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Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes.
Gillies LA
,
Du H
,
Peters B
,
Knudson CM
,
Newmeyer DD
,
Kuwana T
.
???displayArticle.abstract??? Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.
FIGURE 1:. Cryo-EM imaging of OMVs permeabilized with Bax showing a solitary, large pore in many OMVs. (A) Untreated OMVs show round or ellipsoidal shapes with smooth membranes. (B) OMVs treated with Bax and cBid show only one pore per vesicle (arrows). Regions with negative curvature (asterisks) were often observed near the edges of the pores. The black spots are debris.
FIGURE 2:. Bax-induced membrane pores increased in frequency and size with increased Bax concentration and incubation time. Untreated (663) and treated (633) vesicles were visualized by cryo-EM, counted, and categorized as pore-containing or smooth. (A) The frequency of pores in untreated OMVs was negligible, whereas this frequency ranged from 7.7% to 52.5% in Bax-treated OMVs. The difference was statistically highly significant (Mann-Whitney test: p = 0.0012), and thus pores were a feature only seen with Bax/Bid treatment. The frequency of OMVs containing pores was also higher with higher cBid/Bax input. (B) The pore size increased significantly with higher Bax concentration (p = 0.003) and with longer incubation time (p = 0.0005). “Low Bax” denotes the treatment with 812 nM Bax and 420 nM cBid; “High Bax” denotes treatment with 3.25 μM Bax and 2.5 μM cBid. Two-way analysis of variance showed there was no statistically significant synergy between time and Bax concentration.
FIGURE 3:. A flow-cytometry approach can analyze the fluorescence of individual OMVs and reveal trends in the overall population caused by Bax-induced MOMP. (A) Principle of the method. Over time, a leftward shift in the peak center (toward lower fluorescence intensity) would indicate gradual dextran release from the population of vesicles (top diagram). In contrast, a decline in the area under the peak (bottom diagram) would indicate that some of the vesicles had lost their entire content of fluorescent dextrans. At the same time, a low MFI peak corresponding to “empty” OMVs would emerge. (B) Experimental simulation of gradual release using OMVs loaded with varied amounts of F-dex. Reducing the dextran concentrations used for loading resulted in shifts of the OMV peak toward lower fluorescence intensity, as expected. (C) Experimental simulation of all-or-none release by dilution of the OMV suspension. This produced the predicted decline in amplitude of the full OMV peak and emergence of the empty OMV peak.
FIGURE 4:. Bax-permeabilized OMVs shifted gradually toward lower MFI. OMVs loaded with 10-, 70-, or 500-kDa F-dex were treated with 0, 5, 10, or 20 nM Bax and 45 nM cBid and analyzed by flow cytometry every 5 min for 45 min. For clarity, histograms at every 10 min are shown. Data are representative of three independent experiments. Note that there were no discrete peaks at the MFI of the empty OMV peak (in solid gray) as shown in the control experiment for each dextran size (bottom panels). Rather, empty OMVs comprised only a tail to the left of the main peak; the main OMV peak gradually shifted toward lower MFI.
FIGURE 5:. MOMP-associated F-dex release occurs predominantly by the gradual mode but is independent of dextran size. (A) An example of curve-fitting analysis from a 30-min time point. In this approach, we quantified the two forms of release as diagrammed in Figure 3. The MFI (peak center) of empty OMVs was estimated by treating OMVs with a relatively high concentration of Bax (20 nM) for 2 h. We then fitted the sum of two log-Gaussian peaks to the histograms at each time point, constraining one Gaussian curve to the MFI of the empty OMV peak and allowing a variable MFI for the second Gaussian curve representing the partially full OMV peak. Shown are the experimental histogram, the dual-Gaussian fitted curve, and the two component Gaussian distributions. Note that the empty OMVs comprise only a small tail on the left of the main peak and thus correspond to a much smaller Gaussian distribution. (B) Gradual release is predominant, regardless of dextran size and Bax concentration. Shown are the total contributions of gradual release for each dextran size at various Bax concentrations, calculated from the curve-fitting analyses described in Materials and Methods. Gradual release comprised ∼80% of total release, independent of Bax concentrations or dextran size. (C) Example of a set of normalized MFI time courses fitted with simple exponential-decay curves. (D) Summary of the rates of change of MFI. These rates (which are essentially proportional to the rate of dextran release via the gradual mode) increased with Bax concentration and were similar for all F-dex sizes. Values show averages and SDs from three to four independent experiments.
Baker,
Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs.
1999, Pubmed
Baker,
Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs.
1999,
Pubmed
Basañez,
Bax-type apoptotic proteins porate pure lipid bilayers through a mechanism sensitive to intrinsic monolayer curvature.
2002,
Pubmed
Billen,
Bcl-XL inhibits membrane permeabilization by competing with Bax.
2008,
Pubmed
Bleicken,
Molecular details of Bax activation, oligomerization, and membrane insertion.
2010,
Pubmed
Bleicken,
Proapoptotic Bax and Bak proteins form stable protein-permeable pores of tunable size.
2013,
Pubmed
Braun,
Photoreactive stapled BH3 peptides to dissect the BCL-2 family interactome.
2010,
Pubmed
Chen,
Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function.
2005,
Pubmed
Chipuk,
The BCL-2 family reunion.
2010,
Pubmed
Chipuk,
Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis.
2012,
Pubmed
Czabotar,
Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.
2013,
Pubmed
Dejean,
Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel.
2005,
Pubmed
Dewson,
Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis.
2009,
Pubmed
Dewson,
To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.
2008,
Pubmed
Eskes,
Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions.
1998,
Pubmed
Estaquier,
Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis.
2007,
Pubmed
Etxebarria,
Endophilin B1/Bif-1 stimulates BAX activation independently from its capacity to produce large scale membrane morphological rearrangements.
2009,
Pubmed
Gavathiotis,
BAX activation is initiated at a novel interaction site.
2008,
Pubmed
Guihard,
The mitochondrial apoptosis-induced channel (MAC) corresponds to a late apoptotic event.
2004,
Pubmed
Hanahan,
Hallmarks of cancer: the next generation.
2011,
Pubmed
Hilgendorf,
The retinoblastoma protein induces apoptosis directly at the mitochondria.
2013,
Pubmed
Kluck,
The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol.
1999,
Pubmed
,
Xenbase
Kluck,
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
1997,
Pubmed
,
Xenbase
Kushnareva,
Bax activation initiates the assembly of a multimeric catalyst that facilitates Bax pore formation in mitochondrial outer membranes.
2012,
Pubmed
Kuwana,
Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane.
2002,
Pubmed
,
Xenbase
Kuwana,
BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.
2005,
Pubmed
Landeta,
Reconstitution of proapoptotic BAK function in liposomes reveals a dual role for mitochondrial lipids in the BAK-driven membrane permeabilization process.
2011,
Pubmed
Lartigue,
Caspase-independent mitochondrial cell death results from loss of respiration, not cytotoxic protein release.
2009,
Pubmed
Letai,
Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics.
2002,
Pubmed
Liu,
Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.
1996,
Pubmed
Llambi,
A unified model of mammalian BCL-2 protein family interactions at the mitochondria.
2011,
Pubmed
Lucken-Ardjomande,
Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane.
2008,
Pubmed
Martinez-Caballero,
Assembly of the mitochondrial apoptosis-induced channel, MAC.
2009,
Pubmed
Moldoveanu,
BID-induced structural changes in BAK promote apoptosis.
2013,
Pubmed
Montessuit,
Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization.
2010,
Pubmed
Muchmore,
X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.
1996,
Pubmed
Muñoz-Pinedo,
Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration.
2006,
Pubmed
Schafer,
Mitochondrial outer membrane proteins assist Bid in Bax-mediated lipidic pore formation.
2009,
Pubmed
,
Xenbase
Takahashi,
Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis.
2005,
Pubmed
Tanaka,
A chemical inhibitor of DRP1 uncouples mitochondrial fission and apoptosis.
2008,
Pubmed
Terrones,
Lipidic pore formation by the concerted action of proapoptotic BAX and tBID.
2004,
Pubmed
Tilley,
The mechanism of pore formation by bacterial toxins.
2006,
Pubmed
Van Loo,
A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid.
2002,
Pubmed
Volkmann,
The rheostat in the membrane: BCL-2 family proteins and apoptosis.
2014,
Pubmed
Westphal,
Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis.
2014,
Pubmed
Xu,
Three-dimensional structure of Bax-mediated pores in membrane bilayers.
2013,
Pubmed
Yang,
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
1997,
Pubmed
van der Pol,
Single vs. swarm detection of microparticles and exosomes by flow cytometry.
2012,
Pubmed
