A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

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

Summary

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Abstract

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Introduction

The mitochondrial network is a series of interconnected organelles that play a crucial role in energy production1, innate immunity2,3, and cell signalling4,5. Mitochondrial dysregulation has been associated with neurodegenerative diseases such as Parkinson's disease (PD)6,7. PD is a progressive neurodegenerative disorder affecting dopaminergic neurons of the substantia nigra that impacts nearly 10 million people worldwide8. PD has been genetically linked to mitophagy, a mitochondrial quality control pathway necessary for maintaining cellular homeostasis that selectively removes damaged mitochondria9,10. Studies have identified multiple independent mitophagy pathways, including FUN14 domain containing 1 (FUNDC1)-mediated mitophagy, Bcl-2 interacting protein 3 (BNIP3)-facilitated mitophagy, NIX-dependent mitophagy, and the well-characterized PTEN-induced kinase 1 (PINK1)/Parkin-regulated mitophagy10,11. PINK1 (a putative kinase) and Parkin (an E3 ubiquitin ligase) work in tandem to phospho-ubiquitinate damaged mitochondria, which drives the formation of autophagosomes that engulf the damaged organelle and fuse with lysosomes to initiate degradation12,13,14,15,16. Mutations in Parkin have been associated with PD-linked phenotypes such as neurodegeneration via the loss of dopaminergic neurons17,18.

Here, a protocol is described in which HeLa cells, routinely used immortalized cells derived from cervical cancer, are used to investigate the role of Parkin in maintaining mitochondrial network health. HeLa cells express negligible levels of endogenous Parkin and therefore require exogenous Parkin expression19. To study the role of Parkin in mitochondrial network health, HeLa cells are transfected with either wild-type Parkin (ParkinWT), a Parkin mutant (ParkinT240R), or an empty control vector. ParkinT240R is an autosomal recessive juvenile parkinsonism mutation that affects Parkin E3 ligase activity, significantly reducing the efficiency of the mitophagy pathway20. HeLa cells are subject to mild (5 Β΅M) or severe (20 Β΅M) concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. Treatment with severe concentrations of CCCP is routinely used to induce Parkin-mediated mitophagy in various cell lines, such as HeLa and COS-7 cells21,22,23.

Following treatment, the protocol employs live imaging of the mitochondrial network using two currently available mitochondrial-targeted fluorescent dyes. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE) is a cationic dye that fluoresces based on mitochondrial membrane potential24, while MitoSOX is a mitochondrial superoxide indicator where the fluorescence intensity is a function of superoxide concentration25. Finally, the outlined protocol uses a fluorescence-based quantification and simple workflow to effectively quantify the mitochondrial membrane potential and superoxide levels with minimal scope for user bias. Although this protocol was designed to study mitochondrial function in HeLa cells, it can be adapted for additional cell lines and primary cell types to quantitively characterize mitochondrial network health.

Protocol

1. Preparation of biological samples

NOTE: Perform the following steps using sterile technique in a biosafety cabinet. Spray the surface of the cabinet and all materials with 70% ethanol.

