JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Published: May 12th, 2023

DOI:

10.3791/65304

1Department of Cell Biology, Duke University Medical Center

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.

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.

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 bee....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 th.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Chemicals, Peptides, and Recombinant Proteins
CCCP (carbonyl cyanide m-chlorophenyl hydrazone)  Sigma-Aldrich C2759
DMEM (1x) with 4.5 g/L glucose Gibco 11-965-084
DMSO, Anhydrous ThermoFisher Scientific D12345
Fetal Bovine Serum Hyclone SH3007103
FuGENE 6 (Tranfection Reagent) Promega E2691
GlutaMAX 100x (L-Glutamine Solution)  Gibco  35-050-061
Hoescht 33342 ThermoFisher Scientific 62249
MitoSOX  Red  ThermoFisher Scientific M36008
MitoTracker Deep Red ThermoFisher Scientific M7514
Opti-MEM (Redued Serum media) ThermoFisher scientific 31985070
Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)  ThermoFisher Scientific T669
Experimental models: Organisms/Strains
HeLa-M (Homo sapiens) A. Peden (Cambridge Institute for Medical Research) N/A
Recombinant DNA
EYFP Empty Vector N/A N/A
YFP-Parkin T240R This Paper Generated by site-directed mutagenesis from YFP-Parkin
YFP-Parkin WT Addgene; PMID:19029340 RRID:Addgene_23955
Software and Algorithms
Adobe Illustrator Adobe Inc. https://www.adobe.com/products/illustrator (Schindelin, 2012)
Excel (Spreadsheet Software) Microsoft Office  https://www.microsoft.com/en-us/microsoft-365/excel
ImageJ https://imagej.net/software/fiji/
Leica Application Suite (LAS X) Leica https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/
Microsoft Excel Microsoft Office https://www.microsoft.com/excel
Prism9 (Statistical Analysis Software) GraphPad Software https://www.graphpad.com
Other
35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated MatTek P35G-1.5-20-C
Cage Incubator (Environmental Chamber) Okolab https://www.oko-lab.com/cage-incubator
DMiL Inverted Microscope Leica N/A
LIGHTNING Deconvolution Software Leica N/A
STELLARIS 8 confocal microscope Leica N/A

  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).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved