(366a) Characterization of Membrane Potential of Individual Mitochondria by Capillary Electrophoresis and Capillary Isoelectric Focusing

Mitochondrial dysfunction has been associated with many diseases and the aging process. In cells and tissue, mitochondria are heterogeneous organelles forming subpopulations that exhibit differences in morphology, protein expression, respiratory capacity, and oxidative stress. These subpopulations of mitochondria may contribute differently to cellular dysfunction. Mitochondrial membrane potential is known to vary among mitochondria within a cell and among different subpopulations of damaged and dysfunctional mitochondria. Imaging techniques using mitochondrial-specific probes and fluorescence microscopy are useful to evaluate differences in mitochondrial membrane potential, but lack the ability to separate individual mitochondria for further characterization. We have used electrophoretic methods such as capillary electrophoresis with laser-induced fluorescence (CE-LIF) and capillary isoelectric focusing (cIEF) to separate individual mitochondria based on differences in their electrophoretic properties arising from variation in their surface composition. These techniques have the potential to simultaneously characterize mitochondrial electrophoretic properties and membrane potential when mitochondria are treated with membrane potential sensitive probes.

In this work, we are investigating the use of a ratiometric fluorescent probe sensitive for mitochondrial membrane potential, JC-1, for detection of individual mitochondria in CE and cIEF. Mitochondria from cultured L6 rat myoblast cells will be loaded with JC-1, injected into the capillary and separated with either CE or cIEF. Migration time and fluorescence from two channels will be recorded for individual mitochondria; this data will be used to determine electrophoretic mobility (CE) or isoelectric point (cIEF), and mitochondrial membrane potential. Distributions of both measurements will be used to determine the dependence between membrane potential and the electrophoretic parameters. Information on the distributions of membrane potential and the potential purification of mitochondria using electromigration techniques can lead to better understanding of mitochondrial turnover and dysfunction during disease and aging. This work is funded by the National Institutes of Health (R01-AG20866).