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Rab27a and Rab27b Control Different Steps of the Exosome Secretion Pathway

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Rab27a and Rab27b Control Different Steps of the Exosome Secretion Pathway ARTICLES Rab27a and Rab27b control different steps of the exosome secretion pathway Matias Ostrowski1,2, Nuno B. Carmo3,12, Sophie Krumeich1,2,12, Isabelle Fanget10,12, Graça Raposo4,2, Ariel Savina1,2, Catarina F. Moita3, Kristine Schauer4,2, Alistair N. Hume5, Rui P. Freitas3, Bruno Goud4,2, Philippe Benaroch1,2, Nir Hacohen8,9, Mitsunori Fukuda11, Claire Desnos10, Miguel C. Seabra5,6,7, François Darchen10, Sebastian Amigorena1,2, Luis F. Moita3,13 and Clotilde Thery1,2,13 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. Exosomes are membrane vesicles secreted into the extracellular space membrane of MVEs into the lumen of these endosomes14. Observations, by numerous cell types1–4. These nanometre-sized vesicles have a simi- using electron microscopy, of fusion profiles between MVEs and the lar membrane orientation as the plasma membrane, and a canonical plasma membrane and the consequent release of intraluminal vesicles protein composition, mostly including proteins from the endocytic sys- as exosomes has so far been the strongest evidence for the endosomal tem, the plasma membrane and the cytosol. In addition, exosomes also origin of exosomes5,6,15. Nevertheless, in some cell types, such as T lym- contain proteins specific to the cell type from which they originate and phocytes, exosomes bud at the plasma membrane from endosome-like that are probably important for their extracellular functions. Based on domains16. Therefore, identification of molecules that control the intra- in vitro studies, it has been proposed that exosomes could participate in cellular trafficking of MVEs and their fusion with the plasma membrane the induction of immune responses5–7, in the dissemination of viruses will increase our understanding of exosome secretion. or prions8,9, in the pathogenesis of neurodegenerative diseases10 and in Most intracellular transport pathways are controlled by conserved fami- mediating communication between tumour cells and their microenvi- lies of cytosolic proteins, including the Rab family of small GTPases. Rab ronment11–13. However, it has not been conclusively demonstrated that proteins control different steps of vesicular trafficking, including budding, in vivo exosome secretion has a role in any physiological process due to motility and docking and fusion of different vesicular transport intermedi- the inability to specifically inhibit or increase it. Indeed, the molecular ates to acceptor membranes17. Almost 70 Rab proteins and Rab-like pro- mechanisms of exosome secretion are not completely understood. teins have been identified in humans18, and a wide range of specific effector Evidence collected during the last 20 years suggests that, in most cell proteins that execute their diverse roles has also been identified19. types, exosomes correspond to secreted intraluminal vesicles of MVEs4. Here we analyse the possible functions of Rab proteins in exosome Intraluminal vesicles are formed by inward budding of the limiting production or secretion by the human HeLa cells, using a short hairpin 1INSERM U932, 26 rue d’Ulm, 75005 Paris, France. 2Institut Curie, 26 rue d’Ulm, 75005 Paris, France. 3Cell 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. 4CNRS UMR144, 26 rue d’Ulm, 75005 Paris, France. 5Molecular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK. 6CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169-056, Lisboa, Portugal. 7Instituto Gulbenkian de Ciência, 2780-156, Oeiras, Portugal. 8Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Charlestown, MA 02142, USA. 9Broad Institute of Harvard and MIT, Cambridge, MA, USA. 10Institut de Biologie Physico-chimique, Centre National de la Recherche Scientifique, FRE 3146, 13 rue Pierre et Marie Curie, 75005 Paris, France. 11Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Tohoku University, Miyagi 980-9578, Japan. 