Figure 2. ATAD-3 is a mitochondrial protein and its depletion leads to arrest at developmental stages with low mitochondrial activity. (A) Western Blot analysis of C. elegans wild type cytoplasmic and mitochondrial protein fraction using anti-NUO-2, anti-ATAD-3 and anti-GAPDH antibodies to identify mitochondrial NUO-2/ATAD-3 (NUO-2 = homologue of the human NADH ubiquinone oxidoreductase subunit NDUFS3) and cytoplasmic GAPDH (glyceraldehyde-3-phosphate dehydrogenase), respectively. (B) Western blot analysis of protein extracts from wild type (WT) and
atad-3(RNAi) animals reveals a strongly reduced ATAD-3 signal. For comparison of relative protein levels, GAPDH served as a loading control. (C,D) Mitochondrial morphology was analyzed in transgenic strain SJ4103 expressing a mitochondrial targeted GFP under control of the muscle-specific
myo-3 promoter ([25]). Bar: 10 um; N = nucleus. (C) In SJ4103 control animals (fed with HT115 bacteria carrying the empty L4440 feeding-vector), mitochondria in body wall muscle form a well organized interconnected reticulum. (D) After
atad-3(RNAi), mitochondria appear disorganized and thinner. (E) Quantification of mitochondrial filamentation (aspect ratio = AR) and mitochondrial branching (formfactor = F) revealed no significant changes. Number of individual animals analyzed: WT = 11,
atad-3(RNAi) = 11; number of objects analyzed: WT = 780,
atad-3(RNAi) = 797. (F) Enzymatic activities (nmol/min/mg) of complex I (CI) and citrate synthase (CS) of a mixed stage WT population (green), WT L1 larvae (yellow) and
atad-3(RNAi) animals (red). Asterisks indicate significant differences (p<0.05) in comparison to the control.