Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro [MANUSCRIPT].
Lentz SI, Edwards JL, Backus C, McLean LL, Haines KM, Feldman EL
J Histochem Cytochem (2009)
Category: methods, mitochondrial DNA ¤ Added: Nov 05, 2009 ¤ Rating: ◊◊
Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to visualize newly synthesized mtDNA in individual cells in order to study mtDNA replication within subcellular compartments of neurons. The technique combines the incorporation of BrdU and/or EdU into mtDNA together with a tyramide signal amplification protocol. Employing this technique, we visualized and measured mtDNA biogenesis in individual cells. The labeling procedure for EdU allows for more comprehensive results by allowing the comparison of its incorporation with other intracellular markers because it does not require harsh acid or enzyme digests necessary to recover the BrdU epitope. In addition, the utilization of both BrdU and EdU permits sequential pulse-chase experiments to follow the intracellular localization of mtDNA replication. The ability to quantify mitochondrial biogenesis provides an essential tool for investigating alterations in mitochondrial dynamics involved in the pathogenesis of multiple cellular disorders including neuropathies and neurodegenerative diseases.