accumulation of fragmented dysfunctional mitochondria [33]. Indeed, we observed that following BH3 peptides treatment, the mitochondrial of Bax2/2/Bak2/2 MEFs undergo fission as assessed by staining mitochondria with a fluorescent dye or electron microscopy. Thus, we propose that BH3 peptides induce mitochondrial fission resulting in mitochondrial membrane depolarization in the absence of BAX/BAK. The role of mitochondrial fission during the process of cell death still remains unclear [14]. An existing idea is that fragmentation of the mitochondria is important for the release of apoptogenic factors such as cytochrome c to induce cell death [7,8,34]. This is further supported by the observation that OPA-1 mediated cristae remodeling is required for efficient cytochrome c release. Our data indicate that the mitochondria can undergo fission without the release of cytochrome c, indicating that the two processes are discrete events. This observation is consistent with a recent study by Sheridan et al demonstrating that BH3 proteins can induce mitochondrial fragmentation without cytochrome c release in the presence of BAX/BAK inhibition by a pro-survival protein [12]. The mechanism responsible for the activation of mitochondrial fission and its regulation is still not fully understood [4]. However, it is appreciated that the activation of the fission protein DRP1 is increased under apoptotic conditions [6,7]. The fact that BAX and BAK deficient cells can still undergo fission [11] suggests that the BAX and BAK are dispensable for initiating mitochondrial fission. By contrast, BAX and BAK proteins play important roles in the normal morphogenesis of the mitochondria by activating assembly of the fusion GTPase, Mfn2, thereby promoting fusion of the mitochondria [35]. However, we did not find that loss of BAX and BAK altered mitochondrial morphology or dynamics under normal cell culture conditions (Movie S1) compared with wild-type cells (Movie S2). Bid BH3 peptide altered mitochondrial morphology and dynamics (Movies S3) compared to mutant BH3 peptide (Movie S3 and Movie S4). Interestingly, we could not rescue mitochondrial fission by a dominant negative Drp1 or mdivi-1, a pharmacological inhibitor of mitochondrial fission. Since the process of mitochondrial fission is not fully understood [14], we cannot exclude the possibility that unidentified regulator(s) of fission might be responsible for cell death in the absence of BAX/BAK in the presence of BH3 peptides. Much of the previous work has primarily focused on the regulation of mitochondrial fission by BAX and BAK and not by the pro-survival BCL-2 proteins. A recent study did demonstrate that the pro-survival protein BCL-XL binds to DRP1 which leads to an increase in the GTPase activity of DRP1 [27]. We also observed that DRP1 coimmunoprecipitates with BCL-XL. Moreover, this interaction between the two proteins was increased when BH3 peptide was added to the cells. Our current findings, however, does not distinguish whether the BH3 peptide binding to pro-survival proteins is permissive for mitochondrial fission to occur, or whether the BH3 binding to pro-survival protein directly promotes mitochondrial fission. The physiological implication of our findings presently remains unknown. The BAX and BAK deficient animals are not embryonic lethal and do not display severe developmental defects [36] when compared to BCL-XL, MCl-1 or caspase-9, deficient animals which all display severe developmental defects [37–39]. The mechanism by which cells undergo developmental cell death in the absence of BAX and BAK remains unknown. But it suggests that there are other death mechanisms initiated during develop- ment in the absence of BAX and BAK to obtain viable mice. We speculate that BH3 proteins engage prosurvival BCL-2 proteins to trigger mitochondrial fission and cell death in the absence of BAX and BAK during development. The mitochondrial fission results in dysfunctional mitochondria which could trigger cell death in the BAX/BAK Independent Cell Death absence of BAX/BAK. In summary our data provide evidence that pro-survival proteins can regulate mitochondrial fission and death in the absence of BAX and BAK. Methods Cell lines Wild type and Bax2/2/Bak2/2 mouse embryonic fibroblasts were kindly provided by Dr. Craig Thompson. Wild type and Bax2/2/Bak2/2 Baby mouse kidney epithelial cells were kindly provided by Dr. Eileen White. Cells were cultured in Dulbecco’s modified essential media (DMEM), supplemented with 10% heat- inactivated Fetal Bovine Serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 20 mM Hepes. All cell culture reagents were purchased from GIBCO. Peptides Peptides containing 8 D-arginine were synthesized by Tufts University Core Facility and purified by HPLC. The N-terminus and C-terminus of the peptides were blocked by an acetyl and amide group respectively. Peptide sequences are the following: BID BH3: EDIIRNIARHLAQVGDSMDR, Mutant BID BH3: EDIIRNIARHAAQVGASMDR, BIM BH3: MRPEIWIAQELRRIGDEFNA, BAD BH3: LWAAQRYGRELRRMSDEFEGSFKGL, Mutant BAD BH3: LWAAQRYGREARRMSAEFEGSFK- GL, NOXA A BH3: AELPPEFAAQLRKIGDKVYC, NOXA B BH3: PADLKDECAQLRRIGDKVNL Measurement of cell death Cell death was assessed by the release of lactate dehydrogenase (LDH) into the surrounding medium using a cytotoxicity detection kit from Roche Applied Science. Percentage of cell death was calculated by the amount of LDH released in the medium, divided by the total LDH released after treatment of cells with 1% Triton X-100. Flow cytometry was also used to detect PI positive cells stained according to manufacture’s protocol (BD Biosciences). Immunoblot Analysis Protein expression was analyzed in total cell by lysing cells with 16 cell lysis buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride. Protein concentration was deter- mined using the Bio-Rad protein assay. 50 mg of total cell lysate were resolved on a 10% or 12% sodium dodecyl sulfate- polyacrylamide gel (Bio-Rad) and transferred to a Hybond-ECL nitrocellulose membrane (Amersham). Membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 buffer. Primary antibodies used were Bok antibody (Cell signaling), Bcl-XL antibody (Santa cruz), DRP1 antibody (H-300, sc 32898), Flag antibody (Sigma), Cytochrome c antibody (Mitosciences), COX-1 antibody (BD Pharmingen) and alpha-tubulin antibody (Sigma clone B-5-1-2) at 1:2,000. Secondary antibodies used were horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signaling) 1:1000. SuperSignal chemiluminescent substrate (Pierce) was used to develop the blot. A representative blot is shown above of three independent experiments. Live cell imaging Cytochrome c release was monitored using cytochrome c tagged with GFP. Mitochondrial membrane depolarization was assessed PLoS ONE | www.plosone.org 13 May 2009 | Volume 4 | Issue 5 | e5646