Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores

Bax assembles into full and incomplete ring‐like structures at mitochondria of apoptotic cells

To investigate the nanoscale organization of Bax oligomers at the MOM during apoptosis, we used a single‐molecule approach based on image reconstruction by the accurate localization of isolated emitters, called single‐molecule localization microscopy (SMLM). We labeled HeLa cells overexpressing GFP‐Bax with anti‐GFP nanobodies coupled to Alexa Fluor 647 (AF647), a fluorescent probe optimized for super‐resolution imaging (Ries et al, 2012). These nanobodies are smaller (1.5 nm × 2.5 nm) than usual antibodies and bind GFP with high specificity (Rothbauer et al, 2006). In addition, the bright dyes coupled to them are photo‐switchable under specific buffer conditions and allow resolving structures in the 20‐nm range (Ries et al, 2012).

Previous work suggested that in single cells only minimal gathering of Bax is needed to promote mitochondria depolarization and fragmentation (Düssmann et al, 2010) before total translocation of Bax to the MOM is achieved (Zhou & Chang, 2008). We acquired images on fixed cells that had been treated with STS for 2–6 h and qualitatively showed no difference in the number or shape of structures within this range of incubation times with the drug. In particular, the individual cells selected for imaging in the different experiments exhibited a similar apoptotic state, characterized by GFP‐Bax in a dotted distribution, but overall cellular integrity, as judged by the cell shape and the absence of blebs (Figs 2A and EV2). This was important to minimize the background signal of cytosolic Bax because it would interfere with a proper image reconstruction. Therefore, we visualized a specific apoptotic stage corresponding to the organization of Bax signal once almost all molecules are irreversibly bound to mitochondria, but before most cellular components have been disassembled. As the pores induced by Bax are stable and permeabilize mitochondria for long periods of time (Munoz‐Pinedo et al, 2006; Bleicken et al, 2013a), the stage of Bax organization we investigated was associated with the pore‐forming state of Bax assemblies.

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Figure 2

Super‐resolution of Bax by single‐molecule localization microscopy reveals non‐random structures in apoptotic mitochondria

1. Overview of a reconstructed super‐resolution image of GFP‐Bax‐overexpressing HeLa cells stained with AF647‐anti‐GFP nanobodies. Image was acquired on fixed cells 3 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV2). Scale bar, 5 μm.
2. Magnified reconstructed super‐resolution image corresponding to the white rectangle in (A) showing the shapes of GFP‐Bax WT structures (white arrows). Scale bar, 500 nm.
3. Gallery of typical GFP‐Bax WT structures during apoptosis found in all the analyzed cells. Scale bars, 100 nm.
4. Overview (left) and 3 enlarged insets (right) of HeLa cells overexpressing GFP‐Bax 1‐2/L‐6 stained with AF647‐anti‐GFP nanobodies. Figure shows reconstructed super‐resolution images acquired without apoptosis induction. Scale bars, 5 μm (overview) and 500 nm (insets).

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Figure EV2

Cell shape identification in SMLM images

1. Bright field images and SMLM overviews of HeLa and HCT116 DKO cells overexpressing GFP‐Bax or GFP‐Bax 1‐2/L‐6, respectively. Bright field images were taken in the GFP and AF647 channels before acquisition with single‐molecule localization. Scale bars, 500 nm.
2. SMLM images shown in (A) reconstructed with all the localizations and no cutoff applied. Pictures were converted to binary images and a black‐white mask was applied to define the cell shape. Scale bars, 500 nm.

Reconstructed super‐resolution images showed a large fraction of the Bax signal as random dots or big aggregates in which no particular structural organization could be distinguished. But importantly, we also found a significant amount of well‐defined non‐random structures, which seemed to be specific of active Bax on the membrane (Fig 2B). Concretely, we identified both full and incomplete (arc‐shaped) rings of Bax molecules, as well as linear assemblies, which in a few cases appeared as double lines (Fig 2C).

In an effort to validate the connection between these defined structures and Bax role in MOM permeabilization, we examined the signal of inactive Bax at mitochondria under control conditions by SMLM. We acquired super‐resolution images of healthy HeLa cells transfected with the non‐functional mutant GFP‐Bax 1‐2/L‐6 (Fig 2D). Consistent with the lack of activity, we detected a higher proportion of undefined dots and aggregates in GFP‐Bax 1‐2/L‐6 with respect to its wild‐type counterpart (GFP‐Bax WT) (Fig EV3), which were in general smaller. To ensure a systematic, blind analysis, we also classified the non‐random structures detected as lines, arcs, and full rings (we did not detect any double lines). We next calculated different parameters for each structure in order to compare them quantitatively under both conditions. Concretely, we measured: (i) the area of dots and aggregates, (ii) the length of the lines, (iii) the length and the radius of curvature of the arc assemblies, and (iv) the radius of the full rings (Fig 3D).

