Model of mitochondrial repopulation required for cellular recovery from MOMP. Following an apoptotic stimulus, the majority of mitochondria within a cell undergo MOMP. However, MOMP can be incomplete; some mitochondria remain intact due to increased levels of anti-apoptotic Bcl-2 proteins on their outer membranes. Over time, these intact mitochondria repopulate the cell, enabling cell survival and proliferation under conditions where caspase function is inhibited.

Incomplete MOMP is cell-type and caspase independent. (A) MCF-7 cells expressing Smac-GFP were treated with UV (18mJ/cm²) plus Q-VD-OPh (20μM) or TNF (10ng/ml) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs pre- and post-MOMP are shown. (B) MCF-7 cells expressing Smac-GFP and matrix-targeted mCherry were treated with ActD (1μM) plus Q-VD-OPh (20μM) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs pre- and post-MOMP are shown. Line scans indicate co-localization of Smac-GFP and mito dsRed and correlate to the line drawn in the images. (C) MCF-7 cells expressing Smac-GFP and Omi-mCherry were treated with ActD (1μM) plus Q-VD-OPh (20μM) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs pre- and post-MOMP are shown and arrows denote cells undergoing iMOMP. (D) HeLa cells expressing Smac-GFP were treated with TNF (10ng/ml) in the presence of propidium iodide (1μg/ml) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs pre- and post-MOMP are shown. Scale bars represent 10μm. See also Figure S2.

Bax and Bak fail to be activated on non-permeabilized mitochondria. (A) HeLa cells expressing GFP-Bax and Omi-mCherry were treated with UV (18mJ/cm²) plus Q-VD-OPh (20μM) and imaged every 10 minutes by live-cell microscopy. Representative confocal micrographs covering the onset and duration of MOMP are shown. The time of each frame relative to the start of image acquisition is shown. (B) Bax/Bak DKO MEFs expressing GFP-Bax and Omi-mCherry were untreated or treated with UV (18mJ/cm²) or ActD (1μM) plus Q-VD-OPh (20μM). Representative confocal micrographs are shown. Arrows denote intact mitochondria that fail to have GFP-Bax in close proximity. (C) Bax/Bak DKO MEFs expressing GFP-Bak and Omi-mChery were untreated or treated with UV (18mJ/cm²) or ActD (1μM) plus Q-VD-OPh (20μM). Representative confocal micrographs are shown. Arrows denote intact mitochondria. Line scans indicate co-localization of Omi-mCherry and GFP-Bak and correlate to the line drawn in the images. Scale bars represent 10μm.

Characterization of iMOMP (A) HeLa cells expressing Smac-GFP were treated with STS (1μM) and Q-VD-OPh (20μM). At various times post-treatment cells were incubated with TMRE (50nM) and imaged by confocal microscopy. Representative images are shown and arrows denote intact mitochondria. (B) MCF-7 cells expressing Smac-GFP were treated with UV (18mJ/cm²) plus Q-VD-OPh (20μM) and imaged every 20 minutes by live-cell confocal microscopy. Numbered arrows denote the same cell at different time-points. Blue arrow denotes cell that undergoes MOMP at a late time-point. The time of each frame relative to the start of image acquisition is shown. (C) Magnified panels from experiment described in Figure 3B. The time of each frame relative to the start of image acquisition is shown. (D) Left: HeLa cells were transfected with control or Drp-1 targeting shRNA vectors. Four days post-transfection and selection, cells were probed for Drp-1 levels by Western blot, ns denotes non-specific protein used as loading control. Right: HeLa or HeLa Drp-1 knockdown cells expressing mito dsRed were analyzed by confocal microscopy, representative micrographs are shown. (E) HeLa cells expressing mito-dsRed and Smac-GFP treated with DMSO or FCCP (50μM) and Drp-1 knockdown or control cells expressing mito-dsRed and Smac-GFP were treated with STS (1μM) and Q-VD-OPh (20μM). Following overnight treatment, cells were assessed. Scale bars represent 10μm. See also Figure S3.

Anti-apoptotic Bcl-2 proteins promote iMOMP. (A) HeLa cells expressing GFP-Bcl-2 and Omi-mCherry were untreated or treated with UV (18mJ/cm²) plus Q-VD-OPh (20μM). Representative confocal micrographs are shown. Line scans indicate co-localization of Omi-mCherry and GFP-Bcl-2 and correlate to the line drawn in the images. (B) HeLa cells expressing Smac 1-60-mCherry/Cerulean and HeLa cells expressing Bcl-2 and Smac-YFP were fused, treated overnight with UV (18mJ/cm²) plus Q-VD-OPh (20μM) and imaged by live-cell microscopy. Representative confocal micrographs pre- and post-MOMP are shown. Arrow denotes intact mitochondria (C) MCF-7 cells expressing Smac-GFP were treated overnight with ActD (1μM) plus Q-VD-OPh (20μM) then treated the following day with enantiomer or ABT-737 (both 10μM) and imaged every 10 minutes by live-cell confocal microscopy. The time of each frame relative to the start of image acquisition is shown and arrows denote cells undergoing iMOMP (D) HeLa cells expressing Smac-GFP were treated for 16 hours with ABT-737 or enantiomer (both 1μM) then treated for 16 hours with STS (1μM) plus Q-VD-OPh (20μM) or (18mJ/cm²) plus Q-VD-OPh. The percentage of cells displaying iMOMP as a percentage of cells displaying MOMP is shown from three independent experiments (minimum 100 cells counted), error bars represent the S.D. (E) HeLa cells expressing GAPDH and Smac-GFP were treated with STS (0.5 or 1μM) +/− Q-VD-OPh (20μM) for 16 hours. Where indicated, prior to or after STS treatment, ABT-737 or enantiomer was added (1μM) for 16 hours. As indicated, Q-VD-OPh was replenished for a further 3 days and cells were assessed for clonogenic outgrowth. As a control, HeLa cells expressing GAPDH and Smac-GFP were cultured +/− ABT-737 (1μM) for three days. Colony number from three independent experiments +/− S.D. is shown. Scale bars represent 10μm. See also Figure S5.

