Drosophila adPEO Mutations Figure 6. Apoptosis and proliferation levels in wing imaginal discs from third instar larvae. (A–J) Overexpression of the different versions of d-mtDNA helicase with the daughterless-Gal4 driver in wing imaginal discs. (A–E) Monoclonal anti-caspase 3 antibody was used for immunocytochemistry to assess apoptotic levels in lines expressing d-mtDNA helicase. (F–J) Monoclonal anti-phosphohistone 3 antibody was used for immunocytochemistry to evaluate cell proliferation levels in lines expressing d-mtDNA helicase. (K–L) Quantification of apoptosis and cell proliferation levels using a ubiquitous and constitutive driver (da-GAL4). Data represent the mean 6 s.d., *P#0.05, **P#0.005 as compared with controls. (M–P) Overexpression of d-mtDNA helicase with the engrailed (en-GAL4) driver (posterior compartment, P) probed by staining with anti- caspase 3 antibody in third instar larvae’s wing imaginal discs. Overexpression of the K388A mutant shows an increase in apoptosis clusters when compared with the anterior, the control compartment. (M) w;en-GAL4;UAS helicaseWT; (N) w;en-GAL4;UAS helicaseK388A; (O) w;en-GAL4;UAS helicaseW441C; (P) w;en-GAL4;UAS helicaseA442P. doi:10.1371/journal.pone.0043954.g006 apoptosis pathway in mammals [17]. Even though nearly all of the proteins involved in cell death pathways are conserved throughout metazoan evolution, the importance of mitochondria in the Drosophila apoptotic pathway is both a controversial and an active field of research [18,19,20]. Drosophila lacking one of the d-cyt-c genes was reported to exhibit severely delayed apoptosis in the developing retina [43], and mutations in d-cyt-c were found to have reduced caspase activation during spermatid individualization [44]. These and other findings have led to the hypothesis that alterations in mitochondrial morphology might lead to formation of a cytochrome c-dependent apoptosome at the mitochondrial surface as part of a feed-forward signal in caspase activation [44,45]. Although the mechanism remains unclear, the results obtained here and in our previous studies [23,42] allow us to conclude that mitochondrial dysfunction associated with defects in replication and expression of mtDNA induces apoptosis, arguing that an intrinsic mitochondrially-mediated apoptotic pathway is active in Drosophila, and offering the possibility to explore its role in pathophysiological processes. PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43954