  1. HeLa cell culturing and transfection
    1. Culture 30,000 HeLa cells in Dulbecco's modified eagle medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum and 1% L-glutamine solution (HeLa media; see Table of Materials). Plate the cells on 35 mm glass-bottom imaging dishes (see Table of Materials) containing 2 mL of prewarmed HeLa media. Maintain the HeLa cultures at 5% CO2 and 37 Β°C26,27.
    2. The day after plating, inspect the 35 mm imaging dishes using a light microscope to ensure the cells are ~50%-60% confluent. Once confluent, transfect the HeLa cells with empty yellow fluorescent protein (YFP) vector, YFP-ParkinWT, or YFP-ParkinT240R (Figure 1A).
    3. Prepare two separate mixtures in sterile microcentrifuge tubes for each transfection. Mix by tapping the tube or gently pipetting up and down.
      1. In tube 1, mix 200 Β΅L of reduced serum media (see Table of Materials) with 2 Β΅g of the plasmid DNA.
      2. In tube 2, mix 200 Β΅L of reduced serum media with 6 Β΅L of transfection reagent (see Table of Materials)28.
    4. Incubate the tubes for 5 min at room temperature (RT). Add tube 2 to tube 1, mix by pipetting up and down, and incubate for 20 min at RT.
    5. Add the transfection complexes dropwise to the existing imaging dishes containing the HeLa cell cultures, ensuring equal distribution across the entire dish. Place the imaging dishes in the 5% CO2 and 37 Β°C incubator overnight.
      NOTE: Label each dish with the appropriate transfected plasmid. For each experiment, label the three YFP-ParkinWT dishes (dimethyl sulfoxide [DMSO; see Table of Materials], 5 Β΅M CCCP, and 20 Β΅M CCCP). Repeat the labeling with the three YFP-ParkinT240R dishes and the three empty YFP vector dishes.
  2. Preparation of CCCP and fluorescent dyes
    CAUTION: Fluorescent dyes are often light-sensitive. Store the dyes in the dark using aluminum foil or brown centrifuge tubes to reduce exposure to light.
    1. Make a 5 mM working stock of CCCP (see Table of Materials) by dissolving 5 mg of CCCP in 4.89 mL of DMSO. Make a 20 mM working stock of CCCP by dissolving 5 mg of CCCP in 1.22 mL of DMSO.
    2. Dilute 10 Β΅L of a 1 mM MitoTracker Deep Red (see Table of Materials) stock solution in 390 Β΅L of DMSO to make a 25 Β΅M working stock.
    3. Dissolve 50 Β΅g of MitoSOX Red25 (see Table of Materials) in 13 Β΅L of DMSO to create a 5 mM working stock.
    4. Dissolve 10 mg of TMRE24 (see Table of Materials) in 19.4 mL of DMSO to make a 1 mM stock solution. Dilute the TMRE by adding 10 Β΅L of 1 mM TMRE into 990 Β΅L of DMSO to make a 10 Β΅M working stock.

2. CCCP treatment and mitochondrial labeling with fluorescent probes in HeLa cells

CAUTION: Perform the following steps quickly to ensure the HeLa cells are not out of the incubator for an extended period.

  1. The day after transfection, add 2 Β΅L of either 5 or 20 mM CCCP (final concentration: 5 Β΅M and 20 Β΅M, respectively) to each experimental plate. For control plates, add 2 Β΅L of DMSO. Return the plates to the 5% CO2 and 37 Β°C incubator for 1.5 h (Figure 1A).
    NOTE: The CCCP treatment is for a total of 2 h. However, the mitochondria are labeled with fluorescent probes during the final 30 min of the CCCP treatment.
  2. After 1.5 h, remove the plates from the incubator and add fluorescent probes to the imaging dishes. Return the plates to the 5% CO2 and 37 Β°C incubator for 30 min (Figure 1A).
    1. TMRE experiment: Add 2 Β΅L of 25 Β΅M MitoTracker (final concentration: 25 nM) and 2 Β΅L of 10 Β΅M TMRE (final concentration: 10 nM).
    2. MitoSOX experiment: Add 2 Β΅L of 25 Β΅M MitoTracker (final concentration: 25 nM) and 1 Β΅L of 5 mM MitoSOX (final concentration: 2.5 Β΅M).
  3. After the 2 h CCCP treatment, prepare the HeLa cells for imaging (Figure 1A,B).
    1. TMRE experiment: HeLa cells are immediately ready to image; ensure that the TMRE remains in the HeLa cell culture media.
    2. MitoSOX experiment: Wash the cells 3x with 2 mL of prewarmed HeLa media to remove free MitoSOX dye. Add 2 mL of prewarmed media. The HeLa cells are now ready to image.
      ​NOTE: Wash the HeLa cells with fresh, prewarmed HeLa media that contains the same DMSO or CCCP concentration as the experimental condition; do not include MitoSOX or MitoTracker.

3. Confocal microscope image acquisition setup

NOTE: Image the HeLa cells using a confocal microscope (see Table of Materials) equipped with a 63x/1.40 numerical aperture (NA) oil immersion objective and an environmental chamber (see Table of Materials).

