Jul 31, 2023

Live imaging and analysis to investigate phase separation properties of NEMO during mitophagy

 Forked from Live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells
DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkw7rxl5r/v1
  • 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
  • Liv
OLIVIA HARDING
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkw7rxl5r/v1
Protocol Citation: OLIVIA HARDING, Erika L.F. Holzbaur 2023. Live imaging and analysis to investigate phase separation properties of NEMO during mitophagy. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw7rxl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82749
Keywords: Tissue culture, Mitophagy, Fluorescent ligands, Confocal microscopy, Live cell imaging, ASAPCRN, fragmented mitochondria, mitochondrial membrane, outer mitochondrial membrane, damaged mitochondria, mitophagy phase condensation of biomolecular particle, phase separation properties of nemo, mitophagy phase condensation, phase separation, determining phase separation, formation of nemo puncta, phase separation property, biomolecular particle, separated particle, fluorescence, fusion like droplet, fission, nemo punctum, potential for fission
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
Abstract
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.
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.
Materials

Materials/Reagents

1.5 mL capped tubesMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP022364120

Reagents

  • FBS (HyClone)
  • Leibovitz's L-15 MediumThermo FisherCatalog #11415064
  • Dimethyl sulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
  • Ethanol
  • Antimycin A from Streptomyces sp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8674
  • Oligomycin AMerck MilliporeSigma (Sigma-Aldrich)Catalog #75351

Equipment:
  • Vacuum apparatus
  • 37 °C water bath
  • Confocal microscope with 60X objective, heated stage chamber, focus correction system, and associated software
Note
Our system is not equipped with a pressurized stage chamber to sustain 5% CO2 conditions. Thus, we use L-15 in order to buffer the samples in atmospheric CO2 conditions.






Before start

  • The start point for this protocol is after cells grown on 35 mm glass bottom dishes have been transfected with the relevant constructs for 18:00:00 - 24:00:00 .
  • 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.

Note
Will use ~1 mL imaging media per dish.


  • 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.
























Replace Standard Media with Imaging Media and Induce Mitophagy
10m
Wait until imaging chamber is heated to 37 °C .

Aspirate media from sample.
Repeat the following two steps for a total of 2 washes.
  • Add 200 µL Imaging media gently to dish.
  • Aspirate.
Add .75 mL Imaging media and place dish in 37 °C imaging chamber.

00:45:00 prior to imaging, add the .25 mL working solution of AntA/OligA to the dish making a total of 1 mL

Note
Samples should equilibrate to imaging media and microscope chamber for at least 10 min prior to imaging.

45m
Imaging set-up
15m
Place dish on microscope stage, moving stage adjusters if necessary to fit the dish.

Note
Remove the lid of the dish to maneuver it more easily without spilling its contents. Replace the lid after dish is firmly secured on the stage.

Raise objective so that there is an oil interface between the objective lens and glass bottom of the dish.
Allow dish to settle at least 00:05:00 in this position before imaging to minimize drift during imaging.

5m
Using 100X objective and GFP or RFP epifluorescence, find the focal plane.
Note
100X is necessary to achieve the desired resolution for these applications.

Note
Note the health of cells, transfection efficiency of the constructs, and brightness of fluorescence. If you will image multiple samples with varying conditions, you may want to observe all dishes before confirming the imaging parameters so that no images are overexposed or too dim.

Configure 488, 561, and 647 lasers and accompanying exposure times.
Note
Set parameters as low as possible while still detecting the signals in order to avoid phototoxicity and bleaching. For a benchmark, we use <10% power and <200 ms exposure for each channel. Save settings in order to maintain consistency among experiments.

Step case

FRAP assay
11 steps

Cells expressing Parkin (no tag), Mito-sBFP2, EGFP-NEMO, and mCherry-p62 are treated with AntA/OligA for at least 45 min to induce mitophagy and NEMO puncta recruitment. Photobleaching is accomplished with the use of a high-powered laser. This assay should be carried out between 50 min and 1 hr 20 min post-AntA/OligA addition.
Imaging set-up and FRAP
2h 10m
Set FRAP and timelapse imaging parameters and engage auto-focus to maintain Z plane.
Note
For FRAP, I used 18 ms/pixel, 1 cycle of photobleaching. For timelapse, I set acquisition to 1 frame/5 sec. Program acquired 5 frames before commencing photobleach, then acquisition continued for a total of 10 min.




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.

Choose several puncta to photobleach and draw box-shaped ROIs targeting them. Choose NEMO-only and NEMO-p62 puncta. Save the ROI set.


Commence imaging. Repeat FRAP assay as many times as possible within the 30 min span.
FRAP data analysis
Load timelapse image and saved ROI set to ImageJ.
Draw a new ROI to encompass a background area that exhibits no puncta for the whole video. Save to the ROI manager.
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.
Note
Exclude data from puncta that do not stay in the z frame.

Note
Note whether the puncta is positive for only EGFP-NEMO, or EGFP-NEMO and mCherry-p62.

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.
Note
This step accounts for whole-cell bleaching effects.

Identify the minimum intensity value for each channel for each puncta, then subtract the minimum from Sub-bg's to calculate "Sub-min".
Note
At least one timepoint will be recorded as 0.

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".
Note
At least one timepoint will be recorded as 0, and one will be recorded as 1.

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.
Note
NEMO data can be further divided by whether puncta are NEMO-only or NEMO-p62.