Binding and Oligomerization of Bcl-2 Family Proteins on Supported Lipid Bilayers with Single Molecule Resolution

Abstract

Bid, Bax and Bcl-XL are apoptosis-regulating proteins that play a key role in mitochondrial outer membrane permeabilization during programmed cell death. Elucidating the molecular mechanisms regulating the function of these proteins is essential to understanding how apoptosis can be controlled with drugs, i.e. increased in diseases such as cancer, or decreased in the case of stroke. Cleaved Bid (cBid) recruits Bax to the mitochondrial membrane, which forms oligomeric pores and triggers cell death through the release of cytochrome c into the cytoplasm. Bcl-XL is also recruited to the membrane by cBid, but inhibits apoptosis. In this study, fluorescent confocal microscopy is used to characterize the membrane-binding behaviour of cBid to a mitochondria-like supported lipid bilayer in the presence and absence Bax and Bcl-XL. By working at dilute concentrations of fluorescent protein, single molecular complexes could be detected by fitting Gaussian profiles to diffraction-limited spots. The oligomerization state of cBid was determined by normalizing the fluorescence intensity by the brightness of a cBid monomer, as measured using fluorescence fluctuation techniques. This method revealed two main populations of cBid molecules: monomers which diffuse in the plane of the membrane and higher order oligomers which are predominantly immobile. This suggests that Bid goes from a membrane associated to a membrane inserted conformation due to homo-oligomerization.