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, 45 (7-8), 596-602

Oxidative Stress Causes Reversible Changes in Mitochondrial Permeability and Structure

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Oxidative Stress Causes Reversible Changes in Mitochondrial Permeability and Structure

Nelson B Cole et al. Exp Gerontol.

Abstract

Mitochondria are a primary source as well a principal target of reactive oxygen species within cells. Using immunofluorescence microscopy, we have found that a number of mitochondrial matrix proteins are normally undetectable in formaldehyde-fixed cells permeabilized with the cholesterol-binding detergent saponin. However, exogenous or endogenous oxidative stress applied prior to fixation altered the permeability of mitochondria, rendering these matrix proteins accessible to antibodies. Electron microscopy revealed a loss of matrix density and disorganization of inner membrane cristae upon oxidative stress. Notably, the changes in permeability and in structure were rapidly reversed when the oxidative stress was relieved. The ability of reactive oxygen species to reversibly alter the permeability of the mitochondrial membrane provides a potential mechanism for communication within the cell such as between nucleus and mitochondria.

Figures

Fig. 1
Fig. 1
Immunofluorescence of untreated MEFs expressing mouse MsrA. Cells were fixed in formaldehyde/PBS followed by permeabilization with saponin. Staining was with antibodies to MsrA and cytochrome c (A) or MsrA and mtHSP70 (B). Note that MsrA is restricted to the cytosol in saponin permeabilized cells and mtHSP70 is not detectable. In (C) cells were post-fixed with 0.1% Triton X-100 for 1 min prior to antibody staining. Both MsrA and mtHSP70 are visible in mitochondria. Bar = 10 μm.
Fig. 2
Fig. 2
Fractionation of MEFs expressing mouse MsrA. MsrA was detected both in the cytosol (lane 1) and in the mitochondrial fraction (lane 2). The cytosolic marker (α-tubulin) and the mitochondrial marker protein (mtHSP70) are indicated and demonstrate the lack of cross-contamination between compartments
Fig. 3
Fig. 3
Immunofluorescence of MEFs treated with 50 mU/mL glucose oxidase for 1 h before fixing with formaldehyde/PBS followed by permeabilization with saponin. Both MsrA and mtHSP70 are detected in mitochondria and co-localize with cytochrome c. Bar = 10 μm.
Fig. 4
Fig. 4
Immunofluorescence of MEFs treated with (A) 100 ?M rotenone or (B) 100 μM antimycin A for 3 h before fixation with formaldehyde/PBS followed by permeabilization with saponin. Note that not all mitochondria are labeled with antibodies to MsrA. Bar = 10 μm
Fig. 5
Fig. 5
Immunofluorescence of MEFs from untreated (top panel), glucose oxidase-treated (middle panel), and cells in which glucose oxidase was washed out and were allowed to recover for 1h. Although some mitochondrial staining remains in washout cells, there is a general loss of ability to detect mitochondria-localized MsrA in post-fixed cells permeabilized with saponin, similar to untreated cells. Bar = 10 μm
Fig. 6
Fig. 6
Representative electron micrographs from untreated (A), glucose oxidase-treated (B), and glucose-oxidase recovered (C) cells. Note the loss of matrix density as well as narrower, mis-orientated cristae that are reduced in number in glucose oxidase treated cells. Also note the general recovery of mitochondrial structure when glucose oxidase is washed out. Arrows indicate flocculent or woolly densities. In (B), note that the section is tangential to portions of the membranes of the two mitochondria at center and lower left such that the mitochondria appear to overlap with adjacent endoplasmic reticulum. Cells were treated identically as those shown in Fig. 5. Bar = 100 nm.

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