Figure 5.

Increased ROS formation in Coq2 loss-of-function. (A–A”) GCNs expressing a third-copy reporter allele of Coq2 with an insertion of GFP in frame costained for the mitochondrial marker ATP5A indicates localization of Coq2 to the mitochondria. (B) GCN expressing control-RNAi or Coq2-RNAi after 5 minutes of incubation with ROS indicator DHE. Stronger intranuclear signal of DHE upon knockdown of Coq2 indicates increased ROS (dotted circles in right panel without DAPI mark nuclei). (C) Quantitation of nuclear DHE signal indicates significant increase of ROS formation upon knockdown of Coq2. (D) GCN show a decrease of FITC-albumin uptake upon knockdown of ND75, a subunit of the complex I of the respiratory chain, as shown by representative images for one RNAi line and quantitation for two independent RNAis. (E–E’’) Silencing of ND75 results in displacement of Sns/Kirre from the membrane toward the interior of the cell. Areas denuded from labyrinthine channels appear on the cell surface. Knockdown of ND75-RNAi thus results in a phenocopy of Coq2 silencing. (F) TEM analysis of GCN expressing ND75-RNAi reveals loss of slits/labyrinthine channels or elongated channels with multiple slit diaphragms (red arrow heads). (G) Quantitation of slit diaphragm frequency on the full circumference of cells expressing ND75-RNAi. One complete diameter of six cells from three different animals was analyzed. Frequency of slit diaphragms was classified into three groups: normal (>2 slits/µm), reduced (0.5–2 slits/µm), and sporadic (<0.5 slits/µm). Values are presented as mean percentage±SD for each category. Note strong reduction of slit diaphragms upon knockdown of ND75 similar to knockdown of Coq2. Control is identical to control from Figure 4F. (H) Treatment of control or Coq2-RNAi with glutathione for 5 days partially restores uptake of FITC-albumin in Coq2-RNAi nephrocytes but has no effect on control-RNAi–expressing cells. (I) Quantitation of experiments in (H), n=3–5 per genotype and intervention. (J) TEM imaging after treatment of flies expressing Coq2-RNAi with the ROS scavenger glutathione for 5 days partially restores slit diaphragm frequency on the cell surface in ultrastructural analysis. Slit diaphragm localized in labyrinthine channels and elongated labyrinthine channels may still be found. (K) Quantitation of experiments in (J) for six cells from three animals. Control is identical to control from Figure 4O. (L–M’’) Treatment of Coq2-RNAi with glutathione partially restores typical localization including fingerprint pattern of Sns/Kirre (M–M’’). Compare with vehicle-only controls in Figure 4, I–J’’. All scale bars represent 500 nm in electron microscopy images and 5 µm in confocal images unless otherwise stated.