Fig. 5. Autophagy proceeds normally in dark mutant salivary glands. (A-C) Transmission EM of salivary gland cells. (A) A cytoplasm saturated with small vesicles and an electron dense nucleus (N) are indicative of ongoing cell death in wild-type cells at 14 hours APF. By contrast, salivary gland cells appear healthy in 14-hour APF dark⁸² (B) and 24-hour APF dark⁸² (C), showing no sign of cell death (compare the appearance of the nucleus in C with the nucleus in A). Arrows indicate autolysosomes in A-C, demonstrating that dark is not required for autophagy. Insets in panel C show enlargements of representative autophagosomes (top right corner) and autolysosomes (top left corner) seen in mutant glands. N, nucleus; g, secretory granule; asterisks indicate mitochondria. Scale bars: 1 µm; 250 nm for the insets. Arrowheads in C indicate autophagosomes. (D-I) Salivary glands dissected at the indicated time points (25°C) and stained with the acidic marker monodansylcadaverine (MDC) to detect autolysosomes (Munafo and Colombo, 2001). F shows a merged image of MDC staining (red) and detection of GFP-LC3 (green) (Rusten et al., 2004), a transgenic GFP marker for autophagosomes and autolysosomes in wild-type salivary glands (14 hours APF). At this stage, prior to histolysis, the overlap between MDC and GFP-LC3 is extensive, indicating an abundance of autolysosomes. (D-G) Time course of MDC staining in wild-type salivary glands. (D) At 9 hours APF, MDC staining is barely detectable. (E) At 11 hours APF, some punctate MDC-positive staining can be observed. However, by 14 hours APF (F) and in 15-hour APF glands (G), large MDC-positive structures are very conspicuous. Likewise, in comparably staged mutant glands, prominent MDC-positive vesicles are seen, shown here at 12 hours APF (H) and in persisting salivary glands 4 hours later (I).