Bioscience Reports, Vol. 20, No. 6, 2000
MINI REVIEW
Membrane Fusion Induced by Phospholipase C and
Sphingomyelinases
Félix M. Goñi1,2 and Alicia Alonso1
Receiûed July 11, 2000
In the past decade lipid vesicle fusion induced by either bacterial PC-preferring phospholipase C, phosphatidylinositol-specific phospholipase C, sphingomyelinase, or a combination
of phospholipase C and sphingomyelinase has been demonstrated. In the present paper,
the experimental evidence is reviewed, and discussed in terms of the underlying molecular
mechanisms of fusion, and of the possible physiological relevance of these findings.
KEY WORDS: Phospholipase C; sphingomyelinase; membrane fusion; liposomes; model
membranes; non-bilayer lipids; non-lamellar lipid phase.
ABBREVIATIONS: Ch, cholesterol; DAG, diaglyclycerol; HII, inverted hexagonal (phase);
LUV, large unilamellar vesicles PC, phosphatidylcholine; PC–PLC, phosphatidylcholinepreferring phospholipase C; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIPLC, phosphatidylinositol-specific phospholipase C; QII, inverted cubic (phase); SUV,
small unilamellar vesicles; TMC, trans-monolayer contacts.
INTRODUCTION
This review deals with enzymes that generate certain amphilphiles in model bilayer
membranes, and that lead to liposome fusion, through the formation of transient
nonlamellar intermediates. The interplay between enzyme activity, nonlamellar
phase formation and membrane fusion is the connecting thread in this review.
As in so many areas of membrane biology, the use of model membranes has
contributed greatly to our understanding of membrane fusion. The idea that amphipathic molecules might act as intermediates in membrane fusion was first proposed
by Lucy (1970), on the basis of his studies on cell fusion. Early studies on model
membrane fusion induced by amphilphiles include liposome fusion induced by free
fatty acids (Kantor and Prestegard, 1978) or by surfactants (Alonso et al., 1981).
However, liposomes as a tool in the study of membrane fusion only became really
useful when water-soluble fluorescent probes, that could be easily entrapped in the
vesicles, were introduced (Wilschut and Papahadjopoulos, 1979). This was complemented by other fluorescence techniques allowing the assay of mixing of bilayer
1
Unidad de Biofisica (CSIC-UPV兾EHU), and Departamento de Bioquı́mica, Universidad des Paı́s Vasco,
Aptdo. 644, 48080 Bilbao, Spain.
2
To whom correspondence should be addressed. E-mail [email protected]
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0144-8463兾00兾1200-0443$18.00兾0  2000 Plenum Publishing Corporation
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lipids (Struck et al., 1981; Hoekstra et al., 1984), and by further methods for the
mixing of aqueous contents (Ellens et al., 1986a, b). Together with the methodological process, physical studies on the effects of certain aphilphiles, diacylglycerol
(DAG) in particular, on the phase behaviour of phospholipids in water (Das and
Rand, 1986; Siegel et al., 1989) gave support to the hypothesis that amphiphiles
would perturb the lipid bilayer, and membrane-membrane contacts within these relatively unstable regions would in turn lead to bilayer fusion (Siegel, 1986; Rand and
Parsegian, 1986).
In this intellectual climate, our first report on liposome fusion induced by the
catalytic activity of phospholipase C (i.e., in situ generation of DAG) clearly
strengthened the putative role of amphilphiles in membrane fusion. It was also the
first report of fusion induced by the catalytic activity of an agent; previously known
fusogens acted stoichiometrically. The enzyme used in this study was the bacterial
phosphatidylcholine-preferring phospholipase (PC-PLC). Later reports described
that, under certain conditions, sphingomyelinase could fuse sphingomyelin-containing liposomes (Basañez et al., 1997a), and that the mixture PCPLCCsphingomyelinase was fusogenic under conditions where none of the enzymes,
acting separately, would have this effect (Ruiz-Argüello et al., 1998a). More recently,
we have been able to observe liposome fusion induced by phosphatidylinositolspecific phospholipase C (PI-PLC) (Villar et al., 2000). In this review we shall
summarize the above findings and put them in the context of the physiological process of cell membrane fusion.
FUSION INDUCED BY PC-PLC
PC-PLC (EC 3.1.4.1) was obtained from Bacillus cereaus; the enzyme source
was chosen in view of its availability, and also because of the immunological and
functional similarities between this and mammalian PLC (Clark et al., 1986; Graziani et al., 1991). A first series of experiments were aimed at determining the conditions under which, if at all, PLC-promoted liposome fusion occurred (Nieva et al.,
1989). It was found that fusion of large unilamellar vesicles (LUV) [∼100 nm in
diameter, obtained by extrusion, phosphatidylcholine (PC)兾phosphatidylethanolamine (PE)兾cholesterol (Ch) 2:1:1 mole ratio] takes place when treated with PLC, concomitant with the hydrolysis of very small amounts of phospholipid and without
significant release of vesicle contents (Fig. 1). Fusion was detected as the mixing of
vesicle contents and of bilayer lipids respectively in Figs 1(a) and 1(b). The fusion
signal (from mixing of vesicle contents) was not corrected for leakage, since the
latter was undetectable in the time scale of our observations. Vesicle fusion was
accompanied by a concomitant increase in scattered light (Fig. 1b). When the
enzyme was preincubated in the presence of 50 mM O-phenanthroline, a specific
inhibitor of PLC, neither phospholipid hydrolysis nor liposome fusion was observed.
The influence of bilayer composition on the fusogenic effects of PLC was examined by repeating the above experiments using liposomes containing varying proportions of PC, PE and Ch. The optimal enzyme concentration was determined
separately in each case; preliminary experiments showed that under our conditions,
PC and PE were hydrolyzed at similar rates by PLC. It was found that the bilayer
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Fig. 1. Fusion of liposomes (PC兾PE兾Ch, 2:1:1 molar ratio)
in the presence of PC-PLC. (a) Fusion as mixing of aqueous
contents (continuous lines), and phospholipid hydrolysed by
the enzyme (closed circles correspond to experimental values).
