In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Summary

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Abstract

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Introduction

Mitophagy is essential for mitochondrial maintenance. Mitochondria intersect multiple cell signaling pathways and are universal sub-cellular organelles responsible for cellular energy production, cell metabolism, and calcium homeostasis^1,2,3,4. Mitochondria constantly experience challenges from endogenous and exogenous sources, such as reactive oxygen species (ROS) and mitochondrial toxicants, respectively, which lead to the generation of "aged" and dysfunctional mitochondria. Accumulation of damaged mitochondria decreases the effici....

Protocol

Animals (male and female mice) were born and bred in an accredited animal facility, in accordance and approval of the NIH Animal Care and Use Committee. Euthanasia methods must be consistent with all national and institutional regulations.

1. Detection of Mitophagy in Human Cells

1. Detection of mitophagy using mt-Keima plasmid
   NOTE: The Keima protein, fused to a mitochondria target signal, simplified as mt-Keima, has proved useful for in vivo mitophagy detection. mt-Keima is a ratiometric pH-sensitive fluorescent protein which exhibits green fluorescence (excitation 458 nm) in basic or neutral cond....

Results

Detection of Mitophagy in Human Cells:

Using the procedure presented here, human HeLa cells were transfected with mt-Keima plasmid. Healthy cells demonstrated a well-organized mitochondrial network (GFP, 488 nm) with few incidences of mitophagy (RFP, 561 nm). However, cells pretreated with a mitochondrial uncoupler FCCP (30 µM for 3 h) exhibited a profound increase in mitophagy incidence (Figure 1A). M.......

Discussion

Accurate measurement of mitophagy is technically demanding. Here, we have presented several robust techniques which allow for both qualitative detection of mitophagy and quantification of mitophagy levels in the most common laboratory experimental models.

To acquire replicable data, an experimental design with at least three biological repeats is necessary. All researchers involved in experimentation and analysis must be blinded to experimental group identities. Furthermore, imaging fields mus.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
mt-Keima mouse	Jackson Laboratory
Lipofectamine 2000 DNA transfection reagent	Thermofisher	#11668027
Opti-MEM medium (Gibco)	Thermofisher	#31985062	serum-free medium
mtKemia plasmid: pCHAC-mt-mKeima	addgene	#72342
COXII antibody (mouse)	abcam	#ab110258
LAMP2 antibody (rabbit)	NOVUS	#CD107b
goat-anti-rabbit with wavelength 568 nm of red fluorescent protein (RFP)	Thermofisher	#Z25306	Alexa Fluor 568 dye
goat-anti-mouse with wavelength 488 nm of green fluorescent protein (GFP)	Thermofisher	#Z25002	Alexa Fluor 488 dye
prolong gold antifade with DAPI	Invitrogen	#P36931
6-well plate	SIGMA Corning Costar	#CLS3516
4-well chamber slide	THermofisher, Nunc Lab-Tek	#171080
Nunc F 96-well plate	Thermofisher	#152038
LC3B antibody rabbit	NOVUS	#NB100-2220
DNA antibody	Progen Biotechnik	#anti-DNA mouse monoclonal, AC-30-10
DAPI	Thermofisher	#D1306	antifade mounting medium with DAPI
IN Cell analyzer (fluorescent reader )	GE Healthcare Life Sciences	#IN Cell analyzer 2200
Eclipse TE-2000e confocal microscope	Nikon	#TE-2000e
Colocalization software	Volocity	#Volocity 6.3	alternative Zeiss ZEN 2012 software
IN Cell Investigator Software	GE Healthcare Life Sciences	#28408974
cell culture medium	Thermofisher	#DMEM–Dulbecco's Modified Eagle Medium
Penicillin-Streptomycin (10,000 U/mL)	Thermofisher	#15140122
Fetal Bovine Serum	Sigma-Aldrich	#12003C-1000ML
Cell culture Incubator	Thermofisher	#Thermo Forma 3110 CO2 Water Jacketed Incubator
epifluorescence microscope	Zeiss		Zeiss Axio Imager Z2
camera	Olympus		Olympus DP71
confocal microscope	Zeiss		Zeiss Axio Observer Z1
confocal software	Zeiss		ZEN 2012
image analysis software	Image J	colocalization analysis, etc	https://imagej.nih.gov/ij/
statistical analysis software	GraphPad Software Inc., San Diego, USA		GraphPad Prism software package
material to make a worm pick	Surepure Chemetals	#4655	The pick is made of 30 gauge 90% platinum 10% iridium wire
IR: N2;Ex[pmyo-3 TOMM-20::Rosella]	Material inquiry to Tavernarakis Nektarios		Maintain transgenic animals at 20 °C
IR: N2; Ex[pdct-1 DCT-1::GFP; pmyo-3 DsRed::LGG-1]	Material inquiry to Tavernarakis Nektarios
pmyo-3 TOMM-20::Rosella	Material inquiry to Tavernarakis Nektarios
pdct-1 DCT-1::GFP	Material inquiry to Tavernarakis Nektarios
pmyo-3 DsRed::LGG-1	Material inquiry to Tavernarakis Nektarios
Paraquat solution	see supplementary data for preparation
M9 buffer	see supplementary data for preparation
M9-levamisole buffer	see supplementary data for preparation
Glass Microscope Slides and Coverslips	Fisher Scientific	#B9992000
Surgical forceps	STERIS Animal Health	19 Piece Canine Spay Pack Economy
Surgical scissors	STERIS Animal Health	19 Piece Canine Spay Pack Economy
1x PBS	Thermofisher	#AM9625	10x PBS needs to be diluted to 1x PBS by using ddH2O
shaker	Fisher Scientific	#11-676-178	Thermo Scientific MaxQ HP Tabletop Orbital Small Open Air Platform Shaker Package A
2% agarose pad	see supplementary data for preparation
Vibroslice blades	World precision instruments	#BLADES-2	single-edge blade
metal plate	MSC	#78803988	0.012 in thick x 6 in wide x 12 in long, 430 Stainless Steel Sheet
Triton X-100			detergent
Methyl viologen dichloride hydrate	Sigma-Aldrich	#856177	paraquat
Incubator for nematodes	AQUALYTIC		Incubator to maintain 20 °C
Dissecting stereomicroscope	Olympus	SMZ645
Confocal microscope	Zeiss	AxioObserver Z1	For nematodes (step 2)
epifluorescence microscope	Zeiss	AxioImager Z2	For nematodes (step 2)
UV crosslinker	Vilber Lourmat	BIO-LINK – BLX-E365	UV light source; 356 nm