Bioscience Reports, Vol. 20, No. 6, 2000
MINI REVIEW
Mechanisms of Initiation of Membrane Fusion:
Role of Lipids
Paavo K. J. Kinnunen1,2 and Juha M. Holopainen1
Receiûed August 2, 2000
Main emphasis in studies on the mechanisms of fusion of cellular membranes has been in
the roles of various proteins, with far less interest in the properties of lipids. Yet, on a
molecular level fusion involves the merging of lipid bilayers. Studies so far have revealed
lipids forming inverted non-lamellar phases to be important in controlling membrane
fusion. However, the underlying molecular level mechanisms have remained controversial.
While this review is focused on presenting one possible mechanism, involving so-called
extended lipid conformation, we are also advocating the view, that in order to obtain a
more complete understanding of this process it is necessary to merge the relevant physicochemical properties of lipids with the models describing the specific functions of proteins.
To this end, taking into account the central importance of fusion in a wide range of cellular
processes, we may anticipate its control to open novel possibilities also for therapeutic
intervention.
KEY WORDS: Membrane fusion; lipid conformational dynamics; lipid packing;
extended lipid conformation; fluorescence spectroscopy; pyrene.
ABBREVIATIONS: bisPDPC, 1,2-bis[(pyren-1)-yl]decanoyl-sn-glycero-3-phosphocholine;
DOPA, 1,2-dioleoyl-sn-glycero-3-phosphatidic acid; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPH, 1,6-diphenyl1,3,5-hexatriene; HII , inverted hexagonal phase; Ie , pyrene excimer fluorescence emission
intensity; Im , pyrene monomer fluorescence emission intensity; IMI, inverted micellar intermediates; Lα , fluid lamellar phase; Lε , frustrated lamellar phase; LDL, low density lipoprotein; LUVs, large unilamellar vesicles; Me2C, divalent metal cation; NBD, 7-nitro-2,1,3benzoxadiazol-4-yl; P, polarization; PA, phosphatidic acid; PC, phosphatidylcholine;
PDPTPC, 1-[(pyren-1)-yl]decanoyl-2-[(pyren-1)-yl]tetra-decanoyl-sn-glycero-3-phosphocholine; PE, phosphatidylethanol-amine; PEG, poly(ethyleneglycol); PPDPC, 1-palmitoyl2[(pyren)-1-yl]decanoyl-sn-glycero-3-phosphocholine; sn, stereochemical notation; SUVs,
small unilamellar vesicles; X, mole fraction of the indicated lipid.
FUSION OF BIOMEMBRANES: AN OVERVIEW
Membrane fusion is centrally involved in a large number of cellular processes, such
as membrane recycling, protein trafficking within the cell, exocytosis, fertilization,
1
Helsinki Biophysics and Biomembrane Group, Department of Medical Chemistry, Institute of Biomedicine, P.O. Box 8 (Siltavuorenpenger 10A), University of Helsinki, Finland.
2
To whom correspondence should be addressed at; Institute of Biomedicine, Department of Medical
Chemistry, P.O. Box 8 (Siltavuorenpenger 10A), FIN-00014 University of Helsinki, Finland. E-mail:
[email protected]
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0144-8463兾00兾1200-0465$18.00兾0  2000 Plenum Publishing Corporation
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and enveloped virus infection. Fusion involved in the synaptic transmission of
impulses in neurons (Jahn and Südhoff, 1999) now represents probably the most
studied and perhaps the best understood example of biomembrane fusion. In the
pursuit for an understanding of the machineries controlling the above cellular processes proteins have so far received the main interest (e.g., Hoekstra, 1990; Zaks and
Creutz, 1990; White, 1992; Söllner et al., 1993; Jahn and Südhoff, 1999), with less
attention to the possible involvement of lipids. Although several proteins are important in initiating fusion this process can be regarded as a lipid level event culminating
in the merging of the bilayers. To this end, no protein has been found to account
for the Ca2C sensitive step in the fusion of synaptic vesicles, thus readily suggesting
a possible role for acidic phospholipids. A very different example for the importance
of fusion is provided by the metabolism and pathophysiology of plasma low density
lipoprotein, LDL (Kruth, 1997). Although a major fraction of LDL is metabolized
in the cells by the receptor mediated pathway, a significant amount is also taken up
by non-receptor mediated mechanisms, involving fusion between the plasma membrane of e.g., macrophages and the lipid monolayer surrounding the LDL particle.
Similar mechanisms could be involved in the lysosomal processing of LDL (Lauraeus et al., 1998). Fusion of LDL particles in the arterial initima is currently considered to be the major process in the development of atherosclerotic lesions
(Guyton and Klemb, 1989). The development of cationic liposomes as vehicles for
efficient delivery of DNA into cells, transfection has become one of the key areas in
molecular medicine (Felgner and Ringold, 1989). Also this process involves fusion
of the liposome-DNA complexes with cellular membranes. Obviously, deciphering
the underlying detailed molecular level mechanisms can be expected to yield novel
therapeutic means for a variety of disorders.
Membrane fusion is a highly localized event, progressing as a sequence of distinct and specific steps. Prior to fusion the two membranes must first come into close
contact, which requires that short-range repulsive undulation, electrostatic and兾or
hydration forces must be overcome as these factors at atomic distances represent a
large energy barrier, prohibiting fusion (Rand and Parsegian, 1986, 1989; Chernomordik et al., 1987). How this repulsive barrier is overcome in biological membranes
has remained elusive and represents one of the fundamental questions in understanding the mechanisms controlling fusion in living cells. Two different views to account
for this problem have been proposed and are called either the protein or the lipid
theory of membrane fusion. In the former (Lindau and Almers, 1995), the fusion
pore is described as a toroid formed of the involved proteins and spanning the two
adjacent membranes, forming an oligomeric ring structure. In this model lipids
would only be directed into the fusion site subsequent to the formation of the protein
pore. Moreover, the proposed involvement of lipidic non-lamellar fusion intermediates would be governed by proteins. The lipid theory of membrane fusion assigns a
greater role for lipids in forming the fusion intermediate, limiting the role of proteins
to the reduction of the free energy barrier inhibiting membrane fusion.
