V100 is required for the acidification of a subset of neuronal degradative compartments. (A–C’) Lysotracker live measurements in eye–brain cultures. (A and B) Representative scans of v100 MARCM eye discs (mutant cells marked with GFP). (A’ and B’) Only Lysotracker channel. (C) Live scan of a v100 MARCM eye expressing v100^R755A only in the mutant cells marked by GFP. (C’) Only Lysotracker channel. (D) Quantification of Lysotracker data in A–C’ shows a significant reduction in Lysotracker signal after photoreceptor differentiation at P + 40% (orange, control [ctrl]; blue, mutant). Expression of v100^R755A in the mutant (green) does not rescue the Lysotracker signal reduction. Gray, expression of v100^WT in mutant photoreceptors. (E) Identification of Lysotracker-positive compartments by live imaging using GFP-Rab5, GFP-Rab11, GFP-Rab7, and Atg8-GFP. Note that the ratio of Rab7/Lysotracker and Atg8/Lysotracker compartments is unaltered in the mutant despite the 50% Lysotracker reduction shown in D. The inset shows Rab7-positive late endosomes that are filled with Lysotracker (arrows). The arrowhead shows a Rab7-negative, lysotracker-positive ring, presumably a lysosomal or autophagosomal structure. (F–H) Block of all v-ATPase–dependent acidification using bafilomycin A1 in L3 larval eye discs with mutant v100 MARCM clones marked by GFP. Error bars indicate SEM. Bars: (A’ and F’) 10 µm; (E) 1 µm.