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

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

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a PE-conjugated antibodies b

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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

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a b 200 Control shRNA 200 RAB6A shRNA 200 RAB7 shRNA 200 RAB11A shRNA

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100 100 150 150 * 75 75 * * * 100 100 * ** * 50 50 ** ** ** ** *** 50 50 25 25 *** *** (percentage of control) *** ***

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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. Data are mean ± s.d. of measurements performed in five shRNA. Data are mean ± s.d. of three experiments (in each experiment, (Rab2b, Rab9a and Rab5a) or nine (Rab27a and Rab27b) different wells 100% = mean of three independent wells). (b) Exosome and OVA secretion, present in independent plates.(d) Exosome secretion by control and Rab27a- and specific mRNA levels in cells transduced with different shRNAs (each bar or Rab27b-knockdown cells was also evaluated by detection of exposed represents a different shRNA) targeting Rab proteins that were not selected phosphatidylserine with annexin V. Data are mean ± s.d. of measurements as hits in the screen, either because neither exosome nor OVA secretion performed in three independent wells. Arrows in c and d indicate shRNAs were modified (Rab7, Rab11a) or because only OVA secretion was inhibited that were chosen for further characterization. Asterisks indicate statistically (Rab6a). Mean values of two independent wells are shown. (c) Exosome and significant differences from the control. *P < 0.05, **P < 0.01, ***P < 0.001 OVA secretion, and specific mRNA levels in cells transduced with shRNAs (one-way ANOVA followed by Tukey’s post test). Exo, exosome. silencing of Rab27a and Rab27b, respectively, suggesting a preferential exosomes and soluble OVA (through the classical secretory pathway), interaction between these proteins for exosome secretion. allowing differentiation of both pathways. Exosomes present in cell culture supernatants were captured onto beads coated with antibodies to RESULTS CD63, a tetraspanin strongly enriched in late endosomes and exosomes20, Semi-quantitative detection of exosomes in cell culture and were detected by FACS after staining with anti-HLA-DR and anti- supernatants CD81 fluorescent antibodies (CD81 is another tetraspanin abundant To screen for molecules specifically involved in exosome secretion, on exosomes)5,20, or annexin V (which binds to the phosphatidylserine we developed a semi-quantitative fluorescence-activated cell scanning present at the surface of exosomes; Fig. 1a)21. Membrane vesicles with (FACS)-based assay that allowed simultaneous detection of exosomes a diameter ranging from 50 to 100 nm and with the typical cup-shaped and proteins secreted through the classical secretory pathway in small morphology of exosomes were detected on the surface of the beads by volumes of cell culture supernatants. The HeLa B6H4 tumour cell line, electron microscopy (Fig. 1b). Exosome detection was dependent on which stably expresses the transactivator CIITA (an activator of the MHC the number of exosome-secreting cells present in the wells (Fig. 1c, d), class II family genes, including HLA-DR) and a secreted form of chicken with a sensitivity threshold of 75,000 cells per well. When supernatants ovalbumin (OVA), was used. These cells secrete HLA-DR-positive from apoptotic cells (which secrete apoptotic blebs but not exosomes)22 nature cell biology VOLUME 12 | NUMBER 1 | JANUARY 2010 21 © 2010 Macmillan Publishers Limited. All rights reserved.

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a * b 25 ** Control Rab27a KD Rab27b KD CL Exo CL Exo CL Exo cells (µg) 6 20 0.13 20 7.5 0.13 20 7.5 0.13 20 7.5 × 106 cells

15 HLA-DR

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c Control Rab27a KD Rab27b KD CL Exo CL Exo CL Exo 30 10 3 30 10 330103Protein (µg) 0.13 14.3 4.8 0.13 14.3 4.8 0.13 14.3 4.8 × 106 cells HLA-DR Hsc70

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Figure 3 Large-scale purification and characterization of exosomes secreted knockdown cells, normalized to the value obtained with exosomes from by Rab27a and Rab27b-knockdown cells. (a) The total amounts of control cells. Data are the mean ± s.d. of three independent experiments proteins in the exosomal pellet purified from large-scale cultures of stably for HLA-DR and four for Tsg101. Data are the mean of two independent transduced cells were quantified by a Bradford assay, and are presented experiments for Hsc70. (c) Equal amounts of total exosomal proteins as the values per 10 million secreting cells. Data are the mean ± s.d. from (quantified by Bradford assay) secreted by either control or knockdown four independent experiments. (b) Characterization of exosomal proteins by cells were analysed by immunoblotting for presence of CD63, Tsg101, immunoblotting. Purified exosomes secreted by equal numbers of control HLA-DR and Gp96 proteins, and compared with cell lysates. (d) Exosomes or knockdown cells were analysed by immunoblotting for the presence of purified from cell culture supernatants were negatively stained and analysed CD63, Tsg101, HLA-DR, Hsc70 and Gp96 proteins. A total of 130,000 by electron microscopy . Scale bar, 200 nm. *P < 0.05, **P < 0.01, cells were lysed and used for comparison. One representative experiment ***P < 0.001 (one-way ANOVA followed by Tukey’s post test). NS, not out of four is shown. Histogram shows quantification of signal intensity of significant. KD, knockdown. CL, cell lysates. For full scans of blots in b and HLA-DR, Hsc70 and Tsg101 proteins in exosomes secreted by 20 million c, see Supplementary Information, Fig. S6. were used, the FACS signals were significantly lower than those obtained (corresponding to exosomes) and of OVA, produced by living cells, in from supernatants of living cells (Fig. 1e). very small volumes of culture supernatants. Secretion of soluble OVA in cell culture supernatants was detected using beads coated with anti-OVA antibodies and stained with a The role of Rab GTPase family members in exosome secretion fluorescent anti-OVA antibody (Supplementary Information, Fig. S1). The effects of various shRNAs on exosome secretion were analysed by This methodology allowed the quantitative detection of OVA from live screening a lentiviral shRNA library targeting 59 members of the Rab cells, with a sensitivity threshold of 30,000 cells per well. GTPase family, using the methodology described above. Cells express- This FACS-based assay thus allowed simultaneous quantification of ing scrambled shRNA were used as controls for both OVA and exosome CD63/CD81/HLA-DR/phosphatidylserine-positive membrane vesicles secretion pathways (Fig. 2a). Genes for which at least two different shRNAs

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a Surface CD63 Total CD63 b zoom 3.8x 100 100 Control shRNA CD63 DAPI KD 60 60 Isotype control

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) 80 –4

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20 Surface area (nm Rab27a KD 0 Rab27b KD Control Rab27a KD Rab27b KD

