TITLE: Live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
There is no substitute for live cell imaging in the investigation of the kinetics of subcellular biology. Here, we enumerate a protocol to visualize fluorescently-tagged mitochondria, NEMO, OPTN, and p62 during the cellular response to mitochondrial depolarization. Because live cells are sensitive to photo-damage, we used low laser power and short exposure times in our confocal microscopy system. Even with minimally damaging parameters, we were able to collect high-content data that we subsequently analyzed. With this technique, we showed that NEMO and OPTN, despite containing highly similar domains, were recruited to damaged mitochondria with less correlation than NEMO and p62. Furthermore, live imaging of NEMO occupancy on damaged mitochondria was a necessary complement to our parallel fixation studies. Since fixation introduces artifacts, especially in samples with concentrated proteins like those in mitophagy, results from our live cell imaging corroborated our findings in fixation studies. Our reporting of these results would not have been possible without real-time, live cell imaging.

[BEFORE_START]
 

The start point for this protocol is after cells grown on   glass bottom dishes have been transfected with Mito-dsRed2, EGFP-NEMO, HaloOPTN, and Parkin for 1080 min- 1440 min and tagged with Halo ligand.
Prepare 45 millimolar (mM) stock of Antimycin A by suspending 50 mg solid AntA in 2 mL ethanol.
Prepare 10 millimolar (mM) stock Oligomycin A by suspending 5 mg solid OligA in 630 µL DMSO. 
Prepare imaging media by making a 10% FBS solution in L-15 and warm in water bath.

 

Prepare working AntA/OligA solution by adding 1 µL 10 millimolar (mM) OligA and 4 mL  45 millimolar (mM) AntA to 0.5 mL Imaging media in a 1.5 mL tube. Keep warm in bath or imaging chamber.
 Heat microscope imaging chamber to 37 °C.
















[GUIDELINES]
This protocol was developed with the HeLa subtype, HeLa-M. HeLa-M cells are flatter than standard HeLa cells, making them easier to image. They also uptake siRNA better than standard HeLa. Regardless, the protocol would be easily adaptable to standard HeLa cells or other cell culture lines. 
This protocol was created in order to investigate Parkin-dependent mitophagy. Parkin and several other fluorescently-tagged mitophagy components are intended for use in the protocol.

[STEPS]
SECTION: Replace Standard Media with Imaging Media
1.  Wait until imaging chamber is heated to 37 °C.  

SECTION: Replace Standard Media with Imaging Media
2. Aspirate media from sample.
SECTION: Replace Standard Media with Imaging Media
3. Repeat the following two steps for a total of 2 washes.
Add 200 µL Imaging media gently to dish.
Aspirate.
SECTION: Replace Standard Media with Imaging Media
4. Add 1.5 mL Imaging media and place dish in 37 °C imaging chamber.

SECTION: Replace Standard Media with Imaging Media
5. Incubate sample in imaging conditions for at least 10 min before imaging.

SECTION: Imaging set-up
6. Place dish on microscope stage, moving stage adjusters if necessary to fit the dish.

 

SECTION: Imaging set-up
7.  Raise objective so that there is an oil interface between the objective lens and glass bottom of the dish.
SECTION: Imaging set-up
8. Allow dish to settle at least 5 min in this position before imaging to minimize drift during imaging.

SECTION: Imaging set-up
9.  Using 60X objective and RFP epifluorescence, find the focal plane of fixed cells by looking for dsRed2-labeled mitochondria.
 

 

SECTION: Imaging set-up
10. Configure 488, 561, and 647 lasers and accompanying exposure times.
 

SECTION: Imaging set-up
11. 
 

Choose a field of view with multiple cells that look healthy and express all three fluorescent constructs. Set focus on a confocal section approximately between 5 and 7, where 0 is the bottom, attached part of the cell and 10 is the top of the cell.
 

SECTION: Imaging set-up
12. Begin imaging at acquisition rate of 1 frame per min.
SECTION: Imaging set-up
13. After 5-10 frames (5 min-10 min), remove the lid of the imaging dish and add the working AntA/OligA solution for a total of 2 mL (5 micromolar (µM) AntA/10 micromolar (µM) OligA).
 

SECTION: Imaging set-up
14. Replace the lid to the dish and continue imaging for as long as desired.
 

SECTION: Imaging set-up
11. 
 

Choose a field of view with multiple cells that look healthy and express all three fluorescent constructs. Set Z stack to collect volume from the 50th to 75th percentile of the cell (see Figure 1).
  

SECTION: Imaging set-up
12. Collect images in order to record 10-20 cells, depending on the quality of the transfection and cells. Collect images from diverse areas of the coverslip.
SECTION: Imaging set-up
13. Add the working AntA/OligA solution for a total of 2 mL (5 micromolar (µM) AntA/ 10 micromolar (µM) OligA).

SECTION: Imaging set-up
14. Incubate ~50 min or to desired timepoint.

SECTION: Imaging set-up
15. Reposition dish to a new field of view with cells that demonstrate OPTN recruitment.
SECTION: Imaging set-up
16. Collect images in order to record 10-20 treated cells, again collecting images from diverse areas of the coverslip.
 

SECTION: End of imaging
17. Samples may be saved for biochemical analysis by scraping cells into 1.5 mL tube and snap freezing in liquid nitrogen after aspirating any media.