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Identification and characterization of mitochondrial subtypes in C. elegans via analysis of individual mitochondria by flow cytometry Joseph Renner Daniele, Kartoosh Heydari, Edgar Augusto Arriaga, and Andrew Dillin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00542 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016
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Analytical Chemistry
Identification and characterization of mitochondrial subtypes in C. elegans via analysis of individual mitochondria by flow cytometry Joseph R. Daniele1, Kartoosh Heydari2, Edgar A. Arriaga*3 & Andrew Dillin1
1. Department of Molecular & Cellular Biology, University of California, Berkeley Berkeley, CA 94720
2. LKS Flow Cytometry Core, Cancer Research Laboratory, University of California, Berkeley Berkeley, CA 94720
3. Department of Chemistry, University of Minnesota Minneapolis, MN 55455
* = Corresponding Author:
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Analytical Chemistry
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Abstract Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of mitochondrial membrane potential (∆ψ), 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 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 ∆ψ in C. elegans during development and in tissuespecific 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 ∆ψ 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.
Keywords: isolated organelle, mitochondrial subtypes, flow cytometry, individual organelle analysis, tissue-specific organelle analysis
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Introduction Understanding and measuring the bioenergetics of an organism is vital to the analysis and characterization of a wide array of cellular and physiological phenomena. Many of these processes, such as aging, metabolism, and stress-resistance, depend heavily on the functionality of mitochondria. Although it is clear that mitochondrial dysfunction is prevalent in brain, muscle, and liver1,2 in multiple mitochondrial diseases, methodologies that are both widely applicable to diverse animal models and used to characterize mitochondrial heterogeneity and tissue-specific variation in mitochondrial function are lacking. A central feature of mitochondrial function and bioenergetics is their membrane potential (∆ψ). Flow cytometry has been used to measure mitochondrial ∆ψ in single cells and in isolated mitochondria3–12. While the identification and characterization of mitochondrial subtypes is paramount to understanding the pathology and tissue etiology of mitochondrial disease, the ability to isolate and characterize mitochondria and characterize mitochondrial ∆ψ from the nematode Caenorhabditis elegans (C. elegans), one of the most important biological models of disease and aging, has not been demonstrated. In C. elegans ∆ψ perturbation has been implicated in lifespan extension, fertility, apoptosis, and in a related species (C. briggsae) has been found essential to increased survival in tropical environments and resistance to stress13–18. Due to its small size, fast generation time, transparent cuticle, and multicellularity (nervous system, digestive system, musculature), C. elegans is an excellent biological model for various fields of biology, including aging and mitochondrial research. Most of the work linking mitochondrial characteristics to phenotypes in C. elegans has been done via microscopy. Studies utilizing isolated mitochondria are scarce, likely due to the lack of well characterized and effective methods to isolate high quality mitochondria from nematodes, which possess a remarkably tough outer cuticle and distinct morphological features at each stage of development. Although flow cytometry has been applied to analyzing isolated nematode mitochondria for reactive oxygen species 19, here we define a new flow cytometry approach to [1] analyze the membrane potential of C. elegans mitochondria obtained through an improved isolation procedure and [2] demonstrate the potential of this approach to analyze isolated mitochondria from different larval stages and specific nematode tissue. With this approach, we characterize two mitochondrial subtypes, which arise during larval development and conclude that ∆ψ is higher in muscle than in mitochondria pooled from the entire organism. While we applied this approach to C. elegans because of its prevalence as a model for development, disease pathology, toxicity, and aging, it is envisioned that it could also be applied to monitor the ∆ψ of mitochondria isolated from cell cultures, other model organisms, or tissue biopsies thereby providing a new tool to improve our understanding on the role of mitochondrial bioenergetics, which is critical in multiple fields of biomedical research.
