acts with the transcriptional repressor UNC-37/Groucho to protect CEMs from undergoing apoptosis in males. Interestingly, the second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A, the terminal sex determination factor, and UNC-86, a POU-type homeodomain protein that specifies the CEM cell fates. In vitro TRA-1A interacts directly with UNC-86 on intron 2 and may suppress transcriptional activation of ceh-30 by UNC-86, leading to activation of CEM cell death in hermaphrodites that have a high level of TRA-1A. Thus ceh-30 integrates the sex determination signal TRA-1A and the cell fate determination and survival signal UNC-86 to control sex-specific death of CEMs (Figure 34-2). There is evidence that non–egl-1 cell death activator(s) may be directly regulated by CEH-30/UNC-37, although the precise target of CEH-30/UNC-37–mediated transcriptional repression is unclear.

4. EXECUTION

Once the CED-3 caspase is activated, it orchestrates different cell death execution events by cleaving and activating multiple downstream effectors, leading to chromosomal fragmentation, mitochondrial elimination, and removal of apoptotic cells.

4.1. DNA degradation

Fragmentation of chromosomes is a hallmark of apoptosis that aids in dismantling the cell. In dying cells, chromosomes condense and are cleaved between nucleosomes, creating approximately 180 base pair fragments and permanently preventing DNA replication. The contribution of deoxyribonucleases to apoptosis has been enigmatic for many years. It was originally thought that DNA degradation was an expendable event late in apoptosis, because inactivation of the first identified apoptotic nuclease gene, nuc-1 (nuc, abnormal nuclease), did not affect cell death activation or other aspects of apoptosis. Recent studies, however, have shown that DNA degradation actually facilitates apoptosis and clearance of a dying cell.

Currently, at least 10 genes are important for chromosomal degradation, several of which also facilitate clearance of the dying cell by a neighboring cell in C. elegans, which do not have professional phagocytes. These include nuc-1, wah-1 (wah, worm AIF homolog), cps-6 (cps, CED-3 protease suppressor), cyp13 (cyp, cyclophilins), and crn-1 to crn-6 (crn, cell deathrelated nuclease). Loss or reduction of activity in any of these genes results in the accumulation of cells with

3 hydroxyl DNA breaks in C. elegans embryos that can be labeled by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. With the exception of nuc-1 and crn-6, a defect in any of these genes also causes delayed or reduced cell death in sensitized genetic backgrounds. These genes seem to act in two distinct pathways to promote apoptotic DNA degradation, with cps-6, wah-1, crn-1, crn-4, crn-5, and cpy13 acting in one pathway and crn-2 and crn-3 acting in another. Interestingly, disrupting both DNA degradation pathways causes a defect in cell corpse engulfment at all stages of embryonic development. One possible explanation for this is that the DNA fragmentation process may facilitate the generation or exposure of “eat me” signals for phagocytosis. For example, nucleosomes can be presented on the surface of T cells and enhance phagocytosis by dendritic cells. However, the exact mechanism by which chromosomal fragmentation promotes clearance of apoptotic cells in C. elegans is unclear.

Both cps-6 and wah-1 encode mitochondrial proteins that are homologs of human endonuclease G (endoG) and apoptosis-inducing factor (AIF), respectively, which are also important for apoptotic DNA degradation in mammals. WAH-1 is released from the mitochondria by the cell death initiator EGL-1 and translocates to the nucleus in a CED-3–dependent manner. WAH-1 also physically binds and enhances the endonuclease activity of CPS-6. Moreover, CPS-6 may form a large DNA degradation complex with CRN-1, CRN-4, CRN-5, and CYP-13 (named the degradeosome) to promote stepwise DNA fragmentation, starting from generation of DNA nicks to single-stranded gaps to double-stranded breaks. On the other hand, nuc-1 and crn-6 encode type II acidic DNases that affect neither cell death nor engulfment. These two nucleases may act at a later stage of the cell death and DNA degradation process.

4.2. Mitochondrial elimination

As described previously, mitochondria play a central role in the killing phase of apoptosis in mammals. Mitochondria undergo dramatic morphological changes during apoptosis, including fragmentation, reorganization of cristae structures, and increased permeability of the outer mitochondrial membrane, all of which have been proposed to promote release of proapoptotic factors such as cytochrome c and Smac/Diablo from mitochondria of mammals. There are also reports that mitochondria are reduced or lost during apoptosis, which would eliminate cellular energy production and contribute to the demise of the cell. A comprehensive genetic and cell biological analysis of components of the C. elegans