  1. At 1 h before imaging, turn on the CO2 by opening the tank valve. Press the On button to turn on the environmental controller for the microscope. Use the Up and Down Arrows on the touchpad to adjust the temperature to 37 Β°C and the CO2 to 5%. Press Set when complete.
    NOTE: Ensure the doors to the environmental chamber are closed and wait for the conditions to stabilize.
  2. Laser settings
    1. Turn on the White Light Laser (WLL) and set the laser power to 85% and the excitation control to maximum power. Click the Acquire tab, select Add Laser, and in the dialog box that appears, toggle the WLL to On. Click the Laser Power button and enter 85%. Click the Excitation Control button and select Maximum Power from the dropdown menu (Figure 2A).
    2. TMRE experiment: Set the excitation and emission spectra for YFP, MitoTracker, and TMRE.
      1. For YFP, set the excitation laser to 514 nm and the emission spectra window to 524-545 nm. In the Acquire tab, click the Add New Setting button; then, click Add Laser and drag it into Setting 1. Double-click on the Excitation Line and enter 514 as the wavelength in the dialog box. Double-click on the corresponding detector and enter 524 for the beginning and 545 for the end wavelength.
      2. For MitoTracker Deep Red, set the excitation laser to 641 and the emission spectra window to 650-750 nm. In the AcquireΒ tab, click the Add Laser button and drag it into Setting 1. Double-click on the Excitation LineΒ and enter 641 as the wavelength in the dialog box. Double-click on the corresponding detector and enter 650Β for the beginning and 750 for the end wavelength.
      3. For TMRE, set the excitation laser to 555 nm and the emission spectra window to 557-643 nm. In the Acquire tab, click the Add New Setting button; then, click the Add Laser button and drag it into Setting 2. Double-click on the Excitation Line and enter 555 as the wavelength in the dialog box. Double-click on the corresponding detector and enter 557 for the beginning and 643 for the end wavelength.
    3. MitoSOX experiment: Set the excitation and emission spectra for YFP, MitoTracker, and MitoSOX (Figure 2B).
      1. For YFP, set the excitation laser to 507 nm and the emission spectra window to 517-540 nm. In the Acquire tab, click the Add New Setting button; then, click the Add Laser button and drag it into Setting 1. Double-click on the Excitation Line and enter 507 as the wavelength in the dialog box. Double-click on the corresponding detector and enter 517 for the beginning and 540 for the end wavelength.
      2. For MitoSOX, set the excitation laser to 547 nm and the emission spectra window to 564-636 nm. In the Acquire tab, click the Add New Setting button; then, click the Add Laser button and drag it into Setting 2. Double-click on the Excitation LineΒ and enter 547 as the wavelength in the dialog box. Double-click on the corresponding detector and enter 564 for the beginning and 636 for the end wavelength.
      3. For MitoTracker Deep Red, set the excitation laser to 641 nm and the emission spectra window to 652-742 nm. In the Acquire tab, click the Add New Setting button; click the Add Laser button and drag it into Setting 3. Double-click on the Excitation Line and enter 641 as the wavelength in the dialog box. Double-click on the corresponding detector and enter 652 for the beginning and 742 for the end wavelength.
  3. Image acquisition settings (Figure 2C)
    1. Set the format to 1,024 x 1,024, the scanning speed to 600 Hz, and the line average to 3.
      1. Select the AcquisitionΒ tab and click the Format button. From the dropdown menu, select 1,024 x 1,024. Click the Speed button and select 600 from the dropdown menu. Then, click the Line Average button, and from the dropdown menu, select 3.
      2. Turn Bidirectional Scanning on and set the phase and zoom factor to 22.61 and 1.50, respectively.
        1. In the AcquisitionΒ tab, toggle the Bidirectional button to On. Click on the Phase X setting and set it to 22.61. Click on the Zoom Factor setting and set it to 1.50.

