Additional characterization of free and tGA-lysosomes. (A) FIB-SEM images showing free lysosomes (not associated to trans-Golgi) in fat body adipocytes and imaginal wing disc epidermal cells, as well as multivesicular bodies in wing disc cells. We did not find similar multivesicular late endosomal structures in fat body of the L3 feeding larva, but their presence has been reported later, upon developmental autophagy induction (Marusz et al., 2015). Multivesicular bodies in wing disc cells are sometimes trans-Golgi–associated (3 out of 19 examples). (B) Confocal images of wing disc cells at interphase, prophase, metaphase, and telophase stages of the mitotic cycle. Upper images show F-actin stained with phalloidin (white) and DAPI (blue); lower images show lysosome marker Lamp1-YFP (green) and trans-Golgi marker GalT-TagRFP (magenta, driven by rn-GAL4), as well as phalloidin (white). The graphs below quantify Lamp1/GalT puncta proximity measured in individual cells (n = 10, 14, 3, 6, and 1 for prophase, metaphase, anaphase, telophase, and cytokinesis, respectively) or in 6.23 × 6.23 μm areas (interphase, n = 10). (C) Confocal images showing Lamp1-YFP (green) and GalT-TagRFP (magenta, driven by BM-40-SPARC-GAL4) in L3 fat body (feeding stage) after 6 h culture with (left) or without (right) 0.25 mg/ml of actin polymerization inhibitor Cytochalasin D. Magnified insets are shown below. The graphs quantify puncta proximity in 10 cells, as well as average proximity values (n = 10, error bars indicate SD). (D) Confocal images showing distribution of general secretion marker secr-GFP (green, BM-40-SPARC-GAL4–driven) in fat body after 6 h culture with (left) or without (right) 100 μg/ml of secretion inhibitor Brefeldin A. DAPI in blue. (E) Lamp1-YFP (green) and trans-Golgi marker GalT-TagRFP (magenta, driven by BM-40-SPARC-GAL4) in fat body after 6 h culture with (left) or without (right) 100 μg/ml of secretion inhibitor Brefeldin A. Magnified insets are shown below. Graphs quantify puncta proximity as in C.