Figure 5.

Sanpodo promotes Notch endocytosis. (A) apterous-Gal4–driven ectopic expression of GFP-tagged Sanpodo transgenes (green) in wing disc epithelial cells labeled with the Notch^ECD antibody (red). Bar, 100 µm. White box is the approximate region of disc shown in B–G. Merged apical XY confocal sections through wing disc epithelial cells (1.5-µm thickness, the border between GFP+ and GFP− regions are marked by a dashed yellow line). Expression of GFP alone has no effect on NECD localization to apical cell junctions in wing disc epithelial cells (B), whereas expression of Sanpodo^wt-GFP depletes Notch from the apical membrane in epithelial cells and causes Notch to accumulate in large apical vesicles, which in some cases colocalize with Sanpodo-GFP (C, white arrowheads). Both the ΔNPAF mutant (D) and the LHAAA mutant (E) retain the ability to deplete apical Notch, whereas the ΔNPAF, LHAAA mutant (F) or ΔN180 mutant of Sanpodo (G, ΔN180) abrogate Notch apical depletion. Quantification of apical NECD staining represented as a ratio of the area occupied by apical NECD staining in the GFP+/GFP− regions of merged apical XY planes from apterous-Gal4–driven ectopic expression of GFP-tagged Sanpodo transgenes and a GFP control 9H). Sanpodo-GFP and Sanpodo^ΔNPAF-GFP significantly deplete membrane Notch levels, whereas Notch levels are decreased in the LHAAA; they do not attain statistical significance. In contrast, Notch levels in the Sanpodo^{ΔNPAF, LHAAA}–GFP expression region were indistinguishable from GFP control expression, and significantly increased relative to Sanpodo-GFP. Bars, 25 µm.