Periodic Reporting for period 4 - APOSITE (Apoptotic foci: composition, structure and dynamics)
Reporting period: 2022-11-01 to 2024-03-31
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.
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.
So far, we have generated a list of protein and lipid candidates for components of apoptotic foci that we are currently validating. We have 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 and are generating and knock out cell lines.
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 identified changes in lipid composition in apoptotic foci and validated their functional effect. Now we are investigating the mechanism involved and the functional relevance.
In addition, we have also compared the structure and stoichiometry of BAX and BAK assembly during apoptosis using super-resolution and single particle imaging, discovered a new mechanism and established biological relevance, which has now been published in a scientific article. We have implemented a protocol for super-CLEM, which is now being applied to BAX and BAK.
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 identified changes in lipid composition in apoptotic foci and validated their functional effect. Now we are investigating the mechanism involved and the functional relevance.
In addition, we have also compared the structure and stoichiometry of BAX and BAK assembly during apoptosis using super-resolution and single particle imaging, discovered a new mechanism and established biological relevance, which has now been published in a scientific article. We have implemented a protocol for super-CLEM, which is now being applied to BAX and BAK.
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 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 expect to dissect the mechanism involved and the biological relevance until the end of the project.
We have implemented a method for super-CLEM that allows to correlate nano-structures from STED images with the native environment of the mitochondrial ultrastructure. We expect to use this method to understand how BAX/BAK nano-assemblies are linked to mitochondrial alterations.
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 expect to dissect the mechanism involved and the biological relevance until the end of the project.
We have implemented a method for super-CLEM that allows to correlate nano-structures from STED images with the native environment of the mitochondrial ultrastructure. We expect to use this method to understand how BAX/BAK nano-assemblies are linked to mitochondrial alterations.