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

Abstract

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 (Δψ), 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 Δψ 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 Δψ 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.

DGAT1-Dependent Lipid Droplet Biogenesis Protects Mitochondrial Function during Starvation-Induced Autophagy

“…Mitochondria were isolated from four P150 (15 cm) plates of MEFs via a previously described differential centrifugation protocol (Daniele et al, 2016). Briefly, PBS from previous washes was replaced with filtered mitochondrial isolation buffer (MIB) [50 mM KCl, 110 mM Mannitol, 70 mM Sucrose, 0.1 mM EDTA (pH 8.0), 5 mM Tris-HCl (pH 7.4), and Protease Inhibitors (Calbiochem)] and cells were homogenized by five passes through a 27.5 gauge needle.…”

“…Once all “polarized” samples were run, valinomycin [final 12 uM] was added to these set-aside tubes for 3–5 min and then mitochondria were re-analyzed. JC-9 dye, similar to JC-1 dye, is incompatible with CCCP and FCCP, therefore valinomycin was used to depolarize mitochondria (Cossarizza et al, 1993; Daniele et al, 2016; Reers et al, 1991; Wolken and Arriaga, 2014). All “depolarized” sample controls were analyzed in this manner.…”

Mitochondrial Subtype Identification and Characterization

“…These include [1] a tube that consists only of MIB and JC-9 dye to identify pelleted dye aggregates and [2] a tube that consists only of lysate (no dye added) so that unlabeled mitochondria can be identified and [3] if using mitochondria that can be identified in any other way, e.g. with a blue fluorescent protein (see Daniele et al, 2016, Figure 4), include a “no dye” control of this lysate as well. …”

“…for tissue specific studies, as in Figure 4 of Daniele et al, 2016), one should include Pacific Blue (“Blue channel”) (450 nm/50) in the recorded channels. One should also run a sample that contains unlabeled, blue-fluorescent mitochondria (see Figure 2).…”

Sizing lipid droplets from adult and geriatric mouse liver tissue via nanoparticle tracking analysis

“…Flow cytometry and capillary cytometry are well-defined methods for determining heterogeneity among individual organelles[22–24]. To our knowledge, there has been no report of flow cytometry of individual lipid droplets, possibly due to their unique physiochemical properties including low specific gravity and tendency to adsorb to surfaces.…”