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Active Wnt proteins are secreted on exosomes


Julia Christina Gross1,2 , Varun Chaudhary1,2 , Kerstin Bartscherer1,3 and Michael Boutros1,4

Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert
their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors
that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely
unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells.
We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo
receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE
Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.

Wnt proteins act as morphogens during development and can spread complemented by the idea of fat-body-derived lipoprotein particles
over a distance of many cells to induce patterning decisions1,2 . This mediating extracellular transfer of Wnts (refs 25,26). However, it
has remained a paradox because Wnts are lipid modified and rather remains unclear whether lipoprotein-bound Wnts have signalling
hydrophobic3,4 . Different hypotheses are being debated explaining how activity. More recently, the soluble protein SWIM was found to bind
Wnts travel across tissues57 . One possibility could be the existence of secreted Wg at the surface of secreting cells, enabling monomeric
different forms of Wnts that may contribute to short- and long-range forms of Wg to spread through wing discs and influence long-range
signalling8 . Therefore, decoding their secretory paths might be the key signalling27 . Another possibility for Wg transfer could be transport
to understand how Wnts act as morphogens. on exosomes. Exosomes originate from multivesicular bodies (MVBs)
Recently, progress has been made in understanding how Wnts are and MVBs are also required for proper Wnt signalling in receiving
secreted through specialized cargo-receptor interactions and trafficking cells28 . Evi was also shown to transfer from one cell to another on
routes. In the endoplasmic reticulum, Wnts are lipid modified by the exosomes at Drosophila neuromuscular junctions29 , a process requiring
acyl transferase porcupine3,8 , then shuttled to the Golgi with help of the Rab5 and Rab11 (ref. 30).
p24 protein family9,10 , where Evi binds them through their palmitate In this study, we biochemically fractionated extracellular Wnt activity.
modification and transfers them to the plasma membrane1115 . After We found that Wnt proteins are secreted on extracellular vesicles by
endocytosis, the retromer complex recycles Evi to the Golgi, to engage Drosophila and vertebrate cells and that this fraction represents an
the cargo receptor in a next round of Wnt transport1620 . This process active form of secreted Wnts. Furthermore, we identified Ykt6 as a
depends on the endosomal sorting nexin 3 (SNX3), which represents a component that is required for exosomal Wnt secretion in cell culture
nonclassical pathway for cargo retrieval specific for Wnt secretion21,22 . and in vivo. We show that exosomal secretion is an alternative route
Furthermore, Evi and Wnt complexes accumulate on cell surfaces when for secretion of active Wnts.
acidification of trafficking compartments is blocked by inhibition of
vacuolar ATPases (V-ATPases)23 . RESULTS
Several competing hypotheses exist explaining how Wnts are Wnt proteins are present on exosomes from Drosophila
transported extracellularly. Earlier, argosomes were proposed as and human cells
the extracellular spreading vehicle for Wnts in Drosophila wing Extracellular vesicles such as exosomes can be isolated from
imaginal discs24 . Greco et al. demonstrated that Wingless (Wg), the supernatant of cells by a differential centrifugation31 . We
the Drosophila Wnt1 homologue, co-localized in receiving cells purified exosomes from mammalian and Drosophila cell culture
with glycosyl phosphatidylinositolgreen fluorescent protein (GFP) supernatants (Fig. 1a) and characterized the 100,000g pellet (P100)
labelled vesicles, originating from Wg-producing tissue and moving by ultrastructural analysis. We found that this fraction, in both
at speeds comparable to Wg (ref. 24). This hypothesis was later human and Drosophila cells, showed exosome-typical cup-shaped

1
German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Department for Cell and Molecular Biology, Medical
Faculty Mannheim, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. 2 These authors contributed equally to this work. 3 Present address: Max Planck Institute
for Molecular Biomedicine, Research Group Stem Cells and Regeneration, 48149 Mnster, Germany.
4
Correspondence should be addressed to M.B. (e-mail: m.boutros@dkfz.de)

Received 3 April 2012; accepted 8 August 2012; published online 16 September 2012; DOI: 10.1038/ncb2574

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a b P100 HEK293 cells d L cells

Cell extract (CX) Mr (K) CX P100 SN0

300g-10 min 65 TSG101

Wnt3A
30
P0.3 Supernatant I
(floating cells) 2,000g-10 min 80 Calnexin
-discarded- -actin
50
Supernatant II
P2 1 2 3
(dead cells) 10,000g-30 min e L cells
-discarded- c P100 Drosophila Kc167
Mr (K) CX 1 2 3 4 5 6 7 8 9 10
P10 Supernatant III (SN0)
100,000g-3 h TSG101
(cell debris) 65
-discarded-
30 Wnt3A
P100 Supernatant IV (SN)
(exosomes) 115 LRP6

1.08 1.28
Sucrose gradient Sucrose (g ml1)

f Kc167 g Kc167 h P100 Drosophila Kc167


Mr (K) CX P100 SN0 SN Mr (K) CX P100 SN0 SN

Wg Wg
30 30

Anti-Wg
Wg(long exp.)
ApoII
30 65
1 2 3 4 1 2 3 4

Figure 1 Wnt proteins are secreted on exosomes. (a) Differential loaded on top of a step sucrose gradient (1.081.28 g ml1 ; that
centrifugation protocol for the isolation of exosomes from cell culture is, 0.82 M) and ten fractions collected and analysed for Wnt and
supernatants. (b,c) Representative transmission electron microscope exosomal proteins. (f,g) Immunoblot analysis of cell extract, P100 and
image of the 100,000g pellet fraction from supernatant of HEK293 supernatant before and after ultracentrifugation of Drosophila Kc167
cells (b) and of Drosophila Kc167 cells contrast stained with 3% uranyl cells. Full images of blots (dg) are presented in Supplementary
acetate (c). Scale bars, 250 nm. (d) Immunoblot of cell extract (CX), Fig. S3c. (h) Immunogold labelling of Kc167 P100 with anti-Wg
supernatant (SN) and 100,000g pellet (P100) of mouse Wnt3AL antibodies. Scale bar, 100 nm. A representative example of three
cell supernatant. (e) P100 of mouse Wnt3AL cell supernatant was replicates is shown.

structures of 40100 nm whereas contaminating disrupted structures blebs are found at >1.2 mg ml1 and lipoproteins at <1.03 mg ml1
and non-circular membranes were absent (Fig. 1b,c). These vesicular (refs 31,35,36). Immunoblotting demonstrated the presence of Wnt3A
structures purified from human embryonic kidney 293 (HEK293) in four out of ten sucrose fractions, overlapping with the exosomal
cells were immunogold positive for the exosomal marker CD63 marker TSG101 (Fig. 1e). These data support our conclusion that a
(Supplementary Fig. S1a and refs 32,33). To investigate whether fraction of Wnts is secreted on exosomes.
Wnts are present on exosomes, we used human and Drosophila cell We further investigated whether Wg is found on microvesicles
lines that endogenously or stably express Wnts, mouse fibroblast secreted from Drosophila cells. The supernatant from Kc167 cells
L-Wnt3a cells, and human colon cancer Caco-2 cells (Supplementary was fractionated as described (Fig. 1a) and the P100 fraction was
Fig. S1b,c). Immunoblot analysis of the cell lysate, supernatant and analysed by electron microscopy. Similar to the results in mammalian
P100 fractions from Wnt3AL cells showed the presence of Wnt3A cells, the P100 fraction contained purified vesicles with a similar size
and the exosomal marker TSG101 in the P100 fraction, whereas (40100 nm) to mammalian exosomes (Fig. 1c). Whereas we find Wg
contaminating proteins of the secretory pathway such as calnexin in the P100, lipophorins are found in the supernatant and do not
and actin were absent (Fig. 1d). In support of these findings, in co-pellet with exosomes at 100,000g (Fig. 1f,g), indicating that Wg
Caco-2 colon cancer cells, which endogenously express primarily secreted from cultured cells co-purifies with exosomes rather than
non-canonical Wnts, Wnt5A is present in the exosomal fraction lipoprotein particles. A proteomics analysis of the 100,000g pellet
(Supplementary Fig. S1b). These experiments indicate that Wnts identified endogenous markers of Drosophila exosomes. In addition
co-purify with vesicular particles and exosomal markers, consistent to previously identified exosomal proteins including flottilin and Rabs
with a putative localization on exosomes. (refs 30,37), we also found homologues of the human tetraspanin
To extend these observations by a more stringent analysis and to family (Supplementary Table S1 and ref. 38). We used two tetraspanins,
rule out contamination of lipoprotein particles in our preparations, we Tsp42Ef and Tsp96F, as haemagglutinin (HA)-fusion proteins to
loaded the P100 fraction from mouse L cells on a discontinuous sucrose confirm their secretion on exosomes from Kc167 cells (Supplementary
gradient34 . Exosomes migrate in fractions with a refraction index of Fig. S1e) and their presence in MVBs (Supplementary Fig. S1f). Taken
1.141.19 mg ml1 , contaminating cellular membranes or apoptotic together, these experiments indicate that the purification protocol for

