Insights into the Regulation of the Mitochondrial Apoptotic Pore

Abstract

Apoptotic pore formation by the BCL-2 executioners BAX and BAK at the mitochondrial outer membrane represents the decisive step of intrinsic apoptosis. This process is accompanied by extensive mitochondrial fragmentation mediated by the dynamin-like protein DRP1, which colocalizes with BAX on mitochondria during apoptosis. Following outer membrane permeabilization, mitochondrial inner membrane extrusion and permeabilization allows the release of mitochondrial DNA into the cytosol, which can trigger inflammatory signaling. However, the mechanism by which BAX and BAK assemble to form apoptotic pores, the molecular basis and functional consequences of the apoptotic interplay of BAX and DRP1, as well as the processes underlying mitochondrial inner membrane extrusion and permeabilization, remain obscure. Here, we reveal a direct physical interaction between BAX and DRP1 that is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs specifically in the membrane environment, requires the N-terminal region of BAX, and promotes the membrane-remodeling activity of both proteins. Forced dimerization of BAX and DRP1 is sufficient to trigger their activation and mitochondrial translocation, inducing mitochondrial fragmentation and permeabilization in the absence of canonical apoptotic triggers. These findings identify DRP1 as a noncanonical, direct activator of BAX through physical engagement with its N-terminal region. We further uncover that BAX and BAK exhibit distinct properties of apoptotic pore formation. BAK oligomerizes with faster kinetics into smaller structures than BAX, and nucleates and accelerates BAX assembly into oligomers that continue to grow during apoptosis. Both proteins co-assemble into the same apoptotic pores, where their relative abundance determines pore growth rates and the relative kinetics of mitochondrial content release, specifically of the mitochondrial DNA. This cooperative mechanism modulates the activation of the cGAS/STING pathway, linking apoptotic pore formation to inflammatory signaling. In addition, we developed a multimodal super-resolution approach, termed CorreLative Oligomerization STED Electron (CLOSE) microscopy, which enables the correlative assessment of the stoichiometry of individual protein complexes and their nanoscale structural assembly, as well as alterations in the underlying membrane ultrastructure. Using CLOSE, we characterize the oligomeric state and nanoscale structural organization of BAK within individual apoptotic pores in relation to the mitochondrial membrane ultrastructure. We find that the geometry of the outer membrane opening defines the spatial arrangement of BAK assemblies and that mechanical forces drive inner membrane extrusion through the apoptotic pore. Conversely, the inner membrane exerts forces on the outer membrane, promoting further pore expansion. These results demonstrate mechanical coupling between mitochondrial membranes that governs apoptotic pore growth and provide a biophysical framework for inner membrane extrusion, defining the structural organization of the apoptotic pore. Together, our findings identify DRP1 as a noncanonical activator of BAX and uncover fundamental mechanistic principles of apoptotic pore formation by BAX and BAK. We propose a multimodal model in which cooperative BAX/BAK oligomerization nucleates pore opening and expansion through an oligomerization-dependent process. Once the apoptotic pore reaches a critical diameter, inner membrane extrusion and membrane mechanics drive further pore growth, thereby coupling outer and inner membrane permeabilization and modulating downstream immune signaling.