ARTICLES

Syndecan–syntenin–ALIX regulates the biogenesis

of exosomes
Maria Francesca Baietti1,2,3,5 , Zhe Zhang1,3,5,6 , Eva Mortier2,5 , Aurélie Melchior1,3 , Gisèle Degeest1,3 ,
Annelies Geeraerts2,4 , Ylva Ivarsson2 , Fabienne Depoortere2 , Christien Coomans1,3 , Elke Vermeiren2 ,
Pascale Zimmermann2,7 and Guido David1,3,7

The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we
report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of
exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the
intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans,
ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for
syndecan–syntenin–ALIX in membrane transport and signalling processes.

Exosomes are small secreted vesicles thought to play key roles
in cell-to-cell communication1–6 . Exosome formation starts in
endosomes, by budding of the limiting endosomal membrane, away
from the cytosol, generating vesicle-laden endosomes, referred to as
multivesicular endosomes (MVEs) and multivesicular bodies (MVBs).
Fusion of these late endosomes with the plasma membrane releases the
intraluminal vesicles (ILVs) as exosomes1–7 .
The endosomal-sorting complex required for transport (ESCRT) ma-
chinery regulates membrane budding, at cell surfaces8,9 and at the level
of late endosomes10,11 . ESCRT-0, -I and -II contain ubiquitin-binding
subunits that sort ubiquitylated membrane proteins to specific domains
of endosomes and into the endosomal invaginations. In vitro recon-
stitution experiments suggest that ESCRT-I and ESCRT-II together
support budding processes and tentatively identify ESCRT-III as the
complex that cleaves the buds to form ILVs (refs 12,13). Yet, further
mechanisms must exist14–16 . ALIX (also known as PDCD6IP), a protein
that interacts with several ESCRT (TSG101 and CHMP4) proteins is
thought to participate in the budding and abscission processes17,18 .
Importantly, ALIX also interacts directly with Leu-Tyr-Pro-X(n)-Leu
(LYPX(n)L) late domains in retroviral membrane proteins, allowing
enveloped viruses to hijack cellular mechanisms of membrane-domain
biogenesis to egress from cells19,20 . Thus, both topologically and mecha-
nistically, retroviral budding seems related to ILV/exosome formation.
Signalling-induced ubiquitylation of membrane proteins triggers
their sorting to ILVs, thus terminating intracellular signalling by these
proteins. Many of the signalling events that occur at cell surfaces require
the assistance of heparan sulphate, for instance the engagement of
fibroblast growth factor (FGF) with its receptor21,22 (FGFR). Most
of the cell-surface-bound heparan sulphate is supplied by syndecans,
ubiquitous transmembrane proteins that are of key importance
for development, angiogenesis, carcinogenesis, inflammation and
infection23,24 . Syndecans have a short, evolutionarily conserved
cytoplasmic domain, which binds cytosolic factors25–27 such as the
small PDZ scaffolding protein syntenin28,29 (also known as SDCBP).
Here, we show that the connection of the syndecans (also known as
SDC1-4) with syntenin, and through syntenin with ALIX supports the
biogenesis of ILVs and exosomes, and the segregation of signalling
cargo to these vesicles.
RESULTS
Syntenin interacts with ALIX, mimicking retroviral late domains
Syntenin has a simple domain structure: two PDZ domains,
flanked by a 100-amino-acid amino-terminal domain and a small
carboxy-terminal domain (Fig. 1a). The two PDZ domains are
required for high-affinity interaction with the syndecans and for the
recruitment of syntenin to membranes30–33 . Screening yeast two-hybrid

1
Laboratory for Glycobiology and Developmental Genetics, Department of Human Genetics, KULeuven, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium.
2
Laboratory for Signal Integration in Cell Fate Decision, Department of Human Genetics, KULeuven, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium.
3
Center for the Biology of Disease, VIB, 3000 Leuven, Belgium. 4 Laboratory for Biomolecular Dynamics, Department of Chemistry, KULeuven, 3001 Heverlee,
Belgium. 5 These authors contributed equally to this work. 6 Present address: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
7
Correspondence should be addressed to P.Z. or G.D. (e-mail: pascale.zimmermann@med.kuleuven.be or guido.david@cme.vib-kuleuven.be)

Received 15 June 2011; accepted 16 April 2012; published online 3 June 2012; DOI: 10.1038/ncb2502

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 677

© 2012 Macmillan Publishers Limited. All rights reserved.
ARTICLES

a b IP c Syntenin GST–ALIX e f lys exo

5,000 5 µM

Anti-syntenin
100

Response (RU)
2.5 µM SDC1CTF
3,800 1.25 µM

Anti-ALIX

Particles (× 106 ml–1)

Syntenin Syntenin
2,600 GST 5 µM 80
N-ter PDZ-1 PDZ-2 C-ter

Input
ALIX

IgG
1 109 194 273 298
1,400 60
CD63
ALIX 0
40
--LYPSLEDL--------------LYPRLYPELSQYMGL--
Syntenin 400 800 1,200 HSP70
20
Time (s) Calnexin
d Syntenin

Normalized response
ALIX 1.0 0
GM130
CD63 50 150 250

Response (RU)
Bro1 V PRD 400
1 360 702 868 SDC2 Size (nm) LAMP-2

200 0.5
CHMP4 CD63
HIV p6 TSG101
Src SH2 EIAV p9 Src SH3 SDC2
Syntenin ALG-2 0
Endophilin 0
0 100 200 300 0.01 10 1,000
SETA/CIN85
Time (s) Syntenin (µM)
g SDC1CTF
i Percentage
Syntenin 250 j IP Control CD63 0 20 40 60 80 100 IP

Beads

Beads
ALIX CD63

Wash

Wash
200

Control
1 2 3 4 5 6 7 8 9 10

SN

SN
SDC1CTF

h SDC1CTF Diameter (nm) 150 SDC1CTF

Syntenin
Flotillin-1
ALIX Syntenin
100 CD63

CD63
CD63 SDC1CTF
CD63 50 Syntenin

Flotillin-1 Flotillin-1
Flotillin-1 0
1 2 3 4 5 6 7 8 9 10 Beads SN Wash

Figure 1 Syntenin interacts with ALIX, adapting ALIX to syndecan, and blots, illustrating the presence of syndecan 1 CTF (SDC1CTF), syntenin,
co-fractionates with these in exosomes. (a) Schematic representation of ALIX, CD63 and HSP70 in MCF-7 cell lysate (lys) and HSC exosomes
syntenin (top) and ALIX (bottom). Numbers indicate amino-acid residues. (exo). Note that there is no detectable contamination of the exosomes by
LYPX(n)L motifs that mediate interaction with ALIX are highlighted. ALIX GM130 (Golgi), calnexin (endoplasmic reticulum) or LAMP-2 (lysosomes).
binding partners are indicated below the respective binding domains. (g,h) Exosome fractionation by density-gradient centrifugation. SDC1CTF,
(b) ALIX co-precipitates with syntenin. Detergent extracts of microsomal CD63, syntenin and ALIX occur in fractions corresponding to densities
membranes were incubated with control IgG or specific antibody. IP, of 1.094 to 1.143 g ml−1 . Note that flotillin-1 floats at higher buoyancy.
immunoprecipitate. (c) Biacore experiments showing, first, the interaction (i) Ultrastructural appearance (left) and size distribution (right) of particles
of non-tagged recombinant syntenin with immobilized syndecan 2 or co-purifying with syntenin in the density gradient. Scale bar, 50 nm. The
SDC2 (left part of the sensorgram) and, subsequently, that of GST–ALIX diameter is 80 ± 46 nm (mean, s.d.; n = 137). (j) Exosome subfractionation
with SDC2:syntenin complexes (right part of the sensorgram; perfusion by CD63 content. Adsorption to magnetic beads substituted with anti-CD63
of syntenin stops at 600 s; GST or GST–ALIX perfusion starts at 800 s). or control antibody was measured by western blotting (example on the left).
(d) Sensorgrams (left panel) comparing the binding of 2.5 µM syntenin The histogram (right) represents the mean distributions of the markers over
to SDC2 (black line) and CD63 (grey line), and normalized equilibrium the beads, supernatants (SN) and washes (the sum of the three fractions
binding isotherms (right panel) yielding apparent Kd values of 1.8 ± 0.2 µM taken as 100%), calculated from three independent immunofractionation
and 24 ± 4 µM, for SDC2 and CD63, respectively. Data were collected experiments. Note that syndecan and syntenin largely co-fractionate with
from two replicates. (e) Size distribution of the particles that compose CD63 (P < 0.001, t -test). Flotillin-1 does not. Uncropped images of
HSC exosomes, as measured by nanoparticle tracking analysis. (f) Western blots/gels are shown in Supplementary Fig. S6.

experiments, we found that the N-terminal domain of syntenin controlled by syndecan:syntenin:ALIX complexes might ‘resemble’
interacts with ALIX (Supplementary Fig. S1a). The interaction is viral budding and pertain to the formation of ILVs and exosomes.
direct, as shown by the co-immunoprecipitation of the endogenous
proteins (Fig. 1b) and by in vitro binding assays (Supplementary Fig. Syndecan, syntenin and ALIX co-accumulate in exosomes
S1b,c). Importantly, syndecans, syntenin and ALIX form tripartite Proteomic analyses have identified ALIX and syntenin in exosomes of,
complexes in vitro (Fig. 1c). for example, dendritic cells37 . Together with CD63, ALIX and syntenin
The N-terminal domain of syntenin contains three LYPX(n)L are among the 20 proteins most frequently identified in exosomes38 .
motifs (Fig. 1a), resembling ALIX-binding sites identified in HIV-1 CD63 is a tetraspanin that, similarly to syndecans, interacts with the
p6 (LYPLTSL) and in EIAV p9 (LYPDL), late domains involved PDZ domains of syntenin39 . Yet, we found the affinity of syntenin for
in viral budding19,20 . Mutating the YP residues to alanines in the CD63 (Kd = 24 µM) to be ∼10-fold lower than its affinity for syndecans
three motifs was necessary and sufficient to abolish syntenin–ALIX (Fig. 1d). We first investigated whether syndecan, syntenin and ALIX
interaction (Supplementary Fig. S1a). HIV-1 p6 and EIAV p9 interact co-accumulate in exosomes. We used MCF-7 cells, as these cells contain
with the V-domain of ALIX, with the binding centred on Phe readily detectable amounts of SDC1, SDC4 and syntenin, and release
676 (refs 34–36). Similarly, syntenin interacts with the V-domain numerous nanoparticles that are exosome-like in size (diameter =
of ALIX (Supplementary Fig. S1a), and substituting aspartate for 88 ± 13 nm; mean, s.d.; Fig. 1e). As purified by differential high-speed
Phe 676 in ALIX completely abolishes syntenin–ALIX binding centrifugation (HSC), these particles contained membrane-spanning
(Supplementary Fig. S1d). This suggests that cellular processes C-terminal syndecan fragments (CTFs), syntenin, ALIX and several

678 NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012

© 2012 Macmillan Publishers Limited. All rights reserved.

