B.al/rapaav death is caspase- and engulfment gene-dependent

To confirm and extend the observations that B.al/rapaav death is dependent on the engulfment genes ced-1 and ced-2 [10] and the caspase ced-3 [12], we assayed B.al/rapaav death in a variety of cell-death mutant backgrounds. Specifically, we scored the presence of a cell that expressed a reporter specific for the dying B.al/rapaav, P egl-1 ::4xNLS::gfp (see below), in a position consistent with that of an undead B.al/rapaav in late fourth larval stage animals and interpreted such a cell as one that had failed to undergo programmed cell death. In this way, we confirmed that B.al/rapaav cell death is dependent on both the suicide and engulfment pathways. Specifically, strong loss-of-function alleles of the pro-apoptotic genes egl-1, ced-4 or ced-3 or a gain-of-function allele of the anti-apoptotic gene ced-9 almost completely blocked B.al/rapaav death (Fig. 1d). Strong loss-of-function alleles of any of the major engulfment genes were also sufficient to prevent B.al/rapaav death (Fig. 1d). In two engulfment-defective animals that we observed into adulthood, the undead B.al/rapaav cell continued to persist for the duration of observation, indicating that engulfment defects prevent B.al/rapaav cell death rather than temporarily delaying it. While loss of engulfment gene function can weakly contribute to the survival of other cells in C. elegans, the effect is smaller than that of weak reduction-of-function mutations in suicide genes [12]. By contrast, weak reduction-of-function mutations in the pro-apoptotic genes ced-3 or ced-4 had smaller effects on B.al/rapaav survival than did loss of engulfment gene function (Fig. 1d). This finding indicates that B.al/rapaav death is particularly dependent on the function of engulfment genes rather than highly sensitive to any slight perturbation to the cell-death pathway. Mutations in unc-108 Rab2 cause severe defects in phagosome maturation and cell-corpse degradation after engulfment [18], but a mutation in unc-108 did not significantly block B.al/rapaav death, suggesting that the engulfment process and not downstream degradation processes are important for B.al/rapaav death.

The dying B.al/rapaav expresses cell-death genes

The cell-death genes in C. elegans act cell-autonomously to specify and cause cell death [3, 9]. To examine if the presumptive cell-death suicide genes indeed act in the dying B.al/rapaav, we used a transcriptional reporter for egl-1, the most upstream gene in the core cell-death pathway; egl-1 is transcriptionally upregulated to drive programmed cell death [4]. The egl-1 reporter was expressed strongly in the B.al/rapaav cell fated to die and not at all or only weakly in the B.al/rapaav homolog, which is fated to live (Fig. 2a, b). All B.al/rapaav corpses in fourth larval stage wild-type animals were GFP-positive (n = 53), confirming that the GFP expression is associated with the secondary fate of cell death.

Fig. 2 Cell-death genes are expressed in B.al/rapaav. a GFP under the control of the egl-1 promoter is expressed brightly in B.al/rapaav in the tail of an early fourth larval stage “wild-type” male of genotype nIs343; him-8. Scale bar: 10 μm. b The average intensity of P egl-1 ::4xNLS::GFP in the nucleus is higher in the dying B.al/rapaav cell than in the surviving B.al/rapaav homolog in early fourth larval stage animals. The B.al/rapaav fate was assigned based on partial cytoplasmic refractility and/or nuclear position and distance from the midline of the animal. Average fluorescent intensity within the nucleus was measured for confocal images of B.alapaav, B.arapaav and P12.pa in each animal, and the average background intensity was subtracted from each. A.U., arbitrary units. All genotypes include nIs343, and some also contain †: him-5(e1490); ‡: him-8; °: nIs349. c–e Proapoptotic genes egl-1 and ced-3 are not highly expressed in the surviving B.al/rapaav homolog (c) but are highly expressed in the dying B.al/rapaav (d). These two images were taken from the same animal, approximately 1.6 μm apart. Anterior, left; dorsal, top. Scale bars: 10 μm. Full Z-stack of this animal is available as Additional file 2: Movie 2, and two more examples are available as Additional file 3: Movie 3, Additional file 4: Movie 4. These animals were of the genotype nIs343; him-5(e1490); nIs349. e Quantification of nuclear mRNA transcripts of egl-1 and ced-3 in B.al/rapaav and the B.al/rapaav homolog. Only nuclear transcripts were counted, because we could not unambiguously determine to which cell non-nuclear transcripts belonged. Condensed chromatin (as visualized by DAPI, which stains DNA and shows the chromatin in a dying cell to be slightly brighter and smaller in volume than that in a living cell), a position to the left or right of P12.pa, or expression of P egl-1 ::4xNLS::gfp was each interpreted as indicating the secondary cell fate. All animals were of the genotype nIs343, him-5(e1490), nIs349. *: P < 0.005, **: P < 5 × 10−4 Full size image