Figure 4 Effect of Rab27a and Rab27b knockdown on the intracellular compartments (left; mean ± s.d. of 9796, 8247 and 9072 compartments distribution of CD63-positive compartments. (a) Control, Rab27a- and analysed in 14, 15 and 20 independent cells, respectively.) and Rab27b-knockdown cells were stained with anti-CD63 antibodies with percentage of cells with asymmetrical distribution (right; mean ± s.d. of (total) or without (surface) permeabilization, and analysed by FACS. four experiments; a total of 144, 222 and 277 cells, respectively) are Histograms from one representative experiment out of four are shown represented. (d) Left, electron micrographs of ultrathin cryosections of (dashed-lined empty histograms correspond to isotype-controls). (b) control, Rab27a-knockdown or Rab27b-knockdown cells immunogold- Three-dimensional deconvolution fluorescence microscopy of control, labelled with anti-HLA-DR (10 nm of gold) and anti-CD63 antibodies Rab27a- and Rab27b-knockdown cells stained with anti-CD63 (green) (15 nm of gold). Scale bars, 250 nm. Right, the surface area of individual and DAPI (nucleus, blue). Scale bars, 17.5 μm. Zooms are × 3.8. (c) CD63-positive multivesicular compartments on randomly taken electron Quantification of the morphology and distribution of CD63-positive micrographs was measured. Asterisks indicate statistically significant vesicles in control (white bars), Rab27a (black bars)- and Rab27b differences. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA (grey bars)-knockdown cells: mean size of individual CD63-positive followed by Tukey’s post test). KD, knockdown. induced a significant modification in the secretion of exosomes, but not of by quantitiative PCR, qPCR), were finally selected (Fig. 2c): Rab2b, OVA, were selected for two additional rounds of validation. Rab5a, Rab9a, Rab27a and Rab27b. Most of these Rab proteins have Some examples of Rab proteins that did not fulfil the selection been previously associated with endocytic functions, in agreement criteria are shown in Fig. 2b. Silencing of Rab6a inhibited the secre- with the postulated endosomal origin of exosomes. tion of OVA but did not affect exosome secretion, whereas inhibi- We chose to further analyse the mechanism of exosome secretion inhi- tion of Rab7 or Rab11a did not significantly modify either exosome bition caused by silencing Rab27a and Rab27b, two homologous Rab or OVA secretion. In contrast, five Rab proteins whose inhibition proteins (71% amino-acid identity)23 encoded by different genes and asso- by shRNA resulted in decreased exosome secretion in at least five ciated with lysosome-related organelles. To ensure that Rab27 silencing independent wells, and for which most shRNA concomitantly modi- affects total exosome secretion rather than any specific exosome-asso- fied the phenotype and the mRNA expression level (as determined ciated protein, we performed annexin V staining of phosphatidylserine

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a Rab27a CD63 b CD6315 Rab27a-GFP10

Rab6 Merge CD6315 Rab27a-GFP10 GA

MVE

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Figure 5 Differential intracellular distribution of Rab27a and Rab27b. (a) panel)-transfected cells immunogold-labelled with anti-CD63 (15 nm of Three-dimensional deconvolution fluorescence microscopy of cells transiently gold) and anti-GFP (10 nm of gold) antibodies. Scale bars, 200 nm. Arrows transfected with GFP–Rab27a (top panels)- or GFP–Rab27b (bottom panels)- indicate Rab27 association with the limiting membrane of MVEs. Arrowheads encoding plasmids, and subsequently stained with anti-CD63 and anti-Rab6 indicate localization of Rab27b to the Golgi apparatus (GA) area. (c) Confocal antibodies. Arrows indicate CD63-positive compartments with GFP–Rab27 fluorescence microscopy of cells doubly transfected with mCherry–Rab27a- staining. Scale bars, 10 μm. Zoom is × 2.3. (b) Electron micrographs of and GFP–Rab27b-encoding plasmids, and subsequently stained with ultrathin cryosections of GFP–Rab27a (top panel)- or GFP–Rab27b (bottom anti-CD63. Scale bars, 10 μm. Zooms are × 8. on exosomes captured by CD63-coated beads (Fig. 2d). Silencing of both (Supplementary Information, Fig. S2a). Exosomes were purified from Rab27a and Rab27b decreased annexin V staining, thus showing a general cell supernatant by ultracentrifugation24 (at 100,000g). The total amount decrease of secretion of exosome-associated lipids. of secreted exosomes, determined by measuring the total amount of protein in a pellet (Fig. 3a) or by the FACS-based assay (Supplementary Rab27a and Rab27b silencing inhibits exosome secretion Information, Fig. S2b), was significantly reduced in both knockdown without modifying exosome protein composition cells when compared with control cells. Inhibition of exosome secretion in cells knocked down for Rab27a and Consistent with these findings, a reduction in the signals of several Rab27b was confirmed by standard exosome purification techniques24. exosome markers (HLA-DR, CD63, Tsg101 and Hsc70) was observed by First, cells transfected with shRNA 3 for RAB27A or shRNA 1 for RAB27B western blotting of exosomes purified from the supernatant of the same were amplified to produce a large volume of supernatant. Expression of number of Rab27a-knockdown or Rab27b-knockdown secreting cells these genes was specifically and significantly downregulated in these cells when compared with control cells (Fig. 3b; Supplementary Information,

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FigS6a). Nevertheless, all markers were detected in exosomes from 1/5 were Rab27a-positive and b-negative and less than 10% were either knockdown cells when similar amounts of exosomal proteins where Rab27a- and b-positive or Rab27a-negative and b-positive. However, analysed (Fig. 3c; Supplementary Information, Fig.S6b). In addition, the proportion of Rab27-positive MVEs was increased in the subplas- electron microscopy showed that exosomes secreted by Rab27a- and malemmal region (Supplementary Information, Fig. S3). Rab27b-knockdown cells were identical in size and morphology to exo- Rab27a and Rab27b therefore have different subcellular distributions: somes produced by control cells (Fig. 3d). Rab27b predominantly presents an asymmetrical perinuclear distribution These data show that silencing of either Rab27a or Rab27b reduces the in the TGN area and a minor localization to CD63-positive compartments, amount of exosomes released in the cell culture supernatant, but does whereas Rab27a is preferentially associated with CD63-positive MVEs. not modify exosome protein content or morphology. Rab27a or Rab27b silencing reduces MVE docking to the Different phenotypes of MVEs are observed in Rab27a- and plasma membrane Rab27b-knockdown cells To further analyse exosome secretion, we used total internal reflection To gain further insight into the mechanism responsible for the reduced fluorescence (TIRF) microscopy to image MVEs in the subplasmalem- exosome secretion in Rab27a- or Rab27b-knockdown cells, the subcel- mal region of live cells (Fig. 6; Supplementary Information, Fig. S3 and lular distribution and the morphology of MVEs were investigated using Movies 1–3). In this region, around 30% and 40% of CD63-positive CD63 as a marker. We first observed, by FACS analysis, that the propor- vesicles were labelled with GFP–Rab27b and GFP–Rab27a, respectively tion of intracellular to surface CD63 was increased in Rab27a-, but not (Supplementary Information, Fig. S3a), supporting a possible role of Rab27b-, knockdown cells when compared with control cells (Fig. 4a). both Rab27 isoforms in MVE exocytosis. Rab27b silencing significantly Thus, in the absence of Rab27a, CD63 accumulates in internal compart- lowered MVE density (Fig. 6a), in agreement with the perinuclear relo- ments, whereas the absence of Rab27b does not induce such behaviour. calization of MVEs observed by epifluorescence microscopy (Fig. 4b). In The subcellular distribution of CD63 was then analysed by immun- addition, in Rab27a- and Ra27b-knockdown cells, MVEs were signifi- ofluorescence imaging (Fig. 4b, c). In control and Rab27a-knockdown cantly less present near the borders of the cell footprint (Supplementary cells, CD63-positive MVEs appeared to be homogenously distributed Information, Fig. S3b), suggesting that Rab27a and Rab27b promote throughout the cell, but their size was significantly increased upon MVE distribution to the cell periphery. Rab27a silencing (Fig. 4b, upper and middle panels; Fig. 4c). In sharp The movements of MVEs were then analysed by tracking individual contrast, Rab27b-knockdown cells showed a characteristic asymmetrical trajectories25 (Supplementary Information, Movies 1–3). The diffusion perinuclear accumulation of CD63-positive organelles, but without sig- coefficient Dxy, an index of mobility, was calculated along trajectories using 25 nificant alteration of the size of these organelles, compared with control a rolling analysis window . Dxy values were higher in Rab27a- or Rab27b- cells (Fig. 4b, lower panel, Fig. 4c). Consistent with these findings, elec- knockdown cells than in control cells (P < 0.0001), and in Rab27b- than in tron microscopy analysis showed that MVEs from Rab27a-knockdown Rab27a-knockdown cells (P = 0.006; Fig. 6b). Fewer vesicles had very low cells were significantly larger (Fig. 4d) than those from control cells, Dxy values in Rab27a- or Rab27b-knockdown cells, suggesting that both whereas in Rab27b-knockdown cells MVEs were smaller (Fig. 4d) and may participate in MVE attachment (docking) to the plasma membrane, showed a clustered distribution (data not shown). Thus, although silenc- a process that restricts vesicle motion25–27. To set a threshold value below ing of either Rab27a or Rab27b impairs exosome secretion, the induction which vesicles could be considered immobile, we plotted the distribution of of different changes in the size and distribution of CD63-positive MVEs instantaneous Dxy values (Supplementary Information, Fig. S3c). A major suggests that these proteins have different roles in the pathway. peak comprising the lowest values was centered around 10–4 μm2 s–1 (that 26 is, the mean Dxy of immobilized beads on our setup) . Vesicles presenting Rab27a and Rab27b have different subcellular localizations a Dxy less than three times this value were considered immobile. Using this