(b) Fusion as mixing of bilayer lipids (curve 1), change in
scattered light (curve 2), and fusion as mixing of vesicle contents, redrawn here to facilitate comparison (curve 3). Reproduced from Nieva et al. (1989) with permission from the
American Chemical Society.
composition was rather critical and that both PE and Ch were essential, in addition
to PC, for significant fusion to occur with low levels of phospholipid hydrolysis
(Nieva et al., 1989).
Fusion of sonicated unilamellar vesicles (SUV) in the presence of PLC was also
considered. As expected, these vesicles were better substrates than LUV for the
enzyme; i.e., the initial rates of lipid hydrolysis were much higher. Consequently,
fusion also occurred at an earlier stage, mainly because of a shorter lag period;
however, the extent of fusion and the maximum fusion rates were similar to those
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recorded with LUV (Nieva et al., 1989). Luk et al. (1993) later published observations of PLC-induced aggregation and fusion of cholesterol-lecithin SUV that
confirmed our results obtained previously.
Thus PLC, when added to a liposome suspension could induce mixing of bilayer
lipids and, simultaneously or immediately afterwards, leakage-free mixing of vesicle
contents (Fig. 1). These two observations taken together are usually considered as
indicative of liposome–liposome fusion (Ellens et al., 1985). The extent of fusion
(H50%) indicated that each final fusion product was composed of more than two
of the original liposomes (Bentz et al., 1988). None of the above phenomena were
observed when the enzyme activity was inhibited by Q-phenanthroline; thus vesicle
fusion was being promoted specifically by the catalytic activity of PLC. Since this
enzyme cleaves phospholipids releasing diacylglycerols, it could be reasonably
assumed that the latter lipids were responsible for the observed phenomena. This
fusion system was acknowledged (Little et al., 1993) as the first model system in
which fusion was induced by a catalytic agent.
MODULATION OF FUSION BY DAG AND LYSO-PC
Further information on the molecular mechanisms involved in phospholipaseC-promoted membrane fusion was obtained by preparing liposomes containing in
addition to phospholipid and Ch small amounts of DAG or other lipids, such as
lysophosphatidylcholine (lyso-PC) (Nieva et al., 1993). The fusion process could be
modified by substituting for DAG some of the phospholipids in the initial liposome
formulation. At 37°C, when no DAG was present in the substrate, fusion started
⯝10 s after enzyme addition, when the amount of DAG generated was ⯝∈4 mol.%
of the total lipid. The reaction proceeded for another 20 s (up to ⯝15 mol.% DAG),
before end-product inhibition became apparent; still, although at progressively
declining rates, the process continued until virtually 100% mixing occurred without
any significant vesicle leakage (Nieva et al., 1993). When the initial liposome composition was modified, so that 5 mol.% of the phospholipid was substituted by DAG,
the fusion process became faster and the lag time after enzyme addition became
shorter: 10 s after enzyme addition the total DAG content was 12 mol.% and the
reaction rate was already maximal. Saturation occurred ⯝20 s after enzyme addition,
when DAG made up to 20 mol.% of the total lipids. When the substrate liposomes
contained 10 mol.% of the phospholipid substituted by DAG, fusion started as soon
as PLC was added, and the process reached a plateau after 10 s, when the total
amount of DAG was again ⯝10 mol.%. These results appear to indicate that, at
37°C, the enzyme catalyses efficiently the fusion of liposomes containing PC兾PE兾
Ch (2:1:1 mole ratio) plus DAG when the proportion of the latter is in the range 5–
20 mol.% of the total lipid. At higher DAG proportions, fusion becomes inhibited
by this end-product.
The fusion process was equally abolished when lyso-PC was incorporated into
the liposomes at 10 mol.% (Nieva et al., 1993) or when Ca2C concentration was
reduced from 10 to 2 mM while the hydrolytic activity of the enzyme remained
unchange. In these samples, mixing of aqueous contents occurred only at DAG
concentrations greater than 30 mol.%. Lysophospholipids have been described as
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447
inhibiting fusion process very different from the one described here (Chernomordik
et al., 1993.
A detailed analysis considering fusion rate in the various liposome preparations
as a function of temperature showed that fusion varied with temperature in a different way from enzyme activity or vesicle aggregation (Fig. 2). A good correlation
was observed, at all temperatures, between aggregation and enzyme activity for the
liposomes that did not originally contain DAG; both phenomena appeared to be
activated at ⯝45°C. For DAG-containing liposomes, the enzyme activity appeared
to be higher at all temperatures, but the temperature dependence was otherwise
similar to the other substrates. However, aggregation was much more efficient in the
presence of DAG than in its absence, particularly at low temperatures, suggesting
that DAG conferred some degree of adherence on the vesicles. The temperature
dependence of vesicle fusion (as mixing of contents) shows maxima whose absolute
values were higher, and occurred at lower temperatures, the higher the initial DAG
contents. The presence of lyso-PC or low Ca2C concentrations, specifically inhibited
this process.
Moreover, in contrast with aggregation, fusion did not increase monotonically
with temperature, and each liposome composition exhibited a peculiar pattern as a
function of temperature (Fig. 2). At low temperatures, fusion proceeded at a low
rate, but it was very efficient (plateau values near 100%); when the temperature was
increased, the rate increased, but the saturation point decreased. Finally, at the highest temperature tested, both values were decreased. This inhibitory effect, at temperatures at which both enzyme activity and vehicle aggregation were enhanced,
provided the first hint that the existence of a structural intermediate whose formation
would be influenced both by the temperature and by DAG concentration. The fact
that lyso-PC also inhibited the fast fusion process suggested that the fusion intermediate might have a non-lamellar structure (Epand, 1985).
ELECTRON MICROSCOPY OBSERVATIONS
PC-PLC-induced fusion of liposomes composed of PC, PE and Ch was studied
by conventional and fast-freeze freeze-fracture electron microscopy (Burger et al.,
1991), as well as by cryo-transmission electron microscopy (Basañez et al., 1997a).
The conditions described by Nieva et al. (1989) led to a fusion process that was
essentially completed in ca. 30s. To carry out time-resolved ultrastructural studies
new conditions were established, decreasing enzyme concentration, so that the fusion
rate was lowered by about one order of magnitude. Note, however, that the DAG
concentrations marking the onset and termination of fusion were still 5% and 20%
respectively.
