| dc.description.abstracteng | Apoptosis, the most common form of cell death in the human body, is essential for numerous physiological processes. The intrinsic apoptosis pathway can be triggered by a wide variety of stimuli and leads to the insertion of BAX and BAK, the effector proteins of the intrinsic apoptosis pathway, into the mitochondrial outer membrane (MOM). The insertion of BAX and BAK opens a pore in the MOM, which releases proteins from the mitochondrial inter-membrane space into the cytosol, thereby activating the caspase cascade, resulting in the ultimate demise of the cell.
Although the two pore-forming effector proteins, BAX and BAK, have been extensively investigated by in vitro studies, it is still unclear how the two proteins are arranged in the apoptotic pore in the MOM in situ. Using diffraction-limited microscopy, numerous studies have revealed the general sub-cellular localization of BAX and BAK, but the resolution of conventional light microscopes is insufficient to analyze the nanoscale distribution of BAX and BAK.
Therefore, in this work I employed super-resolution microscopy on apoptotic cells in order to investigate the fine structure of the apoptotic BAX-BAK pore in situ with sub-diffractional resolution. My results demonstrate that, when overexpressed and imaged by live-cell STED microscopy, BAX and BAK induce apoptosis, but differ in nanoscale dynamics and ultra-structures. However, the overexpression of the pore- forming proteins proved disadvantageous for the study of the apoptotic pore, because the overabundance of BAX or BAK disturbs the fragile equilibrium of pro- and anti-apoptotic proteins, leading to rapid, unphysiological cell death.
To circumvent the drawbacks of overexpression, I established a dual-color antibody staining of endogenous BAX and BAK in fixed cells suitable for STED microscopy. The super-resolved images demonstrate that BAX and BAK at endogenous expression levels formed mosaic rings together that are lining the apoptotic pore. Quantitative analysis revealed that these differently sized rings contained variable relative amounts of BAX and BAK, which tended to homogenize with the growth of the rings. Furthermore, the rings were not continuously and regularly outlined by proteins, but instead large clusters and voids were irregularly interspersed. Within this irregular arrangement along the ring outline, the two proteins had a tendency to follow similar distribution patterns rather than forming mutually exclusive assemblies. Although in wild type cells most rings were composed of both proteins, knock- out cell lines of BAX or BAK demonstrated that, if one of the partners was absent, the remaining protein was able to form pores independently, corroborating the partial functional redundancy of the two proteins.
Next to immortal cell lines, I furthermore detected BAX-BAK rings in primary human cells, which suggests that the formation of complex BAX/BAK assemblies, especially mosaic rings, is a physiological and essential step during apoptosis.
Altogether, my results display BAX and BAK in apoptotic cells in situ and the data strongly support a toroidal pore model, where BAX and BAK proteins cover the pore rim together in an unordered fashion. | de |