Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Rab27a and Rab27b control different steps of the exosome secretion pathway

Abstract

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Semi-quantitative detection of exosomes in cell culture supernatants.
Figure 2: Modulation of exosome and OVA secretion by members of the Rab family.
Figure 3: Large-scale purification and characterization of exosomes secreted by Rab27a and Rab27b-knockdown cells.
Figure 4: Effect of Rab27a and Rab27b knockdown on the intracellular distribution of CD63-positive compartments.
Figure 5: Differential intracellular distribution of Rab27a and Rab27b.
Figure 6: Effect of Rab27a and Rab27b knockdown on the mobility of CD63-positive compartments in the subplasmalemmal region analysed by TIRF microscopy.
Figure 7: Figure 7 Effect of Rab27 effector proteins on exosome secretion.
Figure 8: Interaction between Slp4 and Rab27a.

References

  1. Thery, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. Nature Rev. Immunol. 2, 569–579 (2002).

    Article  CAS  Google Scholar 

  2. Fevrier, B. & Raposo, G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 16, 415–421 (2004).

    Article  CAS  Google Scholar 

  3. Johnstone, R. M. Exosomes biological significance: a concise review. Blood Cells Mol. Dis. 36, 315–321 (2006).

    Article  CAS  Google Scholar 

  4. Lakkaraju, A. & Rodriguez-Boulan, E. Itinerant exosomes: emerging roles in cell and tissue polarity. Trends Cell Biol. 18, 199–209 (2008).

    Article  CAS  Google Scholar 

  5. Raposo, G. et al. B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 183, 1161–1172 (1996).

    Article  CAS  Google Scholar 

  6. Zitvogel, L. et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nature Med. 4, 594–600 (1998).

    Article  CAS  Google Scholar 

  7. Thery, C. et al. Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nature Immunol. 3, 1156–1162 (2002).

    Article  CAS  Google Scholar 

  8. Wiley, R. D. & Gummuluru, S. Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection. Proc. Natl Acad. Sci. USA 103, 738–743 (2006).

    Article  CAS  Google Scholar 

  9. Fevrier, B. et al. Cells release prions in association with exosomes. Proc. Natl Acad. Sci. USA 101, 9683–9688 (2004).

    Article  CAS  Google Scholar 

  10. Rajendran, L. et al. Alzheimer's disease β-amyloid peptides are released in association with exosomes. Proc. Natl Acad. Sci. USA 103, 11172–11177 (2006).

    Article  CAS  Google Scholar 

  11. Wolfers, J. et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nature Med. 7, 297–303 (2001).

    Article  CAS  Google Scholar 

  12. Iero, M. et al. Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ. 15, 80–88 (2008).

    Article  CAS  Google Scholar 

  13. Zeelenberg, I. S. et al. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Res. 68, 1228–1235 (2008).

    Article  CAS  Google Scholar 

  14. Futter, C. E., Collinson, L. M., Backer, J. M. & Hopkins, C. R. Human VPS34 is required for internal vesicle formation within multivesicular endosomes. J. Cell Biol. 155, 1251–1264 (2001).

    Article  CAS  Google Scholar 

  15. Pan, B. T., Teng, K., Wu, C., Adam, M. & Johnstone, R. M. Electron. microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J. Cell Biol. 101, 942–948 (1985).

    Article  CAS  Google Scholar 

  16. Booth, A. M. et al. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J. Cell Biol. 172, 923–935 (2006).

    Article  CAS  Google Scholar 

  17. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).

    Article  CAS  Google Scholar 

  18. Seabra, M. C., Mules, E. H. & Hume, A. N. Rab GTPases, intracellular traffic and disease. Trends Mol. Med. 8, 23–30 (2002).