We thus investigated whether these two proteins could function in differ- criterion, we categorized Dxy values computed on entire trajectories and ent compartments by analysing their respective subcellular localizations. found that the percentage of docked structures was significantly lowered GFP-tagged versions of both Rab27a and Rab27b presented a dough- by Rab27a or Rab27b silencing (Fig. 6c). Moreover, 82% of the enlarged nut-like pattern surrounding CD63-positive compartments (Fig. 5a). MVEs observed upon Rab27a silencing (Fig. 4b) showed a mean Dxy > However, most Rab27b, but not Rab27a, was mainly concentrated in a 3 × 10–4 μm2 s–1 (50 structures from 5 cells) and were thus not docked. perinuclear area labelled by the Golgi-associated proteins Rab6 (Fig. 5a) To distinguish true vesicle docking from other processes, we meas- and TGN46 (data not shown). Consistent with this, electron microscopic ured the duration of immobilization events along trajectories. Rab27a observation showed that although both Rab27a and Rab27b were associ- or Rab27b silencing reduced the percentage of vesicles that were always ated with the limiting membrane of MVEs, Rab27b was also observed in immobile and increased the percentage of vesicles never immobile the membrane of Golgi stacks and in vesicles located in the trans-Golgi (Fig. 6d). Survival curves of immobilization times were best fitted with network (TGN) area (Fig. 5b). two exponentials, indicating the existence of two different processes When mCherry–Rab27a and GFP–Rab27b were expressed simul- (Fig. 6e). The short-lasting component may reflect transient tethering to taneously in HeLa cells, both Rab27a and Rab27b were partially the plasma membrane or phases of slow diffusion. We considered long- associated with CD63-positive compartments (Fig. 5c), but Rab27b lasting immobilization to reflect MVE docking. Using this refined crite- was predominantly in the perinuclear area and was partially colocal- rion of docking, we conclude that Rab27a or Rab27b silencing reduced ized with TGN46 (data not shown). Four groups of CD63-positive both the number (0.25 and 0.28 docking events per trajectory in Rab27a- organelles could be identified based on their association with the and Rab27b-knockdown cells, respectively, compared with 0.44 in con- Rab27 isoforms: most organelles were Rab27a- and b-negative, about trol cells) and the characteristic time (10.1 and 9.3 s in Rab27a- and nature cell biology VOLUME 12 | NUMBER 1 | JANUARY 2010 25 © 2010 Macmillan Publishers Limited. All rights reserved.

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a b ** ** ** * 0.35 NS 1,000

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0.3 0.01 Velocity (µm s 0.2

events per trajectory 0.1 Remaining immobilization 0.001 0 010203040 Control Rab27a Rab27b Immobilization time (s) KD KD

Figure 6 Effect of Rab27a and Rab27b knockdown on the mobility of CD63- knockdown: 10 cells, 415 trajectories; white bars, Rab27b knockdown: 12 positive compartments in the subplasmalemmal region analysed by TIRF cells, 528 trajectories; *P = 0.0479, **P = 0.011, ***P = 0.0097). (d–f) Dxy microscopy. (a) Right, representative TIRF microscopy images showing was computed along trajectories using a rolling analysis window applied to GFP–CD63 distribution in control, Rab27a- and Rab27b-knockdown cells. 499 trajectories in n = 12 control cells, 415 trajectories in n = 10 Rab27a- Scale bar, 5 μm. Left, vesicle density in the subplasmalemmal region and 528 trajectories in n = 12 Rab27b-knockdown cells. (d) Data show the detected by TIRF microscopy (data are the mean ± s.e.m. number of vesicles mean ± s.e.m. percentage of vesicles that were always (grey bars) or never 2 -4 2 –1 per μm quantified in n = 18 control, n = 19 Rab27a- and n = 20 Rab27b- (black bars) docked (Dxy < 3 × 10 μm s ). *P = 0.0147, **P = 0.0086, knockdown cells. **P = 0.0042, Mann-Whitney test). (b) Effect of Rab27a ***P = 0.0067. (e) Further characterization of docking events. Immobilization and Rab27b knockdown on vesicle motion. Mean diffusion coefficients (Dxy) periods were detected as in d and their duration was measured. Logarithmic values of individual trajectories obtained for at least 400 vesicles in control, plots of survival curves for immobilization times are shown (control, black Rab27a- and Rab27b-knockdown cells are shown. Red bars indicate median line; Rab27a knockdown, dashed line; Rab27b knockdown, grey line). The and interquartile ranges (*P < 0.01, **P < 0.001, Kruskal Wallis test, best fit was obtained with the sum of two exponentials (r2 > 0.998) and was followed by Dunn’s post test). (c) Data show the mean ± s.e.m. of Dxy values significantly different in Rab27 knockdown cells compared to control cells computed on entire trajectories. Rab27a and Rab27b silencing reduced the (P < 0.0001, extra sum-of-squares F‑test). (f) Rab27b knockdown increases –4 2 –1 percentage of vesicles with Dxy < 3 × 10 μm s , which are presumably mobility along microtubules. The mean ± s.e.m. velocity of vesicles during docked (black bars, control: 12 cells, 499 trajectories; grey bars, Rab27a periods of directed motion is shown (*P = 0.011).

Rab27b-knockdown cells, respectively, compared with 15.5 s in control Another alteration was observed in Rab27b-knockdown cells. The cells) of docking events. Altogether, these analyses show that Rab27a and proportion (39 ± 3% versus 29 ± 3% in control cells) and velocity Rab27b promote the occurrence and stability of MVE docking. (Fig. 6f) of vesicles moving rapidly along linear axes (a characteristic

ARTICLES of microtubule-based motion)26 were increased in Rab27b-knockdown These observations suggest that Slp4 mediates the function of Rab27a, (but not in Rab27a-knockdown) cells, compared with control cells. while Slac2b is required for the function of Rab27b. This behaviour accounted for the increased proportion of structures Consistent with this idea, in cells co-transfected with T7-tagged Slp4 or –4 2 –1 with high mean Dxy (> 20 × 10 μm s ; Fig. 6c) observed in Rab27b- Slac2b and GFP-tagged Rab27a or Rab27b, Slp4 colocalized with Rab27a knockdown cells. Therefore, Rab27b also negatively regulates long- to doughnut-like structures containing CD63 (Fig. 8a, top panel). In range movements of MVEs. contrast, Slp4 and Rab27b labelled different CD63-positive structures (Fig. 8a, bottom panel). The Rab27a–Slp4 interaction was also con- Slp4 and Slac2b are two Rab27 effectors involved in exosome firmed by the observation that the Slp4 protein level was decreased in production Rab27a-knockdown cells (but not in Rab27b-knockdown cells; Fig. 8b; Rab proteins exert their functions by interacting with multiple effectors. Supplementary Information, Fig. S6d), whereas the SYTL4 mRNA level We observed that 7 of the 11 Rab27 effector proteins were expressed in was increased (Fig. 8c), suggesting a stabilizing interaction of Rab27a and HeLa B6H4 cells (Supplementary Information, Table 1). Their genes Slp4 proteins. Due to low expression level of endogenous Slac2b, which were then silenced with specific shRNAs, and exosomes and OVA in cell could not be detected by the available antibodies, and to poor expres- culture supernatants were quantified using the FACS-based assay. sion of a T7-Slac2b construct in our cell line, we could neither determine Silencing of EXPH5 (gene encoding Slac2b), SYTL4 (gene encoding its intracellular distribution nor its preferential association with Rab27b. Slp4) or UNC13D (gene encoding Munc13–4) significantly reduced exo- Finally, co-silencing of Rab27a and Slp4 led to a similar phenotype as some secretion, with at least 2 of the 4–5 shRNAs that targeted each gene single silencing of each (that is, enlarged MVEs), whereas co-silencing of (Fig. 7a). In cells transduced with shRNAs against SYTL4 and EXPH5, Rab27b and Slp4 induced mainly the perinuclear redistribution of MVEs the level of exosome secretion reflected the level of mRNA expression typical in Rab27b-knockdown cells, thus confirming that Rab27b and Slp4 (Fig. 7b). In contrast, in cells transduced with UNC13D shRNA, although are not functionally associated (Supplementary Information, Fig. S4). shRNAs 1, 2 and 3 decreased both exosome secretion and mRNA levels, These observations thus show that Rab27a and Slp4 function together shRNAs 4 and 5 strongly inhibited mRNA expression without affecting in the MVE and exosome pathway of HeLa cells. By contrast, although exosome secretion (Fig. 7a, b), suggesting off-target effects of the shRNA. silencing of Slac2b induces a phenocopy of Rab27b-knockdown cells, for Consequently, only Slac2b and Slp4 were chosen for further analysis, technical reasons we could not demonstrate the functional interaction although a role of Munc13–4 in exosome secretion cannot be ruled out. between these two proteins. In addition to reducing exosome secretion, knock down of the effectors increased OVA secretion (Fig. 7a), suggesting that they also have Rab27- Discussion independent roles in the conventional secretion pathway. Three major results are presented here. First, we set up an assay allow- Large-scale cultures of cells expressing lentiviruses 3 (EXPH5 shRNA) ing medium-scale screens of the molecular machinery involved in or 3 (SYTL4 shRNA) were prepared. Specific decrease of Slp4 at the exosome secretion. Second, using this assay, we identified several Rab protein level was confirmed by immunoblotting (Fig. 7c; Supplementary family members, including Rab27a and Rab27b, that function in exo- Information, FigS6c), but we could not specifically detect Slac2b by some secretion. Third, we found that Rab27a and Rab27b serve both western blotting of B6H4 cell lysates, probably due to the low level of common and different roles, most likely through the Slp4 and Slac2b expression. Exosomes secreted by the same amount of cells were puri- effector proteins, respectively, in the intracellular trafficking of MVEs, fied from cell culture supernatants and analysed by western blotting leading to exosome secretion. (Fig. 7d; Supplementary Information, FigS6e): a significant reduction The identification of Rab GTPases that affect both the morphology in the amount of CD63, HLA-DR and Tsg101 secreted in exosomes was and distribution of CD63-positive MVEs and exosome secretion con- observed in Slp4- and Slac2b-knockdown cells. These results show that firms the endocytic origin of exosomes, which was proposed previously two Rab27 effectors, Slp4 and Slac2b, are positive regulators of exosome based on results from elegant electron microscopy studies5,15. In addition, secretion in HeLa B6H4 cells. as reducing mRNA expression of RAB27 genes to less than 20% of the control induced > 50% reduction in exosome secretion, most secreted Slp4 and Slac2b-knockdown cells are phenocopies of Rab27a exosomes originate from a Rab27-dependent secretion event, probably and Rab27b-knockdown cells, respectively due to fusion of late endocytic compartments with the plasma membrane. The distribution and morphology of CD63-positive compartments was In addition to Rab27, three other Rabs related to the endocytic pathway then studied in Slac2b- and Slp4-knockdown cells. In Slp4-knockdown were identified in this study as being potentially involved in exosome cells, an intracellular accumulation of CD63 was observed both by secretion: Rab9 and Rab5 participate in the trafficking of late and early FACS (Fig. 7e) and by immunofluorescence (Fig. 7f, left and middle endosomes, respectively17,28, and Rab2, although generally described panels), resembling the phenotype observed in Rab27a-knockdown cells as allowing trafficking between the endoplasmic reticulum and Golgi (Fig. 4a). In contrast, FACS analysis of Slac2b-knockdown cells did not apparatus, is phylogenetically closer to Rab4, Rab11 and Rab14 (ref. 29), show an accumulation of CD63 either at the cell surface or intracellularly, which are associated with the endocytic pathway. Surprisingly, other Rab but showed an asymmetrical perinuclear accumulation of CD63-positive GTPases associated with the endocytic system, Rab7 and Rab11, did not vesicles of normal size, mimicking the phenotype observed in Rab27b- modify exosome secretion. These observations suggest that a specific knockdown cells (Fig. 7f, right panel). Altogether, loss of function of Slp4 subset of MVEs participates in the generation of exosomes, as recently and Slac2b induces phenotypes that, in terms of exosome secretion as proposed for dendritic cells30. well as morphology and distribution of MVEs, highly resemble those Rab27a and Rab27b have been found previously to be involved in observed in cells knocked down for Rab27a and Rab27b, respectively. the regulated secretion of secretory granules (that is, compartments nature cell biology VOLUME 12 | NUMBER 1 | JANUARY 2010 27 © 2010 Macmillan Publishers Limited. All rights reserved.

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a OVA b Exosome 500 * ** EXPH5 SYTL4 UNC13D 250 *** 100 400 * 200 * 50 150 300 * * * of control) 0 * ** (percentage 1 234 1234 12345 100 200

** mRNA expression * ** 100 50 ** ** 100 c *** ** ***** Secretion level Secretion level 75 0 0 50 (percentage of control) (percentage of control) Control Slac2b KDSlp4 KD 25 Noc2 Noc2 Slp4 EXPH5 SYTL2 SYTL3 SYTL4 EXPH5 SYTL2 SYTL3 SYTL4 0 UNC13D UNC13D Slp4 level Tubulin Control d Slp4 KD

*** * (Percentage of control) Slac2b KD Control Slp4 KD Slac2b KD *** 100 * CL Exo CL Exo CL Exo f Control Slp4 KD Slac2b KD 0.13 9 0.13 9 0.13 9 6 10 cells 75 CD63 HLA-DR DAPI 50

CD63 25 Selection level

(percentage of control) 0 Tsg101

Control Control

Gp96 Slp4 KD Slp4 KD

Slac2b KD Slac2b KD HLA-DR Tsg101

e Surface CD63 Total CD63 80 Control 70 Slp4 KD 75 Slp4 KD 60 Slac2b KD *** 50 50 Control shRNA 40 *** KD 30 Isotype control 25 20 *** *** (percentage) 10 Slac2b KD Vesicles (percentage) 0 0 < 400 400–600 600–800 > 800 Cells with asymmetrical distribution of CD63 staining maximum Vesicle diameter (nm) Percentage of Control Slp4 KD Fuorescence intensity Slac2b KD

