suggesting that they were acidic, including the late endosomes. PI3K (III) products therefore appear to localize to Rab7-positive late endosomes in these cells. Although PI3K (III) has been implicated in the fusion of clathrin-coated vesicles and early endosomes and also in the fusion between early endosomes, it has been unclear whether reduced PI3K (III) activity impairs these endosomes in Drosophila wing disc cells. Knockdown of dVps15 markedly increased the numbers of Rab5-positive and Rab7-positive dot-like structures, as shown in Fig. 2B, suggesting that the inactivation of PI3K (III) leads to the PI3K & Rbsn-5 in Drosophila accumulation of early and late endosomes. Interestingly, knock- down of dVps15 also increased the numbers of endosomes positive for the Rab11 effector Nuf [43,44], indicating that PI3K (III) plays either a direct or an indirect role in the regulation of recycling endosomes. The abnormal accumulation of endosomal markers raises the possibility that PI3K (III) is required for the endosomal trafficking and endosome formation. The question of whether the inactivation of PI3K (III) also affects the vesicular trafficking leading to lysosomes in Drosophila wing cells was then examined, since it has been demonstrated with other cell types [20,28]. To this end, the incorporation and intracellular trafficking efficiencies of Texas Red-labeled dextran (TR-D) in the wing discs was determined. Discs from wild type and knockdown larvae expressing GFP-LAMP1, a lysosomal marker [45], were placed in Schneider’s medium containing TR-D for 5 min and then washed. At 5, 20, 40, or 60 min after washing, discs were fixed and the number of TR-D-positive lysosomes counted (Fig. 2C). At all time points observed the percentage of GFP-LAMP1 and TR-D double-positive vesicles (TR-D-positive lysosomes) among the TR-D positive vesicles was clearly lower in the knockdown cells, compared with the normal cells (Fig. 2C). This suggests that intracellular endosomal trafficking to the lysosome is impaired by knockdown of PI3K (III). PI3K (III) is required for lysosome maturation Lysosomes mature from MVBs, and PI3K (III) is necessary for the formation of internal vesicles within the MVBs in mammalian cells. We therefore examined whether maturation of MVBs also requires PI3K (III) activity in Drosophila. Knockdown cells were observed at high resolution using a TEM (transmission electron microscopy), and it was found that the number of abnormal MVB- like structures markedly increased (Fig. 3B and C), although these compartments were rarely observed in wild type cells (Fig. 3A). Figure 2. Endocytotic trafficking is compromised by knockdown of PI3K (III). (A) GFP-2xFYVE co-localized with Rab7 and Lysotracker but not with GM130, Rab5 or Rab11. Arrowheads point to the GFP-2xFYVE-positive compartments with Rab7 and Lysotracker. (B) The numbers of Rab5-, Rab7- and Nuf-positive endosomes (red) were increased in the dVps15-knockdown cells (left sides of white lines, indicated by double-headed arrows) compared with the wild type cells (right sides of white lines). The GFP-2xFYVE (green) and dsRNA for dVps15 were simultaneously expressed by dpp- Gal4 driver. (C) Upper panels: Trafficking of TR-D from the plasma membrane to the lysosome was monitored by double labeling of TR-D and GFP- LAMP1 (lysosome marker, green) in the wing discs. Wing discs of the wild type or dVps15 knockdown were pulse labeled with TR-D for 5 min and chased in Schneider’s medium for the indicated times. The wing discs were then fixed and analyzed by confocal microscopy. Arrows show the co- localization of TR-D and GFP- LAMP1. The scale bars represent 10 mm. Lower graph: The ratios of co-localized TR-D to GFP-LAMP1 in the wild or dVps15 knockdown discs (n = 89,152). doi:10.1371/journal.pone.0007306.g002 PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7306