Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612
Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this "stunted" fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.
In protein-mediated membrane fusion, lipids reorient
from two bilayers into one (White, 1992
The shape of lipids may be critical not only for outer
leaflets to form stalks, but also for lipids in inner leaflets to
reorient into a fusion pore within an HD (Chernomordik
et al., 1987 Cells expressing the ectodomain of HA that is glycosylphosphatidylinositol (GPI)-linked to outer leaflets (GPIHA) have been shown to hemifuse, but do not fuse, to
RBCs (Kemble et al., 1994 Amphipathic agents that preferentially partitioned into
the external leaflets of cell membranes, such as methochlorpromazine (M-CPZ), did not promote fusion pore
formation. In contrast, membrane-permeable, cationic amphipaths (MPCAs), such as chlorpromazine, partitioned into cytoplasmic leaflets and caused formation of fusion
pores within the hemifusion diaphragm between GPI-HA
cell-RBC pairs. We show that MPCAs lower the energy for
forming pores in phospholipid bilayers. This is consistent
with MPCAs promoting fusion by rendering the spontaneous curvature of cytoplasmic leaflets of plasma membranes more positive, as predicted by the stalk-pore hypothesis
(Kozlov et al., 1989 Chemicals and Solutions
Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine
(DOPE), bovine brain phosphatidylserine (PS), lyso-lauroylphosphatidylcholine (L-LPC), and lyso-stearoylphosphatidylcholine (S-LPC) were
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used as received. Oleic acid, arachidonic acid, and defatted BSA were obtained from Sigma Chemical Co. (St. Louis, MO). Chlorpromazine (CPZ), trifluoperazine (TFP), dibucaine (DB), trinitrophenol, dipyridamole, and squalene were from Aldrich Chemical Co. (Milwaukee, WI). M-CPZ was a generous gift of Smith Kline Beecham Pharmaceuticals (King of Prussia, PA).
Solutions with amphipaths, at the indicated concentrations, were prepared
freshly just before experiments and stored in the dark. All fluorescent
dyes: 6-carboxyfluorescein (CF), octadecylrhodamine B chloride (R18),
NBD-taurine (NBD-t), and tetramethylrhodamine-labeled dextran (mol
wt 40,000) were purchased from Molecular Probes (Eugene, OR).
HA-expressing Cells
All HA-expressing cell lines were received from J.M. White (University
of Virginia, Charlottesville). CHO cells transfected with X:31 influenza
HA (HA300a cell line referred to as WT cells) or with engineered GPIlinked ectodomain of HA (referred to as GPI cells) were maintained as
described (Kemble et al., 1994 Labeling of RBCs with Lipidic and Aqueous Dyes and
Video Microscopy
Fresh human RBCs, obtained from the Rush Blood Gas Laboratory, were
loaded with 1-2.5 mM CF by mild hypotonic lysis (Ellens et al., 1989 Fusion of HA-expressing cells to RBCs was monitored under an upright microscope with fluorescence attachment (Leitz D, Edison, NJ) using a ×20 or ×40 ELWD objective (Nikon Inc., Garden City, NY). Standard rhodamine (for R18, RD) or fluorescein (for CF, NBD-t) filter sets
were used to visualize the spatial redistribution of membrane and aqueous
dyes upon hemifusion and/or fusion. Fluorescence images were obtained
by a videocamera (SIT-66; DAGE MTI, Indianapolis, IN) and recorded
on VHS format videotape. Selected frames from the tape were digitized
off-line by a frame grabber (Meteor; Matrox Electronic System, Ltd.,
Quebec, Canada).
Hemifusion and Fusion of RBCs to
HA-expressing Cells
HA-expressing cells were grown to 30-40% confluency and made fusion
competent, and RBCs were bound as described (Melikyan et al., 1995b To determine the effects of the amphipaths on redistribution of membrane and aqueous fluorescent dyes after hemifusion or fusion was triggered, cells were exposed to these agents for 1 min (in the dark), and then
washed once with solutions free of drug. 20-50 mM raffinose (Sigma
Chemical Co.) was included in the solutions to compensate for the osmotic pressure of the hemoglobin in RBCs, thereby eliminating possible
swelling of RBCs that might lead to transfer of aqueous dye (Melikyan et al.,
1995b Experiments with Planar Lipid Bilayers
Solvent-free planar phospholipid bilayers were formed on an ~250-µm
hole from a solution of DOPE/DOPC/PS (6:3:1 wt/wt) in squalene. The
voltage-clamped membranes were bathed by a 150 mM NaCl, 10 mM Tris
buffer, pH 7.0, solution containing 1 mM ascorbic acid, 1 mM sodium
sulfite, and 1 mM sodium bisulfite to prevent oxidation of CPZ during experiments. The specific capacitance was calculated as the measured membrane capacitance divided by the area of the hole. The bilayer tension ( The line tension of the lipidic pore ( (1) where A is a preexponential factor that is independent of voltage; Complete Fusion of Stably Hemifused Cells Is Induced
by Modification of the Inner but Not Outer Leaflets
Fig. 2 shows a typical fluorescence pattern observed after
GPI cells and RBCs were induced to hemifuse by transiently lowering pH. The membrane dye (R18) redistributed between cells (Fig. 2, A and B), while the aqueous
dye (CF) remained within RBCs (Fig. 2, C and D), consistent with hemifusion (Melikyan et al., 1995b
The MPCAs induced fusion of hemifused cell pairs in a
concentration-dependent fashion (Fig. 3 A). When RBCs
loaded with either CF or NBD-t were hemifused to GPIexpressing cells, increasing concentrations of phenothiazines or local anesthetics resulted in a higher extent of fusion. The creation of pores was confined to the HD: the
aqueous dye transferred into as much as 90% of the GPI
cells decorated by RBCs, without a significant lytic release of dye into the external solution. The relative efficiencies
of TFP, CPZ, and DB in inducing full fusion correlated
well with their pharmacological potency and ability to
cause hemolysis (Seeman, 1972 At low pH, agents with a tertiary amine, such as CPZ, exist predominantly in their membrane-impermeable charged
form and are kinetically trapped within the external leaflets. Fusion was not detected when hemifused cell pairs
were treated with CPZ at pH 4.9 for 1 min. However, the
transition from hemifusion to full fusion increased when
the cell pairs were incubated with 0.4 mM CPZ for 1 min
at higher pH values before removing the drug (Fig. 3 B).
Deprotonated CPZ in the external leaflet translocated
into the inner leaflet where it promoted fusion; this was
validated by comparing the dye transfer to the ratio of the
deprotonated to protonated form of CPZ as a function of
pH , using pKCPZ 9.3 (Perrin, 1965 Membrane-permeable Cationic Drugs Promote
Formation of Pores in Planar Lipid Bilayers
In the hemifusion state, lipids of the outer leaflets are free
to move from one cell membrane to the other, whereas lipids of the inner leaflets remain confined to their original
cells. Integral membrane proteins should not be able to
pass through the junction where the two cell membranes
and the HD meet (Fig. 1, hemifusion) without disrupting
the HD. These proteins should remain confined to their
original membrane, excluded from the HD. Since we conjecture that the HD is devoid of integral membrane proteins, we envision that the mechanism of pore formation
within an HD could be identical to that of pure lipid bilayers (Fig. 1, fusion). Therefore, the physical chemical mechanism by which CPZ and the other amphipaths destabilize
the hemifusion diaphragm was explored by investigating
their effects on planar lipid bilayers. Voltages applied
across the bilayer facilitate the formation and enlargement
of lipid pores in bilayers (Abidor et al., 1979 In the presence of 1 mM CPZ or 4 mM DB added to
both sides of the membrane (Fig. 4, filled diamonds and
filled squares, respectively), lower voltages were required
to cause irreversible breakdown than in the absence of
these drugs (filled circles). The line tension of the pore,
Somewhat higher concentrations of MPCAs were required to substantially lower the line tension of lipidic
pores in bilayers than to induce the hemifusion-to-fusion
transition. The need for higher concentrations with bilayers may simply reflect the differences in experimental systems. Addition of 1 mM CPZ (but not 0.1 mM) or 4 mM
DB to one or both sides of preformed bilayers inevitably led to their breakdown within a few seconds in the absence
of an applied voltage. This indicates that the drugs destabilized the bilayer during their initial incorporation, before
they reached equilibrium between the membrane, aqueous phase, and torus that connects the bilayer to the Teflon partition. This breakdown is probably analogous to the
destabilization of the HD in hemifused cell pairs. Bilayers
that were reformed in the presence of these drugs were
sufficiently stable to allow their lifetimes to be measured
as a function of voltage (Fig. 4).
Suboptimal Fusion Conditions Lead to Transfer
of Membrane but Not Aqueous Dyes from RBCs to
WT Cells
The extent and rate of fusion increase with higher surface
density of HA (Ellens et al., 1990
CPZ Enlarges Fusion Pores between RBCs and WT
Cells but Only Forms Small Pores between Hemifused
RBCs and GPI Cells
As was the case with GPI cells (Fig. 2), MPCAs promoted
transfer of CF in stunted fusion. However, almost an order
of magnitude lower concentration of drug was required to
promote aqueous dye transfer from RBCs to WT cells
than to GPI cells (see below). For example, ~0.1 mM CPZ
greatly facilitated the transfer of CF from RBCs to WT cells
(Fig. 5 E). In contrast, 1 mM of the impermeant M-CPZ
did not promote transfer of CF, although washing out the M-CPZ caused some dye transfer (Fig. 5 F, arrow). The
mechanism by which this removal promoted CF redistribution is not clear.
We assessed the average size of CPZ-induced pores by
coloading RBCs with CF and a larger aqueous marker RD
(mol wt 40,000) (Schoch and Blumenthal, 1993
The transfer of CF and RD into cells was quantified by
plotting the percentage of cells stained by these dyes vs
CPZ concentrations (Fig. 7). CPZ was much more efficient in promoting transfer of both dyes from RBC to WT
cells (Fig. 7 B) than to GPI cells (Fig. 7 A). Also, CF transfer increased with CPZ concentration up to 0.7 mM for
GPI cells, whereas it saturated for WT cells with K1/2 ~ 0.025 mM. The relative size of fusion pores was characterized by plotting the ratio of cells stained with RD to those
that acquired CF as a function of CPZ concentration. The
RD/CF ratios were independent of the CPZ concentration
between 0.1 and 0.7 mM when hemifused GPI cell-RBC
pairs were treated with CPZ (Fig. 7 C, filled circles). In
contrast, the RD/CF ratio increased with CPZ concentration for WT cells (Fig. 7 C, filled squares). This suggests
that for WT cells larger fusion pores became more probable in the presence of higher concentrations of CPZ. For
GPI cells, CPZ promotes pore formation, but the size distribution does not appear to increase with greater concentrations of CPZ. Thus, MPCAs at low concentrations promote large pores in stunted fusion, whereas, even at high
concentrations, they promote relatively small pores in HDs.