12These authors contributed equally to this work. 13Correspondence should be addressed to C.T. or L.F.M. (e-mails: [email protected]; [email protected]). Received 14 August 2009; accepted 10 November 2009; published online 6 December 2009; DOI: 10.1038/ncb2000 NATURE CELL BIOLOGY VOLUME 12 | NUMBER 1 | JANUARY 2010 19 © 2010 Macmillan Publishers Limited. All rights reserved. ARTICLES a PE-conjugated antibodies b CD81 CD63 HLA-DR Annexin V-APC Anti-CD63-coated latex bead Number of secreting cells c Unconditioned culture medium 72,000 120,000 177,000 103 103 103 103 0.59 3.11 11.6 29.3 101 101 101 101 HLA-DR/CD81 0 400 800 0 400 800 0 400 800 0 400 800 Forward Scatter 103 103 103 103 0.3 1.3 3.75 12.6 101 101 101 101 Annexin V 0 400 800 0 400 800 0 400 800 0 400 800 Forward Scatter Beads Beads Beads Beads d e 10,000 10,000 HLA-DR/CD81 Annexin V 1,000 900 300 1,000 100 600 100 200 10 300 10 Annexin V (AU) 100 1 Exosomes (AU) HLA-DR/CD81 (AU) Exosomes (AU) 0 0 0 0 0 75,000 150,000 225,000 0 75,000 150,000 225,000 Live Apoptotic Live Apoptotic Number of cells Number of cells Figure 1 Semi-quantitative detection of exosomes in cell culture supernatants from increasing numbers of cultured cells. Percentages of supernatants. (a) Schematic drawing of the technical approach used to positive beads are indicated inside the boxed regions. (d) Dose-response selectively capture and stain exosomes (small circle) present in cell culture curve of HLA-DR/CD81 (left) and annexin V (right) staining of exosomes supernatants by using anti-CD63-coated beads and FACS detection. PE, captured by beads incubated with 66 different supernatants. (e) Specific phycoerythrin; APC, allophycocyanin. (b) Electron microscopy image of detection of exosomes secreted by live cells, compared with membranes exosomes present on the surface of anti-CD63-coated beads after incubation secreted by apoptotic cells. Supernatants from cell cultures of either UV- with supernatant from B6H4 cells. Scale bar, 250 nm. (c) Representative irradiated (apoptotic) or untreated (live) cells (a total of 50,000 cells) were flow cytometry dot-plots showing HLA-DR/CD81 (top panels) and annexin incubated with anti-CD63-coated beads and analysed by FACS. Data are V (bottom panels) staining of exosomes captured by beads incubated with mean ± s.d. of six independent measurements. AU, arbitrary units. RNA (shRNA)-based screen targeting human Rab genes. We observed roles of Rab27a and Rab27b were analysed further, and we demonstrated that knocking down five Rab proteins (Rab2b, Rab9a, Rab5a, Rab27a and a common function in MVE docking to the plasma membrane, but also Rab27b) inhibited exosome secretion without major modifications in the different and non-redundant roles in the MVE pathway. In addition, secretion of soluble proteins through the regular secretory pathway. The silencing of two known Rab27 effectors, Slp4 and Slac2b, phenocopied 20 NATURE CELL BIOLOGY VOLUME 12 | NUMBER 1 | JANUARY 2010 © 2010 Macmillan Publishers Limited. All rights reserved. ARTICLES a b 200 Control shRNA 200 RAB6A shRNA 200 RAB7 shRNA 200 RAB11A shRNA 150 150 150 150 100 100 100 100 50 50 50 50 Secretion level (percentage of control) (percentage of control) 0 Secretion/expression level 0 0 0 Exo sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 OVA Exo OVA mRNA Exo OVA mRNA Exo OVA mRNA c RAB2B shRNA RAB9A shRNA RAB5A shRNA 200 200 200 ** 150 150 150 100 100 * 100 ** * ** ** * ** ** ** *** ** ** ** *** 50 ** 50 50 (percentage of control) *** Secretion/expression level 0 0 0 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh1 sh2 sh3 sh1 sh2 sh3 Exo OVA mRNA Exo OVA mRNA Exo OVA mRNA d RAB27A shRNA RAB27B shRNA Control shRNA RAB27A shRNA RAB27B shRNA 200 200 125 125 100 100 150 150 * 75 75 * * * 100 100 * ** * 50 50 ** ** ** ** *** 50 50 25 25 *** *** (percentage of control) *** *** Secretion/expression level 0 0 0 0 Secretion (percentage of control) sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 Exo OVA mRNA Exo OVA mRNA Exo Exo Exo (annexin V) (annexin V) (annexin V) Figure 2 Modulation of exosome and OVA secretion by members of the targeting Rab family members that were selected as candidate hits because at Rab family. (a) Exosome secretion (black bar), measured as HLA-DR/CD81 least two shRNAs significantly affected exosome secretion without affecting staining, and OVA secretion (white bar) by cells transduced with a control OVA secretion.
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