mitochondria fission and fusion machinery, such as the dynamin GTPases DRP-1 (drp, dynamin-related protein), FZO-1 (fzo, Fzo mitochondrial fusion protein related), and EAT-3 (eat, eating defective), reveals that defects in mitochondria fission or fusion in C. elegans do not affect apoptosis activation. However, loss of DRP-1 and FIS-2, a homolog of the human Fis1 fission protein, does cause a mild cell death defect that can be detected in sensitized genetic backgrounds, suggesting that fis- 2 and drp-1 have minor proapoptotic roles. Genetic epistatic analysis suggests that fis-2 and drp-1 act independently of one another and downstream of ced-3 to promote apoptosis. Analysis by electron microscopy indicates that mitochondria normally reduced in size or eliminated in apoptotic cells persist in worms deficient in fis-2 or drp-1, suggesting that DRP-1 and FIS- 2 play a role in promoting mitochondrial elimination during apoptosis. Interestingly, active CED-3 protease can cleave DRP-1, and such cleavage is important for DRP-1’s proapoptotic function, but not for its function in mitochondrial fission. Furthermore, the carboxyl terminal cleavage product of DRP-1 appears to be important for activating its proapoptotic function. Therefore, fis-2 and drp-1 represent two novel cell death execution pathways acting downstream of ced-3 to promote mitochondrial elimination (Figure 34-2).

4.3. Engulfment

The timely removal of dying cells is important for development and tissue homeostasis in all organisms, in addition to immune responses and resolution of inflammation in mammals. Apoptotic cells that are not cleared undergo secondary necrosis, which could lead to tissue injury. Genetic analyses have identified many genes that are important for engulfment of apoptotic cells in C. elegans. These genes seem to work in two partially redundant pathways, with ced-1, ced-6, ced-7, and dyn- 1 (dyn, dynamin-related) acting in one pathway and ced-2, ced-5, ced-10, and ced-12 working in another. Most of these genes are required in the engulfing cell, although the activity of ced-7 is also needed in the dying cell. CED-1 is similar to the human scavenging receptor SREC and may function as a receptor on engulfing cells because it clusters around cell corpses in C. elegans. CED-1 clustering requires CED-7, an ATP-binding cassette (ABC) transporter, suggesting that CED-7 may play a role in transporting a signal that CED-1 recognizes or in mediating homophilic interaction between CED-7 on the dying cell and CED-7 on the engulfing cell. The ligands recognized by CED-1 have not been identified,

but CED-6 binds the intracellular portion of CED-1 via a phosphotyrosine binding (PTB) domain. CED-6 may transduce the engulfment signal to reorganize the engulfing cell membrane in response to CED-1 activation, probably through DYN-1, a C. elegans large dynamin GTPase. DYN-1 appears to mediate the internalization and degradation of dying cells by delivering intracellular vesicles to phagocytic cups to support pseudopod extension and to phagosomes to support their maturation and eventual digestion of apoptotic cells. After the internalization of apoptotic cells, multiple Rab GTPases and the HOPS complex mediate the maturation of phagosomes and the degradation of apoptotic cells.

In the other engulfment pathway, ced-2, ced-5, ced10, and ced-12 encode conserved components of the Rac GTPase signaling pathway that are important for rearrangement of the actin cytoskeleton in cell migration and engulfment. CED-2 is a CrkII-like adaptor with one SH2 (Src homology) and two SH3 domains that physically interacts with CED-5, the homolog of human DOCK180. CED-10, the C. elegans homolog of mammalian Rac GTPase, controls cytoskeletal dynamics and cell-shape changes downstream of CED-2 and CED-5. CED-12, a homolog of mammalian ELMO1, contains a potential PH (pleckstrin homology) domain and an SH3binding motif through which it can interact with the CED-2/CED-5 complex. Genetic analyses indicate that ced-2, ced-5, and ced-12 function at the same step but upstream of ced-10 in the engulfment process. Biochemical analyses suggest that CED-2, CED-5, and CED-12 form a ternary complex to activate CED-10 GTPase. The phagocyte receptor for this pathway is poorly characterized. One candidate is PSR-1, the worm phosphatidylserine receptor homolog. PSR-1 binds specifically to cells exposing phosphatidylserine (PS) on their surface and acts in the CED-2/CED-5/CED-12 pathway to promote cell corpse engulfment, probably through interacting with CED-5 and CED-12. However, the engulfment defect of the psr-1(lf) mutant is significantly weaker than that of the ced-2 or ced-5 mutants, suggesting that other phagocyte receptors must also act in this pathway.

Almost all of the genes previously discussed act in phagocytes. Little is known, however, about the “eat-me” signals expressed by apoptotic cells. PS, which normally is restricted to the inner leaflet of the plasma membrane, is externalized during apoptosis and can serve as an “eatme” signal to trigger phagocytosis. Recently, PS exposure on the surface of apoptotic cells in C. elegans has been demonstrated to be important for removal of apoptotic cells. Interestingly, the mitochondrial proapoptotic factor, WAH-1, is involved in promoting PS