Figure 5.

Mitochondrial respiration, ultrastructure, and ATPase activity in cells expressing hFis1. (A) Oxygen consumption in control and hFis1-expressing intact wt MEFs. Forty-eight hours after transfection, cells were harvested, and 10⁸ cells were incubated in HBSS into an oxygen electrode chamber. Where indicated (arrows), the uncoupler FCCP (2 μM) and the complex IV inhibitor NaN₃ (10 mM) were added. (B) Oxygen consumption in control and hFis1-expressing permeabilized MEFs. Experiments were as in A, except that the cells (10⁸) were incubated in 0.5 ml EB containing 0.01% (wt/vol) digitonin. Where indicated (arrows), glutamate plus malate (G/M, 5/2.5 mM) or succinate (Succ, 5 mM in the presence of 2 μM Rotenone, an inhibitor of complex I), ADP (100 μM), oligomycin (2.5 μg/ml), FCCP (60 nM), and NaN₃ (1 mM) were added. (C) In-gel activity assay of F1-ATPase. Mitochondria from hFis1-transfected and untransfected cells were isolated and solubilized in n-dodecylmaltoside. Solubilized samples were then separated by BN-PAGE as described in experimental procedures. The Blue Native gel was then histochemically stained for ATP hydrolysis activity. Mitochondria isolated from human hearth samples were loaded as a control. (D) Representative ultrastructure of mitochondria in hFis1-expressing cells. Cells transfected with hFis1 were fixed, and standard electron microsopy images were acquired as described. Bar, 100 nm.