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Figure EV3

Frequency of Bax assemblies in the cells

Quantification of the frequency of the different types of Bax assemblies in HeLa cells with respect to the total number of assemblies found in each condition (GFP‐Bax WT or GFP‐Bax mutant 1‐2/L‐6). Mean ± SEM of 14 independent cells for each condition.

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Figure 3

Bax wild‐type adopts different structures compared to the inactive mutant Bax 1‐2/L‐6

1. Quantification of the structures found on Bax wild‐type (purple) and on Bax 1‐2/L‐6 (gray) overexpressing cells. Data show the total number of structures in all the measurements. n (Bax wild‐type) = 13, n (Bax 1‐2/L‐6) = 11.
2. Cumulative distribution plots of GFP‐Bax wild‐type (purple) and GFP‐Bax 1‐2/L‐6 (gray) showing differences in amplitude and frequency. Each plot represents a single cell. Thick lines represent average cumulative distributions for all the single cells transfected with GFP‐Bax WT or mutant GFP‐Bax 1‐2/L‐6.

As GFP‐Bax 1‐2/L‐6 is reliant on efficient disulfide‐tethering in the reducing environment of the cytosol, we cannot discard that some of the structures and aggregates observed are due to incomplete linkage despite positive TRMRE staining. Nevertheless, and although the number of clear complete rings of GFP‐Bax WT molecules was lower than that of other structures, they were almost absent in the inactive mutant, which strongly supports their functional character (Figs 3A and EV3). Their radius followed a normal distribution centered on a diameter of 35 nm, in good agreement with Bax pores observed in liposomes (Bleicken et al, 2010). In the case of arc‐shaped assemblies, their length was broadly distributed and two peaks at approximately 100–300 and 400–500 nm could be distinguished for GFP‐Bax WT. Linear structures also followed a wide distribution in the case of GFP‐Bax WT with lengths centered at 200–250 nm. In contrast, the inactive mutant exhibited less and shorter arcs and lines. In agreement with Bax oligomerization during apoptosis, also undefined structures were larger for GFP‐Bax WT than for GFP‐Bax 1‐2/L‐6. In summary, these differences in the signal suggest that the defined structures detected in the case of GFP‐Bax WT are distinct from those of the inactive mutant and confirm that they are related to the functional role of Bax in MOM permeabilization during apoptosis.

Our quantification involved the total number of structures found in all measured cells together, but it is also important to consider cell‐to‐cell heterogeneities. To account for this, single cell cumulative distribution functions for structures including dots, aggregates, and lines were plotted. As shown in Fig 3B, mutant GFP‐Bax 1‐2/L‐6 cells displayed in all cases considerably smaller structures than the active protein. Besides, the cumulative distribution functions of individual cells corresponding to WT or mutant GFP‐Bax were similar and could be distinguished as two different populations. These results confirm that the structures analyzed in the images of wild‐type and mutant GFP‐Bax are significantly different from each other and comparable in the different cells analyzed for each of them.

In the case of GFP‐Bax structures organized as two lines or arcs parallel to each other (Fig 2C), we cannot discard their possible relevance in the molecular mechanism of Bax function at the MOM despite their low numbers. They have a defined shape and their size is comparable to the single lines. Indeed, these double structures, only detected in GFP‐Bax WT images, resembled the spiral assemblies proposed for the dynamin‐like protein Drp1 during mitochondrial fragmentation (Mears et al, 2011).

To rule out potential effects due to the presence of endogenous Bax and Bak in the HeLa cells transfected with GFP‐Bax, we also performed SMLM experiments in Bax/Bak double knockout HCT116 cells transfected with GFP‐Bax. In the absence of endogenous protein, we could also identify a significant fraction of non‐random GFP‐Bax structures including lines, double lines, arcs, and full rings of comparable sizes (Fig 4A and B). In addition, in order to corroborate that our results were not dependent on the sample preparation or imaging technique, we directly measured GFP‐Bax distribution (without staining with nanobodies) during apoptosis with an alternative super‐resolution method, stimulated emission depletion microscopy (STED). This technique has also showed improvements in resolution up to 60 nm for imaging of GFP‐labeled molecules (Willig et al, 2006; Rankin et al, 2011). As observed in Fig 4C, the images obtained with STED showed a substantial resolution increase compared to those acquired with diffraction‐limited imaging. This technique also revealed comparable non‐random assemblies of GFP‐Bax shaped as lines, arcs, rings, and even double lines (Fig 4D), supporting that the structures characterized with localization microscopy reflect the organization of Bax in apoptotic mitochondria.

