The mitochondrial gateway to cell death.

Mitochondria play a key role in death signaling. The intermembrane space of these organelles contains a number of proteins which promote cell death once they are redistributed to the cytosol. The formation of pores in the outer membrane of mitochondria defines a gateway through which the apoptogenic proteins pass during death signaling. Interactions between pro-apoptotic and pro-survival members of the Bcl-2 family of proteins are decisive in the initiation of pore opening. While the specific composition of the pore in molecular terms is still subject to debate and continuing investigation, it is recognized functionally as a passive channel which not only allows egress of proteins to cytosol but also entry in the reverse direction. A variety of constraints may restrict the release of proteins from the intermembrane space to the cytosol. These include trapping in the intercristal spaces formed by the convoluted invaginations of the inner membrane, binding of proteins to the inner membrane or to other soluble proteins of the intermembrane space, or insertion of proteins into the inner membrane. There is a corresponding variety of mechanisms that facilitate release of apoptogenic proteins from such entrapment. Morphological changes that expand the inner membrane enable proteins to be released from enclosure in intercristal spaces, allowing these proteins access to the mitochondrial gateway. Specific cases include cytochrome c molecules bound to inner membrane cardiolipin and released upon oxidation of that lipid component. Further, AIF that is embedded in the inner membrane is released by proteases (caspases or calpains), which enter from the cytosol once the outer membrane pore has opened. The facilitation (or restriction) of apoptogenic protein release through the mitochondrial gateway may provide new opportunities for regulating cell death.

Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.

Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK(-) cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK(-) cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.