Pitfalls in the use of electron microscopy to study the mitochondrial membrane permeability transition in apoptotic cells and pellets: where do we stand in relation to the incidence of mitochondrial swelling in apoptosis?

The importance of apoptosis as a form of programmed cell death was recognized in the 1980s, whereas the central role of mitochondria in controlling this process was identifi ed in the mid-1990s. An important event in apoptosis is the collapse of the mitochondrial transmembrane potential (??m), with the ensuing loss of the selective permeability of the inner membrane resulting in swelling of the hyperosmolar mitochondrial matrix. This event is known as the mitochondrial permeability transition (MPT). After swelling of the intermembrane space, the outer membrane ruptures, exposing the permeable inner membrane. An increasingly swollen matrix covered by the inner membrane eventually herniates into the cytoplasm through the breach formed in the outer membrane (OM). The increase in surface area of the inner mitochondrial membrane (IMM) involves the unfolding of membrane stored in the cristae. This membrane movement is osmotically driven since the cytoplasm has a lower osmolality. The proteins partly embedded in the inner membrane are thus exposed to the cytoplasm. In nine out of ten electron microscopy studies of isolated mitochondria expressing the permeability transition, the existing ruptures of the OMM were overlooked. The MPT can also be recognized in individual mitochondria by using fl uorescent probes that are not retained in these organelles once the ??m is lost. In cases in which there is no rupture of the OMM, cytochrome c must be released from mitochondria with impermeable inner membranes. Examination of several hundred of the more than 61,000 published papers on programmed cell death revealed that the key signaling events of apoptosis, such as the onset of the MPT, mitochondrial swelling and cytochrome c release to the cytoplasm, are infl uenced by factors such as the cell type and presence of apoptogenic agents. These two factors need to be examined in any assertion that non-swollen mitochondria are more frequent than swollen mitochondria in apoptotic cells after opening of the mitochondrial transition pore. This view differs from data obtained so far for human and laboratory animal cells in our laboratory, and also from literature reports for these same cells