Bcl-2 proteins and membranes: insights on their interaction

Abstract

The Bcl-2 family of proteins regulates mitochondrial outer membrane permeabilization (MOMP) – considered as the point-of-no-return in apoptosis. As the gatekeeper for survival or death of cells, this network is tightly regulated by interactions of its members with opposing functions. The embedded together model remains as one of the most accepted models to describe the interactions between family members. This model highlights the role of the mitochondrial membrane to augment these interactions. And while this model has been repeatedly exemplified in vitro, the e↵ect of membranes on Bcl-2 protein interactions has not yet been fully explored in living cells. On the other hand, the Bcl-2 proteins could also a↵ect the physical properties of the membrane. Bax and Bak are the main e↵ectors of apoptosis, believed to form pores on the mitochondrial outer membrane. However, no one has seen an apoptotic pore. Aside from its permeabilizing activity, the e↵ect of Bcl-2 proteins on the physical properties of the membrane is not yet well-elucidated. As such, the main question of this thesis is: how do Bcl-2 proteins a↵ect the membrane (and vice versa)? This work has shown the e↵ect of pro-apoptotic member Bax and its activator, cleaved Bid (cBid), on membranes using atomic force microscopy (AFM). Cleaved Bid was shown to lower the breakthrough force of membranes, but not permeabilize it. This suggests that cBid may have a role in paving the way for Bax insertion and oligomerization. On the other hand, Bax’s ability to change membrane properties depends on its oligomerization state – monomeric Bax does not change the membrane’s properties, but oligomerized Bax lowers the membrane’s breakthrough force. I also show through AFM imaging that Bax forms rings, arcs and lines in model membranes. In instances where pores are visible on the membrane, structures at the edge of the pore, which may correspond to Bax, does not necessarily need to line the edge completely. This further supports previous findings that Bax forms toroidal pores and that these pores can have a varied range of sizes. Furthermore, this work has also shown the potential of scanning Fluorescence Correlation Spectroscopy (FCS) as a tool to measure interactions of Bcl-2 proteins in the mitochondria of living cells. I first validated scanning FCS’ applicability using green fluorescent protein (GFP) targeted to mitochondria. I also evaluated possible models that characterize the di↵usion of molecules in the mitochonodria. Using two color FCS, I report for the first time, to the best of my knowledge, a calculated dissociation constant between truncated Bid and anti-apoptotic member, Bcl-xL, in the mitochondria. Based on these results, I highlight the important interplay between the membrane and the Bcl-2 proteins – in deciding a cell’s fate, both membrane and Bcl-2 proteins work with and change each other.