* The Laboratary of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda Maryland 20892; and Laboratory of Bioelectrochemistry, Frumkin Institute of Electrochemistry,
Russian Academy of Sciences, Moscow, 117071 Russia
While the specificity and timing of membrane fusion in diverse physiological reactions, including virus-cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell-cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH-induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone-shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.
Protein-mediated membrane fusion is a ubiquitous
process in living systems. By far the best characterized example is fusion mediated by the influenza virus hemagglutinin (HA)1 (White, 1992 Since the first detectable event in HA-mediated fusion
is the opening of the fusion pore (Tse et al., 1993
An alternative class of models assumes that activated
fusion proteins bring membrane lipid bilayers into close
contact and decrease the energy barrier for lipids to mix
(Monck and Fernandez, 1992 In contrast to the model of a proteinaceous fusion pore,
early fusion intermediates in the alternative models already involve membrane lipids, so the lipid composition of
membranes may affect early stages of membrane fusion.
To determine which class of models fits HA-mediated
membrane fusion, we decided to systematically study the
effect of exogenous lipids on the fusion of human red blood cells (RBC) with cells expressing influenza HA. We
were guided in our choice of lipids by a specific model, the
"stalk" hypothesis, which suggests that membrane fusion
proceeds via the formation of stalk intermediates, which
are local lipidic connections between contacting monolayers of fusing membranes (Fig. 1 e; Kozlov and Markin,
1983 Stalks are highly bent local hemifusion intermediates,
and thus the elastic energy of monolayer bending is considered in the stalk hypothesis as a very important component of the stalk's energy. Different lipids bend lipid
monolayers in different directions reflecting the dynamic
molecular shape of the lipids (spontaneous curvature of the
monolayer), which in turn depends on the conformation of
individual lipid molecules and on the interactions between lipid molecules in a monolayer. Inverted cone-shaped
lysophosphatidylcholine, LPC, promotes a micellar positive
spontaneous curvature (Epand, 1985 In the present study, we found a lipid-sensitive stage
common to fusion mediated by HA of different strains of
influenza (Japan/305/57 and X:31 strains) and for hemifusion mediated by GPI-anchored HA. This lipid-sensitive
stage was subsequent to the low pH-induced change in
HA conformation but preceded or coincided with lipid mixing as assayed by membrane dye redistribution from
labeled to unlabeled cells upon their fusion. This stage was
also upstream of fusion pore formation assayed both as
aqueous dye transfer between cells and by electrophysiological recording. The lipid sensitivity of fusion, i.e., its inhibition by LPC and promotion by oleic acid (OA), is similar to that reported for some other examples of biological
fusion and for fusion of purely lipid bilayers and is consistent with the stalk mechanism of membrane fusion.
Preparation of Cells
HAb2 cells, a line of stably transfected NIH-3T3 fibroblasts expressing
the A/Japan/305/57 strain of influenza virus HA (Doxsey et al., 1985 Human RBC, freshly isolated from whole blood obtained from the National Institutes of Health blood bank (Bethesda, MD), were labeled with
fluorescent dyes. To label RBC membranes with R18 (Hoekstra et al.,
1984
To label RBC ghosts with the fluorescent water-soluble dyes carboxyfluorescein (CF) or ethidium bromide, we used mild hypotonic lysis followed by resealing as described (Ellens et al., 1989 Fusion Experiments
Expressed HA0 was cleaved into its fusion-competent HA1-S-S-HA2
form with 5 µg/ml trypsin (Fluka AG, Buchs, Switzerland) in the presence
of 250 µg/ml neuraminidase (Sigma Chemical Co.) for 10 min at room
temperature. The reaction was terminated by washing cells twice with
MEM containing 10% fetal serum. Cells were washed twice by PBS and
then incubated for 10 min with a 1 ml suspension of RBCs or RBC ghosts
(0.05% hematocrit) at room temperature (20-22°C). The unbound RBCs
were removed by three washings with PBS. HA-expressing cells with
bound RBCs (~0-2 erythrocytes per cell) were then used for experiments.
In some experiments, we evaluated the effects of exogenous lipids and
some enzymatic treatments on the RBC binding to cells. We randomly selected several areas of the dish and screened at least 200 cells to find the
average number of RBCs bound to one HA-expressing cell.
All fusion experiments were performed at room temperature (20-
22°C). Fusion was triggered by replacing PBS with an isoosmotic buffer titrated by citrate to acidic pH. After incubation of cells at low pH for 2 min
(if not stated otherwise), the acidic solution was replaced by PBS.
The extent of fusion, as defined by dye redistribution from RBCs (or
RBC ghosts) to HA-expressing cells, was quantified using fluorescence
microscopy as the ratio of dye-redistributed bound RBCs to the total
number of the bound RBCs. Extent of fusion was always measured >20
min after low pH application. Longer incubations (up to 1 h) did not increase the extent of fusion. Because extent of fusion and kinetics varied
from day to day, apparently because of variation in the level of HA expression, number of attached cells, differences in blood samples, and temperature, we routinely started the experiments by choosing the precise
conditions of the low pH treatment and concentrations of exogenous lipids to use. Each experiment presented here was repeated at least three times, and all functional dependences reported were observed in each experiment. If not stated otherwise, all data presented in one figure correspond to the same experiment performed in triplicate.
In some experiments, HAb2 cells with bound RBCs were treated by
neuraminidase (0.25 mg/ml, 20 min) or proteinase K (Sigma Chemical
Co.; 0.2 mg/ml, 20 min), or both enzymes together in PBS at room temperature before or after low pH application. Reactions were terminated by
washing cells twice with PBS.
Spectrofluorometric Measurements of Membrane
Fusion and Lipid Incorporation
An SLM Aminco Luminescence Spectrometer (Urbana, IL) was used for
all experiments. Excitation and emission wavelengths were 550 and 590 nm
for R18 and 473 and 515 nm for NBD-taurine. The standard fusion assay
was performed as follows: 2 ml of suspension of HA-expressing cells with
prebound RBCs in PBS were placed into a fluorescence cuvette and
stirred with a teflon-coated flea. The pH in the cuvette was changed to 4.9 by injecting citric acid. The increase in fluorescence was normalized to the
maximum dequenching signal obtained at infinite dilution of the probe by
lysing cells with 0.06% SDS.
Incorporation of exogenously added lipids into R18-labeled RBC
membranes decreased the membrane concentration of R18 and thus caused some relief of dye self-quenching and an increase in fluorescence. A larger fluorescence increase corresponds to more incorporation. To
evaluate how change in R18 concentration affects the extent of R18 selfquenching, we measured the fluorescence of RBCs labeled with different
amounts of R18 before (F ) and after (F0) SDS addition. These data were
used as a calibration curve to roughly estimate the incorporation of different LPCs and OA into cell membranes from the decrease in R18 quenching (F0 /F ratio) caused by adding different lipids to HAb2 cells with prebound R18-labeled RBCs. This experimental approach is based on the
assumption that both R18 and exogenously added lipids are homogeneously and independently distributed in the same monolayers of RBC
membranes, i.e., all these lipids are present only in the outer monolayer or
in both membrane monolayers. Because characteristic times for flip-flop of all LPCs used (~hours; Mohandas et al., 1982 This approach allowed us to evaluate in the same experiment, first, incorporation of exogenous lipids into RBC membranes and then, after low
pH application, fusion of RBCs with HA-expressing cells. Fusion efficiencies found by the R18 dequenching assay were not affected by decreasing
the initial extent of the dye self-quenching (F0 /F) from 3.3 to 2.45 by lowering the amount of R18 solution used to label RBCs from 15 to 10 µl
(data not shown). Thus, the smaller decreases in the extent of R18 selfquenching before low pH application, caused by lipid incorporation, did not interfere with the fusion assay.
In control experiments using RBCs without HA-expressing cells or
with HAb2 cells that were fusion incompetent (not trypsin treated), we
found that low pH application did not affect lipid incorporation. In some
experiments, HAb2 cells with prebound R18-labeled RBCs were incubated with stearoyl LPC and then pelleted. Because all R18 fluorescence
was found to stay associated with cells upon their pelleting, dequenching
of R18 measured after LPC addition appears to be caused by LPC incorporation into cell membranes rather than by extraction of R18 into lysolipid micelles.
Treatment of Cells with Exogenous Lipids
Stock solutions of lysolipids, purchased from Avanti Polar Lipids (Birmingham, AL), were freshly prepared as a 0.5% (wt/wt) aqueous dispersion and vortexed until clear. Stock solutions of oleic and arachidonic acids
(Sigma Chemical Co.), monoolein, and diolein (Nu Chek Prep, Elysian,
MN) were freshly prepared as 25-50 mM ethanolic solutions. In spectrofluorometry experiments, exogenous lipids were added directly to the cell
suspension by rapid injection of a few microliters of the corresponding
stock solutions into the cuvette under constant stirring. In fluorescence
microscopy experiments, the cell medium bathing the plastic or glass attached HA-expressing cells with prebound RBCs was replaced by 0.5 ml of medium supplemented with exogenous lipids 5 min before low pH application to the cell medium, unless stated otherwise. Low pH medium
(used to trigger fusion) and "normal" pH medium (used to terminate the
low pH treatment) were supplemented with the same concentration of
lipid. Note that since exogenous lipids were added in different ways in the
spectrofluorometry and fluorescence microscopy experiments, conditions
of lipid partition into membranes would differ between these two assays.
Added lipids did not change the pH of the medium. Ethanol at the concentrations used to add the exogenous lipids had no effect on fusion.
Some of the lipids tested in this study were toxic for cells at some concentrations. However, no correlation was found between the toxicity of
these compounds, assayed using trypan blue exclusion test, and their ability to affect low pH-induced cell-cell fusion (see also Chernomordik et al.,
1993 In some experiments, treatment of cells with exogenous lipids was followed by removing unbound lipids. Cells were washed by 2 ml PBS three
times. To extract long-chain lipids such as stearoyl and oleoyl LPC or OA,
we washed cells with PBS supplemented by 10 mg/ml of fatty acid-free
BSA (Sigma Chemical Co.).
Electrophysiological Recording
To prepare HAb2 cells for patch clamp experiments, we followed the protocol described above but lifted cells using EGTA/Trypsin (1 mM EGTA;
0.25% trypsin; GIBCO BRL, Gaithersburg, MD) and diluted them in the
external solution: 155 mM N-methyl-glucamine glutamate, 5 mM MgCl2,
2 mM Cs-Hepes, pH 7.4 (Tse et al., 1993 As expected, brief application of mildly acidic pH to HAexpressing cells with bound RBCs caused both lipid mixing (assayed as the transfer of the membrane marker, R18,
from RBCs to HA-expressing cells) and mixing of aqueous
contents (assayed as the redistribution of the content probe
CF from RBC ghosts to HA-expressing cells) (Sarkar et al.,
1989 Because the net curvature of the lipid monolayer in the
hypothetical stalk intermediate has the same sign as that of
the inverted HII hexagonal phase, stalk formation should be
inhibited by inverted cone-shaped LPC and promoted by
cone-shaped OA. Thus, if HA-mediated fusion proceeds
via stalk intermediates, LPC should inhibit and OA should
promote merger of membrane lipid bilayers. In the following experiments, we have studied low pH-triggered hemifusion and fusion mediated by different HA forms to verify these predictions of the stalk hypothesis of membrane
fusion.
Lysolipids Inhibit, and cis-Unsaturated Fatty Acids
Promote HA-Mediated Fusion
Altering membrane lipid composition by adding exogenous lipids to the medium affected the extent of low pH-
triggered fusion, assayed by spectrofluorometry, in a manner consistent with the predictions of the stalk hypothesis.
LPCs, with saturated hydrocarbon chains of lengths varying from 12 to 18 hydrocarbon groups, all decreased the
extent of fusion, although at rather different lysolipid concentrations in the medium
The dependence of fusion extent on membrane concentrations of different LPCs is presented in Fig. 2 b. While
the aqueous concentration of stearoyl LPC required to
cause twofold inhibition of fusion was ~125 times lower
than that of lauroyl LPC (~0.8 vs. ~100 µM) (Fig. 2 a),
the membrane concentration of stearoyl LPC required for equivalent (~50%) inhibition of fusion was approximately six times higher than that of lauroyl LPC (~6 vs.
~1 mol %). Thus, the membrane concentrations of different lysolipids required to cause fusion inhibition vary significantly less than their corresponding aqueous concentrations. Judging from membrane concentrations, shortchained lauroyl and myristoyl LPCs, having more profound inverted cone-shape, appear to be stronger inhibitors than the longer-chained palmitoyl and stearoyl LPCs.
In contrast, cone-shaped molecules OA (Fig. 2 b), arachidonic acid (AA), monoolein, and diolein (not shown)
promoted fusion in a dose-dependent manner. In control
experiments, no fusion was observed in the presence of
50 µM OA without application of low pH or with application of low pH to cells expressing the uncleaved HA0
(data not shown). The R18 dequenching assay was also
used to compare the concentrations at which different
lipid promoters enhanced fusion at pH 5.3 by ~50%: from
29 ± 4% (no lipids added, n = 9) to 43 ± 7% (1.25 µM diolein, n = 3), 45 ± 6% (4.2 µM monoolein, n = 3), and
45 ± 6% (2.5 µM OA, n = 3), respectively. The corresponding membrane concentrations were 2 ± 0.7, 4.5 ± 1.4, and 9.4 ± 3 for diolein, monoolein, and OA, respectively. While the differences between aqueous concentrations of these lipids apparently characterize the differences between their partition into the membrane under our experimental conditions, the membrane concentrations required for fusion promotion correlated with dynamic molecular shape of these lipids judged by their relative effects on inverted hexagonal phase formation.
Diolein is a more potent promoter of HII phase than monoolein, which is in turn a better promoter of HII phase than OA (Epand et al., 1988 Altering membrane lipid composition affected not only
the extent of fusion but also the apparent fusion kinetics
(Fig. 2 c). Oleoyl LPC increased and OA decreased the
delay time between low pH application and the onset of
R18 dequenching due to cell-cell fusion. Also, LPC decreased and OA increased the rate of R18 dequenching.
The lipid effects on fusion inferred from the R18 dequenching assay were verified by direct counting of the
fused cells with fluorescence microscopy. The spectrofluorometry and fluorescence microscopy assays significantly
differ in ways of introducing exogenous lipids and in total
numbers of suspended cells (spectrofluorometry) and attached cells (microscopy) used. Thus, quantitative relationships between aqueous and membrane concentrations
of the exogenous lipids found with R18 dequenching assay
(Fig. 2) cannot be used to directly estimate membrane
concentrations of lipids in fluorescence microscopy experiments.
Inhibition and promotion of fusion by LPC and OA, respectively, were seen with fluorescence microscopy in all
three molecular species of HA studied (Fig. 3). Lipid inhibitors and promoters of fusion were additive and canceled the effects of each other when added together. LPC
and OA added one after another did not affect the incorporation of each other into membranes as evaluated by
R18 dequenching measurements (data not shown).
The more acidic the pH applied to cells was, and thus,
the more fusion proteins activated, the less profound were
the effects of exogenous lipids (Fig. 4). Fusion inhibition
by 19 µM LPC increased from ~1.2 to ~20 times, when
the pH applied to trigger fusion was increased from 4.9 to
5.3. Similarly, the fusion promotion by 25 µM OA increased from ~1.8 to ~8 times when the pH applied was
increased from 5.15 to 5.35. These results suggest that activated HA molecules more strongly affect the spontaneous
curvature of membrane monolayers (and thus the energetics of the bent stalk intermediates) than nonbilayer lipids
such as LPC and OA. This may explain the recent findings
(Stegmann, 1993
Both inhibition and promotion of cell-cell fusion by exogenous lipids were reversible. For all three lines of HAexpressing cells studied, the fusion competence of membranes treated with short-chain lysolipids (lauroyl LPC and
myristoyl LPC) could be completely restored before low
pH application by merely washing the cells with PBS (data
not shown). Thus, the effects of lipids on fusion cannot be
mediated by any irreversible processes such as cell lysis or
solubilization of membrane components (e.g., cholesterol
depletion; Golan et al., 1988 Lipids with longer hydrocarbon chains (e.g., stearoyl
LPC, oleoyl LPC, OA, or AA) remained in the cell membrane even when cells were washed with fresh medium
(Fig. 5 a). To extract these lipids from membranes and to
restore the fusion competence, we had to wash the cells
with solutions containing fatty acid-free BSA. The low
solubility of stearoyl LPC in water allowed us to add LPC
exclusively to only one of the fusion partners, HA-expressing cells (HAb2) or RBC. We treated the given type of
cells with stearoyl LPC, washed the unbound LPC out,
brought treated cells in contact with untreated ones, and
applied low pH. LPC treatment of only one of the two
bound membranes (either HA-bearing membrane or RBC
membrane) inhibited fusion (Fig. 5 b). This experiment allows us to exclude the possibility that LPC was transferred
from treated to untreated cells via the aqueous phase.
Even if all of the LPC present in the treated cells was released into the medium, the corresponding aqueous concentration of the lysolipid would be two orders of magnitude lower than that required for inhibition of fusion. It is
still possible that some LPC had been directly transferred
from treated to untreated membrane in the contact region.
Although both the specific fluorescence of membrane
dyes and their lateral mobility can be changed by altering
membrane composition (Golan et al., 1988 Lipid Action Is Upstream of Membrane Merger and
Fusion Pore Formation
What stage of the fusion reaction is lipid-sensitive? LPC
inhibited and OA promoted mixing of the membrane lipids in fusion mediated by HA (Figs. 2 and 3, a and b) and
in hemifusion mediated by GPI-HA (Fig. 3 c). LPC also
strongly inhibited the transfer of water-soluble dyes such
as CF (Fig. 3 a) and ethidium bromide (not shown) from
RBC ghosts or NBD-taurine (data not shown) from RBCs
into the cytoplasm of HA-expressing cells (HAb2 and
HA300a cells) (Fig. 3 a), indicating that LPC blocked the
mixing of the aqueous contents of fusing cells. These results proved that lipids affect fusion before membrane
merger, including hemifusion, and suggested that the lipidsensitive stage precedes fusion pore formation. Because
the diffusion of some water-soluble dyes is restricted in the
smallest fusion pores (Tse et al., 1993 Patch clamp studies of HAb2 cell fusion to RBCs confirmed that LPC inhibits fusion pore formation at concentrations that inhibit membrane dye redistribution in the
same pair of cells. The HAb2 cell with RBCs, labeled by
fluorescent membrane marker PKH26, was patch-clamped
in the whole-cell configuration in the absence or in the
presence of lauroyl LPC in the external medium, low pH
was applied, and both the capacitance of the membrane of
HAb2 cell and the PKH26 transfer into this membrane
were measured using simultaneous electrical and video recording. In 10 out of 12 control experiments with no LPC
added, we observed changes in capacitance corresponding
to the development of a fusion pore (Fig. 6) and accompanied by PKH26 transfer. The capacitance change was 1.2 ± 0.1 pF (n = 7), which is in agreement with the values of
RBC capacitance reported before (Tse et al., 1993 Lipids Affect Fusion Downstream of HA's
Conformational Change
The lipid-sensitive stage of fusion was downstream of
membrane binding. Both inhibition of fusion by oleoyl
LPC and promotion of fusion by OA were accomplished
without concomitant changes in the RBC's adhesion to
HAb2 and HA300a cells, assayed by direct counting of the
bound R18-labeled RBCs (data not shown).
The extent of fusion depended on the duration of the
low pH pulse (Fig. 7). For short pH pulses, the longer the
pulse, the higher the fraction of RBC-HAb2 contacts
wherein cells were committed to fuse by completion of the
low pH-dependent stages, and thus the higher the fusion
extent. The same dependence of fusion extent on low pH
pulse duration was observed when the low pH medium
was supplemented by lauroyl LPC at concentrations completely inhibiting fusion. The LPC-free, neutral pH medium (used to terminate the pulse) washed LPC out and allowed fusion of cells previously committed to fuse in the
presence of LPC. The results of this experiment indicated
that LPC had not affected the effectiveness of the low pH-
dependent conformational changes of HA required for fusion.
Additional indications that the lipid-sensitive stage was
downstream of the triggering step came from the following
experiments. If cells were exposed to a 2-min pulse of low
pH and then returned to normal pH, fusion occurred (Fig.
8 a). In the presence of LPC, there was no fusion. However, if LPC was removed up to 30 min after giving this
short pulse of low pH, almost the full extent of fusion was
observed. There was a reduction in the extent of fusion
seen upon LPC removal if cells were kept in the LPCarrested fusion stage for longer times (Fig. 8 b). This decrease of the extent of fusion may be caused by slow inactivation or internalization of activated HA. Interestingly,
compensating for LPC by adding OA, instead of washing
out LPC, also led to complete fusion. Thus, adding OA to
LPC-arrested cells removed the block that keeps membranes from merger (Fig. 8 c).
Both low pH application and membrane contact were
required concomitantly to reach the LPC-arrested fusion
stage. We first applied a low pH pulse to HAb2 cells for
1 min and then 10 min later, at neutral pH, added RBCs to
establish cell-cell contact. No fusion was observed in these
experiments (Fig. 8 a). Likewise, no fusion was observed
when this experiment was conducted in the presence of
LPC, even though it was removed after the establishment of cell-cell contact. This eliminated the possibility that
lysolipid stabilized an activated state in the absence of
membrane contact.
Additional evidence that LPC arrests fusion at a stage
after the low pH-triggered conformational change of HA
came from experiments with neuraminidase. Neuraminidase treatment of HAb2 or HA300a cells with prebound
RBC inhibits both HA-mediated fusion (Fig. 9 a) and
RBCs binding to HA-expressing cells (Fig. 9 b), presumably by cleaving the sialic acids required for HA1-receptor
binding (White et al., 1983
The insensitivity of the LPC-arrested stage to neuraminidase treatment suggests that LPC blocks fusion at a
"committed state" where the fusion peptides of HA are already inserted into the target membrane. This means that
the HA1-RBC sialic acid complex is not required for completion of fusion from the LPC-arrested stage and that
the HA-receptor binding energy does not contribute to
the completion of fusion (see also van Meer et al., 1985;
Schoch et al., 1992 The same experiments with proteinase K gave opposite
results. Only HA that has been exposed to low pH is
cleaved by proteinase K (Doms et al., 1985 These data confirm that LPC blocks fusion at a stage
subsequent to low pH-induced changes in HA conformation and control for the neuraminidase experiments by
showing that the relevant HA molecules at a fusion site
are apparently accessible to external macromolecules. We
also conclude that activated fusion proteins are still
needed for the fusion reaction when the system is released from the LPC-arrested stage.
The existing hypotheses on the mechanisms of membrane
fusion differ fundamentally in their predictions on whether
a lipidic connection precedes or follows the establishment
of aqueous continuity (fusion pore formation) (Monck
and Fernandez, 1992 Our data demonstrate that lipids are involved in the
very early fusion intermediates, which is inconsistent with
models where the first stage of fusion is the lipid-independent opening of a proteinaceous fusion pore. The data also
argue against the possibility that while lipids are not directly involved into the initial proteinaceous fusion pore,
they affect the formation of this pore by altering the function of membrane proteins, such as HA. For instance, the composition of HA-expressing membranes can influence
the association of membrane proteins into some multimolecular complex required to drive membrane merger (Gutman et al., 1993 Nonbilayer lipids may modulate fusion by direct interaction with HA. An increase in the hydrophobicity of the
HA fusion peptide upon HA activation manifests itself by
a significant increase in the binding of nonionic detergent
and lipid to the extramembraneous protein domain (Doms
et al., 1985 In contrast, our results corroborate the hypothesis that
HA-mediated membrane fusion includes an early stage of
local hemifusion, when a specific lipid-involving intermediate, the fusion stalk, is formed (Kozlov and Markin,
1983 Fusion Is Affected by Membrane-incorporated Lipids
in Correlation with Their Dynamic Molecular Shape
It is the membrane-incorporated lipid that affects fusion.
Stearoyl LPC inhibited fusion, and OA promoted fusion,
even when cells treated by LPC or OA were thoroughly
washed by fresh medium to remove unbound lipids (monomers, micelles, and mixed micelles) before low pH application (Fig. 5 a). Our finding that inhibition by LPC can be
reversed by adding OA (Figs. 3 and 8), and the lack of any
direct correlation between the critical micelle concentration, CMC, of different lipids and their effects on fusion
also argue against the involvement of monomers and micelles. Lauroyl and stearoyl LPCs inhibited fusion below
and above their CMCs of ~430 and 0.4 µM (Kramp et al.,
1984 Asymmetric intercalation of lysolipids can affect fusion
by causing compression and buckling of membrane monolayers. However, while different LPCs induce echinocytosis at the same membrane concentrations (Fujii and
Tamura, 1984 The net curvature of a strongly bent monolayer in the
stalk has the same sign as in inverted HII phase (Chernomordik et al., 1995b In contrast, inverted cone-shaped LPCs inhibited fusion
(this work and Gunther-Ausborn et al., 1995 Lipids Affect Fusion at a Stage Downstream of
HA Activation
The stalk model suggests that nonbilayer lipids should affect the initial stage of actual membrane merger. By adding LPC and altering the membrane lipid composition to
be nonpermissive for fusion, we have isolated a lipid-sensitive stage of fusion. This stage is upstream of lipid mixing
and fusion pore formation, but downstream of a low pH-
induced conformational change in HA. The presence of
LPC did not affect the low pH-dependent stages of HA
activation (Fig. 7). Release from the LPC-arrested fusion
stage occurred at neutral pH upon altering membrane
lipid composition either by washing LPC out or by adding
OA (Fig. 8), so the inhibited stage was after the event in
which pH triggered fusion. Establishment of this "activated" stage was accompanied by drastic changes in the
system's sensitivity to neuraminidase and proteinase K
(Fig. 9). The initial RBC binding to HA-expressing cells is
mediated by HA1 binding to sialic acids and is sensitive to
neuraminidase (White et al., 1983 HA-mediated Fusion and Many Other Fusion
Reactions Can Involve Formation of Common,
Stalk Intermediates
Lysolipids added between fusing membranes inhibit and
cis-unsaturated fatty acids promote not only diverse biological fusion reactions (this paper and Creutz, 1981 Hypothetical stalk intermediates, if formed, can evolve
in different directions. The connection between membranes can succumb, returning the system to its original
state of two separate membranes. Alternatively, local
hemifusion can rapidly develop into complete fusion via
fusion pore formation. Finally, a local hemifusion intermediate can be stabilized or expand to form an extended
hemifusion structure. Such an extended and long-lasting
hemifusion structure has been reported for cell-cell fusion
mediated by GPI-anchored HA (Melikyan et al., 1995c To conclude, we have identified an early stage of HAmediated fusion that is dependent on membrane lipids in
agreement with the predictions of the stalk hypothesis of
membrane fusion. This hypothesis suggests that fusion involves local bending deformations of lipid monolayers
(Fig. 1 e). The interaction of activated HA molecules with
membrane lipid bilayers may dramatically decrease the
energy of stalk intermediates and, thus, allow spontaneous fusion of membrane lipid bilayers by altering the geometry
of fusion sites and/or the spontaneous curvature of membrane monolayers. The domineering role of fusion proteins in membrane bilayer rearrangement is substantiated
by our findings that the more HA molecules activated, the
less profound is the dependence of fusion on lipids and
that the presence of activated HA was imperative for the
completion of fusion downstream of the lipid-dependent
fusion stage. However, since the stalk would be formed by
bent lipid monolayers, nonbilayer lipids that aid or oppose
the monolayer curvature dominating in the stalk, respectively, promoted and inhibited fusion when added to contacting monolayers of fusing membranes. Altering the
lipid composition of membranes to be nonpermissive for
fusion allowed us to isolate the long-living activated fusion
state in which the fusion proteins are frozen in a fusioncompetent conformation. The fact that this state can last
for hours suggests that our approach may help to find out
whether the low pH conformation of HA determined by
x-ray crystallography is the fusogenic conformation.
; White, 1996
). The
influenza virus enters cells by receptor-mediated endocytosis (Yoshimura et al., 1982
). The low pH environment in
the endosome induces a conformational change in the viral hemagglutinin (HA), through which this protein mediates fusion between the viral and endosomal membranes
to deliver the viral nucleocapsid into host cell cytosol. Two
posttranslational cleavage products of the HA0 precursor
(HA1 and HA2) are responsible for viral binding to sialic
acids of receptors on the surface of host cells and for membrane fusion, respectively (Wiley and Skehel, 1987
). Both
the structure of the nonfusogenic form of HA at neutral
pH and the conformational changes this glycoprotein undergoes at low pH have been studied (Wilson et al., 1981
;
Carr and Kim, 1993
; Bullough et al., 1994
; White, 1996
).
We know that this conformational change results in a
lengthening of a coiled-coil helix in the center of the HA
trimer. At low pH, a highly conserved apolar NH2-terminal HA2 segment, the fusion peptide, is exposed, which
then binds hydrophobically to both the target bilayer and viral membrane and interacts with both of them (Stegmann et al., 1991
; Tsurudome et al., 1992
; Weber et al.,
1994
; Tatulian et al., 1995
). These interactions are thought
to trigger membrane fusion in a process that can be affected both by specific mutations of the fusion protein
(Daniels et al., 1985
; Gething et al., 1986
; Schoch and Blumenthal, 1993
; Kemble et al., 1994
) and by the lipid composition of membranes (White et al., 1982
; Stegmann et al., 1985
; van Meer et al., 1985). The specific mechanisms of
the membrane rearrangements in fusion remain unknown.
; Zimmerberg et al., 1994
), it is natural to imagine that a low pH
conformation of HA (activated HA), spanning both bilayers, starts fusion by promoting the formation of a proteinaceous pore (Fig. 1 c) similar to gap junction channels and,
only later, the expansion of this fusion pore allows mixing
of lipids and actual merger of two fusing membrane bilayers (Tse et al., 1993
; Lindau and Almers, 1995
). In agreement with this model, simultaneous measurements of lipid dye flux and fusion pores in HA-mediated cell-cell fusion
showed that fusion pore formation precedes lipid dye redistribution (Tse et al., 1993
; Zimmerberg et al., 1994
).
However, lipid flow from membrane to membrane before
the expansion of an initial fusion pore can be hindered by
the complexes of integral membrane fusion proteins in the
fusion site (Tse et al., 1993
; Zimmerberg et al., 1993
;
Kemble et al., 1994
).
Fig. 1.
Two hypothetical
pathways in HA-mediated
fusion. (a) HA-expressing
membrane in contact with
the target membrane. (b)
Low pH induces dramatic
changes in HA conformation including insertion of
the HA fusion peptide into
the target membrane. (c)
Formation of a proteinaceous pore. (d) Completion
of the fusion process. Dashed
lines show the boundaries of
the hydrophobic surfaces of monolayers. (e) Local
merger of membrane outer
monolayers. According to
the stalk hypothesis, a transient and local connection
between membranes (stalk)
has a net negative curvature
and its formation should be
facilitated and hindered by cone-shaped lipids (e.g., OA), and by inverted cone-shaped lipids (e.g., LPC), respectively. Dashed lines
show the boundaries of the hydrophobic surfaces of two monolayers.
[View Larger Version of this Image (37K GIF file)]
; Zimmerberg et al., 1993
;
Chernomordik et al., 1995b
; Melikyan et al., 1995c
). In this
scenario, local mixing of membranes' lipids occurs before
fusion pore opening (Fig. 1 e). Recent findings confirm
that membrane merger is not necessarily accompanied by fusion pore formation. A modification of HA, engineered
so that the ectoviral domains are anchored in the outer
leaflet of cell membranes via a glycosylphosphatidylinositol (GPI) tail, rather than via its normal transmembrane
domain, promotes low pH-triggered mixing of lipids but
not water-soluble probes (Kemble et al., 1994
; Melikyan
et al., 1995c
). These results suggest that the ectodomains
of HA mediate hemifusion (i.e., fusion of contacting membrane monolayers without merger of the inner monolayers) and that the transmembrane HA domains then merge
the inner membrane leaflets to open the fusion pore.
; Siegel, 1993a
,b; Chernomordik et al., 1995b
), and
gives specific predictions for the effect of lipids on fusion.
), and cone-shaped
cis-unsaturated fatty acids and phosphatidylethanolamine promote negative spontaneous curvature, as in the inverted hexagonal HII phase (Epand et al., 1991
). The net
curvature of a stalk is negative (Chernomordik et al.,
1995b
). Thus, one may expect LPC present in the contacting monolayers of membranes to inhibit, and phosphatidylethanolamine or cis-unsaturated fatty acids to promote
stalk formation and membrane merger (Fig. 1 e).
Materials and Methods
),
were cultured as described (Melikyan et al., 1995a
). CHO-K1 cells expressing the X:31 strain of influenza virus HA (referred to as HA300a
cells) or expressing the GPI-anchored X:31 HA (BHA-PI cells) (Kemble
et al., 1993
, 1994) were grown as described (Kemble et al., 1994
).
), we rapidly added 15 µl of R18 solution in ethanol (1 mg/ml) to 10 ml
of a RBC suspension (1% hematocrit) in PBS. Unbound R18 was removed by washing RBC with complete medium followed by four washings
with PBS. Under these conditions, R18 incorporated into RBC membranes to a high enough concentration to cause significant self-quenching
of fluorescence. Because of slow fusion-independent redistribution of
R18 between contacting membranes observed in experiments when HAexpressing cells with uncleaved HA were incubated at neutral pH with
prebound R18-labeled RBCs for more than 1 h, in longer experiments
(see Figs. 6 and 8 b) we also used another fluorescent membrane dye
PKH26 (Sigma Chemical Co., St. Louis, MO). Cell labeling with PKH26
was performed as described (Dimitrov and Blumenthal, 1994
).
Fig. 6.
The inhibition of pore formation by LPC. Fusion of a
HAb2 cell with a single RBC. The arrows indicate the beginning
of the application of pH 4.9 solution. Note that these experiments
were performed at 33°C rather than at room temperature to accelerate fusion pore formation upon low pH application (Spruce
et al., 1989). (a) No LPC added. The increase in capacitance reflects the fusion event. The waiting time between the beginning
of low pH application and the onset of the capacitance increase
was 16.8 ± 8.9 s (n = 10). (b) Capacitance trace in the presence of
25 µM lauroyl LPC.
[View Larger Version of this Image (7K GIF file)]
Fig. 8.
Lipids affect fusion downstream of the triggering step.
(a) LPC reversibly arrested fusion downstream low pH-dependent stages. Fusion was triggered by a 2-min application of pH 4.9 medium and assayed by fluorescence microscopy as R18 redistribution. , fusion of HAb2 cells with prebound RBCs, no lipids
added;
, starting 5 min before the low pH pulse, cells were incubated in the presence of 150 µM lauroyl LPC;
, LPC was
washed out by LPC-free PBS 20 min after the end of low pH application;
, the same pulse of low pH was applied to a suspension of HAb2 cells in the absence of RBCs. Then, back at neutral
pH , R18-labeled RBCs were added to the low pH-treated HAb2
cells.
, similar to the previous experiment, but low pH was applied to HAb2 cells in the presence of 150 µM lauroyl LPC, then
R18-labeled RBCs were added at neutral pH but still in the presence of LPC, and, finally, HAb2 cells with bound RBCs were
washed out with LPC-free PBS to remove lysolipid. Fusion was
assayed 20 min later. (b) Slow inactivation of the committed fusion stage arrested by LPC. Fusion of HAb2 cells with prebound
PKH26-labeled RBCs was triggered in the presence of 150 µM
lauroyl LPC by a 2-min application of pH 4.9 medium. At different time intervals after low pH application, LPC was removed by
washing cells with LPC-free PBS. t = 0 corresponds to the end of
the low pH pulse. The extent of fusion observed at t = 2 h in the
experiment when LPC was not removed is shown by dashed line.
Fusion extents assayed by fluorescence microscopy as PKH26 redistribution were normalized to those in control experiments with
no lipids added. Each point is mean ± SEM, n = 3. (c) Adding
OA released cells from the LPC-arrested fusion stage. Cells were
triggered to fuse by a 2-min treatment with pH 4.95 medium in
the absence or in the presence of 9.6 µM oleoyl LPC and then returned to the LPC-free neutral pH medium. The recovery of fusion previously arrested by LPC reached an extent close to that of
the control experiment (no lipids added) upon addition of 5 µM
OA at neutral pH , 5 min after the end of low pH pulse. No fusion
was observed in control experiments when OA was added to cells
not treated by low pH . Thus, OA canceled the LPC block to low
pH-triggered fusion rather than having induced fusion on its
own. Each point is mean ± SEM, n = 3.
[View Larger Version of this Image (17K GIF file)]
; Melikyan et al.,
1995a
). NBD-taurine was loaded into RBC as described (Morris et al.,
1993
).
; Bhamidipati and Hamilton, 1995
) are orders of magnitude slower than those of OA and R18
(~seconds; Broring et al., 1989
; Leenhouts and De Kruijff, 1995; Melikyan
et al., 1996
), this assumption can cause some underestimation of LPC incorporation. In contrast to the binding measurements (Weltzien, 1979
;
Mohandas et al., 1982
; Chernomordik et al., 1993
), this assay gave us an
estimate, although only approximate, of how much of the exogenous lipid
was incorporated, rather than just bound to membranes.
). Even dead cells were able to fuse upon exposure to low pH (see
also Sarkar et al., 1989
). All data presented in the paper were obtained at
nontoxic concentrations of tested compounds.
). Then cells were kept on ice until the experiment (1-3 h). Whole-cell recordings were made on HAb2 fibroblasts with prebound RBC using glass micropipettes (Microcaps, 75 µl;
Drummond Sci. Co., Broomall, PA) coated with Sylgard (World Precision
Instruments, Sarasota, FL). Patch pipettes with resistances of 1-3 M
contained (in mM): 155 Cs-glutamate, 5 MgCl2, 5 BAPTA, 10 Cs-Hepes,
pH 7.4 (Tse et al., 1993
). Capacitance measurements were made by applying 250-, 500-, and 750-Hz sine waves simultaneously (Melikyan et al.,
1995b
). The sine waves were superimposed over a
29 mV holding potential using an in-house software lock-in amplifier (program available upon
request). Partially attached cells were patched at 10 ± 2°C. After wholecell configuration (access resistance: 4.1 ± 1.3 M
[control cells, n = 10],
4.7 ± 1.9 M
[cells in the presence of LPC, n = 15]; capacitances: 20.8 ± 5.3 pF [control cells, n = 10], 12.1 ± 7.3 pF [cells in the presence of LPC,
n = 15]), cells were pulled off the glass cover slip and held in suspension
while the bath temperature was raised to accelerate fusion pore development upon low pH application (Spruce et al., 1989
). We found that this experimental procedure increased the probability of maintaining a stable
whole-cell configuration. Once the cell warmed up to 33 ± 2°C, a pipette
(same size as patch pipette) containing pH 4.9 medium (same as the external solution but with 20 mM succinic acid replacing 2 mM Hepes) was positioned ~40 µm from the cell and a pulse of positive pressure (<10 p.s.i. for 5 min) from a pneumatic picopump (model PV830; World Precision Instruments) forced a flow of pH 4.9 solution around the patched cell.
When LPC was present in the external medium, it was also added to the
pH pipette to avoid washing lysolipids out of the cell by the flow of the
low pH medium. The concentration of LPC in the external solution was
determined for each experiment by finding the concentration providing
maximum inhibition of PKH26 redistribution without significant cell lysis.
During electrical recording, we followed PKH26 redistribution using an
inverted microscope with an intensified camera (model VE1000SIT; Dage
MTI, Inc., Michigan City, IN) connected to a tape recorder (Sony Electronics, Lanham, MD) (Zimmerberg, 1993
).
Results
; Kemble et al., 1994
; Melikyan et al., 1995c
). In contrast, application of low pH to GPI-HA-expressing cells (BHA-PI cells) with bound RBC resulted in hemifusion,
i.e., R18 redistribution with no CF transfer (Kemble et al.,
1994
; Melikyan et al., 1995c
).
the longer the hydrocarbon
chain of the lysolipid, the lower the inhibiting concentration (Fig. 2 a). Because the length of the hydrocarbon
chains of the lipid affects its partitioning into RBC membrane considerably (Weltzien, 1979
), we evaluated the
membrane concentrations of exogenously added lipids
into cell membranes using R18 dequenching. Incorporation of lipids into RBC membrane labeled by self-quenching concentration of R18 resulted in membrane dye dilution and, consequently, in an increase in the fluorescence.
Membrane concentrations of added lipids were estimated
using the calibration curve of R18 self-quenching vs. dye
concentration.
Fig. 2.
Inhibition and promotion of fusion by lipids. (a and b)
The extent of R18-labeled RBC fusion to HAb2 cells in the presence of OA or LPCs with different hydrocarbon chains. In this
representative experiment, fusion was triggered by lowering the
pH to 4.9. Fusion, assayed by spectrofluorometry, was plotted vs.
concentration of exogenous lipids in the medium (a) or in RBC
membranes (b). Membrane concentration of exogenous lipids
was estimated from R18 dequenching as described in Materials
and Methods. (c) Effects of oleoyl LPC (2.5 µM) and OA (10 µM) on the kinetics of membrane mixing. The increase of fluorescence with time after lowering the pH to 5.0 reflects dilution of
self-quenched R18 upon lipid mixing between labeled RBCs and
unlabeled HAb2 cells upon their fusion.
[View Larger Version of this Image (29K GIF file)]
, 1991
). These data indicate
that the more profound the cone shape of the lipid, the
lower is its membrane concentration required for fusion
promotion.
Fig. 3.
LPC and OA added together compensate the effects of
each other on lipid mixing and content mixing mediated by different forms of HA. Fusion of HA-expressing cells (HAb2 [a]
and HA300a [b]) and hemifusion of GPI-HA-expressing cells
(BHA-PI [c]) with prebound RBCs labeled by either membrane
dye R18 or aqueous content marker CF was studied with no
added lipids (Control) or in the presence of oleoyl LPC (50 µM),
OA (50 µM), or both LPC and OA (same concentrations). Fusion was triggered by a 2-min application of low pH medium (pH 5.2 for HAb2 and HA300a cells, and pH 5.25 for BHA-PI cells). Fusion extents, assayed by fluorescence microscopy as R18 redistribution (hatched bars) or as CF redistribution (open bars), were
normalized to those in control experiments: 36.1, 26.8, 13.2, and
17.7% for HAb2, HA300a and BHA-PI (all -R18 assay), and
HAb2- CF assay, respectively. Each bar is mean ± SEM, n = 3. Bars which were not statistically different from the corresponding
controls are labeled by asterisks. In a, fusion extent for HAb2
(CF assay) in the presence of OA alone was significantly higher
than that in the presence of both OA and LPC (P < 0.05).
[View Larger Version of this Image (25K GIF file)]
; Alford et al., 1994
; Gunther-Ausborn
et al., 1995
; Shangguan et al., 1996
) that, although nonbilayer lipids affect HA-mediated fusion of lipid vesicles or
RBCs to influenza virus particles, which presumably have
very high surface density of HA molecules at the acidic pH applied, these effects appear to be less profound than
those found in the present study.
Fig. 4.
The lower the pH applied to trigger fusion, the less profound is the dependence of fusion on lipids. Fusion of HAb2 cells with prebound RBCs was triggered by a 2-min application of low pH medium in the presence of 19 µM stearoyl LPC (a) or 25 µM
OA (b). The extent of fusion assayed by fluorescence microscopy
as R18 redistribution was normalized to those in the corresponding control experiments with no lipids added: (a) 98 ± 0.5%, 68 ± 3%, 53 ± 4%, and 36 ± 4.5% for pH 4.9, 5.15, 5.2, and 5.3, respectively; (b) 39 ± 3%, 15 ± 3%, and 3 ± 0.5% for pH 5.15, 5.3, and 5.35, respectively. Each point is mean ± SEM, n = 3. Note
that because of day-to-day variability (see Materials and Methods), fusion efficiency observed for the same suboptimal pH values with no exogenous lipids added was somehow different in
two independent experiments presented in a and b.
[View Larger Version of this Image (26K GIF file)]
).
Fig. 5.
LPC incorporated into either one or both of two fusing
membranes reversibly inhibits fusion. Fusion was triggered by a
10-min application of pH 4.9 medium and assayed by fluorescence microscopy as R18 redistribution. Each bar is mean ± SEM, n = 3. (a) Fusion inhibition is caused by membrane-incorporated rather than unbound LPC. , fusion of HAb2 cells with
prebound RBCs, no lipids added;
, fusion in the presence of
36 µM stearoyl LPC;
, unbound LPC was removed before low
pH application. Cells were incubated with LPC (same concentration as above) for 10 min and then washed out with LPC-free
PBS;
, LPC was extracted from membranes by BSA before low
pH application. Cells were incubated with LPC as above and then
washed out with LPC-free PBS containing BSA. Each point is
mean ± SEM, n = 3. (b) LPC inhibits fusion when present either in HA-expressing membrane or in RBC membrane.
, fusion of
HAb2 cells with RBCs, no lipids added,
, only HAb2 cells were
treated by stearoyl LPC (42 µM);
, only RBCs were treated by
stearoyl LPC (1 µM/5 × 106 RBCs);
, both RBCs and HAb2
cells were separately treated with stearoyl LPC (same concentrations as above).
[View Larger Version of this Image (24K GIF file)]
; MacDonald,
1990
), the effects presented cannot be explained by some
lipid-dependent changes in the properties of our probes.
Fusion inhibition by LPC was observed with a variety of
different assays including R18 dequenching (Fig. 2), fluorescence microscopy with different membrane dyes (R18
and PKH26, Figs. 3 and 8 B, respectively) and water-soluble probes (e.g., CF; Fig. 3 A), and electrophysiological recording (Fig. 6). The additivity of effects of LPC and OA
observed in the fluorescence microscopy experiments with
R18 (Fig. 3) was also observed with PKH26 (not shown).
; Zimmerberg et al.,
1994
), we have applied the most sensitive assay for fusion pore formation, electrophysiological recording, which allows one to detect a pore as soon as ions can pass through it.
; Zimmerberg et al., 1994
). In 10 out of 15 experiments performed in the presence of LPC, we observed neither membrane dye redistribution nor formation of fusion pores
larger than our resolution limit of 100 pS. In five remaining experiments, we observed both dye redistribution and
pore formation. Thus, when LPC inhibited fusion, it did so upstream of both membrane merger and fusion pore formation.
Fig. 7.
LPC does not affect the low pH-dependent conformational changes of HA required for fusion. HAb2 cells with prebound R18-labeled RBCs were incubated in low pH medium (pH
5.2), LPC-free, or supplemented with 150 µM lauroyl LPC for
different times. At the end of the low pH pulse, the low pH medium was replaced by LPC-free PBS (pH 7.4) to both terminate
the fusion-triggering pulse and to wash out LPC. Fusion was assayed by fluorescence microscopy as R18 redistribution. Each
point is mean ± SEM, n = 3. In the experiments when the PBS
used to terminate the low pH pulse was supplemented with 150 µM
lauroyl LPC, and thus LPC was not washed out, the extent of fusion was 0.2 ± 0.16% (n = 3).
[View Larger Version of this Image (17K GIF file)]
). In contrast, the LPC-arrested
stage was insensitive to neuraminidase. In these experiments, LPC was present during a 2-min application of low
pH. Then cells were incubated with neuraminidase at neutral pH, and LPC was withdrawn. Both fusion and binding
were unaffected by neuraminidase treatment.
Fig. 9.
Effects of neuraminidase and proteinase K on the LPCarrested stage of HA-mediated fusion. Fusion of HAb2 cells with
prebound R18-labeled RBCs was triggered by a 3-min pH 4.9 pulse applied in the presence of 50 µM lauroyl LPC. After a 20min incubation of cells at neutral pH in LPC-containing PBS,
lysolipid was removed by washing cells with LPC-free PBS. Cells
were treated by neuraminidase or proteinase K before low pH
application or at the LPC-arrested fusion stage in the time interval between the end of low pH and removal of LPC. (a) Fusion.
The extent of fusion after removal of LPC was measured as R18
redistribution using fluorescence microscopy, and normalized to
those in the control experiments (, neither LPC nor enzymes
applied).
, the extent of fusion observed at t = 2 hours in the experiments when LPC was not removed.
and
, cells were
treated with neuraminidase or proteinase K, respectively. Each
point is mean ± SEM, n = 3. (b) Binding. In this representative experiment, the effects of neuraminidase (
) or proteinase K (
), or the combination of both enzymes (
) on binding of R18labeled RBCs to HAb2 cells before low pH application and during the LPC-arrested fusion stage were evaluated with fluorescence microscopy by counting RBCs bound to ~100 HAb2 cells.
, An average number of RBCs bound per one HAb2 cell in the
control experiments with neither LPC nor enzymes applied.
[View Larger Version of this Image (28K GIF file)]
).
), so the finding
of normal fusion and binding when cells were treated by
proteinase K before low pH application was expected.
However, the LPC-arrested stage was found to be sensitive to proteinase K, as removal of LPC after both a low pH pulse and proteinase K treatment resulted in a considerably reduced extent of fusion (Fig. 9 a). RBCs binding to
HAb2 cells at LPC-arrested stage was also susceptible to
proteinase K applied alone and, in particular, in combination with neuraminidase (Fig. 9 b), indicating that this
binding is mediated by insertion of HA fusion peptides
into RBC membrane and by HA1 interaction with RBC's
sialic acid.
Discussion
; Zimmerberg et al., 1993
; Tse et al.,
1993
; Lindau and Almers, 1995
). We have now found that
membrane lipids modulate cell-cell fusion mediated by influenza HA at a stage preceding both the merger of membrane lipid bilayers and fusion pore formation but subsequent to the low pH-induced conformational change in
HA. Hemifusion mediated by GPI-anchored HA had the
same dependence on membrane lipids as that observed for
membrane merger in fusion mediated by wild-type HA
(Fig. 3). These data are consistent with the hypothesis that both extended hemifusion and membrane merger in complete fusion involve similar membrane rearrangements
such as local hemifusion (Zimmerberg et al., 1993
; Kemble
et al, 1994; Melikyan et al., 1995c
).
; Danieli et al., 1996
). Alternatively, the
lipid composition of the target membrane can affect the
orientation of the HA fusion peptide inserted into this
membrane (Martin et al., 1993
). Our finding that LPC inhibited fusion regardless of the membrane in which it was
located (HA-expressing membrane or target membrane,
Fig. 5 b) argues against both of these hypotheses.
). Perhaps blocking the incorporation of the HA
fusion peptide into the target membrane by direct binding of monomeric or micellar LPC to the fusion peptide may
account for LPC inhibition of influenza virus-liposome fusion (Gunther-Ausborn et al., 1995
; Shangguan et al.,
1996
). It appears, however, that this mechanism is not
valid here. The LPC-arrested stage was already insensitive
to neuraminidase, indicating that LPC inhibits fusion downstream of fusion peptide insertion into the target
membrane (see below). The effects of lipids on fusion
were dependent on membrane-incorporated lipids rather
than on monomeric or micellar lipids. In addition, the interaction of the fusion peptide with amphiphilic compounds appears to be fairly nonspecific (Doms et al., 1985
),
but cone-shaped lipids promoted fusion instead of inhibiting it. Finally, the similarity of the lipid dependencies between HA-mediated fusion and other examples of biological fusion and even fusion of purely lipid bilayers (see
below) argues against the hypothesis that lipids inhibit or
promote formation of a proteinaceous fusion pore via direct lipid-HA interactions.
; Chernomordik et al., 1987
; Siegel, 1993a
,b; Zimmerberg et al., 1993
). The elastic energy of a strongly bent
monolayer in the stalk depends on the dynamic molecular
shape of the lipids in contacting monolayers. Lipid effects
on fusion found in the present study are consistent with the specific predictions of this model.
), respectively. While the CMC of palmitoyl LPC is
about 10 times higher than that of stearoyl LPC, these two
lysolipids incorporated into biological membranes and inhibited fusion in the same range of concentrations (Fig. 2, a and b).
), membrane concentrations of lauroyl LPC
and stearoyl LPC required for inhibition of fusion differed
significantly (Fig. 2 b).
), and thus stalk formation should be
promoted by the inclusion of cone-shaped lipids into contacting membrane monolayers. Cone-shaped lipids such as
OA, AA, monoolein, diolein (this work), and unsaturated phosphatidylethanolamine (White et al., 1982
; van Meer
et al., 1985; Stegmann, 1993
; Alford et al., 1994
) did promote fusion. Moreover, the fusion-promoting activity of
OA, monoolein, and diolein correlated with their dynamic
molecular shape: the more profound the cone shape of the
lipid judging by its effects on the inverted HII phase formation, the lower the membrane concentration of the lipid
required for fusion promotion.
; Shangguan
et al., 1996
). The fusion-inhibiting activity of different LPCs
was higher for shorter hydrocarbon tails (lauroyl and
myristoyl) and, therefore, for compounds having more
profound inverted cone shapes (Fig. 2 b). Lipids of complementary dynamic shapes (LPC and OA) were found to
cancel the effects of each other with respect to membrane fusion (Fig. 3), in agreement with the known additivity of
lipid effects on the spontaneous curvature of lipid monolayer (Madden and Cullis, 1982
).
). This binding mechanism was apparently superseded at an LPC-arrested stage
by mechanisms based on the low pH-induced insertion of
the HA fusion peptide into RBC membranes (Stegmann et al., 1991
; Tsurudome et al., 1992
), and thus fusion was
insensitive to neuraminidase. In contrast, both membrane
binding and fusion upon release from the LPC-arrested
stage were inhibited by proteinase K treatment (Fig. 9), indicating that activated HA molecules are still required at
this stage to keep membranes together and to drive fusion
to completion. These data indicate that nonbilayer lipids
affect the merger of membrane lipids rather than any
other stage of the fusion process.
; Glick
and Rothman, 1987
; Chernomordik et al., 1993
; Paiement
et al., 1994
; Yeagle et al., 1994
; Chernomordik et al., 1995c
;
Gunther-Ausborn et al., 1995
; but see Nagao et al., 1995
;
Coorssen, 1996
), but also fusion of purely lipid bilayers
(for review see Chernomordik et al., 1995b
). Importantly,
LPC inhibits HA-mediated fusion at membrane concentrations similar to those found to inhibit syncytia formation mediated by the Sendai virus F protein (Yeagle et al.,
1994
) and baculovirus gp64 (Chernomordik et al., 1995c
),
as well as for microsome-microsome fusion (Chernomordik et al., 1993
) and vesicle-planar bilayer fusion
(Chernomordik et al., 1995a). We suggest that fusion mediated by HA and other proteins and fusion of purely lipid
bilayers proceed via a common lipid-involving intermediate
a stalk structure, producing local and transient hemifusion of membranes. Nonbilayer lipids may influence the
energy required for stalk formation by altering some properties of membrane lipid bilayers and, in particular, the
propensity of lipid monolayers to bend into nonbilayer fusion intermediates (Chernomordik et al., 1995b
), the hydrophobicity of membrane surfaces (Ohki, 1988
), and the
repulsive pressures and the distances between the contacting monolayers (Rand and Parsegian, 1989
; McIntosh et al.,
1995
).
).
The wall of an initial fusion pore can be formed at least
partially by lipids (Monck and Fernandez, 1992
; Zimmerberg et al., 1991
, 1993). In this case, as in the case of a
stalk, the energy of the pore will be dependent on lipid composition. However fusion pore formation involves the
bending of different membrane monolayers, and the direction of lipid monolayer bending in a pore and in a stalk
will be opposite (Chernomordik et al., 1995b
). Thus, to
study the lipid effects on fusion, one needs to know which
lipids are added to which membrane monolayers and how
fast the transmembrane redistribution of lipids is. For instance, OA quickly redistributes between outer and inner
membrane monolayers (Broring et al., 1989
). This may explain why in Fig. 3 a the enhancement of fusion by OA was
found to be more profound when fusion was measured
with lipid-mixing assay, which does not distinguish between hemifusion and complete fusion, than with contentmixing assay, which characterizes only complete fusion. Cone-shaped OA in contacting membrane monolayers is
expected to promote stalk formation and hemifusion. The
same lipid in the inner membrane monolayers should inhibit fusion pore development and thus can partially compensate the hemifusion and fusion promotion by OA
present in outer monolayers.
Received for publication 7 August 1996 and in revised form 7 November 1996.
Address all correspondence to Leonid V. Chernomordik, The Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 10, Room 10D04, 10 Center Drive, Bethesda, MD 20892-1885. Tel.: (301) 594-1128. Fax: (301) 594-0813. E-mail: lchern{at}helix.nih.govWe are grateful to Drs. M. Kozlov, G. Melikyan, and J. White for valuable discussions. Special gratitude is due to Dr. J. White for providing us with HA300a and BHA-PI cells. We thank Drs. J. Coorssen and T. Whalley for critical reading of the manuscript.