Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism

https://www.nature.com/articles/s41556-017-0023-x

Arnaud Ahier, Chuan-Yang Dai, Andrea Tweedie, Ayenachew Bezawork-Geleta, Ina Kirmes & Steven Zuryn

• The authors demonstrate a technique called Cell-specific mitochondrial affinity purification (CS-MAP) to yield intact, functional, mitochondrial with >96% enrichment, >96% purity at single-cell and single-animal resolution in C. elegans.
• CS-MAP consists of tagging the outer mitochondrial membrane protein TOMM-20 with a fluorophore and a particular epitope (which is something that an antibody can bind to) called HA. They placed the fusion protein under the control of a tissue-specific promoter so that e.g. muscle-specific mitochondria could be tagged. Mitochondria can then be purified using magnetic beads coated with anti-HA antibody and performing immunoprecipitation.
• The authors crossed worms containing the CS-MAP construct with animals containing mitochondrial DNA deletions, and analysed the heteroplasmy in the individual mitochondria purified from different cell types and compared this to homogenate heteroplasmy across the whole animal. The authors found that intestine and neurons had significantly lower heteroplasmy than the homogenate, whereas the germline had significantly higher heteroplasmy than the homogenate.
• The authors also quantified mtDNA copy number per mitochondrion in a number of different tissues, finding that the germline had ~3.5 mtDNAs per mitochondrion whereas tissues such as neurons and the intestine had ~1.5 mtDNAs per mitochondrion.
• Using three-dimensional reconstruction from fluorescence images, the authors found that individual germ cells contained 71.2+/-6.5 mtDNAs per cell, whereas neurons contained 14.4+/-0.5 mtDNAs per cell.
• The authors suggest that mtDNA turnover is higher in germ cells, which may account for their observations of increased copy number and heteroplasmy in the germline
  

Thoughts:

Check out a pre-print from our group with some ideas on how increases in copy number may be related to heteroplasmy, and thoughts about comparisons between homogenate heteroplasmy and cellular heteroplasmy