We used single-molecule fluorescent in situ hybridization (smFISH) to determine whether, like the P egl-1 ::4xNLS::gfp reporter, endogenous cell-death genes are transcribed in the dying B.al/rapaav. We usually detected egl-1 mRNA expression in B.al/rapaav and only rarely in the B.al/rapaav homolog, consistent with the expression pattern of P egl-1 ::4xNLS::gfp (Fig. 2c–e and Additional file 2: Movies 2, Additional file 3: Movies 3, Additional file 4: Movies 4). We detected low levels of ced-9 and ced-4 transcripts broadly (data not shown). ced-3 transcripts were visible in only a subset of cells, usually including B.al/rapaav, and only rarely in the B.al/rapaav homolog (Fig. 2c–e and Additional file 2: Movies 2, Additional file 3: Movies 3, Additional file 4: Movies 4). Primary- and secondary-fated cells were identified based on nuclear position and morphology (the secondary-fated B.al/rapaav cell nucleus appears condensed after staining DNA with DAPI to visualize nuclei and is closer to P12.pa). Because B.al/rapaav cell death is dependent on suicide genes that are expressed in the dying B.al/rapaav and not in nearby cells, B.al/rapaav death is likely a form of cell suicide rather than a murder.

Engulfment genes do not induce the caspase-mediated suicide pathway

As in wild-type animals, P egl-1 ::4xNLS::gfp was expressed in the undead secondary B.al/rapaav in cell-death suicide and engulfment mutants (Fig. 3a, b). Similarly, endogenous transcripts of the cell-death genes egl-1 and ced-3 were generally present in one, but not both, of B.alapaav and B.arapaav in the engulfment-defective double mutant, ced-12 ced-1 (Fig. 3c–g). These data indicate that the induction of suicide gene expression in B.al/rapaav requires neither engulfment nor signals transduced via the engulfment pathway.

Fig. 3 B.al/rapaav and the B.al/rapaav homolog express pro-apoptotic genes differentially in cell-death mutants. All genotypes include nIs343, and some also contain †: him-5(e1490), ‡: him-8, °: nIs349, §: lon-1(e1820) dpy-17, : lon-1(e185), ★: unc-32. a B.al/rapaav expresses P egl-1 ::4xNLS::gfp in all genetic backgrounds studied. Percentage of animals with P egl-1 ::4xNLS::gfp expression in death-fated B.al/rapaav during the early fourth larval stage, before B.al/rapaav death occurs. b The average intensity of P egl-1 ::4xNLS::GFP in the nucleus is higher in B.al/rapaav than in the B.al/rapaav homolog in early fourth larval stage animals defective for cell death genes. A.U., arbitrary units. egl-1 (c, e) and ced-3 (d, f) transcripts are detectable in the presumptive B.al/rapaav (white arrow) in the tails of early fourth larval stage wild-type (c, d) and engulfment-defective (e, f) males. The genotype in c was nIs343; him-8; d nIs343; him-5(e1490); nIs349; e, f ced-12 ced-1; nIs343; him-8. Scale bars: 10 μm. g ced-3 and egl-1 are differentially expressed between B.alapaav and B.arapaav. Only nuclear transcripts were counted. For each animal, the cell with the larger number of transcripts was classified as “high expression B.al/rapaav” and the other cell was classified as “low expression B.al/rapaav homolog.” When both cells had the same number of transcripts, one was arbitrarily labeled high and one low. This panel includes data from Fig. 2e. Animals contained nIs343 and †: him-5(e1490), ‡: him-8, °:nIs349. *: P < 0.005 Full size image

The dying B.al/rapaav exhibited morphological changes as visualized with Nomarski differential interference microscopy (DIC) similar to those of other dying cells [13, 19]. At the time of their generation, B.alapaav and B.arapaav are indistinguishable using DIC and appear similar to normal healthy cells (Fig. 4a). Later, one (B.al/rapaav) becomes round and condensed with partial cytoplasmic refractility (Fig. 4b) before becoming a highly refractile corpse (Fig. 4c). The other (the B.al/rapaav homolog) remains non-refractile and looks healthy and normal (data not shown).