Figure 7 Effect of Rab27 effector proteins on exosome secretion. (a) FACS and are the mean ± s.d. of three independent experiments. (e) FACS analysis of analysis of exosome (HLA-DR/CD81 staining, black bars) and OVA (white bars) Slp4-, Slac2b-knockdown and control cells stained in parallel with anti-CD63 secretion by cells transduced with shRNA targeting genes for Rab27 effector antibodies with (total) or without (surface) permeabilization. Representative proteins (3–5 viruses per gene). Data are the mean ± s.d. of three independent histograms of three independent experiments are shown (dashed-lined, empty experiments. (b) Specific inhibition of SYTL4, EXPH5 and UNC13D gene histograms correspond to isotype controls). (f) Three-dimensional deconvolution expression, as assessed by quantitative reverse transcription-PCR (qRT-PCR). fluorescence microscopy of control, Slp4-, and Slac2b-knockdown cells stained Results are from two independent experiments, and are expressed as a with anti-CD63 (green) and DAPI (nucleus, blue). Upper panels, representative percentage of mRNA expression in control cells. (c) Immunoblot (left panel) and images of control, Slp4- and Slac2b-knockdown cells. Scale bars, 10 μm. quantification (right panel) of Slp4 protein level in Slp4- and Slac2b-knockdown Zooms are × 2.5. Lower panels, quantification of CD63-positive vesicle size cells. Tubulin was used as a loading control. (d) Exosome secretion by B6H4 (left panel, mean ± s.d. of 10106, 11430 and 11404 vesicles from of 20 cells stably transduced with scrambled shRNA (Control), SYTL4 shRNA 3 control, 21 Slp4- and 23 Slac2b-knockdown cells, respectively) and cells with (Slp4 KD), or EXPH5 shRNA 3 (Slac2b KD). Exosomes (Exo) secreted by 9 asymmetrical distribution of CD63-positive vesicles (right panel, mean of two million cells, or 30 μg of total cell lysates (CL) were analysed by immunoblotting independent experiments, a total of 107 control, 86 Slp4- and 115 Slac2b- for presence of CD63, Tsg101, HLA-DR and Gp96 proteins (left panel). One knockdown cells). Asterisks indicate statistically significant differences from representative experiment of three is shown. The amount of HLA-DR and control cells. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed Tsg101 secreted in exosomes was quantified by signal intensity measurements by Tukey’s post test). KD, knockdown. For full scans of blots in c and d, see (right panel). Data are expressed as secretions relative to those in controls cells Supplementary Information, Fig. S6. of Golgi origin)31–33 and of lysosome-related organelles in mast cells34, The phenotype of MVEs induced by Rab27b silencing (inhibition of platelets35 and cytolytic T lymphocytes36. In this study, we found that docking, clustering in the perinuclear area and high velocity movements Rab27a and Rab27b have a key role in exosome secretion by promoting along microtubules), is similar to the phenotype of secretory granules31 and the targeting of MVEs to the cell periphery and their docking at the melanosomes38,39 induced by silencing myosin Va, a downstream effector plasma membrane. of Rab27a, and suggests that Rab27b mediates the transfer of MVEs from Functional redundancy between Rab27a and Rab27b depends on microtubules to the actin-rich cortex and their retention at the cell periph- the cell type and/or the secretory process studied34,35,37. Interestingly, we ery. In addition, the smaller size of MVEs observed by electron microscopy show here that in the HeLa cell line, Rab27a and Rab27b perform dif- in Rab27b-knockdown cells suggests that Rab27b could participate in the ferent and non-redundant tasks in the exosomal pathway. transfer of membranes from the TGN to MVEs, as proposed previously40.

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CD63 Slp4 Rab27b Merge

CD63 Slp4 Rab27a Merge

b c 200 Slp4 protein SYTL4 mRNA ** ** 200 Control Rab27a KD Rab27b KD 150 * Slp4 150 100 100 50 Tubulin Slp4 expression 50 Slp4 expression (percentage of control) 0 0 Control Rab27a Rab27b (percentage of control) Control Rab27a Rab27b KD KD KD KD

Figure 8 Interaction between Slp4 and Rab27a. (a) Three-dimensional normalized to tubulin levels (right panel) are shown. For full scans of blots deconvolution fluorescence microscopy of cells doubly transfected with see Supplementary Information, Fig. S6. (c) The expression level of Slp4 T7–Slp4 and either GFP–Rab27b (top panels)- or GFP–Rab27a (bottom mRNA in cells knocked down for either Rab27a or Rab27b was analysed panels)-encoding plasmids, and subsequently stained with anti-CD63 by qPCR and normalized to the expression level of GAPDH. Data are the and anti–Slp4 antibodies. Arrows indicate zoomed areas (× 8). (b, c) Slp4 mean ± s.d. of three independent experiments. Asterisks indicate statistically expression levels in cells knocked down for either Rab27a or Rab27b were significant differences from control cells. *P < 0.01, **P < 0.001 (one-way evaluated by immunoblotting. (b). A representative blot (left panel) and ANOVA followed by Tukey’s post test). KD, knockdown. For full scan of blot in quantification of the relative mean intensity ± s.d. of three independent blots b, see Supplementary Information, Fig. S6.

In contrast, Rab27a silencing reduces docking of MVEs and increases with Rab27b, and thus regulates the Rab27a-dependent docking event their size. This could be due to lack of motor linkage, resulting in appar- leading to exosome secretion in HeLa cells. The preferential interaction ently enlarged compartments. Alternatively, either Rab27a is required of Rab27b with Slac2b in HeLa cells could not be demonstrated here, for docking and vesicles fuse with each other instead of fusing with the although it has been previously demonstrated in vitro41. plasma membrane when Rab27a is absent, or Rab27a prevents fusion of Finally, this work was performed using the HeLa model cell line: the CD63-positive compartments with each other or with other vesicular physiological processes at work in HeLa cells may not be identical to intermediates, and its absence leads to the formation of enlarged compart- those of other cell types. However, mouse dendritic cells deficient in ments whose size physically impairs docking to the plasma membrane. both Rab27a and Rab27b secrete half the amount of exosomes that their In addition, we show here that silencing of the Rab27 effectors Slp4 wild-type counterparts do (Supplementary Information, Fig. S5) and our and Slac2b phenocopies the silencing of Rab27a and Rab27b, respec- preliminary data suggest that silencing of Rab27a in a mouse tumour cell tively, and that Slp4 preferentially associates with Rab27a, rather than line also leads to reduced exosome secretion (A. Bobrie, M.O. and C.T., nature cell biology VOLUME 12 | NUMBER 1 | JANUARY 2010 29 © 2010 Macmillan Publishers Limited. All rights reserved.

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7. Thery, C. et al. Indirect activation of naive CD4+ T cells by dendritic cell-derived unpublished data). Thus, the Rab27 subfamily is probably also important exosomes. Nature Immunol. 3, 1156–1162 (2002). in exosome secretion by other cell types. 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). Overall, the potential to specifically inhibit exosome secretion with- 9. Fevrier, B. et al. Cells release prions in association with exosomes. Proc. Natl Acad. out affecting the regular secretory pathway of soluble proteins paves the Sci. USA 101, 9683–9688 (2004). 10. Rajendran, L. et al. Alzheimer’s disease β-amyloid peptides are released in association way for the manipulation of exosome secretion in vivo, to eventually with exosomes. Proc. Natl Acad. Sci. USA 103, 11172–11177 (2006). 11. Wolfers, J. et al. Tumor-derived exosomes are a source of shared tumor rejection anti‑ answer the long-standing question of the physiological functions of gens for CTL cross-priming. Nature Med. 7, 297–303 (2001). exosomes in vivo. 12. Iero, M. et al. Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ. 15, 80–88 (2008). 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 METHODS (2008). Methods and any associated references are available in the online version 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. of the paper at http://www.nature.com/naturecellbiology/. 155, 1251–1264 (2001). 15. Pan, B. T., Teng, K., Wu, C., Adam, M. & Johnstone, R. M. 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Targeting of Rab GTPases to cellular membranes. Biochem. Frontier Science Program and receives support from Fundação Luso-Americana Soc. Trans. 33, 652–656 (2005). para o Desenvolvimento and Fundação para a Ciência e a Tecnologia (PTDC/SAU- 20. Escola, J. M. et al. Selective enrichment of tetraspan proteins on the internal vesicles MII/69280/2006 and PTDC/SAU-MII/78333/2006). We thank PICT IbiSA Imaging of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273, 20121–20127 (1998). Facility at Curie Institute, V. Racine and J.-B. Sibarita for providing a copy of their 21. Morelli, A. E. et al. Endocytosis, intracellular sorting, and processing of exosomes by multidimensional image analysis program, I. Hurbain for help in electron microscopy dendritic cells. Blood 104, 3257–3266 (2004). quantification, P. Simões and M. H. Raquel for some of the lentiviral preparations, 22. Thery, C. et al. 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USA 104, reprintsandpermissions/. 5872–5877 (2007). 36. Stinchcombe, J. C. et al. Rab27a is required for regulated secretion in cytotoxic T 1. Thery, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. lymphocytes. J. Cell Biol. 152, 825–834 (2001). Nature Rev. Immunol. 2, 569–579 (2002). 37. Barral, D. C. et al. Functional redundancy of Rab27 proteins and the pathogenesis of 2. Fevrier, B. & Raposo, G. Exosomes: endosomal-derived vesicles shipping extracellular Griscelli syndrome. J. Clin. Invest. 110, 247–257 (2002). messages. Curr. Opin. Cell Biol. 16, 415–421 (2004). 38. Strom, M., Hume, A. N., Tarafder, A. K., Barkagianni, E. & Seabra, M. C. A family of 3. Johnstone, R. M. Exosomes biological significance: a concise review. Blood Cells Mol. Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melano‑ Dis. 36, 315–321 (2006). some transport. J. Biol. Chem. 277, 25423–25430 (2002). 4. Lakkaraju, A. & Rodriguez-Boulan, E. 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DOI: 10.1038/ncb2000 METHODS

