Characterization of Bax and Drp1 during apoptosis with advanced microscopy techniques

Abstract

Bax is a pro-apoptotic Bcl-2 family member that experiences a net translocation from the cytosol to mitochondria under cell stress. It inserts and oligomerizes irreversibly in the mitochondrial outer membrane (MOM) to mediate its permeabilization, leading to cytochrome c release and cell death. Bax translocation also correlates with massive mitochondrial fragmentation. Indeed, Bax colocalizes with the dynamin-related protein 1 (Drp1) at fission sites during apoptosis. However, the molecular basis behind Bax function and the nature of Bax structures responsible for MOM permeabilization remain poorly understood. In this thesis, we studied the nanoscale spatial organization of Bax at mitochondria of apoptotic cells using dual-color super-resolution microscopy. We also investigated Bax interplay with Drp1 at the single-molecule level by fluorescence cross-correlation spectroscopy (FCCS). We show that active Bax assembled into distinct architectures including full rings, arcs, and lines that localized in discrete foci along mitochondria in close association with Drp1 foci. The physiological relevance of these structures is supported by the different organization adopted by an inactive mutant that constitutively localizes at the MOM. Remarkably, both rings and arc-shaped oligomeric assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Moreover, in vitro FCCS assays with recombinant proteins in single-vesicle approaches threw light upon the interaction between Bax and Drp1, where the membrane environment has an active contribution. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the MOM. Altogether, our results contribute to the understanding of the interplay between the mitochondrial fission machinery and cell death regulation.