Abstract
Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
Change history
28 October 2021
Correction to: Volkmar Weissig and Marvin Edeas (ed.), Mitochondrial Medicine: Volume 3: Manipulating Mitochondria and Disease-Specific Approaches, Methods in Molecular Biology, vol. 2277 https://doi.org/10.1007/978-1-0716-1270-5
References
Frey TG, Mannella CA (2000) The internal structure of mitochondria. Trends Biochem Sci 25(7):319–324. https://doi.org/10.1016/s0968-0004(00)01609-1
Jourdain AA, Boehm E, Maundrell K, Martinou JC (2016) Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression. J Cell Biol 212(6):611–614. https://doi.org/10.1083/jcb.201507125
Pearce SF, Rebelo-Guiomar P, D'Souza AR, Powell CA, Van Haute L, Minczuk M (2017) Regulation of mammalian mitochondrial gene expression: recent advances. Trends Biochem Sci 42(8):625–639. https://doi.org/10.1016/j.tibs.2017.02.003
Jakobs S (2006) High resolution imaging of live mitochondria. Biochim Biophys Acta 1763(5–6):561–575. https://doi.org/10.1016/j.bbamcr.2006.04.004
Opstad IS, Wolfson DL, Øie CI, Ahluwalia BS (2018) Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy. Nanophotonics 7:935. https://doi.org/10.1515/nanoph-2017-0112
Jakobs S, Wurm CA (2014) Super-resolution microscopy of mitochondria. Curr Opin Chem Biol 20:9–15. https://doi.org/10.1016/j.cbpa.2014.03.019
Ma B, Tanese N (2017) RNA-directed FISH and immunostaining. In: Liehr T (ed) Fluorescence in situ hybridization (FISH): application guide. Springer, Berlin, pp 327–335. https://doi.org/10.1007/978-3-662-52959-1_34
Kukat C, Wurm CA, Spahr H, Falkenberg M, Larsson NG, Jakobs S (2011) Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proc Natl Acad Sci U S A 108(33):13534–13539. https://doi.org/10.1073/pnas.1109263108
Silva Ramos E, Motori E, Bruser C, Kuhl I, Yeroslaviz A, Ruzzenente B, Kauppila JHK, Busch JD, Hultenby K, Habermann BH, Jakobs S, Larsson NG, Mourier A (2019) Mitochondrial fusion is required for regulation of mitochondrial DNA replication. PLoS Genet 15(6):e1008085. https://doi.org/10.1371/journal.pgen.1008085
Huang B, Jones SA, Brandenburg B, Zhuang X (2008) Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5(12):1047–1052. https://doi.org/10.1038/nmeth.1274
Stoldt S, Wenzel D, Kehrein K, Riedel D, Ott M, Jakobs S (2018) Spatial orchestration of mitochondrial translation and OXPHOS complex assembly. Nat Cell Biol 20(5):528–534. https://doi.org/10.1038/s41556-018-0090-7
Stephan T, Roesch A, Riedel D, Jakobs S (2019) Live-cell STED nanoscopy of mitochondrial cristae. Sci Rep 9(1):12419. https://doi.org/10.1038/s41598-019-48838-2
Jans DC, Wurm CA, Riedel D, Wenzel D, Stagge F, Deckers M, Rehling P, Jakobs S (2013) STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria. Proc Natl Acad Sci U S A 110(22):8936. https://doi.org/10.1073/pnas.1301820110
Iborra FJ, Kimura H, Cook PR (2004) The functional organization of mitochondrial genomes in human cells. BMC Biol 2:9. https://doi.org/10.1186/1741-7007-2-9
Battich N, Stoeger T, Pelkmans L (2013) Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat Methods 10(11):1127–1133. https://doi.org/10.1038/nmeth.2657
Kotrys AV, Szczesny RJ (2019) Mitochondrial gene expression and beyond-novel aspects of cellular physiology. Cells 9(1):17. https://doi.org/10.3390/cells9010017
Jeandard D, Smirnova A, Tarassov I, Barrey E, Smirnov A, Entelis N (2019) Import of non-coding RNAs into human mitochondria: a critical review and emerging approaches. Cells 8(3):286. https://doi.org/10.3390/cells8030286
Summer S, Smirnova A, Gabriele A, Toth U, Fasemore AM, Forstner KU, Kuhn L, Chicher J, Hammann P, Mitulovic G, Entelis N, Tarassov I, Rossmanith W, Smirnov A (2020) YBEY is an essential biogenesis factor for mitochondrial ribosomes. Nucleic Acids Res 48(17):9762–9786. https://doi.org/10.1093/nar/gkaa148
Brown A, Amunts A, Bai X-C, Sugimoto Y, Edwards PC, Murshudov G, Scheres SHW, Ramakrishnan V (2014) Structure of the large ribosomal subunit from human mitochondria. Science 346(6210):718–722. https://doi.org/10.1126/science.1258026
Rackham O, Shearwood AM, Mercer TR, Davies SM, Mattick JS, Filipovska A (2011) Long noncoding RNAs are generated from the mitochondrial genome and regulated by nuclear-encoded proteins. RNA 17(12):2085–2093. https://doi.org/10.1261/rna.029405.111
Dhir A, Dhir S, Borowski LS, Jimenez L, Teitell M, Rotig A, Crow YJ, Rice GI, Duffy D, Tamby C, Nojima T, Munnich A, Schiff M, de Almeida CR, Rehwinkel J, Dziembowski A, Szczesny RJ, Proudfoot NJ (2018) Mitochondrial double-stranded RNA triggers antiviral signalling in humans. Nature 560(7717):238–242. https://doi.org/10.1038/s41586-018-0363-0
Rizk A, Paul G, Incardona P, Bugarski M, Mansouri M, Niemann A, Ziegler U, Berger P, Sbalzarini IF (2014) Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh. Nat Protoc 9(3):586–596. https://doi.org/10.1038/nprot.2014.037
Glushonkov O, Réal E, Boutant E, Mély Y, Didier P (2018) Optimized protocol for combined PALM-dSTORM imaging. Sci Rep 8(1):8749. https://doi.org/10.1038/s41598-018-27059-z
Ovesný M, Křížek P, Borkovec J, Svindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30(16):2389–2390. https://doi.org/10.1093/bioinformatics/btu202
Acknowledgments
This work was supported by the CNRS (Centre National de Recherche Scientifique); the University of Strasbourg; the IMCBio consortium, the Labex MitoCross (ANR-11-LABX-0057_MITOCROSS) of the National Programme “Investissements d’Avenir” and the Idex UNISTRA. YM is grateful to the Institut Universitaire de France (IUF) for support and providing additional time to be dedicated to research.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Smirnova, A., Richert, L., Smirnov, A., Mély, Y., Tarassov, I. (2021). Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 2277. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1270-5_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1270-5_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1269-9
Online ISBN: 978-1-0716-1270-5
eBook Packages: Springer Protocols