Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA

Abstract

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (>90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by intercellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.

A Comparison of Mitochondrial DNA Isolation Methods in Frozen Post-Mortem Human Brain Tissue—Applications for Studies of Mitochondrial Genetics in Brain Disorders

“…Although Method E provided a greater enrichment than Method C (differential centrifugation) in mouse liver (18) and a similar enrichment to Method A (Percoll) in an osteosarcoma cell line (19), higher levels of mitochondrial enrichment have previously been reported (20). However, this study, like Method B in our study, relies on the circular nature of the intact mitochondrial genome, which, while present in cell lines and blood, may be more degraded in frozen, archived brain.…”

The universal non-neuronal nature of Parkinson's disease: A theory

“…Nevertheless, there is the possibility that a different biological entity is responsible for propagating the PD-state. Recently, the inter-cellular exchange of mitochondrial DNA (mtDNA) has been demonstrated 40,41 . We propose that mtDNA could be the vehicle for the inter-cellular transmission of the PD-state.…”

“…No specific mutations in mtDNA have been consistently associated with PD 48 . However, heteroplasmy of mtDNA, i.e., the presence of multiple mtDNA variants within a cell, is now widely appreciated 41 . Thus, undetected lower frequency mtDNA variants could potentially be involved in the transmission of the PD-state.…”

The complete mitochondrial DNA sequence of Pectenocypris sp. (Actinopterygii: Cyprinidae) from Serkap River, Sumatra, Indonesia

“…A small portion of muscle tissue was excised under the dorsal fin and preserved in ethanol for subsequent DNA extraction by Genomic DNA Mini Kit (Geneaid/ New Taipei City). Mitochondrial DNA was purified by digesting the linear nuclear DNA with the exonuclease V under the Mseek protocol (Jayaprakash et al 2015) and then sequenced using the NEXTflex TM Rapid DNA-Seq kit (Bioo Scientific/ Texas) with the Illumina NextSeq platform. This resulted in a single, circular DNA sequence which was then blasted against the Mitofish database (Iwasaki et al 2013) for the confirmation of a fish mitochondrial DNA.…”