ATG5 Is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major
Figure 5
L. major Δatg5 promastigotes have a dysfunctional mitochondrion.
(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm. (B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106 promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05). (C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm. (D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm. (E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05). (F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotes at 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).