Figure 18–4 DNA fragmentation during apoptosis. (A) In healthy cells, the endonuclease CAD associates with its inhibitor, iCAD. Activation of executioner caspases in the cell leads to cleavage of iCAD, which unleashes the nuclease. Activated CAD cuts the chromosomal DNA between nucleosomes, resulting in the production of DNA fragments that form a ladder pattern (see B) upon gel electrophoresis. (B) Mouse thymus lymphocytes were treated with an antibody against the cell-surface death receptor Fas (discussed in the text), inducing the cells to undergo apoptosis. DNA was extracted at the times indicated above the figure, and the fragments were separated by size by electrophoresis in an agarose gel and stained with ethidium bromide. Because the cleavages occur in the linker regions between nucleosomes, the fragments separate into a characteristic ladder pattern on these gels. Note that in gel electrophoresis, smaller molecules are more widely separated in the lower part of the gel, so that removal of a single nucleosome has a greater apparent effect on their gel mobility. (C) Apoptotic nuclei can be detected using a technique that adds a fluorescent label to DNA ends. In the image shown here, this technique was used in a tissue section of a developing chick leg bud; this cross section through the skin and underlying tissue is from a region between two developing digits, as indicated in the underlying drawing. the procedure is called the tUNEL (tdt-mediated dUtpnick end labeling) technique because the enzyme terminal deoxynucleotidyl transferase (tdt) adds chains of labeled deoxynucleotide (dUtp) to the 3ʹ-Oh ends of DNA fragments. the presence of large numbers of DNA fragments therefore results in bright fluorescent dots in apoptotic cells. (B, from D. McIlroy et al., Genes Dev. 14:549–558, 2000. With permission from Cold Spring harbor Laboratory press; C, from V. Zuzarte-Luís and J.M. hurlé, Int. J. Dev. Biol. 46:871–876, 2002. With permission from UBC press.)
Cell-Surface Death receptors Activate the Extrinsic pathway of Apoptosis
Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis. Death receptors are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain, which is required for the receptors to activate the apoptotic program. The receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family, which includes a receptor for TNF itself and the Fas death receptor. The ligands that activate the death receptors are also homotrimers; they are structurally related to one another and belong to the TNF family of signal proteins.
A well-understood example of how death receptors trigger the extrinsic pathway of apoptosis is the activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte. When activated by the binding of Fas ligand, the death domains on the cytosolic tails of the Fas death receptors bind intracellular adaptor proteins, which in turn bind initiator caspases (primarily caspase-8), forming a death-inducing signaling complex (DISC). Once dimerized and activated in the DISC, the initiator caspases cleave their partners and then activate downstream executioner caspases to induce apoptosis (Figure 18–5). In some cells, the extrinsic pathway recruits the intrinsic apoptotic pathway to amplify the caspase cascade and kill the cell.