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. 2010 Mar;17(3):534-9.
doi: 10.1038/cdd.2009.185. Epub 2009 Dec 4.

The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila

Y Fan  1 , A Bergmann
Affiliations

The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila

Y Fan et al. Cell Death Differ. 2010 Mar.

Abstract

The cleaved-Caspase-3 antibody is a popular tool in apoptosis research in Drosophila. As the antibody was raised against cleaved human Caspase-3, it was assumed that it detects cleaved DRICE and DCP-1, Caspase-3-like effector caspases in Drosophila. However, as shown here, strong immunoreactivity persists in apoptotic models doubly mutant for drICE and dcp-1. In contrast, mutants of the apoptosome components DRONC (Caspase-9-like) and ARK (Apaf-1 related) do not label with the cleaved-Caspase-3 antibody. By peptide blocking experiments and further genetic studies, we provide evidence that the cleaved-Caspase-3 antibody recognizes multiple proteins including DCP-1 and likely DRICE, but also at least one additional unknown protein, all of which require DRONC for epitope exposure. The unknown substrate may be involved in non-apoptotic functions of DRONC. Because the cleaved-Caspase-3 antibody not only detects cleaved Caspase-3-like proteins in Drosophila, but also other proteins in a DRONC-dependent manner, it is more accurate to consider the cleaved-Caspase-3 antibody as a marker for DRONC activity, rather than effector caspase activity.

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Figures

Figure 1
Figure 1. The cleaved-Caspase-3 antibody is a marker for DRONC activity
Shown are eye imaginal discs of third instar larvae. Posterior is to the right. GMR-hid is a transgenic insertion on the X chromosome. (a) Wild-type (wt) disc labeled with cleaved-Caspase-3 antibody. Arrows point to a few immunopositive cells. (b) A disc doubly mutant for the null alleles dcp-1Prev and drICEΔ1 labeled with cleaved-Caspase-3 antibody. Arrows point to a few immunopositive cells. (c) and (d) GMR-hid eye discs in otherwise wild-type background labeled with (c) cleaved-Caspase-3 antibody and (d) TUNEL. Note the strong signals in the posterior half of the eye discs. (e) and (f) GMR-hid eye discs doubly mutant for dcp-1Prev and drICEΔ1 labeled for (e) cleaved-Caspase-3 antibody and (f) TUNEL. While TUNEL labeling is completely blocked, cleaved-Caspase-3 antibody still delivers a strong signal in the posterior half of the eye disc. (g) and (h) GMR-hid eye discs mutant for the null allele droncI24. Both (g) cleaved-Caspase-3 antibody and (h) TUNEL labeling are blocked by loss of DRONC. Genotypes: (a) wild-type (b) dcp-1Prev/dcp-1Prev; drICEΔ1/drICEΔ1 (c) and (d) GMR-hid/GMR-hid; +/+ ; +/+ (e) and (f) GMR-hid/GMR-hid; dcp-1Prev/dcp-1Prev ;drICEΔ1/drICEΔ1 (g) and (h) GMR-hid/GMR-hid; droncI24/droncI24.
Figure 2
Figure 2. A peptide containing ETD blocks cleaved-Caspase-3 immunoreactivity
(a) Amino acid sequence alignment of the residues from catalytic Cys163 to the cleavage site at Asp175 of human Caspase-3 (Caspase-3 peptide) and the corresponding regions of the Drosophila caspases. Residues highlighted in red are identical in the Caspase-3 peptide. For DRICE, DCP-1 and DRONC cleavage has been demonstrated following the last residue (D and E) of the sequence shown. For DECAY, DAMM, DREDD and DREAM cleavage is uncertain and the end of the sequence shown does not imply cleavage (indicated by ...). Underlined sequences were used in blocking peptides (compare with b). (b) Amino acid sequences of the blocking peptides. Only peptide A blocks immunoreactivity of the cleaved-Caspase-3 antibody. (c and d) Preincubation of cleaved-Caspase-3 antibody with peptide A completely abrogates its immunoreactivity in GMR-hid eye discs (c) and GMR-hid eye discs mutant for dcp-1 and drICE (d). (e and f) Preincubation of cleaved-Caspase-3 antibody with peptide B has little or no effect on its immunoreactivity in GMR-hid eye discs (e) and GMR-hid eye discs mutant for dcp-1 and drICE (f). Similar results were obtained for peptides C and D (data not shown).
Figure 3
Figure 3. Expression of a ΔN-dcp-1 transgene in ark mutant clones partially restores cleaved-Caspase-3 immunoreactivity
(a, a’, b, b’) GMR-hid eye discs containing ark mutant clones were labeled with cleaved-Caspase-3 antibody (a, a’) and TUNEL (b, b’). ark mutant clones are marked by absence of GFP. A few clonal boundaries are indicated by stippled lines. Both the cleaved-Caspase-3 and TUNEL signals are lost in ark clones. a’ and b’ are the cleaved-Caspase-3 and TUNEL channels only. Genotype: GMR-hid ey-FLP; FRT42D arkG8 / FRT42D P[ubi-GFP]. (c, c’, d, d’) GMR-ΔN-dcp-1 eye discs containing ark mutant clones were labeled with cleaved-Caspase-3 antibody (c, c’) and TUNEL (d, d’). ark mutant clones are marked by absence of GFP. In ark+ tissue, marked by GFP (green), cleaved-Caspase-3 and TUNEL signals are easily detectable. In ark mutant clones (see outline of clonal boundaries by stippled lines), the number of both cleaved-Caspase-3- and TUNEL-positive cells is reduced, but a few are present (arrows). c’ and d’ are the cleaved-Caspase-3 and TUNEL channels only. Genotype: ey-FLP; FRT42D arkG8 / FRT42D P[ubi-GFP] ; GMR-ΔN-dcp-1.
Figure 4
Figure 4. Specificity of the cleaved-Caspase-3 antibody
HID-mediated release of DRONC from DIAP1-inhibition triggers formation of the apoptosome through association with ARK. After DRONC cleavage, the apoptotic substrates DRICE and DCP-1 form a heterotetramer, exposing the ETD epitope at the C-terminus of the large subunits of these proteins. In addition, DRONC has at least one additional substrate which likely exposes an ETD epitope after cleavage. This substrate is unknown and may mediate non-apoptotic functions of DRONC. The cleaved-Caspase-3 antibody (Y shape) detects the exposed ETD epitope of cleaved DCP-1, DRICE and the unknown DRONC substrate.
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