ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells

INTRODUCTION Mitochondria are endosymbiotic organelles that have their own genome and perform many essential functions in eukaryotic cells, including ATP synthesis via oxidative phosphorylation. Mitochondria evolved from bacteria, which stringently replicate and segregate their genome in a binary fission process. The residual circular ~16-kilobase human mitochondrial genome is essential, as it encodes mitochondrial ribosomal and transfer RNAs and respiratory chain complex proteins. Within human cells, hundreds to thousands of copies of mitochondrial DNA (mtDNA) are packaged into nucleoids, the unit of mtDNA inheritance, and distributed within dynamic mitochondrial networks. The nature of mtDNA transmission in mitochondrial syncytia is relaxed, with replication occurring asynchronously throughout the cell cycle and also in postmitotic cells. If and how nucleoids are actively chosen for mtDNA replication and distributed within mitochondrial networks is not understood. This question is highly relevant for understanding the basis of human metabolic diseases caused by mutations in mtDNA and in nuclear genes that affect mtDNA maintenance. In addition, aging and neurodegenerative disorders are also linked to defective mtDNA maintenance and mitochondrial dysfunction. In this study, we investigated the fundamental process of mtDNA transmission in mammalian cells. RATIONALE To address the cellular mechanism for mtDNA transmission, we examined whether nucleoid and mitochondrial distribution were coupled. Mitochondrial distribution is determined by mitochondrial division, fusion, and motility events. Previous work established that the mitochondrial division site placement is not random and is instead spatially marked by regions of contact between the endoplasmic reticulum (ER) and mitochondria, in a process termed ER-associated mitochondrial division (ERMD). In biological systems, division events serve a fundamental role in the inheritance of genetic material. Thus, we addressed whether ERMD serves to facilitate the transmission of mtDNA by examining the behavior of mitochondria, ER, and nucleoids in mammalian cells via fluorescent microscopy. We marked the subset of nucleoids in cells actively engaged in mtDNA synthesis with a functional green fluorescent protein–tagged version of POLG2, the processivity subunit of the human mitochondrial DNA polymerase holoenzyme. Using this sensitive and highly specific marker for mtDNA synthesis in live cells, we asked whether replicating nucleoids were selectively linked to ERMD for the purpose of ensuring the segregation of nascent mtDNA to daughter mitochondria. RESULTS Our work revealed that nucleoids actively engaged in mtDNA synthesis in mammalian cells were spatially and temporally linked to a small subset of ER-mitochondria contacts destined for mitochondrial division. At division sites, mtDNA replication occurred upstream of mitochondrial constriction and assembly of the division machinery. Nucleoids containing nascent mtDNA localized to mitochondrial tips, and these products of division were preferentially distributed within cells as compared with nonreplicative nucleoids. Our observations also demonstrated that ER structure and mtDNA maintenance were intertwined; ER tubules proximal to nucleoids were necessary but not sufficient for mtDNA synthesis and also functioned in nucleoid distribution. CONCLUSION We propose that, at ER-mitochondria contacts destined for division, the consecutive events of mtDNA replication, mitochondrial division, and mitochondrial motility are connected together to ensure the accurate distribution of nucleoids within cells. Our findings suggest that ER-mitochondria contacts coordinate the licensing of mtDNA replication with downstream mitochondrial division events to distribute newly replicated mtDNA to daughter mitochondria. The connection revealed between ER structure and mtDNA replication and distribution has broad implications for understanding human cellular homeostasis and the cellular pathology underlying human diseases.