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HEMATOPOIESIS
Lipid raft–associated protein sorting in exosomes
Aude de Gassart, Charles Géminard, Benoit Février, Graça Raposo, and Michel Vidal
Exosomes are small membrane vesicles
secreted by cells upon fusion of multivesicular endosomes with the cell surface. The mechanisms underlying the specific sorting of proteins in exosomal membranes are far from being unraveled. We
demonstrate here, using different cells,
that some molecules are released in the
extracellular medium via their association
with lipid raft domains of the exosomal
membrane. Various typical raft-associated
molecules could be detected by immunoblot in exosomes and Triton X-100–insoluble fractions isolated from exosomes
of different origins. Partial localization of
major histocompatibility complex (MHC)
class II molecules with detergent-resistant fractions isolated from Daudi-secreted exosomes was demonstrated by
immunoblot and confirmed by electron
microscopy colocalization of MHC class II
molecules and ganglioside GM1. Moreover, we found that exosome-associated
Lyn (1) had a lower molecular weight
compared with Lyn detected in cellisolated detergent-resistant domains, (2)
was absent from the Triton X-100–insoluble fraction isolated from exosomes,
and (3) had lost its partitioning capacity
in Triton X-114. Exosomal Lyn is probably
cleaved by a caspase-3–like activity contained in secreted vesicles. All together,
the data highlight the presence of lipid
microdomains in exosomal membranes
and suggest their participation in vesicle
formation and structure, as well as the
direct implication of exosomes in regulatory mechanisms. (Blood. 2003;102:
4336-4344)
© 2003 by The American Society of Hematology
Introduction
Exosomes were first described in studies of reticulocyte maturation
about 20 years ago1,2 and more recently found to be released by
other cells.3-7 Exosomes correspond to internal vesicles of multivesicular bodies (MVBs) and are released in the extracellular
medium upon fusion of MVBs with the plasma membrane.
Exosome secretion was shown to be responsible for the loss of
transferrin receptors (TfRs) during reticulocyte maturation.8 On the
other hand, exosomes secreted by antigen-presenting cells (APCs)
contain few TfRs, but are enriched with major histocompatibility
complex (MHC) class II molecules,3,4 which has led to investigations of their possible implication in intercellular communication
during the immune response.9,10 The molecular basis of protein
sorting during exosome formation is not fully understood. However, the recently identified machinery involved in MVB formation
is likely to be relevant for the biogenesis of exosomes.10 A first step
involves segregation of proteins at the limiting membrane of the
endosome. The second step is inward budding with selected cargo
into internal vesicles. At least 3 protein complexes, named endosomal sorting complex responsible for transport 1, 2, and 3 (ESCRT-1, -2,
and -3), were shown to be subsequently involved in these steps.11-14
Among these steps, ubiquitination and deubiquitination of selected
cargo by proteins of these complexes seem to play a key role,15-17
although other proteins follow the same pathway in a ubiquitinindependent manner.18 Finally, lipid metabolism19 and phosphoinositide signaling20-22 are certainly also involved in the
MVB biogenesis.
The study of the exosomal process in reticulocytes highlighted
several points. Sorting by cytosolic machinery is not an obligatory
process or at least not the sole one, since (1) lipids incorporated into
the external leaflet of the plasma membrane are very efficiently
sorted and secreted in association with exosomes,23 (2) acetylcholinesterase (AChE), a glycosylphosphatidylinositol (GPI)–
anchored protein in red cells, is similarly lost by approximately
50% by reticulocytes through exosome release during differentiation in erythrocytes,24 and (3) exoplasmic cross-linking of membrane proteins (TfR, AChE) by antibodies favors their sorting into
exosomes.23 Segregation and association of specific lipids and
proteins in the plasma membrane to form microdomains is now
largely accepted. These membrane subdomains, termed lipid rafts,
can be isolated owing to their insolubility in nonionic detergents
and low buoyant density on sucrose density gradients.25 These
low-density, Triton-insoluble (LDTI) fractions are enriched in
cholesterol and glycosphingolipids, and contain different acylated
proteins such as GPI-anchored proteins and tyrosine kinases of the
Src family.26
Our previous studies on reticulocyte exosomes gave us reason
to suspect the presence of rafts in secreted vesicles: (1) the
cholesterol-phospholipid ratio was found to be similar to that noted
in plasma membrane,27 considered a cholesterol-rich membrane
compared with intracellular compartments; (2) the ganglioside GM1
is found in exosomes28; (3) GPI-anchored proteins such as CD55,
CD58, and CD59 are selectively sorted in exosomes during
reticulocyte maturation,28 possibly explaining the phenotype change
in red blood cells during their final differentiation stage29; (4) Lyn,
a protein kinase of the Src family, was found associated with
exosomes secreted by reticulocytes and erythroleukemic cells30;
From the Unité Mixte de Recherches (UMR) Centre National de la Recherche
Scientifique (CNRS) 5539, Université Montpellier II, Montpellier, France; and
the UMR CNRS 144, Institut Curie, Paris, France.
9580; M.V.); and by an ARC fellowship (C.G.).
Submitted March 21, 2003; accepted July 18, 2003. Prepublished online as
Blood First Edition Paper, July 24, 2003; DOI 10.1182/blood-2003-03-0871.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by grants from the CNRS, the Institut Curie, the Ministère de la
Recherche, and the Association pour la Recherche sur le Cancer (ARC) (no.
4336
Reprints: Michel Vidal, UMR 5539, Univ Montpellier II—cc107, Montpellier
34095, France; e-mail: [email protected].
