Monitoring Stub1-Mediated Pexophagy

Peroxisomes carry out beta oxidation and are prone to ROS damage. This method allows to investigate how cells deal with ROS-stressed peroxisomes. This protocol is not only used to globally target all the peroxisomes within a cell population but can also permit the manipulation of the individual peroxisomes within single cells.

Dye-assisted ROS generation can also be used to target organelles outside of peroxisomes to probe how cells deal with organelle injuries. To begin, seed two times 10 to the fifth human SHSY5Y cells or six times 10 to the fourth mouse NIH H3T3 cells in 840 microliters of culture medium on glass-bottomed 35-millimeter culture dishes with a 20-millimeter diameter glass microwell. Grow the cells under 5%carbon dioxide at 37 degrees Celsius for 24 hours.

Next, transfect the cells with desired plasmids using a transfection reagent or SHSY5Y cell transfection. Dilute plasmids in 55 microliters of serum-free DMEM/F-12 and dilute one microliter of transfection reagent in 55 microliters of serum-free DMEM/F-12. Combine the diluted DNA with the diluted reagent.

Mix gently and incubate for 30 minutes at room temperature. Add the complex to the 20-millimeter microwell previously plated with 100 microliters of culture medium containing SHSY5Y cells without antibiotics. Gently shake the dish back and forth.

Perform NIH 3T3 cell transfection as demonstrated before but substitute reduced serum medium instead of serum-free DMEM/F-12 for plasmid dilution and transfection reagent. After two hours of transfection, remove the transfection reagent-containing medium and add one milliliter of fresh culture medium. Incubate the NIH 3T3 or SHSY5Y cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.

For light-activated ROS production, at 24 hours post transfection remove the culture medium and add 10 microliters of 200 nanomolar HaloTag TMR ligand or 200 nanomolar Janelia Fluor 646 HaloTag ligand onto the 20-millimeter glass microwell. Transfer the dish to a 37 degrees Celsius and 5%carbon dioxide incubator and leave it for one hour to stain the cells. After one hour, remove the staining medium thoroughly and add one milliliter of fresh culture medium.

Place a glass-bottomed dish containing the desired cells on a laser scanning confocal microscope equipped with a stage-top incubator. In the software, click on Tool Window, choose Ocular, and click on the DIA button to turn on the transmitted light. View the dish through the microscope eyepiece and bring the cells into focus by turning the focus knob.

Choose LSM and click on the Dye Detector Select button. Double click on the desired dyes in the pop-up window to select the appropriate laser and filter settings for imaging the cells. Click on Live4x in the Live panel to initiate fast laser scanning to start screening for the fluorescent signals of the cells.

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. Click on Tool Window and choose LSM Stimulation.

Then click on the ellipse button and in the Live Image window use the mouse to draw an ROI of 15 micrometers in diameter containing the desired peroxisomes to which ROS stress will be applied. To apply ROS stress, use 561 nanometers for cells with the diKillerRed VKSKL or TMR-labeled SLP ligand and use 640 nanometers for cells stained with the Janelia Fluor 646-labeled SLP ligand. Select the laser wavelength for applying the ROS stress.

Enter the desired laser percentage and the duration. For ROS production in the peroxisomes, click on Acquire and then the Stimulation button to initiate the scanning with 561 or 640 nanometers of light through the ROIs for 30 seconds each. This stimulation corresponds to approximately 5%laser power settings for 561 and 640 nanometers.

After 30 seconds of laser illumination, peroxisomes within the ROIs lose their diKillerRed VKSKL, TMR, or Janelia Fluor 646 signals. This signal loss allows for distinguishing the illuminated and non-illuminated peroxisomes within the cell. To monitor the pexophagean live cells, follow the translocation of EGFP-tagged Stub1, Hsp70, ubiquitin, p62, or LC3B onto the illuminated or ROS-stressed peroxisomes by time lapse imaging using 10-minute intervals between frames.

Focal illumination leads to instantaneous and localized ROS production within individual peroxisomes as indicated by the fluorescent reporter roGFP2-VKSKL. The diKillerRed VKSKL image was taken after all the roGFP2-VKSKL measurements were done to indicate the damaged peroxisomes'location. The data also show that unilluminated peroxisomes are not affected, indicating the precision of the methodology.

This methodology was used to monitor Stub1-mediated pexophagy. During this process, Hsp70, Stub1, ubiquitinated proteins, autophagy adapters p62 and LC3B appear subsequently in ROS-stressed peroxisomes to drive pexophagy. Using the same strategy, it was possible to simultaneously damage all the peroxisomes on a culture dish for the biochemical characterization of Stub1-mediated pexophagy.

Before LED illumination, EGFP-ubiquitin fluorescence was homogenous in the cytoplasm and did not colocalize with the peroxisomes. After nine hours of LED illumination, the EGFP-ubiquitin signals accumulated onto all the peroxisomes in the cells grown in a confocal dish. Using this protocol, we found that ROS-stressed peroxisomes are removed through a ubiquitin-dependent degradation pathway.

ROS-stressed peroxisomes recruit the ubiquitin E3 ligase Stub1 to allow their removal by pexophagy.