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.
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