1 Laboratorio de Biología Celular y Molecular-Instituto de
Histología y Embriología, Facultad de Ciencias Médicas,
Universidad Nacional de Cuyo-CONICET, Mendozam 5500, Argentina
2 UMR 5539, Université Montpellier II, Montpellier 34095, France
* Author for correspondence (e-mail: mcolombo{at}fmed2.uncu.edu.ar )
Accepted 1 April 2002
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Summary |
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Key words: Exosomes, Multivesicular body, Rab11, Transferrin receptor
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Introduction |
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Rab proteins are small GTPases that play an essential role in the
regulation of membrane traffic (for a review, see
Schimmoller et al., 1998;
Brennwald, 2000
). Different
members of the Rab family are localized to distinct membrane-bound
compartments where they are thought to have a central role in the proper
targeting to and fusion of transport vesicles with the correct destination
membrane (Brennwald, 2000
).
Rab11 has been shown to be associated with post-Golgi membranes, secretory
vesicles (Urbe et al., 1993
)
and the pericentriolar recycling endosome
(Ullrich et al., 1996
;
Ren et al., 1998
), as well as
with the apical recycling system in polarized cells
(Casanova et al., 1999
).
Transfection experiments with CHO or BHK cells indicate that Rab11 regulates
Tf recycling through the pericentriolar endosomal compartment
(Ullrich et al., 1996
;
Urbe et al., 1993
). Rab11
function in exocytic trafficking has been deduced from the Rab11 requirement
for transport from the trans-Golgi network to the plasma membrane
(Chen et al., 1998
). In
addition, in a recent publication it was shown that Rab11 regulates transport
from early endosomes to the trans-Golgi network
(Wilcke et al., 2000
),
suggesting that Rab11 may control the interconnection between the endocytic
and secretory pathways.
K562 cells release exosomes with similar characteristics to reticulocyte
exosomes (Johnstone, 1996) and
are therefore a useful model to study the secretion of these small vesicles
from erythroid cells. One of the most significant similarities is the presence
of the TfR, which is absent in other exosomes (e.g. from platelets, antigen
presenting cells, cytotoxic T lymphocytes), suggesting a biogenesis from an
early endosomal compartment. Moreover, we have shown by subfractionation
analysis that reticulocyte MVBs contain early endosomal markers (e.g. rab4 and
rab5) and not classical markers of late endosomes (e.g. rab7, CI-M6PR)
(Dardalhon et al., 2002
).
Interestingly, K562 cells have been shown to possess high amounts of Rab11
compared with other Rab proteins (Green et
al., 1997
). Its relative abundance in K562 cells led us to
hypothesize that Rab11 may have a specific function in this hematopoietic cell
type, perhaps related to the formation and release of exosomes.
Since Rab proteins are key elements of the molecular machinery that controls membrane traffic, we examined the role of Rab11 in the exosome pathway in the K562 cells. The role of Rab11 in TfR trafficking was also analyzed. For this purpose, K562 cells were stably transfected with GFP-Rab11wt and mutants. As shown in other cell types, expression of the different forms of Rab11 inhibited TfR recycling. Furthermore, the number of TfR present on the plasma membrane of transfected K562 cells was markedly reduced. Expression of Rab11S25N, the dominant-negative mutant of Rab11, substantially inhibited the amount of released exosomes, whereas in cells transfected with the wild-type protein, exosome secretion was slightly enhanced. Therefore, our results indicate that Rab11, probably because of its function of regulating membrane recycling from early endosome and/or trans-Golgi network (TGN) compartments, is involved in the secretion of exosomes in K562 cells. This study provides new insights into the intracellular mechanisms that modulate not only the maturation of red cells but also the mechanism of exosome secretion in other cell types.
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Materials and Methods |
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Cell culture and transfection
K562, a human erythroleukemia cell line, was grown in RPMI supplemented
with 10% FCS, streptomycin (50 µg/ml) and penicillin (50 U/ml).
The cDNA of Rab11a and its mutants (a generous gift from David Sabatini, New York University) were subcloned into the vector pEGFP as fusion proteins with the green fluorescent protein (GFP). K562 cells were transfected with Transfast (Promega) according to the manufacturer's instructions, with pEGFP (control vector), pEGFP-Rab11wt, pEGFP-Rab11Q70L (a GTPase deficient mutant) and pEGFP-Rab11S25N (a GTP-binding deficient mutant). Stably transfected cells were selected with geneticin (0.5 mg/ml) and separated by flow cytometry using a FACS Vantage SE-TSO (INSERM U475, Montpellier).
Antibodies
Rabbit anti-rab11 serum was a generous gift from Bruno Goud (Institut
Curie, Paris, France). The rat monoclanal anti-Hsc 70 antibody was purchased
from StressGen (Victoria, Canada), and the rabbit polyclonal anti-Lyn antibody
was from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse anti-transferrin
receptor was kindly provided by Sebastian Amigorena (Institut Curie, Paris).
Anti-Man6P receptor (CI-MPR) was a generous gift from Stuart Kornfeld
(Washington University, St. Louis, MO). The rabbit antibody against TGN46 was
kindly provided by Franck Perez (Institut Curie, Paris). Peroxidase-conjugated
antibodies were purchased from Jackson Immunochemicals (West Grove, PA).
Fluorescence microscopy
pEGFP-Rab11-transfected K562 cells were analyzed by fluorescence microscopy
using an inverted microscope (Nikon Eclipse TE 300, Germany) equipped with a
filter system (excitation filter 450-490, barrier filter 515). Images were
obtained with a CCD camera (Orca I, Hamamatsu) and processed using the program
MetaMorph 4.5 (Universal Images Corporation).
Labeling with Transferrin-rhodamine
For colocalization studies, cells were incubated in serum-free RPMI
containing 20 µg/ml human Tf (tetramethyl rhodamine-conjugated, Molecular
Probes) for 45 minutes at 37°C and washed twice with ice-cold PBS. Cells
were mounted on coverslips and immediately analyzed by fluorescence microscopy
using the following filters: excitation filter 510-560 nm, barrier filter 590
nm.
Visualization of MDC-labeled vacuoles
Autophagic vacuoles were labeled with monodansylcadaverine (MDC) by
incubating cells with 0.05 mM MDC in PBS at 37°C for 10 minutes. After
incubation, cells were washed four times with PBS, mounted on coverslips and
immediately analyzed by fluorescence microscopy using the following filter
system: excitation filter V-2A 380-420 nm, barrier filter 450 nm.
Labeling of acidic compartments with Lysotracker
Acidic compartments were labeled by incubating the cells with 1 µM Lyso
Tracker (Molecular Probes) in serum-free RPMI medium for 10 seconds at room
temperature. After incubation, cells were extensively washed with PBS, mounted
on coverslips and immediately analyzed by fluorescence microscopy using the
following filter system: excitation filter 510-560 nm, barrier filter 590.
Labeling with BODIPY-TR ceramide
Membranes of the Golgi apparatus were labeled with 2.5 µM BODIPY-TR
ceramide (Molecular Probes), essentially as described by Pagano and
collaborators (Pagano et al.,
1991).
Indirect immunofluorescence for Man-6-P receptor (CI-MPR) and
TGN46
Cells were fixed in suspension with 1 ml of 2% paraformaldehyde solution in
PBS for 30 minutes at room temperature. Cells were washed with PBS and blocked
by incubating with 0.1 M glycine in PBS. Cells were permeabilized with 0.05%
saponin in PBS containing 0.2% BSA and were then incubated with a rabbit
antibody against CI-MPR (dilution 1:50) or with the antibody against TGN46
(dilution 1:100). Bound antibodies were subsequently detected by incubation
with Texas-Red-conjugated goat anti-rabbit secondary antibody. The cells were
mounted with 50% glycerol in PBS and analyzed by fluorescence microscopy.
Exosome isolation
Exosomes were collected from the media of 15 ml K562 cells cultured for 24
hours. The culture media was placed on ice and centrifuged at 800 g
for 10 minutes to sediment the cells and subsequently was centrifuged at
12,000 g for 20 minutes to remove the cellular debris. Exosomes were
separated from the supernatant by centrifugation at 100,000 g for 2
hours. The exosome pellet was washed once in a large volume of PBS and
resuspended in 100 µl of PBS (exosome fraction).
