Programmed cell death is associated with a highly conserved series of morphological changes in the nucleus, cytoplasm and plasma membrane of the dying cell. Some alterations in cellular structure may be required for cell killing per se, and others may be important for preventing lysis of the dying cell prior to engulfment. Genes involved in morphological changes may function genetically downstream of those genes absolutely required for the death process, and loss of function of such downstream effectors might not be sufficient to block cell killing. Little is known about downstream cell-death molecules, but a clue comes from the behavior of mammalian cells transfected with forms of lamin that can no longer be cleaved by caspases, the family of CED-3-like proteases. When these cells are induced to die, death occurs slowly and early changes in nuclear morphology are abnormal (Rao et al., J. Cell Biol. 135: 1441, 1996). Thus, some morphological changes in dying cells, such as the breakdown of the nuclear envelope, are important for normal cell-death kinetics. We previously reported the initial characterization of the C. elegans genes
ced-11 and
ced-8. Mutations in these genes affect the morphology and timing of programmed cell deaths, suggesting that
ced-11 and
ced-8 may represent downstream cell death effectors. In
ced-11 mutants, cell corpse morphology appears abnormal both by Nomarski optics and by electron microscopy. In
ced-8 mutants, cell corpses become visible later than in wild-type embryos. Both
ced-8 and
ced-11 encode novel proteins. If CED-11 and CED-8 function as downstream cell-death effectors, then these proteins might be targets of the CED-3 protease. We are trying to test this hypothesis by testing for cleavage in vitro. We also are trying to use cell-corpse markers developed in other systems to study C. elegans programmed cell deaths. We are examining retention of acridine orange by cell corpses, DNA fragmentation detected by TUNEL (TdT-mediated dUTP Nick End Labeling) and loss of membrane polarity as evidenced by Annexin V labeling. We would like to determine whether specific attributes of cell corpses are abnormal in
ced-11 mutants and whether all aspects of corpse formation are delayed in
ced-8 mutants. Preliminary data suggest that DNA degradation may be less delayed in
ced-8 mutants than is the appearance of a refractile cell corpse as visualized by Nomarski optics. It is possible that cell death is initiated at the appropriate time in
ced-8 mutants and that aspects of the downstream execution process are temporally uncoupled from one another. However, an alternative model is that
ced-8 is involved in the activation of cell killing by
ced-3 and
ced-4. By genetic epistasis,
ced-8 functions downstream of or in parallel to
ced-9. We will try to determine the order of action of
ced-8 with respect to
ced-3 and
ced-4 by examining whether cell killing by ectopic expression of
ced-3 or
ced-4 is delayed in a
ced-8 mutant background.