Subsequently to the contact formation the membranes adhere. This step is
essential yet not sufficient for fusion (Zimmerberg et al., 1993) and may also be
reversible. Partial dehydration of the adhering surfaces precedes the formation of an
intermediate state and ‘‘hemifusion’’ which allows for lipid mixing between vesicles
Lipid Conformational Dynamics and Bilayer Fusion
467
in the absence of coalescence of the aqueous contents (Zimmerberg et al., 1993;
Katsaras et al., 1993; Arnold, 1995). Finally, a fusion pore opens into the septum
separating the internal aqueous cavities of the two merging membrane compartments, allowing their mixing (Zimmerberg et al., 1993).
LESSONS FROM LIPOSOMES
Because of the fact that at some stage in the course of the fusion process merging of the lipid bilayers must take place, liposomes continue to provide a good
model to study the different stages of the mechanism and machinery of fusion. The
properties of lipids have been shown to be of importance. For example, Chernomordik et al. (1997) reported that depending on their 3-D molecular shape different
lipids either inhibited or promoted influenza virus fusion, and also showed that
hemagglutinin initialized membrane fusion by promoting the formation of a lipidintermediate. Corver et al. (1995) found that the fusion of Semliki Forest virus with
cholesterol containing liposomes required 1–2 mol.% of sphingolipid to be present
in the membrane, lending support for a specific role for these lipids. A recent review
on the role of non-bilayer forming lipids in biological fusion (Chernomordik, 1996)
concludes that the lipid composition affects the fusion ‘‘downstream’’ to the activation of the fusion proteins and ‘‘upstream’’ to the fusion pore formation. The
effects of lipids are strongly correlated with their geometrical shapes. In this context
it is relevant to emphasize that it is the effectiûe molecular shape which matters.
Accordingly, characterization of the properties of a lipid in this respect requires
knowledge not only about its chemical structure, determining its van der Waals
volume but also information on its hydration shell, conformation, conformational
dynamics, and intermolecular interactions, involving those due to charges. In other
words, the effective geometry of a lipid can be considered as a soft and diffuse
surface determined by (i) the amplitudes and frequencies of the thermal motion of
the molecule, (ii) fields due to its charges and dipoles, (iii) interactions with its
neighboring lipids (e.g., hydrogen bondings), and (iv) the hydration shell of its
headgroup (Kinnunen, 1996b). The above is completed by information on the distribution of membrane free volume Vf which represents the difference between the
effective and the van der Waals volumes per molecule (Bondi, 1954; Turnbull and
Cohen, 1970). For a phospholipid bilayer Vf arises from short-lived, mobile structural defects due to trans-gauche isomerization of the lipid acyl chains created
because of packing constraints as well as by thermal motion (Xiang, 1993). Vf and
its distribution further depend on factors such as hydration (Lehtonen and Kinnunen, 1994).
Micelle forming lipids with positive spontaneous curvature such as lysophospholipids having a large headgroup relative to the hydrophobic part inhibit fusion
(Chernomordik et al., 1995). Lipids which induce fusion should be cone shaped with
the projected headgroup area occupied in the membrane plane being less than that
occupied by the hydrocarbon chains, in general corresponding to a lipid structure
with a small or weakly hydrated headgroup. Unsaturated 1,2-sn-diacylglycerols
represent an example of the former and phosphatidylethanolamines of the latter
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category. Further, short chain saturated and long-chain unsaturated 1,2-sn-diacylglycerols increase the fusion rate whereas long-chain saturated species do not (Sánchez-Migallón et al., 1995). In cells 1,2-sn-diacylglycerols are produced by
phospholipase C. Given the important role of this enzyme in cellular signaling cascades understanding of the membrane level physicochemical consequences of the
non-equilibrium state generated by product accumulation is of great interest (Nieva
et al., 1989, 1993, 1995). Another example of fusogenic lipids is provided by the
negatively charged species (phosphatidylserine, phosphatidic acid, phosphatidylglycerol, and cardiolipin) which upon binding of divalent cations (most commonly
Ca2C and Mg2C ) can trigger fusion. This is likely to result from dehydration and
charge neutralization of their headgroups changing their effective molecular
geometry.
