Figure 4.

Mitochondrial dysfunction following protein conformational stress results in mitochondrial fragmentation, AMPK phosphorylation and induction of autophagy. (a and b) dOTC expression resulted in mitochondrial fragmentation. Confocal analysis of the posterior compartment of larval wing discs co-expressing OTC or dOTC and a mitochondria-targeted protein (mitoGFP) under the control of the en-GAL4 driver. Red, OTC or dOTC; blue, nuclei; green, mitoGFP. (c) Quantification of mitochondrial length in larval wing discs expressing OTC or dOTC. Mean±S.D. are shown. Statistically significant values relative to OTC are indicated (unpaired t-test). (d) dOTC expression-induced autophagy. Lysates prepared from adult flies expressing OTC and dOTC under the control of the nos-GAL4 driver were subjected to western blot analysis with the indicated antibodies. Non-lipidated LC3 (GFP-LC3-I, 43 kDa) and lipidated LC3 (GFP-LC3-II, 36 kDa) are indicated with arrows. Adult flies were starved for 48 h as a positive control for the activation of autophagy. (e and f) Autophagy was induced upon accumulation of misfolded proteins in the mitochondria. Autophagy was detected in the larval fat body of transgenic animals by confocal microscopy. Induction of dOTC expression led to a redistribution of GFP-LC3 from a uniform to a punctate pattern. (g) Quantification of the GFP-LC3 signal intensity (mean±S.D.; n=3). Statistically significant values relative to OTC are indicated (unpaired t-test). (h) dOTC expression-induced AMPK phosphorylation. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. As a control for the activation of AMPK, control flies were starved for 72 h. (i) RNAi-mediated suppression of AMPK. Downregulation of Ampk was confirmed by measuring the protein levels of total AMPK in the indicated genotypes. Phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC) was used as a marker for AMPK kinase activity. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. Adult flies were starved for 72 h as a positive control for AMPK activation and consequent ACC phosphorylation. (j) AMPK acts as an autophagic-triggering sensor following mitochondrial stress. RNAi-mediated knockdown of Ampk blocked the lipidation of LC3 upon dOTC expression or starvation. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. Non-lipidated LC3 (GFP-LC3-I, 43 kDa) and lipidated LC3 (GFP-LC3-II, 36 kDa) are indicated with arrows. Adult flies were starved for 72 h as a positive control for the activation of autophagy