Fusion experiments were performed as described above. Experiments were first
executed by conventional freeze-fracture, in the presence of 30% (v兾v) glycerol as a
cryoprotectant., Samples were taken from a suspension of LUV and frozen before
and 30, 90 or 300 s after the addition of PLC. The corresponding freeze-fracture
results showed vesicle aggregates that formed and increased in size with time (Fig. 3).
The outer membrane of these aggregates appeared to be continuous and this enveloping membrane must have resulted from fusion processes. At an early time point
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Fig. 2. Maximum activities (rates) measured after addition of
PL-PLC as a function of temperature. (a) maximum rates of
PLC-PLC activity, as percentage of total lipid hydrolysis per
second. (b) Maximum rates of vesicle aggregation as percentage
of total change in light scattering per second. (c) Maximum
rates of vesicle fusion (mixing of aqueous contents), as percentage of total change in fluorescence intensity per second.
Liposome formulation (mole ratio): (䊊) PC兾PE兾Ch (2:1:1);
(●) PC兾PE兾Ch兾DAG (47:23:25:5); (䉮) PC兾PE兾Ch兾DAG
(43:22:25:10); (▼) PC兾PE兾Ch兾lysoPC (43:22:25:10); (䊐) PC兾
PE兾Ch (2;1:1) but 2 mM Ca2C instead of 10 mM. Reproduced
from Nieva et al. (1993).
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Fig. 3. PC-PLC-induced aggregation and fusion of LUV as visualized by conventional freeze-fracturing.
Frozen (a) before, (b) 30 s after, (c) 90 s after, and (d) 300 s after enzyme addition. The outer membrane
of the aggregates appears to be continuous (e.g., arrow in d, inner fracture face). The core of the aggregate
has an amorphous appearance (star in d). Printed at the same final magnification; bar 500 nm. Direction
of Pt兾C-shadowing indicated by encircled arrowhead. Reproduced from Burger et al. (1991) with permission from Elsevier Science.
(30 s) other signs of bilayer destabilization were not observed. At longer incubation
times (90 and 300 s) the vesicular compartments decreased in size and, in addition,
the aggregate core progressively lost its bilayer structure, becoming almost amorphous in appearance.
The images in Fig. 3 suggest that aggregates grew peripherally via fast aggregation and non-leaky fusion, for which only small amounts of DAG are required,
while a slower process of extensive phospholipid hydrolysis and leaky fusion could
be responsible for the changes observed in core structure; the amorous core may
well contain (segregated) DAG and Ch almost exclusively. The fact that aggregates
always appeared to be surrounded by a continuous membrane may explain the
absence of detectable vesicle leakage even when substantial phospholipid hydrolysis
had occurred (Nieva et al., 1989).
In a parallel series of experiments glycerol was omitted and samples were studied using fast-freeze freeze-fracture electron microscopy. Again, vesicle aggregates
were seen to form and to increase in size with time. Lipid structures of variable size,
the smallest one being ⯝14 nm in diameter, were seen to assemble into a honeycomb
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structure. This lipid organization closely resembled that described by Cunningham
et al. (1989) for PC兾DAG dispersions containing 80 mol.% DAG, which was suggested to represent a novel discontinuous inverted cubic phase (Seddon, 1990). The
fact that this honeycomb structure was observed at much lower DAG contents,
especially in fast-frozen samples (20 mol.% DAG) (Burger et al., 1991), was probably
due to the presence of PE and Ch would should favor a type II (‘‘inverted phase’’)
lipid organization (Verkleij, 1984 and Seddon, 1990). Thus the ultrastructural studies
(Burger et al., 1991) suggested that PLC-promoted membrane fusion might involve
a DAG-induced bilayer to inverted non-bilayer’ lipid structure transition.
Figure 4 shows cryo-TEM micrographs corresponding to a LUV preparation
(PC:PE:Ch, 50:25:25) at 0, 90, 180 and 360 s after phospholipase C addition (Fig.
4, A,B,C, and D respectively). The original liposomes were about 100 nm in diameter, and they gave rise to increasingly larger aggregates as the enzyme action proceeded. Figure 4B shows the structure of an aggregate in which, according to the
fluorescence data (not shown) fusion is taking place: the vesicles adopted polygonal
shapes, without any apparent gap between the adjacent membranes. This ‘‘honeycomb’’ structure corresponded to the one that had already been described in identical preparations treated with the freeze-fracture technique (Fig. 3), and was
remarkably similar to the arrangement of PC:PE:Ch:DAG (40:20:25:15) incubated
for a short time at 37°C and shown in Basañez et al. (1997a). Honeycomb structures
were found in virtually any phospholipase C-treated preparation observed at times
during which mixing of aqueous contents was taking place. In some cases, the polygonal compartments appeared to be connected by structures that could correspond
to fusion pores, although this interpretation has to be taken with caution, in the
Fig. 4. Cryo-TEM Micrographs of large unilamellar vesicles of PC:PE:Ch (2:1:1) at various times after
PC-PLC addition. (a) O s; (b) 90 s; (c) 180 s; (d) 360 s). For the experimental conditions see Fig. 2 of
Basáñez et al. (1997a), from where this figure has been reproduced. Bar: 100 nm. Reprinted with permission from The Biophysical Society.
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absence of additional proof. At long times after enzyme addition (e.g., Fig. 4D), the
aggregates progressively lost their honey-comb structure and formed larger compartments, presumably the end point of an extensive series of fusion events.
THE NON-LAMELLAR FUSION INTERMEDIATES
As mentioned in Section 3, the earliest indications of the existence of a ‘‘structural intermediate’’ in phospholipase C-induced liposomal fusion arose from the
observations by Nieva et al. (1993) of a precise range of DAG concentrations outside
of which no fusion was detected. It was hypothesized that an intermediate of a given
lipid composition was involved, at least transiently, in the fusion event. This idea
received considerable experimental support from our 31P-NMR and X-ray diffraction studies (Nieva et al., 1995), and was further reinforced by its accommodation
within the so-called ‘‘stalk hypothesis’’ of membrane fusion (Kozlov and Markin,
1983; Siegel, 1993). The stalk is proposed to be a semitoroidal lipid structure having
a negative curvature (the convention is followed that the curvature of a monolayer
in the inverted hexagonal HII phase is negative) that would allow the merger of the
closest (cis) leaflets of apposed membranes (Chernomordik, 1996).
Moreover, the transient formation of non-bilayer structural intermediates is an
unavoidable requirement of membrane fusion. It is also an essential tenet of the
stalk hypothesis, since the stalk itself is a non-bilayer structure, in which the monolayers have a negative curvature, such as seen in inverted lipid phases, HII hexagonal
or QII cubic. ‘‘Non-lamellar’’ has been equated in practice to ‘‘inverted hexagonal’’
in the context of membrane fusion (Siegel et al., 1989; Siegel, 1993) although isotropic 31P-NMR signals, which may be compatible with, among others, inverted
cubic phases, have also been associated with fusion intermediates (van Gorkom et
al., 1992; Yeagle et al., 1994; Luzzati, 1997). Siegel and Epand (1997) suggested
that TMC (trans-monolayer contacts) intermediates played a role in lamellar-tonon-lamellar phase transitions and that they could either rupture to form fusion
pores that modulate transitions to QII inverted cubic phases, or assemble into
bundles of HIi inverted hexagonal phase tubes. Nieva et al. (1995) showed a direct
correlation between bilayer compositions and temperature giving optimum fusion
and those leading to the formation of an ‘‘isotropic’’ component, which was identified with a bicontinuous inverted cubic phase Q224 by X-ray diffraction (Fig. 5).
Both the stalk and the pore, as predicted by the modified stalk theory, have gheometries that can be related to that of the Q224 phase. In our previous studies of
fusion inhibition by positive-curvature lipids, ganglioside and poly-(ethylene glycol)modified PE, (Basañez et al., 1996b; Basañez et al., 1997b) a good correlation was
shown between the inhibitory effects of those lipids and the increased temperatures
in the corresponding lamellar-to-non-lamellar transitions. This point was also
explored using a fluorescence polarization technique (Basañez et al., 1996a).
The effects of a low concentration of a variety of single-chain lipids on the
lamellar-to-non-lamellar (isotropic, Q224) phase transition of PC:PE:Ch:DAG
(50:25:25:5, mol ratio) mixture have been studied by fluorescence polarization and
31
P-NMR (Basañez et al., 1998). A very good correlation is observed between the
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Fig. 5. A pseudo-phase diagram of PC兾PE兾Ch兾DAG in excess water, constructed from 31PNMR data. L, lamellar; H, hexagonal; I, isotropic. In parentheses the nature of the cubic
phases, as identified by X-ray scattering experiments (sample concentration 50% w兾w). The
shaded area corresponds to the region of temperature and composition at which optimum
liposome fusion induced by PC-PLC is observed. Reproduced from Nieva et al. (1995) with
permission.
modification of phase transition temperature and fusion activity. Squalene and arachidonic acid, which were found to enhance lipid and content mixing, are seen to
facilitate the lamellar-isotropic transition, and the opposite occurs with the positivecurvature lipid lysoPC. Arachidic acid was virtually neutral both with respect to
fusion and with respect to phase transition. These results are in obvious agreement
with the stalk model.
Other data on phospholipase C-induced liposomal fusion could be reinterpreted
in the light of the predictions of the modified stalk theory. Siegel (1993) suggested
that when the lipid in the bilayer was very close to the Th lamellar-to-hexagonal
transition temperature, stalks might form HII phase precursors, and any TMCs that
formed should have a tendency to radially expand, decreasing the driving force for
fusion pore formation. However, the expanded TMC would make a large comparatively stable lipid connection between opposed bilayers, which would promote
extensive lipid mixing. It had been observed that the content mixing rate (Siegel et
al., 1989; Leikin et al., 1996; Ellens et al., 1986a, b) often increased with temperature
and went through a maximum at T ⯝Th, (Siegel et al., 1989; Leikin et al., 1996;
Ellens et al., 1986a, b) decreasing thereafter, while the lipid mixing rate increased
monotonically. Combining our data on phospholipase-induced fusion as a function
of temperature (Nieva et al., 1993) with those on the phase behavior of our lipid
mixtures (Nieva et al., 1995) we could show (Basañez et al., 1998) that vesicle aggregation and lipid mixing increased monotonically with temperature, while content
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453
mixing had a maximum in the temperature region corresponding to the lamellar-tonon-lamellar (in our case cubic) transition, in agreement with the above-mentioned
predictions and observations. Thus the structural ‘‘fusion-intermediate’’ whose existence was predicted from our kinetic studies (Nieva et al., 1993) corresponded probably to the stalk-TMC-pore.
FUSION INDUCED BY SPHINGOMYELINASE
Sphingomyelinase is a phospholipase C specific for sphingomyelin. It cleaves
this phospholipid yielding phosphorylcholine and ceramide. Ceramide is a structural
analog of DAG, although the similarity between both molecules is less than it
appears at first sight, and indeed significant differences exist between the physical
properties of both [see Goñi and Alonso (1999) for a discussion on the properties
of DAG, and Kolesnick et al. (2000) or Krönke (1999) for the properties of ceramides]. The effects of DAG and ceramide were comparatively studied by RuizArgüello et al. (1996). For that purpose, our original system consisting of PC:PE:Ch
was replaced by SM:PE:Ch, and sphingomyelinase was used instead of phospholipase C. The phase behavior of lipid mixtures containing diacylglycerols or ceramides
was also comparatively examined by 31P-NMR. In spite of having similar structure,
both families of compounds displayed quite different effects. Assuming that fusion
would take place through the transient formation of a structural intermediate
(‘‘stalk’’), and that in the generation of this intermediate some of the lipids would
have to adopt the kind of ‘‘negative’’ curvature that is found in inverted hexagonal
or cubic phases, we had pointed out (Nieva et al., 1995) that, for phospholipase
C-induced fusion, this phenomenon would be observed only under conditions of
temperature and composition that allow formation of HII hexagonal and兾or Q224
cubic phases. The 31P-NMR results in Ruiz-Argüello et al. (1996) showed that
SM:PE:Ch bilayers were perfectly stable at 37°C even in the presence of ceramide.
The lack of fusion under those conditions supported the requirement of a certain
plasticity in the lipid mixture for fusion to occur. Interestingly, when vesicles composed of SM:PE:Ch (2:1:1) were treated with low amounts of sphingomyelinase, so
that the rate of sphingomyelin hydrolysis was reduced considerably (Basanez et al.,
1997a), as compared to that of the previously discussed experiments, a situation was
found in which about one-third of the vesicles were clearly larger in size (by ca. 6fold) than the original ones. However, no ‘‘honeycomb’’ intermediates were found
and fusion had taken place together with extensive leakage. In that case, fusion
appeared to occur via a different mechanism from stalk formation. Perhaps vesicle
lysis and reassembly, rather than true fusion, was taking place under low-sphingomyelinase conditions (Basañez et al., 1997a). These observations were confirmed by
Holopainen et al. (1998), who could also ascertain that, in contrast, ceramide-containing LUV (in which ceramide and phospholipds had been mixed in organic solvent prior to hydration and extrusion) did not show any signs of aggregation for up
to 24 h of incubation.
Moreover, in a recent paper, Holopainen et al. (2000) have shown microscopic
images of ‘‘endocytic’’ budding of vesicles composed of phosphatidylcholine and
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sphingomyelin, upon addition of sphingomyelinase. Budding and fusion are probably mirror image-processes of fusion, and they may share the same kind of intermediates. Holopainen et al. (2000) attributed the observed budding to this tendency
of ceramide to separate into domains, and to its negative spontaneous curvature,
that would lead to membrane invagination.
Additional insights on the respective effects of phospholipase C and sphingomyelinase on pure lipid vesicles were obtained from the studies of Ruiz-Argüello et
al. (1998a) in which both enzymes were tested, either separately or together, on lipid
bilayers containing both PC and SM. It was known that addition of phospholipase
C to a suspension of LVU vesicles of PC:PE:Ch (2:1:1, mole ratio) led to rapid
liposomal aggregation and fusion (Nieva et al., 1989). However, when PC was substituted by an equimolar mixture of PC and SM, so that the new liposomal composition was PC:SM:PE:Ch (1:1:1:1), addition of phospholipase C, under otherwise
similar conditions, produced neither fusion (contents mixing) nor lipid mixing (Fig.
6A) even in a time scale that was 1 order of magnitude longer than the one used for
PC-PLC (Nieva et al., 1989). Light scattering did not change either in the experiment
shown in Fig. 6A, which precluded vesicle aggregation under those conditions (not
shown). However, lipid hydrolysis occurred, and the amount of DAG produced in
the experiment shown in Fig. 6A should have been enough to produce vesicle fusion.
At least in the PC:PE:Ch mixture, enzymatically produced DAG in amounts equivalent to 5–20% of the total lipid was found to induce fusion (Nieva et al., 1993).
Similarly, vesicles containing SM:PE:Ch (2:1:1) underwent rapid aggregation
and leakage, but not lipid or contents mixing, when treated with sphingomyelinase
(Ruiz-Argüello et al., 1996). The same enzyme treatment on LUV consisting of
PC:SM:PE:Ch (1:1:1:1) produced lipid hydrolysis, but not aggregation or fusion
(Fig. 6B). Remarkably, however, the joint addition of both phospholipase and
sphingomyelinase to PC:SM:PE:Ch (1:1:1:1) vesicles led to rapid vesicle aggregation, lipid mixing, and contents mixing (Fig. 6C). When both enzymes were added
together, their activities appeared to be mutually potentiated and phospholipase C
became particularly activated (Fig. 6D). None of the effects shown in Fig. 6C and
D were seen when the enzymes had been previously inactivated by heat, or when
phospholipase C had been preincubated with its specific inhibitor, o-phenthanthroline. The behavior of the two enzymes on the four-component lipid vesicles
prompted a reflexion on the role of the enzymes in phospholipase (J sphingomyelinase)-promoted fusion, as detailed in the next section.
THE DOUBLE ROLE OF THE FUSION-PROMOTING ENZYMES
In our systems, and particularly with PC-PLC, fusion occurred as a result of
enzyme activity. This is supported by the following observations:
(a) Neither heat-inactivated enzyme, nor enzyme incubated with the specific
inhibitor o-phenanthroline could induce fusion. The presence of DAG per se, when
this lipid was added to the lipid mixture prior to liposome formation, did not allow
vesicle aggregation or fusion either: the resulting vesicles were stable for days (see
Nieva et al., 1989; Nieva et al., 1993, for experimental details). Moreover, addition
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Fig. 6. Effects of phospholipase C and兾or sphingomyelinase on large unilamellar vesicles. Bilayer
composition was SM:PC:PE:Ch (1:1:1:1). Total lipid concentration was 0.3 mM. (A) PC-PLC,
1.6 units兾ml; (B) sphingomyelinase, 1.6 units兾ml; (C) and (D), PC-PLCCsphingomyelinase, 0.4
units兾ml of each. (■) Enzymically produced diacylglycerol, expressed as molar percentage of
total lipid in the mixture. (●) Id. id. ceramide. ‘‘Fusion’’ indicates mixing of vesicle aqueous
contents. ‘‘Lipid mixing’’ indicates intervesicular mixing of bilayer lipids. Reproduced from RuizArgüello et al. (1998).
of heat-inactivated or o-phenanthroline-treated enzyme to vesicles containing up to
20% DAG did not lead to fusion either (Basañez, unpublished results).
(b) Quantitative variations, either positive or negative, in enzyme activity led
to parallel increases or decreases in the vesicle fusion rates. Some examples are summarized in Table 1. The potencies of the various additives differed considerably.
So did their chemical structures. However, in all cases an increase or decrease in
phospholipase C hydrolytic rate led to a corresponding increase or decrease in vesicle
fusion rate. The changes in both phenomena were not of the same order of magnitude: small changes either positive or negative, in enzyme activity were amplified
when vesicle fusion was measured for reasons that will be discussed in the next
section. All effects described in Table 1 were dose-dependent (data not shown).