    Article  CAS  Google Scholar 

  19. Ali, B. R. & Seabra, M. C. Targeting of Rab GTPases to cellular membranes. Biochem. Soc. Trans. 33, 652–656 (2005).

    Article  CAS  Google Scholar 

  20. Escola, J. M. et al. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273, 20121–20127 (1998).

    Article  CAS  Google Scholar 

  21. Morelli, A. E. et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood 104, 3257–3266 (2004).

    Article  CAS  Google Scholar 

  22. Thery, C. et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol. 166, 7309–7318 (2001).

    Article  CAS  Google Scholar 

  23. Ramalho, J. S. et al. Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene. BMC Genet. 2, 2 (2001).

    Article  CAS  Google Scholar 

  24. Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3 22 (2006).

  25. Desnos, C. et al. Myosin va mediates docking of secretory granules at the plasma membrane. J. Neurosci. 27, 10636–10645 (2007).

    Article  CAS  Google Scholar 

  26. Huet, S. et al. Analysis of transient behavior in complex trajectories: application to secretory vesicle dynamics. Biophys. J. 91, 3542–3559 (2006).

    Article  CAS  Google Scholar 

  27. Nofal, S., Becherer, U., Hof, D., Matti, U. & Rettig, J. Primed vesicles can be distinguished from docked vesicles by analyzing their mobility. J. Neurosci. 27, 1386–1395 (2007).

    Article  CAS  Google Scholar 

  28. Beraud-Dufour, S. & Balch, W. A journey through the exocytic pathway. J. Cell Sci. 115, 1779–1780 (2002).

    PubMed  Google Scholar 

  29. Pereira-Leal, J. B. & Seabra, M. C. Evolution of the Rab family of small GTP-binding proteins. J. Mol. Biol. 313, 889–901 (2001).

    Article  CAS  Google Scholar 

  30. Buschow, S. I. et al. MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways. Traffic 10, 1528–1542 (2009).

    Article  CAS  Google Scholar 

  31. Desnos, C. et al. Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites. J. Cell Biol. 163, 559–570 (2003).

    Article  CAS  Google Scholar 

  32. Chen, X. et al. Rab27b localizes to zymogen granules and regulates pancreatic acinar exocytosis. Biochem. Biophys. Res. Commun. 323, 1157–1162 (2004).

    Article  CAS  Google Scholar 

  33. Imai, A., Yoshie, S., Nashida, T., Shimomura, H. & Fukuda, M. The small GTPase Rab27B regulates amylase release from rat parotid acinar cells. J. Cell Sci. 117, 1945–1953 (2004).

    Article  CAS  Google Scholar 

  34. Mizuno, K. et al. Rab27b regulates mast cell granule dynamics and secretion. Traffic 8, 883–892 (2007).

    Article  CAS  Google Scholar 

  35. Tolmachova, T., Abrink, M., Futter, C. E., Authi, K. S. & Seabra, M. C. Rab27b regulates number and secretion of platelet dense granules. Proc. Natl Acad. Sci. USA 104, 5872–5877 (2007).

    Article  CAS  Google Scholar 

  36. Stinchcombe, J. C. et al. Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J. Cell Biol. 152, 825–834 (2001).

    Article  CAS  Google Scholar 

  37. Barral, D. C. et al. Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J. Clin. Invest. 110, 247–257 (2002).

    Article  CAS  Google Scholar 

  38. Strom, M., Hume, A. N., Tarafder, A. K., Barkagianni, E. & Seabra, M. C. A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport. J. Biol. Chem. 277, 25423–25430 (2002).

    Article  CAS  Google Scholar 

  39. Seabra, M. C. & Coudrier, E. Rab GTPases and myosin motors in organelle motility. Traffic 5, 393–399 (2004).

    Article  CAS  Google Scholar 

  40. Savina, A., Vidal, M. & Colombo, M. I. The exosome pathway in K562 cells is regulated by Rab11. J. Cell Sci. 115, 2505–2515 (2002).

