Final Activity Report Summary - tBid,CL and cyt c (Mitochondrial membrane reorganisation (...): cardiolipin and cytochrome c redistribution analysed by single molecule spectroscopy and microscopy) As a Marie Curie fellow, my work focused on the quantitative study of dynamic processes in membranes via molecular imaging. This included the lateral organisation of membranes, i.e. the formation of lipid domains and rafts, and the study of apoptotic signalling processes, i.e. the pore formation by Bax fragments and the interactions between Bcl-2 proteins. With the aim of quantitative biology, during this period I acquired deep insight in single molecule techniques, such as atomic force microscopy and fluorescence correlation spectroscopy, combined with advanced imaging techniques like confocal microscopy, spinning disc, fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET) and live cell imaging. The Bcl-2 proteins are key regulators of apoptosis, which is a form of programmed cell death. They control the release of the apoptotic factors from the mitochondria that initiate apoptosis. Two important questions about the mechanism of the Bcl-2 proteins involve the interaction network between pro- and anti-apoptotic family members as well as the role of their translocation to the mitochondrial outer membrane during apoptosis. By quantifying the interactions of Bid and tBid with Bcl-xLdeltaCt in solution and membranes using fluorescence correlation spectroscopy, we found that: 1. only the active form tBid bound to Bcl-xLdelatCt, and 2. the membrane strongly promoted binding between them. Particularly, a BH3 peptide from Bid disrupted the tBid and Bcl-xL complex in solution, but not in lipid bilayers. These data indicated that tBid and Bcl-xL interactions in solution and lipid membranes were not equivalent and supported a model in which Bcl-xL inhibition of tBid happened predominantly at the membrane. Our findings implied an active role of the membrane in modulating the interactions between Bcl-2 proteins that was so far underestimated.
