Molecular mapping of the physiological cell death process. Mitochondrial events may be disordered

The mitochondrion plays a central role in Bcl-2-inhibitable physiological cell deaths. The detailed order of mitochondrial and other events during cell death in vivo remains ambiguous, however. As part of an effort to explore this issue, we have asked whether mitochondrial dissolution during physiological cell death occurs in an orderly and concerted process. Here, we describe the characterization of two elements of mitochondrial disintegration on the level of individual cells. Using a novel cytofluorimetric approach, we have assessed simultaneously the release of cytochrome c (specifically a fluorescently tagged transfected construct) from mitochondria and the dissipation of mitochondrial membrane potential. Our results indicate that mitochondrial disintegration does not follow a strictly ordered process and is not concerted. We are extending these studies to further characterize mitochondrial events in the context of Bcl-2 family members and place them definitively within the context of the caspase cascade.