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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).
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