Abstract

Several mitochondrial proteins, such as cytochrome c, are directly involved in the pathway for caspase activation following induction of apoptosis. Release of mitochondrial cytochrome c early in apoptosis is rapid and almost complete. Microinjection of cytochrome c into resting cells induces apoptosis, but the amount needed approaches the total cellular content. These observations suggest that mitochondrial protein release is an all-or-nothing process inside the cell and not an amplifiable apoptotic signal. To test this hypothesis, laser micro-irradiation was used to rupture membranes of individual mitochondria within living rat neural cells. Laser micro-irradiation caused swelling, fragmentation, depolarization, and cytochrome c depletion in targeted mitochondria. These effects were explained by correlative electron microscopic analysis showing local rupture of outer and inner membranes at the site of irradiation. In all cases, there were no detectable changes in the structure, membrane potential, or cytochrome c content of neighboring, non-irradiated organelles. Furthermore, irradiation of up to 15% of the mitochondria in a cell did not induce apoptosis. The results from these laser micro-irradiation experiments prove that local release of mitochondrial proteins does not constitute an amplifiable apoptotic signal in resting neural cells.