Fig 1.
BAX fusion proteins parallel spatial and temporal localization patterns of endogenous BAX.
D407 cells were nucleofected with mito-BFP alone or with GFP-BAX or mCherry-BAX. Cells were treated 24 hours later with 1μM staurosporine (STS) for 1, 2, or 3 hours or DMSO control. (A-C) Endogenous activated BAX was monitored using the BAX 6A7 antibody. In DMSO only treated cells (shown after 3 hour incubation) the 6A7 antibody does not cross-react with BAX. Punctate labeling, co-localized to mitochondria, is detected within 1 hour of STS addition. (D-F) Exogenous GFP-BAX and (G-I) mCherry-BAX demonstrate similar localization changes. Size bar = 5 μm. Three independent experiments were quantified. (J) The percentage of punctate cells (number of punctate cells over the total number of nucleofected cells for cells expressing BAX fusion proteins) increased similarly for each time point among all BAX conditions (p > 0.05).
Fig 2.
Quantitation of BAX recruitment.
(A) Still images from a time-lapse video show a D407 cell expressing mCherry-BAX and a mitochondrial targeted protein, mito-BFP (pseudo-colored teal). The time stamp indicates minutes after the addition of 1 μM staurosporine to induce apoptosis. (B, C) Image analysis software (IMARIS) allows for three-dimensional visualization of identified ‘foci’ within a cell (blue spheres = mitochondrial foci) in the XY (B) or the Z plane (C). The movements of mitochondria are tracked through the Z-stack over time (direction is indicated by red and green lines). (D) A graph of the three mitochondrial foci highlighted in yellow in (B) and (C), demonstrates the accumulation of fluorescent BAX protein at the foci defined by the mito-BFP marker. Raw data is fitted with a sigmoid function. (E) From the sigmoid curve, principal metrics used for analysis can be calculated, including the time of BAX recruitment initiation, the rate of BAX recruitment, and time of BAX recruitment completion.
Fig 3.
Effects of fluorescent tags and endogenous protein on BAX recruitment kinetics.
(A) To determine the effect of fluorescent tags on BAX recruitment rates, the rate of BAX recruitment was calculated for two populations of mitochondria from D407 cells, expressing either mCherry-BAX (red curve) or GFP-BAX (blue curve). A graph depicts the outline of the frequency plot for each population of mitochondria (n = 680 and 727 mitochondria for GFP-BAX or mCherry-BAX, respectively). GFP-BAX recruitment exhibited a statistically faster rate of recruitment than mCherry-BAX (p < 0.05). (B-E) To determine the effect of endogenous BAX protein on GFP-BAX recruitment rates, two populations of mitochondria were compared from wild type HCT116 and HCT116BAX-/-/BAK-/- cells. Box and whisker plots compare the (B) average initiation time for each population (p < 0.001) and (C) the average rate of BAX recruitment. There was no difference in recruitment rates between wild type HCT116 and HCT116BAX-/-/BAK-/- cells. The average rates and initiation times were derived from the population of mitochondria shown in (D and E). Each individual marker represents one mitochondrial focus. Red and yellow markers identify two separate cells from each population. Data from foci originating from the same cell are vertically stacked indicating nearly identical times for the initiation of BAX recruitment within a cell, but that recruitment occurs at different rates at individual foci (n = 424 and 132 mitochondrial foci analyzed for HCT116 and HCT116BAX-/-/BAK-/- cells, respectively).
Fig 4.
Initiation of BAX recruitment and BAX recruitment rates in three different cell types.
(A) GFP-BAX recruitment kinetics were quantified for three different cell types; D407, HeLa, and HCT116. All cells were wild type for endogenous BAX and BAK proteins. Apoptosis was induced by exposure to 1 μM staurosporine (STS). Times given represent the minutes elapsed after STS addition. (A-C) A scatterplot for each cell type shows the time of BAX recruitment initiation on the X-axis and BAX recruitment rate on the Y-axis. Each circular marker represents individual mitochondrial foci from the population (n = 680, 156, 336 mitochondrial foci for D407, HeLa, HCT116, respectively). For each cell type, red and yellow markers show all the mitochondria from two individual cells, demonstrating synchronous recruitment initiation times, but stratified recruitment rates. (D-F) Frequency plots for each cell type (D407, HeLa, HCT116, respectively) show the varied time of BAX recruitment initiation for each population. Each cell type exhibits a distribution of initiation times distinctly different from each other (p < 0.001). (G) A box and whisker plot shows the BAX initiation times for D407, HeLa, and HCT116 cell populations. Each population is significantly different from each other (p < 0.001). (H) A box and whisker plot shows the BAX recruitment rates for D407, HeLa and HCT116 cell populations. HeLa cells had a significantly faster average rate of recruitment compared to either condition (*p < 0.001).
