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(32) MITOCHONDRIAL SWELLING, DEPOLARIZATION, AND PERMEABILITY TRANSITION DURING BAX-INDUCED APOPTOSIS IN HEPATOCYTES A-L. Nieminen, L. Li, P.D. Pichiule,
M.B. Bhat*, J. Ma*, and K.M. Heiskanen
Pro-apoptotic proteins, such as Bax and Bid, translocate from the cytosol to mitochondria in response to apoptotic stimuli to induce mitochondrial depolarization and release of cytochrome c, but the mechanism of cytochrome c release remains controversial. This may be due to differences in methodology used in different experiments. Here, we studied mitochondrial changes in relation to insertion of the pro-apoptotic protein Bax into mitochondria of living mouse hepatocytes. We transfected cultured hepatocytes with a cDNA construct of green fluorescent protein (GFP) fused to the amino terminus of Bax. Transfected cells were also loaded with TMRM, a mitochondrial membrane potential sensitive probe. The green and red fluorescence of GFP-Bax and TMRM were imaged by confocal microscopy. GFP-Bax fluorescence displayed a diffuse cytosolic pattern. GFP-Bax did not localize to mitochondria and appeared as dark oval voids in confocal fluorescence images of green GFP-Bax fluorescence. These voids co-localized with red TMRM fluorescence. Over time, GFP-Bax redistributed to the surface of mitochondria, as indicated by appearance of bright fluorescent rings and spots around individual mitochondria. Subsequently, the shape of the mitochondria changed from oval- and rod-shaped to round, indicating mitochondrial swelling. This was followed by mitochondrial shrinkage, clustering and redistribution to the perinuclear area. Depolarization of a subpopulation of mitochondria paralleled the mitochondrial shrinkage. Simultaneously, the plasma membrane hyperpolarized by about -12 mV, which resulted in increased mitochondrial TMRM uptake by the remaining polarized mitochondria without hyperpolarization of the mitochondria themselves. This plasma membrane hyperpolarization likely explains earlier observations by flow cytometry in which Bax induced increased uptake of potential-indicating fluorophores. Eventually all mitochondria depolarized. This sequence of events preceded the changes in nuclear morphology typical for apoptosis. The data indicate that Bax relocation to mitochondria disrupts mitochondrial volume regulation, causing swelling, and eventually leading to mitochondrial permeabilization, cytochrome c release, depolarization and apoptotic cell death.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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