Syntenin ΔALIX
a b
– Syntenin
Syntenin
ALIX
RNAi
nt
–
SDC1CTF SDC1CTF
Syntenin
Syntenin
ALIX
ALIX
exo
CD63 CD63
exo
HSP70 HSP70
TSG101
TSG101
Flotillin-1 ALIX
Syntenin
lys
SDC1CTF
SDC1CTF
lys
Syntenin
β-actin
Figure 2 Syntenin gain enhances exosome yields. MCF-7 cells were
sham-transfected (-), or transfected to transiently overexpress wild-type
syntenin (syntenin) or (triple LYP-LAA) mutant syntenin defective
for binding to ALIX (syntenin 1 ALIX), and were treated with ALIX
RNAi, non-targeting RNAi (nt) or no RNAi. Total cell lysates (lys)
and corresponding HSC exosomes (exo) were analysed by western
blotting, testing for different exosomal markers, using cognate antibodies.
(a,b) Representative experiments, illustrating the effect of syntenin
overexpression (a,b) and of ALIX RNAi (b). (c) Histograms representing
mean signal intensities (relative to signals in corresponding controls; that
is, cells treated with empty vector, nt RNAi or both empty vector and
nt RNAi), calculated from n independent experiments (as indicated).
other established exosomal markers, including CD63 (Fig. 1f). Some
intact syndecan could be detected when syndecans were overexpressed,
but CTFs predominated (Supplementary Fig. S2a). Exosome formation
depends on ceramide40 . RNA-mediated interference (RNAi) targeting
neutral sphingomyelinase-2 decreased the amounts of syndecan, CD63,
syntenin and ALIX sedimented by HSC, tentatively identifying the
particles that contain these markers as exosomes (Supplementary
Fig. S2b). During density-gradient centrifugation, exosomal syndecan
co-fractionated with syntenin, ALIX and CD63 (Fig. 1g,h), and with
vesicles, mostly with a shape and size characteristic of exosomes
(Fig. 1i). Yet all were slightly offset from flotillin-1 (Fig. 1h).
Immunofractionation indicated that most of the exosomal syndecan,
syntenin and ALIX, yet very little of the exosomal flotillin-1,
accumulates in CD63-positive vesicles (Fig. 1j; ALIX not shown).
In turn, two-colour fluorescence cross-correlation spectroscopy
indicated that most CD63-positive exosomes contain syntenin
(Supplementary Fig. S2c,d). Together this suggests that syndecan,
CD63, syntenin and ALIX largely co-segregate to a distinctive (flotillin-
1-negative) class of vesicles.
Syndecan, syntenin and ALIX support the biogenesis
of exosomes
We then investigated the significance of syndecan, syntenin and
ALIX for exosome formation. When syntenin was overexpressed, the
NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012
© 2012 Macmillan Publishers Limited. All rights reserved.
ARTICLES
c SDC1CTF Syntenin CD63
n= 3 3 3 5 7 4 22 2
500
ALIX
(relative to control)
nt
400
Percentage
300
200
100
Syntenin ΔALIX WT WT
ALIX RNAi – – +
d mGFP–CD63 exosomes
– Syntenin
150
Intensity (kHz)
100
50
1 300 600 900
Peak rank number
WT, wild type. Error bars represent s.d. (d) Exosome quantification by
confocal fluorescence spectroscopy. MCF-7 cells were transfected with
mGFP–CD63, and with or without non-tagged syntenin. HSC exosomes
were examined by fluorescence spectroscopy for 10 s, 50 times. The
graph shows the distributions of the fluorescent peaks (corresponding
to individual exosomes), ranking all collected peaks by increasing
fluorescence intensity (kHz). Note that syntenin overexpression (black
distribution) increases (more than doubles) the number of peaks, but not
their intensities. Data shown are from one representative experiment;
similar results (fold increases) were obtained in three independent
experiments (see Supplementary Fig. S3c). Uncropped images of blots
and gels are shown in Supplementary Fig. S6.
level of exosomal syndecan increased, along with exosomal syntenin
and ALIX, and other exosomal markers such as CD63 and HSP70
(Fig. 2a,c). Similar increases did not occur when syntenin harboured
point mutations in the three ALIX-binding LYPX(n)L motifs (Fig. 2a,c),
or when syntenin was overexpressed in the presence of ALIX RNAi,
confirming a specific involvement of ALIX (Fig. 2b,c). ALIX RNAi
(Figs 2b and 3d) or syntenin RNAi (Fig. 3a,d) led to a significant
decrease in exosomal syndecan, CD63 and HSP70. Conversely,
syndecan depletion reduced the level of exosomal accumulation
of syntenin, ALIX, CD63 and HSP70 (Fig. 3b,d). Knockdown of
CD63, which depends on syndecan and syntenin for accumulating in
exosomes, had little effect on exosomal syndecan, syntenin, ALIX and
HSP70 (Fig. 3c,d). Of note, syndecan or syntenin RNAi had no effect
on the exosomal accumulation of flotillin-1 (Fig. 3a,b), but markedly
reduced the recovery of total biotinylated exosomal protein from
cell-surface-biotinylated cells (Supplementary Fig. S3a). Importantly,
the effects of syndecan RNAi were largely prevented by the expression
of exogenous syndecan (Fig. 3e,f). On the contrary, in combination
with syndecan RNAi, the overexpression of CD63 further decreased the
exosomal yields of syntenin and ALIX (Fig. 3e,f). These results reveal a
specific role for syndecan–syntenin–ALIX in the biogenesis of a major
class of exosomes, the loading of exosomes with specific cargo, or both.
To evaluate these possibilities, we analysed HSC exosomes by
nanoparticle tracking analysis, identifying a specific ∼2-fold increase
679
ARTICLES

a b c e RNAi nt SDC1/4

Syntenin

SDC1/4
SDC1
SDC4

SDC1

SDC1
CD63

CD63
CD63
RNAi RNAi RNAi

nt

nt

nt

–
SDC1CTF SDC1CTF SDC1CTF SDC1CTF
Syntenin Syntenin Syntenin Syntenin
ALIX ALIX Alix

exo
CD63

exo
CD63
exo

exo
CD63 CD63

HSP70 Alix
HSP70 HSP70
TSG101 TSG101
TSG101
Flotillin-1 SDC1
Flotillin-1 Flotillin-1
Syntenin
Syntenin
ALIX

lys
nt

SDC1CTF
SDC1CTF CD63 CD63

lys
lys

SDC4
lys

Syntenin
SDC1 ALIX
ALIX ALIX

d f n= 3 4 3 3 3 1 5 5 2 5 4 2 4 5 1
n=
Percentage (relative to control)
6 6 6 6 4 3 3 4 3 3 4 5
Percentage (relative to control)

250 SDC1CTF
100 Syntenin
SDC1CTF 200 CD63
80
Syntenin
60 150
CD63
40 100

20 50

RNAi ALIX Syntenin SDC1/4 CD63

OE SDC1 CD63 – SDC1 CD63
RNAi nt SDC1/4

Figure 3 Syndecan–syntenin–ALIX depletion impairs exosome yields. experiments (as indicated). Cell treatments are indicated at the bottom.
(a–d) Depletion experiments. MCF-7 cells were treated with various RNAi, Note that the depletion of CD63 has less effect on exosomal markers than
targeting syntenin (a,d), syndecan (b,d), CD63 (c,d) or ALIX (d, and the reductions of syndecan, syntenin or ALIX, and that extra CD63 cannot
Fig. 2b). Non-targeting RNAi (nt) was used as a control. (e,f) Rescue compensate for a loss of syndecan. Note also the significant effects of single
experiments. Cells were sham-transfected (-) or transfected to overexpress syndecan depletions, indicating that the different syndecans have either
(OE) either syndecan (mouse SDC1) or CD63 (mGFP–CD63), and were non-redundant roles in exosome formation, or are individually limiting (that
treated with nt-RNAi or RNAi targeting endogenous syndecans. Total cell is, that the remaining syndecan does not reach the required threshold).
lysates (lys) and HSC exosomes (exo) were analysed by western blotting, Note that while SDC1 levels are maintained in cells, syndecan 4 RNAi also
testing for specific proteins using cognate antibodies. Histograms (d,f) decreases the level of SDC1CTF in exosomes, indicating that the latter or a
represent mean signal intensities (and s.d.) for SDC1CTF (black), syntenin combination of both is the case. Uncropped images of blots/gels are shown
(grey) and CD63 (shaded) in exosomes, calculated from n independent in Supplementary Fig. S6.

in the number of particles with a diameter of 30–110 nm on co-localization of mCherry–syntenin and syndecan intracellular
overexpression of syntenin, and a ∼50% reduction on knockdown domain (ICD), including their accumulation, along with ALIX, ‘inside’
of syndecan, syntenin or ALIX (Supplementary Fig. S3b). By the lumen of enlarged endosomes that were decorated with cerulean
confocal fluorescence spectroscopy, assessing the effects of syntenin (cer)-tagged RAB5Q79L (Fig. 4b and Supplementary Fig. S4). Lumina
overexpression on the detection of mGFP–CD63 in HSC exosomes, we filled with mCherry–syntenin were also filled with CD63, syndecan-
found an increase (2–3-fold) in the number, but no change in the mean ectodomain outlining only the rim of these endosomes (Fig. 4b). When
amplitude, of the fluorescent peaks (Fig. 2d and Supplementary Fig. such cells were incubated with anti-CD63, many of the enlarged
S3c). We concluded that syntenin controls the number of exosomes endosomes were filled with antibody (Fig. 4c). In contrast, without
made/released, rather than the level of (CD63) cargo the modal concomitant mCherry–syntenin expression, internalized anti-CD63
exosome is loaded with. was mostly restricted to the limiting membranes of the endosomes
Next, we examined whether syntenin–ALIX supports budding (Fig. 4c). Together, these results suggest a role for syntenin in the intra-
processes at endosomal membranes. Overexpression of RAB5Q79L , luminal budding of endosomal membrane domains that contain CD63
which impairs early intra-endosomal trafficking and causes endosomes and syndecan, the latter mostly as syndecan-CTF. The knockdown of
to enlarge41 , severely reduced the exosomal release of syndecan, CD63, RAB7, which replaces RAB5 on endosomal membranes42 , regulating
syntenin and ALIX (Fig. 4a). Confocal microscopy revealed extensive late endosomal traffic downstream of MVB (ref. 43), also severely

680 NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012

© 2012 Macmillan Publishers Limited. All rights reserved.
 ARTICLES

a RAB5Q79L – + b Merge CD63 cer–RAB5Q79L mCh–syntenin e RNAi

SDC1CTF Syntenin – + + –
Syntenin RAB7 – – + +
exo

Alix Syntenin
CD63 RAB7
Flotillin-1
Merge SDC1 ICD cer–RAB5Q79L mCh–syntenin
SDC1CTF
f
RAB5Q79L
lys

Flotillin-1

n= 4 3 3
(relative to control)

100
SDC1CTF
Percentage

Syntenin Merge SDC1 ED cer–RAB5Q79L mCh–syntenin

60
CD63

20

RAB5Q79L
RAB7

Syntenin
d c Merge CD63 cer–RAB5Q79L 80 *
RNAi
nt

Percentage of
luminal CD63
SDC1CTF 70 g

Stained MVB area (× 103 nm2)

RNAi nt
Control

100
60 RNAi syntenin
Syntenin 50
40 80
CD63

60
n= 5 5 3 Merge CD63 cer–RAB5Q79L mCh–syntenin
(relative to control)

Syntenin OE

100
SDC1CTF
Percentage

40
Syntenin
60 CD63
20
20 20 40 60 80 100
Total MVB area (× 103 nm2)
RNAi RAB7

Figure 4 Syntenin exosomes are of endosomal origin. (a) Western blots
(top) illustrating the effect of RAB5Q79L on the accumulation of the
indicated proteins in HSC exosomes, relative to levels in control cells
(-). The histogram (below) shows means (and s.d.) from n independent
experiments (as indicated). (b) Confocal micrographs of cells expressing
cer–RAB5Q79L and mCherry–syntenin. Cells are stained with anti-CD63
(upper row), with monoclonal antibody 2E9, detecting the syndecan-1
ICD (middle row), or polyclonal antibodies detecting the syndecan-1
ectodomain (ED; lower row). Molecules as indicated top right were
identified based on their different emission wavelength. In the merge
image, the first molecule is in green, the second in blue and the third
in red. Outlined areas are shown at larger magnifications. Note the
restriction of syndecan-ED to the limiting membranes, and the extensive
co-localization of CD63 and syndecan-ICD with mCherry–syntenin inside
the lumina of the enlarged endosomes outlined by cer–RAB5Q79L . Scale
bars, 10 µm. These lumina are also filled with ALIX (see Supplementary
Fig. S4). (c) Confocal micrographs of cells expressing cer–RAB5Q79L ,
alone (upper row) or with mCherry–syntenin (lower row), and that have
been incubated with anti-CD63 antibody. Cells are stained with anti-IgG.
OE, overexpression. Molecules as indicated in the top right were identified
based on their different emission wavelength. In the merge image, the first
molecule is in green, the second in blue and the third in red. Outlined areas
are shown at larger magnifications. The histogram represents the filling of
cer–RAB5Q79L -decorated vacuoles with antibody. For each endosome, the
luminal fluorescence is expressed as percentage of the total fluorescence
present in the endosome. In the presence of mCherry–syntenin (open
triangles), endosomes contain more luminal fluorescence/anti-CD63 than
in the control (filled triangles). Scale bars, 10 µm. ∗ P < 0.05. (d,e) Western
blots (d, top and e) of HSC exosomes (d) and total cell lysates (e) derived
from cells treated with control (nt), syntenin and/or RAB7 RNAi, and
histogram (d, bottom), illustrating the effectiveness of the RNAi and
the effect of RAB7 RNAi on the accumulation of syntenin, syndecan-1
CTF and CD63 in HSC exosomes (means and s.d.). (f) Representative
electron micrographs, illustrating the morphology of the late endosomal
compartment in MCF-7 cells treated with control, RAB7 and/or syntenin
RNAi, as indicated. Structures were revealed by peroxidase-conjugated
anti-CD63 (internalized for 30 min), and staining with DAB. Scale bar,
200 nm. Note the more peripheral localization of the stain, decrease
in stained ILVs and smaller size of the endosomes in cells treated with
syntenin RNAi. (g) Endosome filling. For each peroxidase/DAB-marked
endosome, the total sectional area occupied by the endosome is plotted
versus the area occupied by the stain in that endosome. Further data
are shown in Supplementary Fig. S5. Uncropped images of blots/gels are
shown in Supplementary Fig. S6.

reduced the level of exosomal release (Fig. 4d). Importantly, RAB7
RNAi also prevented the enhanced exosomal recoveries induced by
syntenin overexpression (Supplementary Fig. S5a). These data indicate
that exosomes supported by syndecan–syntenin–ALIX originate from
endosomal membranes and from the fusion of MVBs with the cell
surface, bypassing lysosomes.
This was further explored by electron microscopy, monitoring the
localization of internalized anti-CD63:HRP conjugates (Fig. 4e–g and
Supplementary Fig. S5b,c,d). In control cells, diaminobenzidine (DAB)
stain was found both on limiting endosomal membranes and in close
association with ILVs, filling up the lumen of endosomes. In contrast,
in cells treated with syntenin RNAi the staining was associated with
limiting membranes and on ILVs that were peripherally located, the
lumen of the endosomes remaining largely empty. Similar results were
obtained in cells treated with both RAB7 RNAi and syntenin RNAi.
Whereas RAB7 RNAi resulted in large endosomes, filled with ILVs,
in cells with a double knockdown of RAB7 and syntenin, staining
remained largely peripheral. Loss of syntenin also significantly reduced
the average sizes of the stained endosomes (Supplementary Fig. S5c).
Empty, yet smaller late endosomes imply that the knockdown of

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 681

© 2012 Macmillan Publishers Limited. All rights reserved.
ARTICLES

TSG101
a b n=3

CHMP4A/B/C

Percentage of CHMP4B
Syntenin
SDC1CTF Syntenin CD63
n = 4 87

(relative to control)
RNAi 234 325 668 334 325 100

CHMP4C
CHMP4B
CHMP4A
nt

TSG101

(relative to control)
RNAi

VPS22

nt
TSG101 100 80

Percentage
β-actin RNAi 80 Syntenin 60

nt

nt
CHMP4B
SDC1CTF 60 CHMP4B 40
40 20
Syntenin
20 CD63
nt

nin
CD63

nt
CHMP4B RNAi
TSG101 VPS22 A B C A/B/C

nte
Flotillin-1 RNAi CHMP4

Sy
lys exo

Percentage (relative to control)

d RNAi RNAi n = 333 333 333 879 333 33 3 344

VPS4AK173Q
c RNAi ALIX
SDC1CTF

CHMP2A

VPS4A/B
I212D

n=3 140

CHMP6

CHMP3
Syntenin

VPS4B
VPS4A

VPS4A
WT

SDC1CTF
Percentage (relative

eYFP–mALIX CD63
–

200 Syntenin 100

SDC1CTF

nt

nt
CD63
to control)

Syntenin 150 SDC1CTF

exo 100 60
Syntenin
CD63 50
20
WT I212D CD63

Q
CHMP6 CHMP2A CHMP3 VPS4A VPS4B VPS4A/B
lys ALIX eYFP-tagged

4A K173
eYFP–mALIX
Endogenous
RNAi RNAi

VPS
Figure 5 The biogenesis of syntenin exosomes requires ESCRTs . (a) Western formation; the mutant form fails. (d) ESCRT-III protein requirements.
blots (left) and histogram (right), illustrating the effectiveness of TSG101 Western blots of HSC exosomes (left) and histogram (right), illustrating the
and CHMP4B RNAi, and the effects of TSG101 (ESCRT-I), VPS22 effect of dominant-negative VPS4AK173Q , versus that of wild-type VPS4A,
(ESCRT-II) and CHMP4 (ESCRT-III) RNAi (relative to that of control, nt and the effects of RNAi targeting CHMP6, CHMP3, CHMP2A or VPS4A/B,
RNAi) on the levels of SDC1CTF, syntenin and CD63 in HSC exosomes. versus that of nt RNAi, on syntenin exosome formation. Note VPS4 and
(b) Effect of syntenin RNAi on CHMP4B levels in HSC exosomes. CHMP2A requirements, contrasting with a lack of dominant-negative
(c) ALIX–CHMP4 interaction requirement. MCF-7 cells were transfected, to VPS4A effects. Similarly, VPS4AE228Q (defective in ATP hydrolysis)46 did
express low levels of eYFP (-) or eYFP fused to wild-type (WT) mALIX or to also not affect the recovery of exosomal syndecan, CD63 and syntenin (data
mALIXI212D , deficient in CHMP4 binding, and were treated with ALIX RNAi not shown). All histograms represent means (and s.d.) from n independent
to suppress endogenous ALIX (left, western blots; right, quantifications). experiments (as indicated). Uncropped images of blots/gels are shown in
Note that for similar levels of expression, wild-type ALIX supports exosome Supplementary Fig. S6.