LPC in Outer Leaflets Inhibits Formation of Pores
by MPCAs in Stable Hemifusion Diaphragms but Does
Not Alter the Effect of MPCAs in Stunted Fusion
Placing LPC into external leaflets of hemifused cell membranes strongly inhibited the ability of MPCAs to induce
the hemifusion-to-fusion transition (Fig. 8). When CPZ
alone was added (i.e., as a control, LPC was not added),
the majority of GPI cells became stained by CF (Fig. 4,
lefthand bar).2 However, pretreating the hemifused cells
with 5 µM S-LPC dramatically inhibited the hemifusion-
fusion transition induced by application of 0.3 mM CPZ,
even when the S-LPC in solution was removed by washing
(Fig. 8, middle bar). The destabilizing effect of CPZ on the
hemifusion diaphragm was completely eliminated by treating the cells with 20 µM S-LPC (not shown). We used S-LPC
(rather than a shorter chained L-LPC or M-CPZ) because
it neither flip-flops nor desorbs from the external leaflet of
membrane after washing out unincorporated S-LPC on
the time scale of our experiment (Chernomordik et al.,
1993
Differences between stunted fusion and hemifusion
were demonstrated by the action of membrane-impermeant amphipaths on transfer of aqueous dye. After the
fusion protocol, treatment of WT-RBC pairs with membrane-impermeant amphipaths (e.g., 1 mM M-CPZ or 0.17 mM L-LPC) for 1 min did not facilitate CF redistribution (not shown), but, in contrast with the GPI cells, removal of
the amphipaths from the external leaflets by washing with
an agent-free solution induced some CF transfer (Fig. 5 F,
arrow). Another difference between stunted fusion and
hemifusion is that treatment of WT-RBC pairs with 20 µM
S-LPC did not inhibit the redistribution of CF induced by
0.1 mM CPZ (not shown). In contrast, pretreatment of GPI-RBC pairs with S-LPC protected the HD against the
action of CPZ (Fig. 8). Thus, the action of the amphipaths
is clearly different for hemifusion induced by GPI-HA and
the stunted fusion mediated by wild-type HA. Stunted fusion was also observed with HAb2 cells (a cell line expressing higher densities of HA than WT cells [Ellens et al.,
1990 We have used amphipathic agents that insert into either
inner or outer leaflets to evaluate how altering the curvature of each of the monolayers affects formation of fusion
pores after stable hemifusion between GPI cells and RBCs.
MPCAs induced pore formation when they were introduced into inner leaflets of hemifused cells and therefore
into HDs. When inserted into outer leaflets, amphipaths
did not promote aqueous dye spread. This study demonstrates that positive spontaneous curvatures of inner leaflets promote pore formation within an HD. Also, these
agents promoted aqueous dye transfer when inserted
within inner leaflets of cell pairs that exhibited stunted fusion.
The high concentrations of amphipaths, however, undoubtedly alter more than spontaneous monolayer curvatures. One can therefore argue that other altered properties
account for our results. For example, the micelle-forming
agents when acting on inner leaflets that constitute the
HD may destabilize it by extracting lipids. Or the amphipaths may interact with cytoskeletal proteins or with integral membrane proteins excluded from the HD. But the
effects of MPCAs on purely lipidic bilayers are consistent
with formation of positive curvature lipidic pores. The theory of spontaneous monolayer curvature and how it is altered therefore provides a simple, unified, and non-ad hoc
explanation for our fusion and bilayer breakdown data.
MPCAs Can Selectively Destabilize
Hemifusion Diaphragms
CPZ and DB when inserted into lipid bilayers have been
directly shown to confer positive spontaneous curvatures
and inhibit HII-phase formation (Hornby and Cullis, 1981 With long times of incubation, MPCAs are lytic at the
concentrations we used (Seeman, 1972 Preferential accumulation of material into one leaflet of
a cell membrane creates mismatches in area between the
two leaflets. According to the well-documented "bilayer
couple hypothesis," the resulting stresses are relieved by
membrane bending toward the leaflet with excess material
(Sheetz and Singer, 1974 In the case of stunted fusion, LPC did not inhibit the
ability of CPZ to promote aqueous dye spread. This shows
that LPC, per se, does not prevent CPZ from reaching inner leaflets. In contrast, LPC in outer monolayers protects
the HD from destabilization by CPZ (Fig. 8). We offer a
possible explanation: before hemifusion, the region of contact between two cells consists of inner and outer leaflets.
After hemifusion, material of the outer monolayer has
been "pushed" out of the region it formerly occupied that is now occupied only by the HD. Since there is conservation of material in the inner and outer leaflets, the material
of the outer monolayers becomes compressed relative to
that of the inner monolayers. This mismatch in areas becomes more accentuated as the HD enlarges, and it should
limit the size of the HD, assuming the mismatch is not relieved by lipid flip-flop. Insertion of LPC into outer monolayers would further increase the compression of these
leaflets. Compression would be relieved by a shrinking of
the HD. If the addition of LPC significantly reduced the
area of the HD, CPZ could be rendered ineffective in destabilizing the small HD because the probability of forming a pore should be proportional to the area of the HD. If
this explanation is correct, the size of an HD can be experimentally manipulated.
