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 \u201cfixable\u201d 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

Mitochondria and Cell Death. Mechanistic Aspects and Methodological Issues.

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