4. Image acquisition

CAUTION: The experimenter must make a visual judgment to select the cells based on the YFP fluorescence signal. Avoid oversaturated pixels, as they can significantly affect the fluorescence intensity quantification. Use an over/under look-up table that indicates pixel saturation to avoid acquiring saturated images.

  1. Click on the Setting of interest and press Fast LiveΒ to provide a live preview of the fluorescence image.
  2. Adjust the gain and intensity by double-clicking on the corresponding detector. In the dialog box that appears, adjust the Gain using the slider. To alter the intensity, double-click on the Excitation LineΒ and use the Up and Down Arrows in the popup window to change the intensity. Click Stop to end the preview.
    1. TMRE experiment: Image the DMSO control plate first. Adjust the gain and intensity of the TMRE signal (Setting 2) so that the mitochondrial network intensity is just below saturation; keep the gain and intensity constant for the experiment. Adjust the gain and intensity of the MitoTracker and YFP (Setting 1) so that the mitochondrial network is visible but dim.
    2. MitoSOX experiment: Image the DMSO control plate first. Adjust the gain and intensity of the MitoSOX (Setting 2) signal so that the fluorescence is visible but dim; keep the gain and intensity constant for the experiment. Adjust the gain and intensity of the YFP (Setting 1) and MitoTracker (Setting 3) so that the mitochondrial network is visible but dim.
      NOTE: Record the gain and intensity of TMRE and MitoSOX, as these values must remain constant throughout the experiment. The fluorescence signals of YFP and MitoTracker are not quantified in this protocol. Therefore, the gain and intensity can be adjusted for each image. It is most effective to have the gain and intensity settings where the cells are easy to see but still dim, to ensure the cells are not exposed to excessive laser intensities that cause cell damage or photobleaching.
  3. Once the gain and intensity settings are complete, click Start to acquire an image. Acquire images of 20 cells per experimental condition (e.g., five images with four cells per image; Figure 2D).

5. Fluorescence intensity quantification using ImageJ

NOTE: Imaging files are saved as ".lif" and are compatible with ImageJ29. Compatible file types with ImageJ are specified on their website. It may be necessary to convert the file type if it is incompatible.

  1. Create and save a region of interest (ROI).
    1. In ImageJ, click File | New | Image. In the dialog box that appears, click OK.
    2. Click the Rectangle Tool button and draw a 6 micron x 6 micron box.Β Open the ROI manager by clicking Analyze | Tools | ROI Manager and wait for a dialog box with the ROI manager to appear (Figure 3A). Add the rectangular ROI to the manager by clicking Add on the manager dialog box. Save the ROI by selecting More, then click the SaveΒ button and OK.
  2. Measure the fluorescence intensity.
    1. In Image J, click File and select Open. In the dialog box, select the imaging files for the experiment and click Open.
    2. Wait for the Bio-formats Import Options window to appear (Figure 3B). Select Split Channels and click Open.
      NOTE: The split channels feature allows each channel in the image to be opened as a separate window. The channel order corresponds to the acquisition order.
      1. TMRE experiments: YFP (Setting 1) is the first channel (c = 0); MitoTracker (Setting 1) is the second channel (c = 1); and TMRE (Setting 2) is the third channel (c = 2).
      2. MitoSOX experiments: YFP (Setting 1) is the first channel (c = 0); MitoSOX (Setting 2) is the second channel (c = 1); and MitoTracker (Setting 3) is the third channel (c = 2).
    3. Adjust the brightness by selecting Image | Adjust | Brightness (Figure 3C).
      NOTE: Do not press SetΒ or ApplyΒ to ensure the image brightness is not permanently altered. Occasionally, the fluorescence intensity is not visible in the TMRE or MitoSOX channels. Adjust the brightness to allow for easier visualization. Altering the brightness does not affect the raw fluorescence intensity values.
    4. Click Analyze and select Set Measurements. Check the Area and Mean gray value boxes and click OK (Figure 3D).
      NOTE: The mean gray value is the fluorescence intensity.
    5. Open the ROI Manager and load the ROI saved in step 5.1 by clicking Analyze | Tools | ROI Manager. In the ROI Manager, click More, then OpenΒ from the list that appears, and select the saved ROI.
    6. Measure the fluorescence intensity of five random regions in a single cell by selecting the saved ROI from the ROI manager and move the ROI to a random location within a cell (Figure 3E). Measure the fluorescence intensity by pressing M. Repeat this step with four additional nonoverlapping regions. When a dialog box with the area and mean gray values appears (Figure 3F), copy and paste the values into a spreadsheet for analysis.
      1. TMRE experiment: Select image (c = 2).
      2. MitoSOX experiment: Select image (c = 2).
    7. Obtain the mean fluorescence intensity values. Using spreadsheet software (see Table of Materials), average the five mean gray values. Repeat this process for each cell.
    8. Analyze and compare the TMRE or MitoSOX mean gray values across experimental conditions using a statistical software program (see Table of Materials; Figure 4 and Figure 5). Present data as bar graphs or violin plots.

