Isolation of Exosome-Like Vesicles from Plants by Ultracentrifugation on Sucrose/Deuterium Oxide (D2O) Density Cushions

  • Christopher Stanly
  • Immacolata Fiume
  • Giovambattista Capasso
  • Gabriella Pocsfalvi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1459)

Abstract

Exosomes are nanovesicles of endocytic origin that are about 30–100 nm in diameter, surrounded by a lipid bilayer membrane, and contain proteins, nucleic acids, and other molecules. Mammalian cells- and biological fluids-derived exosomes have become the subject for a wide range of investigations in biological and biomedical sciences. More recently, a new interest is on the verge of rising: the presence of nanovesicles in plants. Lipoprotein vesicles from apoplastic fluid and exosome-like vesicles (ELVs) from fruit juice have been isolated and shown that they could be loaded with drugs and uptaken by recipient cells. In order to explore and analyze the contents and functions of ELVs, they must be isolated and purified with intense care. Isolation of ELVs can be a tedious process and often characterized by the co-purification of undesired contaminants. Here we describe a method which isolates ELVs based on their buoyant density. The method utilizes differential centrifugation in step 1 and 1 and 2 M sucrose/deuterium oxide double-cushion ultracentrifugation in step 2, to purify two diverse ELV subpopulations. In this method fruit juice is used as an example of starting material, although this protocol can be used for the isolation of vesicles from apoplastic fluid too. The quality and the quantity of ELV preparations have been found appropriate for downstream biological and structural studies, like proteomics, transcriptomics, and lipidomics.

Key words

Extracellular vesicles Exosome-like vesicles Apoplastic vesicles Plant tissue Juice Purification Isolation Ultracentrifugation Differential centrifugation Sucrose gradient centrifugation 

References

  1. 1.
    Yáñez-Mó M, Siljander PR-M, Andreu Z, Zavec AB, Borràs FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, Colás E, Cordeiro-da Silva A, Fais S, Falcon-Perez JM, Ghobrial IM, Giebel B, Gimona M, Graner M, Gursel I, Gursel M, Heegaard NHH, Hendrix A, Kierulf P, Kokubun K, Kosanovic M, Kralj-Iglic V, Krämer-Albers E-M, Laitinen S, Lässer C, Lener T, Ligeti E, Linē A, Lipps G, Llorente A, Lötvall J, Manček-Keber M, Marcilla A, Mittelbrunn M, Nazarenko I, Nolte-‘t Hoen ENM, Nyman TA, Driscoll L, Olivan M, Oliveira C, Pállinger É, del Portillo HA, Reventós J, Rigau M, Rohde E, Sammar M, Sánchez-Madrid F, Santarém N, Schallmoser K, Stampe Ostenfeld M, Stoorvogel W, Stukelj R, Van der Grein SG, Vasconcelos MH, Wauben MHM, De Wever O (2015) Biological properties of extracellular vesicles and their physiological functions. Journal of Extracellular Vesicles 4. doi: 10.3402/jev.v4.27066
  2. 2.
    Yoon YJ, Kim OY, Gho YS (2014) Extracellular vesicles as emerging intercellular communicasomes. BMB Rep 47(10):531–539. doi: 10.5483/BMBRep.2014.47.10.164 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Regente M, Pinedo M, Elizalde M, de la Canal L (2012) Apoplastic exosome-like vesicles: a new way of protein secretion in plants? Plant Signal Behav 7(5):544–546CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Song Y, Zhang C, Ge W, Zhang Y, Burlingame AL, Guo Y (2011) Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE. J Proteomics 74(7):1045–1067CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Regente M, Corti-Monzón G, Maldonado AM, Pinedo M, Jorrín J, de la Canal L (2009) Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins. FEBS Lett 583(20):3363–3366. doi: 10.1016/j.febslet.2009.09.041 CrossRefPubMedGoogle Scholar
  6. 6.
    Gonorazky G, Laxalt AM, Dekker HL, Rep M, Munnik T, Testerink C, de la Canal L (2012) Phosphatidylinositol 4-phosphate is associated to extracellular lipoproteic fractions and is detected in tomato apoplastic fluids. Plant Biol 14(1):41–49. doi: 10.1111/j.1438-8677.2011.00488.x PubMedGoogle Scholar
  7. 7.
    Ju S, Mu J, Dokland T, Zhuang X, Wang Q, Jiang H, Xiang X, Deng ZB, Wang B, Zhang L, Roth M, Welti R, Mobley J, Jun Y, Miller D, Zhang HG (2013) Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol Ther 21(7):1345–1357CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wang Q, Zhuang X, Mu J, Deng Z-B, Jiang H, Zhang L, Xiang X, Wang B, Yan J, Miller D, Zhang H-G (2013) Delivery of therapeutic agents by nanoparticles made of grapefruit-derived lipids. Nat Commun 4:1867. doi: 10.1038/ncomms2886 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Reyes FC, Buono R, Otegui MS (2011) Plant endosomal trafficking pathways. Curr Opin Plant Biol 14(6):666–673. doi: 10.1016/j.pbi.2011.07.009 CrossRefPubMedGoogle Scholar
  10. 10.
    An Q, van Bel AJ, Huckelhoven R (2007) Do plant cells secrete exosomes derived from multivesicular bodies? Plant Signal Behav 2(1):4–7CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Pocsfalvi G, Raj DAA, Fiume I, Vilasi A, Trepiccione F, Capasso G (2015) Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. Proteomics Clin Appl 9(5-6):552–567. doi: 10.1002/prca.201400199 CrossRefPubMedGoogle Scholar
  12. 12.
    Raj DA, Fiume I, Capasso G, Pocsfalvi G (2012) A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes. Kidney Int 81(12):1263–1272. doi: 10.1038/ki.2012.25 CrossRefPubMedGoogle Scholar
  13. 13.
    Joosten MAJ (2012) Isolation of apoplastic fluid from leaf tissue by the vacuum infiltration-centrifugation technique. In: Bolton MD, Thomma BPHJ (eds) Plant fungal pathogens, vol 835, Methods in molecular biology. Humana, New York, NY, pp 603–610. doi: 10.1007/978-1-61779-501-5_38 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Christopher Stanly
    • 1
  • Immacolata Fiume
    • 1
  • Giovambattista Capasso
    • 2
  • Gabriella Pocsfalvi
    • 1
  1. 1.Mass Spectrometry and Proteomics, Institute of Biosciences and BioResources, National Research Council of ItalyNaplesItaly
  2. 2.Division of Nephrology, Department of Cardio-Vascular SciencesSecond University of NaplesNaplesItaly

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