Final Report Summary - APOQUANT (The quantitative Bcl-2 interactome in apoptosis: decoding how cancer cells escape death)
Bax and Bak are required for MOMP and cells lacking these two proteins fail to die in response to apoptotic triggers. In healthy cells, Bax and Bak are inactive monomers that shuttle between the cytosol and the mitochondrial outer membrane (MOM) with different rates, which effectively makes Bax mostly cytosolic and Bak mostly MOM-associated. During apoptosis, they accumulate at the MOM, where they undergo a conformational change, oligomerize and cluster into discrete foci, called here apoptotic foci, which correlate with MOMP. In the frame of the ERC Starting Grant, we shed new light on how Bax mediates MOMP. We proposed that upon activation Bax dimers arrange around the edge of the membrane pore like a “clamp”, which contributes to the stabilization of the high curvature at the pore rim. In addition, we discovered that active Bax at the MOM exists as a mixture of oligomers based on dimer units, which form large, stable membrane pores tunable in size. At the nanoscale, Bax oligomers at apoptotic foci assembled into lines, arcs and rings, with both arcs and rings being able to perforate the membrane. Altogether, our data support a new molecular mechanism in which Bax fully or partially delineates pores of different sizes to mediate MOMP.
We also addressed the question how the intricate, fine-tuned interaction network formed by the Bcl-2 family of proteins determines cell fate using novel, quantitative systems approaches. Using fluorescence cross correlation spectroscopy, We quantified the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, and showed that membrane insertion drastically alters the pattern of Bcl-2 complexes. Our study disentangled the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.
We also addressed the question how the intricate, fine-tuned interaction network formed by the Bcl-2 family of proteins determines cell fate using novel, quantitative systems approaches. Using fluorescence cross correlation spectroscopy, We quantified the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, and showed that membrane insertion drastically alters the pattern of Bcl-2 complexes. Our study disentangled the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.