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human exosomes is applicable to Drosophila and that the purified TSG101, suggesting that Evi and Wnts shuttle together into MVBs
vesicles are exosomes. (Fig. 3a). Intraluminal vesicle (ILV) formation is the critical step of
Next we investigated whether we could observe Wg on secreted endosome maturation that leads to sorting of cargo for its lysosomal
exosomes. Immunogold labelling for Wg on whole-mount Kc167 degradation or secretion on exosomes39 . Segregation of exosomal
exosomes revealed labelling of Wg on their surface (Fig. 1h and proteins into ILVs in MVBs is mediated by either recognition of cargo
Supplementary Fig. S1d). Taken together, we identified canonical receptors by the ESCRT (endosomal sorting complex required for
and non-canonical Wnts on extracellular microvesicles with the transport) machinery or ceramide-dependent sorting40,41 . Therefore,
morphological and biochemical characteristics of exosomes. we tested the requirement of the ESCRT-0 component HGS for
EviWnt3A secretion on exosomes. Depletion of HGS in HEK293
Purified exosomes can induce Wnt signalling activity cells reduced the level of Wnt3A and Evi in the P100 and reduced
We next investigated whether Wnts on exosomes have signal-inducing Wnt activity in the supernatant by 40% as compared with control
activity. Addition of Wnt3A-conditioned medium induces activity of (Fig. 3d,e and Supplementary Fig. S2b). In addition, we tested different
a TCF/Wnt reporter transiently transfected in HEK293 cells (Fig. 2a). components of the endosomal pathway for their role in autocrine and
In comparison to basal reporter level, addition of P100 from cells paracrine Wnt reporter activity (Supplementary Fig. S2d,e). Altogether,
transfected with plasmid encoding Wnt3A led to twofold induction of we conclude that an ESCRT-mediated sorting step is required for
Wnt activity, whereas no induction was observed by the addition of EviWnt complex trafficking into MVBs.
P100 from control-transfected cells (Fig. 2b). Moreover, an eightfold
induction of an Axin2 reporter was observed after addition of P100 Blocking MVB maturation impairs Evi-mediated Wnt trafficking
from mouse L-Wnt3a cells (Fig. 2c). These experiments indicate onto exosomes
that purified exosomes from Wnt-expressing cells have a significant A previous study proposed that endosomal acidification was required
capability to induce Wnt signalling. for the Evi-mediated release of Wnts (ref. 23). Weak bases such as
If exosomal Wnts are a considerable part of the total activity, NH4 Cl and bafilomycin A1, a potent inhibitor of V-ATPases, prevent
we reasoned that it should be possible to deplete Wnt activity acidification and correct organization of endosomal compartments39,42 ,
from supernatant by differential ultracentrifugation. Accordingly, blocking cargo transfer to MVBs. Ceramide-dependent ILV budding
activity in the supernatant of Wnt3AL cells after one hour of into MVBs is blocked by the ceramidase inhibitor GW4869 (ref. 41).
ultracentrifugation was reduced by 20% (Fig. 2d), and comparably In the presence of NH4 Cl, bafilomycin A1 and GW4869, Wnt3A
reduced in supernatant from HEK293 cells transfected with plasmid accumulated intracellularly and decreased in the exosomal fraction
encoding Wnt3A (Supplementary Fig. S2a). Extended centrifugation (Fig. 4a). Blocking acidification (by NH4 Cl or bafilomycin A1) also
times of 3 and 12 h further reduced the remaining activity in the reduced Wnt-inducing activity in the supernatant to 35 and 60%,
supernatant by 40% (Fig. 2d). On the basis of western blot analysis respectively, whereas GW4869 did not have a significant effect (Fig. 4b
from these supernatants, we concluded that roughly 40% of Wnt3A and Supplementary Fig. S2f,g).
is secreted on exosomes from L cells (Fig. 2e). This indicates that a To investigate at which level of the sorting process the trafficking
substantial fraction of secreted Wnt is found on exosomes and suggests of EviWnt3A complexes is halted, we used a constitutively active
that at least two different active extracellular forms of Wnts exist: Rab5 (Rab5QL) to enlarge endosomal compartments, but leaving
one that is depleted by ultracentrifugation (exosomal Wnts), and the ILV formation or sorting of cargo into ILV unaffected39 . Under
residual pool of Wnts that is not. normal conditions, a fraction of Wnt3A localizes in puncta inside
We next investigated whether the Wnt cargo receptor Evi is required enlarged endosomes, similar to its MVB co-localization with CD81 and
for the secretion of both forms of Wnts into the supernatant. To TSG101 (Fig. 3a), whereas endosomes are completely disorganized
this end, Wnt3AL cells were depleted for Evi by short hairpin RNA by the treatment of bafilomycin A1 (Fig. 4c). As bafilomycin A1
(shRNA)-mediated silencing (or with a control shRNA) and the prevents separation of EviWnt complexes23 and leads to mixed
supernatant was tested on HEK293 TCF-reporter cells. Knockdown secretorylysosomal organelles42 , it is likely that inhibition of
of Evi completely abolished Wnt secretion on exosomes and in the acidification by bafilomycin A1 blocks proper sorting of Wnts into
supernatant by reducing its Wnt activity to basal level (Fig. 2f,g). Thus, MVBs. NH4 Cl and GW4869 still show Wnt3A-positive puncta inside
Evi is required for the release of Wnt proteins on exosomes. the enlarged endosomes, signifying that later steps of MVB maturation
and trafficking are impaired and that, in the case of NH4 Cl, exosomal
Evi shuttles Wnt into MVBs and is present on exosomes Wnt secretion is abolished. The effect of GW4869 was unexpected,
Previous studies showed that Evi moves from the pre- to the as we observed Wnts inside MVBs. Surprisingly, the activity in the
postsynaptic cleft in neuromuscular junctions of Drosophila29,30 . In GW4869-treated supernatant cannot be reduced by ultracentrifugation
accordance, we also find Evi present on exosomes in mammalian (Supplementary Fig. S2g), indicating that the inhibitor affects general
HEK293, L cells and Caco-2 cells (Fig. 3b and Supplementary Fig. S1b). membrane segregation at MVBs and thus might shift the equilibrium
By sucrose gradient analysis Evi co-pelleted in density fractions that from exosomal Wnts to another form of Wnt secretion.
contained the exosomal marker TSG101, similar to results obtained We next tested whether blocking acidification also affects Wg
for Wnt3A (Fig. 3c). secretion. Dissected third instar wing discs were short term cultured
We next analysed trafficking of EviWnt complexes from the Golgi ex vivo and treated with bafilomycin A1 or control vehicle. Blocking
into the endosomal compartment for exosomal sorting. A fraction acidification by bafilomycin A1 led to a significant decrease of Wg
of Evi as well as Wnt co-localized with the MVB markers CD81 and (Fig. 4d (left panels),e, also see Supplementary Fig. S3a) and CD63GFP

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a TCF/Wnt reporter b TCF/Wnt reporter c Axin2 reporter d TCF/Wnt reporter


3.0
6
*** 10 1.2 **
2.5 9 ***
5

Wnt reporter activity


Wnt reporter activity ** 1.0

Wnt reporter activity


Wnt reporter activity
8
4 2.0 7 0.8
6
3 1.5
5 0.6
4
2 1.0 0.4
3
1 0.5 2 0.2
1
0 0.0 0 0.0
Basal Wnt3A CTRL +Wnt3A Basal L cell 0 1 3 12
CM exosomes L cell SN
HEK293 exosomes
(P100) hours after ultracentrifugation
(P100)

e L cell SN f g L cells
CX P100
Mr (K) 0 12 h

L
L

TR
TR

i
Mr (K)

Ev
Ev
Wnt3A shRNA

C
C
30
TCF/Wnt reporter Wnt3A
Ponceau
25 30
1 2 *** ***
50
Wnt reporter activity

20 Evi
100
Percentage of of Wnt3A

80 15
50 -actin
60
protein

10
40
5 Calnexin
20 80

0 0
SN0 SN Basal CTRL Evi CTRL Evi shRNA Ponceau
L cells
L cell P100 L cell SN 1 2 3 4

Figure 2 Exosome-bound Wnt is active to induce signalling. HEK293 cells was quantified in Wnt3AL cell supernatant before and after indicated
were transiently transfected with plasmid encoding a luciferase-based hours of ultracentrifugation at 100,000g . SN, supernatant. (f) Loss of
TCF/Wnt reporter. (a) Conditioned medium from Wnt3AL cells was Wnt reporter activity in Wnt3AL cells supernatant and purified exosomes
used to induce reporter activity. (b) Reporter cells were stimulated by shRNA-induced silencing of Evi. (g) Western blot analysis of Wnt3A
with equal amounts of P100 of HEK293 cells transfected with either secretion by Wnt3AL cells treated as in f. CX, cell extract. Full images
empty plasmid (control, CTRL) or plasmid encoding Wnt3A. (c) P100 of blots are presented in Supplementary Fig. S3c. (ag) All experiments
of Wnt3AL cell supernatant was used to induce an Axin2 reporter in were carried out in triplicate (n = 3); error bars, s.d. Significance level as
HEK293 cells. (d,e) Wnt reporter activity and secreted Wnt3A protein indicated: P < 0.05, P < 0.01, P < 0.001.