Fig. 4 The surviving B.al/rapaav is morphologically different in engulfment-gene and suicide-gene mutants. a–c The morphology of a dying B.alapaav changes over time; him-8 fourth larval stage male. Arrows and insets show B.alapaav. Scale bars: 10 μm. a 1 hour after its generation, B.alapaav was non-refractile. b 3.5 hours after its generation, B.alapaav was rounded and the cytoplasm was refractile, but the nucleus was non-refractile. c 4 hours after its generation, B.alapaav was a refractile corpse. d–f Percentage of animals in which B.al/rapaav displayed the morphology seen in images (c), (b), and (a), respectively, in mid-fourth larval stage animals. Animals with no visible secondary B.al/rapaav, presumably because this cell had already been degraded, were excluded from this analysis. All genotypes include nIs343 and some also contain †: him-5(e1490), ‡: him-8, °: nIs349, : lon-1(e185), ★: unc-32, §: lon-1(e1820) dpy-17. d n.s.: P > 0.5, *: P < 0.005. d Wild-type vs. engulfment mutants (ced-1, ced-6, ced-7, ced-2, ced-5, ced-12, and ced-1 ced-12): P < 10−17, wild-type vs. suicide mutants (egl-1, ced-9(gf), ced-4(lf), ced-3(del), and ced-3(lf)): P < 10−15, engulfment vs. suicide mutants: P > 0.5. e Wild-type vs. engulfment mutants: P < 10−11, wild-type vs. suicide mutants: P > 0.5, engulfment vs. suicide mutants: P < 10−31. f Wild-type vs. engulfment mutants: P > 0.5, wild-type vs. suicide mutants: P < 10−12, engulfment vs. suicide mutants: P < 10−30. P values between classes of mutants are by two-tailed Fisher’s exact test using data pooled within genotypic classes (wild-type, engulfment mutants and suicide mutants). g–h Representative images of the morphology of the undead B.al/rapaav in ced-12 ced-1 engulfment (g) and ced-3(lf) suicide (h) mutant backgrounds Full size image

While mutations in either the suicide pathway or the engulfment pathway were sufficient to block B.al/rapaav from acquiring the highly refractile appearance characteristic of programmed cell death, we discovered that the morphology of B.al/rapaav as viewed with DIC optics was different between these two classes of mutants: in the mid-fourth larval stage, B.al/rapaav was a highly-refractile corpse in most wild-type animals (Fig. 4d); the undead B.al/rapaav in engulfment mutants generally was round with a refractile cytoplasm and non-refractile nucleus (Fig. 4e, g) and the undead B.al/rapaav in ced-3 and other suicide mutants generally was non-refractile, similar to other living cells, including the B.al/rapaav homolog (Fig. 4f, h). The morphology of the undead B.al/rapaav in engulfment mutants was indistinguishable from that of the dying B.al/rapaav at an earlier stage of the cell-death process. We interpret this morphology to be that of a cell in which caspases have been at least partially activated, as this morphology is dependent on ced-3 and the rest of the core cell-death pathway. These results further show that the engulfment genes are not required for the initiation of the cell-death process but are rather required in a later process. We examined unc-108 Rab2 mutants, which are defective in phagosome maturation in the engulfing cell and hence in the degradation of cell corpses [18]. B.al/rapaav died and formed a fully refractile corpse in unc-108 mutants, suggesting that the failure of B.al/rapaav to form a fully refractile corpse in engulfment mutants is unlikely to be caused by a block in cell-corpse degradation.

