(Received for publication, October 20, 1994; and in revised form, May 4, 1995)
From the
Since reticulocytes have a high demand for iron, which is
required for heme biosynthesis, these cells are highly specialized in
the endocytosis of the iron carrier transferrin (Tf). From the
resulting endocytic vesicles (EVs), iron is released and the vesicles
rapidly return to the cell membrane where they fuse, causing the
release of the apotransferrin. Due to a lack of other intracellular
compartments, the endocytic vesicles can be readily isolated. In this
study, we have investigated the fusogenic properties of EVs, using
liposomes as target membranes. Membrane fusion was monitored by a lipid
mixing assay based on the relief of fluorescence self-quenching, using
octadecylrhodamine B-chloride (R18). Application of this procedure was
verified and solidified by analysis of the fusion event by an
independent lipid mixing assay, after in situ labeling of EVs,
and by determination of the mixing of aqueous contents. We
demonstrate that the endocytic vesicles are particularly prone to fuse
with target membranes that contain dioleoylphosphatidylethanolamine
(DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or
phosphatidylcholine show a reduced fusion activity with EV. The
specific and strong inhibition of fusion by cyclosporin A and a peptide
known to interfere with the propensity of DOPE to adopt the hexagonal
HII phase suggests that the mechanism of fusion involves the ability of
this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or
cytosol are not necessary to obtain fusion. However, trypsin treatment
of the endocytic vesicles inhibits fusion, indicating the involvement
of (a) protein(s) in the fusion event. Vesicular transport along the endocytic and secretory pathways
involves the budding of vesicles from a donor compartment, specific
targeting of the vesicles to an acceptor compartment, followed by a
fusion event between target and vesicular membranes. To investigate the
mechanisms and regulation of such vesicular transport processes, many
cell-free systems have been developed to reconstitute
intercompartmental transport. This has led to the recognition and
identification of a growing number of molecular factors (proteins,
nucleotides) thought to be involved in the distinct interaction steps (1, 2) . With regard to budding, adaptins and clathrin
have been recognized in the endocytic pathway as the major proteins
involved in receptor sorting and vesicle formation from plasma membrane
and trans Golgi network(3) . As to the subsequent steps in
intracellular trafficking, i.e. vesicle docking and fusion, a
host of molecules have been identified, including small GTPases (rab
proteins), heterotrimeric G proteins, NSF, SNAP, and SNARE. The
function of most of these factors is largely obscure, although these
components may well be part of a machinery, common to all vesicle
fusion events, as unified in the so-called SNARE
hypothesis(4) . According to this hypothesis, SNAPs bind to
their receptors, SNAREs, on the vesicle (v-SNARE) and on the target
membrane (t-SNARE). NSF subsequently binds to SNAPs, which causes ATP
hydrolysis, thus inducing complex dissolution, which may precede or
follow the merging of membranes. Recently, it has been shown that
cellubrevin, a member of the SNARE family(5) , is involved in
the recycling of the Tf Receptor-mediated endocytosis refers to the cell's capacity to
internalize components after their binding to specific receptors.
Depending on the receptor-ligand complex involved, molecules are then
delivered to the lysosomes for degradation. Reticulocytes are highly
exceptional in that they are hyperspecialized for uptake of iron, which
is needed for the biosynthesis of heme. In fact, to meet this
requirement, endocytic membrane traffic consists primarily of the
transferrin-transferrin receptor complex constituting the sole complex
internalized by reticulocytes. As indicated by the quasi-absence of
lysosomes, there is no sorting event in the endosomal compartment.
Rather, after release of iron, Tf rapidly recycles to the cell surface
(5 min for sheep reticulocytes(11) ; less than 4 min for rat
reticulocytes(12) ), making Tf available for another round of
iron transport. Apart from the few lysosomes left, reticulocytes are
also anucleated cells containing only vestigial remnants of Golgi and
endoplasmic reticulum(13) . Hence, the virtual lack of other
internal compartments and their highly specific endocytic function
makes these cells an excellent source and model to isolate and study
the properties, respectively, of endocytic vesicles. Previously, it has
been shown that these vesicles contain several small GTPases, which
presumably regulate the intracellular trafficking in the
cell(14) . In the present work, we have initiated a study aimed
at investigating the fusogenic properties of the endocytic vesicles. To
bypass molecular factors that regulate trafficking and docking,
although essential prerequisites to fusion in vivo, we have
used artificial membranes as target membranes. Various assays based on
lipid mixing and content mixing were employed, allowing us to
conveniently determine initial kinetics of endocytic vesicle fusion.
Thus parameters that affect the fusion process can be readily evaluated
in this manner. We show that fusion is rapid, temperature dependent,
and is markedly affected by the lipid composition of the target
membrane, strongly supporting a role for lipids capable of forming
hexagonal lipid phases. Fusion is mediated by a trypsin-sensitive
factor, which is present on the membrane of endocytic vesicles.
For the gradient analysis
experiments, the EVs were labeled with N-Rh-PE or
Immunoisolation of R18-EVs was carried
out using S. aureus, coated with an antibody raised against a
peptide (24 AA) of the cytosolic domain of the Tf-R(17) ,
generously provided by Rockford Draper (University of Texas, Dallas,
TX).
To confirm that R18 was
specifically incorporated in endocytic vesicles, we immunoisolated the
vesicles via the Tf-R and analyzed the associated fluorescence. The
immunoisolation was carried out using S. aureus coated with an
antibody raised against a peptide contained in the cytosolic domain of
the Tf-R(17) . S. aureus coated with the anti-TfR was
able to pellet three times more R18 fluorescence than S. aureus coated with an irrelevant antibody, thus confirming the
colocalization of lipid probe and Tf-R in EVs.
Figure 1:
Kinetics of fusion between endocytic
vesicles and liposomes. A, aliquots (5 µl) of EVs-R18 in 2
ml of buffer were allowed to equilibrate in a cuvette at 37 °C.
Then, 200 µM (curves a and d), 50
µM (curve b), or 10 µM (curve
c) of liposomes consisting of DOPC/DOPE (6:4), were added (arrow). Saline buffer (150 mM NaCl, 10 mM Tris, EDTA, pH 7.4) was used in curves a-c, and sucrose
buffer (250 mM sucrose, 10 mM Tris, EDTA, pH 7.4) for curve d. B, initial rate of fusion was determined for
each curve in A, as described under ``Experimental
Procedures.''
Figure 2:
Kinetics of R18 fluorescence development
upon fusion between EVs and liposomes. R18-labeled EVs were mixed with
DOPC/DOPE (6:4) liposomes at 37 °C. The kinetics of R18
fluorescence were monitored continuously at conditions that were
otherwise identical to those described in the legend to Fig. 1.
Figure 7:
Temperature dependence of EV-liposome
fusion. EVs-R18 were equilibrated in saline buffer at the indicated
temperatures, and 200 µM of liposomes of DOPC/DOPE at a
molar ratio of 6:4 equilibrated similarly were added. Initial rates of
fusion were determined as described under ``Experimental
Procedures.''
Although the latter observations,
in particular, supported the occurrence of lipid dilution as a result
of fusion, rather than transfer of R18 monomers between labeled and
unlabeled membranes, several additional control experiments were
carried out to corroborate this notion. EVs and liposomes can be
separated on sucrose density gradients because of a difference in
density. After fusion, the fusion product will differ in composition,
and hence density. Indeed, a shift in density was observed when
R18-labeled EVs had been incubated with liposomes for 30 min at 37
°C (not shown). An identical result was obtained when EVs were
labeled endogenously. This was accomplished by labeling the
reticulocyte membrane with the non-exchangeable phospholipid analog N-Rh-PE. Using an analogous procedure as outlined for R18, the
probe can be intercalated at low temperature in the plasma membrane of
cells. Warming of the labeled cells results in internalization of the
probe by endocytosis, as has been shown to occur in BHK cells (19) and HepG2 cells(20) . With this procedure, N-Rh-PE-labeled EVs (note that the probe is asymmetrically
located, i.e. in the inner leaflet only) were isolated after in situ labeling. Subsequent incubation with unlabeled
liposomes for 30 min at 37 °C, dictated by the R18 assay (Fig. 1), showed that a fusion product was obtained with a
density that was less than that of the EVs per se (Fig. 3). Furthermore, measurements of rhodamine
fluorescence before and after addition of Triton X-100 revealed that
relative to the fluorescence measured in fractions 4-6 (Fig. 3), the fluorescence in fractions 8-10 was
dequenched (approximately 50%). Although no efforts were undertaken to
correlate the extent of dequenching to the extent of fusion, it is
apparent that a relative dequenching, in conjunction with a shift in
density, is consistent with the occurrence of dilution due to merging
of labeled EVs with unlabeled liposomes.
Figure 3:
Density gradient analysis of membrane
mixing. N-Rh-PE labeled EVs were prepared as described under
``Experimental Procedures.'' EVs (5 µl) were then
incubated for 30 min at 37 °C with liposomes (DOPC/DOPE, 6:4). The
incubation mixture was loaded on a sucrose gradient (10-50%) and
centrifuged at 35,000 revolutions/min for 1 h. Fractions were collected
and analyzed for fluorescence (
Apart from membrane mixing,
fusion should also lead to the mixing of contents, bounded by the
membranes of the vesicle populations involved. For membrane vesicles of
biological origin, such an approach is commonly impossible. In this
case, however, the exclusiveness of the system could be exploited in
that Tf is the sole substrate carried by the EVs. Therefore, EVs were
isolated that contained
Figure 4:
Density gradient analysis of content
mixing. A,
Figure 5:
Effect of target membrane composition on
EV fusion. R18-labeled EVs (15 µg of protein) were equilibrated in
saline buffer at 37 °C. Fusion was initiated by adding the
appropriate target membranes (130 µM of phospholipid). The
initial rates were calculated as described and the results are
expressed, relative to the rate obtained for DOPC/DOPE (6:4) vesicles
(= 100%).
Taken
together, these results suggest a specific role of DOPE in the fusion
event of the endocytic vesicles. Apart from its low state of hydration,
DOPE has a propensity to form in isolation the hexagonal HII phase.
This bilayer to nonbilayer transition is mediated through formation of
inverted micelles. Evidently, when apposed membranes fuse, membrane
destabilization has to occur(25) , and the potentially
membrane-destabilizing properties of DOPE may therefore be of
relevance, irrespective of the nature of the intermediate structure,
which depends on the experimental conditions. Cyclosporin A and Z-fFG
are known to stabilize membranes by interfering with non-bilayer
transitions(26) . As shown in Fig. 6, both compounds
strongly inhibited the fusion of EVs with DOPE/DOPC liposomes. For both
compounds, the maximal efficiency of inhibition was about 70-80% (Fig. 6). Furthermore, mass action kinetic analysis of the data
obtained in the presence of the peptide reveals that the fusion rate
constant decreases approximately 4-fold at these conditions, with
little effect on the aggregation rate constant.
Figure 6:
A, effect of cyclosporin A and Z-fFG on
EV-liposome fusion. EVs-R18 were equilibrated in saline buffer at 37
°C. DOPC/DOPE (6:4) liposomes (200 µM) preincubated
with cyclosporin A or Z-fFG (30 µM) for 5 min at 20 °C
were then added. Z-GGG and Z-GGA were incubated with the liposomes in a
similar manner and used as control peptides. Initial rates of fusion
were calculated as described under ``Experimental
Procedures.'' B, concentration curve of fusion inhibition
by Z-fFG. Fusion measurements were carried out as described in A with liposomes preincubated with increasing concentration of
Z-fFG. Addition of the same amounts of EtOH did not affect initial
rates of fusion. Data are presented as mean ±
S.E.
Figure 8:
Effect of EV trypsinization on fusion.
EVs-R18 (5 µl) were treated with trypsin (250 µg/ml) in saline
buffer (100 µl) for the indicated time at 37 °C. The reaction
mixture was then mixed with liposomes (DOPC/DOPE, 6:4) and fusion was
monitored as described in Fig. 1. Results are mean ±
S.E.
In this work we have shown that the fusion of endocytic
vesicles can be monitored in a reliable and convenient manner, using an
assay based on lipid mixing. In contrast to the more laborious methods
used so far, and which are largely based on immunoprecipitation
techniques, kinetic and quantitative data are readily obtained by the
procedure described here. To validate the application of a lipid mixing
assay as a measure of fusion, it is imperative to take into account
that monomeric lipid transfer may occur. Therefore, a note of caution
is appropriate. As shown here a variety of control experiments can be
carried out to determine the extent to and conditions at which such a
non-fusion-mediated background transfer interferes with fusion-mediated
lipid dilution. Furthermore, when plotting the initial rate of
fluorescence increase versus acceptor vesicle concentration (Fig. 1), a background i.e. non-fusion-mediated
transfer can be derived from the intercept with the y axis
(0.7-0.8%/min; not shown). Particularly at a low acceptor
concentration, spontaneous transfer can contribute significantly to the
overall lipid dilution. Such an approach further emphasizes the need of
carefully defining the experimental conditions such that the acceptor
vesicle concentration is in a range where the acceptor
concentration-dependent transfer, i.e. fusion, predominates, i.e. well above 10 µM. While this article was in
preparation, Mullock et al.(28) used a similar
approach to characterize the fusion between late endosomes and
lysosomes. This enables them to demonstrate that R18-labeled dense
endosomes fused with lysosomes but not with mitochondria, unlabeled
dense endosomes, or light endosomes. In the present work evidence has
been obtained which supports a specific role of phospholipid in the
target membrane for fusion of endocytic vesicles. This lipid appears to
be unsaturated PE, which, in isolation, readily adopts a nonbilayer
phase. Obviously, such a membrane destabilizing property must be
intimately related to merging of apposed membranes. Our data further
indicate that (a) protein(s), tightly associated with the endocytic
vesicle membrane, is (are) involved in the overall process. From the
literature it is evident that the number of molecules that govern
intracellular fusion steadily increases. However, as far as their role
has been determined, many of these factors seem to be involved in
targeting and docking. To distinguish between both these events and
fusion per se, we have simplified the cell-free system by
using liposomes as target membranes. The process that was reconstituted
in our study represents in essence the fusion susceptibility of the
endocytic vesicles toward the inner leaflet of the reticulocyte
membrane. After their biogenesis upon internalizing the Tf Considering the membrane topology of
the major fusion event, the suceptibility of the EVs for PE in the
target membrane would be in accordance with the asymmetric localization
of this phospholipid. The specificity of this requirement is further
emphasized by the observation that PS vesicles represented a relatively
poor target membrane for the EVs. This is remarkable since fusion of a
variety of biological membranes, including sea urchin egg secretory
granules(22) , rabbit liver Golgi membranes(21) ,
viruses(23) , and spermatozoa(24) , is usually strongly
facilitated when PS is included in the target membrane. The effect of
PE could be explained by the low hydration of its polar headgroup
compared to the repulsive hydration layer associated with the headgroup
of PC. Thus PE would provide a more hydrophobic bilayer surface,
susceptible to energetically more favorable interbilayer interactions.
On the other hand, not only could PE facilitate the close approach of
bilayers, the lipid may also be directly involved in the merging
process. In this context, PE can form the hexagonal HII
phase(29) , the formation of which involves the development of
nonlamellar structures thought to be relevant as intermediates in
membrane fusion(25) . Agents such as the tripeptide Z-fFG or
the immunosuppressive agent cyclosporin A have been shown to inhibit
formation of the HII phase(30) . In fact it has also been shown
that these compounds prevent the formation of highly curved
phospholipid surfaces(31) , a membrane property that, in
analogy to highly curved small unilamellar vesicles, acts as a strong
promotor of membrane fusion. Either one or both of these properties may
be relevant to the fusogenic properties of the EVs. Evidence concerning
the specificity of the effectors was provided by experiments showing
that nonrelated tripeptides do not affect fusion, while cyclosporin
does not affect fusion with PS vesicles. Both the amino acid
composition of the peptide and the carbobenzoxy group appear to be
important structural requirements for the inhibitory effects of
peptides on membrane fusion (32) . It has also been reported
that (Z)-Gly-Phe-amide, but not (Z)-Gly-Gly-amide,
inhibits the recycling of asialoglycoproteins(33) . The effect
was suggested to be related to the involvement of metalloprotease
activity, which was inhibited by the peptide, in the fusion event of
recycling vesicles with the plasma membrane. Moreover, it has been
shown recently that the dipeptide (Z)-Gly-Phe-amide inhibits
homotypic endosome fusion, whereas the control dipeptide (Z)-Gly-Gly-amide was without effect(34) . Although
the dipeptide concentration used (3 mM) was much higher than
in the present work, the results of these studies are consistent with
the present data and strengthen the relevance of the semi-artificial
system as described here to determine molecular parameters that govern
endocytic vesicle fusion. Since enzyme activity cannot play a role in
our system, we therefore propose that properties related to nonbilayer
transitions, as reflected by the molecular properties associated with
DOPE, are the driving force for EV fusion and that agents that
interfere with these bilayer perturbing properties, such as distinct
di- and tripeptides, will inhibit the fusion event. In this context, it
is finally relevant to note that exocytosis in cultured cell lines as
well as the exocrine pancreas from rat, can be inhibited by cyclosporin
A, presumably at the level of the fusion
event(35, 36) . At present the nature of the DOPE
structure that promotes the EV fusion event is unclear. Formation of
the HII phase represents a stable molecular structure reached at
equilibrium. This structure is not likely formed in mixed bilayer
systems and is, conceptually, difficult to reconcile with such a
dynamic, transient event as membrane fusion. A local recruitment, at
sites of interbilayer contact, of lipid molecules susceptible to
bilayer departure seems possible, and protein-triggered changes in
polymorphic properties of PE species have been reported (see e.g.(37) ). The dynamics of such properties may result in
lipid scrambling (38) and leakage of vesicles contents, as
shown for virus-liposome fusion(39, 40) . A protein
triggered change in PE polymorphic properties may represent an
important aspect of the trypsin-sensitive, but N-ethylmaleimide-insensitive, protein (fraction) that we
identified, and which appears to be tightly (transmembrane?) associated
with the EV membrane. For endosome-endosome fusion, involvement of a
similar N-ethylmaleimide-resistant, trypsin-sensitive protein
has been reported(41) . However, this protein was peripherally
associated with the endosomal membrane, since alkaline treatment
effectively abolished endosome fusion. The protein involved is not
AP-2, the clathrin assembly protein, which can induce aggregation of
stripped coated vesicles (42) and, in purified form, the
aggregation of liposomes. We observed, however, that, although AP-2 is
present on the EV surface, addition of anti-AP-2 antibodies did not
inhibit EV-liposome fusion. Furthermore, various rab proteins are
associated with EVs(14) . Although not defined yet, a direct
partitioning in the fusion event of such proteins seems unlikely, their
role being primarily restricted to events (``priming'') that
precede the fusion step(9, 10, 43) .
Concerning the functional role of the protein(s) identified in the
present study, it will be of interest to determine whether this role is
exclusively limited to fusion or that this protein (or perhaps
additional ones) is involved in some aggregation activity as well.
Since fusion is completely inhibited when the incubation is carried out
in an isoosmotic sucrose-containing medium, electrostatic interactions
seem relevant, suggesting a rather nonspecific mode of interaction.
Concerning the characteristics of the potential fusion protein, it is
at present interesting to note that this protein apparently acts
without the need of GTP or its hydrolysis. Based on fusogenic work
involving biological membranes such as viruses or spermatozoa, it is
then pertinent to point out that also in those cases, fusion proceeds
in an energy-independent manner. In fact, existing evidence supports
the notion that GTP hydrolysis is not required for endosome fusion. The
characteristics of the fusion protein associated with EVs are currently
being examined.
(
)receptor, as observed
in permeabilized Chinese hamster ovary cells(6) . NSF, first
described by Rothman and collaborators(7) , has been shown to
be crucial to homotypic fusion of endosomes(8) . The
functioning of rab proteins in the fusion of endocytic vesicles does
not seem to depend on the GTPase activity, as revealed by the use of
rab GTPase mutants (9) and fusion experiments in the presence
of nonhydrolyzable GTP(10) . However, it is evident that the
molecular mechanism of membrane fusion is far from understood.
Materials
Dioleoylphosphatidylcholine (DOPC),
dioleoylphosphatidylethanolamine (DOPE), phosphatidylserine (PS, bovine
brain), cholesterol, and
carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine
(Z-fFG) were purchased from Sigma. Octadecylrhodamine B-chloride (R18), N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (N-Rh-PE) were obtained from Molecular Probes (Eugene, OR).
Cyclosporin A was from Sandoz (Basel, Switzerland)).
1-Palmitoyl,2-[C]oleoyl,3-phosphatidylcholine
(53 mCi/mmol) was purchased from Amersham-France (Les Ulis, France). Staphylococcus aureus (Pansorbin) was a product from
Calbiochem (La Jolla, CA).
Preparation of Liposomes
Large unilamellar
vesicles were prepared as described by Kremer et
al.(15) . Briefly, lipids were mixed from stock solutions,
dried under nitrogen, and redissolved in ethanol. The ethanolic
solution (5 µmol of phospholipids in 50 µl of alcohol) was
injected with a Hamilton syringe into 2.5 ml of saline buffer (150
mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.4),
under vigorous stirring. By quasi-elastic light scattering using a
Coulter particle analyzer (model N4S), it was determined that the
liposome size of the various DOPC/DOPE-containing vesicles varied
between 140 and 160 nm. The size of DOPC and PS vesicles varied between
110 and 130 nm. Note that this similarity in vesicle sizes will exclude
a significant contribution of curvature to the fusion kinetics.
Phospholipid content was quantified as described by
Bartlett(16) .Preparation of Endocytic Vesicles
Anemia was
induced in Sprague-Dawley rats by phenylhydrazine injections. Blood was
withdrawn by heart puncture and centrifuged at 1000 g for 5 min. After removing the buffy coat, the red blood cells
(reticulocyte percentage was generally >70%) were washed three times
with the saline buffer. Endocytic vesicles (EVs) were prepared as
described previously(14) . Briefly, the cells were resuspended
(50% hematocrit) in lysis buffer (50 mM NaCl, 50 mM KCl, 1 mM EGTA, 0.5 mM MgCl
, 0.2
mM phenylmethylsulfonyl fluoride, 3 mM NaN
, 100 mM MES, pH 6.5) and lysed by two
cycles of rapid freezing followed by thawing with gentle agitation at 4
°C. The non-lysed cells and debris were pelleted at 20,000
g for 20 min, and the supernatant was ultracentrifuged at
65,000
g for 60 min. The pellet enriched with EVs was
gently resuspended with 250 mM sucrose, 20 mM
HEPES-KOH, pH 7.0, at a protein concentration of about 5 mg/ml, and
kept in aliquots at -80 °C.
I-Tf. To this end, reticulocytes were preincubated at 4
°C for 1 h in RPMI containing N-Rh-PE (8 µM solubilized in ethanol; final ethanol concentration <1%, v/v))
or
I-Tf (20 µg/ml). To trigger membrane
internalization, the cells were warmed to 37 °C. After 30 min, the
cells were washed three times with the saline buffer, and EVs were
isolated as described above. Tf-labeled EV preparations were loaded
onto a Sepharose 6B column (20
1 cm) equilibrated with saline
buffer to remove free
I-Tf before gradient analysis.
Labeling of Endocytic Vesicles with R18
EVs were
labeled with R18 by injection (under vigorous vortexing) of 10 µl
of an ethanolic solution of R18 (14 mM) into 1 ml of saline
buffer containing the vesicles (1 mg protein/ml, 1.3 mM phospholipid). The mixture was incubated on ice for 1 h in the
dark. Labeled EVs were separated from unincorporated R18 using a
Sephadex G-75 column (20 1 cm), equilibrated with saline
buffer. By measuring R18 fluorescence versus phospholipid
prior to and after gel filtration, it could be determined that the
efficiency of labeling varied from 50-70%. This implies that the
final EV preparations contained approximately 5-7% R18 with
respect to total phospholipid, i.e. roughly 2.5-3.5 mol
% with respect to total lipid. These numbers are similar to the extent
and efficiency of labeling seen for various viruses(18) .
Aliquots of R18-EVs were kept at -20 °C. The probe remained
firmly membrane associated and no release was seen (as determined by
gel filtration) upon prolonged storage nor when the labeled membranes
were incubated at 37 °C for 60 min. Neither was significant
degradation of the probe apparent after a 1-h incubation at 37 °C,
as revealed by lipid extraction and analysis by thin layer
chromatography. This is consistent with the notion that nonspecific
esterase activity is not associated with EVs(44) . Quenching (Q) was calculated according to Q = 1 - F/F
, where F is fluorescence and F
the fluorescence measured after addition
of Triton X-100 (0.5% v/v).
Fusion Assay
Fusion of R18-EVs with liposomes was
measured by the R18 assay(18) . Fluorescence was measured using
a thermostated SLM Aminco Bowman Series 2 luminescence spectrometer
with 1-s time resolution at 560 and 590 nm excitation and emission
wavelengths, respectively. Fusion was initiated by injecting 200 µl
of liposomes (2 mM stock) into a magnetically stirred cuvette
containing the appropriate amount of R18-EVs in saline buffer. The
final incubation volume was 2 ml. The residual fluorescence of R18-EVs
in the cuvette, before addition of liposomes, was set to 0%. The
fluorescence after addition of Triton X-100 (0.5% v/v), corrected for
dilution, was taken as 100%. Data are expressed as the initial rate of
fusion, determined from the fluorescence tracings at time 0.Sucrose Gradient Analysis of Fusion Products
A
discontinuous sucrose density gradient was prepared by sequential
layering of 50, 40, 30, and 20% sucrose, 1 ml each, and 0.5 ml of 10%
sucrose (w/w). The samples (0.25 ml), containing either EVs or
liposomes or both, were layered on top of the gradient. After
centrifugation at 35,000 revolutions/min for 1 h at 4 °C in an SW
50 Beckman rotor, fractions of 450 µl were collected from the
bottom of the tube. Depending on the experiment, aliquots of the
fractions were (i) analyzed for N-Rh-PE fluorescence (after
addition of Triton X-100 (
,
),
(ii) counted in a
-counter (Beckman LS 5000TD) to quantify the
presence of [
C]PC, or (iii) counted in a
-counter (Packard Cobra) to determine
I-Tf.
Labeling of EVs with R18
Endocytic vesicles were
prepared as described under ``Experimental Procedures.'' The
characteristics of the EV preparation were similar to those previously
described(14) , including the enrichment of the Tf-R as
assessed by Western blots. EVs thus prepared were then labeled with R18
using an ethanol injection method(18) . It has been shown that
this method allows the introduction of the probe into biological
membranes, resulting in a concentration-dependent self-quenching of
fluorescence. At the conditions described under ``Experimental
Procedures,'' incorporation of R18 was such that the quenching of
fluorescence was usually higher than 0.9.Lipid Mixing as a Measure of EV Fusion Activity
To
determine the fusion activity of EVs, the R18-labeled vesicles were
mixed with unlabeled liposomes. The procedure implies that fusion of
both membranes will result in dilution of the fluorescent lipid analog,
causing relief of self-quenching. Hence an increase in fluorescence
reports, in principle, the occurrence of fusion. As shown in Fig. 1A (curve a), addition of the liposomes
to R18-labeled EVs resulted in a time-dependent increase of
fluorescence. From the slope of the fluorescence tracing at time 0, the
initial rate of fluorescence increase was calculated (Fig. 1B). Typically, the rate of lipid mixing was in
the order of approximately 5%/min, while the increase of fluorescence
development leveled off after 15-20 min (Fig. 2). As shown
in Fig. 1, both the rate and extent of dilution depend on the
concentration of liposomes, showing an increase when the concentration
increases. Moreover, when the saline buffer was replaced by an
isoosmotic sucrose buffer, lipid mixing was virtually abolished,
showing a residual rate of 0.46%/min, i.e. less than 10% of
the rate in saline buffer (cf.curve aversuscurved in Fig. 1). Neither was
substantial lipid dilution observed when the experiments were carried
out at 10 °C, where an initial rate was seen of 0.15%/min, i.e. less than 5% of that obtained at 37 °C (see Fig. 7).
Taken together, these experiments indicate that if these conditions
reflect conditions at which fluorescence development only occurs as a
result of spontaneous movement of the probe, such monomeric transfer
would contribute at most 5-10% to the fluorescence development
obtained at fusogenic conditions.
). As controls, EVs (
) and
liposomes, labeled with a trace amount of [
C]PC
(
), were run on separate gradients and analyzed
similarly.
I-Tf. As shown in Fig. 4,
when such vesicles had been incubated with liposomes for 30 min at 37
°C, a shift to lower density occurred for the Tf in the gradient.
In fact, the vesicle fraction of lower density was recovered at a
density identical to that observed for the N-Rh-PE-labeled
fusion product (Fig. 3). This result strongly supports the view
that the shift in contents localization reliably reflects a shift due
to membrane mixing as a result of fusion. Furthermore, quantification
of the contents mixing can be approximated by subtracting the gradient
patterns (Fig. 4B). It can thus be estimated that
approximately 20% of the internal contents of the endocytic vesicles is
shifted to lower density due to fusion. This number corresponds very
well with the level of lipid dilution, obtained in case of the R18
assay. Thus, in a parallel experiment, R18-labeled EVs were incubated
with liposomes at otherwise identical conditions. After 30 min at 37
°C, the extent of dilution was determined by measuring fluorescence
prior and after addition of Triton X-100. In three separate experiments
the dilution amounted 18.3 ± 1.4%. The similarity in both these
numbers, derived from contents and lipid mixing, also indicates that
R18 per se does not significantly interfere with the fusion
reaction, as has been noted before(18) . Taken together these
results validate the use of the R18 assay, reflecting the occurrence of
EV fusion in a reliable manner, as monitored by lipid mixing.
I-Tf-labeled EVs were prepared as
described under ``Experimental Procedures.'' EVs (50 µl)
were then incubated for 30 min at 37 °C with liposomes (DOPC/DOPE,
6:4). The incubation mixture was loaded on a sucrose gradient
(10-50%) and centrifuged at 35,000 revolutions/min for 1 h.
Fractions were collected and analyzed for radioactivity (
).
I-Tf-labeled EVs run separately were taken as the control
(
). Approximately 15,000 cpm
I-Tf were loaded on the
gradients. B, values of gradients containing EVs alone were
subtracted from values of gradients containing the reaction mixture.
