Loss of vap alters starvation-induced autophagy-membrane biogenesis.
(A) The autophagosome marker GFP:Atg8a was expressed in control, w- or vap1 fat bodies using a cg-Gal4 driver, and mid-3rd instar larvae were starved for 1h30’. Fat bodies were dissected and stained LysoTracker red for an immediate observation of live tissue. Shown, are comparable images of control, w- and mutant, vap1 cells, which respectively scored 14 yellow vesicles depicting fused autolysosomes (AL) out of 52 total vesicles, and 1 yellow vesicle out of 51 total vesicles, indicating that no or rare autolysosomes had formed in the mutant. LysoTracker red signal area intersecting GFP:Atg8a signal area was 77,3% in control and only 2,3% in vap1 confirming the lack of fused structures. Scale bars = 10 μm. (A’, A”) Green and red vesicular-membrane signals were extracted from similar images and their size (in μm2) and circularity index (1.0 for perfect circles; 0.0 for elongated polygons) graphed as scatter plots (w-: n = 106 green vesicles, n = 49 red vesicles; vap1: n = 43 green vesicles, n = 107 red vesicles). Below are enlarged pictures of green and red single channel images for w- and vap1 respectively. Yellow arrows point to autolysosomes in w-. Green and red arrows point to autophagosome membranes and lysosome respectively, in vap1. Dotted arrows emphasize the absence of vesicular fusion. Scale bar = 10 μm. In A’, control, w- green and red vesicles has relatively packed distribution as a fraction of them derive from same autolysosomes. Mutant vap1 cells has non-fused green and red membranes (or vesicles) which are of larger sizes and wider distributions (green vesicles Mdn = 1,46 μm2 in vap1 vs 1,25 μm2 in controls; red vesicles Mdn = 1,86 μm2 in vap1 vs 1,10 μm2 in controls). These differences correlate with a decreased circularity index of the mutant particles in A” (green vesicles Mdn = 0,47 in vap1 vs 0,63 in controls; red vesicles Mdn = 0,62 in vap1 vs 0,79 in controls), indicating that mutant autophagosomes and lysosome membranes were wider and of uneven shapes. Medians are drawn as lines; significances are from Mann Whitney test. (B) The late endosome Rab7:GFP marker was expressed in control, w- and mutant, vap1 larval fat bodies using a cg-Gal4 driver and 3h-starved fat cells were analyzed. Endogenous p62/SQSTM1 flux marker was detected by immunostaining. In control, w- starved cells, fine p62 bodies (grey or red) are detected over the cytosol whereas Rab7:GFP aggregates are forming independently of them. In vap1 cells both the density (B’) and size range of p62 bodies and Rab7:GFP aggregates (S1D and S1D’ Fig) are increased and the two markers match frequently (B”), suggesting accumulation of unresolved maturation intermediates in the mutant. Scale bar = 20 μm. (B’) The densities for p62 bodies and Rab7:GFP aggregates were compared in a selections of fat body cells samples of defined areas of control and mutant cell in B (w- n = 8, vap1 n = 13). Error bars are standard errors; significances are from ANOVA. (B”) The overlap between Rab7:GFP and p62 staining was determined in the set of cells used in B’. Rab7:GFP signal areas intersecting p62-positive pixels were expressed relative to total p62 staining areas. Significant intersections are only found in the case of starved vap1 cells. Error bars are standard errors; significances is from Student’s t-tests. (D-D”) TEM semi-quantitative analysis of autophagy structures found in fat body cells of 2h-starved early/mid-3rd instar of control, w- and vap1 mutant (Table C in S5 Fig). Control, w- shows typical degradative autolysosomes (AL in D’; scale as in mutant below) of ca. 0.5 μm in size (28 cases of AL out of 41 scored autophagy-related structures). No autolysosomes were detected in vap1 samples. Instead, large hybrid organelles (arrowhead: giant amphisome) of greater than 2 μm are observed (14 giant amphisomes cases out of 38 scored autophagy-related structures). These have single-bilayered membranes (D” inset: white arrow) and are filled with intraluminal, electron-clear vesicles (black arrow) akin those of MVB (multivesicular bodies). These structures appear to match the accumulated maturation intermediate detected in B. Scale bar = 1 μm. Genotypes. (A) Control: w1118/Y; cg-GAL4/ UAS-GFP:Atg8a/+. Assay: vap1/Y; cg-GAL4/ UAS- GFP:Atg8a/+. (B, C, C’) Control: w1118/Y; cg-GAL4/ UAS-Rab7:GFP/+. Assay: vap1/Y; cg-GAL4/ UAS-Rab7:GFP/+. (D) Control: w1118/Y. Assay: vap1/Y.