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Figure 4

Bax non‐random structures shown on HCT116 Bax/Bak^−/− cells by SMLM and on HeLa cells by STED microscopy

1. Overview of a reconstructed super‐resolution image of GFP‐Bax‐overexpressing HCT116 Bax/Bak^−/− cells stained with AF647‐anti‐GFP nanobodies. Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV2). Scale bar, 5 μm.
2. Gallery of typical GFP‐Bax WT non‐random structures during apoptosis on HCT116 Bax/Bak^−/− cells. Scale bars, 100 nm.
3. Comparison of confocal (left) and STED (right) images of GFP‐Bax‐overexpressing HeLa cells 2 h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.
4. Representative GFP‐Bax non‐random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.

Finally, to check whether the appearance of non‐random Bax structures correlated with the opening of pores at the MOM, we analyzed the mitochondria of apoptotic HeLa cells transfected or not with GFP‐Bax using transmission electron microscopy (Fig 5). After 3 h of treatment with staurosporine, cells presented the typical apoptotic phenotype with swollen endoplasmic reticulum and reorganized mitochondrial cristae. In both cases, we detected events of MOM disruption, which exhibited sizes in the same order of magnitude as the Bax structures identified in the super‐resolution images. These pores or defects were absent in the MOM of control healthy HeLa cells. These results support the role of the non‐random structures of GFP‐Bax identified here in the formation of pores at the MOM of apoptotic cells.

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Figure 5

Transmission electron microscopy reveals mitochondrial outer membrane pores on apoptotic cells

1. Representative TEM pictures of HeLa wild‐type cells. Bottom panels show healthy mitochondria. Scale bars, 500 nm (overview) and 200 nm (zooms).
2. Representative TEM pictures of apoptotic HeLa wild‐type cells 4 h after STS treatment (1 μM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500 nm (overview) and 200 nm (zooms).
3. Representative TEM pictures of apoptotic HeLa GFP‐Bax‐transfected cells 4 h after STS treatment (1 μM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500 nm (overview) and 200 nm (zooms).

Bax shows a heterogeneous distribution at mitochondrial foci during apoptosis

To determine the position and orientation of GFP‐Bax structures on the MOM, we carried out dual‐color confocal and super‐resolution experiments. We first performed time series confocal imaging on HeLa cells co‐transfected with GFP‐Bax and mito‐dsRED (mitochondrial marker) following STS treatment. After approximately 1 h, Bax started to accumulate at discrete sites on mitochondria (Fig 6A). However, confocal microscopy cannot provide information about the structure or orientation of Bax assemblies on the membrane at the nanoscale. For this reason, we performed two‐color SMLM on HeLa cells transfected with GFP‐Bax, which were immunostained 3 h after apoptosis induction with AF647‐anti‐GFP nanobodies for Bax signal and CoxIV antibody (with CF680 as secondary antibody) as a mitochondrial marker. As an alternative to CoxIV imaging, we co‐transfected the cells with mMAPLE‐mito, a photo‐switchable fluorescent protein suitable for SMLM that acted as mitochondrial marker (Fig 6C).

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Figure 6

Bax localizes heterogeneously to discrete mitochondrial foci during apoptosis

1. Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP‐Bax (green) and mito‐dsRed (magenta) at 0 and 85 min after apoptosis induction with STS 1 μM. Right panel shows zoomed overlaid images of HeLa cells following GFP‐Bax translocation (green) and mitochondrial fragmentation (magenta) during apoptosis induction with STS 1 μM. Scale bars, 10 μm (left) and 2 μm (right)
2. Reconstructed dual‐color super‐resolution image of GFP‐Bax‐overexpressing HeLa cells. GFP‐Bax was imaged by immunostaining with AF647‐anti‐GFP nanobodies and mitochondria were immunostained with CoxIV (primary) and cf680 (secondary) antibodies. Image was acquired 3 h after STS treatment. Left panel shows the cell overview (scale bar, 5 μm), right panel up shows the overview inset (scale bar, 500 nm), and right panel down shows a non‐random linear GFP‐Bax assembly at mitochondria (scale bar, 100 nm).
3. Reconstructed dual‐color super‐resolution image of HeLa cells co‐overexpressing mMaple‐mito and GFP‐Bax and stained with AF647‐anti‐GFP nanobodies. Image was acquired 3 h after STS treatment. Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel. Scale bars, 1 μm (overview) and 100 nm (insets)

The images qualitatively showed that all GFP‐Bax signal appeared heterogeneously distributed in discrete foci on mitochondria in both cases, as expected. In particular, it localized along the mitochondrial tubules as well as on mitochondrial ends (Fig 6B and C). Non‐random arrangements corresponding to Bax assemblies could be distinguished in structures wrapping mitochondria, in addition to dots and aggregates (Fig 6B and C). When we examined whether the distribution of the non‐random assemblies of GFP‐Bax was detected preferentially at specific sites along the mitochondrial network, we did not observe any clear preference.

Confocal live cell imaging

After transfection in a glass bottom 8‐well μ‐slide (IBIDI, Germany) and staurosporine induction, cells were maintained in phenol red‐free DMEM at 37°C and 5% CO₂ for imaging. Acquisition was made with a Zeiss LSM 710 ConfoCor3 microscope (Carl Zeiss, Jena, Germany) equipped with a temperature and CO₂ using a C‐Apochromat ×40 N.A. 1.2 water immersion objective (Zeiss). Excitation light came from argon ion (488 nm) or HeNe (561 nm) lasers. Images were processed with FiJi.

Cell fractionation and protein cross‐linking

Cells were grown on dishes, transfected with GFP‐Bax as described above, trypsinized, and washed with PBS. For total protein extraction, cell pellet was resuspended in ice‐cold RIPA buffer (150 mM sodium chloride, 1.0% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), incubated for 30 min on ice, and centrifuged at 21,000 g for 30 min. Supernatant was collected and boiled for 5 min in 6× sample loading buffer. For mitochondrial protein extraction, cell pellet was resuspended in mitochondrial isolation buffer (MB) (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM Hepes pH 7.4) and homogenized in a teflon douncer for 100 strokes (cell integrity was checked by trypan blue dye). The samples were centrifuged at 800 × g for 10 min several times (until no pellet was visible). Supernatant was collected and centrifuged at 10,000 × g for 10 min to pellet the mitochondrial fraction. Mitochondria were resuspended in MB Buffer and post‐mitochondrial supernatant was centrifuged at 100,000 × g for 60 min to obtain the cytosolic fraction. Mitochondrial and cytosolic extracts were boiled for 5 min in 6× sample loading buffer and processed for electrophoresis and Western blotting. For the cross‐linking assay, purified mitochondria were incubated with BMH (10 mM final concentration, added from a 50× BMH stock solution in DMSO) at RT for 45 min under gentle agitation. BMH was then quenched in 0.5 M DTT in MB buffer for 15 min at RT.

Mitochondria were centrifuged at 9,000 × g, and mitochondrial pellet was resuspended and washed twice in MB Buffer. Mitochondria were then boiled in 6× sample loading buffer and processed for electrophoresis in 10% acrylamide gels and Western blotting.

Western Blot

Discontinuous SDS–PAGE was performed according to Laemmli in 10% acrylamide gels. Proteins were transferred onto a PVDF membrane (Millipore, no. ISEQ07850) using a semi‐dry Turbo‐blot apparatus (Bio‐Rad). The membrane was blocked with 5% milk in 1× TBST at RT for 1 h and incubated at 4°C overnight with the appropriate antibody in a ratio of 1:1,000 (Bax (Cell Signaling #2772S), Actin (Millipore #691001), VDAC (Abcam #14734)). After washing with 1× TBST, the secondary antibody was added in a ratio of 1:10,000 in 5% milk and incubated from 1 h at RT. Membrane was washed with 1× TBST, and the immunoreactive bands were visualized by ECL chemiluminescence (WESTERN LIGHTNING^™ Plus‐ECL, PerkinElmer).

STED microscopy

Cells were grown on 15‐mm round glass coverslips for 24 h to 70–80% confluency, transfected, and fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. After washing 3× with PBS, coverslips were mounted on slides with Mowiol and dried overnight.

For measurements with the STED microscope, a pulsed diode laser operating at 485 nm (LDH‐P‐C‐485 PicoQuant GmbH, Germany) and a pulsed raman fiber laser operating at 560 nm (PRFL Series, MPB Communication Inc., Canada) were used for excitation and stimulated emission, respectively. The STED beam is spatially formed to a doughnut by a vortex phase plate (VPP‐2, RPC Photonics, USA). On a long‐pass filter (BLP01‐561, Semrock Inc., USA), both excitation and STED beam are overlaid and coupled to the microscope stand (Leica Microsystems, Germany) utilizing an 100× oil objective (1.4NA, HCX PL APO, Leica Microsystems, Germany). Four galvanometer mirrors (6210H, Cambridge Technologies, USA) scan the image field. The fluorescence is separated on a band‐pass filter (ET525/50M, Chroma Technology Corp., USA) and guided to a hybrid photodetector (R10467U‐40 Hamamatsu Cooperations, Japan). A FPGA‐based software controls the hardware and the data acquisition. Export of the raw data into tiff‐Files and the Gaussian filtering was done with ImSpector^® Image Acquisition & Analysis Software v0.10 (http://www.imspector.de).

Single‐molecule localization microscopy

Cells were grown on 24‐mm round glass coverslips for 24 h to 70–80% confluency, transfected, and fixed in 4% PFA in PBS for 15 min at room temperature. Coverslips were then incubated for 15 min in PBS with 50 mM NH₄Cl and permeabilized in PBS with 0.25% Triton X‐100 for 5 min. Cells were washed 3× for 5 min in PBS and blocked for 45 min in PBS with 1% BSA. Labeling was done with AF647‐anti‐GFP nanobodies (1/2,000) and anti‐CoxIV (1/50) primary antibodies (Cell Signaling, #11967) in 1% BSA for 90 min. After extensive washes with PBS, coverslips stained for CoxIV were incubated for 30 min at room temperature with CF680 anti‐mouse secondary antibodies (1:500 in 1% BSA). Samples were mounted and imaged in a custom‐made microscope (Schoen et al, 2011) and covered with 300 μl of imaging buffer (50 mM Tris‐HCl pH 8, 10 mM NaCl, 10% (v/w) glucose, 35 mM cysteamine (MEA), 0.5 mg/ml glucose oxidase (Sigma), and 40 μg/ml catalase (Sigma)). We used an exposure time of 15 ms for single color and 30 s for dual‐color measurements and an EM gain of 100. Imaging laser intensity at 640 nm was 2.5 kW/cm², and the 405‐nm activation laser intensity was automatically adjusted to keep a constant number of localizations per frame. Typically 70,000–100,000 frames were recorded. Analysis was performed using a custom Matlab script. Localizations with uncertainties above 15 nm were discarded. Images were rendered using a Gaussian with a width according to the localization precision. Image analysis of the random and non‐random structures was done with ImageJ. Histogram bin number for GFP‐Bax structures was determined by the square root of the number of events. For clarity, the same bin width was used for GFP‐Bax‐1‐2/L‐6 structures despite the lower number of events.

Transmission electron microscopy

Cells were scrubbed from cell culture dishes, fixed for 1 h at 4°C in glutaraldehyde (2% solution in 0.1 M cacodylate buffer pH 7.4), and very carefully centrifuged (300 rpm for 30 s). Cell precipitate was imbedded in 3% agarose. Cell agarose block was cut in small pieces (1.5 mm × 3 mm) and washed with cacodylate buffer followed by postfixing with 1% osmium tetroxide in 0.1‐M cacodylate buffer. Dehydration was then started by a series of incubations in 30%, 50%, and 70% ethanol. The samples were stained with saturated uranyl acetate. Dehydration was continued by incubations in 70%, 80%, and 96% ethanol, absolute ethanol, and propylene oxide. Each cell agarose block was then embedded in Epon.

Ultrathin sections (70 nm) were made, stained with lead citrate, and observed under an electron microscope (model 900; Carl Zeiss, Oberkochen, Germany).

We thank B. Liebler for technical assistance and N. Brady and F. Edlich for providing the plasmids for GFP‐Bax WT and GFP‐Bax‐1‐2/L‐6, respectively. We thank Dr. Frank Essmann and Prof. Klaus Schulze‐Osthoff for providing the Bax/Bak DKO HCT116 cells. This work was funded by the European Research Council (ERC‐2012‐StG 309966), the Max Planck Society, the German Cancer Research Center, the Deutsche Forschungsgemeinschaft (FOR 2036), the European Molecular Biology Laboratory (M. M., J. R.), and the EMBL International PhD Programme (M. M.).