Destabilization of the bilayer structure must precede fusion. Most ambiguity in
fusion mechanisms concerns the exact nature of this transient intermediate state and
the involved molecular arrangements. As cone shaped and HII phase forming lipids
promote membrane fusion, it has been postulated that fusion and lamellar →HII
phase transition proceed via common intermediate structures (Siegel, 1986; Verkleij
et al., 1979; Hoekstra and Martin, 1982; Ellens et al., 1984, 1986a, b, 1989; Bentz et
al., 1985; Walter et al., 1994; Kinnunen 1992, 1996b; Siegel and Epand, 1997). In
other words, compounds promoting fusion should either form or promote the formation of the HII phase. Likewise, conditions promoting fusion should also favor
the formation of the HII phase. Intervesicle contacts are required for liposomes to
undergo lamellar→HII phase transition (Ellens et al., 1986). The rate of fusion of
PEs is highest for bilayers in the poorly understood intermediate phase (Lε ) existing
between the Lα and HII phases (Ellens et al., 1989). In this intermediate phase thermal motion increases the effective size of the hydrocarbon region and causes negative
spontaneous curvature (Tate et al., 1991). In general, the lamellar →HII phase transition is qualitatively easily comprehended in terms of changes in the effective molecular shapes and can be promoted by decreasing the effective headgroup size by
dehydration or deionization. Accordingly, similarly to fusion the promotion of the
HII phase formation by acidic lipids in the presence of divalent cations, most prominently Ca2C, is likely to reflect both of these processes. The formation of the HII
phase is additionally enhanced by factors which influence the hydrocarbon region of
the lipids, i.e., decrease in pressure, increase in temperature, acyl chain unsaturation,
increase in acyl chain length, and the presence of proper lipophilic additives, surfactants, solvents, metabolites, or chaotropic agents, such as KSCN and urea (Yeagle
and Sen, 1986). The effect of the latter is readily compatible with the suggestion that
the affinity of water to the phosphatidylethanolamine (PE) headgroup should
decrease upon entering the HII phase (Seddon et al., 1983). Accordingly, we may
envisage the effect of chaotropic substances to be due to a diminished affinity of
individual water molecules to the bulk aqueous phase, as this allows for PE to maintain its hydration shell at higher temperatures, counteracting the thermal increase in
the trans → gauche isomerization and the corresponding increase in the effective volume of the acyl chain region (Mantsch et al., 1981). To this end, the sensitivity of
the HII phase for hydration and water activity is of great interest in the light of the
Lipid Conformational Dynamics and Bilayer Fusion
469
increasing recent recognition of the importance of osmotic forces in the regulation
of cellular functions.
Three models for the intermediate structures linking fusion and the
lamellar→HII phase transition have been forwarded. The first one was based on the
formation of inverted micellar intermediates, IMIs (Verkleij et al., 1979, Siegel,
1984). This view was supported by the appearance of intramembrane lipidic particles
IMIs under conditions where fusion or the lamellar →HII phase transition occur. In
addition to electron microscopy, evidence supporting the involvement of IMIs was
also derived from 31P-NMR which reports an isotropic signal under conditions promoting fusion as well as in the so-called isotropic lipid phases present below the
actual onset of the lamellar→HII transition. Yet, direct causality was questioned
(Allen et al., 1990; Verkleij, 1984; Bearer et al., 1982; Papahadjopoulos et al., 1990)
and evidence revealing the lack of involvement of IMIs in fusion has been forwarded
(Caffrey, 1985; Siegel et al., 1994; Hui et al., 1983; Allen et al., 1990; Verkleij, 1984;
Bearer et al., 1982; Papahadjopoulos et al., 1990). The isotropic 31P-NMR signal
may also arise in lipids retaining the bilayer configuration (Thayer and Kohler,
1981). To conclude, it is now rather generally accepted that IMI are not required
for fusion (Papahadjopoulos et al., 1990; Siegel, 1993).
The second mechanism is based on the formation of semitoroidal structures,
‘‘stalks’’ (Markin et al., 1984), estimated to have diameters of >4 nm and lifetimes
<1 ms (Siegel, 1993). However, also these structures have so far escaped direct,
affirmative verification. ‘‘Stalks’’ have been suggested to be involved in ‘‘hemifusion’’
(Siegel, 1993). The lipid organization in the base of the ‘‘stalk’’ would be promoted
by lipids with negative spontaneous curvature (Chernomordik et al., 1995). However, within its medial part a ‘‘stalk’’ requires positive membrane curvature for the
lipids in its outer monolayer. Notably, both IMI and ‘‘stalk’’ arrangements involve
transient increase in free energy and a stochastic nature for the fusion intermediate
has been suggested (Arnold, 1995; Papahadjopoulos et al., 1990). Yet, ‘‘stalks’’ have
been estimated to require less free energy increase than IMI (Siegel, 1993). Due to
the transient nature of the membrane fusion intermediate its identification by
methods that do not allow for fast data collection (in the pico- to nanosec range) is
likely to be experimentally demanding.
Third mechanism is based on the so-called ‘‘extended’’ lipid conformation,
allowing fusion without the exposure of the hydrophobic chains of the lipids to
water and not requiring the formation of intermediate structures such as IMI within
the membranes (Kinnunen, 1992, 1996b). This model also readily connects fusion to
the properties of lipids forming the HII phase and the mechanism of lamellar →HII
phase transition. In brief, in the extended conformation the two acyl chains of a
phospholipid are embedded in the opposing contacting leaflets of the two adjacent
bilayers, with the headgroup remaining in the interface. In the following the physical
basis for this conformation is first described. Subsequently, the molecular mechanism of fusion is outlined, using lipids in the extended conformation as molecular,
amphiphilic ‘‘zippers’’. Finally, recent evidence derived from fluorescence spectroscopy and providing direct support for the existence of lipids in the extended
conformation in the course of fusion and hemifusion is summarized in detail.
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THE ‘‘EXTENDED’’ LIPID CONFORMATION
Phopholipids are customarily illustrated comprising of the headgroup with its
two aligned acyl chains. This conception of lipids seems to deeply influence the
mental framework for any attempt to rationalize the properties of their various
assemblies. Under most conditions surface tension (hydrophobic effect), hydration
of the lipid headgroup, van der Waals forces, and Born energy do make the time
averaged normal parallel chain alignment energetically favorable. Yet, individual
lipids residing in either biological or model membranes undergo rapid lateral,
rotational, and conformational diffusion. The latter involves correlation times ranging from pico- to nanoseconds (e.g., Brown et al., 1995), producing intense fluctuations in membrane organization (Wiener and White, 1992). There are a priori
neither physical nor chemical reasons prohibiting the extended conformation. The
free energy of membrane assembly is not only determined by the arithmetic sum of
its individual constituent molecules but also takes into account constraints due to
lipid packing (Helfrich, 1973; Israelachvili et al., 1980). Accordingly, steric factors
may cause a particular lipid conformation such as the extended conformation to be
energetically allowed under appropriate conditions in a membrane even if it had
only a low probability for occurring in isolation. The inherent dynamics of lipids
are readily compatible with the extended lipid conformation, as discussed below.
