Thursday, 12 March 2015

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

http://nar.oxfordjournals.org/content/43/4/2177.full.pdf

Anitha D Jayaprakash, Erica Benson, Swapna Gone et al.

Deep sequencing allows the measurement of mtDNA heteroplasmy at a single-cell level. However, regions of mtDNA exist in the nuclear genome (nDNA), called Numts. This is due to ancestral transfer of genetic material from the mitochondria to the nucleus. Numts may have variable sequence and copy number, so failure to separate nDNA from mtDNA can cause inaccuracy in heteroplasmy measurement. Although methods currently exist to deal with this, it is unclear whether they are able to resolve heteroplasmies below 5%.

The authors present a method called Mseek, to enzymatically digest linear nDNA and leave behind circular mtDNA, to a purity >98% (whereas endogenously, mtDNA can form <1% of genetic material). Thus ability to resolve different haplotypes is only limited by sequencing depth.  By inspecting multiple cell lines, the authors found that heteroplasmy is a ubiquitous phenomenon, with most mutations being transitions, indicating replication errors of polymerase-γ.

To determine whether heteroplasmy in cull culture originates from a population of cells homoplasmic for different mutations, or a population of heteroplasmic cells, the authors performed the following experiment. They extracted two single cells from a colony, and passaged them for ~25 generations to derive two new colonies. They then measured single-cell heteroplasmy levels for the two new colonies. They found that the derived colonies were heteroplasmic and had similar haplotype distributions. They show that a simple computational model of random genetic drift would shift the haplotype distribution by a large extent over this many generations. Their observations imply that individual cells are heteroplasmic, and this heteroplasmy is relatively stable over a time scale of ~25 generations. There is therefore a mechanism to counteract random drift.

Exchange of mtDNA between cells could bring the haplotype distribution closer to the average across the population. To test this hypothesis, the authors co-cultured cell lines with distinct haplotype distributions, one of which was GFP-labelled. After 4 weeks of co-culture, one of the cell lines was selected for (either with FACS or antibiotic-resistance), and then cultured for a further 4 weeks. In two of four pairs of cell lines tested, they found transfer of mtDNA from one cell line to the other. In the other two pairs, there was no genetic transfer. Thus mtDNA transfer does appear to occur, but is not universal. Furthermore, it is not a necessary mechanism to counteract genetic drift, but it is perhaps sufficient.

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