Perturbations of animal fitness and basal, or starvation-induced autophagy in vap mutants.
(A) The total amount of TAG in aged-matched samples of adult male flies grown in rich food was compared between control, w- and mutant, vap1 and vap2 (n = 4). Mutants showed diminished TAG stores. (B) The mass of adults male flies was compared in aged-matched samples when grown in rich food or poor food (aa-rich or aa-poor conditions, Materials and Methods) (n = 5–8). Mutants had chronic mass deficits in rich conditions. Mutant flies emerged 10h in advance compared to controls but had quite limited mass deficits when grown in poor food conditions. The same effects were observed in females. In A-B, error bars are mean differences; significances are from Student’s t-tests. (C) Acidic lysosomal compartments were revealed by pH-sensitive LysoTracker staining in life tissue. Fat bodies in 96h, mid-3rd vap1 larvae but not control, w- showed punctuate fluorescent particles. Mutant fat bodies also exhibited reduced cell adhesion. Scale bar = 20 μm. Inset: higher magnification images. Scale bars = 10 μm. (C’) LysoTracker-positive punctate densities were evaluated on images obtained in C, using 72h and 96h samples (n = 2). Both time points showed abnormal occurrence of lysosomal staining in vap1. (D) Starvation-induced lysosomal response was analyzed in 72h, 2nd instar larvae subjected to complete food deprivation for 3h. Fat bodies of control, w- showed frequent LysoTracker-positive vesicles but mutant, vap1 fat bodies elicited a stronger response, including the presence of many large acidic particles (compare image in insets; Scales as in C). Similar effects were seen in vap2 and vap3 mutants. (D’) The size distribution of LysoTracker positive punctate in experiment in D was quantified. The size of punctate particles of two wide field images obtained from identically stained tissues, counting 192 particles (w-) and 488 particles (vap1) were analyzed and graphed as boxplots. Mutant tissue shows particles spanning a larger size range (w-, Mdn = 0.80 μm2; vap1, Mdn = 1.68 μm2). See S1A Fig for complete size distribution of the particles. Medians are drawn as thick lines; significance is from Mann Whitney test. Genotypes. (A, B) Control: w1118/Y. Assay: vap1/Y. vap2/Y. (C, C’, D, D’) Control: w1118/Y. Assay: vap1/Y.