METHODS Quantitative reverse transcription‑PCR (qRT-PCR). RNAs were isolated from Cells and reagents. HeLa-CIITA cells42 were stably transfected with pcDNA3 each well at day 5 after lentiviral infection with the Qiagen RNeasy Mini Kit and encoding the OVA cDNA fused to the signal peptide of MFG‑E8/lactadherin13, 200 ng were reverse transcribed with AMV RT (Finnzymes). cDNA (1/10th) was and a subclone named B6H4 was selected. These cells were cultured in DMEM used for each PCR reaction, performed with SYBRgreen (Thermo Scientific) containing 4.5 g l–1 glucose (Invitrogen) 10% fetal calf serum (Abcys), 2 mM on ABI PRISM 7900 real-time thermal cycler (Applied Biosystems). Primer glutamine, 100 IU ml–1 penicillin, 100 μg ml–1 streptomycin, 300 μg ml–1 hygro- sequences for each gene were either retrieved from Primer Bank (http://pga. mycin B (Calbiochem) and 500 μg ml–1 geneticin (Invitrogen). mgh.harvard.edu/primerbank/) or designed using Primer3 (http://frodo.wi.mit. Plasmids used for transient transfections (performed with Bio-Rad gene pulser edu/cgi-bin/primer3/primer3_www.cgi). Cycle thresholds (Ct) were normalized II)42 include pEGFPC3–Rab27a (ref. 43), pEGFPC3–Rab27b (ref. 23), pEGFP– to Ct of GAPDH and fold enrichments were calculated as compared with the bos-CD63 (ref. 44), pT7–Slp4 (ref. 45), mCherry–Rab27a generated by PCR from values from control shRNA-transduced cells. pcDNA3.1mcherry46 and pEGFPC3–Rab27a, and cloned into pCMVMyc vector (A.N.H., unpublished data)38. Exosome purification. Cells were cultured in ‘exosome-depleted medium’ (com- Lentiviruses expressing shRNA were obtained from the RNAi consortium plete medium depleted of FCS-derived exosomes by overnight centrifugation (TRC) and produced as described previously47. Sequences of the shRNA used at 100,000g) for 48 h and exosomes were purified by filtration on 0.22-μm pore 24 for large scale cultures were: RAB27A, 5´-GCTGCCAATGGGACAAACATA-3´; filters, followed by ultracentrifugation at 100,000g as described previously . In RAB27B, CCCAAATTCATCACTACAGTA-3´; SYTL4, 5´-CCTCCCTTTACAT each exosome preparation, the concentration of total proteins was quantified by GGAAAGAT-3´ and EXPH5, 5´-CCCAAACTAGAAGAAGTTCTT-3´. Bradford assay (BioRad). Antibodies used for FACS analysis were: mouse anti-human CD63 (FC-5.01, Zymed) and goat anti-OVA (ICN) for coupling to beads; PE-conjugated anti- Electron microscopy. Exosomes fixed in 2% paraformaldehyde were loaded on Formwar/carbon-coated electron microscopy grids. Samples were post-fixed HLA-DR (L243, BD), anti-CD81 (BD) and rabbit anti-OVA (Sigma) coupled 5 to Alexa488 according to the manufacturer’s instructions (Pierce). Antibodes in 1% glutaraldehyde, contrasted and embedded as described previously . For used for immunoblotting were: goat anti-Tsg101 (Santa Cruz); rat anti-Gp96 and immunolabelling, cells were fixed with 2% paraformaldehyde in 0.2 M phosphate anti –Hsc70 (Stressgen); mouse anti-CD63 (Zymed), anti-HLA-DR (clone 1B5) buffer at pH 7.4 and processed for ultrathin sectioning and immunolabelling with the indicated antibodies and protein A coupled to 10- or 15-nm gold particles and anti-Rab27a (clone 4B12; ref. 43); rabbit anti-Rab27b (ref. 35), anti-Slp4 5 (ref. 33) and anti-Slac2b (ref. 33); HRP-conjugated secondary antibodies (Jackson (Cell Microscopy Center) as described previously . Sections were observed at Immunoresearch). Antibodies used for immunofluorescence and immunoelec- 80kV with a CM120 Twin Philipps electron microscope (FEI Company) and tron microscopy were: mouse anti-CD63 (Zymed); rabbit anti-Rab6 (Santa Cruz) digital acquisitions were made with a numeric camera (Keen View; Soft Imaging and anti-GFP (Invitrogen); Alexa488-anti-mouse and Cy3-anti-rabbit (Jackson system). Quantification of the surface of MVBs was performed on at least 40 Immunoresearch); DAPI (Molecular Probes). CD63-positive multivesicular compartments present in randomly taken pictures, analysed using iTelectron microscopy software (Olympus). Immunoadsorbtion and detection of exosomes by FACS. Anti-human CD63 or anti-OVA antibodies were coupled to 4-μm aldehyde-sulfate beads (Invitrogen) by Immunoblotting. Cells were lysed in 50 mM Tris at pH 7.5, 0.3 M NaCl, 0.5% incubating 35 μg of antibody with 1 × 108 beads followed by blocking of remaining Triton X‑100 and 0.1% sodium azide, with a cocktail of antiproteases (Roche), activated groups with PBS‑4% BSA. and cleared from nuclei by centrifugation at 15,000g for 5 min. Protein extracts Cell culture supernatants were cleared by centrifugations at 300g (for were separated on 4–12% SDS–PAGE, blotted on Immobilon (Millipore), and 5 min) and 2,000g (for 20 min). Cleared supernatants (100 μl) were incu- HRP-conjugated secondary antibodies were revealed using chemoluminescence bated with 20,000 anti-CD63- and 20,000 anti-OVA-coupled beads overnight substrate (Roche). Images were acquired using Chemidoc XRS (BioRad), intensity at room temperature with 6,500 rpm horizontal shaking. Beads were washed of the bands was quantified using QuantityOne software (Biorad) and results were twice in PBS‑2% BSA and incubated with PE‑anti-HLA-DR + PE-anti-CD81 expressed as a ratio to control cells. and Alexa488-anti-OVA (all at a 1:100 dilution) for 30 min on ice. Beads Flow cytometry. For surface labelling, cells were stained with antibodies were washed twice in annexin V buffer (BD Biosciences) and incubated diluted in PBS‑0.5% BSA on ice. For intracellular + surface staining, cells fixed with APC-conjugated annexin V (1:10) for 10 min. Beads were acquired with 1% paraformaldehyde were quenched with PBS‑0.1 M glycine, permea- on a FACSCalibur (BD) and data were analysed with FlowJo software (Tree bilized using PermWash reagent (BD), and stained with antibodies diluted in Star). Threshold of negative staining was obtained with beads incubated with permeabilization buffer. Cells were acquired on a FACScalibur and analysed unconditioned medium, and for each culture condition, arbitrary units (AU) using FlowJo software. of exosome or OVA secretion were calculated as a percentage of positive beads × MFI (mean fluoresence intensity) of positive beads. To rule out detection of Immunofluorescence microscopy. Cells (1 × 105) were seeded on glass cov- apoptotic blebs, a total of 50,000 cells either UV-irradiated or untreated were erslips for 18 h, fixed in 4% paraformaldehyde, quenched with 0.1 M glycine, seeded in a 96-well plate. After 12 h incubation, exosome depleted-medium permeabilized in PBS‑0.2% BSA‑0.05% saponin, and incubated with primary and was added and 24 h later supernatants were incubated with anti-CD63-coated secondary fluorescent antibodies. The coverslips mounted with Fluoromount‑G beads and analysed by FACS. (Southern Biotechnology Associates) were examined under a motorized upright Lentiviral infection and screening procedure. HeLa B6H4 cells were plated wide-field microscope (Leica DMRA2) equipped for image deconvolution. in 96-well round-bottom plates at 2,500 cells per well (day zero). At day 1, cells Acquisition was performed using a ×100 objective (PL APO HCX, 1.4 NA) and a were infected with lentiviral vectors in the presence of 8 μg ml–1 polybrene high-sensitive cooled interlined CCD camera (Roper CoolSnap HQ). z‑position- and 5 μg ml–1 puromycin was added at day 2. Medium was replaced with ing was accomplished by a piezo-electric motor (LVDT, Physik Instrument) and a exosome-depleted medium at day 4. Supernatants were collected 48 h later z-series of images was taken (an image every 0.2 μm). Images were acquired with for OVA and exosome quantification, and the amount of live cells in each Metamorph Software. Vesicle sizes were calculated with the Multidimensional 48 well was determined using Cell titer blue reagent viability assay (Promega). Image Analysis (MIA) interface based on wavelet decomposition , running under OVA and exosome secretions per well were normalized to the number of MetaMorph (Universal Imaging Corporation). Briefly, fluorescent objects corre- viable cells, and only wells containing at least 75,000 cells were analysed. In sponding to MVEs were detected as fluctuations that were 20-fold larger than the each experiment, the basal level of exosome or OVA secretion (control) was noise. The watershed function was routinely applied to precisely detect individual calculated as either AU of cells transduced with control scrambled shRNA, structures in dense regions. The diameter of each structure was calculated from or mean of AU of one entire plate. A candidate gene was defined as a hit if the sum of pixels assuming a spherical shape. Alternatively, images were acquired two or more shRNAs modified AU of exosome secretion, in duplicate, by with Zeiss confocal microscope (LSM Axivert 720) using 6 3 × 1.4 NA oil immer- more than one standard deviation from the control. Results are presented as sion objective. Image acquisition and analysis were performed in the PICT IbiSA percent of AU of control. Imaging Facility (Curie Institute). Brightness and contrast of the images shown

