Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis

Abstract

The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells.

Keywords: Bcl‐2; MICOS; cell death; membrane curvature; superresolution microscopy.

Figure 1. Bax forms ring‐like structures on fragmented mitochondria during apoptosis

1. A

   Left: Overlay image of a confocal recording of an apoptotic U2OS cell surrounded by non‐apoptotic cells labeled with Bax (green) and Tom22 (red). Bax translocates to the mitochondria in apoptotic cells. Right: Individual display of the two color channels as shown in the overlay image. The asterisk denotes the apoptotic cell. Scale bars: 10 μm.

2. B, C

   Dual‐color image of an apoptotic U2OS cell decorated with antibodies against Bax (green) and Tom20 (red). The Bax signal was recorded in the STED mode and the Tom20 signal in the diffraction‐limited confocal mode. (B, C) display the same data set. The small images are magnifications of the areas indicated by the rectangles. In (B), the color table was adjusted to the brightest Bax signal in the image resulting in a clear display of the clusters. In (C), the color table was adjusted such that the dimmer Bax rings are visible and the clusters are saturated. The arrows point to Bax rings. Scale bars: 1 μm.

3. D

   Optical sections of an apoptotic mitochondrion labeled for Bax (green) and Tom22 (red). Shown are three optical sections and a maximum intensity projection of the entire image stack. Scale bar: 0.5 μm.

4. E

   Bax rings on apoptotic mitochondria of various mammalian cell types. Shown are apoptotic mitochondria labeled for Bax (green) and Tom22 (red) in HT1080, SH‐SY5Y, HeLa, and CV‐1 cells, as indicated. Scale bars: 0.5 μm.

5. F

   Quantification of the diameter of Bax rings in apoptotic U2OS cells. Only the long ring‐axis was measured. Bax rings smaller than 200 nm exist, but were not considered.

Source data are available online for this figure.

Figure 2. The area enclosed by a Bax ring is devoid of the mitochondrial outer membrane protein Tom22

Apoptotic mitochondria were decorated with antibodies against Bax (green) and Tom22 (red). The Bax signal was recorded in the STED mode. Shown are the individual color channels as well as an overlay (from top to bottom) of several apoptotic mitochondria. We used Lagrange interpolation to double the number of pixels. Note that throughout this manuscript except for contrast stretching, no image manipulation was applied. Scale bar: 0.5 μm. Source data are available online for this figure.

Figure 3. The formation of Bax rings is strictly correlated to cyto‐chrome c release in apoptotic wild‐type cells

1. A, B

   Three‐color images of mitochondria of unchallenged (A) and apoptotic (B) U2OS cells. From left to right: Tom22, Bax (recorded in the STED mode), cytochrome c, and an overlay of all three channels. The arrows denote Bax rings. Scale bars: 2 μm.

Source data are available online for this figure.

Figure 4. In apoptotic Drp1 knockdown cells, the formation of Bax rings is not sufficient for full cytochrome c release

1. In apoptotic Drp1 knockdown U2OS cells, cytochrome c release is delayed. Confocal overview image of an apoptotic Drp1 knockdown cell labeled with antibodies against Tom20 (red), cytochrome c (blue), and Bax (green). Large image: Overlay of all three channels. Small images: Magnifications of the area indicated by the rectangle in the large image. Shown are the individual channels, as indicated. The arrows point to mitochondria that had not released cytochrome c; the asterisks denote mitochondria that had released cytochrome c. Scale bars: 10 μm (overview) and 2 μm (magnifications).

2. In Drp1 knockdown cells, Bax‐ring formation and cytochrome c release is not correlated. Three‐color images of apoptotic mitochondria of Drp1 knockdown cells. From left to right: Tom22, Bax (recorded in the STED mode), cytochrome c, and an overlay of all three channels. The arrows point to Bax rings. Scale bar: 1 μm.

Source data are available online for this figure.

Figure 5. The redistribution of the MICOS core components Mic27 and Mic60 precedes the release of cytochrome c in apoptotic Drp1 knockdown cells

Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Fig S4). Three‐color images were taken, whereby Mic27 or Mic60 was recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labeled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.

Figure 6. 3D recordings of apoptotic mitochondria suggest the formation of a Bax‐ring‐induced pore in the mitochondrial outer membrane

Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell. Top and middle row: Tom22 and Bax (recorded in STED) channel, respectively. Bottom row: overlay (Tom22: red; Bax: green). Note the absence of Tom22 signal in the area enclosed by the Bax ring. Displayed are raw data. Scale bar: 0.5 μm. Source data are available online for this figure.