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Figure 10: Polarity and membrane protein localization defects of Atg6-knockdown pupal wings. Semiconfocal immunodetection showing XY (marked by a letter p (plan view) at the bottom left corners) and XZ sections (marked by a letter s (side view) at the bottom left corners) in the plane of pupal wings at 32 h APF. Regions of controls are shown in boxes marked by a single character at the upper left side, whereas the regions where dsRNA for Atg6 was expressed by en-Gal4 driver are shown in boxes marked by a character at the upper left side with an apostrophe. (a) Low magnification image of a pupal wing with rhodamine-phalloidin stain showing the regions from where the high magnification images were taken. The expression of the dsRNA by enGal4 is restricted to the posterior compartment of the wing (marked by the coexpression of GFP), and anterior side serves as control. Small boxes surround areas of images (b) and (b′) in which rhodamine-phalloidin stain reveals that control wing hairs are well-developed and very regularly oriented towards the distal end of the wing. In contrast the wing hairs are poorly developed or completely absent in the regions of the RNAi. (c)–(e) In controls Fmi, Arm, and DE-Cad show a very pronounced zonula adherens (ZA) localization; furthermore Fmi shows a very regular planar cell polarity pattern as well, whereas all of them show intracellular punctuation and irregular localization in the Atg6 depleted wing regions. (f), (g) Septate junction proteins Fas III and Dlg show very typical lateral localizations as Dlg is mainly localized at the apical regions of septate junctions (SJ), whilst the domain of Fas III is expanded throughout the whole SJ. In contrast, in Atg6 RNAi cells the amounts of these proteins lowered and both show less pronounced SJ localization. (h) At this developmental stage the prospective wing intervein cells form large, β-integrin (β-int) containing basal junctions between the two epithelial layers of the wing, but this process is dramatically blocked when the level of Atg6 is reduced and wing cells seem to be unable to develop these structures. For genotype, see Table S4. Scale bars represent (a) 10 μm and (b)–(h) 5 μm.