MPCAs Can Be Used to Distinguish Stable Hemifusion
from Stunted Fusion
We found conditions under which CF did not spread from
RBCs to WT cells, but R18 did. While this pattern of dye
movement is that of hemifusion, several lines of evidence
show that the structure that hinders aqueous dye from
transferring in stunted fusion is distinctly different from
the stable hemifusion intermediate induced by GPI-HA.
Notably, the effective concentrations of CPZ and TFP required to promote aqueous dye redistribution between
RBCs and WT cells were substantially lower than those
required for GPI cells. Also, the average size of the fusion
pore connecting WT cells to RBCs could be gradually enlarged by increasing the concentration of CPZ. In contrast,
for GPI cells, more pores formed within an HD with increasing CPZ concentration, but average pore size did not
change (Fig. 7, A and C). This is reminiscent of the action of CPZ on RBC membranes. By measuring sieving of two
different size molecules, it was concluded that increased
CPZ concentration generated additional, but not larger,
pores (Lieber et al., 1984 The structures in stunted fusion are either partially
weakened HDs (weakened perhaps by limited insertion of
the transmembrane domain) and/or fusion pores that remain small for long periods of time (Tse et al., 1993). Thus, lipids
must temporarily leave the bilayer arrangement for a
nonbilayer formation. Nonbilayer structures are known to
be favored by certain lipids (Tilcock and Cullis, 1987
; Seddon, 1990
). For example, phosphatidylethanolamine and
cis-unsaturated fatty acids, with head group areas small
compared with tails, have shapes referred to as "cone
shapes." They promote formation of the HII phase (a negative curvature surface) and favor fusion (Tilcock and Cullis, 1987
; Epand et al., 1991
). In contrast, lysophosphatidylcholine (LPC)1, with its single acyl chain and large area
headgroup, has the shape of an "inverted cone" (Tilock
and Cullis, 1987). Inverted cone-shaped amphipaths pack
to form micelles (a positive curvature surface) (Epand,
1985
) and inhibit fusion when inserted in external leaflets
in a wide variety of protein-mediated fusion systems, including fusion mediated by hemagglutinin (HA) of influenza virus (Vogel et al., 1993
; Chernomordik et al., 1995b
;
Günther-Ausborn et al., 1995; Shangguan et al., 1996
).
These results suggest that the initial structure connecting
membranes has a net negative curvature. The simplest
structure, requiring the fewest lipids to reorient, is a
"stalk," a local and transient "hourglass"-shaped nonlamellar formation (Fig. 1, stalk) that joins the external, facing monolayers of apposed membranes (Kozlov et al.,
1989
; Siegel, 1993
; Chernomordik et al., 1995a
). When the
stalk forms, the membranes become locally hemifused.
Hemifusion is the merger of outer monolayers without the
mixing of inner monolayers or formation of fusion pores.
The expansion of a stalk, promoted by cone-shaped lipids (Kozlov et al., 1989
), brings the inner, distal monolayers
into contact and a hemifusion diaphragm (HD) forms (Fig. 1,
hemifusion). In this model, fusion is completed when a
pore forms within the HD (Fig. 1, fusion). If hemifusion is,
in fact, a universal intermediate stage, it would explain
how fusion can occur in a vast number of disparate systems without leakage of aqueous contents.
Fig. 1.
Schematic representation of fusion progressing from a
bound state to membranes connected by a stalk, followed by stalk
expansion to form an HD, and then to pore formation that completes fusion. The lipids curve in opposite directions for the stalk
and pore. A cone-shaped agent (as shown) facilitates formation
of a stalk. Inverted cone-shaped, positive curvature agents (shown
between hemifusion and fusion) inhibit formation of a stalk but
promote formation of a pore within the HD. The HD is composed of cytoplasmic leaflets of both cells and separates the aqueous compartments of the two fusing cells.
[View Larger Version of this Image (33K GIF file)]
; Kozlov et al., 1989
) (Fig. 1, fusion). A pore in
an HD (Fig. 1, fusion) has net positive curvature. Thus, inverted cone-shaped agents would, upon inserting in the inner leaflets, promote pore formation; cone-shaped agents,
when in the inner leaflets, would inhibit pore formation.
This expectation of the stalk-pore hypothesis has been verified in pure lipidic fusion systems (Chernomordik et al.,
1987
; Kozlov et al., 1989
). LPC acting on distal leaflets has
been shown to promote pore formation in an HD that connects two bulged planar bilayer membranes (Chernomordik et al., 1987
) and in an HD that connects a liposome to
a planar membrane (Chernomordik et al., 1995c
). The role
of lipid shape of distal leaflets in promoting the transition
from hemifusion to fusion has not, until now, been experimentally tested for protein-mediated fusion because a system demonstrating hemifusion was not available and because of the technical difficulty of gaining access to the
inner monolayers.
; Melikyan et al., 1995b
). The
cytoplasmic compartments of hemifused cells remain separated by a stable HD. We have found a means to overcome the problem of how to gain access to the inner
monolayers by using membrane-permeable amphipaths
that are surface active and insert into inner leaflets of
RBCs and other cells (Seeman, 1972
; Sheetz and Singer,
1974
; Browning and Nelson, 1976
; Steck, 1989
). Exogenous amphipathic agents were selected to modify the external and/or cytoplasmic leaflets of plasma membranes of
hemifused cells to test the role of the different leaflets in
fusion pore formation. Using GPI-HA-mediated hemifusion as an experimental model, we have been able to demonstrate that increases in the positive curvature of inner
monolayers promote correspondingly more pore formation within an HD.
). We also characterize connections between pairs of RBCs and cells expressing wild-type HA
when membrane, but not aqueous, probes redistribute. We
show that these connecting structures are different from the
diaphragms between RBCs and GPI-HA-expressing cells.
Materials and Methods
) in a glutamate-deficient medium supplemented with 400 µM l-methionine sulfoximine (Sigma Chemical Co.),
250 µM 1-deoxymannojirimycin (Calbiochem-Novabiochem Corp., San Diego, CA), and defined calf serum (Hyclone Laboratories, Inc., Logan, UT).
) and
labeled with the lipidic dye R18 essentially according to Morris et al.
(1989)
. Typically, only ~70% of the RBCs became loaded with CF; all of
them were labeled by R18. To monitor fusion pore enlargement, separate
batches of RBCs were colabeled with CF and a large aqueous marker,
rhodamine-dextran (RD), using the same mild hypotonic lysis protocol as
described above. CF and RD were dissolved in a loading solution to a final concentration of 1 mM and 0.2 mM, respectively. Both dyes were efficiently trapped in the RBCs after resealing. To study the fusion of HAexpressing cells to intact RBCs, RBCs were loaded with NBD-t (Sarkar et
al., 1989
). RBCs were labeled just before the experiment and stored on ice
for no longer than 6 h.
).
Hemifusion/fusion between HA-expressing cells and RBCs was triggered
at room temperature by exposing them to a pH 4.9 solution for 2 min and
reneutralizing with a pH 7.4 solution supplemented with 2 mg/ml glucose
as a metabolic substrate. Cells were incubated at room temperature for
the indicated period of time (usually 4-5 min) before examining the redistribution of the fluorescent dye under the microscope. A fusion index was
calculated for each dish by randomly selecting several areas and normalizing the number of HA-expressing cells stained with a given dye by the total number of cells decorated with at least one RBC. About 200 cells were
screened for each dish.
). The amphipaths had previously been dissolved in PBS containing
raffinose that was made isotonic and the pH was adjusted to 7.0. The presence of raffinose did not affect the transition from hemifusion to fusion
for intact RBCs, but slightly lowered the fusion index for ghosts (not
shown). The same fusion indices were obtained when stachyose or dextran (mol wt 10,000) was substituted for raffinose. This demonstrates that
aqueous dye transfer was caused by direct action of the amphipaths on the
HD or on small fusion pores, not by concomitant cell swelling. R18 facilitated pore enlargement: a smaller percentage of wild-type (WT) cells were stained by CF when the RBCs were not labeled with R18. Therefore, for
studies quantifying transfer of CF and/or RD, RBCs were not labeled by
R18. Unless otherwise stated, experiments were carried out at room temperature.
)
was calculated from the increment in its area (measured by the increase in
capacitance) that resulted from application of hydrostatic pressure differences between the two bathing solutions (Sukharev et al., 1982
). This technique was verified by independently measuring with a stereo microscope
the area increment of a bulged bilayer formed on a conical Teflon partition in response to hydrostatic pressures (Tien, 1974
). Both methods gave
similar results. We routinely used the electrical technique because it is
more convenient.
) was calculated using a technique
based on dielectric breakdown of the bilayer. The dependence of the lifetime (
) of the membrane on the applied voltage (V) can be described by
(Abidor et al., 1979
)
= Aexp {
·
2 /[ k T (
+ C m(
w /
m
1) V 2 / 2 )]} ,
w = 80 and
m = 2 are the dielectric constants of water and of the hydrophobic
core of the membrane, respectively; Cm is the specific membrane capacitance; k is the Boltzmann constant; T is temperature in °K; and
has its
usual numerical meaning. A and
were determined by nonlinear curvefitting (SigmaPlot; Jandel Scientific, San Rafael, CA) the above equation
to the experimental dependence of the bilayer lifetime on voltage (Chernomordik et al., 1987
, 1994). To measure this dependence, voltage pulses
of varying amplitude were applied to planar bilayers, and the times from
application of the pulse to the fast and irreversible increase in current
across the bilayer were measured (on average, 10 experiments for each
voltage).
Results
). Subsequent
application of 0.5 mM CPZ for 1 min resulted in a fast redistribution of the aqueous dye (Fig. 2 E) without altering the fluorescence pattern of the already spread R18.
Likewise, addition of other MPCAs
trifluoperazine and
dibucaine
to hemifused cells resulted in fast aqueous dye
redistribution (i.e., in fusion, see Fig. 3 A). (While we typically present data for CPZ in this paper, equivalent results
were obtained with TFP and DB at their appropriate concentrations.) A similar effect of MPCAs was observed when GPI cells were hemifused to intact RBCs loaded
with NBD-t (not shown). The cells had to be hemifused
for these agents to promote fusion. In control experiments, fusion was not observed when cationic amphipaths
were applied to cell-RBC pairs not exposed to acidic pH
or when pH was lowered but HA0 was not cleaved into its
fusion-competent HA1-HA2 form (not shown). In contrast to the case of MPCAs partitioned into inner leaflets,
treatment of hemifused cells with agents that preferentially or exclusively partitioned into outer monolayers
such as M-CPZ (Fig. 2 F), L-LPC, trinitrophenol, dipyridamole, oleic acid, and arachidonic acid
did not result
in a transition from hemifusion to fusion. Even at slightly
lytic concentrations of these compounds, CF from ghosts or NBD-t from intact RBCs did not redistribute. Thus,
agents did not promote fusion when inserted in outer leaflets, independent of whether they conferred positive
(LPC; Epand, 1985
) or negative (oleic and arachidonic
acid; Hope and Cullis, 1981
; Epand et al., 1991
) curvature.
Fig. 2.
Chlorpromazine induces complete fusion between hemifused GPI cells and RBCs. RBCs were colabeled with CF and R18. 5 min after fusion was triggered by a brief exposure of cell-RBC pairs to an acidic pH, the redistribution of R18 between RBCs and GPI
cells was completed (A and B), whereas CF did not spread into the GPI cells (C and D). Transiently (for 1 min) exposing cells in the lefthand panels to 0.5 mM CPZ resulted in efficient CF redistribution without detectable lysis (E, compare with C). In contrast, similar treatment of cells shown in the righthand panels with 1 mM of membrane-impermeable M-CPZ did not lead to any changes in CF fluorescence pattern (F, same as D). The CPZ did not cause a redistribution of R18, but it did strongly quench the R18 fluorescence in a
concentration-dependent fashion.
[View Larger Version of this Image (137K GIF file)]
Fig. 3.
(A) Concentration dependence of hemifusion-to-fusion
transition induced by phenothiazines (CPZ and TFP) and a local
anesthetic (DB). The fusion efficiency at pH 7.0 was calculated as
the ratio of cells stained with NDB-t to the total number of cells
decorated with RBCs. The functional forms of extent of fusion vs
concentration (note semilogarithmic scale) were similar for all
three drugs, despite the differences in effective concentration
ranges. Error bars show the standard errors for four to eight experiments. (B) Efficiency of hemifusion-to-fusion transition induced by CPZ as a function of pH. GPI cells hemifused to RBCs
were briefly exposed to 0.4 mM CPZ buffered at the indicated
pH values. Cells were returned to a CPZ-free solution at the
same pH, and the fraction of cells stained with NBD-t was
counted (circles, lefthand scale). The standard error was smaller than
the size of the symbols. The ratios of the neutral (B, membranepermeable) and protonated (BH+) forms of CPZ were calculated
assuming pK 9.3 and are shown by the solid line (righthand scale).
[View Larger Version of this Image (21K GIF file)]
).
) (Fig. 3 B, solid line and
righthand scale). The extent of fusion correlated well with
the fraction of the neutral, nonprotonated form of CPZ.
Both CPZ at low pH and M-CPZ at all pH values partitioned into the external leaflet of membrane where they
did not induce the hemifusion-to-fusion transition. In short, full fusion was observed only when CPZ had access to the
cytoplasmic leaflets.
), resulting in
irreversible breakdown: once a lipidic pore exceeds a critical radius in a planar membrane, it enlarges indefinitely in
the microsecond time scale. Line tension, the energy per
unit length of the edge of a pore, imposes a contracting
force. Line tension arises, in large part, because lipid
monolayers are bent into highly curved hydrophilic pores
in a bilayer. Micelle-forming lipids, such as LPC, readily
insert into the circumference of the highly curved pore
(Fig. 1, fusion) and lower its line tension (Chernomordik
et al., 1987
). Such lipids therefore reduce the contracting
force and thereby decrease the mean lifetime,
, of a bilayer at a given applied voltage.
,
was estimated by curve-fitting the theoretical equation for
electrical breakdown (Eq. 1) to experimental data.
decreased from 16.0 ± 1.0 pN for a bilayer not exposed to
drugs to 3.7 ± 0.2 and 4.1 ± 0.6 pN in the presence of CPZ
and DB, respectively. Thus, the partitioning of cationic
drugs into the planar bilayer lowered the energetic barrier
for lipidic pore formation and enlargement. This parallels
the action of the amphipaths on HDs connecting hemifused GPI cell-RBC pairs. Lowering of
by MPCAs in an
HD would facilitate lipid arrangement into the highly
curved pore and thus would account for their ability to induce the hemifusion-to-fusion transition. The lowering of
and facilitation of pore formation also provides a possible mechanism by which high concentrations of CPZ and
DB promote hypotonic lysis of RBCs (Seeman, 1972
). Adding a lower concentration of CPZ (0.1 mM, open diamonds) to the membrane did not appreciably affect the
line tension of the pore (17.1 ± 0.4 pN). This low concentration of CPZ was also not efficient in inducing fusion of
stably hemifused cell pairs (Fig. 3 A).
Fig. 4.
The effect of CPZ and DB on the propensity of lipids to
form a pore in planar lipid bilayers. (Upper panel) Schematic illustration of the current (top trace) upon application of a voltage
step (bottom trace) that results in irreversible breakdown of a planar membrane. The lifetime of the planar bilayer () is defined as the time from stepping to a given voltage to the moment of the irreversible increase of current signifying membrane breakdown. (Lower panel) Semilogarithmic plot of
as a function of applied voltage (V). Standard errors are shown for 10-12 experiments. The line tensions of lipidic pores were estimated by curve-fitting (solid lines) Eq. 1 to experimental data. The specific capacitance, Cm, and surface tension,
, of DOPE/DOPC/PS bilayers were
measured in the absence (control) and presence of amphipathic
agents. Cm = 0.66 ± 0.05 µF/cm2 was not affected by the agents,
but
= 0.42 ± 0.04 mN/m was lowered significantly by 1 mM
CPZ or 4 mM DB to 0.21 ± 0.02 and 0.15 ± 0.03 mN/m, respectively.
= 0.36 ± 0.04 mN/m in the presence of 0.1 mM CPZ was
not appreciably lower than control. (Filled circles) Control,
= 16.0 pN (R2 = 0.96); (open diamonds) 0.1 mM CPZ,
= 17.1 pN
(R2 = 0.99); (filled squares) 4 mM dibucaine,
= 4.1 pN (R2 = 0.85); (filled diamonds) 1 mM CPZ,
= 3.7 pN (R2 = 0.98).
[View Larger Version of this Image (14K GIF file)]
; Clague et al., 1991
; Melikyan et al., 1995a
; Danieli et al., 1996
), higher temperature (Stegmann et al., 1990
), and lower pH over a narrow
range below the fusion threshold (Wharton et al., 1986
).
For example, the kinetics of R18 transfer is slower at room
temperature (22-23°C) than at 37°C (Morris et al., 1989
).
At room temperature the majority of WT cells became
stained by R18 within a few min of triggering fusion (Fig.
5, A and B), whereas the aqueous dye (CF) transferred
into only a small fraction of the cells (Fig. 5, C and D). The
fraction of cells labeled with aqueous dye increased with
time after fusion was triggered. However, even 30 min after reneutralization, less than half of the WT cells with
bound RBCs received CF (data not shown). The WT cells
stained by R18 but not CF may have hemifused to RBCs
or may have become connected by small and/or transient fusion pores that did not allow passage of a detectable
amount of aqueous dye (Zimmerberg et al., 1994
). Raising
the temperature to 37°C promoted transfer of CF without
appreciably increasing spread of R18, which was already
high. We refer to the situation where wild-type HA promotes easy spread of membrane dye but poor transfer of
aqueous dye as "stunted fusion."
Fig. 5.
The transfer of membrane and aqueous dye between CF/R18-colabeled RBCs and WT cells. R18 redistributed efficiently between cells upon transient exposure to low pH (A and B), but only a small amount of CF (C and D) transferred into only a few cells (arrows) 5 min after reneutralization. A transient (1-min) exposure to 0.1 mM CPZ induced substantial amounts of CF to transfer to almost
all the WT cells (E). There was additional spread of R18 during the time between A and E (not shown). In contrast, treatment with 1 mM M-CPZ did not lead to full fusion, although washing it out induced some CF transfer (F, arrow) by an unknown mechanism.
[View Larger Version of this Image (140K GIF file)]
). These
RBCs were bound to either WT cells or GPI cells, and fusion/hemifusion was triggered at room temperature. Neither dye spread from RBCs into GPI cells, consistent with
a hemifusion phenotype (not shown). For WT, 5 min after
acidification, few cells were stained by CF (Fig. 6 A) and
were rarely labeled by RD (Fig. 6 B). A low concentration of CPZ (0.1 mM) resulted in substantial transfer of CF
(Fig. 6 C). This was accompanied by RD movement into
some WT cells (Fig. 6 D, arrowhead). More cells became
stained by both CF and RD with 0.5 mM CPZ (Fig. 6, E
and F). The transfer of RD clearly shows that relatively
large pores resulted from the CPZ treatment, either by
growth of previously existing pores or formation and enlargement of new pores. Much higher concentrations of
CPZ were required to induce CF and RD transfer between
hemifused GPI-RBC pairs. However, even at high concentrations of CPZ, the large marker, RD, did not move
into GPI cells as well as it did into WT cells (see below).
Fig. 6.
The extent of transfer of small (CF) and large (RD) aqueous probes between RBCs and WT cells. Within 5-10 min after fusion was triggered, a few cells were stained by CF (A), whereas only a rare cell was stained by RD (B). Even 30 min after acidification, a
substantial fraction (about one-half) of the WT cells were stained with CF, whereas virtually all the RD was still confined to RBCs (not
shown). A brief (1-min) exposure to 0.1 mM CPZ dramatically increased the transfer of CF (C), but only slightly facilitated the spread of
RD (D, arrowhead). Subsequent transient treatment of these cells with 0.5 mM CPZ resulted in virtually complete spread of CF (E) and
significantly higher transfer of RD (F). The fluorescence of CF was brighter after CF spread into WT cells (compare C and E to A): RD
probably quenched CF fluorescence within RBCs by resonance energy transfer.
[View Larger Version of this Image (152K GIF file)]
Fig. 7.
Transfer of a small (CF, filled symbols) and large (RD,
open symbols) probe from RBCs to GPI (A, circles) and WT cells
(B, squares) as a function of CPZ concentration 5 min after reneutralizaion. These results are replotted as the ratio of WT
(squares) and GPI (circles) cells stained with RD to those stained
with CF vs CPZ concentration (C). Error bars represent standard
errors of three to seven independent experiments.
[View Larger Version of this Image (19K GIF file)]
). Thus, by using S-LPC, we avoided possible interaction between the external leaflet agent and CPZ in bulk
solution through micellization or other processes. Removing the adsorbed S-LPC by adding delipidated BSA restored the destabilizing effect of CPZ (Fig. 8, righthand
bar). The fact that incorporating agents into external leaflets inhibited the action of agents on inner leaflets is consistent with the state of the outer leaflet affecting the action
of agents on the hemifusion diaphragm (see Discussion).
Fig. 8.
LPC incorporated into external leaflets of hemifused
cells inhibits CPZ-induced fusion. The fraction of cells stained
with NBD-t after exposure to 0.3 mM CPZ is shown (lefthand bar).
