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Fig. 3. Alteration of a caspase cleavage site produces a hypermorphic Dark variant. (A) Recombinant Dark protein (lane 1) was incubated with cytosolic S20 fractions prepared from control S2 cells (lane 2) or cycloheximide (CHX)-treated S2 cells (lane 3). Asterisk denotes the small Dark C-terminal fragment after cleavage. (B) Consistent with in vitro studies (A), stimulus-dependent cleavage of Dark is detected here in Drosophila S2 cells. Samples from unchallenged (Ctrl) S2 cells or cells treated with 20 µM Cycloheximide (CHX) or 200 mJ/cm² UV were harvested after 4 hours. (C) Cleavage of Dark as seen in panel B with CHX treatment, is reversed by the caspase inhibitor z-VAD (100 µM), shown here 5 hours post-treatment. In A,B and C, Dark was visualized with an anti-Dark polyclonal antibody. (D) The cleavage site, detected in vitro at residue 1292, is shown (arrow) in the schematized domain structure of the Dark protein. (E) Illustration of the defective anatomy of dark⁸² flies rescued by leaky expression of UAS-dark^V, which mutates the caspase site mapped in D. The notum of a dark⁸² homozygote rescued to viability by UAS-dark^V, shown here next to a wild-type fly notum (left), exhibits a `split thorax' phenotype and bristle abnormalities. (F) Levels of transgenic Dark protein in various UAS-dark transgenic lines in the absence of any driver or under Tubulin-Gal4 were examined by immunoblot using an anti-Myc antibody. Arrowhead denotes Dark-myc; asterisk indicates an irrelevant cross-reacting band showing equal loading on each lane. Note that the levels of wild-type Dark and Dark^V are comparable when expressed from the Tubulin-Gal4 driver or when examined for basal expression. (G) Hemocyte aspirates from dark⁸²; Hml-Gal4:UAS-dark^WT (Hml:dark^WT) and dark⁸²; Hml-Gal4:UAS-dark^V (Hml:dark^V) L3 larvae were treated with DMSO or the Smac mimetic (Li et al., 2004), a potent apoptotic inducer. Expression of UAS-dark^WT in dark⁸² hemocytes only mildly restored apoptosis after Smac mimetic treatment. However, UAS-dark^V almost completely restored this apoptotic response to dark⁸² hemocytes.

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