Periodic Reporting for period 5 - APOSITE (Apoptotic foci: composition, structure and dynamics)
Reporting period: 2024-04-01 to 2025-09-30
APOSITE aims to understand the molecular structure and dynamic mechanism of the BAX/BAK apoptotic foci, which mediate the permeabilization of the outer mitochondrial membrane during apoptosis. This cellular process constitutes the point-of-no-return in the cellular commitment to apoptosis, which plays a key role in essential biological processes like organ sculpture during embryo development, tissue homeostasis and the correct functioning of the immune system. In addition, dysregulation of apoptosis has been associated with diseases relevant for society, including cancer, stroke or neurodegenerative disorders.
To this aim, the project is divided into three main goals that will define the composition, assembly dynamics and structural organization of the supramolecular complexes underlying the apoptotic foci, as well as their functional relevance.
APOSITE has advanced our understanding of the mechanisms that mediate mitochondrial permeabilization during apoptosis. We found that BAX and BAK present distinct assembly properties, so that their balance determines the growth rate of the apoptotic pore and with it, the downstream consequences for inflammatory signaling. We have also dissected the protein and lipid composition in the environment of the apoptotic pores formed by BAX and BAK, and defined their role in mitochondrial permeabilization. We discovered that both proteins outside the BCL-2 family, as well as lipids, can modulate the growth of the apoptotic pore and its inflammatory function. We have developed a new microscopy method that correlates the copy number of individual apoptotic pores with their nanoscale organization and the underlying mitochondrial ultrastructure. Thanks to this, we could visualize that the BAX and BAK assemblies localize to disruptions at the mitochondrial outer membrane. Our data suggest that it is the membrane shape that defines the shape of the BAX and BAK structures and that the outer and inner membrane cooperate to drive the growth of the apoptotic pore.
To this aim, the project is divided into three main goals that will define the composition, assembly dynamics and structural organization of the supramolecular complexes underlying the apoptotic foci, as well as their functional relevance.
APOSITE has advanced our understanding of the mechanisms that mediate mitochondrial permeabilization during apoptosis. We found that BAX and BAK present distinct assembly properties, so that their balance determines the growth rate of the apoptotic pore and with it, the downstream consequences for inflammatory signaling. We have also dissected the protein and lipid composition in the environment of the apoptotic pores formed by BAX and BAK, and defined their role in mitochondrial permeabilization. We discovered that both proteins outside the BCL-2 family, as well as lipids, can modulate the growth of the apoptotic pore and its inflammatory function. We have developed a new microscopy method that correlates the copy number of individual apoptotic pores with their nanoscale organization and the underlying mitochondrial ultrastructure. Thanks to this, we could visualize that the BAX and BAK assemblies localize to disruptions at the mitochondrial outer membrane. Our data suggest that it is the membrane shape that defines the shape of the BAX and BAK structures and that the outer and inner membrane cooperate to drive the growth of the apoptotic pore.
We generated a list of protein and lipid candidates for components of apoptotic foci and implemented a pipeline for genome editing of the candidates so that they are tagged with fluorescence proteins at endogenous levels using knock in CRISPR/Cas 9 approaches. We have implemented PLA for the characterization of these candidates. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release.
We have validated the interaction with BAX/BAK of the top candidates MTCH2 and DRP1 by PLA and other methods and are working on the characterization of their effect and mechanism on the apoptotic foci, as well as their functional relevance. We have also discovered that MTCH2 promotes the growth of BAX and BAK foci, and that it does so by modulating the lipid composition of mitochondria. We have implemented a protocol for correlating stoichiometry, nanoscale organization by STED superresolution microscopy and electron microscopy, which we have applied to BAK. Using this method, we have found that BAK assemblies localize to mitochondrial outer membrane disruptions and that the membrane edges define the shape of the assemblies.
We have validated the interaction with BAX/BAK of the top candidates MTCH2 and DRP1 by PLA and other methods and are working on the characterization of their effect and mechanism on the apoptotic foci, as well as their functional relevance. We have also discovered that MTCH2 promotes the growth of BAX and BAK foci, and that it does so by modulating the lipid composition of mitochondria. We have implemented a protocol for correlating stoichiometry, nanoscale organization by STED superresolution microscopy and electron microscopy, which we have applied to BAK. Using this method, we have found that BAK assemblies localize to mitochondrial outer membrane disruptions and that the membrane edges define the shape of the assemblies.
We have discovered that during apoptosis BAK organizes into lines, arcs and rings of similar shape to BAX, but with smaller size. We also visualized for the first time that BAX and BAK form part of the same individual structures. We found that BAK assembles faster into smaller oligomers than BAX, while BAX oligomers continue to grow during apoptotic progression. Our results revealed that BAX and BAK exhibit distinct assembly properties, and that they regulate each other, so that the balance of BAX and BAK molecules regulates the growth rate of the apoptotic pore and its permissiveness to mitochondrial content, like mitochondrial DNA. We also found that this has consequences for the downstream inflammatory outcome of apoptosis.
We have also identified changes in lipid composition in apoptotic foci and validated their functional effect. We have discovered that polyunsaturated lipids accumulate in the vicinity of the apoptotic pore and promote its growth, revealing a role for lipids in pore dynamics regulation. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.
We have discovered that MTCH2 strongly affects the dynamics of BAX/BAK assembly in foci, which has consequences for the growth of the apoptotic pore and the release of mitochondrial contents. We dissected the mechanism involved, which relies on the function of MTCH2 regulating mitochondrial lipid composition. Interestingly, MTCH2 depletion has functional consequences for the survival and phenotype of persister cancer cells, as well as for sublethal DNA damage downstream of mitochondrial permeabilization during bacterial infection.
We have implemented a method for super-CLEM that allows to correlate the stoichiometry and nano-structural organization of protein oligomers with the native environment of the mitochondrial ultrastructure. We used this method to understand how BAX/BAK nano-assemblies are linked to mitochondrial alterations. In addition, this led us to discover that osmotic swelling contributes to mitochondrial inner membrane extrusion through the apoptotic ore and that both mitochondrial membranes interplay to determine the apoptotic pore growth.
We have also identified changes in lipid composition in apoptotic foci and validated their functional effect. We have discovered that polyunsaturated lipids accumulate in the vicinity of the apoptotic pore and promote its growth, revealing a role for lipids in pore dynamics regulation. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.
We have discovered that MTCH2 strongly affects the dynamics of BAX/BAK assembly in foci, which has consequences for the growth of the apoptotic pore and the release of mitochondrial contents. We dissected the mechanism involved, which relies on the function of MTCH2 regulating mitochondrial lipid composition. Interestingly, MTCH2 depletion has functional consequences for the survival and phenotype of persister cancer cells, as well as for sublethal DNA damage downstream of mitochondrial permeabilization during bacterial infection.
We have implemented a method for super-CLEM that allows to correlate the stoichiometry and nano-structural organization of protein oligomers with the native environment of the mitochondrial ultrastructure. We used this method to understand how BAX/BAK nano-assemblies are linked to mitochondrial alterations. In addition, this led us to discover that osmotic swelling contributes to mitochondrial inner membrane extrusion through the apoptotic ore and that both mitochondrial membranes interplay to determine the apoptotic pore growth.