Preincubation of hemifused cell pairs with 5 µM S-LPC for 2 min
followed by washing out unincorporated S-LPC resulted in a strong
inhibition of complete fusion induced by a subsequent application of 0.3 mM CPZ (middle bar). When S-LPC was removed
from the external leaflets by incubating the cells with 4 mg/ml of
delipidated BSA for 5 min before CPZ treatment, the extent of
fusion was restored (righthand bar). Standard errors are shown
for three experiments.
[View Larger Version of this Image (48K GIF file)]
]) when fusion was triggered with suboptimal conditions: room temperature rather than 37°C. Low concentrations of CPZ promoted pore enlargement with these cells
in the same described manner.
Discussion
).
TFP, along with CPZ and DB, exhibit phenomena expected of positive curvature amphipaths: they have high
critical micelle concentrations (MacDonald, 1986
; Binford
and Palm, 1994) and lower the energy of pores in lipid bilayers (Fig. 4). At low concentrations, the partition coefficient of CPZ from aqueous solution into membranes is
roughly 3,000 (Lieber et al., 1984
). At equilibrium, CPZ
could thus constitute as much as 50% of the membrane
lipid for 1 mM in solution. However, significantly less amphipath probably inserted into membranes in our experiments. Equilibrium may not have been reached for the
1-min incubation with CPZ (the drug was incubated for
several hours to measure the partition coefficient). Also,
the partitioning of CPZ and M-CPZ into membranes saturates with increasing aqueous concentration (Zachowski
and Durand, 1988
).
; Lieber et al., 1984
).
By exposing hemifused cell pairs to MPCAs only briefly,
however, lipidic pores subsequently formed in only the
HD without causing cell lysis. The most likely explanation
for selective targeting to the HD is that the agents preferentially accumulated within inner monolayers. This accumulation causes MPCAs to be present in both leaflets of
the HD; these agents are therefore at a higher concentration than in the plasma membrane where they reside in
only a single leaflet.
; Browning and Nelson, 1976
;
Steck, 1989
). In other words, accumulation of material into
inner leaflets of RBCs causes their membranes to bend inward into cup shapes; accretion of molecules into outer leaflets causes the RBCs to crenate outward. The MPCAs
are cup-formers whereas M-CPZ and LPC are crenating
agents (Deuticke, 1968
; Sheetz and Singer, 1974
). Even if
MPCAs did not preferentially partition into inner leaflets,
the fraction of the membrane-permeable amphipaths that
did do so promoted the hemifusion-to-fusion transition:
membrane-impermeant agents that only partition into
outer leaflets, such as M-CPZ and LPC, did not promote
the hemifusion-to-fusion transition. Also, the percentage
of cell pairs undergoing the transition correlated with the
fraction of CPZ in the neutral membrane-permeable form.
). The pores created by CPZ in
the lipidic HD and in the protein-containing plasma membrane of RBCs may be fundamentally the same. It appears to be energetically more favorable for increased concentrations of CPZ to create new pores in membranes rather
than to enlarge the small pores already generated by the
amphipath.
; Zimmerberg et al., 1994
). If they are HDs, this would provide
strong evidence that hemifusion is a bona fide intermediate of full fusion because improved conditions (e.g., lower
pH or higher temperature) induce transfer of aqueous dye. The membrane-active MPCAs may promote and/or enlarge pores by making the spontaneous curvature of inner
leaflets more positive or by interacting with the transmembrane domain of HA. If the latter is true, it would indicate
that HA is part of the fusion pore and, in addition to inducing pore formation, is important in pore enlargement.
For topological reasons, the transmembrane domain of
HA should normally be excluded from the HD. Therefore, any tendency for the domain to be driven into the HD as a
result of conformational changes of HA should tend to destabilize the HD (Melikyan et al., 1995b
). In this circumstance, fusion pores would be protein-lipid complexes
(Zimmerberg et al., 1991
) rather than purely lipidic structures. Lipids and proteins could then act synergistically to
enlarge pores: the insertion of the transmembrane domain
into the HD could coax the spontaneous curvature of the inner leaflets to become more positive and to promote the
formation of fusion pores.
Received for publication 16 September 1996 and in revised form 27 November 1996.
Address all correspondence to Fredric S. Cohen, Department of Molecular Biophysics and Physiology, Rush Medical College, 1653 W. Congress Parkway, Chicago, IL 60612. Tel.: (312) 942-6753. Fax: (312) 942-8711. e-mail: fcohen{at}rpslmc.eduWe are grateful to Dr. Judith White for providing us with the HA-expressing cell lines, to Smith Kline Beecham Pharmaceuticals for the generous gift of methochlorpromazine, and to the Rush Blood Gas Lab for supplying us with fresh RBCs. We thank Drs. Leonid Chernomordik and Yvonne Lange for valuable suggestions on the manuscript.
This work was supported by National Institutes of Health grant GM27367.
CF, 6-carboxyfluorescein; CPZ, chlorpromazine; M-CPZ, metho-chlorpromazine; DB, dibucaine; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; HD, hemifusion diaphragm; LPC, lysophosphatidylcholine; L-LPC, lyso-lauroylphosphatidylcholine; S-LPC, lyso-stearoylphosphatidylcholine; MPCA, membranepermeable, cationic amphipaths; NBD-t, NBD-taurine; PS, phosphatidylserine; RD, tetramethylrhodamine-dextran; R18, octadecylrhodamine B chloride; TFP, trifluoperazine; WT, wild type.