Fig. 4.

Impaired V-ATPase activity prevents the signaling activation and overproliferation observed in ESCRT endocytic tumors. (A-C′) Ubiquitin staining of Vha55, vps22 and Vha55 vps22 double-mutant Drosophila eye discs expressing mβ-lacZ. As in Vha68-2^R6, mβ-lacZ expression is low in Vha55 discs and ubiquitin accumulation is not observed (A-A′). By contrast, vps22 mutants express high levels of mβ-lacZ and display accumulation of ubiquitin (B-B′). The Vha55 vps22 double mutant expresses levels of mβ-lacZ that are similar to those of Vha55 mutants, but displays an accumulation of ubiquitin that is similar to that of vps22 mutants (C-C′). (D-F′) Vha55, vps22 and Vha55 vps22 double-mutant eye discs colabeled to the detect the ICD of Notch and Hrs. As in Vha68-2^R6, Notch does not colocalize with Hrs in Vha55 discs (D-D′). By contrast, in both vps22 mutants (E-E′) and Vha55 vps22 double mutants (F-F′) partial colocalization of Notch and Hrs is observed. (G-I) mβ-lacZ expression and proliferation in eye discs. As in Vha68-2^R6, Vha55 mutant discs underproliferate and express low levels of mβ-lacZ (G), whereas vps22 mutants express high levels of mβ-lacZ and overproliferate (H). The Vha55 vps22 double mutant expresses levels of mβ-lacZ and exhibits levels of proliferation that are similar to those of Vha55 mutants (I). (J-L) Mosaic eyes generated by MARCM stained to detect cortical actin. Compared with GFP-positive vps22 mutant cells, which display Notch-dependent overgrowth (K), GFP-positive Vha55 mutant cells (J) and GFP-positive Vha55 vps22 double-mutant cells (L) do not overgrow. (M) Quantification of the experiment shown in J-L, indicating that lack of V-ATPase function rescues the Notch-dependent overproliferation observed in vps22 mutant cells. Scale bars: 10 μm in A-F′; 100 μm in G-L.