Skip to main content
SpringerLink
Log in

MERLIN: A BRET-Based Proximity Biosensor for Studying Mitochondria–ER Contact Sites

  • Protocol
  • First Online:
Bioluminescence

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2525))

  • 1334 Accesses

Abstract

The contacts between the endoplasmic reticulum (ER) and mitochondria play a fundamental role in a wide variety of cellular processes, like the exchange of calcium and lipids between both organelles, as well as in apoptosis and in autophagy signaling. Despite their importance, due to their dynamic and heterogeneous nature, we still lack understanding of the molecular composition, structure, and regulation of these structures. In this chapter, we introduce a new bioluminescence resonance energy transfer (BRET)-based biosensor for the quantitative analysis of mitochondria–ER interorganellar distances without perturbing their natural environment, which we call MERLIN (mitochondria ER length indicator nanosensor). Here, we describe the rationale behind the MERLIN biosensor, detail the experimental setup and methodology, and provide tips for troubleshooting.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hirabayashi Y, Kwon SK, Paek H, Pernice WM, Paul MA, Lee J, Erfani P, Raczkowski A, Petrey DS, Pon LA, Polleux F (2017) ER-mitochondria tethering by PDZD8 regulates Ca(2+) dynamics in mammalian neurons. Science 358(6363):623–630. https://doi.org/10.1126/science.aan6009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sassano ML, van Vliet AR, Agostinis P (2017) Mitochondria-associated membranes as networking platforms and regulators of cancer cell fate. Front Oncol 7:174. https://doi.org/10.3389/fonc.2017.00174

    Article  PubMed  PubMed Central  Google Scholar 

  3. Csordas G, Weaver D, Hajnoczky G (2018) Endoplasmic reticular-mitochondrial contactology: structure and signaling functions. Trends Cell Biol. https://doi.org/10.1016/j.tcb.2018.02.009

  4. Friedman JR, Dibenedetto JR, West M, Rowland AA, Voeltz GK (2013) Endoplasmic reticulum-endosome contact increases as endosomes traffic and mature. Mol Biol Cell 24(7):1030–1040. https://doi.org/10.1091/mbc.E12-10-0733

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Friedman JR, Lackner LL, West M, DiBenedetto JR, Nunnari J, Voeltz GK (2011) ER tubules mark sites of mitochondrial division. Science 334(6054):358–362. https://doi.org/10.1126/science.1207385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Phillips MJ, Voeltz GK (2016) Structure and function of ER membrane contact sites with other organelles. Nat Rev Mol Cell Biol 17(2):69–82. https://doi.org/10.1038/nrm.2015.8

    Article  CAS  PubMed  Google Scholar 

  7. Csordas G, Varnai P, Golenar T, Roy S, Purkins G, Schneider TG, Balla T, Hajnoczky G (2010) Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface. Mol Cell 39(1):121–132. https://doi.org/10.1016/j.molcel.2010.06.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Csordas G, Renken C, Varnai P, Walter L, Weaver D, Buttle KF, Balla T, Mannella CA, Hajnoczky G (2006) Structural and functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol 174(7):915–921. https://doi.org/10.1083/jcb.200604016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Area-Gomez E, Schon EA (2016) Mitochondria-associated ER membranes and Alzheimer disease. Curr Opin Genet Dev 38:90–96. https://doi.org/10.1016/j.gde.2016.04.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schon EA, Area-Gomez E (2013) Mitochondria-associated ER membranes in Alzheimer disease. Mol Cell Neurosci 55:26–36. https://doi.org/10.1016/j.mcn.2012.07.011

    Article  CAS  PubMed  Google Scholar 

  11. Area-Gomez E, de Groof AJ, Boldogh I, Bird TD, Gibson GE, Koehler CM, Yu WH, Duff KE, Yaffe MP, Pon LA, Schon EA (2009) Presenilins are enriched in endoplasmic reticulum membranes associated with mitochondria. Am J Pathol 175(5):1810–1816. https://doi.org/10.2353/ajpath.2009.090219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pera M, Larrea D, Guardia-Laguarta C, Montesinos J, Velasco KR, Agrawal RR, Xu Y, Chan RB, Di Paolo G, Mehler MF, Perumal GS, Macaluso FP, Freyberg ZZ, Acin-Perez R, Enriquez JA, Schon EA, Area-Gomez E (2017) Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease. EMBO J 36(22):3356–3371. https://doi.org/10.15252/embj.201796797

