Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development
Figure 6
Impaired endocytic degradation of Notch in Atg6 depleted cells. (a) In 3rd instar larval wing discs Atg6 null mutant clones (marked by the absence of GFP, asterisks) accumulate the extracellular domain of Notch (NECD). (b) Quantification of (a). (c), (e), and (g) Imaginal wing discs expressing dsRNA (Atg6, Atg14) or siRNA (UVRAG) by enGal4 were immunostained against NECD. The region of RNAi is marked by the coexpression of GFP and the borderline of the region is indicated with a dashed white line. Immunostain revealed that NECD accumulated in small, numerous puncta in the case of (c) Atg6 RNAi and (e) UVRAG RNAi, (g) but not in Atg14 RNAi. (d), (f), and (h) Quantification of (c), (e), and (g), respectively. (i) Live trafficking assay for Notch in cultured wing imaginal discs. Atg6 RNAi region is marked by the coexpression of the lysosome marker Lamp1-GFP. In controls, surface-bound Notch was internalized then degraded after 3 h. In contrast, in Atg6 depleted cells Notch was internalized normally but trapped in vesicular structures even at 3 h of chasing. Arrows indicate Notch signal colocalizing with Lamp1-GFP. 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 50 μm.