In the extended conformation the acyl chains of the lipid in question are pointed
towards opposite directions from the headgroup, instead of their usual parallel alignment (Fig. 1). Although the probability distribution of chain conformations in
amphiphile assemblies shows that the majority of the hydrocarbon chains are to be
found in the interior of the hydrophobic core they partition also into the oil兾water
interface. Xu and Cafiso showed by 1H-NMR magnetization transfer between the
terminal methyl group and the polar headgroup in sonicated phosphatidylcholine
SUVs (1986). This coupling was suggested to result from acyl chain interdigitation
or chain reversal for a fraction of lipids in the vesicles. Recently, Gawrisch and
his coworkers confirmed intermolecular cross-relaxation in liquid crystalline bilayers
between headgroup choline moiety and the terminal methyl groups of the hydrocarbon chains using nuclear Overhauser enhancement spectroscopy (NOESY)
(Huster and Gawrisch, 1999; Huster et al., 1999). Importantly, these authors could
exclude spin diffusion along the chains as the mechanism of magnetization transfer,
thus necessitating contacts between the terminal methyl groups and the headgroup.
These data require either major shifts in the location of neighboring lipid segments
by several Ångstroms along the bilayer normal or acyl chain reversal (Ben-Shaul et
al., 1984). The latter is a prerequisite for the adoption of the extended conformation.
Evidence for the extended conformation of phospholipids is provided by high resolution NMR data on the glycerol backbone configuration (Hauser et al., 1980; 1988).
Accordingly, in normal and inverted micelles for a small proportion of molecules
the antiperiplanar conformation of the C(2)–C(3) glycerol bond is evident and was
shown to be on the NMR time scale in rapid exchange with the predominant gauche
conformations of the torsion angle Q4. Extended conformation has been observed
in lipid crystals (Hybl and Dorset, 1971; Jensen and Mabis, 1966; Larsson, 1963;
1986). Evidence for the extended conformation of triacylglycerol has been obtained
Lipid Conformational Dynamics and Bilayer Fusion
471
Fig. 1. Panel A: A schematic illustration allowing to compare the
projected areas of the headgroups and the hydrocarbon chains on
the membrane surface for (A) phospholipid in the ‘‘extended’’ conformation and (B) in the chain aligned conformation. Panel B: A
schematic view of the contact site between the outer monolayers
of two adhering vesicles, with a small fraction of the lipids in the
‘‘extended’’ conformation. Because of the rapid equilibrium of conformational fluctuations this membrane contact allows for fast
exchange of lipids between the leaflets. See text for details.
by the lipid monolayer technique (Fahey and Small, 1986). As shown for NBD-acyl
chain containing lipid probes, the hydrophilicity of this aromatic moiety favors chain
reversal so as to bring it into the interface (Chattopadya and London, 1987; Abrams
and London, 1993). Similar behavior was recently suggested for a dansyl moiety
containing phospholipid probe (Bartlett et al., 1997), as well as for other fluorescent
lipid analogs (Epand et al., 1996). Finally, due to conformational restrictions
imposed by the glycerophospholipid structure it is readily conceivable that it is the
sn-2 acyl chain which should be more amenable to extension out from the bilayer.
This property should be greatly augmented by unsaturation and may thus explain
the specificity of positional distribution of saturated and unsaturated fatty acids at
sn-1 and sn-2 carbons, respectively, of the glycerol backbone. Furthermore, the utility of the extended lipid conformation in a variety of fundamental membrane processes would explain also the reason for the selection of lipids with two acyl chains
as major structural constituents for biomembranes.
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Kinnunen and Holopainen
What is the driving force promoting the ‘‘extended’’ lipid conformation? Based
on considerations arising from lipid packing constraints the ‘‘extended’’ conformation is anticipated to be promoted by decrease in the effective size of the lipid
headgroup by dehydration, for instance, as well as by lipids favoring inverted nonlamellar phases, i.e., lipids with negative spontaneous curvature (Helfrich, 1973;
Gruner, 1985, 1989). The latter provides a quantatitive description of the consequences of the molecular geometries of lipids (see below). A membrane in a state
with a high degree of propensity to form the HII phase will be referred to be in
Lε phase. Upon increasing temperature, for instance, the degree of trans → gauche
isomerization as well as the rates and amplitudes of chain motion of diacylphospholipids increase, so as to increase the effective size of the lipid acyl chains. The formation of the Lε phase and, accordingly, changes in the surface properties of the
membrane should start several degrees below the onset of the transition as has been
previously observed (Ellens et al., 1986b). Similarly, as repulsion between the headgroups is reduced due to charge neutralization and兾or dehydration, the packing
pressure within the hydrocarbon interior will increase. The driving force for chain
reversal is most easily understood in the context of the lateral pressure profile (Cantor, 1997). Accordingly, the lateral pressure within the membrane interior can be
first relieved by chain reversal provided that the membrane free volume distribution
is such that there is allocating space in the interfacial region. As was pointed out by
Cantor (1997) on a molecular level the magnitudes of forces acting in membranes
are enormous.
Israelachvili et al. (1980) emphasized the (headgroup area)兾(projected area of
the hydrocarbon chains) in the interface in providing a qualitative approach to the
correlation between lipid packing and the formation of distinct 3-D structures, i.e.,
micelles, lamellar, and inverted phases by different lipids. These authors introduced
a parameter P describing the molecular geometry and defined as:
PGV兾(a ∗ lc )
where:
VGeffective volume of the hydrophobic part of the molecule,
aGlimiting surface area of its hydrophilic part, and
lc Glength of its hydrophobic part.
