Advertisement

Detection of mRNA Transfer Between Mammalian Cells in Coculture by Single-Molecule Fluorescent In Situ Hybridization (smFISH)

  • Gal HaimovichEmail author
  • Jeffrey E. Gerst
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

Abstract

In eukaryotic cells, a small percentage of mRNA molecules can undergo transfer from one cell to another. mRNA transfer occurs primarily via membrane nanotubes, which are long thin protrusions that are produced by numerous cell types and can connect cells that can be up to hundreds of microns apart. Potentially, mRNAs might also transfer via extracellular vesicles (EVs). Here we describe a method to detect transferred mRNA in cocultures of two different cell types and to distinguish between nanotube- and EVs-mediated transfer. This method uses single molecule fluorescent in situ hybridization (smFISH) to provide an accurate and quantitative detection of transferred mRNA molecules and their subcellular localization. Following the guidelines presented here will allow the user to investigate mRNA transfer of most transcripts in any co-culture system. In addition, we present modifications that improve nanotube preservation during the smFISH procedure.

Key words

mRNA Membrane nanotubes Extracellular vesicles Exosomes Single-molecule fluorescent in situ hybridization β-Actin Glutaraldehyde 

Notes

Acknowledgments

G.H. was funded by the Koshland Foundation and McDonald-Leapman Grant Senior Postdoctoral Fellowships. This work was funded by grants to J.E.G. from the Joel and Mady Dukler Fund for Cancer Research, the Jean-Jacques Brunschwig Fund for the Molecular Genetics of Cancer, a Proof-of-Principle Grant from the Moross Integrated Cancer Center (Weizmann Institute of Science), and the US-Israel Binational Science Foundation-National Science Foundation (#2015846).

References

  1. 1.
    Buxbaum AR, Haimovich G, Singer RH (2015) In the right place at the right time: visualizing and understanding mRNA localization. Nat Rev Mol Cell Biol 16(2):95–109CrossRefGoogle Scholar
  2. 2.
    Pichon X, Lagha M, Mueller F, Bertrand E (2018) A growing toolbox to image gene expression in single cells: sensitive approaches for demanding challenges. Mol Cell 71(3):468–480CrossRefGoogle Scholar
  3. 3.
    Haimovich G, Gerst JE (2018) Single-molecule fluorescence in situ hybridization (smFISH) for RNA detection in adherent animal cells. Bio-protocol 8(21):e3070CrossRefGoogle Scholar
  4. 4.
    Haimovich G, Ecker CM, Dunagin MC, Eggan E, Raj A, Gerst JE, Singer RH (2017) Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells. Proc Natl Acad Sci U S A 114(46):E9873–e9882CrossRefGoogle Scholar
  5. 5.
    Nawaz M, Fatima F (2017) Extracellular vesicles, tunneling nanotubes, and cellular interplay: synergies and missing links. Front Mol Biosci 4:50CrossRefGoogle Scholar
  6. 6.
    Mittal R, Karhu E, Wang JS, Delgado S, Zukerman R, Mittal J, Jhaveri VM (2019) Cell communication by tunneling nanotubes: implications in disease and therapeutic applications. J Cell Physiol 234(2):1130–1146CrossRefGoogle Scholar
  7. 7.
    Sisakhtnezhad S, Khosravi L (2015) Emerging physiological and pathological implications of tunneling nanotubes formation between cells. Eur J Cell Biol 94(10):429–443CrossRefGoogle Scholar
  8. 8.
    Austefjord MW, Gerdes HH, Wang X (2014) Tunneling nanotubes: diversity in morphology and structure. Commun Integr Biol 7(1):e27934CrossRefGoogle Scholar
  9. 9.
    Sartori-Rupp A, Cordero Cervantes D, Pepe A, Gousset K, Delage E, Corroyer-Dulmont S, Schmitt C, Krijnse-Locker J, Zurzolo C (2019) Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. Nat Commun 10(1):342CrossRefGoogle Scholar
  10. 10.
    Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659CrossRefGoogle Scholar
  11. 11.
    Tomasoni S, Longaretti L, Rota C, Morigi M, Conti S, Gotti E, Capelli C, Introna M, Remuzzi G, Benigni A (2012) Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells. Stem Cells Dev 22(5):772–780CrossRefGoogle Scholar
  12. 12.
    Batagov AO, Kurochkin IV (2013) Exosomes secreted by human cells transport largely mRNA fragments that are enriched in the 3′-untranslated regions. Biol Direct 8:12CrossRefGoogle Scholar
  13. 13.
    Hung ME, Leonard JN (2016) A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. J Extracell Vesicles 5:31027CrossRefGoogle Scholar
  14. 14.
    Shurtleff MJ, Yao J, Qin Y, Nottingham RM, Temoche-Diaz MM, Schekman R, Lambowitz AM (2017) Broad role for YBX1 in defining the small noncoding RNA composition of exosomes. Proc Natl Acad Sci U S A 114(43):E8987–e8995CrossRefGoogle Scholar
  15. 15.
    Svensson V, Natarajan KN, Ly LH, Miragaia RJ, Labalette C, Macaulay IC, Cvejic A, Teichmann SA (2017) Power analysis of single-cell RNA-sequencing experiments. Nat Methods 14(4):381–387CrossRefGoogle Scholar
  16. 16.
    Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Ferrante TC, Terry R, Turczyk BM, Yang JL, Lee HS, Aach J, Zhang K, Church GM (2015) Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc 10(3):442–458CrossRefGoogle Scholar
  17. 17.
    Wang G, Moffitt JR, Zhuang X (2018) Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy. Sci Rep 8(1):4847CrossRefGoogle Scholar
  18. 18.
    Moffitt JR, Zhuang X (2016) RNA imaging with multiplexed error-robust fluorescence in situ hybridization (MERFISH). Methods Enzymol 572:1–49CrossRefGoogle Scholar
  19. 19.
    Ginart P, Kalish JM, Jiang CL, Yu AC, Bartolomei MS, Raj A (2016) Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant. Genes Dev 30(5):567–578CrossRefGoogle Scholar
  20. 20.
    Hansen CH, van Oudenaarden A (2013) Allele-specific detection of single mRNA molecules in situ. Nat Methods 10(9):869–871CrossRefGoogle Scholar
  21. 21.
    Levesque MJ, Ginart P, Wei Y, Raj A (2013) Visualizing SNVs to quantify allele-specific expression in single cells. Nat Methods 10(9):865–867CrossRefGoogle Scholar
  22. 22.
    Mellis IA, Gupte R, Raj A, Rouhanifard SH (2017) Visualizing adenosine-to-inosine RNA editing in single mammalian cells. Nat Methods 14(8):801–804CrossRefGoogle Scholar
  23. 23.
    Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8(2):165–170CrossRefGoogle Scholar
  24. 24.
    Kanada M, Bachmann MH, Hardy JW, Frimannson DO, Bronsart L, Wang A, Sylvester MD, Schmidt TL, Kaspar RL, Butte MJ, Matin AC, Contag CH (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci U S A 112(12):E1433–E1442PubMedPubMedCentralGoogle Scholar
  25. 25.
    Mueller F, Senecal A, Tantale K, Marie-Nelly H, Ly N, Collin O, Basyuk E, Bertrand E, Darzacq X, Zimmer C (2013) FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4):277–278CrossRefGoogle Scholar
  26. 26.
    Tsanov N, Samacoits A, Chouaib R, Traboulsi AM, Gostan T, Weber C, Zimmer C, Zibara K, Walter T, Peter M, Bertrand E, Mueller F (2016) smiFISH and FISH-quant—a flexible single RNA detection approach with super-resolution capability. Nucl Acids Res 44(22):e165CrossRefGoogle Scholar
  27. 27.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682CrossRefGoogle Scholar
  28. 28.
    Lou E, Fujisawa S, Morozov A, Barlas A, Romin Y, Dogan Y, Gholami S, Moreira AL, Manova-Todorova K, Moore MA (2012) Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PLoS One 7(3):e33093CrossRefGoogle Scholar
  29. 29.
    Buxbaum AR, Wu B, Singer RH (2014) Single beta-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343(6169):419–422CrossRefGoogle Scholar
  30. 30.
    Leyton-Puig D, Kedziora KM, Isogai T, van den Broek B, Jalink K, Innocenti M (2016) PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins. Biol Open 5(7):1001–1009CrossRefGoogle Scholar
  31. 31.
    Shaffer SM, Wu MT, Levesque MJ, Raj A (2013) Turbo FISH: a method for rapid single molecule RNA FISH. PLoS One 8(9):e75120CrossRefGoogle Scholar
  32. 32.
    Sinigaglia C, Thiel D, Hejnol A, Houliston E, Leclere L (2018) A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species. Dev Biol 434(1):15–23CrossRefGoogle Scholar
  33. 33.
    Wheeler JR, Matheny T, Jain S, Abrisch R, Parker R (2016) Distinct stages in stress granule assembly and disassembly. elife 5:e18413Google Scholar
  34. 34.
    Long X, Colonell J, Wong AM, Singer RH, Lionnet T (2017) Quantitative mRNA imaging throughout the entire Drosophila brain. Nat Methods 14(7):703–706CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular GeneticsWeizmann Institute of ScienceRehovotIsrael

Personalised recommendations