Quantification of Protein Distribution on Liposomes using Confocal Microscopy: A Single Mobile Fluorescent Particle Detection Method

Abstract

Bcl-2 family proteins play a crucial role in the regulation of the permeabilization of the outer mitochondrial membrane (OMM), which is one of the main steps leading to cell death through apoptosis.To a first approximation, apoptosis is controlled by the balance between pro-apoptotic and anti-apoptotic Bcl-2 family proteins. Deregulation of this balance induces a dysfunction of the apoptosis process leading to different pathologies. The molecular mechanism leading to OMM permeabilization is not yet understood in details. This is in part because of the multiplicity and complexity of the molecular interactions involving Bcl-2 family proteins, since each of them can often interact with itself (homo-oligomerization), with other members of the family (hetero-oligomerization) and with the lipids of the OMM.Whereas in most studies ensemble methods have been used to study these proteins, we have used a single mobile particle detection method to characterize the distribution of different Bcl-2 family proteins on a population of large unilamellar liposomes, in order to better understand the interplay between different family members and lipid membranes during the process of membrane permeabilization. We used large unilamellar liposomes (∼ 200 nm in diameter) with a lipid composition mimicking that of mitochondrial membranes and fluorescently labeled recombinant proteins, which were imaged by confocal microscopy. The specific brightness of each particle detected in the images can then calculated for each separate emission channel.As a first application, we studied the distribution of Bax and Bid on a population of liposomes. These proteins are both pro-apoptotic, promoting pore formation in liposomes, and they are known to interact with one another. We measured the probability that a liposome carried Bax, Bid, or both proteins, allowing us to quantify their colocalization on the same liposome.