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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 diabetes3,4,5. An essential process in the maintenance of peroxisomal operation is pexophagy. Pexophagy is a catabolic process for the selective turnover of peroxisomes by autophagy. Cells use pexophagy to help control the quantity and quality of peroxisomes, thereby ensuring proper peroxisomal function. A recent study demonstrated that peroxisome loss caused by mutations in PEX1 peroxisomal biogenesis factors 1 and 6 results from uncontrolled pexophagy6. Notably, 65% of all peroxisome biogenesis disorder (PBD) patients harbor deficiencies in the peroxisomal AAA ATPase complex, composed of PEX1, PEX6, and PEX26 in mammalian cells7.

A number of methods can be used to initiate and study pexophagy. In yeast, pexophagy is triggered when the supplied nutrients are switched from peroxisome-dependent carbon sources to peroxisome-independent carbon sources (to lower cellular peroxisome numbers)8. For example, the transfer of methanol-grown Pichia pastoris cells from methanol medium to glucose medium and ethanol medium induces micropexophagy and macropexophagy, respectively8,9,10. Micropexophagy sequesters clustered peroxisomes for degradation by remodeling the vacuole to form cup-like vacuolar sequestration membranes and a lid-like structure termed the micropexophagy-specific membrane apparatus (MIPA). In macropexophagy, individual peroxisomes are engulfed by double-membrane structures known as pexophagosomes, followed by fusion with the vacuole for degradation8,9,10. The phosphorylation of pexophagy receptors, such as Atg36p in Saccharomyces cerevisiae and Atg30p in Pichia pastoris, is critical for the receptors to recruit core autophagy machinery and to facilitate peroxisomal targeting to autophagosomes8,11.

In mammalian cells, pexophagy can be induced by ubiquitination. Tagging the peroxisomal membrane proteins PMP34 or PEX3 with ubiquitin on the cytosolic side induces pexophagy12. The overexpression of PEX3 induces peroxisome ubiquitination and peroxisome elimination by lysosomes13. In addition, the fusion of PEX5 with a C-terminal EGFP impairs the export of monoubiquitinated PEX5 and results in pexophagy14. On the other hand, pexophagy can also be triggered by H2O2 treatment. Peroxisomes produce reactive oxygen species (ROS); specifically, the peroxisomal enzyme Acox1, which catalyzes the initial step of beta-oxidation of very long-chain fatty acids (> 22 carbon), produces not only acetyl-CoA but also peroxisomal ROS. In response to the heightened ROS levels under H2O2 treatment, mammalian cells activate pexophagy to lower the ROS production and alleviate stress. It has been reported that H2O2 treatment drives the recruitment of ataxia-telangiectasia mutated (ATM) to peroxisomes. ATM then phosphorylates PEX5 to promote peroxisomal turnover by pexophagy15.

Since peroxisomes are ROS-generating centers, they are also prone to ROS damage. ROS-elicited peroxisomal injuries force cells to activate pexophagy to initiate peroxisome quality control pathways (removal of the damaged peroxisome by autophagy). Here, we outline an approach for the on-demand triggering of ROS-elicited peroxisomal injury. The protocol takes advantage of light-activated ROS production within organelles16,17,18,19,20 (Figure 1). Dye-labeled peroxisomes are illuminated, leading to ROS production within the peroxisomal lumen, which specifically triggers peroxisomal injury. Using this protocol, it is shown that ROS-stressed peroxisomes are removed through a ubiquitin-dependent degradation pathway. ROS-stressed peroxisomes recruit the ubiquitin E3 ligase Stub1 to allow their engulfment into autophagosomes for individual removal by pexophagy16. One can use this protocol to compare the fate of injured and healthy peroxisomes within the same cell by time-lapse microscopy. The method can also be used to globally damage all the peroxisomes (in all cells) on a culture dish, allowing for the biochemical analysis of the pexophagy pathway.