secretion (Fig. 4d (right panels),f). Taken together, MVB maturation This suggests that a subset population of CD63GFP-positive exosomes
is required for proper distribution of EviWnt complexes, sorting onto also contains Wg, which is not easily detectable by total Wg staining.
ILVs and exosomal secretion of active Wnts. Other reports have suggested that exosomes contain different
sets of marker proteins44,45 . We therefore investigated whether Wg
Wg and Evi are secreted on exosomes in vivo from is present on distinct exosomal populations in vivo. On the basis
Drosophila wing disc of proteomic studies and immunohistochemistry (Supplementary
Next, we investigated whether Wg is secreted on exosomes in vivo. Fig. S4fh) in Drosophila cells, we analysed Wg co-localization with
Panakova et al. previously concluded that Wg is absent from exosomes Rab4, which is enriched in exosomes from human reticulocytes46 . A
as no co-localization was found with overexpressed CD63GFP by total substantial (29% 11) co-localization of extracellular Wg with Rab4
Wg staining (which primarily labels intracellular Wg; ref. 26). However, was observed (Supplementary Fig. S4d,e). These results demonstrate
in addition to the presence of ILV-containing MVBs throughout that extracellular Wg co-localized with different exosomal markers and
the wing disc (Supplementary Fig. S3b), we found that CD63GFP indicate that distinct exosomal populations might exist.
expressed at low levels clearly co-localized with intracellular Wg We next investigated whether Evi is secreted from wing disc cells,
(Fig. 5a, insets), suggesting that both proteins are present in the similar to its export at the neuromuscular junction. Both enhanced
same MVBs. We confirmed this co-localization using Lamp1GFP GFP (eGFP)- and Cherry-tagged Evi proteins are secreted from
as another MVB marker (Fig. 5b, insets). Further, image analysis the Wg-expressing cells (Supplementary Fig. S4a), and co-localize
and quantification showed that at least 8% of extracellular Wg with CD63GFP puncta outside the expression domain (Fig. 5c,
(ref. 43) co-localized with CD63GFP outside the producing cells insets), suggesting that Evi is secreted from wing disc cells on
(Supplementary Fig. S4c), conforming to observations in cell culture30 . exosomes. Interestingly, we found that only 10 6% of extracellular

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a CD81 Wnt TSG101 b


Mr (K) CX P100 SN
65 TSG101

65 Evi-C1

Wnt3A
30

CD81 Evi TSG101 80 Calnexin

50 -actin

e TCF/Wnt reporter
12
**
10

Wnt reporter activity


c Mr (K) CX 1 2 3 4 5 6 7 8 9 10 d CTRL HGS siRNA
65 TSG101 Mr (K) CX P100 CX P100 8

65 Evi 6
65
Evi-C1
4
30 Wnt3A
50 GAPDH 2

65 Ponceau 0
1.08 1.28 Basal CTRL HGS siRNA
1 2 3 4
Sucrose (g ml1) HEK293 SN

Figure 3 The secretory factor Evi is found on exosomes/shuttles Wnts on co-transfected with plasmids encoding EviGFP and Wnt3A. Cell extract
exosomes. (a) Representative images from immunofluorescence analysis and P100 were analysed for protein content by western blotting. Full
of MVB localization: HeLa cells were co-transfected with Wnt3AGFP images of blots (bd) are presented in Supplementary Figs S3c and
and TSG101Cherry (upper panel) or EviGFP and TSG101Cherry (lower S6a. (e) HEK293 cells transiently transfected with a luciferase-based
panel), and stained for CD81. Scale bars, 10 m. (b) HEK293 cells were TCF/Wnt reporter were induced by addition of conditioned medium from
co-transfected with plasmids encoding EviGFP and Wnt3A; representative HGS siRNA- or control-treated HEK293 cells (n = 3). CX, cell extract;
immunoblot of cell extract, P100 and supernatant. (c) As in b; P100 P100, 100,000g pellet; SN0 , supernatant before and SN1 , supernatant
was loaded on top of a step sucrose gradient (1.081.28 g ml1 ) and the after 100,000g centrifugation. Experiments were carried out in triplicate;
resulting ten fractions analysed for their protein content. (d) HEK293 cells error bars, s.d. Significance level as indicated: P < 0.05, P < 0.01,
were seeded on short interfering RNA (siRNA) against HGS or control, then
P < 0.001.

Wg co-localized with EviCherry (Fig. 5d, inset and Supplementary Next, we examined if Ykt6 is required for exosomal Wg secretion
Fig. S4b for total Wg), suggesting that in general Evi and Wg proteins in vivo by reducing Ykt6 levels in the Wg expression domain. Both
separate in MVBs and are not secreted on the same exosomes. the exosomal marker CD63GFP and Wg accumulated inside the
expressing cells (Fig. 6e,f). As a control, evi RNAi showed accumulation
Ykt6 is required for exosome-dependent Wnt secretion of Wg but normal secretion of CD63GFP (Fig. 6g,h), suggesting that
Although secretion of exosomes is blocked by perturbing endosomal defects in CD63GFP secretion in Ykt6 RNAi were not caused by
maturation, more specific blocking mechanisms exist. For example, defective Wg secretion. Different studies suggested that Ykt6 functions
members of the Rab protein family, Rab27a, Rab27b and Rab35, have at various steps during vesicular transport in yeast, for example in
been shown to block exosome secretion in a cell-type-dependent endoplasmic reticulum to Golgi transport49 and autophagocytosis50 ;
manner47,48 . To systematically identify proteins involved in secretion of therefore, we investigated whether Ykt6 affected general secretion in
Wnts on exosomes, we carried out an in vivo RNA interference (RNAi) Drosophila cells. First, we analysed the expression of the Hedgehog
screen of exosome-associated proteins in Drosophila (Supplementary target gene patched (ptc) in wing discs. Depletion of Ykt6 in the dorsal
Tables S1 and S2). This screen identified Ykt6 (CG1515) as a candidate compartment of the discs showed unaltered expression and membrane
gene to block Wg secretion. localization of the transmembrane protein Ptc (Supplementary
First, we analysed whether Ykt6 affects Wg secretion in vivo. Fig. S5a,b), indicating normal Hh signalling and proper trafficking of
Depletion of Ykt6 by RNAi in the posterior part of the wing disc Ptc to the membrane. Similarly, Ykt6 RNAi did not affect localization of
(marked with GFP) showed accumulation of Wg inside the producing the multipass transmembrane protein Flamingo (Fmi; Supplementary
cells (Fig. 6a). Concordantly, extracellular Wg was reduced in the RNAi Fig. S5c). These experiments indicate that, in contrast to other
transgene-expressing domain (Fig. 6b). In addition, depletion of Ykt6 transmembrane proteins we analysed, depletion of Ykt6 affects the
in Wg-producing cells reduced the expression of the Wg target gene secretion of exosomal marker and Wg in vivo.
senseless in the neighbouring target cells when compared with control To assess whether this role of Ykt6 is conserved, we tested its require-
discs and led to an adult wing notch phenotype (Fig. 6c,d), consistent ment for Wnt secretion and endosomal sorting in human cells. Whereas
with a defect in Wg signalling. Taken together, we conclude that Ykt6 knockdown of the retromer components VPS26/35 significantly re-
is required for Wg secretion and signalling. duced Wnt3A and EviGFP on exosomes, the amount of the exosomal

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a c EEA1 Wnt3A Evi

H ycin
ci
N my

69
DM 69

om
DMSO

SO

SO

G l
G l

48
4C
4C

48
o
fil

fil

W
DM
Mr (K)

W
H
Ba

Ba

N
50
Wnt3A

Ponceau Bafilomycin

1 2 3 4 5 6 7 8

CX P100

b TCF/Wnt reporter NH4Cl


n.s.
1.2 **
Wnt reporter activity

1.0
0.8 GW4869
0.6
0.4
0.2
0.0
Basal

DMSO

Bafilomycin

NH4Cl

GW4869

e 1,000 f 1,400

Number of secreted CD63GFP puncta


900
Number of secreted Wg puncta

1,200
d 800
DMSO DMSO
700 1,000
600
800
500
400 600
Wg (total) wg::CD63GFP
300 400
Bafilomycin Bafilomycin 200
200
100
0 0
in

in
SO

SO
yc

yc
DM

DM
om

mo
fil

wg::CD63GFP

fil
Wg (total)
Ba

Figure 4 Mechanisms of MVB sorting of Wnt proteins. (a) Wnt3AL (d) Ex vivo treatment of Drosophila wing discs with V-ATPase inhibitor Ba
cells were treated with 20 nM bafilomycin A1, 50 mM NH4 Cl, 5 M bafilomycin A1 (200 nM in DMSO) for 2 h at room temperature causes a
GW4869 or control vehicle. The amount of Wnt3A was determined block in Wg secretion. Shown (lower left panel) is the total Wg staining
in the cell extract and P100. Full images of blots are presented in for secreted Wg protein puncta. Similarly, bafilomycin A1 also blocked
Supplementary Fig. S6a. (b) Wnt activity of cell culture supernatants CD63GFP secretion (lower right panel). Control treatment with DMSO
from a was determined in HEK293 cells transfected with plasmids did not have a significant effect on Wg or CD63GFP secretion (upper
encoding TCF/Wnt reporter. n = 3; error bars, s.d.; significance values panels). Scale bars, 20 m. (e) Quantification of the secreted Wg puncta

