Cell and Tissue Biology

, Volume 12, Issue 2, pp 146–152 | Cite as

Establishment of the HeLa Cell Line with Stable Expression of CD63 Exosome Marker Fused with Fluorescent Protein TagRFP and HTBH Tag

  • V. A. Kulichkova
  • A. V. Selenina
  • A. N. Tomilin
  • A. S. Tsimokha
Article
  • 5 Downloads

Abstract

Exosomes are small microvesicles released into the cellular environment by different types of cells. These vesicles have been found in the blood serum and in other extracellular fluids of body. There are a number of different proteins, mRNA and microRNA, in these exosomes. Exosomes take part in cellular communication, excretion of proteins, immune response, and are also involved in development of some neurodegenerative diseases and cancer. The mechanism through which they get in and out of cells is not clear. To address this issue, we have generated a stable HeLa cell line expressing exosomal marker CD63 fused with a tagRFP and HTBH tag. These and other cells harboring the CD63-tagRFP-HTBH structure constitute a valuable tool that should allow real-time observations of exosomal transport

Keywords

red fluorescence protein exosome tetraspanin CD63 

Abbreviation

PAGE-SDS

polyacrylamide gel

PCR

polymerase chain reaction

TagRFP

modified red fluorescent protein

HTBH

complex polypeptide with two sequences of six histidine (H) specific site for TEV-protease cleavage (T) and site for biotinylation in vitro (B)

