Figure 3.
Distinct roles of the conjugation pathways in midgut autophagy and histolysis. (a) Schematic diagram showing the multiple components of the conjugation pathways. Compared with (b) control (NP1-GAL4/+ pmCherry-Atg8a/+), knockdown of (c) Atg3-IR (NP1-GAL4/+ UAS-Atg3 RNAi/pmCherry-Atg8a), (d) Atg4a-IR (NP1-GAL4/+ UAS-Atg4a RNAi/pmCherry-Atg8a) and (e) Atg7-IR (NP1-GAL4/+ UAS-Atg7 RNAi/pmCherry-Atg8a) did not affect gastric caeca contraction at both 0 h RPF (left) and +4 h RPF (middle) (arrow). The knockdown of (f) Atg5-IR (NP1-GAL4/+ UAS-Atg5 RNAi/pmCherry-Atg8a), (g) Atg12-IR (NP1-GAL4/+ UAS-Atg12 RNAi/pmCherry-Atg8a) and (h) Atg16-IR (NP1-GAL4/+ UAS-Atg16 RNAi/pmCherry-Atg8a) led to an increase in the size of gastric caeca at both 0 h RPF (left) and +4 h RPF (middle) compared with (b) control. Scale bar represents 200 μm. (b–h, right) Autophagy puncta examined using pmCherry-Atg8a (red) and DNA stained with Hoechst (blue) in +2 h RPF midgut gastric caeca. The level of autophagy at +2 h RPF was not affected by knockdown of (c) Atg3-IR, (d) Atg4a-IR, (e) Atg7-IR, (f) Atg5-IR, (g) Atg12-IR or (h) Atg16-IR (right). Scale bar represents 10μm. (i) Quantitation of gastric caeca size (average pixels±S.D.) at +4 h RPF from (b–h) (N≥10, ***P<0.001). (j) Quantitation of the pmCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell at +2 h RPF from (b–h) using ImageJ, error bars represent S.E.M., (N≥10)