In engulfment mutants the caspase pathway is activated, but B.al/rapaav death is not completed. The undead B.al/rapaav cell in engulfment mutants is similar to living cells and unlike other unengulfed cells fated to die, which form fully refractile corpses. Like B.al/rapaav cells that have not yet died or undead B.al/rapaav cells in suicide-defective mutants, the undead B.al/rapaav cell in engulfment mutants was not fully refractile by DIC optics, retained nuclear localization of P egl-1 ::4xNLS::GFP and membrane localization of the cytoplasmic membrane marker P evl-20 ::mCherry::PH (see below), and had diffuse chromatin as revealed by DAPI staining (Fig. 5a–c, g–m, o, p); by contrast, in fully-refractile B.al/rapaav corpses and other cell corpses in the male tail P egl-1 ::4xNLS::GFP and P evl-20 ::mCherry::PH were distributed throughout the cell corpse, and chromatin after DAPI staining appeared condensed (Fig. 5d–f, n). The distribution of P egl-1 ::4xNLS::GFP throughout the cell corpse likely reflects nuclear disruption and/or inactivation of nuclear transport; nuclear disruption and chromatin condensation are two hallmarks of apoptotic cell death [20]. Additionally, during time-course observations, we occasionally saw fluctuations in the level of B.al/rapaav cytoplasmic refractility in engulfment mutants, as was previously reported for other cells that had initiated but not completed the cell-death process [12]. These other cells sometimes survived and differentiated, establishing that cells with partial cytoplasmic refractility are not dead. Similarly, B.al/rapaav is not dead, as it can return to a non-refractile morphology and lacks two canonical hallmarks of apoptotic dead cells, nuclear disruption and chromatin condensation. We conclude that ced-3 activity does not inevitably lead to the death of B.al/rapaav and that the B.al/rapaav cell fated to die in engulfment mutants is abnormal but alive.

Fig. 5 The undead B.al/rapaav cell in engulfment mutants fails to display characteristics of dead cells. a–c A living B.al/rapaav cell (cell boundary indicated by dotted line) in the early fourth larval stage before cell death is complete (a) lacks refractility in the nucleus, (b) has P egl-1 ::4xNLS::GFP in the nucleus but not in the rest of the cell and (c) has P evl-20 ::mCherry::PH localized to the membrane. d–f A B.al/rapaav cell corpse has (d) refractility throughout the cell, (e) P egl-1 ::4xNLS::GFP throughout the cell, and (f) P evl-20 ::mCherry::PH throughout the cell. g–i An undead B.al/rapaav cell in the late fourth larval stage of an engulfment-defective mutant (g) lacks refractility in the nucleus, (h) has P egl-1 ::4xNLS::GFP in the nucleus but not the rest of the cell, and (i) has P evl-20 ::mCherry::PH localized to the membrane. j–l An undead B.al/rapaav cell in the late fourth larval stage of a suicide-defective mutant (j) lacks refractility, (k) has P egl-1 ::4xNLS::GFP in the nucleus but not the rest of the cell, and (l) has P evl-20 ::mCherry::PH localized to the membrane. Animals were of genotypes nIs343; him-5(e1467ts) unc-76; nIs349; nEx2344 (a–f), nIs343; ced-10 him-8; nIs735 (g–i), or nIs343; ced-3 him-8; nIs735 (j–l). Scale bars: 10 μm. m A B.al/rapaav cell (arrow) in the early fourth larval stage has diffuse chromatin similar to neighboring living cells. n A dying or dead B.al/rapaav cell in the mid-fourth larval stage has condensed chromatin. o An undead B.al/rapaav cell in the late fourth larval stage of an engulfment-defective mutant has diffuse chromatin similar to neighboring living cells. p An undead B.al/rapaav cell in a suicide-defective mutant has diffuse chromatin similar to neighboring living cells. Scale bars: 10 μm. Animals were of the genotypes nIs343; him-5(e1490); nIs349 (m, n), ced-12 ced-1; nIs343; him-8 (o), or nIs343; ced-3 him-8 (p). a–l depict different animals than in (m–p), since the fixation required for visualizing DAPI staining precludes the use of DIC optics Full size image

The engulfing cell P12.pa is not required for the B.al/rapaav death

It has been reported that the engulfing cell P12.pa is required for B.al/rapaav cell death [16]. To examine the role of P12.pa in B.al/rapaav cell death, we ablated P12.pa or its precursor P12.p early in development, about 15–20 hours prior to the generation of B.alapaav and B.arapaav, and looked for signs that B.al/rapaav had initiated the cell-death program. In the absence of P12.pa, B.alapaav and B.arapaav adopted normal primary and secondary fates and one initiated the cell-death process, as evidenced by egl-1 reporter gene expression and by the positions and morphologies of the two cells (Fig. 6a–c). Thus, P12.pa is not required for B.alapaav and B.arapaav to differentially adopt the primary and secondary fates or to initiate the death process in the secondary B.al/rapaav.

Fig. 6 P12.pa ablation does not prevent initiation of B.al/rapaav death. a Percentages of P12.pa-ablated and mock-ablated males that have cytoplasmic refractility in B.al/rapaav, P egl-1 ::4xNLS::gfp expression in B.al/rapaav, or death of B.al/rapaav. Percentage of survival of B.al/rapaav in mock-ablated animals is not significantly different from that of untreated animals in Fig. 1d (P = 0.3). b, c A P12.pa-deficient animal in which the secondary B.al/rapaav (arrow) had cytoplasmic refractility (b) and had P egl-1 ::4xNLS::gfp expression (c), indicating that the cell-death process was initiated. Scale bars: 10 μm. All animals in panels (a–c) were of genotype nIs343; him-8. d A round, undead B.al/rapaav that was not engulfed in a P12.pa-deficient animal. White arrow points to the undead B.al/rapaav; the only membrane (red) visible around the B.al/rapaav nucleus (green) is that of the undead cell (since only one nucleus is located within that membrane), and no other cell’s membrane appears to enclose it in any plane. Full Z-stack of this animal is available as Additional file 5: Movie 5. e A B.arapaav corpse that was engulfed by B.alapaav in a P12.pa-deficient animal. The B.arapaav corpse (white arrow) appears to be completely inside the boundaries of the B.alapaav membrane (black in right panel). This membrane can be assigned to B.alapaav, because it contains the B.alapaav nucleus (black arrow). Full Z-stack of this animal is available as Additional file 6: Movie 6. Scale bars: 10 μm. f Outcomes of the secondary B.al/rapaav in P12.pa-ablated animals, showing that all surviving cells were unengulfed while most dying cells were engulfed. All animals in panels (d–f) were of the genotype nIs343; him-5(e1467ts) unc-76; nIs349; nEx2344 Full size image

To our surprise, we found that B.al/rapaav died in many of the animals that lacked P12.pa (Fig. 6a), contrary to the previous report [16]. Thus, although B.al/rapaav death is almost completely dependent on engulfment genes, it is only partially dependent on the presence of the normal engulfing cell. To reconcile this difference, we postulated that other cells might engulf B.al/rapaav in the absence of P12.pa. We designed a reporter protein with mCherry fused to the pleckstrin homology domain, which binds to lipids in the plasma membrane [21], and expressed this construct (P evl-20 ::mCherry::PH) using a promoter from the evl-20 gene [22], which is widely expressed throughout the animal, including in P12.pa, B.alapaav and B.arapaav (Fig. 6d, e). This construct highlights cell membranes and allowed us to visually determine if a corpse is internalized by another cell. We ablated P12.pa in this strain and found again that B.al/rapaav died in many of the animals. In animals in which B.al/rapaav survived, the undead B.al/rapaav was not engulfed (Fig. 6d, f, and Additional file 5: Movie 5). B.al/rapaav died in 16 P12.pa-ablated animals, and in 14 of these 16 cases the B.al/rapaav corpse was engulfed by a neighboring cell (Fig. 6e, f and Additional file 6: Movie 6). We conclude that the discrepancy between the essentially complete dependence on engulfment genes and the weaker dependence on the engulfing cell P12.pa is a consequence of the compensatory ability of other cells to engulf B.al/rapaav in the absence of P12.pa.

In nine of the 14 animals, the engulfing cell was the surviving primary B.al/rapaav homolog, and in five animals it was a more lateral cell. The identities of the other engulfing cells are uncertain. Based on position, likely candidates are K.a or the left rectal gland cell, B.alapaad and F.lvv. The B.al/rapaav homolog, K.a, the rectal gland cells, B.al/rapaad and F.l/rvv are cells that do not normally engulf corpses, since the only nearby dying cell is B.al/rapaav, which is engulfed by P12.pa. These findings demonstrate that other cells are competent to engulf B.al/rapaav in the absence of P12.pa and that even cells that do not normally engulf cell corpses can have a cryptic ability to recognize and engulf dying cells to promote cell death.

