Mechanisms of Apoptosis

Caspase Activation Pathways

Extrinsic pathway

The extrinsic pathway is regulated by signaling through receptors of the Tumor Necrosis Factor receptor (TNFR) superfamily. These receptors include TNFR, Fas, and TNF-related apoptosis inducing ligand (TRAIL) receptors. Binding of a ligand to its respective receptor activates adaptor proteins that contain death domains, such as FADD and TRADD. Adaptor proteins recruit and facilitate the dimerization of initiator caspases like caspase 8 and 10, which are assembled at the death inducing signaling complex (DISC). This pathway can be negatively regulated by decoy TRAIL receptors (TRAIL R3 and R4) which compete for TRAIL binding. 

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Recombinant human TRAIL (TNFSF10) induces cytotoxicity in mouse L929 cells.

Intrinsic pathway

The intrinsic pathway is triggered by detection of cellular stresses, such as DNA damage and hypoxia, or by the absence of signaling from growth factors, hormones, and cytokines. This results in the increased permeability of the mitochondrial membrane, which is regulated by pro- and anti-apoptotic members of the Bcl-2 family (e.g. Bax, Bak, Bad). Cytochrome c is then released from mitochondria to activate adapter protein apoptotic protease-activating factor-1 (APAF1), which forms the apoptosome with dimerized initiator caspase 9. Extrinsic pathway signaling can also cross-activate mitochondrial release of cytochrome c through caspase 8-mediated cleavage of Bid, a pro-apoptotic member of the Bcl-2 family.

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HeLa cells stained with purified anti-Cytochrome c antibody, DyLight 594™-conjugated secondary antibody, and DAPI.

Granzyme pathway

Cytotoxic CD8⁺ T lymphocytes (CTLs) and natural killer (NK) cells can kill target cells (e.g. cancer cells, infected cells) through FasL/Fas binding, and through secretion of granules containing proteases granzyme A and B. These granules penetrate the target cell membrane through pores formed by perforin. FasL/Fas interaction can trigger extrinsic apoptosis, and is the primary killing mechanism used by NKT cells, a subset of T cells that recognize CD1d. Granzyme B can directly cleave and activate initiator and executioner caspases. Granzyme B can also cleave Bid to induce cytochrome c release and the intrinsic apoptotic pathway. In contrast, granzyme A induces cell death through mechanisms independent of caspases.

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Human PBMC stained with anti-CD8 Pacific Blue™ and anti-Granzyme B APC recombinant antibody.