Analysis of individual mitochondria and acidic organelles by capillary electrophoresis with laser-induced fluorescence detection.

摘要

Reported herein is the application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) to separate and detect individual mitochondria and acidic organelles (a term referring collectively to lysosomes and endosomes) from an array of mammalian cultured cells and tissues. Due to the presence of charged groups in their outer membranes, isolated organelles have intrinsic electrophoretic mobilities that are influenced by variations in size, morphology, membrane composition and damage caused during the isolation procedure. Histogram analysis of mitochondrial electrophoretic mobilities gives distributions that vary depending on the sample analyzed and the method used to release the mitochondria from the cells. In addition to characterizations based on mobility, depending on the fluorescent tag used, peak heights can be correlated to physical parameters of the individual organelles. Mitochondria that were stained with a fluorescent dye that stoichiometrically binds to cardiolipin were separated and detected by CE-LIF and the cardiolipin contents of individual mitochondrial events were determined. Similarly, acidic organelles were labeled via endocytosis of 0.1 μm i.d. fluorescent microspheres. The localization of the microspheres within acidic organelles was confirmed using confocal microscopy by the co-localization of the endocytosed microspheres with a lysosomally targeted fluorescent probe. CE-LIF experiments show that the fluorescence content of individual acidic organelles is dependent upon the concentration of microspheres within the cell growth media. CE-LIF with dual channel detection was used to separate and simultaneously detect mitochondria and acidic organelles from cell lysate. Finally, molecular biology techniques were developed to amplify mtDNA from small pools of mitochondria (typically less than five mitochondria) collected following CE-LIF separation and evaluate the prevalence of mutations.