(c) Enzyme activity was also modified by other procedures, such as changes in
enzyme concentration, temperature or specific inhibitors. Lowering the temperature
from 37 to 25°C, or decreasing the enzyme concentration below 0.16 U mL—1 (the
standard concentration in our experiments) diminished considerably the rate of
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Goñi and Alonso
Table 1. Parallel Changes in PC-PLC Hydrolytic Activity and Rate of PC-PLC-Induced Liposome Fusion (Content Mixing) as a Result of Small Changes in Vesicle Lipid Composition
Lipid composition
control
+ GM3 ganglioside
+ GM1 ganglioside
+ GT1b ganglioside
+ hexadecane
+ squalene
+ arachidic acid
+ arachidonic acid
+ lysoPC
+ palmitoylcarnitine
% additive
Hydrolysis rate
Fusion rate
Reference
0
1
1
1
2
2
5
5
5
5
100
2
14
4
108
125
101
119
88
87
100
3
2
F1
215
271
122
232
22
18
Nieva et al. (1989)
Basáñez et al. (1996b)
Id.
Id.
Basáñez et al. (1998)
Id.
Id.
Id.
Id.
Id.
Data are expressed as percentages with respect to a control lipid mixture consisting of PC兾PE兾Ch (2:1:1,
mole ratio).
DAG production and, correspondingly, the rate of vesicles contents mixing (fusion)
(Basañez et al., 1998).
From our studies on phospholipase C-induced liposomal fusion it was concluded that DAG played two different roles. First, a significant amount (between 5
and 20 mol.%) of more or less symmetrically distributed DAG was required to allow
the formation of nonlamellar structures, which are essential for fusion to occur
(Nieva et al., 1993; Nieva et al., 1995; Basañez et al., 1996a). This ‘‘bulk’’ DAG
might be included from the start in the liposomal composition, causing a decrease
in the lag time between enzyme addition and fusion, although phospholipase C
activity was always essential for membrane destabilization (Nieva et al., 1993). The
essential role of phospholipase C consisted of generating the pool of DAG responsible for the second of its two roles: namely the rapid, localized, and asymmetric
synthesis of DAG that was the ‘‘trigger’’ for fusion to occur (Nieva et al., 1993 and
1995). Under similar conditions, but using vesicles containing SM, sphingomyelinase
produced aggregation, but not fusion of liposomes (Ruiz-Argüello et al., 1996). As
described in the latter study, ceramide was similar to DAG though less potent in
the induction of nonlamellar phases, and it might substitute for DAG in reducing
the lag time of phospholipase C-induced fusion.
This double role of PC-PLC, or rather of DAG, explains that, both at very low
and very high enzyme activities, the behavior of the system departs from the above
description. The first description of phospholipase C-induced liposomal fusion
(Nieva et al., 1989) included an experiment in which fusion was measured as a function of enzyme concentration (rate of phospholipid hydrolysis). An optimum enzyme
concentration was found, for the vesicle concentration used in those measurements,
while both above and below certain values fusion was virtually abolished. The lack
of vesicle contents mixing at high enzyme concentrations was explained later (Nieva
et al., 1995), when it was found that the bicontinuous cubic structure that would
allow intervesicular mixing of aqueous contents could only be formed within certain
limits of DAG concentration, namely between ⯝5 and 20 mol% at 37°C. Beyond a
certain enzyme activity, the upper limit of DAG concentration was reached before
any significant fusion could be detected.
Membrane Fusion and Phospholipases
457
The reason why low enzyme activities never lead to vesicle fusion even after
incubation times that allow the formation of appropriate concentrations of DAG
(i.e., between 5 and 20%) is of a kinetic nature. The phenomenon was clearly shown
in Fig. 6 of Ruiz–Argüello et al. (1998a) in which phospholipase C activity and
vesicle fusion were measured in the presence of increasing concentrations of the
specific enzyme inhibitor o-phenanthroline. Both phenomena decreased notoriously
in the presence of inhibitor. However, they did not change in parallel: as soon as
the enzyme activity decreased below 25% of the native value, fusion was virtually
abolished. Our interpretation of this phenomenon is that, as stated above, one of
the essential roles of the enzyme (perhaps the most essential one) is to act as a
trigger for the fusion process. The enzyme triggers fusion by producing a high local
concentration, asymmetrically (the enzyme is present only on one side of the bilayer)
and in a short time. The latter point is essential to overcome the spontaneous diffusion of DAG in the membrane, that will act against the formation of a DAG
patch, in turn a hot point for vesicle aggregation (Basañez et al., 1996b). Low
enzyme concentrations lead naturally to low rates of DAG production, that cannot
compete with the surface dilution rates of the lipid (Carman et al., 1995).
In summary, in systems showing enzyme-induced vesicle fusion, the rate of
fusion increases with enzyme activity within a certain range of activities, beyond
which either kinetic or thermodynamic reasons prevent the formation of the structural intermediates that are required for fusion to occur. In other words, certain
phosphohydrolases catalyze vesicle fusion if and when they allow the formation of
fusion structural intermediates.
In view of these observations, the joint behavior of phospholipase C and
sphingomyelinase in the experiments in Fig. 6 can be explained by assuming that:
(a) the rate of glycerophospholipd hydrolysis by phospholipase C acting on
PC:SM:PE:Ch (1:1:1:1) vesicles (Fig. 6A) is too slow to permit the buildup of a
localized, asymmetric pool of diacylglycerol that may act as a fusion trigger. A local
high concentration of diacylglycerol in one of the monolayers can only build up
competition with the phenomena of lateral and transbilayer (flip-flop) diffusion. (b)
Sphingomyelinase acts in much the same way as in the ternary mixture SM:PE:Ch
(1:1:1) (Ruiz-Argüello et al., 1996), and consequently no fusion occurs. However, a
significant proportion of ceramide is generated in the bilayers, thus facilitating an
eventual lamellar transition. (c) When both enzymes are acting together, the rate of
generation of ceramide and particularly of diacylglycerol is high enough to overcome
diffusion, and vesicle fusion occurs (Fig. 6, C and D). These points were experimentally tested and confirmed by Ruiz-Argüello et al. (1998a).
