Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species

Abstract

Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

Figure 1. Detection and calibration of single BaxG fluorophores.

(a) Schematic representation of the experimental setup showing a supported lipid bilayer with BaxG bound to it. (b) Epifluorescence image of BaxG molecules immobilized on a supported lipid bilayer. Scale bar, 1 μm. Each particle was detected and the fluorescence intensity measured over time. Detected particles are shown in circles to help visualization. (c,d) Fluorescence intensity of two representative individual particles showing photobleaching steps (arrow). (e) Histogram of the fluorescence intensity distribution of individual BaxG particles with a single bleaching event, fitted with a Gaussian to obtain the fluorescence intensity of a single fluorophore. (f) Mean fluorescence intensity (μ) and s.d. (σ) values calculated for individual particles containing one (orange), two (green), three (blue), four (magenta), five (cyan) and six (red) fluorophores.

Figure 2. BaxG binds as a monomer to membranes containing CL in presence of cBid.

Binding of BaxG particles to SLBs is negligible for membranes composed of phosphatidylcholine:cardiolipin (PC:CL) (8:2) in absence of cBid (a) and for membranes composed of pure PC in presence of cBid (b), but it is very efficient for membranes composed of PC:CL (8:2) in presence of cBid (c). The images show similar scaling to compare them visually. Scale bar, 1 μm. (d) Fluorescence intensity distribution of single BaxG particles added to SLBs made of PC:CL (8:2) in presence of cBid. Approximately 500 particles were analysed. The resulting histograms were fitted with a linear combination of four Gaussians to estimate the occurrence of particles containing one (orange), two (green), three (blue) and four (magenta) labelled molecules. The cumulative fit is shown in black. The area of the fitted Gaussians is proportional to the fraction of each species. (e) Percentage of BaxG monomers, dimers, trimers and tetramers calculated from the averaged distributions of species from three different experiments, after correction for partial labelling. The error bars correspond to the average error for each species.

Figure 3. Bax exists in the membrane as a mixture of species based on dimer units.

(a) Intensity distribution of individual BaxG particles bound to SLBs prepared from proteoliposomes after 1 h of incubation with BaxG and cBid. The resulting histogram was fitted with a linear combination of six Gaussians to estimate the occurrence of particles containing one (orange), two (green), three (blue), four (magenta) and six (red) labelled molecules. The cumulative fit is shown in black. (b) Percentage of occurrence of different oligomeric species calculated from the averaged distributions of species from three different experiments, after correction for partial labelling. The error bars correspond to the average error for each species. (c) Intensity distribution of a high number of individual BaxG particles (n=2,440) bound to SLBs prepared from proteoliposomes after 1 h of incubation with BaxG and cBid. The obtained brightness distribution was plotted as a probability density function (black line). The fit is shown in red and the N-mer contributions in blue. (d) Percentage of occurrence of different oligomeric species calculated from c after correction for partial labelling. (e) Representative photobleaching trace (blue) of a BaxG particle bound to SLBs prepared as in a. The green line shows the identified photobleaching steps after noise reduction. (f) Distribution of the number of photobleaching steps for 500 BaxG particles analysed as in e. (g) Percentage of occurrence of different BaxG oligomeric species calculated from f after correction for partial labelling.

Figure 4. The extent of Bax oligomerization is not affected by cBid, while Bcl-xL induces the disassembly of Bax oligomers.

(a) Intensity distribution of individual particles of heat-activated BaxG bound to the SLBs prepared from proteoliposomes after 1-h incubation at 40 °C. The resulting histogram was fitted with a linear combination of six Gaussians to estimate the occurrence of particles containing one (orange), two (green), three (blue), four (magenta), five (cyan) and six (red) labelled molecules. The cumulative fit is shown in black. Percentage of occurrence of different oligomeric species when BaxG is activated by heat (b) or when Bcl-xL is present in the membrane (d), calculated from the averaged distributions of species from three different experiments, after correction for partial labelling. The error bars correspond to the average error for each species. (c) Intensity distribution of single BaxG particles bound to SLBs prepared from proteoliposomes incubated for 1 h with BaxG and cBid, following addition of Bcl-xL and incubation during 1 h more. The colour code for Gaussian fitting is the same as in a.

Figure 5. Particle-based simulation of Bax oligomerization on the surface of LUVs.

Model for Bax self-assembly in the membrane. (a) Snapshots of the particle-based simulation at the indicated time points, showing the species presented in the system. The cubic box has a side length of 7 μm. Initially (0 s), 1,163 soluble Bax monomers, 2,326 cBid molecules and 1,453 LUV particles are uniformly distributed inside the box and are all represented as black spots. In the following time points, for clarity, only free LUVs (black), Bax dimers (magenta), Bax tetramers (green) and Bax hexamers (cyan) are represented as big particles. The other species are represented as small, black dots. (b) Time course of the simulated number of soluble Bax (yellow), soluble cBid (blue) and LUVs containing Bax (magenta) particles. The dashed line indicates the initial number of free LUVs. Only a subset of the LUVs in solution contains Bax molecules. (c) Time course of the simulated number of membrane-bound Bax species during 10 min. (d) Schematic view of the proposed autoactivation model for Bax oligomerization.