The proteins of the Bcl-2 family are essential regulators of apoptosis. They control a key event in apoptosis that is the permeabilization of the mitochondrial outer membrane (MOM). This leads to the release of the so-called apoptotic factors (like cytochrome c (cyt c), AIF or Smac/DIABLO) into the cytosol, which induces the activation of caspases and cell death. Because of the essential role of apoptosis during development, tissue homeostasis or functioning of the immune system, the regulation of apoptosis is a fundamental question in biology. In addition, since the Bcl-2 proteins have an important role in tumorigenesis and in the cellular responses to anti-cancer therapies, understanding their functioning is of great therapeutic interest.
The Bcl-2 proteins are classified into three subgroups: i) antiapoptotic proteins, like Bcl-2 or Bcl-xL; ii) proapoptotic proteins like Bax or Bak that are likely to participate directly in MOM permeabilization; iii) and the BH3-only proteins, including Bid or Bim, which act like sensors for the different apoptotic stimuli and initiate apoptosis. Under normal conditions, many of the proteins of the Bcl-2 family remain in an apoptosis-inactive form in the cytosol or associated to the outer mitochondrial membrane. In the presence of apoptotic stimuli, the BH3-only proteins are activated first. They are considered the initial sensors that recognize the diverse apoptotic signals in the cell. For example, Bid is activated by proteolytic cleavage by caspase-8. Once induced or activated, the Bcl-2 proteins insert into the MOM, where they engage with other members of the family to regulate cytochrome c release and cell death.
Despite their high degree of homology, the reasons why the members of the Bcl-2 family have opposite functions are still obscure. Also, how the different members of the family engage with each other to decide whether or not apoptosis is induced needs better understanding. Another intriguing step in the pathway is their translocation to the mitochondrial membrane, since the role of the membrane environment on their function is unknown. Because of these and other questions, the mechanism of action of the Bcl-2 proteins remains unclear. Our research focuses on the role of the membrane and of specific lipids, and on the interaction network between the Bcl-2 members that decides whether or not apoptosis is induced.
FCS is a technique with single molecule sensitivity that analyzes the fluctuations in fluorescence intensity within a tiny volume (in the order of fL). It can be used to measure diffusion coefficients, fluorophore concentrations, particle sizes, chemical reactions, conformational changes and binding/unbinding processes, among other. All this makes FCS an excellent technique for the investigation of dynamic processes.
Two-color FCS or fluorescence cross-correlation spectroscopy (FCCS) is a variant of FCS extremely convenient for the study of molecular interactions, both in vitro and in vivo. It measures dynamic co-localization and therefore can be used for binding or dissociation studies. In this case, two particles of interest are labeled with spectrally different fluorophores and excited with overlapping laser beams. If the labeled species interact, they will diffuse together through the focal volume, inducing simultaneous fluorescence fluctuations and positive cross-correlation.
In the case of membrane applications of FCS, some issues related to the sample characteristics delayed the standardization of the technique. A very interesting approach is scanning FCS (SFCS), which refers to a group of FCS variants in which the detection volume is scanned through the sample. They can be classified in strategies where the scan path is on the membrane or where the beam is scanned across the membrane. In the first case, the residence time of the fluorophores in the focal volume is shortened, so that photo-bleaching decreases. It also increases statistical accuracy due to parallel acquisition. In such an approach, information about the scanning speed substitutes the calibration of the detection volume and absolute diffusion coefficients and area concentrations of the fluorophores are obtained. In the second strategy the laser beam illuminates the membrane only when it passes through and longer acquisition times are possible. This is especially relevant in the case of slow diffusing species typical of lipid membranes and importantly, it corrects for membrane movements.