Central to the understanding of our two-enzyme fusion system is the steadystate equilibrium between the generation of membrane destabilizing lipids (diacylglycerol, ceramide) in the bilayer and their lateral and transmembrane diffusion.
Numerous lines of evidence (Nieva et al., 1989, 1993, 1995) point toward the requirement of a localized asymmetric generation of fusogenic lipid for the onset of fusion.
Such a ‘‘hot spot’’ can only arise if the synthesis of diacylglycerol, and to a smaller
extent ceramide, is fast enough to overcome the diffusion of these lipids along the
bilayer. This is an example of what has been called ‘‘surface dilution kinetics’’
458
Goñi and Alonso
(Dennis, 1973; Carman et al., 1995). Unfortunately, direct measurements of the lateral diffusion of DAG and ceramide, that would be extremely helpful in confirming
the role of phospholipase C as a trigger in enzyme-induce diffusion, are not available
at present.
8. FUSION INDUCED BY PI-PLC
A novel model system for membrane fusion has been developed by Villar
(2000), in which vesicles containing PI, PC, PE and Ch underwent aggregation and
fusion as a result of the catalytic activity of bacterial PI-PLC. Unlike the case of
ionically neutral-liposomes when fused by PC-PLC, the negatively-charged PI-containing vesicles aggregated and fused in groups of only 2–3 vesicles, and fusion was
accompanied by leakage of vesicular aqueous contents. In experiments in which the
PC:PE:Ch molar ratio was kept constant at 2:1:1, but PI varied between 5 and
40 mol% in the liposome composition, the difference in PI concentration led to different physical effects of PI-PLC. Interestingly, 30–40 mol% PI led to vesicle fusion,
while with 5–10 mol % PI only hemifusion was detected, i.e., mixing of outer monolayer lipid without mixing of aqueous contents. Obtaining these stable hemifusion
intermediates will no doubt be useful in experimental studies on the mechanism of
bilayer fusion. Moreover, it was observed that, when 10 mol% DAG was included
in the bilayer formulations, PI-PLC activity led to compete fusion even with PI
concentration as low as 5 mol%.
9. THE CONDUCTING THREAD
From the results discussed in the previous sections, it was apparent that a causeeffect relationship existed between phospholipase activity, non-lamellar phase formation, and membrane fusion. We suggested that these three phenomena were connected by a conducting thread’’ (Goni et al., 1998). An interesting question is
whether this thread allows all or part of the way to be walked in both directions, i.e.,
formation of non-lamellar intermediates led to membrane fusion, but, did budding
somehow give rise to no-lamellar structures that in turn led to vesicle fission?
Although fusion and fission are widely considered to be in many aspects two sides
of the same phenomenon, formation of non-lamellar intermediates prior to fission
has not been explored in detail, to the authors’ knowledge. However, the fact that
sphingomyelinase could also induce budding in otherwise stable bilayers (Holopainen et al., 2000) suggests that the answer to that question may be positive.
The reversibility of the ‘‘enzyme activity-non-lamellar phase formation’’ stretch
is more clearly understood. One aspect of this problem, i.e., whether enzyme activity
was responsible for the lamellar-to-non-lamellar transitions, is rather straightforward. In the phospholipase C-induced fusion system (Goñi et al., 1994), as well as
in the sphingomyelinase-based systems (Ruiz-Argüello et al., 1998a; Basañez et al.,
1997a) it was obvious that the enzymes were instrumental in modifying the chemical
composition of the bilayer, so that new equilibrium conditions settled in, and a
phase transition ensued. For example, at 37°C, the equilibrium phase structure of
PC:PE:Ch (50:25:25, mol ratio) in excess water was lamellar, but when 10% of the
Membrane Fusion and Phospholipases
459
phospholipid had been converted into diacylglycerol, and the new composition was
PC:PE:Ch:DAG (43:22:25:10), then the predominant phase structure was nonlamellar (HIICQII) (Nieva et al., 1995). It is thus clear that, in these systems, the
non-lamellar phase appeared precisely as a result of the enzyme activity.
However, what about the reverse question? Do lamellar-to-non-lamellar transitions somehow modify the activity of interfacial enzymes, such as phospholipase
C? The answer to this came from a series of experiments in which phospholipase C
activity was studied on egg PC bilayers doped with small amounts of lipids that
were not substrates for the enzyme. Some of these additional lipids (e.g. cholesterol
or squalene) were known to facilitate the lamellar-to-inverted hexagonal phase transition of phospholipids, while others would stabilize the lamellar phase, e.g.,
sphingomyelin, or even favor micelle formation, e.g. lysolecithin. An extensive number of tests (Table 1, and references therein; Goñi et al., 1998; Sáez-Cirión et al.,
2000) agree in showing that phospholipase C was activated, the rates increases, and
the lag times shortened, in the presence of those lipids that favour the lamellarto-inverted hexagonal transition, and inhibited by those with opposite structural
tendencies.
It should be noted that, under our conditions, all the mixtures were lamellar at
the onset of the enzyme assay, and that, along the assay, actual formation of nonlamellar phases was not correlated with higher or lower phospholipase C activities
(Ruiz-Argüello et al., 1998b; Goñi et al., 1998), i.e., the presence or absence of nonlamellar phases and the rates of phospholipase C activity, while being both very
sensitive to lipid composition, were shown to be unrelated phenomena.
What is, then, the explanation for the repeatedly observed phenomenon of the
activation of phospholipase C by lipids that favor non-lamellar phase formation,
and, conversely, its inhibition by lamellar lipids? Epand (1985) suggested a hypothesis according to which a number of enzymes, that interact with membranes as
peripheral proteins, would be activated by a certain propensity of lipid bilayers to
adopt the inverted hexagonal disposition, while remaining in the lamellar phase.
Such propensity would be given by the presence of non-bilayer lipids in the membrane, that would induce a frustrated lamellar state. Our results could certainly be
interpreted in the light of this hypothesis. Thus PLC would join in a large group of
enzymes, reviewed in Kinnunen et al. (1996) whose activities are enhanced by the
presence of non-bilayer lipids in essentially lamellar systems. This hypothesis appears
to be physiologically relevant since it allows the possibility of enzyme regulation in
cell membranes without loss of the bilayer structure or its barrier properties.