Analysis of exosomes
Acetylcholinesterase assay
Acetylcholinesterase activity was assayed by standard procedures
(Ho and Ellman, 1969).
Briefly, 15 µl of the exosome fraction were suspended in 100 µl
phosphate buffer and incubated with 1.25 mM acetylthiocholine and 0.1 mM
5,5'-dithio-bis(2-nitrobenzoic acid) in a final volume of 1 ml. The
incubation was carried out in cuvettes at 37°C, and the change in
absorbance at 412 nm was followed continuously.
Fluorescent N-Rh-PE measurement
The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)
phosphatidyl ethanolamine (N-Rh-PE) was inserted into the plasma membrane as
previously described (Willem et al.,
1990). Briefly, an appropriate amount of the lipid, stored in
chloroform/methanol (2:1), was dried under nitrogen and subsequently
solubilized in absolute ethanol. This ethanolic solution was injected with a
Hamilton syringe into serum-free RPMI (<1% v/v) while vigorously vortexing.
The mixture was then added to the cells and they were incubated for 60 minutes
at 4°C. After this incubation period, the medium was removed and the cells
were extensively washed with cold PBS to remove excess unbound lipids. Labeled
cells were cultured in complete RPMI medium to collect exosomes. 50 µl of
the exosomal fraction were solubilized with 1.5 ml PBS containing 0.1% Triton
X-100 to measure N-Rh-PE using a SLM Aminco Bowman Series 2 luminescence
spectrometer at 560 nm and 590 nm excitation and emission wavelengths,
respectively.
Transferrin recycling assay
Cells were pre-incubated for 1 hour at 37°C in serum-free RPMI medium
supplemented with 1% BSA to deplete endogenous transferrin (Tf). They were
then incubated with 125I-labeled human Tf (2 µg/ml,
2x106 cpm/µg protein) in serum-free RPMI medium
supplemented with 1% BSA for 1 hour at 37°C. Cells were then washed four
times with cold PBS and once with an acetate buffer (200 mM sodium acetate and
150 mM NaCl, pH 4.5) for 2 minutes on ice to remove surface-bound Tf. Cells
were aliquoted (0.5x106 cells per tube) and incubated at
37°C in 0.5 ml of serum-free RPMI medium containing 100 µM deferoxamine
mesylate and 20 µg/ml unlabeled human Tf. At different times, the
incubations were stopped by adding 1 ml ice-cold PBS and immediately placing
the samples on ice. The samples were centrifuged for 20 seconds at 14,000
g to sediment the cells, and the medium was collected. The cell
pellet was then washed with the acetate buffer (see above) to remove
surface-bound Tf. The radioactivity in both the medium and the cell pellet was
determined using a -counter (Packard Cobra). Nonspecific radioactivity
(not competed by incubation with a 100-fold excess of unlabeled Tf) did not
account for more than 10% of the total cell-associated radioactivity.
Transferrin receptor measurements
Ligand-binding studies were performed as previously described
(Sainte-Marie et al., 1991).
Cells were suspended in ice-cold serum-free RPMI medium at
3x106 cells/ml. Binding reactions were prepared with 100
µl of this cell suspension containing 50-500 nM 125I-Tf, with or
without 25 µM unlabeled Tf. Samples were incubated at 4°C for 90
minutes, after which 1 ml of cold PBS-BSA was added and the cells pelleted at
14,000 g for 1 minute. The medium was removed and the cells
resuspended in 50 µl PBS-BSA and centrifuged through a water-impermeable
layer of dibutyl phtalate on 15% sucrose at 14,000 g for 3 minutes.
The tubes were quickly frozen using liquid nitrogen, and the tips containing
the cell pellets were cut off and counted in a
-counter. The amount of
nonspecifically bound 125I-Tf associated in the presence of excess
unlabeled Tf was subtracted from samples incubated in the absence of cold
ligand to determine the amount of specific 125I-Tf bound. Specific
binding data were analyzed by the Scatchard method to determine the number of
Tf binding sites. The total number of transferrin receptors (TfRs) was
determined by western blot of whole cell fractions.
SDS-PAGE and western blotting
Samples of the total cell pellet (100 µg protein) or exosomal fraction
(15 µl) were solubilized in reducing SDS loading buffer and incubated for 5
minutes at 95°C. Samples were run on 7.5% or 10% polyacrylamide gels and
transferred to Immobilon (Millipore) or BioBlot-NC (Costar) membranes. The
membranes were blocked for 1 hour in Blotto (5% non-fat milk, 0.1% Tween 20
and PBS) and subsequently washed twice with PBS. Membranes were incubated with
primary antibodies and peroxidase-conjugated secondary antibodies. The
corresponding bands were detected using an enhanced chemiluminescence
detection kit from Pierce.
Electron microscopy
K562 cells were incubated with Tf-coated colloidal gold particles (10 nm)
for 45 minutes at 37°C. Cells were washed twice with cold PBS to remove
unbound particles and processed for electron microscopy as previously
described (Harding et al.,
1984). Briefly, the samples were fixed in 2% glutaraldehyde in
0.15 M sodium cacodylate buffer for 20 minutes, washed, post fixed in 2%
OsO4 in cacodylate, pH 7.4, rinsed, stained and embedded as
previously described (Harding et al.,
1983
).
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Results |
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In order to determine if the transfected cells were expressing similar levels of the Rab11 GFP-tagged proteins, cells were analyzed by western blotting using an antibody against the cterminal domain of Rab11. As shown in Fig. 2, equivalent levels of overexpressed Rab11, quantified by densitometry, were observed in the transfected cells with either wt or mutants. We were unable to detect the endogenous Rab11 by this technique using the amount of protein loaded in the gel. The higher molecular weight band that appears in the vector-transfected cells is a nonspecific band that is also present in the Rab11-transfected cells. To work with similar levels of overexpressed proteins, the transfected cells were frequently checked by FACS.
|
As it is known that Rab11 associates with transferrin-containing recycling
compartments (Ullrich et al.,
1996; Ren et al.,
1998
; Green et al.,
1997
), we studied the localization of the chimeric Rab11 proteins
with rhodamine-transferrin internalized by endocytosis for 45 minutes at
37°C. Consistent with previous observations, a marked colocalization with
GFP-Rab11wt was observed in the perinuclear and peripheral small vesicles
(Fig. 3), however no
colocalization was observed in the large ring-shaped structures in the case of
cells overexpressing Rab11wt or the mutant Rab11Q70L
(Fig. 3, bottom panels). In the
case of the mutant Rab11S25N, colocalization was exclusively observed in the
pericentriolar region.
|
The observation that the large vesicles in the transfected cells do not colocalize with internalized transferrin might be explained by the fact that only a very small amount of this protein, which is unable to be detected by fluorescence microscopy, is directed to this compartment.
The large ring-shaped vesicles are labeled by TGN markers
In order to determine the nature of the large vesicles observed in cells
overexpressing the mutant Q70L, colocalization studies with markers of
different vesicular compartments were carried out. As shown in
Fig. 4, the large vesicles were
neither labeled with Lysotracker, a marker of acidic compartments, nor with
monodansylcadaverine (MDC), an autofluorescent compound that specifically
accumulates in autophagic vacuoles
(Biederbick et al., 1995).
Also, no colocalization with the mannose-6-phosphate receptor was observed,
indicating that these structures are not late endocytic compartments. In
contrast, the ring-shaped vesicles were labeled with the fluorescent compound
Bodipy-TR ceramide, a lipid that preferentially accumulates in the Golgi,
suggesting that these structures may originate from specialized regions of the
Golgi apparatus, perhaps the TGN.
|
In order to address this possibility, we have used monensin, a ionophore
that alters transport from the TGN to the plama membrane
(Tartakoff and Vassalli, 1977;
Tartakoff, 1983
) and also
blocks transferrin recycling in K562 cells
(Stein et al., 1984
). Monensin
causes a marked distention of the Golgi membranes and also the formation of
dilated MVBs in the perinuclear region of the cell
(Stein et al., 1984
). Exosomes
were labeled with the fluorescent lipid analog N-(lissamine rhodamine B
sulfonyl) phosphatidyl ethanolamine (N-Rh-PE). This lipid is efficiently
internalized via endocytosis but does not return to the cell surface. Sucrose
gradient analysis and immunoisolation experiments have demonstrated that
N-Rh-PE accumulates in exosomes that are eventually secreted into the
extracellular medium (Vidal et al.,
1997
). Cells overexpressing GFP-Rab11Q70L were incubated with
monensin and the lipid N-Rh-PE to label exosomes for 4 hours. As shown in
Fig. 5A, the large
multivesicular bodies were clearly decorated with GFP-Rab11 Q70L and contained
numerous red dots that probably represent exosomes. Images of two typical
cells are shown.
|
To confirm that the large MVBs originated, at least in part, from the Golgi apparatus, we have used an antibody generated against TGN46, a specific TGN marker for human cells. TGN46 was detected by indirect immunofluorescence and, as depicted in Fig. 5B, there was a marked colocalization of this protein with the ring-shaped structures decorated by GFP-Rab11Q70L. It is important to mention that for immunofluorescence studies, cells are fixed and fixation alters the size and shape of the GFP-labeled structures, therefore they are not visualized as clearly as in nonfixed cells.
Taken together these results clearly indicate that the TGN contributes to the biogenesis of the MVBs involved in the formation and release of exosomes.
Transferrin recycling and cell surface delivery of Tf-receptors is
impaired by overexpression of Rab11wt and mutants
The colocalization of Rab11 with rhodamine-transferrin in the recycling
compartment suggests a role for Rab11 in directing the transport of Tf through
this compartment in K562 cells, as described in other cell types
(Ullrich et al., 1996;
Ren et al., 1998
). Therefore,
we examined the function of Rab11 in the Tf pathway by studying the recycling
of this protein and the delivery of TfRs to the cell surface. To study the
kinetics of Tf recycling, K562 cells stably transfected with the vector alone
or the chimeric Rab11wt and mutants were incubated with
125I-labeled human Tf for 1 hour at 37°C and then transferred
to fresh medium to measure the return of the labeled Tf to the medium. As
shown in Fig. 6 in K562 cells
transfected either with Rab11 wt (Fig.
6A) or with the mutants Rab11 Q70L and Rab11S25N
(Fig. 6B,C) transferrin
recycling was partly inhibited. In the case of the dominant-negative mutant
although the effect was small it was consistently observed.
|
Transferrin (apotransferrin) recycles back to the cell surface bound to the TfR. Since recycling of transferrin was partly inhibited by overexpression of Rab11 constructs, it is possible that part of the TfRs remains trapped in internal compartments and is unable to reach the plasma membrane. In order to test this possibility we measured the number of TfRs on the cell surface. As shown in Fig. 7, the number of TfRs on the plasma membrane was markedly reduced in K562 cells overexpressing Rab11 wt or the mutants Rab11 S25N and Rab11 Q70L. In contrast, the total number of TfRs was not modified (data not shown). These results suggest that TfRs are probably trapped in internal compartments.
|
Overexpression of mutant Rab11S25N causes inhibition of the amount of
exosomes released
Like reticulocytes, K562 cells release small vesicles termed exosomes into
the extracellular medium after fusion of MVBs with the plasma membrane
(Johnstone, 1996).
Reticulocyte exosomes are enriched in proteins such as the TfR, Hsc70 and
acetylcholinesterase (AChE) (Johnstone et
al., 1989
). They also contain the tyrosine kinase Lyn (C.
Géminard and M. V., unpublished). Therefore, we have quantified the
amount of exosome secretion by measuring the levels of TfR, Lyn and Hsc70 by
western blot analysis. For this purpose, exosome fractions were harvested from
the culture medium of Rab11-transfected K562 cells as described in the
Materials and Methods. As shown in Fig.
8A, there was an increase in exosome release by K562 cells
transfected with Rab11wt compared with cells transfected with the
dominant-negative mutant of Rab11 (Rab11S25N), where the secretion of exosomes
was inhibited. A slight inhibition was also observed with the mutant Q70L. The
inhibitory effect caused by overexpression of Rab11S25N was observed with all
the exosome markers used. The quantification by densitometry of several
western blots is shown in Fig.
8B.
|
We also measured the activity of AChE in the harvested exosomes (see the Materials and Methods). Consistent with the results obtained by western blot, there was a decrease of exosome secretion in cells overexpressing the mutant Rab11S25N, whereas overexpression of Rab11wt stimulated the amount of exosomes released as determined by AChE activity (Fig. 8C). Similar results were obtained when the exosomes were labeled with the fluorescent lipid analog N-Rh-PE (see above). As shown in Fig. 8D, there was a marked decrease in the amount of exosomes collected from cells transfected with Rab11 S25N, as determined by measuring the fluorescence of N-Rh-PE, whereas Rab 11wt increased exosome secretion.
The results indicate that overexpression of dominant-negative Rab11 mutant inhibits exosome release. As mentioned above, exosomes originate from inward budding of the endosomal compartment leading to the formation of MVBs, which by fusion with the plasma membrane induces the release of the included vesicles. Therefore, the inhibition in exosome secretion observed in the Rab11S25N transfected cells may have various causes, such as impairment of molecule sorting or inward membrane budding during MVBs formation, or it may involve a latter step such as fusion of the MVBs with the plasma membrane. In order to get insights into some of these possibilities, we have analyzed by electron microscopy cells transfected with either Rab11wt or the mutant Rab11S25N. The cells were incubated with transferrin-coated colloidal-gold particles (Tf-gold) to label the TfR present in the small inclusion vesicles. As shown in Fig. 9, MVBs labeled with Tf-gold are clearly observed in the K562 cells overexpressing Rab11wt (Fig. 9A,B). In cells overexpressing the mutant Rab11S25N, similar MVBs were observed (Fig. 9C). This suggests that overexpression of this mutant protein does not highly impede the formation of the MVBs. However, we can not discard the possibility that membrane inward budding or the biogenesis of certain membrane domains might be perturbed. It is likely that small changes in membrane-flow steady state, induced by transfection of the Rab11 constructs, are resulting in modification of exosome secretion.
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Discussion |
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In this report we used K562 cells, a human erythroleukemic cell line, to
study the exosome pathway in relation to the expression of Rab11.
Interestingly, K562 cells have been shown to possess high amounts of Rab11
compared with other Rab proteins, and Rab11 is associated with compartments
involved in TfR recycling (Green et al.,
1997). K562 cells stably transfected with GFP-Rab11wt and mutants
support the involvement of Rab11 in TfR recycling. Consistent with previous
observations (Ren et al.,
1998
), GFP-Rab11wt and GFP-Rab11Q70L presented a typical vesicular
distribution in the perinuclear region, whereas the dominant-negative form
GFP-Rab11S25N was diffusely distributed in the cytoplasm, although some
perinuclear structures were also labeled. The distribution of the
dominant-negative mutant is in accordance with the observation that this
chimeric protein is enriched in the TGN
(Chen et al., 1998
). Our
results indicate that transfection of K562 cells with either Rab11wt or
mutants partly inhibited Tf recycling. This inhibition is concordant with data
obtained from CHO cells (Ren et al.,
1998
) and BHK cells (Ullrich
et al., 1996
), although less pronounced. Consistent with this
inhibitory effect, Scatchard analysis revealed a marked decrease in the TfR
number on the surface of cells overexpressing the different forms of Rab11.
This decrease could be caused by retention of TfR in intracellular
compartments and/or by a loss of this receptor via exosomes. Indeed, K562
cells have been demonstrated to release exosomes that contain TfR
(Ahn and Johnstone, 1993
;
Baynes et al., 1994
). We
recently found that Hsc70, AChE and Lyn are also associated with K562
exosomes, as well as vesicles released by reticulocytes (C. Géminard
and M.V., unpublished). These four endogenous proteins were used as markers to
evaluate exosome secretion by K562 cells transfected with the Rab11 alleles
together with the fluorescent phospholipid analog (N-Rh-PE), which is
incorporated into the surface of cells and is efficiently sorted after
endocytosis and directed towards lysosomes
(Willem et al., 1990
) and
exosomes (Vidal et al., 1997
).
The five markers indicated an inhibition of exosome secretion by K562 cells
transfected with Rab11S25N. Conversely, we have observed an increase in
vesicle release in Rab11wt overexpressing cells. Therefore it is likely that
in the Rab11wt cells the decrease in the number of TfR on the cell surface is
a consequence of re-routing TfR from the recycling pathway toward the exosome
pathway. The results reported here suggest that exosome secretion could be at
least one of the mechanisms involved in TfR downregulation from the cell
surface. In contrast, in cells transfected with the dominant-negative mutant,
exosome secretion was inhibited. Since exosomes originate from fusion of MVBs
with the plasma membrane and subsequent release of the inclusion vesicles, the
inhibition of exosome secretion observed in Rab11S25N transfected cells may be
a consequence of a reduction in MVB formation or in fusion with the plasma
membrane. The electron microscopy studies show that Tf-gold labeled MVBs are
present in Rab11S25N-transfected cells, thus indicating that sorting of TfR
and inward endosomal budding during MVBs formation are not completely blocked.
However, we can not discard the possibility that the biogenesis of MVBs,
including the formation of the internal vesicles, is somehow affected by
overexpression of this Rab11 mutant. Further experiments are necessary to
determine the exact step at which Rab11 regulates exosome secretion. It is
important to mention that in our system we are measuring the final product of
the exosome pathway, that is the released vesicles. Overexpression of Rab11
and its mutants may disturb either the formation of these small vesicles or
the process of release of exosomes during the fusion with the cell
surface.
Interestingly, Rab11 has also been reported to be involved in vesicle
transport from the TGN to the plasma membrane
(Urbe et al., 1993;
Chen et al., 1998
) and from the
recycling endosome to the TGN (Wilcke et
al., 2000
). Consistently, overexpressing GFP-Rab11Q70L in K562
cells induced the formation of large ring-shape structures that were negative
for Tf but Golgi related, as demonstrated by colocalization studies with a
fluorescent ceramide. Furthermore, we have shown that these structures are
also labeled with the antibody TGN46, a TGN marker. Lastly, in the presence of
monensin the large ring-shape structures generated in cells overexpressing
GFP-Rab11Q70L accumulated the fluorescent lipid N-Rh-PE internalized via
endocytosis. Therefore, our results strongly suggest that these Rab11-labeled
structures originate from the TGN, but they also receive material from the
endocytic pathway. It is noteworthy that a close relationship between the
endosomal compartment and the Golgi has previously been demonstrated in K562
cells (Stein and Sussman,
1986
). Two distinct pathways involved in TfR recycling were
described, one of which was found to be monensin sensitive. Treatment of K562
cells with 10-5 M monensin induced a marked reduction in cell
surface TfR that accumulated in MVBs. Similarly, we have observed that
overexpression of Rab11 markedly reduced the TfR present at the plasma
membrane. This led us to the conclusion that, as previously stated by Sussman
and collaborators (Stein et al.,
1984
), a coordinated interaction between MVBs and the Golgi
apparatus is involved in the recycling of TfR in K562 cells. We have also
observed by electron microscopy that in Rab11S25N the Golgi cisternae were
markedly dilated (data not shown). Therefore, the evidence presented here
suggest that this coordinated TfR traffic between MVBs and the trans-Golgi is
probably modulated by Rab11.
It is thus tempting to speculate that the secretion of exosomes in K562 cells is caused by the fusion of these MVBs, described to be involved in TfR recycling, with the plasma membrane. This would be consistent with our results showing that the exosome pathway in these cells is regulated by Rab11 expression. However, the involvement of Rab11 in traffic between the recycling endosome and Golgi makes the picture more complex, and further experiments are necessary to unravel the precise mechanism by which Rab11 regulates exosome secretion. Thus, the hypothesis is that exosome release occurs in some cell types (e.g. erythroid cells) in which membrane outflow from the recycling endosome is used to secrete material. In circulating reticulocytes, this process would be a remnant process from the erythroid precursors. Moreover, it is also interesting to draw parallels with exosomes enriched with MHC class II molecules secreted by antigen presenting cells: (i) MHC class II molecules are major proteins in the function of antigen presenting cells as are TfR in the erythroid cell lineage, (ii) they are both `receptors' transported from the endosomal compartment to the plasma membrane, (iii) for reasons that are as yet unknown, they both can be sorted in vesicles contained in MVBs, and finally (iv) these MVBs are connecting points between the endocytic and secretory pathways. Elucidation of variations around a common theme could be of great interest for the further development of exosomes as immunotherapeutic vehicles.
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Acknowledgments |
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References |
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