Fig. 3.

Notch accumulates in endosomes of ESCRT mutant cells leading to overactivation of signaling. (A-F) ESCRT-I, ESCRT-II and ESCRT-III mosaic eye discs stained with an antibody recognizing the intracellular domain of Notch. Compared with WT tissue, Notch is depleted from the cellular surface and accumulates in enlarged endosomes (labeled Avl in A′-F′) in all ESCRT mutants except vp32 mutants, in which less Notch accumulates (F). (G-L) Notch localizes in intracellular puncta in vps25 mutants and is diffuse in vps32 mutants. Notch immunodetection (Notch ECD; G-I) and 5 hour Notch internalization assay (inter. NECD; J-L) using antibodies directed to the extracellular portion of Notch in WT (G,J), vps25 (H,K) and vps32 (I,L) entirely mutant eye disc tissue. In vps32 mutants, Notch accumulation in intracellular puncta is reduced, whereas cortical localization is increased compared with vps25 (especially visible on fixed tissue in I). Note that in J, very little signal is present because most Notch is degraded 5 hours after labeling. (M-R) ESCRT-I, ESCRT-II and ESCRT-III mosaic eye discs stained with an antibody to detect expression of the Notch target unpaired (Upd). Compared with WT tissue, Upd is ectopically expressed to varying degrees in mutant cells. Notably vps32 mutants appear to express the lowest amounts of Upd. Mutant tissue is encircled by dashed pink lines in A-F, M-R (white in A′-F′). Scale bars: 10 μm.