Starvation-induced autophagy is initiated faster in the buffy mutant fat body in comparison to wild-type fat body. (A) Timecourse showing LysoTracker Red (LTR) staining following 0, 2 and 4 h of amino-acid starvation. Blue represents Hoechst-stained nuclei (note that not all nuclei are accessible to Hoechst in live tissue). Robust LTR-positive staining is visible after 2 h of starvation in the buffy mutant fat body, whereas very few LTR-positive punctae are visible at this time point in wild-type. Scale bar = 47 μm. (B) Quantification of density of LTR punctae from 5 to 10 animals per genotype after 2 and 4 h of starvation. Mean ± SEM. (C) Fat bodies from 2 h-starved larvae expressing mCherry-Atg8a (red) and stained with LTR (green). Both autophagosomes (arrows) and early autolysosomes (arrowheads) were labeled with mCherry-Atg8a. Scale bar = 38 μm. (D) Transmission electron microscopy (TEM) images of fat bodies from larvae starved for 0, 2 and 4 h demonstrating detection of autophagosomes (yellow arrowheads) and autolysosomes (yellow arrows). Scale bars are as indicated. (E) Quantification of autolysosomes in TEM images from 4 h-starved larval fat bodies.