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Rab27a and Rab27b control different steps of the exosome secretion pathway

Nature Cell Biology volume 12, pages 19–30 (2010)Cite this article

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.

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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.

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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.

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Author notes

  1. Nuno B. Carmo, Sophie Krumeich and Isabelle Fanget: These authors contributed equally to this work.

Authors and Affiliations

  1. INSERM U932, 26 rue d'Ulm, 75005, Paris, France

    Matias Ostrowski, Sophie Krumeich, Ariel Savina, Philippe Benaroch, Sebastian Amigorena & Clotilde Thery

  2. Institut Curie, 26 rue d'Ulm, 75005, Paris, France

    Matias Ostrowski, Sophie Krumeich, Graça Raposo, Ariel Savina, Kristine Schauer, Bruno Goud, Philippe Benaroch, Sebastian Amigorena & Clotilde Thery

  3. Cell Biology of the Immune System Unit, Instituto de Medicina Molecular Sala P3-B-40, Edifício Egas Moniz, Avenida Professor Egas Moniz, 1649-028, Lisboa, Portugal

    Nuno B. Carmo, Catarina F. Moita, Rui P. Freitas & Luis F. Moita

  4. CNRS UMR144, 26 rue d'Ulm, 75005, Paris, France

    Graça Raposo, Kristine Schauer & Bruno Goud

  5. Molecular Medicine, National Heart and Lung Institute, Imperial College London, SW7 2AZ, London, UK

    Alistair N. Hume & Miguel C. Seabra

  6. CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, 1169-056, Portugal

    Miguel C. Seabra

  7. Instituto Gulbenkian de Ciência, Oeiras, 2780-156, Portugal

    Miguel C. Seabra

  8. Division of Rheumatology, Center for Immunology and Inflammatory Diseases, Allergy, and Immunology, Massachusetts General Hospital, Charlestown, 02142, MA, USA

    Nir Hacohen

  9. Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Nir Hacohen

  10. Institut de Biologie Physico-chimique, Centre National de la Recherche Scientifique, FRE 3146, 13 rue Pierre et Marie Curie, 75005, Paris, France

    Isabelle Fanget, Claire Desnos & François Darchen

  11. Department of Developmental Biology and Neurosciences, Laboratory of Membrane Trafficking Mechanisms, Tohoku University, 980-9578, Miyagi, Japan

    Mitsunori Fukuda

Authors
  1. Matias Ostrowski
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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.

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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

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