METHODS DOI: 10.1038/ncb2000

(–t/ 1) (–t/ 2) in Figs 4b, 5a, 7f and 8a were adjusted for better visualization according to Nature fN(t) = N1 exp τ + N2 exp τ , using SigmaPlot (Systat Software). N1 and N2

Cell Biology guidelines for the presentation of digital data. represent the abundance of each component and τ1 and τ2, their time constants. Quantification of asymmetrical distribution of CD63-positive vesicles was Portions of trajectories showing directed motion were detected by inspection of performed manually in a double-blind setting. Asymmetry was defined as a x vs t and y vs t plots and selected using locally written routines (MatLab, The concentration of the CD63-staining in one side of the nucleus (covering less MathWorks). Frame-to-frame rates of movement were calculated and averaged than 50% of the nucleus diameter). over each period of directed motion.

Total internal reflection fluorescence (TIRF) microscopy. B6H4 cells stably Statistical analyses. Data were analysed using GraphPad Prism (GraphPad expressing shRNA were transfected with a GFP–CD63-encoding plasmid 36 h Software). Normality of the data was tested using the Kolmogorov-Smirnov test. before analysis by TIRF microscopy. TIRF imaging was performed on a custom Based on the normality test, either one-way ANOVA followed by Tukey’s post test setup as described previously26. The penetration depth (δ) of the evanescent or Kruskal Wallis followed by Dunn’s post test were used for multiple comparison field used to excite the fluorophore was set to 150 nm. Frames were acquired at analyses. Values are given as mean ± s.d. in all figures except Fig. 6 where values 10 Hz in stacks of 450 images with an exposure time of 100 ms. One pixel cor- are mean ± s.e.m. responds to 107.5 nm. The density of GFP–CD63 structures was evaluated using 42. Stumptner-Cuvelette, P. et al. HIV-1 Nef impairs MHC class II antigen presentation MIA software48. The quantification was performed on regions devoid of TGN- and surface expression. Proc. Natl Acad. Sci. USA 98, 12144–12149 (2001). like labelling and expressed as the number of observed CD63-positive structures 43. Hume, A. N. et al. Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J. Cell Biol. 152, 795–808 (2001). divided by the size of the cell footprint. From stacks of images, two‑dimensional 44. Blott, E. J., Bossi, G., Clark, R., Zvelebil, M. & Griffiths, G. M. Fas ligand is targeted to (x, y) trajectories were obtained by single particle tracking using MIA software. secretory lysosomes via a proline-rich domain in its cytoplasmic tail. J. Cell Sci. 114, Mean square displacement (MSD) in the x, y plane was calculated as described 2405–2416 (2001). previously25,49. The diffusion coefficient D was then calculated as D = s/4, s 45. Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y. & Kuroda, T. S. Slp4-a/granuphilin-a xy xy regulates dense-core vesicle exocytosis in PC12 cells. J. Biol. Chem. 277, 39673– being the slope of the linear fit of the first 15 points of the MSD curve. To meas- 39678 (2002). 46. Shu, X., Shaner, N. C., Yarbrough, C. A., Tsien, R. Y. & Remington, S. J. Novel chromo‑ ure Dxy variations along a given trajectory, a rolling analysis window of 2.5 s was used as described previously25. Using this method, a D value was attributed to phores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 xy (2006). each point of the trajectory. Immobilization periods correspond to portions of 47. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an –4 2 –1 arrayed viral high-content screen. Cell 124, 1283–1298 (2006). trajectories during which Dxy < 3 × 10 μm s . Survival curves for immobilization times (that is, the number of events per trajectory lasting at least the indicated 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). immobilization time) were computed for each cell and averaged over the differ- 49. Steyer, J. A. & Almers, W. Tracking single secretory granules in live chromaffin cells by ent cells. The best fit for the curves was obtained with the sum of 2 exponentials, evanescent-field fluorescence microscopy. Biophys. J. 76, 2262–2271 (1999).

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DOI: 10.1038/ncb2000

a b Number of secreting cells

6000 0 74,000 115,000 174,000 4 10 2.31% 61.1% 59% 65.9% 3 4000 10 MFI: 31 MFI:33 MFI:44 MFI:84 Factor: Factor: Factor:

Factor: /6!!5 102 71,61 2016,3 2596 5535,6 2000 1

OVA 10

100 0 200 600 1000 0 75,000 150,000 225,000 Cell number Forward Scatter

beads beads beads beads c OVA detection

4500

3000

/6!!5 1500 **

0 Live Apoptotic

Figure S1 Semi-quantitative detection of OVA in cell culture supernatants. Ig. (b) Dose-response curve of OVA staining, obtained by quantification of 66 (a) Representative flow cytometry dot-plots showing the detection of OVA present experimental conditions. (c) Comparison of the signal obtained following OVA in the supernatants collected from cultures of increasing numbers of cells, staining in supernatants collected from cultures of either live or UV-irradiated after capture onto anti-OVA-coated beads and staining with Alexa488-anti-OVA cells collected 24 h after seeding 50,000 cells/well in a 96-well plate.

