Development of an Optical Tweezers Methodology to Separate Single Cells and Single Mitochondria to Determine the Location of Heteroplasmy in Mitochondrial DNA

Abstract

Mitochondrial DNA heteroplasmies are well documented at the multi-cell level. Some have been linked to various degrees of chronic symptoms of mitochondria-based diseases. However, the mechanism producing heteroplasmy is undetermined. The presence of heteroplasmy may differ depending on the tissue examined (e.g., somatic cells may be heteroplasmic, while germ cells can be homoplasmic). The presence and degree of heteroplasmy is important in both the forensic and medical communities. The question of whether heteroplasmy is present in the single cell and in the single mitochondrion remains. We developed an effective protocol to isolate single human leukocyte cells and single mitochondria from those cells. The cells from an HL-60 cell culture were labeled with Mitotracker Green FM (tested and shown not to interfere with PCR), and individually separated using optical tweezers (5 watt, CW, Ytterbium (Yb) fiber laser) and an Axiovert 100 M fluorescent microscope. Once it was determined that the individual cells were heteroplasmic (PCR and sequencing showed a 50/50 C/T heteroplasmy at nucleotide position 12071), individual dyed mitochondria were separated by the optical tweezers method and tested in the same manner. Preliminary results suggest that single mitochondria also contain the heteroplasmy.