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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Published: May 19th, 2023

DOI:

10.3791/65337

1Department of Biochemistry and Molecular Biology, Faculty of Medicine, Institute for Medical Research, Israel-Canada (IMRIC), The Hebrew University of Jerusalem

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes.

Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Mitochondria are essential for all aerobic animals, including humans. They convert the chemical energy of biomolecules to adenosine triphosphate (ATP) via oxidative phosphorylation1, synthesize heme2, degrade fatty acids through β oxidation3, regulate calcium4 and iron5 homeostasis, control cell death by apoptosis6, and generate reactive oxygen species (ROS), which play a vital role in redox homeostasis7. Two complementary and opposite processes maintain the integrity and proper function o....

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NOTE: For the convenience of the readers, we have divided the protocol into two parts: one focuses on the protocol for measuring mitophagy in C. elegans, and the other focuses on the protocol for measuring mitophagy in liver cells. The list of materials can be found in the Table of Materials provided.

1. The C. elegans protocol

  1. Preparing the nematode growth medium (NGM) plates and Escherichia coli OP50 bacterial stock

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Induction of a robust mitophagy response in both C. elegans worms and Hep-3B cells with VL-850
VL-850 protects C. elegans worms and human keratinocytes (HaCaT cells) from oxidative stress23. To further explore its mechanism of action, we examined whether VL-850 induces mitophagy in C. elegans and other human cells. To test this, we exposed C. elegans worms (young adults, 3 days post-L1) to 62.5 µM VL-850, 5 µM FCCP (positive cont.......

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Multiple mitophagy pathways involve various proteins and biomolecules (e.g., cardiolipin29). However, the endpoint of these pathways is similar-the degradation of mitochondria by lysosomal enzymes12,13. Indeed, several methods use this endpoint to quantify mitophagy. However, some methods, such as electron microscopy, demand access to costly equipment, trained experts, and an extended preparation time for the specimens and analysis. Furthe.......

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We thank members of the Gross laboratory for the critical reading of the manuscript and their comments and advice. We thank the Caenorhabditis Genetics Center (CGC), which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), for providing some of the strains. This research was supported by a grant from Vitalunga Ltd and the Israel Science Foundation (grant No. 989/19). The graphical abstract figure (Figure 1) was generated with BioRender.com.

....

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NameCompanyCatalog NumberComments
Reagent or resource
Analytical balanceMettler-Toledo
Bacto AgarBD-Difco214010
Bacto PeptoneBD-Difco211677
Bacto TryptoneBD-Difco211705
Bacto Yeast extractBD-Difco212750
Calcium chlorideSigmaC1016
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)SigmaC2920
Chemicals
CholestrolThermo FisherC/5360/48
DMEM high glucoseBiological Industries01-055-1A
Double distilled water (DDW)
Dulbecco's Phosphate Buffered Saline (PBS)Biological Industries02-023-1A
FBS heat inactivatedInvitrogenM7514
Gluteradehyde (25%)SigmaG5882
HEPES Buffer 1 MBiological Industries03-025-1B
L-gluatamineBiological Industries03-020-1B
Lysosome/Mitochondria/Nuclear Staining Cytopainter ReagentAbcamab139487
Magnesium SulfateSigmaM7506
Nonidet P 40Sigma74385
Paraformalydehyde (16%)Electron Microscopy Sciences15720
Poloxamer 188 SolutionSigmaP5556
Potassium dihydrogen phosphateMillipore1.04873.1000
Potassium phosphate dibasicSigmaP3786
SeaKem LE AgaroseLonza50004
Sodium ChlorideBio-Lab1903059100
Sodium HydroxideGadot1310732
Sodium phosphate dibasic dodecahydrateSigma4273
Tetracycline hydrochlorideSigma87128-25G
Trypsin-EDTABiological Industries03-052-1A
VL-850: 1,8-diaminooxy-octanePatented
Glass/Plastic Disposables
0.22 μm syringe filterMillex GVSLGV033RS
1.7 mL Micro Centrifuge TubesLifegeneLMCT1.7B-500
10 cm Petri platesCorning430167
1,000 mL Erlenmeyer FlaskIsoLab, Germany
15 mL Sterile Polypropylene tubeLifegeneLTB15-500
35 mm Petri dishesBar NaorBN9015810
500 mL vacuum filter/storage bottle system, 0.22 μmLifegeneLG-FPE205500S
50 mL Sterile Polypropylene tubeLifegeneLTB50-500
Deckgläser Microscope cover glass 24 x 60 mmMarienfeld101152
Glass test tubes (10 mL- 13 x 100 mm) Borosilicate glassPyrex99445-13
iBiDi 8 well μ-slidesiBiDi80826
Microscope cover glass 24 x 40 mmBar NaorBN1052421ECALN
Platinum iridium 0.25 mM wireWorld Precision InstrumentsPT1002
Instruments
Cell counter CellDrop BFDeNovixCellDrop BF-UNLTD
Microspin FV-2400BiosanBS-010201-AAA
Nikon Yokogawa W1 Spinning Disk confocal microscope with DAPI, FITC, and TRITC filters and bright-field, with a 60x CFI Plan-Apochromat Lambda type lens (air lens) and NIS-Elements softwareNikonCSU-W1
Olympus SZ61 stereo microscopeOlympusSZ61
pH meterMettler-ToledoMT30019032
Revolver Adjustable Lab RotatorLabnetH5600

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