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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 the glass microwell area when using glass bottom dishes of other specifications.
  2. Grow the cells under 5% CO2/37 °C for 24 h.
  3. Transfect the cells with desired plasmids using a transfection reagent.
    1. Use PMP34-TagBFP to label the peroxisomes in live cells. Use either peroxisome-targeting diKillerRed-VKSKL or SLP-VKSKL (+ dye-labeled SLP ligands) for ROS generation in the peroxisome lumen (through light-activated ROS production). In this protocol, we use the self-labeling protein HaloTag-VKSKL21. To monitor the translocation of Stub1/Hsp70, the accumulation of ubiquitinated proteins, and the formation of autophagosomes on ROS-stressed peroxisomes, transfect EGFP-Stub1, EGFP-Hsp70, EGFP-Ub, or EGFP-LC3B, respectively. To check ROS generation in the peroxisomal lumen, co-transfect diKillerRed-VKSKL with the peroxisome-localized fluorescent redox probe roGFP2-VKSKL22.
    2. For SHSY5Y cell transfection, dilute plasmids in 55 µL of serum-free DMEM/F-12, and dilute 1 µL of transfection reagent in 55 µL of serum-free DMEM/F12. Combine the diluted DNA with the diluted reagent. Mix gently, and incubate for 30 min at room temperature.
    3. Add the complex to the 20 mm microwell previously plated with 100 µL of culture medium for SHSY5Y without antibiotics (DMEM/F-12 with 10% fetal bovine serum). Gently shake the dish back and forth.
    4. For NIH3T3 cell transfection, substitute reduced serum medium for the serum-free DMEM/F-12 for diluting the plasmids and transfection reagent. Replace the plating culture medium with SHSY5Y with culture medium for NIH3T3 cells without antibiotics (DMEM with 10% bovine serum).
  4. After 2 h of transfection, remove the transfection reagent-containing medium, and add 1 mL of fresh culture medium. Incubate the NIH3T3 or SHSY5Y cells at 37°C and 5% CO2 for 24 h.
    NOTE: Other cell lines can also be used to study Stub1-mediated pexophagy (e.g., HeLa cells).

2. Staining peroxisomes with dye-labeled SLP ligands (for light-activated ROS production)

  1. At 24 h post-transfection, remove the culture medium, and add 100 µL of 200 nM HaloTag TMR ligand or 200 nM Janelia Fluor 646 HaloTag ligand onto the 20 mm glass microwell.
    NOTE: The dye-labeled SLP ligands should be diluted with the respective culture medium for each cell line. Choosing Janelia Fluor 646-labeled ligands will free up the blue, green, and red channels for downstream fluorescence imaging.
  2. Transfer the dish to a 37°C, 5% CO2 incubator. Stain for 1 h. After 1 h, remove the staining medium thoroughly, and add 1 mL of fresh culture medium.

3. ROS-stressing of peroxisomes on a laser-scanning confocal microscope

  1. Place a glass-bottomed dish containing the desired cells on a laser-scanning confocal microscope equipped with a stage-top incubator.
  2. Click on Tool Window, choose Ocular, and click on the DIA button (Figure 2A) to turn on the transmitted light. View the dish through the microscope eyepiece, and bring the cells into focus by turning the focus knob.
  3. Choose LSM, and click on the Dye & Detector Select button (Figure 2B). Double-click on the desired dyes in the pop-up window to select the appropriate laser and filter settings for imaging the cells (Figure 2C). Click on Livex4 in the Live panel to initiate fast laser scanning to start screening for the fluorescence signals of the cells (Figure 2D).
  4. Move the stage around using the joystick controller, and locate the medium diKillerRed-VKSKL- or SLP-VKSKL-expressing cells to apply ROS stress (on the peroxisomes). Acquire an image of the desired cell.
  5. Click on Tool Window, and choose LSM Stimulation (Figure 2E). Click on the ellipse button, and in the live image window, use the mouse to draw a circular ROI of 15 µm in diameter (Figure 2F) containing the desired peroxisomes acquired in step 3.4 to which ROS stress will be applied (see Figure 3 for an example).
  6. To apply ROS stress, use 561 nm for cells with the diKillerRed-PTS1- or TMR-labeled SLP ligand, and use 640 nm for cells stained with the Janelia Fluor 646-labeled SLP ligand.
  7. Select the laser wavelength for applying the ROS stress. Enter the desired laser percentage and the duration (Figure 2G).
  8. Click on Acquire, and click on the Stimulation button (Figure 2H) to initiate the scanning with 561/640 nm light through the ROIs for 30 s each. This corresponds to a ~5% laser power setting for both 561 nm and 640 nm on the confocal microscope used here. This operation will lead to ROS production in the peroxisomes.
    NOTE: One can select ROIs of different sizes. One should proportionally adjust the illumination time for each ROI according to its area.
  9. After 30 s of laser illumination, peroxisomes within the ROIs will have lost their diKillerRed-PTS1/TMR/Janelia Fluor 646 signals. This signal loss allows one to distinguish the illuminated from the non-illuminated peroxisomes (damaged vs. healthy) within a cell.

