PI3K & Rbsn-5 in Drosophila Figure 7. The number of basolateral large PI(3)P-positive endosomes was decreased in Rbsn-5 knockdown pupal wings. Ratios (upper) or numbers (lower) of small (,0.5 mm, blue), middle (0.5–1.0 mm, red) and large (1.0–1.5 mm, yellow) endosomes are shown for repeated 1 mm sections from the apical to basal region in the wild type (left), or Rbsn-5-knockdown (right) wings. doi:10.1371/journal.pone.0007306.g007 Discussion In this study, we have demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and b-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study. We have also demonstrated that Rbsn-5, a FYVE domain- containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and b-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5C241 mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface. PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes [15]. However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. Our results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III). Materials and Methods Fly strains Flies were reared in vials containing a standard cornmeal medium at 25uC. Conton–S flies were used as the wild type. The transgenic strains used were Tub . YFP Rab7 [47]. Several transgenic strains were expressed by using the Gal4/UAS system [48]. UAS strains were UAS-GFP-Myc-26FYVE [40] and UAS- GFP-LAMP1 [45], UAS-dVps34 (CG5373) dsRNA, UAS-dVps15 PLoS ONE | www.plosone.org 9 October 2009 | Volume 4 | Issue 10 | e7306