Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark -organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of 'autophagic cell death', dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a 'killing event' and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.KEY WORDS: Autophagy, Apoptosis, Drosophila, Histolysis, dark Development 133, 1457Development 133, -1465Development 133, (2006 DEVELOPMENT 1458 per se is not the mechanism of cell killing but lies upstream, or parallel to dark. These data establish that common effector pathways, regulated by the apoptosome, specify apoptotic and histolytic forms of PCD. MATERIALS AND METHODS MutagenesisTo isolate deletions that eliminate dark without compromising the function of adjacent neighboring genes, a P insertion associated with dark CD4 (Rodriguez et al., 1999) was remobilized and candidates were tested in trans to existing alleles and against lethal mutations in flanking genes. Promising 'hits' were screened by PCR. dark 82 failed to complement dark CD4 , but complements adjacent lethal alleles in the neighboring genes, RhoGEF and a new lethal P mutation in CG8963 that we fortuitously obtained in our first round of mutagenesis. Genomic PCR across the deletion junction and RT-PCR were used to validate the mutation and define the dark 82 lesion. yw was the parental wild-type strain for molecular analysis and for ex vivo hemocyte studies. RNA extraction and QRT-PCR were conducted as described by Gorski et al. (Gorski et al., 2003). Ages at 25°C were normalized from 18°C (Park et al., 1996). Genomic PCR and RT-PCR were performed as described by Chew et al. (Chew et al., 2004), with relevant gene-specific primers. Transgenic 'rescue' and genetic manipulationFull-length dark with 8ϫHis-tags at the N terminus and 3ϫMyc-tags at the C terminus was cloned into the BamHI/XhoI sites of the pFastBac1 vector (Invitrogen). The BamHI/XhoI insert was then subcloned into the pUAST vector to produce pUAST-dark WT . pUAST-dark V was generated by changing Aspartate 1292 to Alanine using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). The ...

The Drosophila malate dehydrogenase, or malic enzyme (ME) encoded by the Men gene, is a non-mitochondrial enzyme recovered in the cytosolic fraction. By using mutation in the Men gene and deficiencies uncovering this locus, we could show that the ME activity recovered in cytosolic fractions originates exclusively from the Men gene located at map position 87D-1 on the right arm of the 3rd chromosome. We found that juvenile hormone (JH) can induce ME activity by two mechanisms. One mechanism corresponds to a direct effect of JH on the enzyme, whose activity was enhanced by a twofold factor in the absence of transcription and translation. This enhancement can be noticed 1 h after JH treatment and lasts for approx 3-4 h. The other mechanism involves the transcription of the MEN gene. In the absence of inhibitors the induction of ME activity by JH is increased by a three to fourfold factor and extends over a period of 10-16 h. Since induction of ME activity by JH and JH analogs displayed a dose-response curve, specific for each tested component, we concluded that the hormonal action could be mediated through a receptor. The use of two temperature sensitive mutations deficient in the production of ecdysteroid, ecd1 and su(f)ts67g revealed that ME response to JH requires the presence of a minimal level of the steroid hormone ecdysone, showing a complex hormonal regulatory circuit in the execution of the JH response.