© 2003 by The American Society of Hematology
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
and (5) the heterotrimeric G protein ␣-subunit (G␣i2) is present in
reticulocyte exosomes.31 The finding that these molecules—
considered to be typical lipid raft markers—are present in reticulocyte exosomes suggests that lipid raft domains may participate in
molecule segregation during reticulocyte exosome formation. In
fact, raft domain involvement in molecule sorting in exosomes (ie,
internal vesicles of MVBs) may occur in various cells since
electron microscopy studies using perfringolysin, a cholesterolbinding toxin, recently showed the presence, in B lymphocytes, of
cholesterol in intralumenal vesicles of MVBs and in exosomes
present in the intercellular space.32
We now present data showing that GM1, Lyn, flotillin-1, and
stomatin, which were previously shown to be present in detergentresistant domains in several cell types,25,33,34 are also present in
LDTI fractions isolated on sucrose gradient from reticulocytes,
K562, and Daudi cells. Importantly, these molecules are further
sorted as raft components in exosomes. Moreover, our results
suggest that these raft domains may support the sorting of other
proteins, such as MHC class II molecules, that we found partially
associated with detergent-resistant domains isolated from Daudi
exosomes. Finally, we provide evidence that, after sorting in
exosomal membranes, raft-associated Lyn is probably processed by
a caspase-3–like protease in the exosomal cytosol.
Materials and methods
Cells
Reticulocyte production in Sprague-Dawley rats was induced by phenylhydrazine, as previously described.35 Human reticulocytes (always exceeding
6%) were obtained from patients during routine follow-up visits to the
Hôpital St Eloi (Montpellier, France). The lymphoid B-cell line Daudi and
human erythroleukemia cell line K562 were maintained in RPMI 1640
supplemented with 10% fetal calf serum (FCS), streptomycin (50 ␮g/mL),
and penicillin (50 U/mL).
EXOSOMES CONTAIN RAFT PROTEINS
4337
Exosome isolation
Exosomes were isolated by differential centrifugation as described previously.35 For further exosome purification, pellets were resuspended in 4 mL
2.55 M sucrose, 20 mM HEPES (N-2-hydroxyethylpiperazine-N⬘-2ethanesulfonic acid)/NaOH, pH 7.4. A linear sucrose gradient (2.25 to 0 M
sucrose, 20 mM HEPES/NaOH, pH 7.4) was layered on the top of exosome
suspension in a Beckman SW41 tube (Beckman Coulter, Fullerton, CA).
Gradients were centrifuged for 15 hours at 30 000 rpm, after which 500 ␮L
fractions were collected from the top of the tube. After trichloroacetic acid
(TCA) precipitation, fractions were analyzed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.
Isolation of LDTI membranes
Cells. LDTI fractions were isolated as previously described.25 Briefly,
K562 or Daudi cells (1 ⫻ 109) were washed in phosphate-buffered saline
(PBS) (pH 7.3) and lysed in 2 mL ice-cold MBS lysis buffer (25 mM Mes,
150 mM NaCl, pH 6.5) containing 1% Triton X-100 for 30 minutes on ice
and homogenized with 10 strokes of a dounce homogenizer. The lysate was
adjusted to 40% (wt/vol) sucrose (6 mL), placed in the bottom of an
ultracentrifuge tube (Beckman SW40), and overlaid with 4 mL 30% sucrose
and 2.5 mL MBS. Ultracentrifugation was performed at 39 000 rpm for 16
hours at 4°C. Then, 500 ␮L fractions were collected from the top of the
gradient. For red cells, LDTI membranes were isolated from ghosts
prepared from a 250 ␮L packed cell volume of rat reticulocytes (exceeding
70%)35 with the same protocol scaled down by means of a Beckman SW55
rotor. For Western blot analysis, fractions were precipitated with TCA
before SDS-PAGE; for dot blot analysis, 100 ␮L of each fraction was dotted
onto nitrocellulose filters; for bead adsorption, LDTI fractions were pooled,
mixed with a 5-fold volume of MBS, and centrifuged at 200 000g for 1 hour
at 4°C. Pellets were then resuspended in PBS.
Exosomes. Exosomes were isolated from 1 L culture supernatant and
washed once with MBS. The pellet was then resupended in 1 mL ice-cold
MBS 1%, Triton X-100, at 4°C overnight and homogenized with 10 strokes
of a dounce homogenizer. The lysate was adjusted to 40% (wt/vol) sucrose
(2 mL), placed in the bottom of a tube (SW55, Beckman), and overlaid with
2 mL 30% sucrose and 1 mL MBS. Ultracentrifugation was performed at
39 000 rpm for 16 hours at 4°C. Fraction collection and analysis were
carried out as described.
Antibodies
FACS analysis of exosomes and LDTI fractions
Rat monoclonal antibody (MoAb) anti–heat shock cognate protein 70
(anti-hsc70) (SPA-815), and mouse MoAb antihuman TfR (H68.4) were
from Stressgen Biotechnologies (Victoria, BC, Canada) and Zymed Laboratories (South San Francisco, CA), respectively. Mouse MoAb antihuman
leukocyte antigen–DR␣ (HLA-DR␣) chain (clone TAL.1B5) was obtained
from Dako (Hamburg, Germany). Mouse MoAb raised against human
HLA-DR (clone BL2) was from Immunotech (Marseilles, France). Mouse
MoAb anti–flotillin-1 (clone 18) was from BD Pharmingen (San Diego,
CA); rabbit anti-Lyn, goat anti-tsg101, and rabbit anti–caspase-3 (H-277)
were from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidaseconjugated goat antirat immunoglobulin G (IgG), peroxidase-conjugated
donkey antimouse IgG, and donkey antigoat IgG were obtained from
Jackson ImmunoResearch Laboratories (Bar Harbor, ME). Peroxidaseconjugated donkey antirabbit IgG and fluorescein isothiocyanate (FITC)–
conjugated donkey antimouse IgG were from Rockland Immunochemicals
(Gilbertsville, PA). Mouse antihuman stomatin (glutamic acid/alanine-rich
protein 50 [GARP-50]) was a generous gift from Rainer Prohaska
(University of Vienna, Austria).
Magnetic beads with covalently bound antimouse IgG (Dynal, Oslo,
Norway) were incubated with a mouse anti–HLA-DR antibody (clone BL2)
as described by the manufacturer. Exosomes or LDTI fractions were
incubated at room temperature for 2 hours with these activated magnetic
beads. The beads were then separated magnetically and washed 3 times
with PBS, 0.1% bovine serum albumin (BSA).
In parallel, exosomes or LDTI membranes were incubated with 25 ␮L
of 4-␮m-diameter aldehyde/sulfate latex beads (Interfacial Dynamics,
Portland, OR) for 2 hours at room temperature in a 30 to 100 ␮L final
volume; 50 ␮g BSA was then added to each sample, and the incubation was
prolonged for 15 minutes. This was followed by overnight incubation in
1 mL PBS with gentle shaking. After the reaction was stopped with glycine,
membrane-coated beads were then washed 3 times and resuspended in
fluorescence-activated cell sorter (FACS) wash (1% FCS in PBS).
Membrane-coated latex or magnetic beads were incubated for 45
minutes with anti–HLA-DR antibody or biotin-labeled cholera toxin B
subunit, followed by incubation with FITC-conjugated antibody or PEconjugated streptavidin, and analyzed on a FACSCalibur flow cytometer
(BD Biosciences, Le Pont de Claix, France), with a 488-nm argon laser and
standard band pass filters for FL-1 (530/30 nm) and FL-2 (585/42 nm).
Reagents
Biotinylated cholera toxin-B subunit and Triton X-114 were from Sigma (St
Louis, MO); peroxidase-conjugated streptavidin was from Amersham
(Arlington Heights, IL). Phycoerythrin (PE)–conjugated streptavidin and
the EnzChek caspase-3 assay kit were obtained from Molecular Probes
(Leiden, The Netherlands). Protein concentrations in samples were determined by the bicinchoninic acid (BCA) method (Pierce, Rockford IL).
Triton X-114 partition
First, 50 ␮L aliquots of purified exosomes or LDTI membranes were
diluted in 200 ␮L 1% Triton X-114 in 10 mM Tris (tris(hydroxymethyl)aminomethane)/HCl, 0.15 M NaCl, pH 7.4. The samples were incubated on ice
for 5 minutes and then overlaid onto a 6% sucrose (wt/vol), 0.06% (vol/vol)
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4338
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
DE GASSART et al
Triton X-114, 10 mM Tris/HCl, 0.15 M NaCl, pH 7.4 cushion (300 ␮L);
incubated for 3 minutes at 30°C; and centrifuged for 3 minutes at 3000g.
The top aqueous phases were transferred to new tubes and re-extracted with
0.5% Triton X-114, left for 5 minutes on ice, and then overlaid onto the
same sucrose cushion, incubated for 3 minutes at 30°C, and centrifuged for
3 minutes at 3000g. The aqueous phase was re-extracted with 2% Triton
X-114 as described. The final aqueous phase was about 150 ␮L, and the
detergent phase was made up to the same volume by adding 10 mM
Tris/HCl, 0.15 M NaCl, pH 7.4.
France). Rabbit antibodies were detected with protein A coupled to 10- or
15-nm gold particles (Department of Cell Biology, Utrecht University, The
Netherlands). The sections were contrasted and embedded in a mixture of
methylcellulose and uranyl acetate and viewed under a CM120 Philips
electron microscope (Eindhoven, The Netherlands).
After removal of sucrose by ultracentrifugation in PBS, membrane
fractions were loaded on formvar-carbon–coated grids for 30 minutes and
immunogold labeled and constrasted as described.
Western blot analysis
Results
Samples were separated by SDS-PAGE with the use of 10%, 12%, or 15%
polyacrylamide gels, and the proteins were electrophoretically transferred
to polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore,
Billerica, MA). Membranes were blocked and incubated with primary
antibodies and peroxidase-conjugated secondary antibodies. The corresponding bands were detected by means of an enhanced chemiluminescence
detection kit from Amersham. Western blot quantification was carried out
by densitometry scanning with ImageQuaNT (Molecular Dynamics, Piscataway, NJ) image analysis software.
Dot blot analysis
Density gradient fractions were dotted onto nitrocellulose filters (Schleicher
& Schuell, Düren, Germany) by means of a dot blot apparatus (Bio-Rad,
Hercules, CA). Membranes were then treated as described for Western blot
or by overlay with the horseradish peroxidase (HRP)–streptavidin and
enhanced chemiluminescence (ECL) method.
Caspase-3
Caspase-3 activity was measured in purified exosomes with the EnzChek
Caspase-3 assay kit (Molecular Probes) according to the manufacturer’s
instructions. Fluorescence was detected by means of a spectrofluorometer
with excitation/emission at 496/520 nm.
Exosomes (3 mg protein) from K562, Daudi cells, and (0.6 mg protein)
reticulocytes, were solubilized with RIPA buffer, and caspase-3 immunoprecipitation was carried out with the use of 2 ␮g rabbit anti–caspase-3
antibody (Santa Cruz Biotechnology) recognizing p11, p17, and p20
subunits and the full-length precursor of caspase, and 60 ␮L of a 50% slurry
of protein A-sepharose (Amersham). As control, Jurkat human Tlymphoblastoid cells (107 cells), cultured in the absence or presence of
5 ␮M actinomycin D for 7 hours, were lysed with the use of RIPA buffer,
and caspase-3 was immunoprecipitated as described. After washing,
samples were loaded on 15% SDS-PAGE, and Western blotting was carried
out with the use of the same anti–caspase-3 antibody.
Inhibition of caspase-3 activity during exosome formation was carried
out with the use of z-DEVD–fluoromethyl ketone (Z-DEVD-FMK), a
cell-permeable inhibitor (VWR International, Poole, Dorset, United Kingdom). First, 106 cells were cultured in 1 mL medium for 24 hours in the
presence of 20 ␮M Z-DEVD-FMK or 0.2% dimethyl sulfoxide (DMSO);
the supernant was discarded; and cells were incubated in the absence or
presence of inhibitor for 24 hours longer. Exosomes were then collected and
analyzed for Lyn by Western blotting.
Electron microscopy
Cells and exosome-coated beads were fixed with a mixture of 2%
paraformaldehyde and 0.125% glutaraldehyde in 0.2 M phosphate buffer,
pH 7.4 (PB) for 2 hours at room temperature. Fixed cells were processed for
ultrathin cryosectioning as described previously.36 After washing with PB
and PB containing 50 mM glycine, cells were embedded in 7.5% gelatin.
Small blocks were infiltrated with 2.3 M sucrose at 4°C for 2 hours and then
frozen in liquid nitrogen. Ultrathin cryosections were prepared with a Leica
(Vienna, Austria) ultracut FCS, retrieved with a mixture of 2% methylcellulose and 2.3 M sucrose (vol/vol), and immunogold labeled. After thawing,
sections were indirectly immunogold labeled with FITC or biotin-coupled
cholera toxin B subunit (Sigma), followed by a rabbit anti-FITC antibody
(Molecular Probes) or a rabbit antibiotin (Biovalley, Marne La Vallee,
To verify the presence of lipid rafts on the reticulocyte cell surface,
we isolated these subdomains from rat reticulocyte ghost membranes on the basis of their insolubility in Triton X-100 and low
buoyant density in sucrose gradients. Acetylcholinesterase activity
was present in the fractions corresponding to a density of approximately 15% to 20% (wt/vol) sucrose (not shown). The addition of
60 mM n-octyl-␤-D-glucopyranoside during membrane solubilization markedly decreased the percentage of AChE activity present in
the low-density fractions. The Src tyrosine kinase Lyn and flotillin-1 revealed by Western blot, and the ganglioside GM1 detected by
dot blot with the use of peroxidase-cholera toxin B, were found in
the same fractions with a similar effect of n-octyl-␤-D-glucopyranoside on their distribution (not shown). Interestingly, it was recently
shown that flotillin-1 and stomatin, another raft protein, are
differently sorted within vesicles released from erythrocytes after
treatment with Ca2⫹ and ionophore A23187.37 We were thus very
interested in whether these proteins were present in reticulocyte
exosomes. First we compared the selective enrichment of TfR and
Lyn in 3 subcellular fractions: ghosts, exosomes, and LDTI
membranes of rat reticulocytes (Figure 1A). As already shown,35
TfR was found to be highly enriched in exosomes as compared
with the plasma membrane, and in agreement with the literature,
traces of TfR were found in the LDTI fraction. As expected, Lyn
was highly enriched in LDTI membranes as compared with the
plasma membrane, but was also present in high amount in
reticulocyte exosomes. Surprisingly, however, Lyn was detected in
exosomes as a doublet of lower molecular weight as compared with
the Lyn doublet (53 to 56 kDa) detected in total cell LDTI membranes.
As shown in Figure 1B, flotillin-1 and stomatin were detected in
secreted vesicles, pointing out another difference with vesicles
shed from the erythrocyte plasma membrane after Ca2⫹ treatment,
which are devoid of flotillin-1.37 In agreement with the endosomal
processing of exosomes, flotillin-1 was described as a constituent
of lipid subdomains present at the plasma membrane38 as well as in
recycling endosomes.39 To further investigate the sorting step at the
MVB-formation level, we incubated reticulocytes with the biotinylated B subunit of cholera toxin (bCTXB) and analyzed the
distribution of bCTXB by electron microscopy. Consistent with
GM1 sorting in MVB, bCTXB was found predominantly on the
internal vesicles and not on the limiting membrane of the multivesicular structures (Figure 1C).
We have shown that K562 cells, an erythroleukemic cell line, secrete
exosomes with characteristics similar to those of reticulocyte exosomes;
for example, TfR, hsc70, and AChE are present.30 Interestingly, K562
cells were also able to sort N-Rh-PE (N-(lissamine rhodamine B
sulfonyl)dioleoylphosphatidylethanolamine) incorporated on the plasma
membrane, leading to lipid-labeled exosome secretion, suggesting that
lipid sorting may be a general feature of exosome processing. To
investigate this hypothesis, we used cells from another lineage, such as
B-lymphoblastoid Daudi cells, known to secrete exosomes containing
MHC class II molecules.40 We thus first examined whether the 4 raft
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
EXOSOMES CONTAIN RAFT PROTEINS
4339
Figure 1. Presence of raft markers in reticulocyte exosomes. (A) Equivalent protein amounts of ghosts, exosomes, and LDTI membranes from rat reticulocytes (exceeding
70%) were loaded on 10% SDS-PAGE and analyzed by Western blotting for Lyn and the TfR. (B) Because of GARP-50 antibody specificity, human red cells (6% reticulocytes)
were subcultured in vitro. Red cell ghosts and exosomes collected from the medium were analyzed by Western blotting for human stomatin (GARP-50) and then for flotillin.
(C) Rat reticulocytes (40 ␮L packed cell volume) were incubated with (50 ␮g/mL) biotinylated B-subunit of cholera toxin (bCTXB) for 3 hours at 4°C and chased for 60 minutes
at 37°C. Cells were then fixed in 2% paraformaldehyde and processed as described in “Materials and methods.”
markers GM1, Lyn, flotillin-1, and stomatin were present in LDTI
fractions isolated from K562 and Daudi cells. The cells were lysed in
Triton X-100 and fractionated as described to isolate raft and nonraft
proteins. Figure 2 shows that the distribution of the 4 raft markers
in sucrose density gradient was similar for both kinds of cells. GM1,
Lyn, flotillin-1, and stomatin were detected mainly in low-density
fractions, with only traces of Lyn and flotillin-1 in the bottom 40%
sucrose fractions.
On the contrary, EEA1 and TfR (not shown), which were taken
as nonraft protein markers, were detected only in the heavier
sucrose fractions. The distribution of MHC class II molecules over
the gradient was investigated for Daudi cells. In agreement with
previous studies on B cells,41 HLA-DR␣ chain was partly found to
be localized in low-density sucrose fractions. Taking into account
the volume of each fraction loaded onto SDS-PAGE, we roughly
Figure 2. Characterization of LDTI membranes isolated from K562 and Daudi
cells. K562 and Daudi cells were lysed in Triton X-100 and subjected to sucrose
gradient centrifugation. Aliquots of fractions collected from the top of the gradient
were analyzed by Western blotting with the use of antibodies against Lyn, flotillin-1,
early endosomal antigen 1(EEA1), and MHC class II (␣ chain) molecules, and by dot
blot for GM1 content with the use of peroxidase-coupled cholera toxin B subunits.
Threefold lower amounts of the 4 bottom gradient fractions were loaded on
SDS-PAGE.
estimated, by quantitative densitometry of the blots, that approximately 20% of HLA-DR␣ chain was present in LDTI fractions.
The addition of 60 mM n-octyl-␤-D-glucopyranoside during cell
lysis almost abolished the detection of the various raft markers in
low-density fractions (not shown).
We thus wondered if the same raft markers were present in
exosomes secreted by K562 or Daudi cells. Exosomes collected
from culture media of Daudi cells by differential centrifugation
were fractionated by flotation on sucrose gradient and analyzed for
the presence of the various proteins by Western blot, and for the
presence of GM1 by CTXB overlay (Figure 3A).
As already described for exosomes secreted by the human
B-cell line RN,3 MHC class II molecules were distributed mainly in
fractions between 1.08 and 1.18 g/mL. The hsc70, which is a major
component of reticulocyte exosomes, was also found in vesicles
secreted by Daudi cells. The 3 raft markers (flotillin-1, Lyn, and
GM1) were found to be distributed with perfect overlapping with
fractions containing MHC class II and hsc70 molecules. Immunoadsorption of exosomes on magnetic beads through the HLA-DR␣
chain was first carried out to control the size and heterogeneity of
the vesicles. As shown in Figure 3B, the size of the exosomes was
very homogenous (about 60 to 80 nm). Adsorption of membrane
fractions also allows the cytofluorometric detection of molecules.
We previously demonstrated the enrichment of different GPIanchored proteins on reticulocyte exosomes using this approach.28
We thus isolated LDTI membranes from K562 and Daudi cells and
from K562 and Daudi exosomes. These membrane fractions and
exosomes from the 2 cell types were adsorbed on the surface of
latex beads, as previously described.42 We then analyzed the
presence of GM1 on the different immobilized fractions using
biotinylated cholera toxin B subunit and phycoerythrin-streptavidin, and colabeling with an anti–HLA-DR antibody for Daudi
fractions. The 3 fractions isolated from K562 cells (not shown) and
from Daudi cells adsorbed on latex beads were labeled with
phycoerythrin through CTXB (Figure 3C, upper panel). MHC class
II molecules were also detected on the surface of the 3 Daudi
membrane–coated beads (lower panel).
However, to assess colocalization of the 2 markers on the same
membrane portion, fractions were immunoadsorbed on magnetic
beads through HLA-DR␣ chain as described and were analyzed for
bCTXB and PE-streptavidin binding. As shown in Figure 3D, GM1
was detected on the 3 immunoadsorbed membrane fractions,
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4340
DE GASSART et al
Figure 3. Analysis of exosomes secreted by Daudi cells. (A) Exosomes were
obtained by differential centrifugation as described in “Materials and methods” and
deposited on a linear sucrose gradient. Fractions were collected and analyzed by
Western blot and dot blot for the indicated markers. Indicated densities (grams per
milliliters) were obtained for each fraction by refractometry. (B) Alternatively, exosomes were immunoadsorbed on the surface of magnetic beads coated with
anti–HLA DR (clone BL2) and processed for electron microscopy (EM) as described
in “Materials and methods.” (C) FACS analysis of beads coated with different
membrane or subdomains from Daudi cells. Latex beads were coated with exosomes
(Exos), or with LDTI membranes isolated from cells (LDTI) or from secreted vesicles
(LDTI from exos) as described in “Materials and methods.” Membrane-coated beads
were then analyzed for the presence of the ganglioside GM1 through bCTXB binding
and PE-streptavidin detection (FL-2, upper panel), and for the presence of MHC class
II molecules through anti–HLA-DR antibody binding and FITC-antimouse IgG
detection (FL-1, lower panel). CTRLs are beads incubated with PE-streptavidin but
without bCTXB (FL-2, upper panel) and beads incubated with FITC–antimouse IgG
but without anti–HLA-DR (FL-1, lower panel). (D) The same membrane fractions
were immunoadsorbed on magnetic beads through the MHC class II molecules as
described in “Materials and methods” and analyzed for the presence of GM1 as in
panel C. CTRL represent beads incubated with PE-streptavidin without bCTXB.
although more faintly on the surface of exosomal LDTI membranes
(lower panel). These experiments, although not quantitative, highly
suggested the presence of MHC class II molecules in LDTI
membranes isolated from exosomes. To confirm this point, an
electron microscopy study was carried out on the LDTI membrane
fraction prepared from exosomes. LDTI membrane fractions from
Daudi exosomes were thus isolated on sucrose density gradient and
first analyzed by dot blot to characterize a fraction enriched with
MHC class II molecules and flotillin-1 (Figure 4A).
Fraction no. 3 was then pelleted and used for EM colocalization
of GM1 and MHC class II molecules. As shown in Figure 4B (upper
panel), gold particles (Au10) detecting bCTXB (small arrows)
were found on almost all membrane fragments present in the
preparation. Some of these fragments were also immunogold
labeled (Au15) with anti–HLA-DR␣ (large arrows), confirming the
presence of MHC class II molecules in raft domains of exosomes. It
should be noted that LDTI membranes isolated from cells (Figure
4, lower panel) appeared as larger vesicles (200 to 500 nm) as
compared with membrane fragments obtained from exosomes
(about 30 to 60 nm). This may be due to the fact that, as already
proposed, raft subdomains present on the cell surface are assembled together during membrane solubilization with Triton
X-100 to form large membrane vesicles. In contrast, the amount of
raft domain on the surface of exosomes (60 to 80 nm diameter) is
not sufficient to form such large vesicles. In agreement with this,
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
small membrane fragments labeled with bCTXB or anti–HLADR␣ were also observed in LDTI membranes isolated from cells.
To further study the involvement of raft domains in molecule
sorting toward exosomes, we more carefully analyzed the enrichment of some components in LDTI membranes isolated from K562
and Daudi exosomes (Figure 5). In both cell types, the whole
flotillin-1 pool was distributed in low-density fractions, overlapping with most of the ganglioside GM1 detected in the sucrose
gradient (Figure 5A).
On the contrary, Lyn was distributed only in the detergentsolubilized high-density fraction of K562 and Daudi exosome
preparations. As previously (Figure 4A), MHC class II molecules
were found to be partly distributed in LDTI fractions. We estimated
that about 10% to 20% of the total HLA-DR␣ chain detected on the
gradient was associated with low-density fractions. Western blot
analysis of known amounts of different subcellular fractions from
K562 and Daudi cells was carried out to confirm these dot blot data
(Figure 5B).
In both cell types, flotillin-1 was enriched in fractions corresponding to purified raft domains, confirming results obtained
earlier in this study. The other raft protein marker, Lyn, was found
in high amounts in LDTI fractions isolated from both kinds of cells,
confirming data obtained on the different sucrose gradient distribution. As described earlier for reticulocyte exosomes (Figure 1A),
Figure 4. EM colocalization of MHC class II molecules and GM1 in exosomal
LDTI domains. (A) Daudi exosomes were lysed in 1% Triton X-100 and subjected to
sucrose gradient centrifugation. Aliquots of fractions collected from the top of the
gradient were analyzed by dot blot for the distribution of flotillin-1 and HLA-DR␣.
(B) Fraction no. 3 of the sucrose gradient was used for the EM study (upper panel).
Detergent-resistant membrane isolated from Daudi cells was also analyzed for GM1
and MHC class II molecule colocalization (lower panel). Large arrows indicate gold
particles (protein A gold 15) detecting anti–HLA-DR␣; small arrows, gold particles
(protein A gold 10) detecting bCTXB.
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
EXOSOMES CONTAIN RAFT PROTEINS
4341
Figure 5. Comparison of protein enrichment in the
different membrane subdomains. (A) Dot blot characterization of exosomal LDTI membranes. K562 and
Daudi exosomes were lysed in 1% Triton X-100 and
subjected to sucrose gradient centrifugation. The distribution of the 3 raft markers (GM1, Lyn, and flotillin-1) in the
gradients was assessed by dot blot. Aliquots of the
fractions were analyzed for HLA-DR␣ distribution in the
gradient from Daudi exosomes. (B) Known protein
amounts of exosomes, cells, and LDTI membranes
isolated from cells or exosomes were loaded on SDSPAGE to get roughly comparable amounts of flotillin and
were analyzed by Western blot for the indicated proteins.
the tyrosine kinase in K562 and Daudi exosomes was detected as a
lower–molecular weight (lower-MW) doublet, and was absent
from LDTI fractions isolated from both K562 and Daudi exosomes,
confirming previous data (Figure 5A). As expected, TfR was
excluded from LDTI membranes and poorly enriched in Daudi
exosomes,40 contrary to what was found in K562 secreted vesicles.30
The hsc70 had the same pattern as TfR in K562 fractions, but was
found in all Daudi fractions except exosomal LDTI membranes.
Finally, the presence of MHC class II molecules was confirmed in
all Daudi fractions, including exosomal LDTI membranes.
The lower MW of the Lyn doublet found in exosomes,
suggesting proteolytic cleavage, together with its absence of
purified exosomal LDTI membrane, was highly reminiscent of the
cleavage of Lyn that occurs in apoptotic cells.43 Indeed, it was
demonstrated that activated caspase-3 induced Lyn cleavage after
Asp18 at the N-terminal domain, which is critical for the membrane association of Lyn through double acylation. This proteolytic
cleavage induces the relocation of the protein, which becomes
cytosolic. We thus compared the hydrophobic characteristics of
Lyn present in LDTI membranes isolated from Daudi cells and Lyn
present in Daudi exosomes using Triton X-114 partitioning.
As shown in Figure 6, Lyn was recovered mainly in the
detergent phase after Triton X-114 extraction of Daudi LDTI
membranes. On the contrary, Lyn, with a lower MW in exosomes,
was recovered almost exclusively in the aqueous phase after Triton
X-114 extraction. Note that a small amount of Lyn was detected in
the detergent phase, but with a higher MW, corresponding to the
native form. As a control, the HLA-DR␣ chain present in both cell
Figure 6. Partition in Triton X-114 of Lyn and HLA-DR␣ present in cell LDTI
membrane or secreted within exosomes. Exosomes and LDTI membranes
isolated from Daudi cells were extracted by Triton X-114 as described in “Materials
and methods.” Aliquots (50 ␮L) of the aqueous and detergent phases and of the
starting fractions were mixed with Laemmli buffer, loaded on SDS-PAGE, and
analyzed for the presence of Lyn and HLA-DR␣ by Western blot.
LDTI membrane and exosomes was distributed exclusively in the
detergent phase after extraction. This confirmed that Lyn cleavage
in exosomes induced a loss of membrane association and relocation
in the lumen of vesicles. To explain this relocation in exosomes,
and only in exosomes, this suggested that proteolytic cleavage of
Lyn occurred in exosomes. We thus looked for caspase-3 activity in
secreted vesicles. Exosomes from the 3 kinds of cells were
consequently solubilized and assayed for caspase-3–like activity.
As shown in Figure 7A, low DEVDase activity was detected in
the 3 types of exosomes, with much higher activity (about 10-fold
more) in reticulocyte exosomes. This activity was dependent on the
incubation time and was negligible when the vesicles were not
solubilized (not shown). Although very faint, a 19-kDa band
potentially corresponding to the activated form of caspase-3 was
detected by Western blot after immunoprecipitation from the 3
types of exosomes, consistent with the presence of low caspase-3
activity in the vesicles (Figure 7B). Finally, the presence of a
caspase-3 inhibitor during Daudi cell culture led to recovery of
exosomal Lyn with an unchanged molecular weight (Figure 7C).
Discussion
In this report, we investigated the presence of lipid raft domains in
exosomes. We show that typical raft components were associated in
detergent-resistant membrane on the surface of secreted exosomes
in the 3 cell types studied. These components belong to various
families of molecules, such as glycolipids (ganglioside GM1), Src
tyrosine kinases (Lyn), GPI-anchored proteins (AChE), and proteins containing prohibitin domains (stomatin and flotillin-1). The
existence of such raft domains on the exosome surface was
suspected from our previous data, especially on lipid and GPIanchored protein sorting in reticulocyte exosomes.23,28 The fact that
other cells, such as erythroleukemic K562 cells and lymphoblastoid
Daudi cells, secrete exosomes containing similar organized lipid
subdomains suggests that lipid domains may play a general role in
exosome biogenesis and structure. Note that lipid rafts have already
been described as key elements in the vesiculation process from the
red cell membrane when there is a rise in cytosolic Ca2⫹. More
recently, the shed vesicles were demonstrated to contain other raft
components such as GM1, cholesterol, and stomatin.37 It was also
suggested that lipid rafts are involved in virus budding from the cell
surface, since GPI-anchored proteins are incorporated in virion
membrane.44 These raft domains may therefore represent weak
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4342
DE GASSART et al
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
Figure 7. Evidence of caspase-3–like activity in exosomes secreted by reticulocytes, K562, and Daudi cells. (A) Left panel: Reticulocyte exosomes were solubilized,
and various protein amounts were assayed for caspase-3–like activity (1-hour incubation) with the use of Z-DEVD–rhodamine 110. Data shown (means of duplicate
experiments) represent total DEVDase activities (䡺) or activities measured in the presence (o) of the inhibitor (Ac-DEVD-aldehyde [Ac-DEVD-CHO]). Right panel: Similar
protein amounts (250 ␮g) of exosome from K562 cells and Daudi cells were assayed for caspase-3–like activity (1-hour incubation) as described. (B) Caspase-3 was
immunoprecipitated by means of rabbit anti–caspase-3 from exosomes of the 3 cell types: Daudi (3 mg protein), K562 (3 mg protein), and reticulocytes (0.6 mg protein).
Controls were carried out with the use of Jurkat cells incubated or not for 7 hours with 5 ␮M actinomycin D to activate caspase-3. The p20 subunit was revealed by Western blot
with the use of the same anti–caspase-3 antibody. (C) Daudi cells were cultured in the presence of Z-DEVD-FMK (20 ␮M) or DMSO (0.2%), as described in “Materials and
methods.” Exosomes were collected from the medium and analyzed for the presence of Lyn by Western blot. Cell lysate and exosomes from Daudi cells were also loaded on
the gel as migration controls.
points on the membrane surface, prone to outward bending
or budding.
Another possible role of lipid rafts during exosome formation
could be related to the intrinsic low lateral diffusion of the proteins
present in these subdomains.45 The presence of such lipid rafts in
recycling endosomes has been suggested to slow down recycling of
GPI-anchored proteins to the plasma membrane.46 Variable recycling capacities were also described for lipid molecules such as
fluorescent-labeled lipids that, depending on the length and saturation of fatty acids, present different recycling aptitudes.47 Slowly
recycling molecules would thus be more prone to be packaged in
intralumenal vesicles, whereas fast recycling molecules would
more easily escape MVB budding. Accordingly, we found that the
fast recycling lipid (C6-NBD-SM [1-acyl-2[6-[N-(7-nitro-2,1,3benzoxadiazol-4-yl) amino]caproyl] phosphatidylcholine]) was not
incorporated in reticulocyte exosomes, unlike N-Rh-PE, which is
known to be present on membrane in small clusters48 that are sorted
in exosomes during red cell maturation.23 We show here that
proteins such as flotillin-1 and stomatin, which have an intrinsic
affinity for lipid rafts, are sorted in exosomes secreted by various
cell types. It could be possible that other proteins interacting with
raft components may be also sorted in exosomes. Note here that the
glucose transporter Glut-1 found in reticulocyte exosomes was
demonstrated to associate with stomatin in red cells.49 Stomatin,
like caveolin and possibly flotillin-1, is an acylated protein found in
oligomeric forms in lipid domains.34,50 This is highly reminiscent
of another protein family, the tetraspan proteins (TSPs), which are
also acylated and interact to form a protein web presenting some
affinity for detergent-resistant domains,51 and which were found to
be enriched in B-cell exosomes.52 Certain TSPs were described to
interact with MHC class II molecules, while others can associate
with integrins.53 While we were writing this manuscript, Wubbolts
et al54 reported the colocalization of MHC class II molecules and
TSP in a CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonic acid)–resistant domain in B cell–derived exosomes. In addition, the integrin ␣4 chain was identified by mass
spectrometry as a component of B-cell–secreted exosomes. Interestingly, we previously detected very late activation antigen–4
(VLA4) integrin (␣4␤1) in reticulocyte exosomes.55 In this scenario, interaction of MHC class II molecules and possibly integrins
with TSP would reduce lateral diffusion of these molecules on
endosomal membranes, favoring their incorporation in MVB buds.
This hypothesis is strengthened by the observation that TfR
recycles more slowly when interacting with oligomerized Tf,56 and
is more efficiently sorted in exosomes when clustered by antibodies,23 which are thought to reduce their diffusion in the membrane.
Finally, lipid raft domains may serve as a ubiquitin-based
sorting platform in exosomal protein sorting. Indeed, ubiquitination
of cargo proteins was shown to be a sorting mechanism during
MVB formation,18 involving various effectors that associate with
lipid domains. For example, the ubiquitin ligases Cbl and Nedd4
become associated with lipid rafts upon activation of IgE signaling
on mast cells.57 Similarly, upon insulin binding on adipocytes, Cbl,
in association with the adapter c-Cbl–associated protein (CAP),
translocates to lipid rafts through the interaction of the sorbin
homology domain of CAP with flotillin-1.58 Very interestingly, Cbl
was demonstrated to be present in exosomes produced by activated
T cells.5 Tsg101 (tumor susceptibility gene 1), the mammalian
homolog of vacuolar protein sorting–23 (Vps23), has also been
found in dendritic cell-derived exosomes.59 Vps23 is a component
of the endosomal sorting complex ESCRT-1, which recognizes
ubiquinated cargoes and participates in their inclusion in MVB
vesicles.11 Interestingly, tsg101 also interacts specifically with
HIV-1 Gag protein and Ebola virus matrix viral protein 40 (Vp40)60
and recruits the Vp40 protein into lipid rafts.61 In agreement with
this, we found that tsg101, which was present in the 3 types of
exosomes tested, was also found in LDTI domains isolated from
Daudi exosomes (data not shown).
A second major finding in this study is that the Src tyrosine
kinase Lyn was selectively cleaved in exosomes secreted from the 3
cell types, leading to a 3-kDa truncated form that was no longer
membrane bound. This is a major finding since such Lyn cleavage
was already described as being induced by caspase-3 in apoptotic
cells.43 Accordingly, we documented DEVDase activity in solubilized exosomes. The presence of caspase-3–like activity in reticulocytes is not really surprising since transient activation of several
caspases was shown to be required during erythroid differentiation.62 Moreover, the remodeling occurring during red cell
maturation is very favorable for caspase activation. Mitochondria
destabilization by a 15-lipoxygenase63,64 expressed during reticulocyte maturation induces cytochrome C release,65 which can activate
the caspase cascade. Accordingly, we found much higher DEVDase activity in reticulocyte exosomes relative to the 2 other cell
types of vesicles. The presence of caspase activity in exosomes
from nucleated cells is much more surprising because the cells
were not undergoing apoptosis.
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
However, caspase-3 activation has already been reported to
occur in cells that are not undergoing apoptosis, such as activated B
or T cells66 and proliferative lymphoid cells.67 In fact, a growing
body of evidence suggests that caspase-3 may be activated under
physiologic states other than terminal apoptosis. Moreover, cells
stained with annexin V, the early indicator of apoptosis, were
shown to be able to re-enter the cell cycle and continue to
proliferate.68 This suggests that mechanisms could rescue cells
before commitment to apoptotic death. Packaging activated caspase
within exosomes might be a mechanism developed by cells to
ensure survival. At this point, it should be noted that members of
the inhibitor-of-apoptosis protein family could regulate apoptosis
by binding to and targeting caspase for ubiquitination.69,70 On the
other hand, caspase-3 proenzyme and its activated counterpart have
been shown to copurify with caveolin-enriched microdomains.71
The presence of other apoptosis-related proteins in exosomes has
EXOSOMES CONTAIN RAFT PROTEINS
4343
already been reported,59 in agreement with a possible role of
exosomes in apoptosis regulation.
A major consequence of membrane sorting at the endosomal
level is fine regulation of cellular processes. Lipid raft domains
present on the endosomal membrane might be involved in setting
up sorting platforms to concentrate cargo and effector proteins
within MVB intralumenal vesicles that could be destined for
lysosomal degradation or extracellular secretion.
Acknowledgments
We thank Dr Rainer Prohaska for kindly providing MoAb antistomatin (GARP-50) and Dr Herisoa Rabesandratana (Hôpital St Eloi,
Montpellier, France) for obtaining the blood samples.
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2003 102: 4336-4344
doi:10.1182/blood-2003-03-0871 originally published online
July 24, 2003
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