    CAS  PubMed  Google Scholar 

  41. Kondo, H. et al. Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets. J. Biol. Chem. 281, 28657–28665 (2006).

    Article  CAS  Google Scholar 

  42. Stumptner-Cuvelette, P. et al. HIV-1 Nef impairs MHC class II antigen presentation and surface expression. Proc. Natl Acad. Sci. USA 98, 12144–12149 (2001).

    Article  CAS  Google Scholar 

  43. Hume, A. N. et al. Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J. Cell Biol. 152, 795–808 (2001).

    Article  CAS  Google Scholar 

  44. Blott, E. J., Bossi, G., Clark, R., Zvelebil, M. & Griffiths, G. M. Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail. J. Cell Sci. 114, 2405–2416 (2001).

    CAS  PubMed  Google Scholar 

  45. Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y. & Kuroda, T. S. Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells. J. Biol. Chem. 277, 39673–39678 (2002).

    Article  CAS  Google Scholar 

  46. Shu, X., Shaner, N. C., Yarbrough, C. A., Tsien, R. Y. & Remington, S. J. Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 (2006).

    Article  CAS  Google Scholar 

  47. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

    Article  CAS  Google Scholar 

  48. Racine, V. et al. Visualization and quantification of vesicle trafficking on a three-dimensional cytoskeleton network in living cells. J. Microsc. 225, 214–228 (2007).

    Article  Google Scholar 

  49. Steyer, J. A. & Almers, W. Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophys. J. 76, 2262–2271 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by post-doctoral salaries from Institut National du Cancer and Institut Curie to M.O., grants from Association pour la Recherche sur le Cancer and Fondation de France to C.T., and from the Agence Nationale de la Recherche to F.D. (ANR-06-BLAN-0211-02). L.F.M. is a Young Investigator from the Human Frontier Science Program and receives support from Fundação Luso-Americana para o Desenvolvimento and Fundação para a Ciência e a Tecnologia (PTDC/SAU-MII/69280/2006 and PTDC/SAU-MII/78333/2006). We thank PICT IbiSA Imaging Facility at Curie Institute, V. Racine and J.-B. Sibarita for providing a copy of their multidimensional image analysis program, I. Hurbain for help in electron microscopy quantification, P. Simões and M. H. Raquel for some of the lentiviral preparations, T. Tolmachova for bones of Rab27-knockout mice, J. Mordoh for providing an anti-CD63 FC-5.01 antibody and R. Allan for critical reading of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

M.O. conceived, designed, performed and analysed data from all experiments, and wrote the paper. N.B.C. performed initial lentivirus screens. S.K. performed immunoblotting, RNA analyses and prepared cell cultures. I.F. performed and analysed data from TIRF microscopy experiments. G.R. performed and analysed data from electron microscopy experiments and edited the manuscript. A.S. analysed data and edited the manuscript. C.F.M. designed the lentivirus library and prepared lentiviruses. K.S. analysed data from and quantified immunofluorescence experiments. A.N.H performed immunoprecipitation experiments. P.B., M.F., R.P.F. and N.H. contributed essential reagents. B.G. conceived the Rab effector-related experiments. M.C.S. contributed essential reagents, analysed data and conceived the Rab effector-related experiments. C.D. and F.D. analysed data from TIRF microscopy experiments and wrote the paper. S.A. conceived the project and edited the manuscript. L.F.M. conceived the screen, designed and supervised the lentivirus library production and use, analysed data and edited the manuscript. C.T. conceived and supervised the project, analysed data and wrote the manuscript.

Corresponding authors

Correspondence to Luis F. Moita or Clotilde Thery.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information Figures and table (PDF 1537 kb)

Supplementary Information

Supplementary Information Movie 1 (MOV 2768 kb)

Supplementary Information

Supplementary Information Movie 2 (MOV 3778 kb)

Supplementary Information

Supplementary Information Movie 3 (MOV 3111 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ostrowski, M., Carmo, N., Krumeich, S. et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol 12, 19–30 (2010). https://doi.org/10.1038/ncb2000

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2000

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing