Phase Separation of Mitochondrial DNA in the Premature Aging Disease Hutchinson-Gilford Progeria Syndrome

Abstract

Increased mutations and damage to mitochondrial DNA (mtDNA) have been linked to aging. Within the mitochondria, mtDNA is coated by proteins forming nucleoprotein complexes or nucleoids, which serve as the sites of mtDNA replication and transcription. Mitochondrial nucleoids are tightly regulated as they contain only 1-2 molecules of mtDNA and maintain a fixed size of approximately 100 nm. However, the biophysical properties of mtDNA organization and their contributions to aging are unknown. Here, we probe the phase separation of mtDNA nucleoids in a model of premature aging. We hypothesize that mtDNA nucleoids represent phase separated nucleoprotein complexes and that age-associated oxidative stress further drives nucleoid condensation in the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Using several microscopy techniques, we find that mitochondria in HGPS cells are swollen and damaged, exhibiting remarkably enlarged, spherical nucleoids, with diameters greater than 300 microns. Moreover, in response to photoinduced ROS generation, mitochondria begin to swell, while the nucleoids undergo rapid homotypic fusion events with neighboring nucleoids. Nucleoid coalescence and coarsening are consistent with the phase behavior of liquid-liquid phase separation. Additionally, we perform several assays to understand the functional consequences of nucleoid phase separation, including next generation sequencing of the entire mtDNA genome as well as mitochondrial functional markers for membrane potential and oxidative stress. We postulate that increased oxidative damage in HGPS cells alters the phase behavior of mitochondrial nucleoids contributing to mitochondrial dysfunction. These results shed light on how the physical properties and organization of mitochondrial nucleoids are influenced by oxidative stress, with potential implications for normal aging.