Ostrowski et al. Supp. Fig 1

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Rab27a and Rab27b mRNA expression 150 Rab27a Rab27b 100

OFCONTROL 50

0 Rab27a Rab27b KD cells KD cells

#ONTROL 2ABA+$SH2.! 2ABB+$SH2.! .ON CONDITIONEDCULTUREMEDIUM

103 66.1 22.4 46.8 1.66

101 HLA/DR/CD81 400 800 Forward Scatter

67.2 21.7 41.8 0.45 103

101 Annexin V 400 800 Forward Scatter

beads beads beads beads

Figure S2 Further characterization of cells stably transduced with Rab27a- expression, as shown by quantitative RT-PCR with primers specific for each or Rab27b-specific shRNAs. (a) Specific extinction of RAB27A (respectively gene. (b) Representative dot plot showing HLA-DR/CD81 (top panel) and RAB27B) gene expression by Rab27a-specific virus 3 (respectively Rab27b- Annexin V (bottom panel) staining on large-scale supernatants from cells specific virus 1), with no effect on RAB27B (respectively RAB27A) gene transduced with Rab27a-specific virus 3 and Rab27b-specific virus 1.

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a zoom: 2.5x zoom: 2.5x CD63 CD63 Rab27b-GFP Rab27a-GFP merge merge

20 % of colocalization with CD63

0 Rab27a Rab27b

b control Rab27a KD Rab27b KD 80 70 60 *** 50 *** 40 30 20 10

area of vesicles / total cell (%) 0 Control Rab27a KD Rab27b KD c 10 80 70 /s) 2 60

8 µm 50

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6 xy D values 10 0 xy 105 15 20 25 4 time (s)

% of D 2

0 5 10 15 20 25 30 -4 2 Dxy (10 µm /s) Ostrowski et al. Supp. Fig. 3

Figure S3 Morphology of MVEs in the subplasmalemmal region. (a) shown. The ratio of the vesicles area / total cell area was calculated Colocalisation of GFP-Rab27a or GFP-Rab27b and CD63 (labeled with in 23 control cells, 23 Rab27a KD cells and 25 Rab27b KD cells. (c) anti-mouse Cy3) observed by TIRFM. Quantification of colocalisation GFP-CD63 vesicles were tracked and diffusion coefficients Dxy were was performed on 10 cells per condition. Scale bar: 5 µm. Zoom: x2.5. computed along the trajectories. Shown is the distribution of Dxy pooled (b) Rab27a and Rab27b silencing increase the distance of the vesicles from 118 trajectories monitored in 7 control cells. Values above 30 x to the border of the cells. The areas of the cell footprints (yellow), and 10-4 µm2.s-1 were omitted. An example of a vesicle exhibiting a directed of the minimal polygon encompassing all vesicles (red) were measured. motion (0-5 s) followed by a docking period (5-10 s, 15-25 s) is presented Representative images of control, Rab27a KD and Rab27b KD cell are in the inset.

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100

20 % of cells with large CD63+ compartments 0 control 27a KD 27b KD Slp4 KD 27a+Slp4 27b+Slp4 KD KD

100

20 % of cells with asymmetrical distribution of CD63+ compartments

0 control 27a KD 27b KD Slp4 KD 27a+Slp4 27b+Slp4 KD KD

Figure S4 Effect of double-silencing of Rab27a or Rab27b and Slp4 on the compartments. Single KD of Rab27a and double KD of Rab27a and Slp4 morphology of CD63-positive MVEs. B6H4 stably expressing shRNA to either induce the same phenotype (enlarged MVEs). (b) Quantification of cells Rab27a or Rab27b were transfected with a plasmid encoding EGFP and a presenting asymmetrical perinuclear accumulation of CD63+ compartments. shRNA to Slp4. Four days later, cells were fixed and stained with anti-CD63, Slp4 KD does not induce asymmetrical distribution of MVEs, whereas both and morphology of CD63 compartments was manually quantified in GFP- single KD of Rab27b and double Rab27b+Slp4 KD induce this phenotype. expressing cells. (a) Quantification of cells presenting enlarged CD63+ For each condition, 20 cells were evaluated in a blind manner.

/STROWSKIETAL3UPP&IG3

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Control mice Rab27a/b KO mice Cell lysate Exosomes Cell lysate Exosomes 30 Mg 10 Mg 5.8M 1.4M 30 Mg 10 Mg 5.8M 1.4M

CD9

tsg101

MFG-E8

MHC II

gp96

120 100 80 * 60 * 40 ** % of wild type 20 0 CD9 tsg101 MFG-E8 MHC II

Figure S5 Exosome secretion by bone marrow-derived dendritic cells Purified exosomes secreted by equal amounts of control or KO cells were (BMDCs) from Rab27a/Rab27b double knock-out mice. Dendritic cells analysed by immunoblotting for presence of CD9, tsg101, MFG-E8, MHC were differentiated from bone marrow precursors in the presence of class II, and gp96 proteins. A total of 30 or 10 µg cells were lysed and used GM-CSF. At day 10 after the onset of culture, cells were cultured in for comparison. One representative experiment of four is shown. Histogram exosome-depleted medium for 24h, before collection of supernatant and shows quantification of signal intensity of CD9, tsg101, MFG-E8, and purification of exosomes by ultracentrifugation. WT and Rab27a/b-KO MHC II proteins in exosomes secreted by 5.8 million cells from the KO cells contained more than 75% of BMDCs, and less than 10% of mature mice, normalized to the value obtained with exosomes from control cells BMDCs, as assessed by FACS staining for CD11c, Gr1, MHC Class II. (mean±SD of 4 experiments).

Ostrowski et al. Supp. Fig. S5

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Figure S6 Uncropped Western blots corresponding to Figures 3b, 3c, in the displayed order. Position of molecular weight markers (kDa) is 7c, 8b, and 7d. Western blots corresponding to Figures 3b (a), 3c (b), indicated. Bands corresponding to the specific protein are highlighted by 7c (c), 7d (e) and 8b (d) were hybridized consecutively with antibodies an arrow.

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Figure S6 continued

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Figure S6 continued

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Figure S6 continued

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Supplementaty movies

Movie S1 for a CTRL cell. Movie S2 for a Rab27a KD cell. Movie S3 for a Rab27b KD cell.

Images were acquired by TIRFM at 10 Hz for 40 s. Movies are accelerated 4 times.

Supplementary Table 1 : qPCR analysis of Rab27 effector proteins in HeLa B6H4 cells

Official Official name Also known as Delta Ct HeLa Delta Ct Expression symbol B6H4 cells1 Fon cells in HeLa cells SYTL3 synaptotagmin-like 3 Slp3 12 >20 Yes RPH3A rabphilin 3A homolog 0 >9 No (mouse) SYTL2 synaptotagmin-like 2 Slp2 >20 13.79 Yes UNC13D unc-13 homolog D Munc13-4 >20 ND Yes MLPH Melanophilin Slac2a 0.5 7,76 No SYTL1 synaptotagmin-like 1 Slp1 0.28 >16.3 No RPH3AL rabphilin 3A-like Noc2 >13 >17 Yes (without C2 domains) EXPH5 exophilin 5 Slac2b 10.8 5.63 Yes SYTL5 synaptotagmin-like 5 Slp5 14.5 2 Yes SYTL4 synaptotagmin-like 4 Granuphilin, 15.85 19.6 Yes Slp4 MYRIP myosin VIIA and Rab Slac2c 0 0 ND interacting protein

1 Delta Ct was calculated as the difference between the Ct obtained by quantitative RT-PCR using primers specific for the indicated gene, minus the Ct obtained in the same conditions in samples in which no RT was used for the reverse transcription reaction.