Real-time visualization of membrane permeabilization by Bcl-2 proteins during apoptosis

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Apoptosis is a cellular self-destruction program that is executed either after external death signals or internal stimuli, e.g. DNA damage. Mitochondrial outer membrane permeabilization (MOMP) occurs during the so-called intrinsic pathway of apoptosis and leads to the release of signaling molecules like cytochrome c from the intermembrane space of mitochondria to the cytosol. This process is controlled by both pro- and anti-apoptotic members of the Bcl-2 family of proteins. In particular, interaction with BH3-only proteins like tBid leads to the targeting of the pro-apoptotic protein Bax to the outer mitochondrial membrane, its homo-oligomerization and, finally, to the formation of pores and loss of outer membrane integrity. These rearrangements are accompanied in vivo by extensive fission of mitochondria. Interestingly, the formation of fusion or fission intermediates accelerates Bax-induced pore formation. As biophysical models suggest, these intermediates might display intrinsically instable membrane areas that could become permanently destabilized by Bax oligomers. The goal of this project thus is to clarify the connection between membrane intermediates and pore formation. Using giant unilamellar vesicles undergoing fusion and fluorescently labeled recombinant proteins, the localization of Bax oligomers with respect to hemifusion stalks will be determined by confocal videomicroscopy with high temporal resolution. Moreover, with the help of fluorescent dextrans encapsulated inside these vesicles and released upon membrane permeabilization, the spatial and temporal details of membrane leaks will be demonstrated. This direct visualization of MOMP in vitro will lead to a comprehensive picture regarding the role of Bcl-2 proteins in this important signaling event.