TITLE: Live imaging and analysis to investigate phase separation properties of NEMO during mitophagy
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
Phase condensation of biomolecular particles is increasingly examined for its relevance in physiological contexts. When we observed formation of NEMO puncta associated with the outer mitochondrial membrane of damaged mitochondria, we wondered whether these puncta were incorporated into phase separated particles. Three standards of the field for determining phase separation are: (A) particles undergo fission and fusion like droplets of water, (B) individual puncta recover fluorescence after photobleaching, and (C) particles dissipate upon exposure to the organic compound 1,6-Hexanediol. Our system wherein NEMO puncta appeared to be attached to fragmented mitochondria precluded a systematic assessment of standard A. I.e., one NEMO punctum would so rarely encounter another, nor would it be prompted to split, that we could not confirm or rule out the potential for fission and fusion of droplets. This protocol describes our approach to investigating standards B and C. 

[BEFORE_START]
 

The start point for this protocol is after cells grown on   glass bottom dishes have been transfected with the relevant constructs for 1080 min- 1440 min .
Prepare 45 millimolar (mM) stock of Antimycin A by suspending 50 mg solid AntA in 2 mL ethanol. On day of use, prepare a secondary dilution of 0.45 millimolar (mM) 
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 0.5 µL 10 millimolar (mM) OligA and 22.5 mL  045. millimolar (mM) AntA to 0.25 mL Imaging media in a 1.5 mL tube. Keep warm in bath or imaging chamber.
Prepare 2X (10%) 1,6-Hexanediol in imaging media and warm to 37 °C 
 Heat microscope imaging chamber to 37 °C.
Calibrate microscope FRAP capability. 

























[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 and Induce Mitophagy
1.  Wait until imaging chamber is heated to 37 °C.  

SECTION: Replace Standard Media with Imaging Media and Induce Mitophagy
2. Aspirate media from sample.
SECTION: Replace Standard Media with Imaging Media and Induce Mitophagy
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 and Induce Mitophagy
4. Add .75 mL Imaging media and place dish in 37 °C imaging chamber.

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 100X objective and GFP or RFP epifluorescence, find the focal plane.
 

 

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

SECTION: Imaging set-up and FRAP
12. Choose a field of view with cell(s) that look healthy and express all three fluorescent constructs, and exhibit visible NEMO/p62 puncta recruitment to mitochondria. 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 and FRAP
13. Choose several puncta to photobleach and draw box-shaped ROIs targeting them. Choose NEMO-only and NEMO-p62 puncta. Save the ROI set.


SECTION: Replace Standard Media with Imaging Media and Induce Mitophagy
5. 45 min prior to imaging, add the .25 mL working solution of AntA/OligA to the dish making a total of 1 mL 

 

SECTION: Imaging set-up and FRAP
11. Set FRAP and timelapse imaging parameters and engage auto-focus to maintain Z plane.
 



SECTION: Imaging set-up and 1,6-HD treatment
11. Set timelapse imaging parameters to acquire frames every 5 sec and engage auto-focus to maintain Z plane. 


SECTION: Imaging set-up and 1,6-HD treatment
12. Choose a field of view with cell(s) that look healthy and express both fluorescent constructs, and exhibit visible NEMO puncta recruitment to mitochondria. 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 and 1,6-HD treatment
13. Begin image acquisition and wait 5 min 

SECTION: Imaging set-up and 1,6-HD treatment
14. Gently add 1 mL 10% 1,6-Hexanediol dropwise during image acquisition and allow acquisition to continue for 1 min .
 



SECTION: Imaging set-up and 1,6-HD treatment
15. End image acquisition. 
 

SECTION: FRAP data analysis
15. Load timelapse image and saved ROI set to ImageJ. 
SECTION: Imaging set-up and FRAP
14. Commence imaging. Repeat FRAP assay as many times as possible within the 30 min span. 
SECTION: FRAP data analysis
16. Draw a new ROI to encompass a  background area that exhibits no puncta for the whole video. Save to the ROI manager. 
SECTION: FRAP data analysis
17. Measure average fluorescence for NEMO channel and p62 channel for each ROI (including background) for every timepoint. Paste fluorescence values to a new spreadsheet in Excel. 
 

 

SECTION: FRAP data analysis
18. Subtract the respective background value from each puncta ROI measurement for each timepoint to calculate "Sub-bg", i.e. NEMO puncta value at t1 minus NEMO background value at t1 equal Sub-bg. 
 

SECTION: FRAP data analysis
19. Identify the minimum intensity value for each channel for each puncta, then subtract the minimum from Sub-bg's to calculate "Sub-min". 
 

SECTION: FRAP data analysis
20.  Identify the maximum intensity Sub-min values for each channel for each puncta, then divide each Sub-min value by the maximum to calculate "Norm-to-max". 
 

SECTION: FRAP data analysis
21. Plot these Norm-to-max values in Prism using the XY format with replicate values in side-by-side columns where NEMO values populate group 1 and p62 values populate group 2.