P < 0.05, P < 0.01, P < 0.001. (c) HeLa cells were co-transfected (n = 4). (f) Quantification of CD63GFP secretion (n = 4); error bars, s.d.
with plasmids encoding EviV5, Wnt3AGFP and Rab5QL, stained for CX, cell extract; P100, 100,000g pellet; SN0 , supernatant before; SN1 ,
EEA1 and analysed by immunofluorescence microscopy. Scale bars, 1 m. supernatant after 100,000g centrifugation.

marker CD81 was unchanged, indicating that retromer function is not and Supplementary Fig. S5eg). Ultrastructural characterization of
required for exosome formation (Fig. 7a,b). In contrast, knockdown YKT6 depletion in HEK293 cells indicates an increased sorting of the
of the ESCRT-0 components HGS/TSG101 shows their requirement exosomal marker CD63 from MVBs into lysosomes (Supplementary
for secretion of CD81, Wnt3A and EviGFP. Strikingly, YKT6 siRNA Fig. S6b). Thus, the role of Ykt6 in exosome-associated Wnt secretion is
has a similar effect, significantly blocking all three proteins in the P100 evolutionarily conserved. In summary, the data show that active Wnts
exosomal fraction, indicating that YKT6 siRNA reduces the amount of are secreted on exosomes, a process that requires shuttling through
Wnt-containing exosomes (Fig. 7a,b and Supplementary Fig. S5d). Evi, and that Ykt6 blocks exosome-marker and Wnt secretion in vivo,
In addition, we analysed the Wnt activity of supernatant from these resulting in reduced Wnt signalling.
cells. Loss of retromer, ESCRT-0 and YKT6 significantly decreased Wnt
activity in the supernatant to 50% of the control supernatant (Fig. 7c; DISCUSSION
also compare Fig. 3e). Yet, depletion of Ykt6 had no significant effect In the present study, we demonstrate by biochemical and genetic
on general secretion in human and Drosophila reporter assays, whereas approaches that a portion of functional Wnts is secreted on exosomes.
Evi and Wnt3A were still found in endosomal compartments (Fig. 7d The discrepancy between the hydrophobic properties of Wnts and their

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ARTICLES

Wg (total) CD63GFP Merge wg::CD63GFP

Wg (total) Lamp1GFP Merge wg::Lamp1GFP

wg::eviCherry
EviCherry CD63GFP Merge wg::CD63GFP

EviCherry Wg (ex) Merge wg::eviCherry

Figure 5 Wingless is secreted on exosomes during Drosophila (c) Co-expression of EviCherry with CD63GFP and wgGal4 shows
imaginal disc development. (a) The exosomal marker CD63GFP co-localization outside the expression domain. (d) Extracellular (ex) Wg
was expressed for 3 h using wgGal4; tubGal80 ts . Total Wg staining staining on discs overexpressing EviCherry shows little co-localization
shows co-localization with the CD63GFP-positive vesicles inside (inset). Inset: Magnified and enhanced image of the indicated box. The
the Wg-producing cells (inset). (b) MVB marker Lamp1GFP was image represents a merge of two confocal sections at 0.5 m distance,
expressed for 4 h. Total Wg staining shows co-localization with the except for d, which is a merge of three confocal sections. Scale bars,
Lamp1GFP-positive vesicles inside the Wg-producing cells (insets). 20 m (ad).

trafficking over longer distances has been a puzzling observation, lead- alternative route of Wnt secretion in vivo that is independent of lipopro-
ing to different hypotheses on how Wnts disperse. Our results demon- tein particles. The biochemical fractionation method differs from
strate that Wnts are secreted on exosomes in vivo and ex vivo. These the parameters used to biochemically isolate lipoprotein particles35,54 ;
secreted microvesicles originate through inward budding in MVBs and in addition, lipoprotein particles were not found by ultrastructural
influence intercellular trafficking5153 . Several lines of evidence support analysis in our preparations. Taken together, we believe that our study
our conclusions. First, by biochemical fractionation we find Wnt provides solid evidence for a Wnt secretion route through exosomes.
co-segregating with exosomes derived from mammalian and Drosophila Ykt6 belongs to the longin type of R-SNARE involved in various
cells. Ultrastructural analysis demonstrated that Wg is found on the trafficking events. Studies in yeast have suggested that Ykt6 localizes to
outer membrane of exosomes. Second, we demonstrate that Wnts are the Golgi and to endosomal and vacuolar membranes49,55 . We show
shuttled to MVBs by their cargo receptor Evi; this step is impaired by that in Drosophila Ykt6 does not affect secretion of transmembrane
inhibition of MVB maturation or depletion of the ESCRT-0 complex, proteins such as Ptc and Fmi, and that on loss of Ykt6 in HeLa cells Evi
which mediates cargo recognition of exosomal proteins. Third, we show accumulates in early endosomes but not in the Golgi. This finding is
that a fraction of extracellular Wg co-localizes with several exosomal consistent with the role of Ykt6 in early/recycling endosomes described
markers in vivo. Fourth, we identify Ykt6 as a protein required for in mammalian cells56 ; however, further functional roles for Ykt6 might
secretion of Wnts and exosomal proteins. Our results argue for an exist and might also be modulated in a cell-type-specific manner.

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 7


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ARTICLES

Rel. intensity of Wg (total)


2.0

1.5

1.0

0.5
RNAi
0.0
Wg (total) en::Ykt6 RNAi

1
31
61
91
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241
271
301
331
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391
Wg (total) GFP en::GFP
b x position

Rel. intensity of Wg (ex)


2.5
2.0
1.5
1.0
0.5
Wg (ex) en::Ykt6 RNAi RNAi
Wg (ex) GFP en::GFP 0.0

1
31
61
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121
151
181
211
241
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391
c
x position

Sens wg::Rel RNAi Sens wg::Ykt6 RNAi wg::Rel RNAi wg::Ykt6 RNAi

e f

Wg (total)

GFP

wg::Ykt6 RNAi
Wg (total) GFP Merge wg::CD63GFP Merge
g h

Wg (total)

GFP

wg::evi RNAi
Wg (total) GFP Merge wg::CD63GFP Merge

Figure 6 Ykt6 is a required regulator for exosomal secretion of Wingless. wgGal4>UAS Ykt6 RNAi discs when compared with discs expressing
(a) RNAi-mediated knockdown of Ykt6 using an enGal4,UASGFP relish RNAi (Wg-unrelated gene); compare arrow heads in right and
driver line shows accumulation of total Wg inside the GFP-positive left panels. (d) Adult wing notch phenotype (arrowheads) seen in the
producing cells. (a, right panel) Plot profile of the total Wg staining wgGal4>Ykt6 RNAi. (e,f) Knockdown of Ykt6 with wgGal4 shows
in the shown inset, comparing Wg in the anterior half of the discs accumulation of total intracellular Wg (e, left panel) and CD63GFP (e,
(no GFP) with the posterior RNAi-expressing (GFP-positive) half. middle panel), which can also be seen at higher magnification shown in
(b) Extracellular Wg staining on the discs expressing Ykt6 RNAi f. (g,h) Knockdown of Evi only shows accumulation of total intracellular
with the enGal4,UASGFP driver show reduced Wg (in the RNAi Wg (g, left panel) whereas CD63GFP was normally secreted (g, middle
domain). (b, right panel) Plot profile of the extracellular Wg staining panel), also visible in higher magnification h. n 4 in all the above
in the inset, similar to a. (c) Wg target gene Sens is reduced in the experiments. Scale bars, 20 m.

Interestingly, our results imply that the retromer complex acts supported by the finding that depletion of retromer leads to lysosomal
upstream of MVB sorting of EviWnt complexes, as demonstrated sorting, thereby reducing the pool of Evi as well as inhibiting the
by the effect of ESCRT and Ykt6 RNAi on secretion of Wnt/Evi secretion of Wnts on exosomes (Supplementary Fig. S2c). It has
and the exosomal marker CD81. The retromerSNX3 sorting route been shown that SNX3 together with HGS functions in sorting
might decide whether unloaded Evi is recycled to the Golgi or and membrane invagination at the MVB level57 . In yeast, SNX3,
whether EviWnt complexes are packaged onto exosomes. This is similar to Ykt6 (ref. 55), retrieves cargo receptors to Golgi before

8 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION


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ARTICLES

a b c TCF/Wnt reporter

YK 6/ 01

YK 6/3 01
S2 G1

1
***

S2 G
Wnt3A

T6 5
T6 35
VP TS

VP /TS
1.4 ***
CD81 ***

S/
L

H L
S
TR

TR
G

G
Mr (K) siRNA

Wnt reporter activity


1.2
C

C
1.2

Rel. amount of
50
1.0 1.0
Wnt3A

protein
0.8 *
* ** 0.8
0.6
25 0.4 ** ***
0.6
0.2
CD81 0.4
siRNA

T6
35
0.2

TR

10

YK
6/
65

G
C

S2
Evi

TS
0.0

VP
S/

CTRL

VPS26/35

HGS/TSG

YKT6
siRNA

G
H
Ponceau P100

1 2 3 4 5 6 7 8
SN added
CX P100
e
Exosomes
d SWIM Lipid-modified Wnt
EEA1 EVI TGN46
CTRL siRNA

