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to investigate the role of dmfn in the interaction between reaper and bax, dmfn was overexpressed in hela cells. cells were transfected with vector control, myc-dmfn or myc-dmfn-n and then treated with bax or reaper. cells were then fixed and stained with anti-bax or anti- reaper antibodies. bax and reaper are shown in green and cytochrome c is shown in red in the merged images. arrows point to cytochrome c punctae.treatment with caspase inhibitors can block diap-1 ubiquitylation and reaper-induced cytochrome c release, but not apoptosis, in drosophila cells. 2, 23 although caspase inhibitors can block apoptosis in vertebrate cells, they have been shown to induce mitochondrial fission, and drp1 has been shown to promote mitochondrial fission in mammalian cells. 26, 27 to examine the role of mitochondrial fission in diap-1 ubiquitylation and apoptosis, we used a dominant negative drp1 mutant, drp1k38a. 27 to assess the role of drp1 in reaper-induced apoptosis, we co-transfected hek293 cells with flag-reaper or flag-reapergh3 and a plasmid expressing either full-length drp1, drp1k38a or empty vector as control. cells were treated with cycloheximide (chx) to block translation, or etoposide to induce apoptosis. as shown in figure 9, overexpression of drp1k38a blocked reaper-induced apoptosis in cells that were treated with chx but not in cells treated with etoposide. the cell death induced by etoposide was also completely blocked by expression of drp1k38a. as shown in figure 9b, the induction of diap-1 ubiquitylation by reaper was completely blocked by drp1k38a. these data suggest that reaper can induce mitochondrial fragmentation, which in turn inhibits mitochondrial fusion. the inhibition of mitochondrial fusion by reaper can block diap-1 ubiquitylation, which leads to cytochrome c release and apoptosis.
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