CED-3 and its human homolog, Interleukin-1-beta Converting Enzyme (ICE), are known to play a central role in a cell's decision to undergo apoptotic death. However, the downstream effectors of these cysteine proteases are not well understood. In an effort to understand the processes by which cysteine protease activation leads to apoptotic death, we are conducting screens for supressors of ICE-induced cell death in C. elegans. In previous work with Cori Bargmann (UCSF), we fused the
glr-1 promoter to the human ICE sequence and integrated this construct.
glr-1::ICE animals are touch-defective due to the ICE-induced apoptotic death of interneurons. We mutagenized this strain with EMS and screened for suppression of the touch phenotype. Four suppressor strains were isolated from this screen. These strains are currently being characterized. Since the pathway that leads from ICE to apoptosis is likely to be extensively branched, we are taking a parallel approach that will boost our screening efficiency. We obtained the
ceh-28 promoter from Brian Harfe and Andrew Fire, who showed that
ceh-28::gfp is expressed only in the M4 pharyngeal neuron. Leon Avery has shown by laser ablation that M4 is required for survival beyond L1. M4 innervates the posterior half of the isthmus; when M4 is killed, the isthmus remains closed and the animal starves. Avery also found that M4-ablated animals are viable on media containing arecoline, a drug which depolarizes M4's post-synaptic target. We have fused the
ceh-28 promoter to the human ICE sequence. We expect
ceh-28::ICE animals to be non-viable on normal media due to apoptotic death of M4, but viable on arecoline. This will allow us to select for suppressors of the M4-ablated phenotype by growing
ceh-28::ICE animals on arecoline, mutagenizing, and then moving F2's to normal media. This selection is likely to yield several broad classes of genes. In addition to downstream effectors of ICE, we expect to find mutations that allow
ceh-28::ICE animals to survive by depolarizing the isthmus.