Fluorescensbaseret kvantificering af mitokondriemembranpotentiale og superoxidniveauer ved hjælp af levende billeddannelse i HeLa-celler
Summary
Denne teknik beskriver en effektiv arbejdsgang til at visualisere og kvantitativt måle mitokondriemembranpotentiale og superoxidniveauer i HeLa-celler ved hjælp af fluorescensbaseret levende billeddannelse.
Abstract
Mitokondrier er dynamiske organeller, der er kritiske for metabolisk homeostase ved at kontrollere energiproduktionen via ATP-syntese. For at understøtte cellulær metabolisme samarbejder forskellige mitokondrie kvalitetskontrolmekanismer for at opretholde et sundt mitokondrienetværk. En sådan vej er mitofagi, hvor PTEN-induceret kinase 1 (PINK1) og Parkin phospho-ubiquitination af beskadigede mitokondrier letter autofagosomsekvestrering og efterfølgende fjernelse fra cellen via lysosomfusion. Mitofagi er vigtig for cellulær homeostase, og mutationer i Parkin er forbundet med Parkinsons sygdom (PD). På grund af disse resultater har der været en betydelig vægt på at undersøge mitokondrieskader og omsætning for at forstå de molekylære mekanismer og dynamikker i mitokondriel kvalitetskontrol. Her blev levende cellebilleddannelse brugt til at visualisere mitokondrienetværket af HeLa-celler til at kvantificere mitokondriemembranpotentialet og superoxidniveauerne efter behandling med carbonylcyanid m-chlorphenylhydrazon (CCCP), et mitokondrielt afkoblingsmiddel. Derudover blev en PD-bundet mutation af Parkin (ParkinT240R), der hæmmer Parkin-afhængig mitofagi, udtrykt for at bestemme, hvordan mutant ekspression påvirker mitokondrienetværket sammenlignet med celler, der udtrykker vildtype Parkin. Protokollen skitseret her beskriver en simpel arbejdsgang ved hjælp af fluorescensbaserede tilgange til effektivt at kvantificere mitokondriemembranpotentiale og superoxidniveauer.
Introduction
Mitokondrienetværket er en række indbyrdes forbundne organeller, der spiller en afgørende rolle i energiproduktion1, medfødt immunitet2,3 og cellesignalering 4,5. Mitokondriel dysregulering har været forbundet med neurodegenerative sygdomme såsom Parkinsons sygdom (PD)6,7. PD er en progressiv neurodegenerativ lidelse, der påvirker dopaminerge neuroner i substantia nigra, der påvirker næsten 10 millioner mennesker verden over8. PD har været genetisk forbundet med mitofagi, en mitokondriel kvalitetskontrolvej, der er nødvendig for at opretholde cellulær homeostase, der selektivt fjerner beskadigede mitokondrier 9,10. Undersøgelser har identificeret flere uafhængige mitofagiveje, herunder FUN14-domæne indeholdende 1 (FUNDC1)-medieret mitofagi, Bcl-2-interagerende protein 3 (BNIP3)-faciliteret mitofagi, NIX-afhængig mitofagi, og den velkarakteriserede PTEN-inducerede kinase 1 (PINK1)/Parkin-regulerede mitofagi:10,11. PINK1 (en formodet kinase) og Parkin (en E3 ubiquitin ligase) arbejder sammen for at phospho-ubiquitinere beskadigede mitokondrier, som driver dannelsen af autofagosomer, der opsluger den beskadigede organel og smelter sammen med lysosomer for at indlede nedbrydning 12,13,14,15,16. Mutationer i Parkin har været forbundet med PD-bundne fænotyper såsom neurodegeneration via tab af dopaminerge neuroner17,18.
Her beskrives en protokol, hvor HeLa-celler, rutinemæssigt anvendte udødeliggjorte celler afledt af livmoderhalskræft, bruges til at undersøge Parkins rolle i at opretholde mitokondrienetværkets sundhed. HeLa-celler udtrykker ubetydelige niveauer af endogen Parkin og kræver derfor eksogen Parkin-ekspression19. For at studere Parkins rolle i mitokondrienetværkets sundhed transfekteres HeLa-celler med enten vildtype Parkin (ParkinWT), en Parkin-mutant (ParkinT240R) eller en tom kontrolvektor. ParkinT240R er en autosomal recessiv juvenil parkinsonismemutation, der påvirker Parkin E3-ligaseaktivitet, hvilket signifikant reducerer effektiviteten af mitofagivejen20. HeLa-celler udsættes for milde (5 μM) eller svære (20 μM) koncentrationer af carbonylcyanid m-chlorphenylhydrazon (CCCP), et mitokondrielt afkoblingsmiddel. Behandling med alvorlige koncentrationer af CCCP anvendes rutinemæssigt til at inducere Parkin-medieret mitofagi, i forskellige cellelinjer, såsom HeLa- og COS-7-celler21,22,23.
Efter behandling anvender protokollen levende billeddannelse af mitokondrienetværket ved hjælp af to aktuelt tilgængelige mitokondriemålrettede fluorescerende farvestoffer. Tetramethylrhodamin, ethylester, perchlorat (TMRE) er et kationisk farvestof, der fluorescerer baseret på mitokondriemembranpotentiale24, mens MitoSOX er en mitokondriel superoxidindikator, hvor fluorescensintensiteten er en funktion af superoxidkoncentrationen25. Endelig bruger den skitserede protokol en fluorescensbaseret kvantificering og enkel arbejdsgang til effektivt at kvantificere mitokondriemembranpotentialet og superoxidniveauerne med minimal plads til brugerbias. Selvom denne protokol blev designet til at studere mitokondriefunktion i HeLa-celler, kan den tilpasses til yderligere cellelinjer og primære celletyper for kvantitativt at karakterisere mitokondrienetværkets sundhed.
Protocol
Representative Results
Discussion
Den arbejdsgang, der er skitseret her, kan bruges til at kvantificere mitokondriemembranpotentiale og superoxidniveauer robust og reproducerbart ved hjælp af fluorescensbaseret billeddannelse30. Der er vigtige tekniske begrænsninger at overveje, når man designer disse eksperimenter. HeLa-celler blev transfekteret med en tom YFP-vektor, YFP-ParkinWT eller YFP-ParkinT240R. Den tomme YFP-vektor blev brugt som kontrol for at bekræfte, at de eksperimentelle fund var specifikke…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker medlemmerne af Evans-laboratoriet for deres tankevækkende feedback på dette manuskript. Dette arbejde støttes af Duke Whitehead Scholars, Duke Science and Technology Scholars og Howard Hughes Medical Institute (HHMI) Hanna Gray Fellowship. Figur 1A blev lavet ved hjælp af BioRender.com.
Materials
| 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 |
References
- Spinelli, J. B., Haigis, M. C. The multifaceted contributions of mitochondria to cellular metabolism. Nature Cell Biology. 20 (7), 745-754 (2018).
- West, A. P., Shadel, G. S., Ghosh, S. Mitochondria in innate immune responses. Nature Reviews. Immunology. 11 (6), 389-402 (2011).
- 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).
- Tait, S. W. G., Green, D. R. Mitochondria and cell signalling. Journal of Cell Science. 125, 807-815 (2012).
- 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).
- 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).
- 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).
- 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).
- Martinez-Vicente, M. Neuronal mitophagy in neurodegenerative diseases. Frontiers in Molecular Neuroscience. 10, 64 (2017).
- Youle, R. J., Narendra, D. P. Mechanisms of mitophagy. Nature Reviews. Molecular Cell Biology. 12 (1), 9-14 (2011).
- Villa, E., Marchetti, S., Ricci, J. E. No Parkin zone: mitophagy without Parkin. Trends in Cell Biology. 28 (11), 882-895 (2018).
- Geisler, S., et al. The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations. Autophagy. 6 (7), 871-878 (2010).
- 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).
- Koyano, F., et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature. 510 (7503), 162-166 (2014).
- 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).
- 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).
- Kitada, T., et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature. 392 (6676), 605-608 (1998).
- Valente, E. M., et al. PARK6 is a common cause of familial parkinsonism. Neurological Sciences. 23, S117-S118 (2002).
- 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).
- 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).
- 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).
- 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).
- 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).
- Crowley, L. C., Christensen, M. E., Waterhouse, N. J. Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harbor Protocols. 2016 (12), (2016).
- 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).
- 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).
- Evans, C. S., Holzbaur, E. L. F. Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons. eLife. 9, e50260 (2020).
- Jacobsen, L. B., Calvin, S. A., Colvin, K. E., Wright, M. FuGENE 6 Transfection Reagent: the gentle power. Methods. 33 (2), 104-112 (2004).
- Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
- Mitra, K., Lippincott-Schwartz, J. Analysis of mitochondrial dynamics and functions using imaging approaches. Current Protocols in Cell Biology. , 1-21 (2010).
- 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).
- 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).
- Thorn, K. Genetically encoded fluorescent tags. Molecular Biology of the Cell. 28 (7), 848-857 (2017).
- Pavel, M., et al. Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis. Nature Communications. 9 (1), 2961 (2018).
- Rossignol, R., et al. Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Research. 64 (3), 985-993 (2004).
- 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).
- 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).
- 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).
- 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).
- Demine, S., Renard, P., Arnould, T. Mitochondrial uncoupling: a key controller of biological processes in physiology and diseases. Cells. 8 (8), 795 (2019).
- 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).
- 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).
- 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).
- 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).
- Baloyannis, S. J. Mitochondrial alterations in Alzheimer’s disease. Journal of Alzheimer’s Disease. 9 (2), 119-126 (2006).
- Wallace, D. C. Mitochondria and cancer. Nature Reviews. Cancer. 12 (10), 685-698 (2012).
- Middleton, P., Vergis, N. Mitochondrial dysfunction and liver disease: role, relevance, and potential for therapeutic modulation. Therapeutic Advances in Gastroenterology. 14, 17562848211031394 (2021).