Results

In this protocol, fluorescence-based quantification was used to measure the membrane potential and superoxide levels of the mitochondrial network following CCCP treatment (Figure 1). This workflow used HeLa cells, an immortalized cell line derived from cervical cancer. HeLa cells are routinely used to study mitochondrial biology and are relatively flat, making it easy to visualize the mitochondrial network using microscopy. To investigate the role of Parkin in maintaining mitochondrial netwo...

Discussion

The workflow outlined here can be used to quantify mitochondrial membrane potential and superoxide levels robustly and reproducibly using fluorescence-based imaging30. There are important technical limitations to consider when designing these experiments. HeLa cells were transfected with an empty YFP vector, YFP-ParkinWT, or YFP-ParkinT240R. The empty YFP vector was used as a control to confirm that the experimental findings were specific to Parkin. For the transient transfec...

Disclosures

The authors have no competing interests to declare.

Acknowledgements

We thank the members of the Evans lab for their thoughtful feedback on this manuscript. This work is supported by Duke Whitehead Scholars, Duke Science and Technology Scholars, and Howard Hughes Medical Institute (HHMI) Hanna Gray Fellowship. Figure 1A was made using BioRender.com.

Materials

NameCompanyCatalog NumberComments
Chemicals, Peptides, and Recombinant Proteins
CCCP (carbonyl cyanide m-chlorophenyl hydrazone)Β Sigma-AldrichC2759
DMEM (1x) with 4.5 g/L glucoseGibco11-965-084
DMSO, AnhydrousThermoFisher ScientificD12345
Fetal Bovine SerumHycloneSH3007103
FuGENE 6 (Tranfection Reagent)PromegaE2691
GlutaMAX 100x (L-Glutamine Solution)Β GibcoΒ 35-050-061
Hoescht 33342ThermoFisher Scientific62249
MitoSOXΒ  RedΒ ThermoFisher ScientificM36008
MitoTracker Deep RedThermoFisher ScientificM7514
Opti-MEM (Redued Serum media)ThermoFisher scientific31985070
Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)Β ThermoFisher ScientificT669
Experimental models: Organisms/Strains
HeLa-M (Homo sapiens)A. Peden (Cambridge Institute for Medical Research)N/A
Recombinant DNA
EYFP Empty VectorN/AN/A
YFP-Parkin T240RThis PaperGenerated by site-directed mutagenesis from YFP-Parkin
YFP-Parkin WTAddgene; PMID:19029340RRID:Addgene_23955
Software and Algorithms
Adobe IllustratorAdobe Inc.https://www.adobe.com/products/illustrator(Schindelin, 2012)
Excel (Spreadsheet Software)Microsoft OfficeΒ https://www.microsoft.com/en-us/microsoft-365/excel
ImageJhttps://imagej.net/software/fiji/
Leica Application Suite (LAS X)Leicahttps://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/
Microsoft ExcelMicrosoft Officehttps://www.microsoft.com/excel
Prism9 (Statistical Analysis Software)GraphPad Softwarehttps://www.graphpad.com
Other
35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, UncoatedMatTekP35G-1.5-20-C
Cage Incubator (Environmental Chamber)Okolabhttps://www.oko-lab.com/cage-incubator
DMiL Inverted MicroscopeLeicaN/A
LIGHTNING Deconvolution SoftwareLeicaN/A
STELLARIS 8 confocal microscopeLeicaN/A

