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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

Discussion

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 enzymes^12,13. 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.......

Materials

Name	Company	Catalog Number	Comments
Reagent or resource
Analytical balance	Mettler-Toledo
Bacto Agar	BD-Difco	214010
Bacto Peptone	BD-Difco	211677
Bacto Tryptone	BD-Difco	211705
Bacto Yeast extract	BD-Difco	212750
Calcium chloride	Sigma	C1016
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)	Sigma	C2920
Chemicals
Cholestrol	Thermo Fisher	C/5360/48
DMEM high glucose	Biological Industries	01-055-1A
Double distilled water (DDW)
Dulbecco's Phosphate Buffered Saline (PBS)	Biological Industries	02-023-1A
FBS heat inactivated	Invitrogen	M7514
Gluteradehyde (25%)	Sigma	G5882
HEPES Buffer 1 M	Biological Industries	03-025-1B
L-gluatamine	Biological Industries	03-020-1B
Lysosome/Mitochondria/Nuclear Staining Cytopainter Reagent	Abcam	ab139487
Magnesium Sulfate	Sigma	M7506
Nonidet P 40	Sigma	74385
Paraformalydehyde (16%)	Electron Microscopy Sciences	15720
Poloxamer 188 Solution	Sigma	P5556
Potassium dihydrogen phosphate	Millipore	1.04873.1000
Potassium phosphate dibasic	Sigma	P3786
SeaKem LE Agarose	Lonza	50004
Sodium Chloride	Bio-Lab	1903059100
Sodium Hydroxide	Gadot	1310732
Sodium phosphate dibasic dodecahydrate	Sigma	4273
Tetracycline hydrochloride	Sigma	87128-25G
Trypsin-EDTA	Biological Industries	03-052-1A
VL-850: 1,8-diaminooxy-octane	Patented
Glass/Plastic Disposables
0.22 μm syringe filter	Millex GV	SLGV033RS
1.7 mL Micro Centrifuge Tubes	Lifegene	LMCT1.7B-500
10 cm Petri plates	Corning	430167
1,000 mL Erlenmeyer Flask	IsoLab, Germany
15 mL Sterile Polypropylene tube	Lifegene	LTB15-500
35 mm Petri dishes	Bar Naor	BN9015810
500 mL vacuum filter/storage bottle system, 0.22 μm	Lifegene	LG-FPE205500S
50 mL Sterile Polypropylene tube	Lifegene	LTB50-500
Deckgläser Microscope cover glass 24 x 60 mm	Marienfeld	101152
Glass test tubes (10 mL- 13 x 100 mm) Borosilicate glass	Pyrex	99445-13
iBiDi 8 well μ-slides	iBiDi	80826
Microscope cover glass 24 x 40 mm	Bar Naor	BN1052421ECALN
Platinum iridium 0.25 mM wire	World Precision Instruments	PT1002
Instruments
Cell counter CellDrop BF	DeNovix	CellDrop BF-UNLTD
Microspin FV-2400	Biosan	BS-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 software	Nikon	CSU-W1
Olympus SZ61 stereo microscope	Olympus	SZ61
pH meter	Mettler-Toledo	MT30019032
Revolver Adjustable Lab Rotator	Labnet	H5600