Monitoring Stub1-Mediated Pexophagy

Summary

This protocol provides instructions for triggering and monitoring Stub1-mediated pexophagy in live cells.

Abstract

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality of peroxisomes. During this process, heat shock protein 70 and the ubiquitin E3 ligase, Stub1, translocate onto peroxisomes to be turned over to initiate pexophagy. The Stub1 ligase activity allows the accumulation of ubiquitin and other autophagy-related modules on targeted peroxisomes. Elevating reactive oxygen species (ROS) levels within the peroxisomal lumen can activate Stub1-mediated pexophagy. One can, therefore, use dye-assisted ROS generation to trigger and monitor this pathway. This article outlines the procedures for using two classes of dyes, fluorescent proteins and synthetic fluorophores, to initiate pexophagy within mammalian cell cultures. These dye-assisted ROS generation-based protocols can not only be used to target all the peroxisomes within a cell population globally but can also permit the manipulation of individual peroxisomes within single cells. We also describe how Stub1-mediated pexophagy can be followed using live-cell microscopy.

Introduction

Peroxisomes are single-membrane-bound organelles present in most eukaryotic cells. Peroxisomes are a metabolic compartment essential for carrying out the beta-oxidation of very long-chain fatty acids, purine catabolism, and ether phospholipid and bile acid synthesis¹. Peroxisome-derived acetyl-CoA controls lipid homeostasis by regulating the central signaling in metabolism². Therefore, it is no surprise that compromised peroxisomal functions are implied in various diseases, including neurodegenerative disorders, aging, cancers, obesity, and diabetes^3,

Protocol

1. Preparation of cells expressing diKillerRed or self-labeling proteins (SLPs) in the peroxisome lumen

1. Seed the desired cells on glass-bottomed cell culture dishes. For the experiment here, seed 2 x 10⁵ human SHSY5Y cells in 840 µL of culture medium (DMEM/F-12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) or 6 x 10⁴ mouse NIH3T3 cells in 840 µL of culture medium (DMEM supplemented with 10% bovine serum and 1% penicillin/streptomycin) on a 35 mm culture dish with a 20 mm diameter glass microwell.
   NOTE: The number of cells seeded should be adjusted proportionally according to....

Results

The Stub1-mediated pexophagy induction scheme shown here takes advantage of dye-assisted ROS generation within the peroxisome lumen. This operation requires minimal light intensities. Peroxisomes containing fluorescent proteins or dyes can, therefore, be illuminated using standard laser-scanning confocal microscopes. Focal illumination leads to instantaneous and localized ROS production within individual peroxisomes, as indicated by the fluorescent reporter roGFP2-VKSKL (Figure 9). We did no.......

Discussion

This protocol details how to trigger Stub1-mediated pexophagy within cell cultures by elevating peroxisomal ROS levels with light. As the protocol relies on dye-assisted ROS generation, one needs to ensure sufficient expression of diKillerRed-VKSKL or dye-labeled SLP ligand staining within the cells of interest. Given that different cell types or cells of different genetic backgrounds can harbor peroxisomes with slightly different properties, one may need to tune the exact illumination conditions to ensure the induction .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm culture dish with a 20 mm diameter glass microwell	MatTek	P35G-1.5-20-C	20 mm glass bottomed
3-amino-1,2,4-triazole (3-AT)	Sigma Aldrich	A8056
bovine serum	ThermoFisher Scientific	16170060
Cell culture incubator	Nuaire	NU-4750
diKillerRed-PTS1	Academia Sinica		made by appending the KillerRed tandem dimer with PTS1(VKSKL)
Dulbecco's Modified Eagle Medium (DMEM)	ThermoFisher Scientific	11965092
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)	ThermoFisher Scientific	11330032
EGFP-C1	Clontech	pEGFP-C1	The backbone of EGFP-C1 was used for cloning EGFP-Stub1, EGFP-Hsp70, EGFP-p62
EGFP-Hsp70	Academia Sinica		Hsp70 gene (HSPA1A) PCR amplified from HeLa cDNA and cloned into EGFP-C1
EGFP-LC3B	Addgene	11546
EGFP-p62	Academia Sinica		generated by inserting the human SQSTM1 gene (through PCR amplification of the HeLa cell cDNA) into EGFP-C1
EGFP-Stub1	Academia Sinica		generated by inserting the mouse Stub1 gene (through PCR amplification of the total mouse kidney cDNA) into EGFP-C1
EGFP-Ub	Addgene	11928
fetal bovine serum	ThermoFisher Scientific	10437028
HaloTag TMR ligand	Promega	G8252
HaloTag-PTS1	Academia Sinica		PTS1 appended and cloned into EGFP-C1 backbone
HEPES	ThermoFisher Scientific	15630080
Inverted Confocal Microscope	Olympus	FV3000RS	405 nm Ex, 488 nm Ex, 561 nm Ex,  microscope with a TOKAI HIT chamber incubator and the UNIV2-D35 dish attachment
Janelia Fluor 646 HaloTag Ligand	Promega	GA1120
LED	VitaStar	PAR64	80 W, 555-570 nm
lipofectamine 2000	ThermoFisher Scientific	11668	transfection reagent
NIH3T3 cell	ATCC	CRL-1658	adherent
Opti-MEM	ThermoFisher Scientific	319850	reduced serum media
penicillin/streptomycin	ThermoFisher Scientific	15140
PMP34-TagBFP	Academia Sinica		PMP34 PCR amplified from HeLa cDNA and cloned intoTagBFP-C (Evrogen FP171)
roGFP2-PTS1	Academia Sinica		generated by appending eroGFP (taken from Addgene plasmid 20131) with the amino acid sequence VKSKL, and cloned into the EGFP-C1
SHSY5Y cell	ATCC	CRL-2266	adherent