There has been an explosion of interest in the role of cell death pathways and damage-associated molecular pattern (DAMP) signaling in shaping inflammatory and immune responses. Mitochondria are central to the intrinsic apoptosis pathway, the classical form of programmed cell death. Several mitochondrial constituents have been implicated as DAMPs, including mitochondrial DNA (mtDNA). Recent work has shown that activation of intrinsic BAK and BAX–mediated apoptosis results in mtDNA-dependent triggering of the innate immune cGAS/STING pathway, resulting in type I interferon production by dying cells. The apoptotic caspase cascade normally functions to suppress this mtDNA-induced cGAS/STING signaling, rendering apoptosis “immunologically silent.”

It is thought that during apoptosis, mtDNA is released into the cytoplasm. In addition to apoptosis, loss of mtDNA from the matrix has been associated with conditions including HIV and dengue infection, calcium overload, irradiation, or inflammatory diseases such as systemic lupus erythematosus or rheumatoid arthritis. However, mtDNA escape from the mitochondria has not been documented in real time.

Using a combination of live-cell lattice light-sheet microscopy, 3D structured illumination microscopy, correlative light electron microscopy, and electron cryotomography, we found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner membrane an outlet through which it herniated, carrying with it mitochondrial matrix components, including the mitochondrial genome. A subset of the herniated inner membranes lost their integrity, allowing mtDNA to be exposed to the cytoplasm.

An extensive literature suggests that mtDNA is found outside the mitochondria—and, indeed, outside the cell—in a wide range of circumstances. Our study provides a mechanistic description of its release from the mitochondria. mtDNA release from mitochondria during apoptosis occurs irrespective of caspase activity, but in normal cells, caspases attenuate the subsequent cGAS/STING-mediated interferon response by driving rapid cellular collapse and clearance. Mitochondrial herniation might represent a general mechanism of mtDNA escape. In addition to BAK and BAX oligomerization, there may be alternative triggers—for example, other pore-forming proteins (host- or pathogen-derived) or mitochondrial stresses—that lead to the occurrence of this phenomenon.

Inset images show the key stages as captured by lattice light-sheet microscopy, structured illumination microscopy, and correlative light and electron microscopy.