Bioanalytical techniques for the analysis of mitochondrial heterogeneity

In this thesis, techniques are described for the measurement of individual mitochondrial isoelectric point (pI) using capillary isoelectric focusing (cIEF) with laser-induced fluorescence detection, simulation of the contribution of mitochondrial surface compositions to p I, and determination of individual mitochondrial membrane potential and electrophoretic mobility using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). These techniques provide insight into mitochondrial heterogeneity, which could increase fundamental understanding of the role played by mitochondria in aging.;A method was developed to determine the pIs of individual mitochondria by cIEF. This method provides reproducible distributions and accurate determination of individual mitochondrial pI by the use of internal standards, and was able to detect changes in mitochondrial pI distributions caused by changes to the mitochondrial surface by treatment with trypsin. Application of this method demonstrated the heterogeneity of mitochondrial pI, which reflects the heterogeneity of mitochondrial surface compositions.;To model the effect of surface composition on mitochondrial p I heterogeneity, a method was developed to predict mitochondrial pI values using simulated surface compositions consisting of different percentages of amino acids and phospholipids found in the mitochondrial outer membrane. This method was validated by predicting the pI values of known mitochondrial outer membrane proteins then extended to isolated mitochondria and used to model a pI distribution determined experimentally by cIEF. Significant changes in the percentages of some amino acids and phospholipids were predicted for observed pI differences between individual mitochondria. This model provides insight into the heterogeneity of mitochondrial pI and contribution of surface compositions.;Distributions of individual mitochondrial membrane potential and electrophoretic mobility were measured using CE-LIF. Mitochondria from cultured cells and mouse muscle and liver tissue were labeled with JC-1, a ratiometric dye which indicates membrane potential. Analysis of specific regions of interest defined by performing CE-LIF of depolarized samples makes this method capable of analyzing mitochondrial membrane potential even in preparations where depolarized mitochondria may be present due to biological variation or experimental factors that result in damage to mitochondria or may be insufficient to keep all mitochondria polarized. This analysis revealed additional differences between samples and an effect of membrane potential on electrophoretic mobility. This method allows for the characterization of mitochondrial heterogeneity in membrane potential and surface properties.;In the future, these methods can be applied to biological models of aging to elucidate the role that mitochondrial heterogeneity plays in age-related dysfunction and disease.