Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry - PubMed (original) (raw)

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

Joseph R Daniele et al. Anal Chem. 2016.

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.

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Figures

Figure 1

Figure 1

Collagenase treatment enhances yield in C. elegans (worm) mitochondrial isolation. (A) Schematic of C. elegans life cycle to illustrate size and morphology differences. (B) Mitochondria were isolated from the L1 and L4 larval stages of worm development using either a metal dounce grinder (Metal), a glass dounce homogenizer (Glass), or a glass homogenizer following 1 h of collagenase 3 treatment (1 mg/mL) at 20 °C (Glass and Collag.). Bar plots depicting flow cytometric counts of MitoTracker Red (10 µM) positive mitochondrial yield (number of mitochondria per minute) from three technical replicates of mitochondrial isolations from larval L1 (white bars) and L4 (gray bars) worms using the isolation techniques described above. Error bars are SEM from three technical replicates. * = p < 0.05, *** = p < 0.0001, and ns = not significant.

Figure 2

Figure 2

JC-9 can label both polarized and depolarized isolated mitochondria. (A) JC-9 is a mitochondrial (Mito) dye that exists in a monomeric (green fluorescent) state but forms red fluorescent aggregates when mitochondria are polarized. Red fluorescence intensity increases as the mitochondrial membrane potential (Δψ) increases. (B) Contour plot showing separation of mitochondria from specific JC-9 dye aggregates alone (Blue) and L4 stage isolated mitochondria labeled with (Red) or without (Orange) 10 µM JC-9. (C) Contour plot showing distribution of isolated mitochondria from L4 stage worms labeled with JC-9 (Red) or with both JC-9 and the depolarizing ionophore valinomycin (10 µM) (Blue). (D) Representative use of the Red/Green JC-9 fluorescence ratio in a comparison of polarized and depolarized mitochondria. Median Red/Green JC-9 fluorescence ratio is ~1.3× higher for polarized mitochondria due to formation of more aggregate (red). N = 26274 mitochondria for polarized and N = 5455 mitochondria for depolarized. *** = p < 0.0001.

Figure 3

Figure 3

Characterization of mitochondrial subpopulations in worm development. (A) Mitochondrial Δψ is relatively high during larval L1–L3 in C. elegans development and then falls. D1 and D2 stand for adult Day 1 and Day 2, respectively. Depolarization (gray bars) by valinomycin (12 µM) treatment reduces Δψ. (B) Mitochondrial granularity (side scatter, SSC) is relatively high during L1 to L3. For parts (A) and (B), # = p < 0.0001 relative to previous stage (e.g., L2 vs L1), ‡ = p < 0.0001 relative to polarized, mitochondria within a stage. (C) Using the L4-D2 JC-9 distribution profiles, and Δψ range under depolarizing conditions (see Figure S3F), one can set a division point (white dotted line) to distinguish between mH (variable) and mL (consistent) mitochondrial subtypes. (D) Frequencies (% of JC-9 positive mitochondria) of high Δψ mitochondria (mH) and low Δψ mitochondria (mL) at each developmental stage. Values are averages from at least 3 biological replicates. Error bars are SEM, + = p < 0.01 relative to previous stage e.g. L2 to L1, and ns = not significant relative to previous stage. (E,F) Membrane potential, Δψ, of both the mH (3E, gray bars) and mL (3F, white bars) throughout worm development. Values in bar charts are medians unless otherwise specified. # = p < 0.0001 relative to previous stage (e.g., L2 vs L1), + = p < 0.01 relative to previous stage e.g. L2 to L1, and ns = not significant relative to previous stage. Bars are median absolute deviation (MAD). For polarized mitochondria, N = 34280 for Eggs, 7963 for L1, 18586 for L2, 22313 for L3, 23497 for L4, 23476 for D1, and 45393 for D2. For depolarized mitochondria, N = 11613, 8137, 9965, 16035, 27394, 30069, and 29851 for Eggs – D2, respectively.

Figure 4

Figure 4

Analysis of mitochondrial properties from specific C. elegans tissues. (A) At the L4 stage, unlabeled worms (empty worm) and worms expressing a blue fluorescent protein in their muscle mitochondria (blue worm) were lysed and mitochondria were isolated. (B) Flow cytometric analysis enabled the cutoff point (in the blue fluorescence channel) where only blue fluorescent mitochondria (which are also labeled with JC-9, light yellow) were detected. Relevant controls of JC-9 and valinomycin alone (blue), unlabeled mitochondria (orange), nonblue fluorescent mitochondria labeled with JC-9 (red), blue fluorescent mitochondria alone (dark green) are included in the plot to demonstrate the heightened blue and green fluorescent signal of JC-9-positive muscle mitochondria (light yellow). (C) Measurement of polarized (white bars) and depolarized (gray bars) mitochondrial membrane potential (Δψ) in muscle mitochondria compared to mitochondria isolated from all tissues (Total). Muscle (#4), Muscle (#8), and Muscle (#10) refer to different extrachromosomal lines expressing blue-fluorescently labeled mitochondria specifically in muscle tissue. *** = p < 0.0001 relative to all tissues Total mitochondria control, ‡ = p < 0.0001 relative to polarized, mitochondria within an isolate. For polarized mitochondria, N = 54839 for Total. 1235 for Muscle(#4), 823 for Muscle(#8), and 618 for Muscle(#10). For depolarized mitochondria N = 1176 for Total, 435 for Muscle(#4), 467 for Muscle(#8), and 392 for Muscle(#10).

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