Fig 5.
Coefficient of variation analysis.
The coefficient of variation describes the variability for each metric within an individual cell (n = 41, 24, 42 cells for D407, HeLa, and HCT116, respectively). This statistic is normalized to a percentage of the means, which allows for direct comparison between different metrics. Each cell is represented by a single red and blue point. The average coefficients of variation of all cells within a cell type for BAX recruitment rates were 57%, 150%, and 67% for D407, HeLa, and HCT116, respectively. The average coefficient of variation of all cells within a cell type for the initiation of BAX recruitment were 2%, 5%, and 1% for D407, HeLa, and HCT116 respectively, with a statistical difference between D407 cells when compared to HCT116 (p < 0.05) and between HeLa cells when compared to any other cell type (p < 0.001). The mean of each metric for all cells is shown as a solid red or blue line. Comparison between the two metrics for all cells, regardless of cell type, showed a statistical difference (p < 0.001) by the two-tailed covariance test. These data indicate that while BAX recruitment is initiated at unique times in different cell types (Fig 4), once activated, recruitment is initiated relatively synchronously within a given cell. The rate of recruitment within the same cell, however, can vary dramatically. These features appear to be conserved across different cell lines.
Fig 6.
Cytochrome c, SMAC and AIF are released at different times during BAX recruitment.
Time-lapse videos were collected from D407 cells expressing mCherry-BAX, mito-BFP and one of three GFP-tagged apoptotic molecules that are released from mitochondria after being challenged with 1 μM staurosporine. Representative graphs indicate the release of apoptotic molecules (A) cytochrome c-GFP, (E) SMAC-GFP, and (I) AIF-GFP, and the recruitment of mCherry-BAX at one mitochondrial focus. (B-D) Representative images show (B) cytochrome c-GFP localized at the mitochondria (pre-initiation), and (C) diffuse cytochrome c-GFP four minutes after BAX recruitment initiation and (D) 30 minutes after BAX recruitment initiation. (F-H) Images from a time-lapse video of SMAC-GFP and mCherry-BAX (F) pre-initiaton, (G) four minutes and (H) 30 minutes after BAX recruitment initiation, show the same localization patterns as cytochrome c-GFP. (J-L) Images from a time-lapse video of AIF-GFP and mCherry-BAX show (J) AIF-GFP localized at the mitochondria (pre-initiation), (K) AIF-GFP partially retained in the mitochondria four minutes after BAX recruitment initiation and (L) diffuse AIF-GFP 30 mins after BAX recruitment initiation. For all three datasets, the time of molecule release was compared to the time of BAX recruitment initiation. (M) A box and whisker plot shows that cytochrome c-GFP and SMAC-GFP are released -2.5 ± 3 mins and -1.3 ± 1.6 mins before mathematical assignment of the initiation of BAX recruitment, respectively, while AIF is released at 10.3 ± 11 mins after recruitment initiation. Each molecule’s relative release time was significantly different from the others by one-way ANOVA (p** < 0.01; n = 53, 52, 46 mitochondria foci for cytochrome c, SMAC, and AIF, respectively). Cytochrome c and SMAC release times were also significantly different (p* < 0.05). (N) A bar graph showing the rate of apoptotic molecule release for each molecule. Cytochrome c was released at a rate of -0.78 ± 0.46 RFU/min, SMAC at -1.36 ± 0.72 RFU/min and AIF at -0.04 ± 0.07 RFU/min (n = 54, 52, 51 mitochondria foci respectively). All were significantly different (p < 0.001), while SMAC exhibited a significantly faster release rate when compared to the other molecules (*p< 0.001).