Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development
Figure 2
Knockdown of Atg6 and UVRAG. (a) The PI3P marker GFP-2xFYVE localized to dots and vesicle-like structures in wild type pupal wings. (b), (d) In Atg6 or UVRAG depleted wings the GFP-2xFYVE became dispersed within the cytoplasm, indicating a failure in PI3P production. (c) In contrast to Atg6 or UVRAG RNAi wings the localization of GFP-2xFYVE did not differ from controls in Atg14 RNAi wings. (e) Quantification of (a)–(d). (f), (g) In control wings clathrin coated vesicle marker Clc-GFP showed punctate pattern which was unaffected by Atg6 RNAi. (h) Quantification of (f), (g). (i)–(w) Images from pupal wings expressing endosomal and lysosomal fluorescent markers controlled by a constitutive promoter (tubulin promoter for Rab-FP-s and Lamp1-GFP, cathD promoter for CathD-mCherry) and expressing RNAi constructs by enGal4. The region of RNAi is marked by the coexpression of RFP or GFP and the borderline of the region of RNAi is indicated with a dashed white line. (i)–(p) The number of structures positive for endosomal markers ((i): Rab4-YFP; (k): Rab5-CFP; (m): Rab7-YFP, except (o): Rab11-YFP) was significantly increased in Atg6 RNAi cells. (j), (l), (n), and (p) Quantification of (i), (k), (m), and (o), respectively. (q)–(t) The number of lysosomes marked by Lamp1-GFP or CathD-mCherry was significantly increased in Atg6 depleted cells. (r), (t) Quantification of (q), (s), respectively. (u) UVRAG RNAi resulted in the accumulation of Lamp1-GFP structures very similar to Atg6 RNAi. (v) Quantification of (u). (w) RNAi of Atg14 did not alter the number of Lamp1-GFP positive dots. (x) Quantification of (w). On images from (i) to (w), the intensity of the markers is enhanced by immunostaining the wings with an anti-GFP or anti-mCherry. Note the presence of RFP positive hemocytes under the epithelia on (i), (m), and (o) (marked by asterisks). On box plots, bars show the data ranging between the upper and lower quartiles; median of the signal covered areas is indicated as a horizontal black line within the box. Whiskers plot the smallest and largest observations, while dots and asterisks indicate outliers. NS means and means . For details and exact values of statistical analyses, see Table S3. For genotypes see Table S4. Scale bars represent 15 μm.
