Figure 4. Autophagic vesicles expand under prolonged starvation in V-ATPase-depleted cells.

(a) Representative images of LAMP1-GFP and mCh-Atg8a-positive vesicles in fat body cells depleted for Syx17 or V1H under fed, 4 h starvation or 24 h starvation conditions. Autophagic vesicles progressively expand in V-ATPase-depleted but not Syx17-depleted cells. (b) Quantification of LAMP1-positive vesicle diameter in cells depleted for Syx17 or V1H as shown in a. Error bars mark s.e.m.; fat body cells from a minimum of eight larvae were analysed for each genotype and time point. ***P<0.001, Student's t-test. (c) TEM images showing representative autolysosomes in control and V1H-depleted fat body cells following 0, 4 and 24 h starvation. Scale bar, 1 μm. (d) Quantification of individual autolysosome area in control and V1H-depleted fat body cells as shown in c. Vesicles from 15 sections from three larvae were analysed for each genotype and time point. Error bars mark s.e.m.; ***P<0.001, Student's t-test. (e) Mosaic fat body containing a GFP-marked clone of cells depleted for Syx17 and the V-ATPase subunit V0d, following 4 h starvation. mCh-Atg8a-marked autophagic vesicles in these cells are not enlarged relative to those in the surrounding GFP-negative control cells. N=7. In a and e, nuclei are labelled in blue with DAPI; scale bars, 10 μm. (f) High-magnification confocal images of mCh-Atg8a-positive vesicles in fat body cells depleted of V1H under starvation-induced autophagy (4 h). Variation of fluorescence intensity is observable within individual single vesicles. N≥10. Scale bar, 1 μm. (g) High-magnification TEM image illustrates a non-degradative autolysosome containing four autophagic bodies (1–4) in V0c-depleted fat body. Scale bar, 0.3 μm.