Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry

Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (δ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of δ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in δ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.