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