Plasma membrane

Rab5QL
Retromer/SNX3
YKT6 siRNA

EEA1 EVI TGN46


ESCRT
Evi

Rab5QL Lipid-modified
YKT6
EEA1 EVI TGN46 Golgi
HGS siRNA

MVB

Rab5QL Endoplasmic reticulum

Figure 7 YKT6 blocks Wnt3A and exosome secretion. (a) HEK293 cells were level, P < 0.05, P < 0.01, P < 0.001. (c) Wnt activity of cell culture
co-transfected with plasmids encoding EviGFP and Wnt3A, then exosomes supernatants from a was determined in HEK293 cells transfected with
were purified by ultracentrifugation and cell-number-normalized amounts plasmids encoding TCF/Wnt reporter. n = 4; error bars, s.d. (d) Representative
loaded onto an SDSpolyacrylamide electrophoresis gel. Representative images from immunofluorescence analysis of control and YKT6 siRNA HeLa
western blot; n = 3. Full images of blots are presented in Supplementary cells, co-transfected with plasmids encoding Rab5QL and EviGFP and
Fig. S6a. CX, cell extract; CTRL, control. (b) Densitometric quantification stained for Golgi marker TGN46 and early endosome marker EEA1. n = 3;
of a. Error bars, s.d.; significance level as compared with control protein scale bars, 10 m. (e) Model of exosome-dependent Wnt secretion.

homotypic fusion of vacuoli5860 . Thus, it is tempting to speculate that a ACKNOWLEDGEMENTS


retromerSNX3-dependent step retrieves empty Evi to Golgi, whereas We thank T. Soellner, T. Buechling and D. Kranz for helpful comments on the
manuscript. We would like to thank T. Vaccari, S. Eaton, S. Cohen, C. Niehrs,
Wnts (and Evi) are packaged onto exosomes for functional release. H. Bellen and the Bloomington stock centre for fly strains and reagents. We thank
Different extracellular forms of Wnts might not be mutually VDRC for the RNAi lines. We are grateful for help from the CellNetworks Electron
exclusive. Our results show that removal of vesicle-bound Wnt only Microscopy Core Facility (EMCF), the Light Microscopy Core Facility at the DKFZ
and the Mass Spectrometry Core Facility at the DKFZ. J.C.G was supported by the
partially reduced Wnt activity, implying that other secretion routes CellNetworks postdoctoral fellowship programme. The DFG Wnt Research Group
exist, possibly through the direct release from the plasma membrane, FOR1036 supported research in the laboratory of M.B.
which might have different extracellular trafficking properties.
It is tempting to speculate that morphogens provide spatial AUTHOR CONTRIBUTIONS
J.C.G. designed experiments, carried out the biochemical and cell-biological
information in other forms than concentrations of signalling molecules, experiments and wrote the manuscript. V.C. designed experiments, carried out
involving a digital rather than analogue mode of encoding signals. the in vivo experiments and wrote the manuscript. K.B. provided essential advice,
Different packaging modes of Wnt molecules and their control in generated in vivo reagents and contributed to writing the manuscript. M.B. designed
experiments and wrote the manuscript.
Wnt-producing cells might have a profound effect on spreading and
signalling properties of morphogens.  COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
METHODS Published online at www.nature.com/doifinder/10.1038/ncb2574
Reprints and permissions information is available online at www.nature.com/reprints
Methods and any associated references are available in the online
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2. Zecca, M., Basler, K. & Struhl, G. Direct and long-range action of a wingless
Note: Supplementary Information is available in the online version of the paper morphogen gradient. Cell 87, 833844 (1996).

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2012 Macmillan Publishers Limited. All rights reserved.
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DOI: 10.1038/ncb2574 METHODS

METHODS control and evi shRNA (Sigma). Both UASevi-PBCherry and UASevi-PBeGFP
Exosome purification. Exosomes were purified by differential centrifugation as were generated by cloning the PB isoform of evi into the pCherry-N1 and
described previously31 . In short, supernatants from mammalian and Drosophila pEGFP-N1 vectors, respectively, and then cloned into pUAST vector (EcoRI, XbaI).
cells were subjected to sequential centrifugation steps of 300g , 2000g and 10,000g Constructs were confirmed by sequencing. pET21a-Sens-N-term (a gift from H.
before pelleting exosomes at 100,000g in a SW41Ti swinging bucket rotor for 3 h Bellen, Baylor College of Medicine, Houston, USA, ref. 62) shRNA against Evi, 50 -
(Beckman). Supernatant was discarded and exosomes were taken up in 1/100 of GATCTACAAGTTGACCCGCAA-30 , control, 50 CCCGTGTAAATATGTACATTT-
their original volume in H2 O. For sucrose gradient purifications, P100 was loaded 30 , from Sigma.
on top of a sucrose step gradient (0.82 M) and centrifuged at 100,000g for 3 h Dharmacon siRNA SMARTpools were used for HGS (No. 9146), TSG101
(ref. 34). Ten fractions of 1 ml were collected and protein content enriched by (No. 7251), VPS26a (No. 9559), VPS35 (No. 25479), YKT6 (No. 10652), GL3
acetone precipitation (1:4, v/v). To purify exosomes for mass spectrometry, Kc167 (D-001400-01-20).
supernatant was treated as described above. In addition, exosomes were purified on VPS26a: 50 -CCACCUAUCCUGAUGUUAA-30 ; 50 -GCUAGAACACCAAGGA-
a 30% sucrose cushion, as described by Stoeck and colleagues34 . AUU-30 ; 50 -CCAAUUUGUGAGAUCGAUA-30 ; 50 -AAACCUAGCCUUUAAGC-
AA-30 .
Antibodies. Antibodies were used against Wg, 1:5 for extracellular staining VPS35: 50 -UUACAAGUCUCUCUGAUUA-30 ; 50 -GUUGUUAUGUGCUUA-
and 1:50 for total (mouse, 4D4, DSHB), Fmi, 1:50 (mouse, Flamingo no 74, GUAA-30 ; 50 -AAAUACCAGUUGACACUUA-30 ; 50 -GAACAUAUUGCUACCAG-
DSHB), Ptc, 1:10 (mouse, Apa-1, DSHB), Wnt3A, 1:500 (WB; rabbit (2391), UA-30 .
CST), Wnt5A, 1:500 (WB; rabbit (2530), CST), Evi, 1:500 (WB; rabbit, ref. 61), TSG101: 50 -AAACUGAGAUGGCGGAUGA-30 ; 50 -GAACCUCACUGGAA-
beta-actin (AC-15), 1:20,000 (WB; mouse (ab6276), Abcam), TSG101, 1:1,000 (WB; CAAUC-30 ; 50 -CCGUUUAGAUCAAGAAGUA-30 ; 50 -UCCCACAGCUCCCUUAU-
rabbit (HPA006161), Sigma), CD81 (1.3.3.22), 1:1,000 (WB; mouse (DLN-09707), AC-30 .
Dianova), GM130, 1:300 (IF; mouse (610823), BD), LRP6, 1:2,000 (rabbit, a kind HGS: 50 -GCACGUCUUUCCAGAAUUC-30 ; 50 -AGAGAGCGAUGCCAUG-
gift from C. Niehrs, DKFZ, Heidelberg, Germany), ApoII, 1:5,000 (WB; rabbit, a UUU-30 ; 50 -GAUAUUCUGUGGAAAGUGU-30 ; 50 -GUAAACGUCCGUAACAA-
kind gift from S. Eaton, MPI, Dresden, Germany), GAPDH (6C5), 1:5,000 (WB; GA-30 .
mouse (AM4300), Ambion), EEA1, 1:300 (IF; mouse (610456), BD), calnexin, YKT6: 50 -GCUCAAAGCCGCAUACGAU-30 ; 50 -GUGAGAAGCUAGAUGAC-
1:1,000 (WB; rabbit (sc-11397), St Cruz), and TGN46, 1:200 (IF; rabbit (ab50595), UU-30 ; 50 -GAAGGUACUAGAUGAAUUC-30 ; 50 -AAGCUGAUCCCAUGACUAA-
Abcam). Antibodies against Sens were generated by expressing and isolating Sens 30 .
amino-terminal fusion protein using the previously published pET21a-Sens-N-term Dharmacon siRNA SMARTpools were used for beta-catenin (No. 1499), Evi (No.
(ref. 62) plasmid (see plasmids) from BL21 (DE3)-pLysS cells and injected into 79971), Rab5A (No. 5868), Rab7A (No. 7879), VPS4B (No. 9525), Rab27A (No.
guinea pig (Eurogentec). Purified serum was used at 1:500 dilution. Secondary 5873) and Rab27B (No. 5874).
antibodies used were anti-guinea pig-Alexa594 (1:500 (A11076), Invitrogen), Beta-catenin: 50 -GCTGAAACATGCAGTTGTA-30 ; 50 -GATAAAGGCTACT-
anti-mouse-Alexa594 (1:500, (A11005), Invitrogen), anti-mouse-Alexa488 (1:500 GTTGGA-30 ; 50 -CCACTAATGTCCAGCGTTT-30 ; 50 -ACAAGTAGCTGATATTG-
(A11001), Invitrogen) and HRP-labelled anti-mouse, anti-rabbit (1:20,000 (NA931, AT-30 .
NA934), Amersham). Evi: 50 -ACGAATCCCTTCTACAGTA-30 ; 50 -TAACGGAAGGCCATTGGAA-30 ;
50 -TAAAGGATATCCGGTTGGT-30 ; 50 -GAACCACATCGCAGGGTAT-30 .
Cell culture. Drosophila Kc167 and S2 cells were maintained at 25 C in Schneiders Rab5A: 50 -GAACTGCGCCACTTCGTCA-30 ; 50 -CAACCCAAGATGTTCGTTA-
medium (Invitrogen) supplemented with 10% fetal calf serum (Biochrom) and 30 ; 50 -GACACAAGAGATTGAGTAA-30 ; 50 -TGAACACAATTCTGGTGAA-30 .
50 g l1 penicillin/streptomycin (Invitrogen). Cells were transiently transfected Rab7A : 50 -CTAGATAGCTGGAGAGATG-30 ; 50 -GTACAAAGCCACAATAGGA-
with Effectene (Qiagen) according to the manufacturers instructions. HEK293, 30 ; 50 -AAACGGAGGTGGAGCTGTA-30 ; 50 -CGAATTTCCTGAACCTATC-30 .
Wnt3AL and Caco2 cells were maintained in DMEM (Gibco) supplemented with VPS4B: 50 -CCTCTGATCTTGTTTCTAA-30 ; 50 -GAGAACAAGCCCTCCATTA-
10% fetal calf serum (Biochrom) and 50 g l1 penicillin/streptomycin (Invitrogen) 30 ; 50 -GAAGCCGCACGTAGAATTA-30 ; 50 -CGATAGATCTGGCTAGCAA-30 .
at 37 C in a humidified atmosphere with 5% CO2 . Human cells were transiently Rab27A: 50 -GAGCAAAGTTTCCTCAATG-30 ; 50 -GGAGAGGTTTCGTAGCT-
transfected with Dharmafect1 (ThermoFisher) for siRNA and TransIT (Mirus) for TA-30 ; 50 -CCAGTGTACTTTACCAATA-30 ; 50 -CGACAGCGTTCTTCAGAGA-30 .
plasmids according to the manufacturers instructions. Rab27B: 50 -CAACAGAGCTTCTTAAATG-30 ; 50 -GGAACTGGCTGACAAATAT-
30 ; 50 -GCAAATGCTTATTGTGAAA-30 ; 50 -GGGAACTGGCTGACAAATA-30 .
Cell mixing experiments. Kc167 cells were transiently transfected with plasmid Kc167 cells were seeded on double-stranded RNA (dsRNA) and incubated for
encoding Actyellow fluorescent protein (YFP)Rab4 construct; 48 h later, cells 45 min in serum-free medium at 25 C. dsRNAs were designed and generated as
were mixed for 5 h with another population of Kc167 cells which were treated described previously9,68 .
with mitotracker-580 (50 nM) live cell dye for one hour and were washed with Forward (F) and reverse (R) primers for dsRNA for Drosophila cells (with T7
PBS before mixing. Cells were then fixed and imaged using a Leica SP5 confocal promoter sequences for in vitro transcription in lower case):
microscope. Sar1, F 50 -taatacgactcactatagggGACGCGTCTGGAAGGACTAC-30
R 50 -taatacgactcactatagggTAGCTGATACAGTCCGAACACG-30
Drosophila stocks. The following Drosophila stocks were used in this study: Evi, F 50 -taatacgactcactatagggGTTCAAGGGTGGTCCCATTT-30
UASLamp1GFP (a gift from T. Vaccari, IFOM-FIRC, Milan, Italy; ref. 63), R 50 -taatacgactcactatagggTGGGCAGAGTCCTCTGGAT-30
UASGFP;CD63 (a gift from S. Eaton, MPI, Dresden, Germany, ref. 26) , wgGal4 HGS, F 50 -taatacgactcactatagggTCATTGATGTGGCAGCACTA-30
(second chr., a gift of S. Cohen, IMCB, Singapore), apGal4 (second chr.; ref. 64), R 50 -taatacgactcactatagggTTGTCGAAACTTGATCGAAACA-30
enGal4, UASGFP (second chr., ref. 65), UASYFPrab4 (Bloomington Stock TSG101, F 50 -taatacgactcactatagggTTTGAATCGAGAGCTTGAAAGAG-30
no 23269) and UASrab4mRFP (Bloomington Stock no 8505). The following R 50 -taatacgactcactatagggCAACGCTTTTCTCCCTGGTA-30
RNAis were obtained from Vienna Drosophila RNAi Center: UASYkt6 RNAi VPS35, F 50 -taatacgactcactatagggTCAGGACGTCCAATGTGTCT-30
(KK no 105648) and UASrel RNAi (KK no 108469). UASeviPBCherry (on R 50 -taatacgactcactatagggAGGAATTTCGACTTCTGGCG-30
second and third chr.) and UASeviPBeGFP (on second chr.) were generated as VPS26, F 50 -taatacgactcactatagggATATGGCAACGCCGTAGTGT-30
previously described12 . R 50 -taatacgactcactatagggGATGGAATGTCGACGCTGT.

Wnt reporter activity assay. HEK293 cells were transfected with TCF/firefly Statistics. All experiments were carried out at least in biological triplicate. Error
(a kind gift from K. Demir, Boutros Laboratory, DKFZ) or Axin2/firefly Wnt bars indicate s.d. Statistical significance was calculated by carrying out Students
reporter and actinRenilla luciferase and Wnt3A (50 ng DNA) and 20 nM siRNA t -test.
for autocrine assays in 384-well plates using TransIT (Mirus) and Dharmafect1
(Dharmacon), respectively. For quantification of Wnt activity of exosomes and Immunostainings, microscopy and image analysis. Extracellular Wg staining
the activity of Wnt secreted into the supernatant, HEK293 cells were transfected (4D4, 1:5) was carried out as described in ref. 43. Other immunostainings were
with plasmids encoding TCF/firefly Wnt reporter and actinRenilla luciferase and carried out as per standard procedures. Staining and microscopy conditions were
reporter activity induced by addition of equal amounts of supernatant or exosomes kept identical for discs used for comparisons. Images were taken with a Leica SP5
from different samples. confocal microscope and were analysed using ImageJ software. Quantification of
co-localization was carried out using CellProfiler 2.0 software69 , where ten single
Plasmids, shRNA, siRNA and dsRNA. pCMV-hEviGFP, pCMV-Wnt3AGFP, confocal scans from each disc were analysed and punctate structures of size 1100
pCMV-Wnt3A, pCMV-TSG101Cherry (Addgene plasmid no 21505; ref. 66), pAc- pixels were counted. For immunofluorescence assays in S2 cells, the cells were
Wg, pAc-PB-EviGFP, Tsp96FHA, Tsp42EfHA (DGRC), Evi/WIsV5 (ref. 16), transiently transfected with EviCherry and Tsp42Ef or Tsp96F and induced with
Rab5QL (ref. 67). Axin2-promotor luciferase, (Addgene no 21275), pLKO shRNA 0.4 mM CuSO4 ; 48 h later cells were plated on Concavalin A coated slides and left

NATURE CELL BIOLOGY


2012 Macmillan Publishers Limited. All rights reserved.
METHODS DOI: 10.1038/ncb2574

to attach for 2 h (ref. 70), before standard immunofluorescence staining was carried medium, after which Wg antibody (4D4, 1:5) was added to the disc-containing
out as described above. solutions and shifted on ice for a further 1 h to enable antibody binding. After this,
discs were briefly washed with ice cold PBS and fixed with 4% paraformaldehyde for
Transfection. Cells were reverse transfected with siRNAs, seeded onto 12 mm secondary antibody labelling.
glass coverslips, 24 h later transfected with indicated plasmids, and 4872 h later
fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Electron microscopy. Purified exosomes were left to settle on carbon-coated grids.
For immunofluorescence labelling, cells were blocked in 10% FCS/PBS. Primary After staining with 3% uranyl acetate, grids were air-dried and visualized using
antibodies in 10%FCS/PBS were incubated for 1 h at room temperature and a transmission electron microscope (Zeiss EM900). For immunogold labelling,
antibody binding visualized by fluorochrome-conjugated secondary antibodies. pelleted exosomes were plated on grids, blocked and stained in anti-Wg antibody
(4D4, 1:5), then incubated in secondary anti-mouse antibody and labelled with
Real-time PCR with reverse transcription Total RNA was extracted using an protein Agold particles (10 nm). Each staining step was followed by five PBS washes
RNeasy extraction kit (Qiagen) according to the manufacturers instructions. Re- and finally ten washes in H2 O before contrast staining with 3% uranyl acetate.
verse transcription and quantitative PCR were carried out with 25 ng complementary Cryosectioning was carried out as described previously72 .
DNA and LightCycler 480 Probes Master as described (Roche). Relative mRNA
expression was calculated as a fold change versus control71 . Human primers were 61. Augustin, I. et al. The Wnt secretion protein Evi/Gpr177 promotes glioma
used against HGS, TSG101, VPS35, VPS26, YKT6, Evi and GAPDH (human tumourigenesis. EMBO Mol. Med. 4, 3851 (2012).
Universal Probe Library). Human quantitative PCR primers with probe number 62. Nolo, R., Abbott, L. A. & Bellen, H. J. Senseless, a Zn finger transcription factor,
from Universal Probe Library: is necessary and sufficient for sensory organ development in Drosophila. Cell 102,
HGS Left No. 17 50 -gtggccaacaagcagacc-30 349362 (2000).
Right No. 17 50 -cggacgtttacctccacttg-30 63. Vaccari, T., Duchi, S., Cortese, K., Tacchetti, C. & Bilder, D. The vacuolar ATPase
is required for physiological as well as pathological activation of the Notch receptor.
TSG101 Left No. 64 50 -gataccctcccaatcccagt-30
Development 137, 18251832 (2010).
Right No. 64 50 -tgttgtggcaggatatggac-30
64. Montagne, J. et al. Drosophila S6 kinase: a regulator of cell size. Science 285,
VPS35 Left No. 31 50 -accctgaccagcagtacttga-30 21262129 (1999).
Right No. 31 50 -gtggcagtgtgaagcgaat-30 65. Thompson, B. J. & Cohen, S. M. The Hippo pathway regulates the bantam microRNA
VPS26 Left No. 18 50 -tgcttgttgatgaggaagacc-30 to control cell proliferation and apoptosis in Drosophila. Cell 126, 767774 (2006).
Right No. 18 50 -cctcagtttttcaggagctttt-30 66. Lee, H. H., Elia, N., Ghirlando, R., Lippincott-Schwartz, J. & Hurley, J. H. Midbody
YKT6 Left No. 67 50 -ttgctgacaatgaatacccatc-30 targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55. Science
Right No. 67 50 -ttgcttggagaattcatctagtacc-30 322, 576580 (2008).
Evi Left No. 38 50 -gcgctccagcacctctgaagt-30 67. Jullien, J. & Gurdon, J. Morphogen gradient interpretation by a regulated trafficking
Right No. 38 50 -gtgactgtgacaactgcaaatgc-30 step during ligand-receptor transduction. Gen. Dev. 19, 26822694 (2005).
GAPDH Left No. 60 50 -agccacatcgctcagacac-30 68. Horn, T. & Boutros, M. E-RNAi: a web application for the multi-species design of
Right No. 60 50 -agccacatcgctcagacac-30 . RNAi reagents2010 update. Nucleic Acids Res. 38, W332-339 (2010).
69. Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and
quantifying cell phenotypes. Gen. Biol. 7, R100 (2006).
Wing imaginal disc treatment and culturing. For treatment of discs with
70. Rogers, S. L., Rogers, G. C., Sharp, D. J. & Vale, R. D. Drosophila EB1 is important
inhibitor bafilomycin A1, discs were dissected in Schneiders medium supplemented for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol.
with 10% FCS and incubated for 2 h at room temperature with either dimethyl- 158, 873884 (2002).
sulphoxide (DMSO) or 200 nM of bafilomycin A1 in Schneiders medium. Discs 71. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative
were then briefly washed with PBS and fixed with 4% paraformaldehyde for im- C(T) method. Nat. Protoc. 3, 11011108 (2008).
munostaining. However, for extracellular Wg staining, discs were incubated for 1 h 72. Slot, J. W. & Geuze, H. J. Cryosectioning and immunolabeling. Nat. Protoc. 2,
at room temperature with either DMSO or 200 nM bafilomycin A1 in Schneiders 24802491 (2007).

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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

DOI: 10.1038/ncb2574

a b c
P100 Hek293 cells P100 P100 Caco-2 cells
kDa CX AP BL

Apical supernatant
anti-CD63 calnexin
80

65 Tsg101

Wnt5A
30
50 Evi

30

Basolaateral
CD81

d Ponceau
Wg antibody
unspecific control 1 2 3
100
***
80
% Gold particles

60 e
40 Kc167 cells
CX P100
20
kDa Tsp42ef Tsp96F Tsp42ef Tsp96F
0 30
<40 40-100 HA
size of
labelled structures
(um)

f Drosophila S2 cells

Merge TSP96F-HA Evi

Merge TSP42ef-HA Evi

Supplementary Fig. S1
Figure S1 Characterisation of exosomes from human and Drosophila cells. of Drosophila exosomes from Fig.1h. Immunostanings were performed
(a) Immunogold labeling of Hek293 P100 with anti-CD63 antibodies. in triplicates from three independent exosomal preparations. Error bars
Scale bar 100 nm. (b) Immunoblot of cell extract (CX) and 100,000g represent s.d. Significance level as indicated: * P<0.05, ** P<0.01, ***
pellet (P100) of polarized Caco-2 cells grown on transwell filter support P<0.001 (e) Immunoblot of cell extract (CX) and 100,000g pellet (P100)
(AP) apical and (BL) basolateral supernatant. (c) Representative of Drosophila Kc167 cells transfected with Tsp42ef-HA or Tsp96F-HA (f)
transmission electron microscope image of the P100 fraction from Representative images from immunofluorescence analysis of Drosophila
supernatant of Caco-2 cells Scale bars is 100 nm (d) Quantification of S2 cells cotransfected with Tsp42ef-HA or Tsp96F-HA and Evi-GFP. Scale
Wg specific and unspecific control labeling of the immuno-gold staining bars 5 m.

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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

a b
Tcf/Wnt-reporter
qPCR
** 1

knock down efficiency


6

Wnt reporter activity


5 0.8
4
0.6
3
0.4
2
1 0.2

0 0
basal CTRL Wnt3A CTRL Wnt3A
siRNA

L
DNA DNA

S
TR

G
H
C
SN0 SN

c GM130 Evi-C2 Lysotracker


L cells

GM130 Evi-C2 Lysotracker


L cells siVPS26

Tcf/Wnt-reporter
d 1.2 Tcf/Wnt-reporter
f
0.018
rel. Wnt reporter activity

1
Wnt reporter activity

* 0.014
0.8
0.01
0.6 ***
** *** 0.006
0.4 *** ***
0.002
0.2 *** 0
GW4869
Bafilomycin

NH4Cl
DMSO
basal

0
basal GL3 bcat Evi HGS Tsg101 rab5 rab7 VPS26 VPS35 siRNA

e
1.4 Tcf/Wnt-reporter g Tcf/Wnt-reporter
1.2 1.4 ***
Wnt reporter activity

SN0 SN
rel. Wnt reporter activity

1 1.2 n.s.
* ***
0.8 *** 1 *
0.6 *** 0.8
0.4 0.6
0.2
*
0.4
0 0.2
siRNA
rab5A
CTRL

VPS4B

rab27AB
VPS35/26

HGS/TSG

0
Bafilomycin
DMSO

GW4869
CTRL

NH4Cl

SN added

Supplementary Fig. S2
Figure S2 Components regulating Wnt activity in the supernatant. (a) (n=3) (e) Paracrine Wnt Assay: Supernatant from Hek293 cells treated with
Hek293 cells were transiently transfected with a luciferase-based TCF/Wnt indicated siRNA were used to induce TCF/Wnt reporter cells. Supernatants
reporter and stimulated with supernatant from Wnt3A/Evi-GFP transfected from three independent experiments were used, error bars are s.d.,
Hek293 cells before and after ultracentrifugation. n=3, error bars are significance level as indicated: * P<0.05, ** P<0.01, *** P<0.001 (d and
s.d. , significance level as indicated: * P<0.05, ** P<0.01, *** P<0.001 e) Control experiments for MVB maturation: (f) TCF/Wnt reporter transfected
(b) Quantitative PCR validation of HGS knockdown efficiency from three Hek293 cells were treated with 20nM bafilomycin A1, 50mM NH4Cl, 5mM
independent knockdown experiments, error bars are s.d. (c) Representative GW4869 or control vehicle alone. (g) Wnt activity of cell culture supernatants
images from immunofluorescence analysis of control (upper panel) or from inhibitor treated Wnt3A-L cells before and after ultracentrifugation
siVPS26 (lower panel) transfected L cells stained with Evi, Golgi marker was determined in TCF/Wnt reporter transfected Hek293 cells. (f and g)
GM130 and Lysotracker, (n=3) Scale bars 10 m (d) Autocrine Wnt Assay: Supernatants from three independent experiments were used, error bars are
Hek293 cells based TCF/Wnt reporter were transfected with indicated siRNA, s.d., significance level as indicated: * P<0.05, ** P<0.01, *** P<0.001

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a b

DMSO Bafilomycin
MVB

Wg (ex) Wg (ex)

Fig.1 d/3b
Mit
c Evi-C2 kDa
185 Fig.1 e Fig.1 f
115
kDa
80
Tsg101 Wg
65 115
185
50 115 80
30 80 65
65 50
Tsg101 kDa 50
185 30
115 30

P100
25

SND
SN0
80 25

CX
65 CX 1 2 3 4 5 6 7 8 9 10 15
Fig.1 g
50
Wnt3A ApoII
30 185 115
25 115 80
80 65
Wnt3A kDa 65 50
185 50
115 30
80 30
65 25
* CX 1 2 3 4 5 6 7 8 9 10
50 Wg
LRP6 115
30 80
25 185
115 65
15
80 50
Calnexin kDa 65
30
185 50 25
115 15

P100

SND
SN0
80 CX 1 2 3 4 5 6 7 8 9 10 30
65 CX
-actin 50
30 Fig. 2 d Fig.2 f/g
25
15 Ponceau -actin
50
Urea CX
RIPA CX
Urea CX
RIPA CX

185 30
P100
P100

SND
SND

SN0
SN0

115
25
80
Hek293 Wnt3A-L cells 65
50
Evi
30
25 50

30
25
Wnt3A
185
115
80
Wnt3A Calnexin
65
50
115
30
25 80
SND

SND

SND

65
SN0

SN0

SN0

50
CTRL P100
shEvi P100
shEvi CX
CTRL CX

Wnt3A-L cells 30
CTRL P100
shEvi P100

25
shEvi CX
CTRL CX

Supplementary Fig. S3 Wnt3A-L cells


Wnt3A-L cells

Figure S3 Control experiments for MVB maturation. (a) Ex vivo treatment of not show a significant effect on Wg secretion (left panel), scale bars 20 m. (b)
wing discs with V-ATPase inhibitor bafilomycin A1 (200 nM in DMSO) for 1 hour Electron micrograph of wing imaginal discs shows ILVs /exosomes (indicated by
at room temperature and then 1 hour on ice with Wg antibody shows reduced arrow-heads) containing MVB with mitochondrium. Scale bar 100 nm (c) Full
levels of the extracellular Wg (right panel). Control treatment with DMSO did immunoblots with indicated area of selection as shown in main figure 1 and 2.

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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

a b c d

Cherry Wg (total) GFP YFP

Enhanced

Cherry wg::Evi-Cherry Cherry Wg (ex) Wg (total)

Enhanced

EGFP wg::Evi-EGFP Merge wg::Evi-Cherry Merge wg::CD63-GFP Merge wg::YFP-rab4

e f g h

20

Nr. of mitotracker 580 positive cells


18
16
YFP YFP-Rab4/BF Dsh-YFP/BF also positive for YFP 14
12
10
8
6
4
Wg (ex) Mitotracker 580/BF Mitotracker 580/BF 2
0
b4 -YFP
P -Ra h
YF Ds

Merge wg::YFP-rab4 Merge Merge

Supplementary Fig. S4

Figure S4 Rab4 is secreted on exosomes and colocalises with Wg. (a) positive punctae (insets). (e) Extracellular Wg also colocalised with YFP-
Evi-Cherry was overexpressed with wg-GAL4 and shows Cherry-positive Rab4 outside the wg-GAL4 expression domain (insets). (f) YFP-Rab4
punctae secreted outside the expression domain, the middle panel shows expressing cells mixed with mitotracker-580 labeled cells for 5 hours
enhanced images of the upper panel. Similarly Evi-EGFP expressed show YFP-positive punctae in red cells (arrows). Moreover, the YFP-Rab4
with wg-GAL4 is secreted (lower panel). (b) Total Wg staining on discs expressing cells did not show mitotracker-580 staining (arrow-head). (g)
overexpressing Evi-Cherry did not show significant colocalisation (inset). Dsh-YFP expressing cells mixed with mitotracker-580 labeled cells for 5
(c) Extracellular Wg staining shows partial colocalisation of Wg with the hours did not show YFP-positive punctae in red cells (open arrow-heads).
exosomal marker CD63-GFP expressed with wg-GAL4 (insets). (d) Wild (h) Quantification of (f) and (g) showing number of Mitotracker-580
type YFP-Rab4 expressed using wg-GAL4 shows presence of YFP-positive positive cells that contain YFP-Rab4 positive punctae. Scale bars 20 m
punctae outside the expressing cells, which colocalises with Wg (total)- (a g).

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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

a single plane

WT

RNAi

Ptc ap::Ykt6 RNAi


b single plane

WT

RNAi

Ptc ap::Rel RNAi


c d 1.2 qPCR
knock down efficiency

1
WT
0.8

0.6

0.4

0.2
ap::Ykt6 0
Fmi RNAi siRNA
H L

VP 101

5
t6
VP A
TS GS

RNAi
TR

S3
6

Yk
S2
G
C

Hek293 cells
e secreted Gluc-reporter g
1.6
1.4 GM130 Evi Tsg101
Gluc activity

1.2
1
0.8
0.6
0.4
siCTRL
0.2
0 GM130 Evi Tsg101
S

siRNA
L

t6
G
TR

10

S2

S3

Yk
H

VP

VP
C

TS

f
Kc167 cells
secreted Fluc-reporter
1.6 siYkt6
1.4 GM130 Evi Tsg101
sFluc activity

1.2
1
***
0.8
0.6
0.4
0.2 siHGS
0
dsRNA
RL

VP 1
6

5
t6
r

10
G
Sa

S2

S3
CT

Yk
H

VP
TS

Supplementary Fig. S5
Figure S5 Ykt6 appears not to strongly affect general secretion. (a) Ykt6 in the RNAi domain (compare insets from the wild type internal control and
was knocked down in the dorsal compartment of the wing discs using ap- RNAi domain). (d) Quantitative PCR validation of knockdown efficiency from
GAL4. Immuno-staining for Ptc shows expression of Ptc in the RNAi domain Fig. 7. from three independent knockdown experiments, error bars are s.d.
(compare arrow-head to the arrow in the left panel). Single confocal plane (e) Secretion assay in Hek293 cells with secreted Gaussia luciferase, cells
shows presence of Ptc at the membrane (arrow-heads, right panel showing were tansfected with indicated siRNA. (n=3), error bars are s.d. (f) Secretion
higher zoom of the middle panel square). (b) Knockdown of gene Relish assay in Kc167 cells with secreted Firefly luciferase. (n=3), error bars are
was used as a control for Ykt6 knockdown. As can be seen in b middle s.d., significance level as indicated: * P<0.05, ** P<0.01, *** P<0.001 for
panel, Ptc is expressed and localised at the membrane, similar to Ykt6 (e and f).(g) Cellular localisation of Evi-GFP and Tsg101-Cherry in control,
knockdown. (c) Ykt6 was knockdown similarly as in a, further shows normal siYkt6 and siHGS stained for Golgi marker GM130. Scale bars 10 m for (g)
membrane localisation of another transmembrane protein Flamingo (Fmi) and 20 m (a c).

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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

a Fig.3 d
Fig.3 c Fig.4 a
kDa Tsg101 Wnt3A Wnt3A
80
kDa
65 kDa 185
50 185 115
115
30 80 80
25 65 65
CX 1 2 3 4 5 6 7 8 9 10 50
50
GFP 30
25 30
25
Evi Ponceau
kDa
185
185 115
CX 1 2 3 4 5 6 7 8 9 10
115
kDa 80
GAPDH 80
65

Bafilomycin
80 65 50

GW4869
65 50 30

siHGS P100

NH4Cl
siHGS CX

DMSO
CTRL CX
CTRL P100
50 25
30
30 25
25
CX 1 2 3 4 5 6 7 8 9 10
Hek293 Ponceau 185
115
80
Fig. 7 a 65
50
Ponceau 30
185 25
115
80
65
Hek293
50
30
25

b siCTRL CD63 siYkt6 CD63


CD81
siHGS/Tsg101

siHGS/Tsg101

185
siVPS35/26

siVPS35/26

115
80
siYkt6
siGL3

65
siYkt6
siGL3

50
30
25
20

Wnt3A 60 GL3
50
% of gold particles

185 Ykt6
115 40
80
65 30
50 20
30
10
25 0
es

20
es

VB

ia
ER

r
cl

an
om

he
dr
M

si

on
br

ot
ve
os

GFP
ch
em
s

as ated
Ly

ito
so +

185
as olgi

M
ci

a
m

115
G

80
pl

65
50

30
Hek293
25
Supplementary Fig. S6

Figure S6 (a) Full immunoblots with indicated area of selection as shown in main figure 3-7. (b) CD63-immunogold labeling of ultrathin sections of control
and Ykt6-depleted Hek293 cells and quantification of labeling for different organelles. n= 1000 gold particles of three grids were counted, scale bar 250 nm.

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Supplementary Table S1: Mass spectrometry results


Exosomes from Kc167 cells were prepared as described in the Main Methods sections, separated on an SDS-PAGE Gel and gel slices were analysed by
nanoLC-ESI-MSMS (Orbitrap), for proteins listed at least two peptides per proteins were identified.

Supplementary Table S2: In vivo RNAi screening results.


Based on the exosomal protein profile, the above shown genes were selected for an in vivo RNAi screen for Wg secretion. All RNAi lines (KK or GD) were
crossed with wg-GAL4 and crosses were kept at 25C. Adult wings were scored for notch phenotypes. Ykt6 (shown in red) was identified as a strong candidate
for Wg secretion defect.

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