Experimental Section Worm Strains and Reagents The worm strain used in most experiments was N2 wild-type obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). The ratiometric mitochondrial membrane potential sensor dye JC-9 (3′dimethyl-\u03B1-naphthoxacarbocyanine iodide) (D-22421) and mitochondrial labeling dyes MitoTracker Red CMXRos (M-7512) and MitoTracker Green FM (M-7514) were purchased from Life Technologies (now Thermo Fisher) while valinomycin (V0627) and Levamisole Hydrochloride (31742) were from Sigma Aldrich, and Collagenase Type 3 (“collagenase 3”, CLS 3- LS004182) was purchased from Worthington. Reagents used in worm lysis included Protease Inhibitor (539134, Calbiochem), glass 3 ACS Paragon Plus Environment
Analytical Chemistry
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pasteur pipettes (9 inch, VWR), a metal dounce grinder (2 mL, Wheaton), and a glass homogenizer (2 mL with small clearance pestle (0.07 mm), Kimble chase). Mitochondrial Isolation and Flow Experiments Worm staging was performed at 20º C in line with previously published developmental timing and worm growth media 20,21. Before lysis, worms of the specified stages were washed off plates and resuspended in filter-sterilized Collagenase 3 buffer [100 mM Tris-HCl, pH 7.4, 1 mM CaCl2] with or without Collagenase 3 enzyme (1 mg/mL) and gently agitated for 1 hour at 20º C 22. Following treatment, the active collagenase was diluted out with 3 washes of autoclaved M9 [42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, and 1mM MgSO4.7H2O]. Mitochondria were isolated via a differential centrifugation protocol modified from Gandre & van der Bliek 200723. To allow for depolarization, the mitochondrial isolation buffer was modified to include K+ but still preserve osmolarity; filter sterilized Mitochondria 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), Protease Inhibitor]. N.B. It has been reported previously that isolated mitochondria can polarize and respire normally in the absence of an ETC substrate (e.g. succinate) as long as a “permeant ion” such as K+ is present in the buffer24–26. Further, the presence of this ion facilitates valinomycin (a K+ ionophore) mediated depolarization of mitochondria. Of note, we found JC-9 dye to be incompatible with CCCP (or FCCP) since treatment with this depolarizing agent appeared to dramatically affect the amount of JC-1 in solution which ultimately could alter the red/green ratio of mitochondria27. Perhaps this is why valinomycin has often been used in conjunction with JC dyes27–29. Briefly, M9 from previous washes was replaced with MIB and worms were homogenized using a glass homogenizer for 18 strokes. In Figure 1, a metal dounce grinder was used to homogenize worms (also 18 strokes). Lysates were then transferred to Eppendorf tubes (using a glass Pasteur pipette) and JC-9 dye [final 10 µM] (or MitoTracker Red [final 10 µM] for Figure 1) was added. Tubes were then spun at 200xG for 5 minutes at 4º C on a tabletop centrifuge. Supernatant was removed and transferred to new tubes and subsequently spun at 800xG for 10 minutes at 4º C. The supernatant from these tubes was spun once more at 12,000xG for 10 minutes at 4º C. Finally, this supernatant was removed and mitochondria pellets were resuspended in filtered MIB and kept on ice before running on the flow cytometer (or prepared for Western blotting). For flow experiments, a portion of the resuspended mitochondria was reserved and set aside on ice. Once all samples were run, valinomycin [final 10 uM] was added to these set-aside tubes 3-5 minutes before each tube was run. All “depolarized” sample controls were run in this manner. A tube containing filtered MIB and JC-9 (or MitoTracker Red) alone was also included in these spins to use as a dye-alone control to run during flow experiments. Another tube with lysate but no JC-9 was also included in the spins to control for unlabeled mitochondria. Data Acquisition and Statistical Analysis Flow data was acquired using an LSR Fortessa Analyzer using the forward scatter (FSC), side scatter (SSC, “Granularity”) (488 nm/10), Pacific Blue (“Blue channel”) (450 nm/50), FITC (“Green channel”) (525 nm/50 with a 500 nm Long Pass filter), and PE-Tx-Red YG (“Red channel”) (610 nm/20 with a 600nm Long Pass filter) filters. Although all data points were recorded, lasers and acquisition settings were calibrated to nullify any signal from unlabeled mitochondria in the fluorescent channels used. For all experiments, only singlets (or single mitochondria) were used when creating any plots or performing any statistical analyses. FlowJo v10 was used to process the data and prepare contour plots. A black background provided a better contrast for visualization of multiple contour lines. For the experiments using blue-labeled (“BFP positive”) mitochondria, only blue fluorescent mitochondria that had blue signal and green signal (to indicate JC-9 labeling) above background were counted as BFP positive mitochondria. Microsoft Excel (with the Sigma XL add-on) was used to perform statistical analyses. 4 ACS Paragon Plus Environment
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Significance testing was performed using the t-test for comparison between means and the non-parametric Mann Whitney test was performed for comparisons between medians. Error bars were included in select figures to represent the standard error of the Mean (SEM) or the median absolute deviation (MAD) when means or medians, respectively, were the main output. Error bars were included when ‘not significant ‘(ns) comparisons prevailed (see Figures 1B, 3D, 3E, 3F, S1B, S1C, S1D, and S3E). For comparisons that proved statistically significant we included only symbols (e.g. ‘*’ = p
