UC San Diego

Cell death is an essential biological process in the metazoans and the mitochondrial pathway of apoptosis is the major mechanism of physiological cell death in the vertebrates. In this pathway, pro-apoptotic members of the Bcl-2 family cause mitochondrial outer membrane permeabilization (MOMP), allowing the release of cytochrome c, which interacts with APAF-1 to trigger formation of an apoptosome leading to caspase activation and apoptosis. It has been widely suggested that apoptosis is conserved throughout the metazoans, however despite conservation of Bcl-2 proteins, APAF-1, and caspases there is no biochemical evidence of the existence of the mitochondrial pathway in either C. elegans or Drosophila apoptosis. The hypothesis of this study is that the mitochondrial pathway of apoptosis is an ancient mechanism regulating apoptosis in the metazoans and that all or part of the pathway has been lost in some lineages. To test this hypothesis, activation of caspases by cytochrome c in cytosolic extracts from animals representing six different phyla was analyzed. This research demonstrates for the first time that cytochrome c activates caspases in cytosolic extract from non-vertebrate animals, including the purple sea urchin, an echinoderm, and the freshwater planaria, a platyhelminth. In response to various stimuli delivered to intact planaria and isolated planaria cells, characteristic features of the mitochondrial pathway of apoptosis were observed, including cytochrome c release to the cytosol, caspase activation, loss of mitochondrial membrane potential, and phosphatidylserine externalization without loss of plasma membrane integrity. In addition, this study demonstrates that MOMP and cytochrome c release in planaria can be regulated by human Bcl-2 proteins. These results suggest that MOMP and cytochrome c activation of caspases are components of an ancient mechanism of apoptosis that was present in the metazoans prior to the divergence of the lophotrochozoans, edysozoans and deuterostomes, and that this mechanism was lost, in whole or in part, in ecdysozoan lineages