Data are mean ± S.D. of three
experiments.
Effect of Lipid Composition
As target membranes,
liposomes represent a convenient tool for initial characterization of a
fusion event because their lipid composition can be readily modified.
The effect of liposomal lipid composition on endocytic vesicle fusion
was thus investigated. As shown in Fig. 5, the presence of DOPE
in the liposomal bilayer strongly promotes fusion of the endocytic
vesicles with artificial bilayers. As a function of the molar ratio of
DOPE to total phospholipid, the fusion efficiency increased as
reflected by the increase of the initial rate. At 10 mol %, a rate of
2.7%/min was obtained, which further increased to 3.4%/min at 20 mol %,
up to 5.1%/min at 40 mol % PE. Interestingly, PS vesicles, which have
been reported to fuse very efficiently with a variety of biological
target membranes(21, 22, 23, 24) ,
represent a relatively poor target for endocytic vesicles, as shown in Fig. 5. Inclusion of PS (10 mol %) in DOPE/DOPC bilayers did not
significantly affect the fusion efficiency, as obtained for DOPE/DOPC
vesicles per se. Neither did addition of cholesterol or
cholesterol hemisuccinate affect the fusion efficiency. Finally, it is
relevant to note that the similarity in vesicle sizes (see
``Experimental Procedures'') excludes that curvature effects
contribute to the observed differences in fusion kinetics.
(
)Hence, the peptide interferes with the fusion step per se rather than the close approach of EVs and liposomes.
The specificity of the inhibitory effect is apparent by the observation
that the tripeptides Z-GGG and Z-GGA were without effect on fusion (Fig. 6). Moreover, cyclosporin A did not interfere with the
fusion between EVs and PS vesicles, implying that identical kinetics
were obtained in the presence or absence of the drug (not shown).
Modulators of EV Fusion
To further characterize
the fusogenic properties of EV with DOPE/DOPC membranes, various
parameters were examined which were thought to play a role in the
overall fusion event in vivo. Addition of ATP (1 mM),
GTP (1 mM), and GTPS (20 µM) did not affect
the fusion reaction, implying that the fusogenic interaction
between EV and liposomes does not depend on energy and/or GTPase
activity. Intriguingly, addition of cytosol (final concentration 0.6
mg/ml) strongly inhibited the rate of lipid dilution by approximately
90%, while the addition of AlF
(10 mM NaF, 20
µM AlCl
) did not affect the fusion between EVs
and liposomes. When increasing concentrations of Ca
were included in the incubation medium (up to 5 mM), no
effect on both the rate or extent of fluorescence increase was seen,
suggesting that the fusion event was Ca
-independent.
Finally, fusion between EV and DOPE/DOPC liposomes is strongly
dependent on temperature. As shown in Fig. 7, lowering the
temperature from 37 to 14 °C gradually inhibited the fusion, the
initial rate becoming virtually negligible around 14 °C.
Involvement of Protein(s) in EV Fusion
In contrast
to pretreatment with N-ethylmaleimide, treatment of the
endocytic vesicles with trypsin strongly inhibited the fusion activity
of the vesicles. Fig. 8shows the effect of trypsinization time
on the initial rate of fusion. Treatment for 30 min with the enzyme
resulted in an inhibition of approximately 80%. This effect would
support the role of (a) protein(s) in the fusion reaction. To determine
whether these proteins represented a peripheral or integral protein
fraction, EVs were extensively washed with 0.1 M NaCO
, pH11. This treatment has been
reported before to remove peripheral proteins from
membranes(27) . However, the fusogenic capacity of the vesicles
was not affected after such treatment, implying that the potential
protein(s) involved are of an integral nature.
iron
complex, the majority of the vesicles rapidly returns to the cell
surface, after expulsion of the iron. Consistent with this notion is
the observation (not shown) that fusion of EVs among themselves is
minimal, proceeding with an initial rate of approximately 0.5%/min.
These experiments were carried out at conditions identical to those at
which the fusion with phospholipid vesicles was examined. It is
possible, however, that fusion of the vesicles among themselves
requires an advanced level of fine regulation of recognition and
docking (``priming''), provided by molecular factors (e.g. rab proteins, nucleotides, and other cytoplasmic
components) that were not included in the incubation medium. It should
also be noted that the EVs fused with liposomes, provided that an
excess of target membranes was used, i.e. to supersede the
targeting and docking steps.
S, guanosine
5`-O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.