10. THE DATA FROM CELL PHYSIOLOGY
Vesicle fusion induced by phospholipase C or sphingomyelinase is indeed an
exciting model system, that can give us powerful insights into several aspects of cell
membrane fusion. However, the physiological relevance of these studies can be more
readily appreciated by examining the various lines of evidence that connect those
enzymes with in ûiûo fusion processes. When examining the vast cell biological literature on fusion, two facts must be taken into account: (a) that cell fusion is in all
cases much more complex than model membrane fusion, in particular because the
460
Goñi and Alonso
process has to be carefully regulated, so that many different proteins and enzymes
are expected to take part, and (b) that phospholipases C may be involved in signalling events, so that their implication in cell fusion may be unrelated to structural
changes caused by DAG.
With these cautions in mind, there are data from in ûiûo experiments that relate
DAG and phospholipase C with fusion events. Of these, exocytosis has been studied
in particular detail as a DAG-activated process. The putative involvement of PIphospholipase C and DAG in exocytotic secretion was already put forward by Allan
et al. (1978) and by Hawthorne and Pickard (1979). More recently, Haines et al.
(1991) studied the activation of human neutrophils in response to the chemoattractant and phlogistic agent Met-Leu-Phe. One of the steps of activation is the exocytotic release of digestive enzymes by these cells. Haines et al. (1991) observed that
exposure to the chemoattractant induced a biphasic rise in DAG (also observed
in other DAG-mediated phenomena, see Hodgkin et al., 1998), a rapid increase
corresponding to PI-derived DAG, that acted through PKC stimulation in nonexocytotic events of cell activation, and a slower, sustained rise, tightly correlated
within exocytosis, that required DAG from a PC-specific phospholipase C.
PC-derived DAGs appear to be equally involved in a particular and important
case of exocytosis, namely the acrosome reaction in spermatozoa. Roldan and Murase (1994), using ram sperm cells, found that treatment with the Ca2C-ionophore
A23187 and Ca2C led to an increase in cell DAG levels and to exocytosis of the
acrosomal granule. Moreover, they found that, as a result of the biphasic response,
PI-derived DAG simulated PC-phospholipase C in a process that was not mediated
by protein kinase C. These observations have been extended to the cases of human
sperm acrosomal exocytosis (O’Toole et al., 1996). Further data (Vázquez and Roldán, 1997) indicate that peak levels of DAG with saturated and monosaturated acyl
chains in the sn-1 and sn-2 positions respectively, i.e., the DAG species typically
derived from PC, were tightly coupled to the onset of visible exocytosis. Data from
an independent line of evidence obtained with a cell-free system that mimics sperm
exocytosis (Spungin et al., 1995) also point to a role of phospholipase C in the
membrane fusion step of exocytosis. Thus it could be provisionally concluded that,
at least in mammals, PC-derived DAG produced in the slow phase of DAG response
may be related to exocytosis.
Secretory vesicles involved in exocytosis are generated in the Golgi complex
through budding and scission, a process that can be considered as a mirror image
of membrane fusion. It is interesting in this context that the Golgi membranes
appear to require a constant pool of DAG for the generation of secretory vesicles
(Kearns et al., 1997). The requirement of DAG in Golgi membranes is such that
activation of the CDP-choline pathway of PC synthesis, that consumes DAG,
impairs the vesicle budding and scission process.
Other physiological observations point to an involvement of sphingomyelinase
and ceramides in cell fusion兾fission events. Entry of Neisseria gonorrhoeae into nonphagocytic cells is mediated by the activation of an acidic sphingomyelinase
(Grassmé et al., 1997). Also a protein secreted by Listeria spp., that has both phospholipase C and sphingomyelinase activities, helps the bacterium to escape phagosomes and spread from cell to cell (Zuckert et al., 1998; Gonzalez-Zorn et al., 1999).
Membrane Fusion and Phospholipases
461
Mammalian sphingomyelinases may induce fusion of low-density lipoprotein particles during atherogenesis (Oorni et al., 1998), although it is not clear to what extent
fusion of lipoprotein particles shares a mechanism with membrane fusion.
Also relevant in this context are the data according to which endocytotic vesicles were formed in the absence of ATP when fibroblasts or macrophages were
treated with exogenous sphingomyelinase or ceramides that induced the rapid formation of vesicles, ca. 400 nm in diameter, not enriched in clathrin or caveolin, that
pinched off from the plasma membrane and went into the cytosol. The authors
speculated that hydrolysis of sphingomyelin on the plasma membrane caused inward
curvature and subsequent formation of sealed vesicles. Ceramide, because of its relatively small polar head group, induces a ‘‘negative curvature’’, that is, inward curvature of the outer monolayer of the plasmalemma. Higher ceramide concentrations,
particularly if localized and asymmetric, will lead to vesicle fission by the mirrorimage mechanism of their facilitation of membrane fusion (Veiga et al., 1999). Interestingly, Li et al. (1999), instead of using exogenous sphingomyelinase, added C6ceramide to fibroblasts. NO toxic effect was detected; instead, ceramide caused the
formation of endocytotic vesicles in the cytosol. These vesicles enlarged with time,
fusing with one another and with preexisting cytosolic structures, late endosomes
and lysosomes. Vesicle size reverted to control values upon removal of ceramide
from the culture medium. the effect is clearly parallel to that induced by sphingomyelinase in the studies by Zha et al. (1998).
Thus, a variety of reports from the field of cell physiology point towards a
structural role of phospholipase C兾sphingomyelinase in the membrane fusion兾fission
events. The biophysical data indicate that a specific investigation of this point is
certainly deserved.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the continuing support from the Spanish
Ministerio de Educación y Cultura, the Basque Government, and the University of
the Basque Country. This review was prepared while on sabbatical leave at the
University of Victoria, B.C., Canada, where the authors enjoyed the hospitality of
Professor J. T. Buckey, of the Department of Biochemistry and Microbiology.
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