Parameter P thus emphasizes the relative sizes of the different parts of the molecule,
and its values for lipids forming bilayers and HII phases are 1兾2HPH1, and H1,
respectively. For HII forming lipids the headgroup is small compared to the hydrophobic part of the molecule. This view is readily compatible with the known properties of lipids adopting the HII phase. Accordingly, upon approaching the
lamellar→HII transition temperature or upon introducing lipid species with negative
spontaneous curvature P → 1 and pressure within the center of the membrane
increases. Due to augmented bending stress the free energy of the system increases
as the intrinsic curvature for a single monolayer should increase. In other words,
maintaining the lipid bilayer at free energy minimum would require change in membrane curvature. For an isolated bilayer, such as present in large unilamellar vesicles
this is not allowed, as increasing the curvature of one monolayer would require
Lipid Conformational Dynamics and Bilayer Fusion
473
decrease in the curvature of the other leaflet. On the other hand, simultaneous
increase in the curvature of the two leaflets is also not possible. Therefore, the planar
configuration would be favored in spite of the augmenting packing pressure in the
hydrocarbon region of the membrane. However, to maintain planarity may require
due to packing strain part of the lipid molecules to adopt the extended conformation, one of the acyl chains sticking out into the aqueous phase. Accordingly,
packing pressure in the membrane interior is relieved. The exposure to water of the
acyl chains increases the free energy of the system and is compensated by free energy
gain due to avoidance of membrane bending.
Largest relief in packing within the hydrocarbon region is achieved when the
chains can extend into the two opposing leaflets forming the contact site between
two adhering bilayers (Kinnunen, 1992; 1996b), as in this case the projected interfacial area for the acyl chains is halved. In terms of the geometric parameter P the
above can be analyzed as follows. The effective volume Ve of the hydrocarbon region
of a lipid in the extended conformation is defined as
Ve GV兾2
Thus, for a lipid in the extended conformation Pe is defined as:
Pe GP兾2
Due to the additivity (Kumar, 1991) of the values for P for composite membranes
(or lipids adopting different conformations) it follows that for a lamellar membrane
containing lipids in the extended conformation we have:
[nPeC(NAn)Pa]兾NF1
where:
NGtotal number of lipids, and
nGnumber of lipids in the extended conformation.
In other words the system value of P can be maintained F1 when a sufficient number of lipids adopt the extended conformation. Upon further increase in temperature, however, the system of adhered bilayers collapses. Volume of the acyl chains
increase further so that also Pe → 1 and the system value for P exceeds unity. This
necessitates reorganization of the asssembly into the HII phase, concomitantly with
the realignment of the acyl chains. Accordingly, there is a gain in free energy upon
lipids adopting the extended conformation at the contact site of adhering vesicles.
To summarize, similarly to the (i) introduction of lipids with negative spontaneous curvature into lamellar phases, (ii) dehydration, (iii) decrease in vesicle size
(i.e., positive curvature of the outer monolayer), and (iv) acyl chain unsaturation
(Kinnunen, 1996a, b), all enhance chain–chain interactions, increase the pressure
within the hydrocarbon region of the bilayer, and promote fusion (Yang et al., 1997),
providing that the vesicles are adhering, i.e., there is no barrier for contact caused
by hydration, electrostatic repulsion, or undulation. Importantly, factors (i) to (iv)
are all anticipated to also promote chain reversal (thus increasing the hydrophobicity
of the liposome surface) as well as ultimately the ‘‘extended’’ conformation.
Increased surface hydrophobicity by stressing the membranes has been concluded to
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Kinnunen and Holopainen
be essential for membrane adhesion and fusion (Arnold, 1995; Düzgünes et al., 1987;
Helm et al., 1992; Leikin et al., 1987; Rand and Parsegian, 1986; Ohki and Arnold,
1990; Ohki and Düzgünes, 1979; Portis et al., 1979) whereas even strong van der
Waals or electrostatic forces alone are not sufficient (Helm et al., 1992; Marra,
1986a, b). This has been suggested to result from the latter forces acting on the
headgroup region and not on the hydrophobic parts of the membrane. Fusogenic
state of the plasma membrane having a lifetime of seconds to minutes has been
demonstrated after exposure of cells to a high voltage pulse used to trigger electrofusion. Reorganization in the headgroup region of the cell membrane concomitant
with an increase in hydrophobicity of the surface was observed (reviewed in Dimitrov, 1995).
THE EXTENDED LIPID ‘‘ZIPPER’’ MECHANISM FOR
BILAYER FUSION
The merging of the bilayers must be preceded by a hemifusion of the contacting
lipid surfaces. In this hemifusion state the extended lipid acts like a molecular ‘‘zipper’’, linking the two leaflets together. The essentials of this model are schematically
depicted in Fig. 1, illustrating the membrane level molecular reorganization. Initially
the opposing bilayers are separated by an aqueous layer. The contributions from
repulsive forces vary depending on the composition of the membranes and the water
phase. Upon adhering the contact site between the two bilayers becomes to a large
extent dehydrated. Due to the fact that the hydration layer of the membrane is
strongly reduced at the contact site of the adhering liposomes there should be very
low energy barrier for chain extension (lack of hydrophobic effect). This allows for
the intercalation of one of the phospholipid alkyl chains into the opposing bilayer
while retaining the headgroup in the interface. Accordingly, there can be a gain in
free energy upon lipids adopting the extended conformation at the contact site of
adhering vesicles. The concluded changes in the orientation of the lipid acyl chains
are schematically illustrated. Similarly, the extended conformation should be present
for instance in the isotropic phase existing between the Lα and HII phase for
dioleoylPE (Ellens et al., 1986b).
A system in fast equilibrium and involving the extended conformation readily
allows for lipid mixing between the outermost monolayers of the opposing membranes without fusion. The postulated mechanism of lipid exchange in hemifusion
is thus closely related to lipid transfer between vesicles. It has been demonstrated
that there is a lipid concentration dependent exchange process at higher concentrations of liquid crystalline vesicles, mediated by transient vesicle–vesicle associations (Jones and Thompson, 1989; 1990). This mode of lipid transfer is enhanced
in the presence of phosphatidylethanolamine (Wimley and Thompson, 1991). In this
connection it is also relevant to note the difference by approx. 106 to 109 orders of
magnitude in the time scales of lipid conformational fluctuations and the contacts
between vesicles. Conformational flexibility of the acyl chains is required for the
adoption of the extended conformation during fusion. This process should be more
effective for unsaturated lipids, in accordance with results from studies on the fusion
of cells as well as vesicles (Zaks and Creutz, 1990; Roos and Choppin, 1985). In
Lipid Conformational Dynamics and Bilayer Fusion
475
keeping with the requirement of the ‘‘extended’’ lipid conformation for fusion
inability to fuse has been recently shown for pure bolaform lipids which have polar
headgroups at both ends of the same molecule (Relini et al., 1994). However, the
presence of small amounts of monopolar lipids was sufficient to allow fusion to be
induced by Ca2C or PEG (Relini et al., 1994, 1996). Ca2C and PEG did induce also
lipid mixing between liposomes composed of the bolaform lipids (Relini et al., 1994)
when measured with lipid probes with the normal two chain configuration for which
the extended conformation is possible. For further details of the above model the
reader is referred to the original publications (Kinnunen, 1992, 1996b).
EVIDENCE FOR THE EXTENDED LIPID ‘‘ZIPPER’’ IN FUSION
AND HEMIFUSION
Metal cation induced fusion of PA containing vesicles provides a well established and thoroughly investigated model system (e.g., Düzgünes et al., 1987; Hong
et al., 1982; Liao and Prestegard, 1979; Ohki and Zschörnig, 1993; Papahadjopoulos
et al., 1990; Simmonds and Halsey, 1985; Smaal et al., 1987). Interestingly, upon
decreasing the mole fraction of PA (XPA) below 0.50 in binary DOPA兾DOPC liposomes only ‘‘hemifusion’’, i.e., lipid mixing between vesicles is induced by Mg2C
whereas neither leakage nor mixing of their aqueous contents is manifested. In contrast, Ca2C induces complete fusion (Leventis et al., 1986). When XPA in PC vesicles
exceeds 0.50 both Ca2C and Mg2C induce membrane fusion (Leventis et al., 1986).
Vesicle size has a large effect on membrane fusion and sonicated PA兾PC vesicles
undergo fusion more easily than LUVs (Liao and Prestegard, 1979). Compared to
LUVs the packing pressure within the hydrocarbon region of the outer monolayer
and chain–chain interactions are increased in SUV’s. Decreasing vesicle size also
increases lipid mixing between vesicles (Leventis et al., 1986). PA complexed with
divalent cations undergoes at elevated temperatures transition into the HII phase
(Miner and Prestegard, 1984). In keeping with previous studies on similar systems
(Farren et al., 1983; Verkleij et al., 1982) these authors presented evidence from
31
P-NMR and low angle X-ray scattering experiments for an unidentified, nonbilayer phase which was not a mixture of lamellar and hexagonal phases as had been
suggested previously (Farren et al., 1983). Miner and Prestegard (1984) concluded
that the Me2C-PA complexes adopt a fundamentally different phase at intermediate
temperatures which somehow resembles but is not identical to the HII phase and
proposed this intermediate to be equivalent to that formed during fusion.
We recently reported evidence for the extended lipid conformation in the course
of fusion and ‘‘hemifusion’’ of DOPA兾DOPC vesicles induced by Ca2C and Mg2C,
respectively (Holopainen et al., 1999), using intramolecular excimer forming pyrenelabeled phospholipids. The rationale for their use is based on the photophysics of
pyrene fluorescence (Duportail and Lianos, 1996; Kinnunen et al., 1993). In brief,
monomeric excited state pyrene may relax back to the ground state by emitting with a
maximum at ≈380 nm (Im ), the exact peak energy and spectral fine structure
depending on solvent polarity. During its lifetime the excited state pyrene may also
form a characteristic short-lived complex, excimer (excited dimer) with a ground state
pyrene. This complex relaxes back to two ground state pyrenes by emitting quanta
476
Kinnunen and Holopainen
as a broad and featureless band centered at ≈480 nm (Ie ). At sufficiently low probe
concentrations excimer fluorescence of dipyrene phospholipids such as 1,2-bis[(pyren-1)-yl]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) and also1-[(pyren-1)yl]decanoyl-2-[(pyren-1)-yl]tetradecanoyl-sn-glycero-3-phosphocholine (PDPTPC),
containing pyrene moieties at the ends of both acyl chains, is intramolecular and
concentration independent (Sunamoto et al., 1980). Accordingly, for these probes
the rate of excimer formation depends on the alignment of the acyl chains (Thurén
et al., 1984; Eklund et al., 1992), intramolecular thermal motion (Sunamoto et al.,
1980), and changes in membrane free volume (Lehtonen and Kinnunen, 1994). In
addition to the above also the ‘‘extended’’ phospholipid conformation would
strongly reduce the excimer emission.
