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
Open Access articles citing this article.
Biological Research Open Access 26 November 2022
Cell Communication and Signaling Open Access 31 October 2022
Biological Research Open Access 01 October 2022
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Thery, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. Nature Rev. Immunol. 2, 569–579 (2002).
Fevrier, B. & Raposo, G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 16, 415–421 (2004).
Johnstone, R. M. Exosomes biological significance: a concise review. Blood Cells Mol. Dis. 36, 315–321 (2006).
Lakkaraju, A. & Rodriguez-Boulan, E. Itinerant exosomes: emerging roles in cell and tissue polarity. Trends Cell Biol. 18, 199–209 (2008).
Raposo, G. et al. B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 183, 1161–1172 (1996).
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).
Thery, C. et al. Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nature Immunol. 3, 1156–1162 (2002).
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).
Fevrier, B. et al. Cells release prions in association with exosomes. Proc. Natl Acad. Sci. USA 101, 9683–9688 (2004).
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).
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).
Iero, M. et al. Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ. 15, 80–88 (2008).
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).
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).
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).
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).
Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).
Seabra, M. C., Mules, E. H. & Hume, A. N. Rab GTPases, intracellular traffic and disease. Trends Mol. Med. 8, 23–30 (2002).
Ali, B. R. & Seabra, M. C. Targeting of Rab GTPases to cellular membranes. Biochem. Soc. Trans. 33, 652–656 (2005).
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).
Morelli, A. E. et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood 104, 3257–3266 (2004).
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).
Ramalho, J. S. et al. Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene. BMC Genet. 2, 2 (2001).
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).
Desnos, C. et al. Myosin va mediates docking of secretory granules at the plasma membrane. J. Neurosci. 27, 10636–10645 (2007).
Huet, S. et al. Analysis of transient behavior in complex trajectories: application to secretory vesicle dynamics. Biophys. J. 91, 3542–3559 (2006).
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).
Beraud-Dufour, S. & Balch, W. A journey through the exocytic pathway. J. Cell Sci. 115, 1779–1780 (2002).
Pereira-Leal, J. B. & Seabra, M. C. Evolution of the Rab family of small GTP-binding proteins. J. Mol. Biol. 313, 889–901 (2001).
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).
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).
Chen, X. et al. Rab27b localizes to zymogen granules and regulates pancreatic acinar exocytosis. Biochem. Biophys. Res. Commun. 323, 1157–1162 (2004).
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).
Mizuno, K. et al. Rab27b regulates mast cell granule dynamics and secretion. Traffic 8, 883–892 (2007).
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).
Stinchcombe, J. C. et al. Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J. Cell Biol. 152, 825–834 (2001).
Barral, D. C. et al. Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J. Clin. Invest. 110, 247–257 (2002).
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).
Seabra, M. C. & Coudrier, E. Rab GTPases and myosin motors in organelle motility. Traffic 5, 393–399 (2004).
Savina, A., Vidal, M. & Colombo, M. I. The exosome pathway in K562 cells is regulated by Rab11. J. Cell Sci. 115, 2505–2515 (2002).
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).
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).
Hume, A. N. et al. Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J. Cell Biol. 152, 795–808 (2001).
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).
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).
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).
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).
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).
Steyer, J. A. & Almers, W. Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophys. J. 76, 2262–2271 (1999).
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.
The authors declare no competing financial interests.
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
This article is cited by
Exosomal circKDM4A Induces CUL4B to Promote Prostate Cancer Cell Malignancy in a miR-338-3p-Dependent Manner
Biochemical Genetics (2023)
Recent advances in conventional and unconventional vesicular secretion pathways in the tumor microenvironment
Journal of Biomedical Science (2022)
Emerging role of exosomes in the pathology of chronic obstructive pulmonary diseases; destructive and therapeutic properties
Stem Cell Research & Therapy (2022)
Cell Communication and Signaling (2022)
Biomarker Research (2022)