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Paillusson S, Stoica R, Gomez-Suaga P, Lau DHW, Mueller S, Miller T, Miller CCJ (2016) There’s something wrong with my MAM; the ER-mitochondria axis and neurodegenerative diseases. Trends Neurosci 39(3):146–157. https://doi.org/10.1016/j.tins.2016.01.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hertlein V, Flores-Romero H, Das KK, Fischer S, Heunemann M, Calleja-Felipe M, Knafo S, Hipp K, Harter K, Fitzgerald JC, Garcia-Saez AJ (2020) MERLIN: a novel BRET-based proximity biosensor for studying mitochondria-ER contact sites. Life Sci Alliance 3(1). https://doi.org/10.26508/lsa.201900600

  15. Pfleger KD, Seeber RM, Eidne KA (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 1(1):337–345. https://doi.org/10.1038/nprot.2006.52

    Article  CAS  PubMed  Google Scholar 

  16. Kocan M, See HB, Seeber RM, Eidne KA, Pfleger KD (2008) Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells. J Biomol Screen 13(9):888–898. https://doi.org/10.1177/1087057108324032

    Article  CAS  PubMed  Google Scholar 

  17. Vance JE (2014) MAM (mitochondria-associated membranes) in mammalian cells: lipids and beyond. Biochim Biophys Acta 1841(4):595–609. https://doi.org/10.1016/j.bbalip.2013.11.014

    Article  CAS  PubMed  Google Scholar 

  18. Malinow R, Hayashi Y, Maletic-Savatic M, Zaman SH, Poncer JC, Shi SH, Esteban JA, Osten P, Seidenman K (2010, 2010) Introduction of green fluorescent protein (GFP) into hippocampal neurons through viral infection. Cold Spring Harb Protoc (4):pdb prot5406. https://doi.org/10.1101/pdb.prot5406

  19. Uyaniker S, van der Spek SJF, Reinders NR, Xiong H, Li KW, Bossers K, Smit AB, Verhaagen J, Kessels HW (2019) The effects of Sindbis viral vectors on neuronal function. Front Cell Neurosci 13:362. https://doi.org/10.3389/fncel.2019.00362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pfleger KD, Eidne KA (2003) New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary 6(3):141–151

    Article  CAS  Google Scholar 

  21. Prinz A, Diskar M, Herberg FW (2006) Application of bioluminescence resonance energy transfer (BRET) for biomolecular interaction studies. Chembiochem 7(7):1007–1012. https://doi.org/10.1002/cbic.200600048

    Article  CAS  PubMed  Google Scholar 

  22. Arai R, Nakagawa H, Kitayama A, Ueda H, Nagamune T (2002) Detection of protein-protein interaction by bioluminescence resonance energy transfer from firefly luciferase to red fluorescent protein. J Biosci Bioeng 94(4):362–364

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This project was funded by the Deutsche Forschungsgesellschaft (DFG, German Research foundation), SFB1218, project no. 269925409, and partially by SFB1403, project no. 414786233, and the European Research Council (ERC) (ERC-CoG grant agreement no. 817758). We thank Louise King for carefully reading the manuscript and providing input.

Author information

Authors and Affiliations

  1. Institute for Genetics, University of Cologne, Cologne, Germany

    Hector Flores-Romero & Ana J. García-Sáez

  2. Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

    Hector Flores-Romero & Ana J. García-Sáez

  3. Interfaculty Institute of Biochemistry, Eberhard-Karls-Universität Tübingen, Tübingen, Germany

    Hector Flores-Romero & Ana J. García-Sáez

Authors
  1. Hector Flores-Romero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ana J. García-Sáez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ana J. García-Sáez .

Editor information

Editors and Affiliations

  1. Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

    Sung-Bae Kim

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Flores-Romero, H., García-Sáez, A.J. (2022). MERLIN: A BRET-Based Proximity Biosensor for Studying Mitochondria–ER Contact Sites. In: Kim, SB. (eds) Bioluminescence. Methods in Molecular Biology, vol 2525. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2473-9_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2473-9_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2472-2

  • Online ISBN: 978-1-0716-2473-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation