Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

Abstract

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.

Figure 1

CED-4 is not localized on mitochondria in the germ line. (a) Representative pictures of late-stage pachytene germ cells were taken by Deltavision microscopy. CED-4 staining was done with the CED-4 9104.1 antibody. Mitochondria were stained with anti-ATP synthase and anti-cytochrome c antibodies. The corresponding 3D stacks of the images are shown in Supplementary Movie 1 (CED-4) and Supplementary Movie 2 (CED-4/mitochondia); scale bar: 6 μm. (b) CED-4 perinuclear accumulation upon treatment with IR. A representative healthy cell, and corpses indicative of different stages of apoptosis progression are indicated in the right panel (1–6). A blow up of these cells is shown in Figure 6a. Note that the CED-4 signal intensity in (b) is lowered by 15% compared with (a) to allow for better visualization. (c) OMX microscopy of single germ cells before and after IR (CED-4 green, nuclear pore complex red). Scale bar: 4 μm

Figure 2

Perinuclear localization of CED-4::GFP. (a) The ced-4 gt1767 Ex[pced-4::ced-4::gfp::ced-4 3′UTR, unc-119(+)] construct rescues the ced-4(n1162) apoptosis defect. Only worms carrying the CED-4::GFP transgene were scored. (b) CED-4::GFP staining using anti-GFP antibodies. A white arrow indicates a very-late-stage corpse, where mitochondria are fragmented, chromatin is condensed and CED-4 staining is absent (see Figures 1b and 6). (c) Direct visualization of CED-4::GFP using fluorescence microscopy. The left panel shows DIC image of pachytene-stage germ cells and the right panel overlaps with fluorescence image. Scale bar: 10 μm

Figure 3

Staining of an embryo undergoing morphogenesis indicates that CED-4 does not colocalize with CED-9 and mitochondria. CED-4 is visualized using anti-GFP antibodies (see Materials and Methods). The image shown is a projection of six Z stacks. The 3D scans are shown in Supplementary Movies 3–5. Scale bar: 10 μm

Figure 4

(a) Punctate CED-9 staining is associated with mitochondria. (b) A blow up of mitochondrial branches (indicated by arrows) is shown in the lower right panels. Scale bar: 4 μm. A 3D scan of the picture is shown in Supplementary Movie 6. (c) Subcellular fractionation. Mixed-stage C. elegans extracts were generated and fractionated as described in Materials and Methods. Enrichment of subcellular compartments was confirmed using antibodies against the indicated marker proteins

Figure 5

CED-4 and CED-9 staining overlaps in secondary spermatocytes. The CED-4 (10147.1) antibody was used

Figure 6

Various stages of germ cell apoptosis. Cells/corpses shown are indicated in Figure 1b. Scale bar: 6 μm

Figure 7

CED-4 perinuclear accumulation upon IR is absent in egl-1 loss-of-function and ced-9 gain-of-function mutants. The CED-4 (9104.1) antibody was used. Scale bar: 10 μm