Contributions of secretory and endosomal traffic to the growth of autophagic vesicles. (A) Confocal images of fat body cells from wil- type, BM-40-SPARC>Rab5ⁱ, >Rab7ⁱ, and >Rab1^DN white pupae (0–1 h after puparium formation) showing lysosomal marker Lamp1-YFP (green). Magnified insets are shown below each image. (B) Autophagic vesicles marked with Atg8a-mCherry (magenta) in fat body from wild-type, BM-40-SPARC>Rab5ⁱ, >Rab7ⁱ, >Rab1^DN, >Rab2ⁱ, >Rab6ⁱ, >Rab9ⁱ, >Rab10ⁱ, and >Rab9Fb(X3)ⁱ white pupae. (C) Autophagic vesicle diameter in wild-type, BM-40-SPARC>Rab5ⁱ, >Rab7ⁱ, >Rab1^DN, >Rab2ⁱ, >Rab6ⁱ, >Rab9ⁱ, >Rab10ⁱ, and >Rab9Fb(X3)ⁱ white pupae. For each genotype, vesicle diameters (dots) inside eight square areas (21.255 × 21.255 μm) are represented. Horizontal lines indicate mean diameter in each area. P values from nested t tests of differences with the wild type are reported. n.s., P > 0.05; *, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, 0.001 > P > 0.0001; and ****, P < 0.0001. (D) Model depicting the location and role of tGA-lysosomes. In normal conditions, basal microautophagy occurs in tGA-lysosomes, including degradation of excess or misrouted secretory cargo (crinophagy). Basal microautophagy failure or external macroautophagy induction results in formation of autophagic vesicles (autolysosomes) with contributions from endocytic and secretory routes, ER, and autophagosomes formed elsewhere.