BAX/BAK Independent Cell Death Figure 5. BH3 Peptides do not induce release of cytochrome c in Bax2/2/Bak2/2 MEFs. (A and B) Wild type and Bax2/2/Bak2/2 MEFs were treated with DMSO, Mutant BID BH3 peptide (100 mM), BID BH3 peptide (100 mM) or BIM BH3 peptide (100 mM) for 1 hour. Cytochrome c localization was assessed in the mitochondrial and cytosolic fractions by Western Blot analysis. Cytochrome c oxidase subunit 1 (COX-1) antibody was used as a control to ensure proper fractionation and loading of mitochondrial pellet. doi:10.1371/journal.pone.0005646.g005 BH3 peptides induce an increase in BCL-XL binding to dynamin-related protein 1 (DRP1) Previous published findings showed that the pro-survival protein BCL-XL interacts with the fission machinery protein DRP1 and that this interaction increases the GTPase activity of DRP1 [27]. We therefore investigated whether BCL-XL and DRP1 interact upon treatment with BH3 peptides in Bax2/2/Bak2/2 MEFs. Bax2/2/ Bak2/2 MEFs overexpressing FLAG-tagged BCL-XL were treated with peptides, followed by immunoprecipiation with a FLAG antibody and immunoblotted for DRP1 (Figure 10A). Treatment with the BID BH3 peptide caused a twofold increase in the interaction between DRP-1 and BCL-XL compared to cells treated with the mutant BID BH3 peptide (Figure 10B). To test whether DRP1 is required for the BH3 peptide induced cell death, Bax2/2/ Bak2/2 MEFs were infected with a retrovirus containing a dominant negative DRP1 (K38A). BIM or BID BH3 peptide induced cell death in Bax2/2/Bak2/2 MEFs expressing the dominant negative DRP1 (Figure 10C). The dominant negative DRP1 also failed to prevent staurosporine induced cell death in wild-type MEFs (Figure 10D). Dominant negative DRP-1 also did not prevent mitochondrial fission upon treatment with BH3 peptides (data not shown). Furthermore, the small molecule inhibitor of DRP1, mdivi-1, also did not inhibit cell death in the presence of BIM BH3 peptides (Figure 10E). However, Mdivi-1 did partially inhibit staurosporine induced cell death in wild-type MEFs (Figure 10F). These results suggest that BH3 peptides bind to pro-survival BCL-2 proteins to engage the DRP-1 dependent fission machinery in the absence of BAX and BAK. However, the DRP1 induced fission is not required for BH3 peptide induced cell death. There might be other yet unidentified regulators of fission machinery that might participate in the BH3 peptide induction of cell death. Discussion The BCL-2 protein BAX and BAK induce the mitochondrial outer membrane permeabilization (MOMP) for the release of cytochrome c. Current models indicate that the BH3 only proteins, a subset of the BCL-2 family proteins, are initiators of BAX/BAK dependent MOMP. How BAX and BAK are activated to induce MOMP remains controversial. One model states that a subset of BH3-only proteins bind pro-survival BCL-2 proteins to release other BH3 only proteins that can directly activate BAX/BAK [21,28]. By contrast, another model states that binding of all pro-survival BCL-2 family proteins by BH3 only proteins is sufficient to activate BAX/BAK [20,29]. A common feature of the two models is that negation of pro-survival BCL-2 family members is required for BAX/BAK to induce MOMP. Here we demonstrate that cell death can be induced in the absence of the multi BH3 proteins BAX and BAK, upon treatment with peptides corresponding to the BH3 domain of BH3 only pro- apoptotic proteins. Previous studies have characterized that the BH3 peptides indeed bind to pro-survival proteins with strong affinities and specificities. Our data indicates that the BH3 peptides can induce cell death in the combined absence of BAX and BAK through binding of all the pro-survival proteins. Our observation is reminiscent of the death process observed in C. elegans, whereby the neutralization of the pro-survival protein CED-9 by the BH3 only protein EGL-1 can induce both mitochondrial fission and cell death [15]. C. elegans do not contain BAX or BAK. A recent report indicates that mitochondrial fragmentation and cell death are distinct events in C. elegans [17]. In mammalian cells where BAX and BAK are present, the neutralization of pro-survival proteins by BH3 only proteins triggers rapid activation of BAK and BAK to induce MOMP. However, in mammalian cells deficient for BAX and BAK, we can unmask the evolutionarily conserved mechanism of cell death, in which BH3 only proteins bind pro-survival BCL-2 proteins to initiate both mitochondrial fission and cell death in the absence of BAX and BAK. Although our findings seem to be in contradiction with previous data that indicate that Bax2/2/Bak2/2 MEFs do not undergo cell PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5646