Fig. 4.

The Opa1^TetraCys mutant that is not oxidized upon H₂O₂ treatment restores cristae shape but not mitochondrial fusion.

(A) Cells of the indicated genotype treated with 1 mM H₂O₂ for 30 min where indicated were lysed and equal amounts of proteins (15 μg) were separated by reducing or non-reducing SDS-PAGE and immunoblotted using the indicated antibodies. Asterisks indicate the ∼180 KDa Opa1 reduction-sensitive oligomer.

(B) Representative confocal images of mitochondrial fusion experiments. MEFs of the indicated genotype were co-transfected with mtPAGFP and mtRFP. At time 0, mtPAGFP was activated in a region of interest (white circle) and cells were imaged by real time confocal microscopy and the frame acquired at time = 30 min is shown. Bar, 20μm.

(C) Quantification of mitochondrial fusion in experiments as in (B). Data represents mean of ±SEM of 4 independent experiments.

(D) Representative electron micrographs of MEFs of the indicated genotype. The boxed area of the top images is magnified in the bottom images. Scale bars: 1 μm (top) and 500 nm (bottom).

(E) Average ± SEM of morphometric analysis of cristae width in 105 (EV), 309 (Opa1^WT) and 287 (Opa1^TetraCys) mitochondria of the indicated genotype from 4 independent experiments. *, p < 0.001 in a two-sample t-Test.