Incomplete mitochondrial outer membrane permeabilization during apoptosis. (A) HeLa cells expressing Omi-mCherry, Smac-GFP or cytochrome c-GFP were treated with STS (1μM) plus Q-VD-OPh (20μM) and imaged every 5 minutes. Regions were drawn around cells, punctate/diffuse and brightness indices were plotted and displayed relative to the onset of MOMP (time point 0). Data was scaled and represents the average of 15 cells, error bars represent S.E.M. (B) HeLa cells expressing Smac-GFP were treated with STS (1μM) or ActD (1μM) plus Q-VD-OPh (20μM) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs covering the onset and duration of MOMP are shown. The time of each frame relative to the start of image acquisition is shown. Arrows denote cells undergoing iMOMP. (C) HeLa cells expressing Smac-GFP were treated for 16 hours with STS (1μM), ActD (1μM) or UV (18mJ/cm²) plus Q-VD-OPh (20μM) and quantified for the occurrence of iMOMP. The percentage cells displaying iMOMP as a percentage of cells that had undergone MOMP (minimum 100 cells counted) is shown and error bars represent the S.D. from 3 independent experiments. (D) HeLa cells expressing Smac-GFP and mito dsRed were treated with UV (18mJ/cm²) plus Q-VD-OPh (20μM) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs covering the onset and duration of MOMP are shown. Arrows denote cells undergoing iMOMP. Line scans indicate co-localization of Smac-GFP and mito-dsRed and correlate to the line drawn in the images (E) HeLa cells expressing Smac-GFP and Omi-mCherry were treated with ActD (1μM) plus Q-VD-OPh (20μM) and imaged every 10 minutes by live-cell confocal microscopy. Representative confocal micrographs pre- and post-MOMP are shown. Arrows denote cells undergoing iMOMP. Line scans indicate co-localization of Smac-GFP and Omi-mCherry and correlate to the lines drawn in the images (F) HeLa cells expressing Smac-GFP were treated for 16 hours with UV (18mJ/cm²) plus Q-VD-OPh (20μM), fixed and immunostained with anti-cytochrome c antibody. Representative confocal micrographs pre- and post-MOMP are shown. Line scans indicate co-localization of Smac-GFP and cytochrome c and correlate to the line drawn in the images. Scale bars represent 10μm. See also Figure S1.

Incomplete MOMP correlates with cellular recovery from CICD. (A) Left/center: representative confocal micrographs from single-cell cultures showing complete (left) and incomplete (center) MOMP in HeLa cells expressing GAPDH and Smac-GFP following 16 hours treatment with STS (1μM) plus Q-VD-OPh (20μM) Right: widefield micrograph of a proliferating colony 14 days later. Inset numbers refer to the number of proliferating colonies that arose/the number of cells analyzed that display either complete or incomplete MOMP. The difference between groups was assessed by Chi-square analysis (p<.001). (B) Schematic experimental outline. Hela cells expressing Smac-GFP following STS treatment leading to complete MOMP will display less relative fluorescence than those displaying iMOMP. These cell populations are sorted by flow cytometry and assessed for their ability to exhibit clonogenic survival (C) HeLa cells expressing Smac-GFP were treated for 16 hours with STS (0.5μM-4μM) plus Q-VD-OPh (20μM) and analyzed by flow cytometry for GFP fluorescence intensity (inset, MFI-mean fluorescence intensity). Gates denote populations covering 20% lowest and highest Smac-GFP fluorescent populations that were sorted. Quantification of Smac-GFP MFI of STS plus Q-VD-OPh treated cells from three independent experiments expressed as percentage relative to control untreated cells, error bars represent the S.D. (D) HeLa cells expressing Smac-GFP were treated for 16 hours with STS (0.5μM-4μM) plus Q-VD-OPh (20μM) and sorted into the 20% highest and lowest GFP expressing cells and analyzed by microscopy for iMOMP. Number of cells displaying iMOMP as a percentage of cells displaying MOMP is shown, a minimum 100 cells per condition were counted. Results are from three independent experiments +/− S.D. (E) HeLa cells expressing GAPDH and Smac-GFP were treated for 16 hours with STS (0.5μM-4μM) plus Q-VD-OPh (20μM) and sorted into 20% highest and lowest GFP expressing populations. 2 × 10⁵ cells from each population were plated down in the presence of Q-VD-OPh (20μM) and examined for clonogenic outgrowth. As a control, 1 × 10⁶ cells were treated with STS (0.5μM-4μM) for 16 hours and examined for clonogenic outgrowth (middle panel). Colony number from three independent experiments, error bars represent the S.D. Scale bars represent 10μm. See also Figure S4.