ced-3 encodes a member of the caspase family of cysteine proteases and is essential for almost all programmed cell death in C. elegans. However, a quantitatively low level of programmed cell death still appears to occur in the absence of CED-3 protease function. Such
ced-3-independent cell death is indicated by the presence of unengulfed cell corpses in heads of L1 larvae containing strong loss-of-function mutations in both
ced-3 and
ced-1. (
ced-1 is necessary for cell-corpse engulfment.) To investigate if any known cell-death genes are involved in this
ced-3-independent death, we are examining whether mutations in other ced genes affect the number of corpses in
ced-1;
ced-3 animals. To date, we have observed that loss-of-function mutations in
ced-7, and possibly in
ced-9 and
ced-4, decrease the number of corpses in heads of L1 triple mutants. Thus, these genes might play a role in
ced-3-independent killing.In addition to CED-3, CED-4S, one of the two alternatively spliced
ced-4 products, also has killing activity. By overexpressing CED-4S in
ced-1;
ced-3 animals, we observed that CED-4S can induce cell death in the absence of CED-3. Overexpression of another cell death activator, EGL-1, however, cannot induce cell death in a
ced-1;
ced-3 background.We are now using electron microscopy and Annexin V and TUNEL (TdT-mediated dUTP digoxgenin Nick End Labeling) staining to better characterize
ced-3-independent cell death. Annexin V specifically labels apoptotic cells by binding to phosphatidylserine exposed on cell surface, while TUNEL labels free DNA ends in apoptotic cells. We are also examining if the caspase homologs
csp-1,
csp-2 and
csp-3 are involved in
ced-3-independent cell death.