Mitochondrial pathway of apoptosis is ancestral in metazoans

Abstract

The mitochondrial pathway of apoptosis is the major mechanism of physiological cell death in vertebrates. In this pathway, proapoptotic 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 caspase activation and apoptosis. Despite conservation of Bcl-2, Apaf-1, and caspases in invertebrate phyla, the existence of the mitochondrial pathway in any invertebrate is, at best, controversial. Here we show that apoptosis in a lophotrochozoan, planaria (phylum Platyhelminthes), is associated with MOMP and that cytochrome c triggers caspase activation in cytosolic extracts from these animals. Further, planarian Bcl-2 family proteins can induce and/or regulate cell death in yeast and can replace Bcl-2 proteins in mammalian cells to regulate MOMP. These results suggest that the mitochondrial pathway of apoptosis in animals predates the emergence of the vertebrates but was lost in some lineages (e.g., nematodes). In further support of this hypothesis, we surveyed the ability of cytochrome c to trigger caspase activation in cytosolic extracts from a variety of organisms and found this effect in cytosolic extracts from invertebrate deuterostomes (phylum Echinodermata).

Fig. 1.

γ-radiation induces phosphatidylserine externalization, caspase activation, and cytochrome c release in planaria. (A) A cell fraction enriched in planaria neoblasts (S. mediterranea) was subjected to 50 Gy of γ-radiation (γ-rad). Twenty-four hours later, cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. Representative dot plots are shown. (B) As in A, except that cells were incubated in the presence or absence of 10 μM qVD-OPh 1 h before γ-radiation. Each data point represents two or three independent experiments. Error bars represent SD. (C–E) Planaria (D. dorotocephala) were subjected, or not, to 100 Gy of γ-radiation, and cytosolic extract was prepared 24 h later. (C) Rate of cleavage of Ac-DEVD-afc by cytosolic extract from untreated or irradiated planaria, preincubated in the presence or absence of a broad protease inhibitor mixture (Materials and Methods) and incubated with or without 10 μM qVD-OPh. RFU, relative fluorescence units. Error bars represent SD. (D) ³⁵S-labeled wild-type iCAD or mutant iCAD (D117E/D224E) (m-iCAD) was incubated with extract from untreated or irradiated planaria preincubated with a protease inhibitor mixture, in the presence or absence of 10 μM qVD-OPh (Left) or with activated human recombinant caspase 3 (rCasp3) (Right). Samples were resolved by SDS/PAGE and analyzed by autoradiography, and percent cleavage was determined by densitometry analysis. Arrowhead indicates cleaved product. (E) Cytosolic extracts from untreated and irradiated planaria were examined for cytochrome c (cyt c) and actin content by Western immunoblot.

Fig. 2.

Cytochrome c activates caspases in planaria cytosolic extract (D. dorotocephala). (A) Planaria cytosolic extract was preincubated in the presence or absence of 10 μM mammalian (horse heart) cytochrome c. Ac-DEVD-afc was added, and cleavage was measured. (B) Rate of cleavage of Ac-DEVD-afc by planaria cytosolic extract preincubated in the presence or absence of 10 μM cytochrome c and either DMSO (vehicle), zVAD-fmk (1 nM to 100 μM), or qVD-OPh (1 nM to 100 μM). (C) ³⁵S-labeled wild-type iCAD or mutant iCAD (D117E/D224E) was incubated with planaria cytosolic extract preincubated with a protease inhibitor mixture in the presence or absence of 10 μM cytochrome c and 10 μM qVD-OPh (Left) or with activated human recombinant caspase 3 (Right). Samples were resolved by SDS/PAGE and analyzed by autoradiography, and percent cleavage was determined by densitometry analysis. Arrowhead indicates cleaved product. (D) Wild-type or mutant PARP (D214A) was incubated with planaria cytosolic extract in the presence or absence of 10 μM cytochrome c and 10 μM qVD-OPh or with activated human recombinant caspase 3, resolved by SDS/PAGE, and examined by immunoblot. Arrowhead indicates cleaved product. (E) Rate of cleavage of Ac-DEVD-afc by planaria extract in the absence or presence of 1, 10, or 100 μM mammalian (horse heart), insect (Manduca sexta), or yeast (S. cerevisiae) cytochrome c. (F) Rate of cleavage of Ac-DEVD-afc by planaria cytosolic extract preincubated or not with supernatant from enriched planaria (D. dorotocephala) mitochondria disrupted with water. Data are representative of at least three independent experiments. Error bars represent SD. (G) Cytochrome c-induced DEVDase activation in mammalian cytolic extract is inhibited by the CARD domains of human APAF1 or caspase-9, but not by the CARD domain of planaria APAF1. Cytosolic extract from 293 was incubated with 10 μM cytochrome c plus doubling dilutions of the indicated recombinant proteins (starting concentrations were equivalent before addition, as indicated by immunoblot; Fig. 2H, Right). (H) Cytochrome c-induced DEVDase activation in planaria cytolic extract is inhibited by the CARD domain of planaria APAF1, but not by the CARD domains of human APAF1 or caspase-9. Cytosolic extract from planaria was incubated with 10 μM cytochrome c plus doubling dilutions of the indicated recombinant proteins (starting concentrations were equivalent before addition, as indicated by immunoblot; Right).

Fig. 3.

Planaria Bak localizes to mitochondria and induces MOMP and cell death. (A and B) S. cerevisiae were transformed with the indicated constructs, plated in serial dilution, and induced to express the transformed gene(s). (C and D) Bax/Bak-deficient MEFs stably expressing Omi-mCherry were transfected with the indicated constructs in the presence of qVD-OPh (32 μM). At 24 h, cells were analyzed by confocal microscopy (C) or flow cytometry (D) for MOMP. Displayed is the percentage of cells that have undergone MOMP relative to the transfected population, averaged over three independent experiments ± SD. (E) HeLa cells were transfected with Venus-Smed Bak and mitochondrial targeted Cerulean (fused to the C-terminal 20 amino acids of human Bcl-xL). Cells were permeabilized with digitonin and imaged by confocal microscopy. In the merged image, the cerulean is false colored red.

Fig. 4.

Cytochrome c activates caspases in sea urchin egg cytosolic extract (S. purpuratus). (A) Cleavage of Ac-DEVD-afc by sea urchin egg cytosolic extract preincubated in the presence or absence of 10 μM mammalian (horse heart) cytochrome c and 1 mM dATP. (B) Rate of cleavage of Ac-DEVD-afc by sea urchin egg cytosolic extract preincubated in the presence or absence of 10 μM cytochrome c/1 mM dATP and either DMSO (vehicle), zVAD-fmk (1 nM to 100 μM), or qVD-OPh (1 nM to 100 μM). (C) ³⁵S-labeled wild-type or mutant iCAD (D117E/D224E) (m-iCAD) was incubated with sea urchin egg cytosolic extract preincubated with a protease inhibitor mixture in the presence or absence of 10 μM cytochrome c/1 mM dATP and 10 μM qVD-OPh or with activated human recombinant caspase 3. Samples were resolved by SDS/PAGE and analyzed by autoradiography, and percent cleavage was determined by densitometry analysis. Arrowhead indicates cleaved product. Note that the rightmost four lanes are also used as controls in Fig. 2C, Right, as they derive from the same experiment. (D) Rate of cleavage of Ac-DEVD-afc by sea urchin egg extract in the absence or presence of 1, 10, or 100 μM mammalian (horse heart), insect (Manduca sexta), or yeast (S. cerevisiae) cytochrome c and 1 mM dATP. Data are representative of at least three independent experiments. Error bars represent SD.