Figure 1.
gcn-1(n4827) and abcf-3(n4927) cause a defect in M4 sister cell death.
(A) Schematic representation of the M4 cell lineage in the wild type and mutants defective in M4 sister cell death. X, programmed cell death. (B–E) Merged epifluorescence and Nomarski images of the pharynx in wild-type, ced-3(n717), gcn-1(n4827) and abcf-3(n4927) animals expressing Pceh-28::gfp. Arrow, M4 neuron. Arrowhead, surviving M4 sister. Scale bar, 20 µm. Panels B and C from ref. 15. (F) The percentages of M4 sister survival in animals of the indicated genotypes. (G) Genomic organizations and protein structures of gcn-1 and abcf-3, including the locations and natures of the mutations n4827 and n4927. Orange box, EF3-like domain. Green boxes, AAA domains. Black bars, sequences deleted in alleles gcn-1(nc40Δ) and abcf-3(ok2237Δ). (H) Comparison of amino acid sequences of the entire protein or the EF3-like domain of GCN-1 and of the entire protein, the N-terminal domain, the first AAA domain or the second AAA domain of ABCF-3 among yeast, C. elegans and humans. Clustal W was used to align amino-acid sequences and to calculate identity and similarity.
Figure 2.
GCN-1 and ABCF-3 proteins are evolutionarily conserved functionally.
(A) Schematic representations of the GCN-1 and ABCF-3 proteins used in the yeast two-hybrid binding assays. Orange box, EF3-like domain. Green boxes, AAA domains. The growth of yeast on histidine-minus medium is summarized. +, wild-type growth. −, no or little growth. (B) Western blot analysis of immunocomplexes purified from wild-type animals with an anti-ABCF-3 antibody or control IgG. Both ABCF-3 and GCN-1 are present in the immunocomplex purified with an anti-ABCF-3 antibody but not with the control IgG. (C) Western blot analysis showing levels of ABCF-3 and GCN-1 proteins in fourth-larval stage animals of the indicated genotypes. (D) Western blot analysis showing the levels of eIF2α with phosphorylated serine 49 and total eIF2α of fourth-larval stage animals of the indicated genotypes. (E) Relative intensities of phosphorylated eIF2 to total eIF2 of fourth-larval stage animals of the indicated genotypes. Errors, standard deviations.
Figure 3.
gcn-1 and abcf-3 are ubiquitously expressed.
(A–E) Expression pattern of gcn-1. (A–C) Expression of GFP under the control of the gcn-1 promoter in second-larval stage animals and (D and E) FISH of gcn-1 mRNA in third-larval stage animals. Green, gcn-1 mRNA. Blue, DAPI staining of nuclei. (F–J) Expression pattern of abcf-3. (F–H) Expression of GFP under the control of the abcf-3 promoter in the second-larval stage animals and (I and J) FISH of abcf-3 mRNA in third-larval stage animals. Green, abcf-1 mRNA. Blue, DAPI staining of nuclei. Arrows, M4 neuron. Arrowheads, intestinal cells. Scale bars, 50 µm in A and F; 10 µm in B, C, G, H; and 5 µm in D, E, I and J.
Table 1.
gcn-1(n4827) and abcf-3(n4927) enhance the cell-death defects of partial loss-of-function ced-3(n2427) mutants.
Figure 4.
gcn-1(n4827) and abcf-3(n4927) cause a defect in radiation-induced germline cell death.
(A–D) Acridine orange (AO) staining of apoptotic germ cells in the posterior gonads of animals of the indicated genotypes. Arrowheads, AO-positive apoptotic germ cells. Scale bar, 10 µm. (E–H) Nomarski DIC images corresponding to A–D. Arrowheads, refractile apoptotic cells. (I) Numbers of AO-positive apoptotic germ cells in the posterior gonads of animals of the indicated genotypes. White bars, means and standard errors of the means without ionizing radiation (IR). Black bars, means and standard errors of the means with IR.
Table 2.
gcn-1(n4827) and abcf-3(n4927) enhance the M4 sister-cell death defect of ced-9(n2812) mutants.
Figure 5.
GCN-1 and ABCF-3 act cell-autonomously in a pathway distinct from the canonical cell-death execution pathway.
(A) The percentages of PLM survival in animals of the indicated genotypes. All strains had the Pmec-4::gfp transgene that expressed GFP in the touch neurons, including the PLM neurons. (B) A model for the pathways that regulate M4 sister cell-specific programmed cell death. gcn-1 and abcf-3 act in a pathway distinct from the canonical ced-9-dependent cell-death execution pathway to promote M4 sister cell death. See text for details.
Table 3.
ceh-34, eya-1, sptf-3 and pig-1 function independently of gcn-1 and abcf-3.