Abstract
Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis-induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl-2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl-2 family proteins and pharmacological agents. Protocols include the use of time-lapse video-microscopy to monitor the onset of apoptosis in living cells and patch-clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.
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