The proteins of the Bcl-2 family function as key regulators of apoptosis by controlling the permeabilization of the mitochondrial outer membrane. They form an intricate, fine-tuned interaction network which is altered in cancer cells to avoid cell death. Currently, we do not understand how signaling within this network, which combines events in cytosol and membranes, is orchestrated to decide the cell fate. The main goal of this proposal is to unravel how apoptosis signaling is integrated by the Bcl-2 network by determining the quantitative Bcl-2 interactome and building with it a mathematical model that identifies which interactions determine the overall outcome. To this aim, we have established a reconstituted system for the quantification of the interactions between Bcl-2 proteins not only in solution but also in membranes at the single molecule level by fluorescence correlation spectroscopy (FCS).(1) This project aims to quantify the relative affinities between an reconstituted Bcl-2 network by FCS.(2) This will be combined with quantitative studies in living cells, which include the signaling pathway in its entirety. To this aim, we will develop new FCS methods for mitochondria.(3) The structural and dynamic aspects of the Bcl-2 network will be studied by super resolution and live cell microscopy.(4) The acquired knowledge will be used to build a mathematical model that uncovers how the multiple interactions within the Bcl-2 network are integrated and identifies critical steps in apoptosis regulation.These studies are expected to broaden the general knowledge about the design principles of cellular signaling as well as how cancer cells alter the Bcl-2 network to escape cell death. This systems analysis will allow us to predict which perturbations in the Bcl-2 network of cancer cells can switch signaling towards cell death. Ultimately it could be translated into clinical applications for anticancer therapy.