4. Monitoring pexophagy in live cells by time-lapse imaging

  1. Follow the translocation of EGFP-tagged Stub1, Hsp70, Ub, p62, or LC3B onto the illuminated/ROS-stressed peroxisomes by time-lapse imaging using 10 min intervals between frames (for examples, see Figures 3-7).
    NOTE: EGFP-Stub1 or EGFP-Hsp70 signals start to appear 10-20 min post illumination and stay on all the damaged peroxisomes until ~40 min post illumination. The EGFP-Ub signals appear 30 min post illumination and last for 2-3 h. EGFP-p62 translocation appears 60 min post illumination, and EGFP-LC3B signals become visible ~3 h after illumination.
  2. Quantify the EGFP signals on the damaged peroxisomes (from Stub1, Ub, p62, or LC3B) using ImageJ. Perform the following steps for using ImageJ to quantify a three-channel image (PMP34-TagBFP/EGFP-LC3B/HaloTag-VKSKL).
    1. Open the image, and apply Elliptical selections or Freehand selections to enclose the region containing all the illuminated peroxisomes (PMP34-TagBFP signal positive and HaloTag ligand signal bleached; Figure 8A).
    2. Click on Duplicate to pick out the PMP34-TagBFP channel (Figure 8A). Set a threshold using the drop-down menu Image > Adjust > Threshold to mark the peroxisomes (Figure 8B).
    3. Select Analyze > Analyze particles to determine the exact regions of the illuminated peroxisomes. ImageJ records these regions of interest (ROIs) in the ROI manager (Figure 8C).
    4. Click on Duplicate to pick out the EGFP channel for analyzing the fluorescence signal from EGFP-conjugated Ub, p62, or LC3B (Figure 8D). Select all the ROIs in the ROI Manager (Figure 8E).
    5. Apply Measure in the ROI Manager to measure the EGFP signals on the peroxisomes (Figure 8E). Find the value of the area and integrated density (the sum of all the pixel intensities) for each ROI in the Results table (Figure 8E).
    6. To obtain the average EGFP fluorescence intensities at different time points after illumination, divide the integrated EGFP intensity by the total area of illuminated peroxisomes (PMP34-TagBFP signal positive and HaloTag ligand signal negative areas). To obtain the accumulated signal on the peroxisomes, subtract the averaged intensity at a time point by the averaged intensity at time = 0, and then normalize by the averaged intensity at time = 0.