References

  1. Spinelli, J. B., Haigis, M. C. The multifaceted contributions of mitochondria to cellular metabolism. Nature Cell Biology. 20 (7), 745-754 (2018).
  2. West, A. P., Shadel, G. S., Ghosh, S. Mitochondria in innate immune responses. Nature Reviews. Immunology. 11 (6), 389-402 (2011).
  3. Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell. 122 (5), 669-682 (2005).
  4. Tait, S. W. G., Green, D. R. Mitochondria and cell signalling. Journal of Cell Science. 125, 807-815 (2012).
  5. Antico Arciuch, V. G., Elguero, M. E., Poderoso, J. J., Carreras, M. C. Mitochondrial regulation of cell cycle and proliferation. Antioxidants and Redox Signaling. 16 (10), 1150-1180 (2012).
  6. Grunewald, A., Kumar, K. R., Sue, C. M. New insights into the complex role of mitochondria in Parkinson's disease. Progress in Neurobiology. 177, 73-93 (2019).
  7. Borsche, M., Pereira, S. L., Klein, C., Grunewald, A. Mitochondria and Parkinson's disease: clinical, molecular, and translational aspects. Journal of Parkinson's Disease. 11 (1), 45-60 (2021).
  8. Ou, Z., et al. Global trends in the incidence, prevalence, and years lived with disability of Parkinson's disease in 204 countries/territories from 1990 to 2019. Frontiers in Public Health. 9, 776847 (2021).
  9. Martinez-Vicente, M. Neuronal mitophagy in neurodegenerative diseases. Frontiers in Molecular Neuroscience. 10, 64 (2017).
  10. Youle, R. J., Narendra, D. P. Mechanisms of mitophagy. Nature Reviews. Molecular Cell Biology. 12 (1), 9-14 (2011).
  11. Villa, E., Marchetti, S., Ricci, J. E. No Parkin zone: mitophagy without Parkin. Trends in Cell Biology. 28 (11), 882-895 (2018).
  12. Geisler, S., et al. The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations. Autophagy. 6 (7), 871-878 (2010).
  13. Kane, L. A., et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. The Journal of Cell Biology. 205 (2), 143-153 (2014).
  14. Koyano, F., et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature. 510 (7503), 162-166 (2014).
  15. Ordureau, A., et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proceedings of the National Academy of Sciences. 112 (21), 6637-6642 (2015).
  16. Ordureau, A., et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Molecular Cell. 56 (3), 360-375 (2014).
  17. Kitada, T., et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature. 392 (6676), 605-608 (1998).
  18. Valente, E. M., et al. PARK6 is a common cause of familial parkinsonism. Neurological Sciences. 23, S117-S118 (2002).
  19. Matsuda, N., et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. The Journal of Cell Biology. 189 (2), 211-221 (2010).
  20. Sriram, S. R., et al. Familial-associated mutations differentially disrupt the solubility, localization, binding and ubiquitination properties of parkin. Human Molecular Genetics. 14 (17), 2571-2586 (2005).
  21. Vives-Bauza, C., et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proceedings of the National Academy of Sciences. 107 (1), 378-383 (2010).
  22. Wong, Y. C., Holzbaur, E. L. F. Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proceedings of the National Academy of Sciences. 111 (42), E4439-E4448 (2014).
  23. Bertolin, G., et al. Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-beta hydroxysteroid dehydrogenase type 10. Cell Death and Differentiation. 22 (10), 1563-1576 (2015).
  24. Crowley, L. C., Christensen, M. E., Waterhouse, N. J. Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harbor Protocols. 2016 (12), (2016).
  25. Kuznetsov, A. V., et al. Mitochondrial ROS production under cellular stress: comparison of different detection methods. Analytical and Bioanalytical Chemistry. 400 (8), 2383-2390 (2011).