In short, B.al/rapaav likely becomes fated to die and begins to die cell-autonomously but generally requires engulfment by P12.pa or another neighboring cell to fully execute the death process. The death of B.al/rapaav thus does not seem to be an induced suicide, the process proposed by Reddien et al. [12].

Engulfment by P12.pa precedes the B.al/rapaav death

If engulfment is required for the B.al/rapaav death, B.al/rapaav might be engulfed early in the cell-death process, before it becomes a fully refractile corpse. To test this hypothesis, we imaged B.al/rapaav in fourth larval stage animals with cell membranes labeled by P evl-20 ::mCherry::PH. We characterized the morphology of each B.al/rapaav as either non-refractile, round with cytoplasmic refractility (suicide-initiated) or fully refractile (corpse). Then, we imaged P evl-20 ::mCherry::PH to determine the boundaries of B.al/rapaav and P12.pa. We found that B.al/rapaav corpses were always (42/42) internalized by P12.pa, cell-death initiated cells were usually (24/29) internalized by P12.pa, and cells with no sign of cell death initiation were never (0/26) internalized (Fig. 7a). These data indicate that P12.pa engulfs B.al/rapaav in the early stages of its death, but probably not before the cell death has already been initiated (as evidenced by cytoplasmic refractility). These findings are consistent with our observations that the suicide pathway acts before the engulfment pathway. We also confirmed that the identity of the engulfing cell was always P12.pa in intact animals (n = 66): other cells engulf B.al/rapaav only if P12.pa is absent.

Fig. 7 Engulfment precedes the B.al/rapaav death. a All non-refractile B.al/rapaav cells are unengulfed, most cells that are round with partial cytoplasmic refractility are engulfed by P12.pa and all fully refractile cells are engulfed by P12.pa. Representative DIC and fluorescent images corresponding to each of the B.al/rapaav morphology classes are shown, with the P12.pa (dashed red line) and B.al/rapaav cell membranes (dashed blue line) outlined (based on P evl-20 ::mCherry::PH signal). All animals were in the fourth larval stage, but were at slightly different ages for visualization of B.al/rapaav at different stages of the death process. b, c A B.arapaav cell with partial cytoplasmic refractility (white arrow) visualized by (b) DIC and (c) electron microscopy (blue arrowheads). c The B.arapaav cell is engulfed by P12.pa (red arrows) but otherwise does not display obvious ultrastructural signs of cell death. The animal was of genotype him-8. d B.al/rapaav does not die in a weak engulfment mutant without being engulfed. Engulfment and cell-killing are not independent events (P = 0.0075, Fisher’s exact test). B.al/rapaav was classified as being dead or undead based on morphology as seen with DIC optics and was then examined to determine whether it was inside of P12.pa based on P evl-20 ::mCherry::PH. Animals were of the genotype nIs343; ced-10 him-8; nIs735 Full size image

We confirmed that engulfment can precede the completion of cell death using electron microscopy. We observed a dying B.arapaav cell using DIC microscopy and fixed and stained the animal at the stage at which the B.arapaav cell was round with cytoplasmic refractility (Fig. 7b). B.arapaav had already been internalized by P12.pa but, ultrastructurally, it lacked signs of cell death such as nuclear envelope dilation or crenulation, extensive chromatin condensation, and membrane whorling (Fig. 7c) [19].

It is conceivable that engulfment genes act to promote B.al/rapaav cell death by cell-killing activities independent of their roles in engulfment per se. The weak loss-of-function allele ced-10(n1993rf) stochastically prevents B.al/rapaav cell death in only some animals (Fig. 1d). We hypothesized that if ced-10 promoted B.al/rapaav death through a cell-killing activity independent of its role in engulfment, these two functions might stochastically fail in different animals. For example, B.al/rapaav might be killed without being engulfed or engulfed without being killed. However, if the cell-killing activity of ced-10 were mediated through engulfment, B.al/rapaav would not be killed unless it were engulfed. We found that B.al/rapaav corpses in ced-10(n1993rf) were always engulfed by P12.pa (Fig. 7d). Undead B.al/rapaav cells were sometimes unengulfed and sometimes engulfed. These data are inconsistent with the hypothesis that engulfment and cell-killing are independent events (P = 0.0075). We conclude that engulfment precedes B.al/rapaav death and that the killing activity of ced-10 is mediated through its engulfment function.