Figure 4.

Acute Ablation of Opa1 Alters Mitochondrial Morphology, Cristae Shape, and RCS Assembly

(A) Opa1^flx/flx MAFs were infected with bicistronic adenoviruses carrying the indicated insert (EV, empty vector; CRE, Cre recombinase) upstream of GFP and, after 24 hr, equal amounts of proteins (20 μg) were separated by SDS-PAGE and immunoblotted with the indicated antibodies.

(B) Opa1^flx/flx MAFs were infected with the indicated adenoviruses and, after 24 hr, fixed, immunostained using an anti-TOM20 antibody, and representative confocal images acquired. The green cytoplasmic staining identifies the coexpression of GFP. The scale bar represents 20 μm.

(C) Average mitochondrial major axis length. Experiments were as in (B). Data represent average ± SEM of four independent experiments (five mitochondria per cell, at least 50 cells/experiment). The asterisk denotes p < 0.05 in a paired sample Student’s t test versus ad-EV.

(D) Opa1^flx/flx MAFs were infected with the indicated adenoviruses and, after 24 hr, fixed and processed for electron microscopy. The scale bars represent 2 μm (top) and 200 nm (bottom).

(E) Morphometric analysis of cristae width in 40 randomly selected mitochondria of Opa1^flx/flx MAFs infected with the indicated adenoviruses. Data represent average ± SEM of three independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus ad-EV.

(F) Mitochondrial DNA copy number quantification. mtDNA was amplified by RT-PCR from total DNA of Opa1^flx/flx MAFs infected with the indicated adenoviruses. Data are normalized to MAFs infected with control adenovirus and represent average ± SEM of four independent experiments.

(G) mtDNA translation assay. Opa1^flx/flx MAFs infected as indicated were metabolic labeled in presence of emetine and lysed after 30 min. Protein samples (40 μg) were separated by SDS-PAGE, and the radioactivity was detected in the fixed and dried gels for 3 days. The mtDNA encoded proteins are indicated.

(H) Densitometric analysis of the mtDNA-encoded proteins. Experiments are as in (G). Data represent average ± SEM of four independent experiments.

(I) RCS assembly assay. Opa1^flx/flx MAFs were infected as indicated and, after 24 hr, metabolically labeled for 2 hr and then chased for the indicated times. Equal amounts of protein (100 μg) were separated by BN PAGE, and radioactivity was detected in the fixed and dried gels for 1 week. RCC and RCS of the respiratory chain are indicated.

(J) Densitometric analysis of the incorporation rate of radioactivity into supercomplexes. Values are normalized for the autoradiographic signal of complex V. Data represent average ± SEM from three independent experiments performed as in (H).

(K) RCR of mitochondria isolated from livers of Opa1^flx/flx mice 3 days after tail-vein injection of the indicated adenoviruses, energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus ad-EV.

See also Figure S4.