syntenin might lead to a compound failure of (a specific subset of) ILVs the formation of syndecan–syntenin exosomes, but RNAi targeting
to form or persist, and of endosomes to mature. CHMP3 and CHMP6 had no effect (Fig. 5d). These results are
reminiscent of the ESCRT-III protein requirements for HIV-1 release47 ,
Mechanisms of exosome biogenesis and might indicate that syntenin/LYPX(n)L viral late domains recruit
The activities of ALIX in membrane trafficking probably depend on its CHMP4 through ALIX, circumventing the need for CHMP6 (whereas
interactions with multiple proteins, including CHMP4 and TSG101. in canonical pathways, VPS22 in ESCRT-II recruits CHMP6, and
The knockdown of CHMP4A/B/C or TSG101 significantly reduced CHMP6 recruits CHMP4). Besides recycling ESCRT-III components,
the exosomal release of syndecan-CTF, CD63 and syntenin (Fig. 5a). VPS4 may also play a direct role in the process of membrane
TSG101 downregulation has strong effects on general endosomal abscission11,48 . Consistent with this, and in contrast to dominant-
trafficking44,45 , arguing against a specific role for TSG101 in the negative VPS4, RNAi targeting VPS4A/B markedly reduced the level
biogenesis of syndecan–syntenin exosomes. Of note, gain (Fig. 2a) or of exosomal release, supporting the involvement of ESCRTs (Fig. 5d).
loss (Fig. 3a) of syntenin left the TSG101 signals in exosomes unaffected. Interestingly, whereas HIV-1 release does not depend on ESCRT-II (for
In contrast, loss of syntenin significantly decreased the recovery recruiting ESCRT-III; ref. 20), RNAi targeting VPS22 (ESCRT-II) also
of CHMP4B from exosomes (Fig. 5b). Low levels of eYFP-tagged significantly reduced the production of syndecan–syntenin exosomes
murine ALIX (mALIX) (approximating the levels of endogenous (Fig. 5a). Thus, the processes of membrane bending and abscission
ALIX in control cells) rescued the exosomal yields of syndecan, that direct syndecan and syntenin to ILVs and exosomes are likely
CD63 and syntenin in cells treated with ALIX RNAi; in contrast, to involve ESCRTs and/or to occur in endosomal compartments that
eYFP–mALIXI212D , deficient in CHMP4 binding, failed to rescue require ESCRTs for their architectural and functional integrity.
exosome production (Fig. 5c). Thus, a role for ESCRTs, in particular Recent reports have revealed roles for higher-order oligomerization
ESCRT-III in the biogenesis of syntenin–ALIX exosomes is likely. A of vesicular cargo49 . We therefore surmised that heparan sulphate,
requirement for the ATPase VPS4 is considered as a defining feature and heparan sulphate cargo impacting on syndecan clustering and
of the ESCRT pathway11–13 . Surprisingly, but as observed by others oligomerization25–27 , might be required for exosome formation.
for CD63-containing exosomes40 , expression of dominant-negative Consistently, disrupting the heparan sulphate structure, by the
VPS4A (VPS4AK173Q , defective in ATP binding46 ), did not affect the knockdown of EXT1/2 (heparan sulphate polymerase), the knockdown
recovery of exosomal syndecan, CD63 and syntenin (Fig. 5d). As this of NDST1/2 (an enzyme required for the creation of sulphated,
might indicate that the ESCRT-III components whose availability ligand-binding domains in heparan sulphate) or growing the cells
critically depends on VPS4-mediated recycling are not required for in culture media supplemented with heparitinase, markedly reduced
the biogenesis of syntenin exosomes (and, conversely, the required the level of exosomal release (Fig. 6a). Anti-syndecan ectodomain
ESCRT components not limited by recycling), we also tested other monoclonal antibodies reduced exosomal syntenin release, possibly
ESCRT-III components. RNAi targeting CHMP2A markedly reduced because the ensuing clusters include syndecan that is bound to the

682 NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012

© 2012 Macmillan Publishers Limited. All rights reserved.
 ARTICLES
a SDC1CTF Syntenin CD63 b

Percentage (relative to control)

n= 6 3 3 6 6 5 4 4 3 3 2 2 n=3
RNAi EXT1/2 – +
RNAi nt NDST1/2
100 120

Percentage of syntenin
80

Percentage of
nd

total eluted

(relative to control)
SDC1 60 80 100
d
40 60 80
SDC1CTF
20 40 60
β-actin
20 40
0.2 0.4 0.6
20
NaCl (M) RNAi EXT1/2 NDST1/2 – NDST1/2
Hpt – – + + Anti-SDC mAb + – +
c d RNAi nt EXT1/2
CTF
WT
–

SDC3 PSI – +
SDC1CTF SDC1CTF
RNAi SDC1/4 + + + Syntenin RNAi SDC1/4 + + Syntenin
n=3 e Cath. I – + – +
SDC1/3CTF CD63 CD63
SDC1CTF

Percentage (relative to control)

RNAi SDC1/4 – – + +
n= 8 8 9
(relative to control)

Syntenin 400 Syntenin

exo

Syntenin

exo

exo
Percentage

300 200 Flotillin-1

CD63 CD63
150 SDC1CTF
200

lys
100 ALIX
SDC1/3CTF 100 SDC1
50 n=3
lys

Percentage of syntenin
Syntenin 150
lys
SDC1CTF

(relative to control)
ALIX SDC3 WT CTF
Syntenin PSI
100
f g 1h 2h 4h 1h 2h 4h
FGF2 – + – + n= 2 3 3 2 3 3 2 3 3 + + – – –
50
FGF2 + + + + + + + +
RNAi SDC1/4 – – + + RNAi SDC1/4 – + – + + – + + – + + – +
300 SDC1CTF
(relative to control)

Syntenin Syntenin OE + + + – + + – + + – + + + Cath. I + – +

CD63 250
Percentage

CD63 HA–FGFR OE – + + + + + + + + + + + +
RNAi nt SDC1/4
exo

200
FGFR
exo

Syntenin 150
SDC1CTF 100
FGFR
ALIX 50
SDC1
lys

FGF2 + – + p-ERKs
lys

SDC4 RNAi nt SDC1/4

Syntenin
ERKs

Figure 6 Syntenin exosomes require syndecan oligomerization and cleavage. as 100%; left, western blots; right, quantifications). (e) The cell-permeable
(a) Heparan sulphate requirement. MCF-7 cells were treated with nt (-), cysteine protease/cathepsin-inhibitor Z-Phe-Gly-NHO-Bz (Cath. I, 2 µM)
EXT1/2 or NDST1/2 RNAi, or grown in the presence of heparitinase reduces syndecan-CTF in lysates, and when syndecans are near limiting
(Hpt). Left: cell lysates digested (d) or not digested (nd) with heparitinase. (syndecan RNAi), strongly reduces exosomal syntenin (top, western blots;
In EXT -RNAi-treated cells, syndecan-1 is detected without digestion, bottom, quantifications). (f) MCF-7 cells were treated with nt RNAi, or with
demonstrating reduced heparan sulphate polymerization. Middle: cell RNAi targeting endogenous syndecans, and serum-starved, overnight, in the
lysates were loaded on DEAE-Sepharose, and eluted by step increments presence or absence of 2 ng ml−1 of FGF2. The histogram (right) indicates
in salt concentration. In NDST -RNAi-treated cells, SDC1 elutes at lower exosomal mean signal intensities (relative to nt RNAi, no FGF2, taken as
salt concentrations, demonstrating reduced sulphation/negative charge of 100%) as detected in western blotting (left). Note that FGF enhances the
the heparan sulphate on syndecan. Right: exosomal releases. (b) Syndecan production of syndecan–syntenin exosomes, pending syndecan support.
clustering. RNAi-treated cells incubated with or without anti-syndecan (g) MCF-7 cells expressing HA-tagged FGFR were stimulated with FGF2
(-1 and -4) ectodomain monoclonal antibodies (mAbs); HSC exosomes (2 ng ml−1 ) for different lengths of time, or left untreated. The cells
were tested for syntenin contents. (c) Cells transfected with empty vector expressed syntenin at endogenous levels or overexpressed (OE) syntenin,
(-), wild-type SDC3 (WT) or N-truncated SDC3 (CTF) and treated with and were treated with control or syndecan RNAi. Syntenin-stimulated
syndecan RNAi (left, western blots; right, quantifications). Note that CTF exosomal FGFR release depends both on syndecan and on FGF, but not
fails to sustain exosome formation (taking the release by sham-transfected on FGF signalling (pERK activation) per se (which persists in the absence
cells as 100%). (d) Presenilin inhibitor (PSI, 0.1 µM), raising CTF levels of syndecan). All histograms represent means (and s.d.) from n independent
in cells with a partial knockdown of syndecans, fails to stimulate exosomal experiments (as indicated). Uncropped images of blots/gels are shown in
syntenin and CD63 release (taking the release in the absence of inhibitor Supplementary Fig. S6.

extracellular matrix (through heparan sulphate), subtracting syndecan syndecan, an N-truncated syndecan (mimicking syndecan-CTF;
from internalization routes. In contrast, in cells treated with EXT1/2 ref. 50) failed to stimulate the exosomal accumulation of syntenin
RNAi, anti-syndecan antibody rescued exosomal release (Fig. 6b). and CD63 in syndecan-depleted cells (Fig. 6c). Syndecan-CTFs are
Together, these data suggest that heparan sulphate-mediated syndecan substrates for presenilin, which releases the syndecan-ICD for turnover
clustering matters for syntenin exosome formation. by the proteasome50 . Thus, presenilin activity might prevent CTF
As syndecans accumulate in exosomes as CTFs, we also assessed accumulation in cells and compromise rescue attempts by CTF
the significance of CTF production. Compared with full-length overexpression. Presenilin inhibition, raising the levels of CTF in

