Caenorhabditis elegans genes required for the engulfment of apoptotic corpses function in the cytotoxic cell deaths induced by mutations in lin-24 and lin-33

Abstract

Two types of cell death have been studied extensively in Caenorhabditis elegans, programmed cell death and necrosis. We describe a novel type of cell death that occurs in animals containing mutations in either of two genes, lin-24 and lin-33. Gain-of-function mutations in lin-24 and lin-33 cause the inappropriate deaths of many of the Pn.p hypodermal blast cells and prevent the surviving Pn.p cells from expressing their normal developmental fates. The abnormal Pn.p cells in lin-24 and lin-33 mutant animals are morphologically distinct from the dying cells characteristic of C. elegans programmed cell deaths and necrotic cell deaths. lin-24 encodes a protein with homology to bacterial toxins. lin-33 encodes a novel protein. The cytotoxicity caused by mutation of either gene requires the function of the other. An evolutionarily conserved set of genes required for the efficient engulfment and removal of both apoptotic and necrotic cell corpses is required for the full cell-killing effect of mutant lin-24 and lin-33 genes, suggesting that engulfment promotes these cytotoxic cell deaths.

Figure 1.—

The Pn.p cells of lin-24(n432) and lin-33(n1043) mutants are morphologically abnormal. Nomarski photomicrographs of abnormal Pn.p cells in lin-24(n432) and lin-33(n1043) L1 larvae are shown. White arrows indicate Pn.p cells with an abnormal refractile appearance. Anterior, left; ventral, down.

Figure 2.—

Ultrastructural characteristics of the abnormal Pn.p cells of lin-24(n432) and lin-33(n1043) mutants. (A) Electron micrograph of a refractile P10.p cell in a lin-33(n1043) mutant. Four ultrastructural characteristics of the abnormal Pn.p cells observed in lin-24(n432) and lin-33(n1043) animals are indicated: dilated mitochondrion with disrupted internal architecture, electron-dense puncta in the nucleus, dilation of the lumen of the nuclear envelope, and dense membranous whorls. Note that these four abnormal characteristics are not observed in the adjacent cells, which all have normal cellular architecture. For comparison, a normal nucleus and normal mitochondria in neighboring cells are indicated. (B) Increased magnification of A showing a disrupted mitochondrion. (C) Increased magnification of A showing a mitochondrion with normal architecture. (D) Increased magnification of A showing the electron-dense nuclear puncta and membranous whorls.

Figure 3.—

Molecular identification of lin-24 and lin-33. (A) Genetic and physical maps of the lin-24 region. lin-24 was mapped to the ∼300-kb interval between unc-22 and an RFLP on C41D9. The four cosmids shown were tested for their abilities to rescue the recessive vulvaless phenotype of lin-24 mutants. The overlapping cosmids B0001 and T20H7 and an NcoI-HindIII fragment containing the gene B0001.1 could rescue the Vulvaless phenotype. (B) The structure of the lin-24 gene as deduced from genomic and cDNA sequences. Exons (solid boxes), 5′ and 3′ untranslated regions (open boxes), the predicted transcriptional start, and the SL1 splice leader sequence are indicated. The location of the deletion lin-24(n4294Δ) and the scale are indicated by the labeled horizontal lines. (C) Sequences of lin-24 mutations. Mutated bases are underlined. Alleles were designated loss-of-function if they suppressed the semidominant vulvaless phenotype of lin-24(n432). (D) Genetic and physical maps of the lin-33 region. lin-33 was mapped to the ∼156-kb interval between a polymorphism on C10G6 and unc-44. PCR products generated from lin-33(n1302) genomic DNA spanning almost all of the shown interval were injected into lin-33(+) animals and tested for the ability to phenocopy the semidominant vulvaless phenotype of the lin-33(n1302) animals. The 26-kb PCR product no. 6 and a 9-kb PCR product containing only the gene H32C10.2 generated a vulvaless phenotype. (E) The structure of the lin-33 gene as deduced from genomic and cDNA sequences. Exons (solid boxes), 5′ and 3′ untranslated regions (open boxes), the predicted transcriptional start, and the SL1 splice leader sequence are indicated. The location of the deletion lin-33(n4514Δ) and the scale are indicated by the labeled horizontal lines. (F) Sequences of lin-33 mutations. Mutated bases are underlined. Alleles were designated loss-of-function if they suppressed the semidominant vulvaless phenotype of lin-33(n1043).

Figure 4.—

LIN-24 contains a domain similar to that in bacterial toxins. Sequence alignment of amino acids 62–176 of LIN-24 and the Mtx3 toxin domain of the 35.8-kDa mosquitocidal toxin from B. sphaericus (GenBank accession no. AAB36654). Numbers at the right and left indicate amino acid positions within the respective proteins. Identical residues are colored red, and similar residues are colored blue (29% identity and 45% similarity). Missense mutations found in lin-24 within the region of homology are indicated.

Figure 5.—

The cytotoxicity of lin-24(n432) and lin-33(n1043) is partially dependent on genes that mediate the removal of apoptotic cell corpses. The penetrances of the vulvaless phenotypes of lin-24(n432); ced and lin-33(n1043); ced strains were analyzed and the percentage of suppression was calculated as described (see materials and methods). At least 56 animals were assayed for each genotype. (A) Mutations in some of the genes that act in programmed cell death partially suppress the vulvaless phenotype of lin-33(n1043) animals. Genes are organized in the following order along the x-axis: cell-killing genes in order of action (egl-1, ced-9, ced-4, and ced-3); one of two partially redundant engulfment pathways (ced-1, ced-6, and ced-7); and the other partially redundant engulfment pathway (ced-2, ced-5, ced-10, and ced-12). (B) Mutations in some of the genes required for programmed cell death partially suppress the vulvaless phenotype of lin-24(n432) animals. (C) A ced-5 and ced-6 double mutant was more strongly suppressed for the vulvaless phenotype caused by lin-33(n1043) than was either single mutant. (D) A ced-5 and ced-6 double mutant was more strongly suppressed for the vulvaless phenotype caused by lin-24(n432) than was either single mutant.

Figure 6.—

A model for the effects of engulfment on lin-24- and lin-33-induced cytotoxicity. Gain-of-function mutations in lin-24 and lin-33 make the Pn.p cells sick, possibly by forming membrane complexes that alter membrane permeability in a process that is similar to that of the bacterial toxins that share similarity with LIN-24. A consequence of this sickness is that a signal is presented on the cell membrane and recognized by a neighboring cell. The neighboring cell then promotes the cytotoxicity in a process that requires the engulfment genes ced-2, ced-5, ced-10, and ced-12, which likely act by reorganizing the actin cytoskeleton (adapted from Reddien and Horvitz 2004). Additional genes that function in programmed cell death, such as ced-6 and egl-1, might also play a role in lin-24- and lin-33-induced cytotoxicity.