In order to allow for unambiguous interpretation of the data collected for the
above dipyrenylphospholipids, we addressed changes induced in DOPA兾DOPC liposomes by the metal cations by measurements of emission polarization for 1,6diphenyl-1,3,5-hexatriene, DPH. As expected from dehydration and screening of the
charges of DOPA both Ca2+ and Mg2+ caused enhanced lipid packing (see e.g.,
Arnold, 1995). Our results demonstrated that these divalent cations reduce membrane free volume Vf , increase lateral lipid packing and acyl chain order, evident as
augmented DPH polarization. In concordance with earlier data this effect was somewhat smaller for Mg2+ (Leventis et al., 1986). We also measured changes in lipid
dynamics induced by Mg2+ and Ca2+, measuring the response of the intermolecular
excimer forming probe, PPDPC. In the absence of possible quantum mechanical
effects (Kinnunen et al., 1987) and the formation of superlattices, Ie 兾Im for a single
pyrene moiety containing phospholipid analog such as PPDPC, depends on the
intermolecular collision frequency of pyrenes (Förster, 1969). Consequently, the
value for Ie 兾Im reflects the lateral mobility (Galla et al., 1979) as well as the local
concentration of the fluorophore in the membrane (Eklund et al., 1988; Galla and
Hartmann, 1980; Hresko et al., 1986; Somerharju et al., 1985). Addition of Ca2+
enhanced Ie 兾Im for PPDPC whereas Mg2+ had no effect. These changes in Ie 兾Im
were complete within the same time range, approx. 10 s, as also observed for the
intramolecular excimer forming probes (see below). Accordingly, either enhanced
rate of lipid lateral diffusion or segregation of the probe into microdomains must be
induced by Ca2+. As DPH polarization revealed increase in membrane lipid packing
enhanced lateral diffusion can be excluded. As a consequence, for Ca2+ enrichment
of PPDPC must occur, in keeping with previous studies reporting Ca2+ induced
phase separation of PA in binary alloys with PC (Leventis et al., 1986; Eklund et
al., 1988; Kouaouci et al., 1985; Silvius, 1990). The lack of effect by Mg2C could be
concluded to be apparent only. More specifically, as DPH revealed reduced membrane free volume to be caused by Mg2C it follows that also for Mg2C the rate of
lateral diffusion of PPDPC must be attenuated. Therefore, as no changes in Ie 兾Im
are evident, accompanying slower lateral diffusion microscopic enrichment of
PPDPC due to Mg2C is required, the opposite signals balancing each other out. Yet,
compared to Ca2C the extent of segregation of lipids by Mg2C must be less pronounced, in keeping with previous reports (Leventis et al., 1986). This is readily
explained by the different coordination numbers for Ca2C and Mg2C as well as by
the more efficient lipid surface dehydrating effect exerted by Ca2C (Arnold, 1995).
Lipid Conformational Dynamics and Bilayer Fusion
477
To conclude, both cations caused the formation of microscopic domains
enriched in PPDPC, and, conversely, domains enriched in PA. In the latter the
charges of PA are neutralized by Me2C and the surface is dehydrated thus overcoming the repulsion between the vesicles and allowing for the formation of a contact
site between the vesicles (Arnold, 1995). Similarly to their effect on the emission on
bisPDPC slightly higher concentrations of Mg2C were required and also the final
increase in P induced by Mg2C was somewhat less than for Ca2C.
Crucial finding was decrease in Ie 兾Im for the intramolecular excimer forming
probe bisPDPC in the course of fusion and ‘‘hemifusion’’. The addition of mmolar
Ca2C decreased Ie 兾Im for bisPDPC in DPA兾DOPC liposomes by approx. 10%. The
same effect and of nearly equal magnitude was observed for Mg2C. Compared to
Ca2C, slightly higher concentrations of Mg2C were needed, in keeping with somewhat less efficient dehydration of the anionic phospholipid by the latter cation
(Düzgünes et al., 1987). The above changes in Ie 兾Im were rapid, being completed
within approx. 10 s and is identical to the timescale for the kinetics of fusion and
hemifusion of DOPA兾DOPC vesicles reported by Leventis et al. (1986). The
enhanced lipid packing and increased acyl chain order induced by Mg2C and Ca2C
and revealed by augmented DPH polarization excludes the possibility that an
increase in membrane free volume (Lehtonen and Kinnunen, 1994) was causing the
decrease in Ie 兾Im for bisPDPC. Importantly, contact site was necessary for the above
decrement in Ie 兾Im for bisPDPC. More specifically, dehydration of the surface of
DOPC vesicles by PEG at concentrations not causing their fusion increased Ie 兾Im
for this probe (Lehtonen and Kinnunen, 1994), in contrast to the conditions where
fusion or hemifusion were observed. The magnitude of the decrease in Ie 兾Im for
bisPDPC induced by Ca2C and Mg2C did depend on the content of PA in the binary
PA兾PC vesicles. More specifically, for neat DOPC LUVs no change in Ie 兾Im after
the addition of 5 mM Ca2C or Mg2C was observed in concordance with previous
reports on lack of either fusion or ‘‘hemifusion’’ under these conditions (Leventis et
al., 1986). Upon increasing XDOPA in LUVs a progressively augmenting decrease in
Ie 兾Im was evident due to the divalent metal cations (5 mM) and at XDOPA G1.0 an
approx. 30% decrease in Ie 兾Im was seen.
