Simard 2018 J Vis Exp
|Simard CJ, Pelletier G, Boudreau LH, Hebert-Chatelain E, Pichaud N (2018) Measurement of mitochondrial oxygen consumption in permeabilized fibers of Drosophila using minimal amounts of tissue. J Vis Exp 134.|
Abstract: The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, Drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.
• Keywords: Biochemistry, Mitochondria, Electron transport system, Metabolism, High-resolution respirometry, Drosophila melanogaster, Permeabilization • Bioblast editor: Plangger M • O2k-Network Lab: CA Moncton Boudreau L, CA Moncton Hebert-Chatelain E
Labels: MiParea: Respiration, Instruments;methods
Preparation: Permeabilized tissue
Coupling state: LEAK, OXPHOS Pathway: N, Gp, CIV, NS, ROX HRR: Oxygraph-2k