Interactions of Chloromethyltetramethylrosamine (Mitotracker OrangeTM) with Isolated Mitochondria and Intact Cells.

Activation of the apoptotic program by cytochrome c and apoptosis inducing factor (AIF) requires release of these intermembrane proteins into the cytosol, and the mechanism(s) by which this occurs is the subject of intense investigation. One of the most studied targets is the mitochondrial permeability transition (PT), a sudden increase of the inner membrane to solutes that has been extensively studied. A specific issue is whether or not a mitochondrial PT is a requisite for the release of apoptogenic proteins, an issue that has generated conflicting results and a major controversy in the literature. A specific, PT-independent release mechanism has been proposed based on the finding that cytochrome c release occurs without measurable decrease of the mitochondrial membrane potential. This point remains very controversial, however. Other groups have indeed reported that cytochrome c release matches membrane depolarization, and that it is mediated by a PT, which has been indicated as the major determinant of AIF release. Many of these studies were performed with the “fixable” probe chloromethyltetramethyl rosamine (CMTMRos) to monitor changes of mitochondrial membrane potential in situ. Here, we have carried out a characterization of the interactions of this probe with mitochondria.