5. Globally damaging all the peroxisomes (in every cell) on a culture dish

  1. Perform step 1 (preparation of cells expressing peroxisome-targeted diKillerRed [diKillerRed-VKSKL] or SLP [SLP-VKSKL]) and step 2 (staining peroxisomes with the SLP ligand [for light-activated ROS production]).
  2. After 1 h of TMR-labeled ligand staining, remove the staining medium, and add 1 mL of culture medium containing 30 mM 3-AT (3-amino-1,2,4-triazole; a catalase inhibitor).
    NOTE: Catalase is a ROS scavenger expressed in the peroxisome lumen, so 3-AT treatment helps to augment the light-elicited oxidative damage on the peroxisomes.
  3. Place the culture dish above an LED (555-570 nm). Illuminate all the cells with a power density of 1.8 W/cm2 for 9 h in an incubator.
    NOTE: To perform this step outside an incubator, place the culture dish on a 37°C heating plate. Add 20 mM HEPES to the 3-AT-containing medium, and cover the culture dish with a transparent film to minimize gas exchange. HEPES is a buffering agent that maintains physiological pH in cell culture outside a CO2 incubator during LED illumination.
  4. Validate the success of LED-elicited damage by imaging the EGFP-Ub, EGFP-p62, or EGFP-LC3B signals on the peroxisomes. Using the illumination condition outlined above, 82% of SHSY5Y cells stably expressing HaloTag-VKSKL displayed Stub1-mediated pexophagy.
  5. Harvest the cells for downstream biochemical analysis.

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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 d...

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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 ...

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Disclosures

The authors declare no competing financial interests.

Acknowledgements

This work was supported in part by a MOST 111-2311-B-001-019-MY3 research grant from the National Science and Technology Council in Taiwan.

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Materials

NameCompanyCatalog NumberComments
35 mm culture dish with a 20 mm diameter glass microwell MatTekP35G-1.5-20-C20 mm glass bottomed
3-amino-1,2,4-triazole (3-AT)Sigma AldrichA8056
bovine serumThermoFisher Scientific16170060
Cell culture incubatorNuaireNU-4750
diKillerRed-PTS1Academia Sinicamade by appending the KillerRed tandem dimer with PTS1(VKSKL)
Dulbecco's Modified Eagle Medium (DMEM)ThermoFisher Scientific11965092
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)ThermoFisher Scientific11330032
EGFP-C1ClontechpEGFP-C1The backbone of EGFP-C1 was used for cloning EGFP-Stub1, EGFP-Hsp70, EGFP-p62
EGFP-Hsp70Academia SinicaHsp70 gene (HSPA1A) PCR amplified from HeLa cDNA and cloned into EGFP-C1
EGFP-LC3BAddgene11546
EGFP-p62Academia Sinicagenerated by inserting the human SQSTM1 gene (through PCR amplification of the HeLa cell cDNA) into EGFP-C1
EGFP-Stub1Academia Sinicagenerated by inserting the mouse Stub1 gene (through PCR amplification of the total mouse kidney cDNA) into EGFP-C1
EGFP-UbAddgene11928
fetal bovine serumThermoFisher Scientific10437028
HaloTag TMR ligand PromegaG8252
HaloTag-PTS1Academia SinicaPTS1 appended and cloned into EGFP-C1 backbone
HEPESThermoFisher Scientific15630080
Inverted Confocal Microscope OlympusFV3000RS405 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 LigandPromegaGA1120
LEDVitaStarPAR6480 W, 555-570 nm
lipofectamine 2000 ThermoFisher Scientific11668transfection reagent
NIH3T3 cellATCCCRL-1658adherent
Opti-MEMThermoFisher Scientific319850reduced serum media
penicillin/streptomycinThermoFisher Scientific15140
PMP34-TagBFPAcademia SinicaPMP34 PCR amplified from HeLa cDNA and cloned intoTagBFP-C (Evrogen FP171)
roGFP2-PTS1Academia Sinicagenerated by appending eroGFP (taken from Addgene plasmid 20131) with the amino acid sequence VKSKL, and cloned into the EGFP-C1
SHSY5Y cellATCCCRL-2266adherent

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PexophagyPeroxisomesROS DamageSHSY5Y CellsNIH 3T3 CellsCell TransfectionTransfection ReagentCulture MediumHaloTag TMR LigandConfocal MicroscopeLive cell ImagingROS GenerationDye assisted TargetingBeta Oxidation

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