An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

Abstract

Source: Fang, E. F., et al. In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice. J. Vis. Exp. (2017).

This video demonstrates an in vivo assay to study mitophagy in Caenorhabditis elegans. Upon exposing the nematodes to a mitophagy-inducing drug, the occurrence of mitophagy is assessed by co-localization between DCT-1, a mitophagy receptor on the damaged mitochondria, and the autophagosomal membrane protein LGG-1.

Protocol

1. Detection of Mitophagy in C. elegans NOTE: The nematode C. elegans provides a platform to assay mitophagy at the organismal level. Two strains can be used to monitor mitophagy: (1) mitochondria-targeted Rosella (mtRosella) or (2) mitophagy receptor DCT-1 fused with GFP along with autophagosomal marker LGG-1 fused with Discosoma sp. red fluorescent protein (DsRed). Mitophagy monitoring using Rosella biosensor<br…

Materials

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
M9 buffer
M9-levamisole buffer
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
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