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© 2012 Macmillan Publishers Limited. All rights reserved.
ARTICLES
lysates and exosomes of cells with partial syndecan knockdown, did
not stimulate the exosomal accumulation of CD63 and syntenin
(Fig. 6d), further documenting that syndecan-CTFs are not sufficient
for exosome formation. Treating cells with cathepsin inhibitor,
reducing the accumulation of syndecan-CTF in cell lysates by ∼40%
(without significant effects on the levels of intact syndecan), had
no significant effect on exosomal syntenin (Fig. 6e). Yet, cathepsin
inhibitor and syndecan RNAi synergized in suppressing exosomal
syntenin releases (Fig. 6e), suggesting that, although non-sufficient,
syndecan-CTF production might be required for exosome formation.
Thus, the syntenin–ALIX axis is likely to be driven by cargo-induced
oligomerization of syndecan, with also a major role for the cleavage
of endosomal syndecan.
The heparan sulphate-assisted docking of FGF to FGFR and ensuing
dimerization of the receptor represents one example of physiological
cargo that can induce heparan sulphate clustering22 . This implies
that FGF–FGFR signalling potentially approximates syndecans to
each other, recruiting syntenin. Consistent with that notion, FGF2
stimulation enhanced exosome production (that is, exosomal CD63
and syntenin), and syndecan downregulation, not interfering with
FGF signalling per se (as indicated by pERK levels; see Fig. 6g),
attenuated this FGF effect (Fig. 6f). By extension, we examined
whether syntenin, adapting ALIX to syndecan, might direct associated
FGF:FGFR cargo to exosomes (Fig. 6g). Syntenin overexpression
stimulated exosomal release of FGFR. This stimulation depended on
syndecan, as it was prevented by syndecan RNAi. Consistent with
the ligand-induced approximation model, exosomal accumulation
of FGFR depended on FGF.
DISCUSSION
Our study establishes a direct mechanistic link between syndecans
and ILV/exosome formation, identifying an as yet unsuspected
role for heparan sulphate proteoglycans in vesicular trafficking and
endocytic controls on signalling processes. The cytosolic adaptor
syntenin connects syndecans to ALIX, an auxiliary component of
the ESCRT machinery that supports endosomal membrane budding
and abscission. This connection not only segregates syndecans and
syndecan cargo, for example FGF:FGFR complexes assembled on
the heparan sulphate chains of syndecan, to budding endosomal
membranes and exosomes; it also supports budding processes and
the production of exosomes. Thus, syndecans emerge as key regulators
of post-engagement receptor trafficking26 . The sheer abundance of the
syndecans (up to 106 copies on the surface of an adherent cell) and the
functional versatility of their heparan sulphate chains imply that the
signalling and trafficking of a large number and variety of receptors
might be regulated in that way.
Together our data suggest that syndecan cargo creates syndecan
assemblies that can recruit syntenin–ALIX and support membrane
budding. Although syntenin can be detected at the plasma
membrane32,33 , the formation or the stability of these assemblies
might require the concentration of syndecan in endosomal domains,
where syndecan cleavage occurs. This cleavage, removing the highly
negatively charged and auto-repulsive syndecan ectodomains, might
allow syndecan-ICD to remain clustered and syntenin bound to
syndecan (as CTF), stabilizing the recruitments and sustaining the
membrane budding process. Of note, although endosomal syndecan
684
© 2012 Macmillan Publishers Limited. All rights reserved.
cleavage potentially also disconnects endocytosed cargo from budding
machineries, once ESCRT-III proteins have assembled on the necks
of budding membranes11 , syndecan ectodomains, with their heparan
sulphate, would become unnecessary for sequestering cargo to budding
membranes. Syndecan ligation (for example, by FGF2) causing
syndecan to become associated with detergent-resistant membrane
domains51 , the lateral segregation of lipids in endosomal membranes
may also be a stabilizing factor. Possibly this pertains to the importance
of ceramide for exosome formation40 . Clearly, further investigations
are required to clarify the spatiotemporal and mechanistic relationships
between syndecan clustering, syntenin recruitment, syndecan cleavage
and consolidated cargo sequestration in membrane buds.
We found that syntenin–ALIX also controls exosome formation
in HeLa cells (M.F.B., unpublished results). Yet, the specificity and
regulation of this pathway remain to be explored. Reportedly, syntenin
has multiple interaction partners, and clusters of membrane and
membrane-associated proteins that represent alternative bait for
the PDZ domains of syntenin might thus function along with or
independently of syndecan (for example, ephrin-B1/2; refs 52,53
and Fz signalling54 ). The functional property of heparan sulphate
depends on its fine structure, which is highly plastic and is set
by modifying enzymes that are cell-type- and differentiation-stage-
specific23,24 . This might select what cargo is trafficking through
syndecan–syntenin vesicles. As the syndecan cytoplasmic domain25–27 ,
syntenin30,31 and ALIX55 are subject to modifications (phosphorylation,
‘open’ and ‘closed’ configurations) that might regulate their mutual
engagement, the model outlined here in MCF-7 cells might be
modified by cellular context.
Taken together, the above findings identify syndecan–syntenin–ALIX
as an important regulator of membrane trafficking and heparan
sulphate-assisted signalling. By extension, activation or deregulation of
this axis might also influence heparan sulphate-associated pathological
processes, including cancer, the propagation of prions, inflammation,
amyloid deposition and neurodegenerative disease. 
METHODS
Methods and any associated references are available in the online
version of the paper at www.nature.com/naturecellbiology
Note: Supplementary Information is available on the Nature Cell Biology website
ACKNOWLEDGEMENTS
We are grateful to H. Ceulemans, G. Reekmans and J. Grootjans for technical
support, and to P. Baatsen for access to the electron microscopy facility at
the Department for Human Genetics, VIB-KULeuven. We thank C. Dotti and
P. Courtoy for critical feedback on the manuscript. This work was supported by
the Fund for Scientific Research-Flanders (FWO), the Concerted Actions Program
of the K.U. Leuven, the VIB, the Belgian Federation against Cancer (STK), the
Interuniversity Attraction Poles of the Prime Ministers Services (IUAP) and the
EMBO young investigator programme (P.Z.). A.M. was supported by a Marie Curie
postdoctoral fellowship of the EU (FP7); Y.I. by an EMBO-long term postdoctoral
fellowship; and A.G. by a PhD-student fellowship of the Agency for Innovation by
Science and Technology (IWT), Flanders.
AUTHOR CONTRIBUTIONS
The work is an equal contribution of two laboratories. M.F.B., Z.Z. and A.M.
performed the cellular experiments; E.M. and Y.I. did the Biacore, A.G. the
fluorescence spectroscopy, G. Degeest the electron microscopy, and F.D. the
co-immunoprecipitation and the Y2H analyses; E.V. made the various expression
constructs and C.C. assisted with cell culture and biochemical analyses; P.Z. and
G. David designed experiments, analysed data and wrote the manuscript. All authors
discussed results and commented on the manuscript.
NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012

COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at www.nature.com/naturecellbiology
Reprints and permissions information is available online at www.nature.com/reprints
1. Bobrie, A., Colombo, M., Raposo, G. & Théry, C. Exosome secretion: molecular
mechanisms and roles in immune responses. Traffic 12, 1659–1668 (2011).
2. Fevrier, B. & Raposo, G. Exosomes: endosomal-derived vesicles shipping extracellular
messages. Curr. Opin. Cell Biol. 16, 415–421 (2004).
3. Couzin, J. Cell biology: the ins and outs of exosomes. Science 308,
1862–1863 (2005).
4. Liegeois, S., Benedetto, A., Garnier, J. M., Schwab, Y. & Labouesse, M. The
V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related
proteins in Caenorhabditis elegans. J. Cell Biol. 173, 949–961 (2006).
5. Al-Nedawi, K. et al. Intercellular transfer of the oncogenic receptor EGFRvIII by
microvesicles derived from tumour cells. Nat. Cell Biol. 10, 619–624 (2008).
6. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel
mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).
7. Thery, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and
function. Nat. Rev. Immunol. 2, 569–579 (2002).
8. Nabhan, J. F., Hu, R., Oh, R. S., Cohen, S. N. & Lu, Q. Formation and release of
arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma
membrane by recruitment of TSG101 protein. Proc. Natl Acad. Sci. USA 109,
4146–4151 (2012).
9. Wehman, A. M., Poggioli, C., Schweinsberg, P., Grant, B. D. & Nance, J. The
P4-ATPase TAT-5 inhibits the budding of extracellular vesicles in C. elegans embryos.
Curr. Biol. 21, 1951–1959 (2011).
10. Raiborg, C. & Stenmark, H. The ESCRT machinery in endosomal sorting of
ubiquitylated membrane proteins. Nature 458, 445–452 (2009).
11. Henne, W. M., Buchkovich, N. J. & Emr, S. D. The ESCRT pathway. Dev. Cell 21,
77–91 (2011).
12. Wollert, T. & Hurley, J. Molecular mechanism of multivesicular body biogenesis by
ESCRT complexes. Nature 464, 864–869 (2010).
13. Hurley, J. & Hanson, P. Membrane budding and scission by the ESCRT machinery:
it’s all in the neck. Nat. Rev. Mol. Cell Biol. 11, 556–566 (2010).
14. Babst, M. MVB vesicle formation: ESCRT-dependent, ESCRT-independent and
everything in between. Curr. Opin. Cell Biol. 23, 452–457 (2011).
15. Stuffers, S., Sem Wegner, C., Stenmark, H. & Brech, A. Multivesicular endosome
biogenesis in the absence of ESCRTs. Traffic 10, 925–937 (2009).
16. Theos, A. C. et al. A lumenal domain-dependent pathway for sorting to intralumenal
vesicles of multivesicular endosomes involved in organelle morphogenesis. Dev. Cell
10, 343–354 (2006).
17. Odorizzi, G. The multiple personalities of Alix. J. Cell Sci. 119, 3025–3032 (2006).
18. Matsuo, H. et al. Role of LBPA and Alix in multivesicular liposome formation and
endosome organization. 303, 531-534 (2004).
19. Fujii, K., Hurley, J. H. & Freed, E. O. Beyond Tsg101: the role of Alix in ’ESCRTing’
HIV-1. Nat. Rev. Microbiol. 5, 912–916 (2007).
20. Martin-Serrano, J. & Neil, S. J. Host factors involved in retroviral budding and release.
Nat. Rev. Microbiol. 9, 519–531 (2011).
21. Rapraeger, A. C., Krufka, A. & Olwin, B. B. Requirement of heparan sulfate
for bFGF-mediated fibroblast growth and myoblast differentiation. Science 252,
1705–1708 (1991).
22. Schlessinger, J. et al. Crystal structure of a ternary FGF–FGFR-heparin complex
reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6,
743–750 (2000).
23. Bishop, J. R., Schuksz, M. & Esko, J. D. Heparan sulphate proteoglycans fine-tune
mammalian physiology. Nature 446, 1030–1037 (2007).
24. Hacker, U., Nybakken, K. & Perrimon, N. Heparan sulphate proteoglycans: the sweet
side of development. Nat. Rev. Mol. Cell Biol. 6, 530–541 (2005).
25. Couchman, J. Transmembrane signaling proteoglycans. Annu. Rev. Cell Dev. Biol.
26, 89–114 (2010).
26. Lambaerts, K., Wilcox-Adelman, S. A. & Zimmermann, P. The signaling
mechanisms of syndecan heparan sulfate proteoglycans. Curr. Opin. Cell Biol. 21,
662–669 (2009).
27. Morgan, M. R., Humphries, M. J. & Bass, M. D. Synergistic control of cell adhesion
by integrins and syndecans. Nat. Rev. Mol. Cell Biol. 8, 957–969 (2007).
NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012
© 2012 Macmillan Publishers Limited. All rights reserved.
ARTICLES
28. Grootjans, J. J. et al. Syntenin, a PDZ protein that binds syndecan cytoplasmic
domains. Proc. Natl Acad. Sci. USA 94, 13683–13688 (1997).
29. Beekman, J. M. & Coffer, P. J. The ins and outs of syntenin, a multifunctional
intracellular adaptor protein. J. Cell Sci. 121, 1349–1355 (2008).
30. Grootjans, J. J., Reekmans, G., Ceulemans, H. & David, G. Syntenin-syndecan
binding requires syndecan-synteny and the co-operation of both PDZ domains of
syntenin. J. Biol. Chem. 275, 19933–19941 (2000).
31. Zimmermann, P. et al. Characterization of syntenin, a syndecan-binding PDZ protein,
as a component of cell adhesion sites and microfilaments. Mol. Biol. Cell 12,
339–350 (2001).
32. Zimmermann, P. et al. PIP(2)-PDZ domain binding controls the association of
syntenin with the plasma membrane. Mol. Cell 9, 1215–1225 (2002).
33. Zimmermann, P. et al. Syndecan recycling [corrected] is controlled by syntenin-PIP2
interaction and Arf6. Dev. Cell 9, 377–388 (2005).
34. Gottlinger, H. G. How HIV-1 hijacks ALIX. Nat. Struct. Mol. Biol. 14,
254–256 (2007).
35. Lee, S., Joshi, A., Nagashima, K., Freed, E. O. & Hurley, J. H. Structural basis for
viral late-domain binding to Alix. Nat. Struct. Mol. Biol. 14, 194–199 (2007).
36. Zhai, Q. et al. Structural and functional studies of ALIX interactions with YPX(n)L
late domains of HIV-1 and EIAV. Nat. Struct. Mol. Biol. 15, 43–49 (2008).
37. Thery, C. et al. Molecular characterization of dendritic cell-derived exosomes.
Selective accumulation of the heat shock protein hsc73. J. Cell Biol. 147,
599–610 (1999).
38. Mathivanan, S. & Simpson, R. J. ExoCarta: a compendium of exosomal proteins and
RNA. Proteomics 9, 4997–5000 (2009).
39. Latysheva, N. et al. Syntenin-1 is a new component of tetraspanin-enriched
microdomains: mechanisms and consequences of the interaction of syntenin-1 with
CD63. Mol. Cell Biol. 26, 7707–7718 (2006).
40. Trajkovic, K. et al. Ceramide triggers budding of exosome vesicles into multivesicular
endosomes. Science 319, 1244–1247 (2008).
41. Stenmark, H. et al. Inhibition of rab5 GTPase activity stimulates membrane fusion
in endocytosis. EMBO J. 13, 1287–1296 (1994).
42. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of
progression from early to late endosomes. Cell 122, 735–749 (2005).
43. Vanlandingham, P. A. & Ceresa, B. P. Rab7 regulates late endocytic trafficking
downstream of multivesicular body biogenesis and cargo sequestration. J. Biol.
Chem. 284, 12110–12124 (2009).
44. Doyotte, A., Russell, M., Hopkins, C. & Woodman, P. Depletion of TSG101 forms a
mammalian ‘Class E’ compartment: a multicisternal early endosome with multiple
sorting defects. J. Cell Sci. 118, 3003–3017 (2005).
45. Razi, M. & Futter, C. E. Distinct roles for Tsg101 and Hrs in multivesicular body
formation and inward vesiculation. Mol. Biol. Cell 17, 3469–3483 (2006).
46. Bishop, N. & Woodman, P. ATPase-defective mammalian VPS4 localizes to
aberrant endosomes and impairs cholesterol trafficking. Mol. Biol. Cell 11,
227–239 (2000).
47. Morita, E. et al. ESCRT-III protein requirements for HIV-1 budding. Cell Host Microbe.
9, 235–242 (2011).
48. Jouvenet, N., Zhadina, M., Bieniasz, P. D. & Simon, S. M. Dynamics of ESCRT
protein recruitment during retroviral assembly. Nat. Cell Biol. 13, 394–401 (2011).
49. Fang, Y. et al. Higher-order oligomerization targets plasma membrane proteins and
HIV gag to exosomes. PLoS Biol. 5, e158 (2007).
50. Schulz, J. et al. Syndecan 3 intramembrane proteolysis is presenilin/gamma-
secretase-dependent and modulates cytosolic signaling. J. Biol. Chem. 278,
48651–48657 (2003).
51. Tkachenko, E. & Simons, M. Clustering induces redistribution of syndecan-4 core
protein into raft membrane domains. J. Biol. Chem. 277, 19946–19951 (2002).
52. McClelland, A. C., Sheffler-Collins, S. I., Kayser, M. S. & Dalva, M. B. Ephrin-B1
and ephrin-B2 mediate EphB-dependent presynaptic development via syntenin-1.
Proc. Natl Acad. Sci. USA 106, 20487–20492 (2009).
53. Xu, N. J., Sun, S., Gibson, J. R. & Henkemeyer, M. A dual shaping mechanism
for postsynaptic ephrin-B3 as a receptor that sculpts dendrites and synapses. Nat.
Neurosci. 14, 1421–1429 (2011).
54. Luyten, A. et al. The postsynaptic density 95/disc-large/zona occludens protein
syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling.
Mol. Biol. Cell 19, 1594–1604 (2008).
55. Zhou, X., Si, J., Corvera, J., Gallick, G. E. & Kuang, J. Decoding the intrinsic
mechanism that prohibits ALIX interaction with ESCRT and viral proteins. Biochem.
J. 432, 525–534 (2010).
685
METHODS
METHODS
Expression vectors and reagents. Expression vectors for GST (pGEX-5X,
Amersham Pharmacia Biotech) and fluorescent protein (pmCherry-C and peYFP-C,
Clontech) fusion proteins, and for yeast two-hybrid experiments (pGAD10 and
pAS2, Clontech), were constructed from syntenin complementary DNAs (described
previously28,30,31 ) and from full-length cDNA clones encoding mouse (ID 4038684)
and human (ID 2455231) ALIX (obtained from the IMAGE consortium, through
Invitrogen). Mutant forms were made by QuickChange site-directed mutagenesis
PCR (Stratagene). For gain-of-function studies in cells, cDNA encoding full-length
non-tagged syntenin was cloned in pcDNA3.1/Zeo(+) (Invitrogen). Expression
vector for mGFP–CD63 was a gift from J. Klumperman (Utrecht University,
The Netherlands), cer–RAB5Q79L was received from W. Annaert (K.U. Leuven,
Belgium; ref. 56) and constructs for expressing GFP–VPS4A were kindly provided
by M. Simons (University of Göttingen/Max-Planck-Institute for Experimental
Medicine, Germany). All other expression vectors were described previously28,30–33 .
All constructs were verified by sequencing. Except for RNAi targeting human
nSMase-2 (Flexitube short interfering RNA (siRNA), Qiagen), all RNAi targeting
human sequences (ON-Target plus Smartpools) and the non-targeting control
RNAi (siSTABLE) were purchased from Dharmacon. Anti-syntenin, and anti-SDC
(-1/3 ICD and -4 ectodomain) antibodies were as described previously31,32,57,58 .
GST–ALIX (full length, mouse) fusion protein and GST were used to generate
affinity-purified ALIX-specific rabbit polyclonal antisera (Eurogentec). Other
antibodies were from commercial sources and were used as recommended by the
manufacturers. Antibodies directed against TSG101 (C-2), HSP70 (W27), RAB7
(H-50), CHMP4B (C-12) and β-actin (AC-15) were from Santa Cruz; antibody
against SDC1 ectodomain (BB4) was from Serotec; antibody against SDC4 ICD
was from Abnova; antibodies against CD63 (MEM-259) and Calnexin (ab10286)
were from Abcam; antibody against HA (16B12) was from Eurogentec; anti-GM130
(clone 35) and anti-flotillin-1 were from BD Biosciences; anti-LAMP-2 (H4B4) was
from the Developmental Studies Hybridoma Bank of the University of Iowa, USA;
anti-phospho-p44/42 MAPK (9101) was from Cell Signaling. Cathepsin inhibitor
I (Z-FG-NHO-Bz) and InSolution γ-Secretase Inhibitor X were from Calbiochem.
See Supplementary Table S1 for more information on antibodies and siRNA.
Recombinant proteins, overlay and surface plasmon resonance experiments.
Recombinant proteins for in vitro binding studies were prepared as GST fusions,
6xHis-tagged or from self-splicing intein fusion protein (non-tagged) and used in
overlay and surface plasmon resonance experiments, as described previously30,54,59 .
Surface plasmon resonance was measured using a Biacore T200 instrument for
Kd determination and a Biacore 2000 for the other experiments. For the triple
binding experiments a total of 1,000 response units (RU) of biotinylated SDC2
cytoplasmic domain peptide, and for Kd determinations 100 RU of biotinylated
peptides, were immobilized on streptavidin biosensor chips. For the ALIX–syntenin
binding experiments, a total of 9,000 RU of GST–syntenin full length, GST–syntenin
peptides corresponding to amino acids 1–30 or 31–60, or GST as a negative control,
were immobilized on a CM-5 biosensor chip.
Immunoprecipitation from microsome extracts. Preparation of microsomal
membranes and immunoprecipitations were as described previously30,31 .
Cells and transfections. MCF-7 cells were obtained from ATCC. Cells were
routinely grown in DMEM/F12 (1:1) medium (Life Technologies) supplemented
with 10% fetal bovine serum (Hyclone). Serum used for experiments was depleted of
exosomes, by prior overnight centrifugation at 140,000g . For transient expressions,
the cells were transfected the day after plating using Fugene 6 reagent (Roche). For
RNAi experiments, cells were transfected at 50% confluency with 100 nM siRNA
using Oligofectamine (Invitrogen); cells were analysed 48 h after RNAi treatment.
Exosomes and total cell lysates. For comparative analyses, in gain- and loss-
of-function studies, exosomes were collected from equivalent amounts of culture
medium, conditioned by equivalent amounts of cells in triplicate cultures. When
reaching 80% of confluency, the cell layers were rinsed with DMEM/F12 and
refreshed with DMEM/F12 containing 10% exosome-depleted FBS. The cell-
conditioned media were collected 16 h later, pooling media from corresponding
triplicate cultures. Exosomes were isolated from these media by three sequential
centrifugation steps at 4 ◦ C: 15 min at 500g , to remove cells; 30 min at 10,000g ,
to remove cell debris; and 3 h at 140,000g , to pellet exosomes, followed by
one rinse (suspension in PBS/centrifugation at 140,000g ), to remove soluble
serum and secreted proteins. Exosomal pellets prepared by differential HSC (HSC
exosomes) were then re-suspended in PBS or lysis buffer (PBS supplemented
with 1% NP40, 1 mM EDTA, 5 µg ml−1 leupeptin, 1 µg ml−1 pepstatin and 1 mM
phenylmethylsulphonyl fluoride). Corresponding cell layers were washed twice
in cold PBS, and scraped on ice in lysis buffer. Lysates from corresponding
triplicate cultures were pooled, and cleared by centrifugation at 16,000g , for
10 min at 4 ◦ C. Cell lysates were normalized by protein content, and corresponding
© 2012 Macmillan Publishers Limited. All rights reserved.
DOI: 10.1038/ncb2502
exosome preparations were adjusted accordingly. Except for RNAi targeting nSMase,
none of the treatments (chemicals, enzymes), transfections or RNAi significantly
affected cell survival or growth, requiring major (>10%) adjustments of the
samples.
Exosome fractionation. For further fractionation by buoyant density, HSC
exosomes were suspended in 2 ml 60% Optiprep (Axis-Shield PoC), and a
6–40% gradient of Optiprep in DMEM/F12 was carefully deposited on top. After
centrifugation for 16 h at 140,000g , at 4 ◦ C, fractions were collected from the top.
The density of each fraction was extrapolated from its attenuance, measured at
340 nm. Then, the different fractions were diluted with DMEM/F12 and further
spun for 3 h at 140,000g , at 4 ◦ C, to collect the exosomes. For fractionation by CD63
content, HSC exosomes were suspended in 1 ml of PBS-0.1%BSA and incubated,
for 30 min at 4 ◦ C, under gentle agitation, with 1.5 µg of anti-CD63 monoclonal
antibody or monoclonal antibody 3G10 (specific for a neo-epitope, generated by
bacterial enzyme digestion, not occurring in non-enzyme-treated mammalian cells,
and used as a control). After spinning (140,000g , 1 h, 4 ◦ C) to remove unbound
antibody, pellets were suspended in 1 ml of PBS-0.1%BSA, supplemented with
20 µl of magnetic beads with covalently bound anti-mouse IgG (Dynal) and further
incubated for 30 min at 4 ◦ C, under gentle agitation. Collected beads were washed
twice with PBS-0.1%BSA, once with PBS, and then resuspended in lysis buffer.
Western blotting. To allow detection of the syndecans, the samples were
digested with (0.006 U ml−1 ) heparitinase and (0.166 U ml−1 ) chondroitinase ABC
(Seikagaku Corporation) for 3 h at 37 ◦ C in heparitinase buffer (0.1% Triton, 0.1 M
NaCl, 1 mM CaCl2 , 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).
Samples were fractionated in 4–12% gradient gels (NuPAGE Novex Bis-Tris gels,
Invitrogen), electro-transferred to Protran nitrocellulose membrane (0.2 µm), and
incubated with the indicated antibodies. Signals were visualized with enhanced
chemiluminescence detection reagent (Amersham Pharmacia Biotech) and were
quantified by densitometric scanning, using Quantity One software (Biorad).
Cell-surface-biotinylation experiments. MCF-7 cells were biotinylated with
reducible sulpho-NHS-SS-biotin (Pierce) at 4 ◦ C, as described previously33 .
Biotinylated cells were allowed to resume their membrane trafficking for 16 h.
Exosomes and cells were then collected as described above. Biotinylated proteins
were detected with streptavidin–HRP and signals were quantified as described above.
Microscopy. For microscopic analysis, cells were plated on non-coated eight-well
chamber slides (Nalge Nunc International). The day after transfection, cells were
immunostained as described previously31 . Images were recorded with a Leica
AS MDW microscopy workstation (Leica Microsystems) or a confocal Olympus
Fluoview 1000 instrument. The filling of RAB5Q79L endosomes was quantified with
ImageJ (National Institutes of Health, USA), and analysed for statistical significance
using Graphpad Prism 5.
Electron microscopy. For assessing MVB morphology, cells were incubated for
30 min at 37 ◦ C with anti-CD63 antibody (Abcam) labelled with HRP (Z 25054
Molecular Probes). Cells were then washed with PBS and fixed for 1 h in 3%
glutaraldehyde in 0.1 M sodium cacodylate (NaCaC) buffer. After several washes
in 0.1 M NaCaC, cells were incubated with DAB (Sigma) in 0.05 M Tris–HCl, at
pH 7.6. The enzymatic reaction was started by adding H2 O2 to a final concentration
of 0.01%, and allowed to proceed for 30 min. After washing in Tris–HCl buffer,
cells were postfixed for 1 h with 1% osmium tetroxide in 0.1 M phosphate buffer,
washed, scraped, re-suspended in 2% agar and pelleted. Sliced agar blocks were
dehydrated in a series of ethanol, and embedded in Agar 100 resin polymerized
for 48 h at 60 ◦ C. Sections (60 nm; Ultracut E Leica) were counterstained with
5% uranyl acetate and lead citrate (Reynold’s), and were examined with a Jeol
(JEM-2100) instrument. MVE/MVB sectional areas were measured using ImageJ
software (National Institutes of Health, USA) and data analysed using Graphpad
Prism 5.
Exosomes purified on a continuous gradient as described above were re-
suspended in PBS containing 4% paraformaldehyde, and negatively stained with 5%
uranyl acetate.
Fluorescence spectroscopy and two-colour fluorescence coincidence
detection. Freshly isolated HSC exosomes were diluted in PBS. Single-colour
fluorescence detection, and two-colour fluorescence coincidence detection mea-
surements on these samples were performed at room temperature (∼21 ◦ C), using
an LSM 510 Confocor-II combination system equipped with argon ion (488 nm)
and HeNe (543 nm) lasers. The excitation light was focused into the sample by
a Zeiss 40x/1.2 Plan-Apochromat water-immersion objective and a 488/543 main
beam splitter. The detected light was split over two channels using a 570 nm dichroic
mirror. To narrow the emission spectrum, 600–650 nm and 505–530 nm band-pass
filters were set before channel 1 and 2 respectively. Fluorescence fluctuations
NATURE CELL BIOLOGY
DOI: 10.1038/ncb2502
were measured 50 times for 10 s, and each experiment was repeated at least three
times, independently. Data were corrected for bleeding through, background and
noise. The percentage of bleeding through in channel 1 (red) was calculated
using exosomes containing eGFP–syntenin. A signal of intensity fivefold above
background fluorescence was defined as a peak; in addition, when a peak was
detected in channel 2 (green), any coincident signal in channel 1 (red) had to be
higher than 5 times the background plus the bleeding through level to be considered
as a bona fide (red) peak.
Syndecan clustering experiments. To reduce proteoglycan recruitment/cluster-
ing, by preventing heparan sulphate engagements, the culture media were
supplemented with bacterial heparitinase (0.006 U ml−1 ). For antibody-induced
clustering, the culture media were supplemented with the monoclonal anti-
syndecan antibodies BB4 (specific for syndecan 1; 0.1 µg ml−1 ) and 8G3 (specific for
syndecan 4; 1 µ g ml−1 ). Exosomes were collected after 16 h of medium conditioning.
Growth factor receptor trafficking. To test for FGFR transfer to exosomes
by western blotting, MCF-7 cells were transiently transfected with HA-tagged
FGFR-1 (ref. 60). When reaching near 80% of confluency, cells were serum-
starved overnight in DMEM/F12 supplemented with 0.1% BSA. Starved cells were
exposed to 2 ng ml−1 of FGF2 in 0.1%BSA-DMEM/F12, supplemented with 0.1 mM
Na3 VO4 , for the indicated lengths of time. Exosomes were prepared by HSC as
NATURE CELL BIOLOGY
© 2012 Macmillan Publishers Limited. All rights reserved.
METHODS
described above. Cells were lysed on ice, in lysis buffer supplemented with 1 mM
Na3 VO4 .
Statistical analysis. P values were calculated by two-tailed t test. For the data on
MVE size, the Mann–Whitney test for non-normal distributions was used. All data
were analysed using Graphpad Prism 5.
56. Sannerud, R. et al. ADP ribosylation factor 6 (ARF6) controls amyloid precursor
protein (APP) processing by mediating the endosomal sorting of BACE1. Proc. Natl
Acad. Sci. USA 108, E559–E568 (2011).
57. David, G., van der Schueren, B., Marynen, P., Cassiman, J. J. & van den Berghe,
H. Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate
proteoglycan expressed by epithelial and fibroblastic cells. J. Cell Biol. 118,
961–969 (1992).
58. Lories, V., Cassiman, J. J., Van den Berghe, H. & David, G. Multiple distinct
membrane heparan sulfate proteoglycans in human lung fibroblasts. J. Biol. Chem.
264, 7009–7016 (1989).
59. Ivarsson, Y. et al. Cooperative phosphoinositide and peptide binding by PSD-95/Discs
Large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin. J. Biol. Chem. 286,
44669–44678 (2011).
60. Zhang, Z., Coomans, C. & David, G. Membrane heparan sulfate proteoglycan-
supported FGF2-FGFR1 signaling: evidence in support of the ‘cooperative end
structures’ model. J. Biol. Chem. 276, 41921–41929 (2001).
S U P P L E M E N TA R Y I N F O R M AT I O N
DOI: 10.1038/ncb2502
Figure S1 Mapping the syntenin-Alix interaction. (a) Syntenin-Alix
interaction, as detected by yeast two-hybrid experiments. Numbers indicate
amino-acid residues. FL, full-length; m, mouse; h, human. Bait constructs
(left column) were cloned in pAS2, prey constructs (rows) in pGAD10.
Positive clones were selected on triple minus plates (Leu-, Trp, His-)
containing 5 mM 3-aminotriazol. mAlix (383-869) corresponds to the prey
isolated from a mouse whole embryo library in the original screen, using
full-length mouse syntenin as bait. Alix was the sole prey found to interact
with the N-Ter domain of syntenin in subsequent mapping experiments,
and also in separate unbiased screens using the N-Ter domain as bait.
Note that truncated forms of Alix containing the V domain interact with
the two parts of the N-Ter domain of syntenin that contain LYPXL motifs,
(1-30) and (31-60), and that mutation of the three LYPXL motifs (YP to
AA) abolishes the interaction. Full-length Alix constructs do not interact
with syntenin in this assay. Possible reasons might be trivial, but include an
intrinsic auto-inhibitory mechanism (closed protein conformation, supported
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by intra-molecular Bro1-PRD interactions) that prohibits Alix interactions
with ESCRT and viral proteins55. (b) Overlay experiment with recombinant
GST-fusion proteins, showing that full-length Alix interacts with full-length
syntenin (FL), but not with the PDZ domains and C-terminus of syntenin
(DN-Ter). (c) Biacore experiments showing the direct interaction of GST-
Alix, used as analyte, with various immobilized GST-syntenin constructs,
as indicated, but not with GST alone, used as a negative control (lower
sensorgram). (d) Biacore experiments showing the interaction of non-tagged
recombinant syntenin with immobilized syndecan-2 (initial part of the
sensorgrams) and, subsequently, that of syndecan-2:syntenin complexes
with GST-Alix (wild-type), but not with mutant GST-Alix F676D (later parts of
the sensorgrams). In both sensorgrams, perfusion of syntenin stops at 400
sec; GST-Alix perfusions start at 1000 sec. All data together indicate that
syntenin-Alix interaction requires the LYPXL motifs in the N-terminal domain
of syntenin and the V domain of Alix, with a critical role for residue F676 of
Alix. Uncropped data are shown in Fig. S6.
S U P P L E M E N TA R Y I N F O R M AT I O N
Figure S2 Syndecan, syntenin and CD63 release in exosomes. (a) Syndecan
over-expression. Left panesl: Western blots of heparitinase-digested total cell
lysates (lys) and HSC exosomes (exo) of MCF-7 cells, transiently transfected
with a construct encoding syndecan-1 or an empty vector (-), as indicated on
top of the lanes, showing the relative abundance of the intact (~65kDa) core
protein and its C-terminal fragment (CTF) (~10kDa). Upper right panels: intact
forms and CTFs of over-expressed syndecan-1 co-fractionate during density-
gradient centrifugation, and fractionate as in Fig. 1g-h. Lower right panels:
also in cells that over-express syndecan-1, the over-expression of wild-type
syntenin stimulates the exosomal accumulation of syndecan-1 (intact and CTF).
Over-expression of (triple LYP-LAA) mutant syntenin, deficient for Alix binding
(synteninDAlix), in contrast, has no or little effect. Compared to non-targeting
(nt) RNAi, syntenin RNAi and Alix RNAi decrease exosomal syndecan-1 (intact
and CTF). (b) Ceramide requirements. Western blots of HSC exosomes derived
from MCF-7 cells treated with non-targeting (nt) RNAi or with RNAi targeting
neutral sphingomyelinase-2 (SMase2), assaying for syndecan-1 CTF, syntenin,
CD63 and flotillin-1. Signals measured by densitometry were expressed as
percentage of the corresponding signals obtained in controls (nt RNAi); bars
represent mean values obtained from 6 independent experiments. Error bars
represent standard deviations (SD). (c) Panels illustrating simultaneous green
and red fluorescence recordings over 10 seconds, obtained for a mixture of
2
© 2012 Macmillan Publishers Limited. All rights reserved.
exosomes derived from MCF-7 cells that express, separately, either mCherry-
syntenin or eGFP-syntenin (left; negative control), and for exosomes derived
from MCF-7 cells co-expressing mCherry-syntenin and mGFP-CD63 (right).
*
Note the extensive coincident detection ( ) of green and red peaks in the right
panel. (d) Similar fluorescence data were collected for exosomes isolated from
MCF-7 cells, as indicated. For each experiment, the extent of co-appearance
(number of peaks recorded in the green channel with co-incident peak in the red
channel, expressed as percentage of the total number of spikes recorded in the
green channel) was calculated from 50 datasets (50 recordings of 10 seconds).
The mean percentages of co-appearance (± SD), calculated from independent
experiments are listed. Coincident fluorescent peaks were barely detectable in
exosome mixtures derived from cells separately expressing mCherry-syntenin
and mGFP-CD63, or in exosomes isolated from mixed spent culture media
of such cells, excluding coincidence due to exosome compaction (as a result
of HSC) and incomplete resuspension. Adding detergent (octylglucoside),
during the isolation procedure or to the final exosome preparation, disrupted
the fluorescent signals (data not shown). Such effect is consistent with the
detection of fluorescence contained in individual vesicles, rather than individual
molecules. Of note, we found that, unlike endogenous flotillin, FP-labelled
flotillin(s) did not incorporate in exosomes, excluding similar experiments for
that exosomal marker. Uncropped data are shown in Fig. S6.
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Figure S3 Exosome production. (a) Cell surface-biotinylated MCF-7 cells were
treated with various RNAi, targeting syndecan, syntenin or Alix, as indicated
on top. Non-targeting RNAi (nt) was used as control. Total cell lysates (lys) and
HSC exosomes (exo) were analysed by Western blotting, testing for biotinylated
protein, using streptavidin. Representative Western blots are shown on the
left. Right, bar graphs representing mean signal intensities (relative to signals
in controls), and standard deviations (SD, error bars), calculated from several
(n) independent experiments, for each RNAi, as indicated at the bottom. (b)
Nanoparticle tracking. HSC exosome preparations from MCF-7 cells treated
with control RNAi, or with RNAi targeting syntenin, Alix or syndecan, and
also from cells that over-express (OE) syntenin were analyzed by nanoparticle
tracking analysis (NanoSight), providing information on the concentration
and size of particles. The number of particles of exosomal size (30-110
nm) recovered from culture media at the end of the experiment (16h of
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S U P P L E M E N TA R Y I N F O R M AT I O N
conditioning), corrected for the number of particles of similar size present in
media at the beginning of the experiment (i.e. recovered by a rinse of parallel
cultures), was considered as the number of exosomes produced. Means (and
SD) of three independent experiments; courtesy of NanoSight. (c) Confocal
fluorescence spectroscopy. MCF-7 cells were transfected with mGFP-CD63,
and with or without syntenin expression constructs. Green fluorescence present
in HSC exosomes was monitored over time (10 seconds, 50 times). The
number of peaks, total fluorescence in peaks, and modal peak fluorescence
intensity, were measured in three independent experiments. Bars represent
means (and SD) of the fold stimulations in syntenin-transfected cells, relative
to values obtained in mock-transfected cells (set as one). Note that syntenin
over-expression more than doubles the total amount of (CD63) fluorescence
released, by increasing the number of peaks (exosomes), but barely affects the
modal signal intensity (amplitude). Uncropped data are shown in Fig. S6.
3
S U P P L E M E N TA R Y I N F O R M AT I O N

Figure S4 Syndecan, syntenin and Alix co-localize in Rab5Q79L endosomes. Merged images represent the merged mCh-syntenin (red channel), Alix (green
Confocal sections of MCF-7 cells transfected with cerulean-Rab5Q79L, mCherry- channel) and syndecan-1 ICD (blue channel) signals. The cer-Rab5Q79L signal
syntenin, syndecan-1 and Alix. Syndecan-1 intracellular domain (ICD) and is only shown as a separate image. Bar, 20µm. Boxed areas show Rab5Q79L
Alix were detected with, respectively, cognate mouse and rabbit antibodies. endosomes at high magnification. Uncropped data are shown in Fig. S6.

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Figure S5 Endosomal membrane budding. (a) Western blots of HSC
exosomes derived from MCF-7 cells that were sham-transfected (-) or
over-expressing syntenin. Cells were treated with control (nt) or Rab7
RNAi, as indicated on top. (b) Gallery of electron micrographs, illustrating
the size, filling and morphology of the late endosomal compartment, as
revealed by peroxidase-conjugated anti-CD63 internalization (30 min) and
staining with DAB, in MCF-7 cells treated with control (nt), syntenin or
Rab7 RNAi, or a combination of both syntenin RNAi and Rab7 RNAi, as
indicated. Bar, 200nm. (c) MVB/endosome size. The plot documents the
distributions and mean values (bars) of the total sectional areas occupied by
endosomes that contain DAB stain (n, indicating the number of endosomes
examined). Note that in the absence of syntenin, residual MVB/endosomes
are significantly smaller in size than in corresponding controls. This result
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S U P P L E M E N TA R Y I N F O R M AT I O N
is counter-intuitive, as a defective membrane budding process, consuming
lesser amounts of the limiting endosomal membrane, would tend to yield
larger endosomes. Thus, syntenin depletion reduces endosome filling with
vesicles, but may also affect the progression of membranes and cargo
through the endosomal compartment, slowing down the maturation of this
compartment. (d) Electron microscopic measurements of MVB/endosome
filling, in cells treated with Rab7 RNAi, or both Rab7 RNAi and syntenin
RNAi. For each individual peroxidase/DAB-marked MVB/endosome, the
total sectional area occupied by the MVB/endosome is plotted versus the
area occupied by the stain in that MVB/endosome. The right panel shows
the data points enclosed by the insert, at higher resolution. Note reduced
endosome filling in syntenin-depleted cells. Uncropped data are shown in
Fig. S6.
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Figure S6 Uncropped data. The uncropped illustrations used to prepare the main and supplementary figures of this manuscript are shown with reference to
their specific sub-figures indicated on each panel.

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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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Figure S6 continued

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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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Figure S6 continued

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Table S1 Additional information about antibodies and siRNA.

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