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S., and Wood, M. J., Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes, Nature Biotechnol., 2011, vol. 29, pp. 341–345.CrossRefGoogle Scholar
  2. Boucheix, C. and Rubinstein, E., Tetraspanins, Cell. Mol. Life Sci., 2001, vol. 58, pp. 1189–1205.CrossRefPubMedGoogle Scholar
  3. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem., 1976, vol. 72, pp. 248–254.CrossRefPubMedGoogle Scholar
  4. Feng, D., Zhao, W.L., Ye, Y.Y., Bai, X.C., Liu, R.Q., Chang, L.F., Zhou, Q., and Sui, S.F., Cellular internalization of exosomes occurs through phagocytosis, Traffic, 2010, vol. 11, pp. 675–687.CrossRefPubMedGoogle Scholar
  5. Ikawa, M., Yamada, S., Nakanishi, T., and Okabe, M., Green fluorescent protein (GFP) as a vital marker in mammals, Curr. Top. Dev. Biol., 1999, vol. 44, pp. 1–20.PubMedGoogle Scholar
  6. Janowska-Wieczorek, A., Wysoczynski, M., Kijowski, J., Marquez-Curtis, L., Machalinski, B., Ratajczak, J., and Ratajczak, M.Z., Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer, Int. J. Cancer, 2005, vol. 113, pp. 752–760.CrossRefPubMedGoogle Scholar
  7. Kalra H., Adda C. G., Liem M., Ang C. S., Mechler A., Simpson R. J., Hulett M. D., Mathivanan S. 2013. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics. 13: 3354–3364.CrossRefPubMedGoogle Scholar
  8. Kleijmeer, M.J., Stoorvogel, W., Griffith, J.M., Yoshie, O., and Geuze, H.J., Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes, J. Biol. Chem., 1998, vol. 273, pp. 20121–20127.CrossRefPubMedGoogle Scholar
  9. Koumangoye, R.B., Sakwe, A.M., Goodwin, J.S., Patel, T., and Ochieng, J., Detachment of breast tumor cells induces rapid secretion of exosomes which subsequently mediate cellular adhesion and spreading, PLoS One, 2011, vol. 6, p. e24234.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Lakhal, S. and Wood, M.J., Exosome nanotechnology: an emerging paradigm shift in drug delivery, Bioessays, 2011, vol. 33, pp. 737–741.CrossRefPubMedGoogle Scholar
  11. Latysheva, N., Muratov, G., Rajesh, S., Padgett, M., Hotchin, N.A., Overduin, M., and Berditchevski, F., Syntenin-1 is a new component of tetraspanin-enriched microdomains: mechanisms and consequences of the interaction of syntenin-1 with CD63, Mol. Cell. Biol., 2006, vol. 26, pp. 7707–7718.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Merzlyak, E.M., Goedhart, J., Shcherbo, D., Bulina, M.E., Shcheglov, A.S., Fradkov, A.F., Gaintzeva, A., Lukyanov, K.A., Lukyanov, S.,and Gadella, T.W., Bright monomeric red fluorescent protein with an extended fluorescence lifetime, Nature Methods, 2007, vol. 4, p. 555.CrossRefPubMedGoogle Scholar
  13. Pan, B.T. and Johnstone, R.M., Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor, Cell, 1983, vol. 33, pp. 967–978.CrossRefPubMedGoogle Scholar
  14. Raposo, G. and Stoorvogel, W., Extracellular vesicles: exosomes, microvesicles, and friends, J. Cell Biol., 2013, vol. 200, pp. 373–383.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Runz, S., Keller, S., Rupp, C., Stoeck, A., Issa, Y., Koensgen, D., Mustea, A., Sehouli, J., Kristiansen, G., and Altevogt, P., Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM, Gynecol. Oncol., 2007, vol. 107, pp. 563-571.CrossRefPubMedGoogle Scholar
  16. Shtam, T.A., Burdakov, V.S., Landa, S.B., Naryzhny, S.N., Bairamukov, V.Yu., Malek, A.V., Orlov, Yu.N., and Filatov, M.V., Aggregation by lectins as an approach for exosome isolation from biological fluids: validation for proteomic studies, Cell Tissue Biol., 2017, vol. 11, no. 2, pp. 172–180.CrossRefGoogle Scholar
  17. Simpson, R.J., Jensen, S.S., and Lim, J.W., Proteomic profiling of exosomes: current perspectives, Proteomics, 2008, vol. 8, pp. 4083–4099.CrossRefPubMedGoogle Scholar
  18. Smalheiser, N.R., Exosomal transfer of proteins and RNAs at synapses in the nervous system, Biol. Direct., 2007, vol. 2, pp. 1–15.CrossRefGoogle Scholar
  19. Smith, J., Leonardi, T., Huang, B., Iraci, N., Vega, B., and Pluchino, S., Extracellular vesicles and their synthetic analogues in aging and age-associated brain diseases, Biogerontology, 2015, vol. 16, pp. 147–185.CrossRefPubMedGoogle Scholar
  20. Thory, C., Zitvogel, L., and Amigorena, S., Exosomes: composition, biogenesis and function, Nat. Rev. Immunol., 2002, vol. 2, pp. 569–579.CrossRefGoogle Scholar
  21. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J., and Lutvall, J.O., Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat. Cell Biol., 2007, vol. 9, pp. 654–659.CrossRefPubMedGoogle Scholar
  22. Wang, X., Chen, C.F., Baker, P.R., Chen, P.L., Kaiser, P., and Huang, L., Mass spectrometric characterization of the affinity-purified human 26S proteasome complex, Biochemistry, 2007, vol. 46, pp. 3553–3565.CrossRefPubMedGoogle Scholar
  23. Wubbolts, R., Leckie, R.S., Veenhuizen, P.T., Schwarzmann, G., Mobius, W., Hoernschemeyer, J., Slot, J.-W., Geuze, H.J., and Stoorvogel, W., Proteomic and biochemical analyses of human B cell-derived exosomes potential implications for their function and multivesicular body formation, J. Biol. Chem., 2003, vol. 278, pp. 10963–10972.CrossRefPubMedGoogle Scholar
  24. Zhang, H., Zhuang, X., Sun, D., Liu, Y., Xiang, X., and Grizzle, W., Exosomes and immune surveillance of neoplastic lesions: a review, Biotech. Histochem., 2012, vol. 87, pp. 161–168.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Zomer, A., Vendrig, T., Hopmans, E.S., van Eijndhoven, M., Middeldorp, J.M., and Pegtel, D.M., Exosomes: fit to deliver small RNA, Commun. Integr. Biol., 2010, vol. 3, pp. 447–450.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • V. A. Kulichkova
    • 1
  • A. V. Selenina
    • 1
  • A. N. Tomilin
    • 1
    • 2
  • A. S. Tsimokha
    • 1
  1. 1.Institute of CytologyRussian Academy of SciencesSt. PetersburgRussia
  2. 2.Institute of Translational BiomedicineSt. Petersburg State UniversitySt. PetersburgRussia

Personalised recommendations