A nanoscale, multi-parametric flow cytometry based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics

https://www.nature.com/articles/s42003-019-0513-4

Julie A. MacDonald, Alisha M. Bothun, Sofia N. Annis, Hannah Sheehan, Somak Ray, Yuanwei Gao, Alexander R. Ivanov, Konstantin Khrapko, Jonathan L. Tilly, and Dori C. Woods

• The authors describe a new technology for isolation and analysis of single mitochondria using flow cytometry, called "fluorescence-activated mitochondria sorting" (FAMS).
• Mitochondria isolated from liver tissue exhibited intact outer and inner membranes, and cristae structure, when evaluated by electron microscopy.
• Staining samples with the DNA stain DAPI, the authors found correlation between side-scatter of organelles and DNA content, suggesting that larger organelles, containing larger amounts of DNA, have larger side-scatter. 
• The authors used the membrane potential sensor dye JC-1 to categorise mitochondria into high/low membrane potential populations. They found that whilst both low and high-membrane potential populations generated ATP when provided with ADP, high-membrane potential mitochondria produced approximately x6 more ATP, and approximately x3 more Mt-ND1 and Mt-Nd4, than low-membrane potential mitochondria. The low-membrane potential mitochondria had ~2.5x lower FSC-PMT, potentially indicating their smaller size [Question: do differences in mitochondrial size confound the inference of differential membrane potential using the JC-1 dye, due to the surface area to volume ratio affecting the aggregation rate? If smaller mitochondria have a higher surface area to volume ratio then perhaps the true difference in mitochondrial membrane potential is even larger.]
• The authors generated mixed samples for two mouse strains, with two different mtDNA haplotypes, and performed single-molecule PCR. Of 54 organelles measured, 2 showed mixtures of mtDNA sequences, suggesting a relatively low rate of artificial fusion of mitochondrial in mixed samples.
• The authors measured the median number of mtDNAs per mitochondrion was 3, ranging from 1 to 22 molecules per sorted organelle.
• The authors used beads to calibrate FSC-PMT and SSC to define two gates: ~0.22-0.5 um, and 0.5-1um, and found that the small gate had approximately 1-2 mtDNAs per organelle, whereas the large gate had 6.5-7.5 mtDNAs per organelle.