Interpretation of the above data set is not trivial and three distinct mechanisms
have to be considered as causes for the decrement in the intramolecular Ie 兾Im for
bisPDPC, as follows:
(i) Changes in the alignment of phospholipid acyl chains have been shown to
alter Ie 兾Im for dipyrenylphospholipids chain (Thurén et al., 1984). This could result
from augmented lateral lipid packing causing a conformation in which the glycerol
backbone parallels the sn-1 acyl chain, which further causes this chain to lie deeper
in the membrane. The sn-2 chain starts perpendicular to the glycerol backbone,
whereafter the subsequent carbon segments bend and align with the sn-1 chain
(Thurén et al., 1984). However, combination of probes (bisPDPC and PDPTPC)
allowed to exclude changes in chain alignment as a cause for decrease in Ie 兾Im for
bisPDPC. PDPTPC contains a pyrene decanoyl chain at sn-1 and pyrene tetradecanoyl chain at sn-2 position, the length of the sn-2 chain exceeding that of the
sn-1 chain by four methylene segments. Compared to bisPDPC approx. 19% lower
Ie 兾Im was measured for PDPTPC in DOPA兾DOPC vesicles in the absence of the
478
Kinnunen and Holopainen
metal cations. This difference has been interpreted to be due to the pyrene at the
end of tetradecanoyl chain being accommodated deeper in the bilayer than the
pyrene moiety of the decanoyl chain (Eklund et al., 1992). If altered chain alignment
was causing the decrement in Ie 兾Im for bisPDPC, using PDPTPC one should instead
observe an increase because the pyrene moieties in the latter lipid would reside
approximately at the same level. This was not the case but a decrement in Ie 兾Im was
evident also for PDPTPC. Interestingly, the decrease in Ie 兾Im for PDPTPC was
approx. 3-fold compared to bisPDPC, and for PDPTPC approx. 30% decrease in
Ie 兾Im was induced by mmolar Ca2C while for Mg2C an approx. 10% decrease was
evident. Dehydrating DOPC bilayers by 10 w兾w% PEG decreases membrane free
volume and causes an approx. 2-fold increase in Ie 兾Im for bisPDPC (Lehtonen and
Kinnunen, 1994). Instead, under these conditions a minor (<5%) decrease in Ie 兾Im
was evident for PDPTPC. This is in keeping with increasing acyl chain order in the
membrane upon dehydration (Arnold, 1995; Rand and Parsegian, 1989) which in
turn increases the effective length of the acyl chain. As a consequence the pyrene
moiety at the end of the tetradecanoyl chain in the sn-2 position becomes embedded
deeper into the membrane so as to diminish the excimer formation with the pyrene
at the end of the decanoyl spacer in the sn-1 chain. Accordingly, although decrease
in free volume augments Ie 兾Im concomitant decrement in the number of gauche
conformers decreases Ie 兾Im for PDPTPC, resulting in a minor net effect in Ie 兾Im .
To summarize, changes in chain alignment can be excluded as a cause for the metal
cation induced decrease in Ie 兾Im for bisPDPC.
(ii) The second possibility causing the decrease in Ie 兾Im for bisPDPC is chain
reversal (Ben-Shaul et al., 1984). Chain reversal could further be promoted by the
polarizability of pyrene, allowing under proper conditions to accommodate this aromatic group into the interface. Dehydration of the phospholipid surface results in
an increased packing within the hydrocarbon region of the bilayer (Lehtonen and
Kinnunen, 1994). However, this does not decrease Ie 兾Im for bisPDPC as would be
expected if chain reversal was taking place. Instead, increase in Ie 兾Im for this probe
was measured, in keeping with diminished membrane free volume and location of
the pyrene moieties in the membrane interior, i.e., the absence of chain reversal
(Lehtonen and Kinnunen, 1994).
(iii) Third possibility explaining the observed decrease in Ie 兾Im for bisPDPC is
the ‘‘extended’’ conformation, in which the acyl chains of the lipid are spread apart
from each other so that the time-averaged angle between them may maximally
approach 180° (Kinnunen, 1992). At XDOPA G0.50 the reduction in Ie 兾Im for
bisPDPC induced by mmolar Ca2C and Mg2C was only approx. 10%. Although part
of the probe could be partitioned into PA enriched domains and adopt the
‘‘extended’’ conformation at the contact sites it is likely that some of the bisPDPCs
would also reside in the membrane domains enriched in PC. Due to generally augmented lipid packing (revealed by DPH) Ie 兾Im for bisPDPC should be increased by
the divalent cations. Not excluding the partitioning of the probe into the
Ca2C-DOPA domains it is also possible that bisPDPC favors localization in the
interface between regions enriched in PC and PA. Upon increasing XDOPA from 0 to
1.0 the relative values for intramolecular Ie 兾Im for bisPDPC decreased from 1.0 to
0.70 and 0.74 for 5 mM Ca2C and Mg2C, respectively. These data indicate that the
Lipid Conformational Dynamics and Bilayer Fusion
479
number of bisPDPC molecules in the ‘‘extended’’ conformation should increase
upon increasing the extent of vesicle aggregation and thus the fraction of the surface
constituted by Me2C-DOPA enriched domains (Leventis et al., 1986). Interestingly,
compared to bisPDPC the decrease in Ie 兾Im for PDPTPC induced by Ca2C is
approx. 3-fold whereas for Mg2C a similar decrement is observed for both probes.
In keeping with PDPTPC being more perturbing one may also expect larger free
energy gain upon adoption of the ‘‘extended’’ conformation by this probe. Taking
into account the more efficient dehydration of PA by Ca2C this difference is readily
comprehensible. Reducing Vf attenuates the amplitude of the thermal motion of the
pyrenedecanoyl chains, i.e., decreases the extent of their splaying. When the thermal
excitation remains constant (i.e., the frequency of chain motion is not reduced) the
probability of collisional intramolecular excimer formation during the lifetime of the
excited state of pyrene increases upon reduction in Vf (Lehtonen and Kinnunen,
1994). As decrease in Vf should increase Ie 兾Im for bisPDPC and because contact site
between adhering liposomes was required we concluded the most feasible explanation to be the adoption of the ‘‘extended’’ conformation by bisPDPC.
CONCLUDING REMARKS
The mechanisms involved in cellular fusion processes remain a perplexing question. This review summarizes some of the aspects derived from studies emphasizing
the properties of lipids, which are in general mostly neglected by cell biologists.
Accordingly, this area represents a great opportunity for physicochemists and physicists to have a major impact on biosciences and pharmaceutical research. Lastly, for
the other consequences of the extended lipid conformation and the significance of
the Lε phase the reader is referred to the other publications from our laboratory
(Kinnunen, 1996a, b).
ACKNOWLEDGMENTS
Helsinki Biophysics and Biomembrane group is supported by Tekes, Finnish
State Medical Research Council, and Biocentrum Helsinki.
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