  26. Moore, A. S., Holzbaur, E. L. F. Dynamic recruitment and activation of ALS-associated TBK1 with its target optineurin are required for efficient mitophagy. Proceedings of the National Academy of Sciences. 113 (24), E3349-E3358 (2016).
  27. Evans, C. S., Holzbaur, E. L. F. Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons. eLife. 9, e50260 (2020).
  28. Jacobsen, L. B., Calvin, S. A., Colvin, K. E., Wright, M. FuGENE 6 Transfection Reagent: the gentle power. Methods. 33 (2), 104-112 (2004).
  29. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
  30. Mitra, K., Lippincott-Schwartz, J. Analysis of mitochondrial dynamics and functions using imaging approaches. Current Protocols in Cell Biology. , 1-21 (2010).
  31. Lin, H. C., Liu, S. Y., Lai, H. S., Lai, I. R. Isolated mitochondria infusion mitigates ischemia-reperfusion injury of the liver in rats. Shock. 39 (3), 304-310 (2013).
  32. Kholmukhamedov, A., Schwartz, J. M., Lemasters, J. J. Isolated mitochondria infusion mitigates ischemia-reperfusion injury of the liver in rats: mitotracker probes and mitochondrial membrane potential. Shock. 39 (6), 543 (2013).
  33. Thorn, K. Genetically encoded fluorescent tags. Molecular Biology of the Cell. 28 (7), 848-857 (2017).
  34. Pavel, M., et al. Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis. Nature Communications. 9 (1), 2961 (2018).
  35. Rossignol, R., et al. Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Research. 64 (3), 985-993 (2004).
  36. Schornack, P. A., Gillies, R. J. Contributions of cell metabolism and H+ diffusion to the acidic pH of tumors. Neoplasia. 5 (2), 135-145 (2003).
  37. Christensen, M. E., Jansen, E. S., Sanchez, W., Waterhouse, N. J. Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release. Methods. 61 (2), 138-145 (2013).
  38. Muller, B., et al. Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos. Scientific Reports. 9 (1), 16778 (2019).
  39. Connolly, N. M. C., et al. Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases. Cell Death and Differentiation. 25 (3), 542-572 (2018).
  40. Demine, S., Renard, P., Arnould, T. Mitochondrial uncoupling: a key controller of biological processes in physiology and diseases. Cells. 8 (8), 795 (2019).
  41. Narendra, D., Tanaka, A., Suen, D. F., Youle, R. J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. The Journal of Cell Biology. 183 (5), 795-803 (2008).
  42. Kwak, S. H., Park, K. S., Lee, K. U., Lee, H. K. Mitochondrial metabolism and diabetes. Journal of Diabetes Investigation. 1 (5), 161-169 (2010).
  43. Reddy, P. H. Role of mitochondria in neurodegenerative diseases: mitochondria as a therapeutic target in Alzheimer's disease. CNS Spectrums. 14 (8), 8-13 (2009).
  44. Wang, W., Zhao, F., Ma, X., Perry, G., Zhu, X. Mitochondria dysfunction in the pathogenesis of Alzheimer's disease: recent advances. Molecular Neurodegeneration. 15 (1), 30 (2020).
  45. Baloyannis, S. J. Mitochondrial alterations in Alzheimer's disease. Journal of Alzheimer's Disease. 9 (2), 119-126 (2006).
  46. Wallace, D. C. Mitochondria and cancer. Nature Reviews. Cancer. 12 (10), 685-698 (2012).
  47. Middleton, P., Vergis, N. Mitochondrial dysfunction and liver disease: role, relevance, and potential for therapeutic modulation. Therapeutic Advances in Gastroenterology. 14, 17562848211031394 (2021).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Mitochondrial Membrane PotentialSuperoxide LevelsLive ImagingHeLa CellsNeurodegenerative DiseasesParkinson s DiseaseALSSTEDSIMExpansion MicroscopyMitophagyPINK1ParkinMitochondrial Quality ControlATP SynthesisCCCPParkin T240R Mutation

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright Β© 2025 MyJoVE Corporation. All rights reserved