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Biophys J, February 2002, p. 555-557, Vol. 82, No. 2
and
*Molecular and Cellular Biophysics, Department of Biochemistry and
Biophysics, University of North Carolina at Chapel Hill, North
Carolina, Givaudan Inc, Cincinnati, Ohio, and
Elan Pharmaceuticals, Princeton, New Jersey 08540 USA
Membrane fusion is at the heart of eukaryotic
cell life. Carefully regulated membrane fusion is required for cell
compartmentalization, for the import of large molecules into the cell,
and for the export both of waste molecules and of signaling molecules
that carry information to other cells in a community. The complex
cellular machinery that carries out these functions is beginning to
come into focus (Brunger, 2001 The widely accepted model for this process derives from the original
proposal that two bilayers brought into close contact can merge their
contacting (cis) monolayers in a torroidal "stalk" that
joins half the lipid components of the two original bilayers (Fig. 2 B from Markin and Albanesi (2001) Two papers appear in this month's issue of the Biophysical
Journal (Kozlovsky and Kozlov, 2001 The other two articles both ask whether it is appropriate to
estimate the interstice energy in terms of the surface of the interface
between a void and smoothly bent lamellar structures. Although it is
unlikely that a true macroscopic void exists in intermediate structures
leading to fusion, this construct was originally used as a
self-consistent and convenient way to estimate the free energy
associated with the inherent nonlamellar nature of the stalk (Siegel,
1993 Kozlovsky and Kozlov (2001) In summary, one essential finding of both teams Markin/Albanesi and
Kozlovsky/Kozlov is that the energy required to deform the monolayers
into a stalk is substantially lower than calculated with the approach
used previously. Basically, the source of the overestimate in previous
models was the use of a simplistic geometric model. Both sets of
authors show that a more realistically shaped stalk is likely to have
considerably lower bending energy, which, relative to planar bilayers,
can even become negative for bilayers of substantial negative intrinsic
curvature. However, it seems that the interstice free energy still
dominates the stalk free energy and we may still have to fill the
potholes in the pathway to fusion pores.
There are tough problems that remain to model successfully the
mechanism of fusion. In both these manuscripts, the authors calculate
only the lower bound to the true activation energy for formation of
fusion intermediates. Present models account only for the energy of
intermediates viewed as static structures. Experimentally, the process
of converting closely contacting membranes (initial static structure)
to a fusion pore (final static structure) involves at least three
kinetic steps and two intermediates (Lentz et al., 2000 How do these new results help us understand the process of
protein-induced membrane fusion? First, the resolution of the energy crisis increases our confidence that structures such as stalks do
represent low-energy pathways to membrane fusion. Second, the dominant
nature of the void or interstice energy suggests that we look to how
parts of fusion proteins that reside in or interact with membrane
bilayers might lower this free energy. This will require combinations
of experimental and theoretical approaches. In doing so, it is
appropriate to keep in mind that the stalk hypothesis is an hypothesis
and that the actual mechanism of biomembrane fusion may be even more
complex than suggested by this model.
ARTICLE
), but a detailed molecular view of how these machines work on membrane lipid bilayers to accomplish a change
in topology of cellular compartments is still lacking. The difficulty
in dissecting this process lies not just in the complexity of the
machines, but also in the task of defining experimentally a dynamic
process operating in a semi-ordered system such as the lipid bilayer.
Although experimental approaches are available, it has been popular for
some time to treat this problem theoretically, in terms of simple
models in which complex arrangements of lipid molecules are treated in
terms of the material properties of arrays of lipid molecules as they
occur in macroscopic semicrystalline lipid phases. Although this is
clearly a vast simplification, it is probably, if properly
parameterized, a useful zeroth order approach to the very difficult
problem of estimating the free energies of presumed intermediates on
the molecular path to fusion.
). The distal
(trans) monolayers of this structure are not merged, which
prevents continuity between the trapped aqueous compartments of the two
fusing membranes. This has been termed the stalk hypothesis
(Markin et al., 1984). Later, it was recognized that the stalk may
exist in two different forms, an initial stalk and a transmembrane
contact (Siegel, 1999). It was universally accepted that such
structures must have a free energy substantially larger than that of
the lipid bilayers from which it was proposed to arise. A systematic
estimate of this energy was first made in terms of two major
contributions: the energy associated with bending planar monolayers
into toroids, and the interstice ("void") energy associated with
the junctions where monolayers are peeled away from one another
(Siegel, 1993). The latter was, in the initial level of approximation,
estimated as proportional to the surface area of the interface between
lamellar structures and a hypothetical void that represented the space unfilled by uniformly packed monolayers distorted to match the hypothesized nonlamellar stalk structure (Siegel, 1993). The bending energy was estimated in terms of the free energy required to distort a
uniform lamellar monolayer from its spontaneous or "intrinsic" curvature to match the assumed stalk shape. The energy of the void was
parameterized based on the properties of the phase transition from a
lamellar to a nonlamellar phase (hexagonal phase) that was also viewed
as composed of bent lamellar structures and voids. Based on this
treatment, the free energy of the stalk was estimated to be so large
(~200 kT) that there has been question as to whether such
a structure could be part of the fusion process, although these
calculations suggested that the stalk was the lowest-energy intermediate of several proposed fusion mechanisms (Siegel 1993). Thus,
the stalk hypothesis was found to face an "energy crisis."
; Markin and Albanesi, 2001) that, along with another recent paper (Kuzmin et al., 2001), propose solutions to this energy crisis. Markin and Albanesi (2001) propose that the assumption of circular torroidal geometry for the stalk produced too large a bending energy. These authors show that it is
possible to relax this assumption and define, in terms of two geometric
parameters, a stress-free stalk that has the same average intrinsic
curvature as the lipid mixture of which it is composed. Just as for the
original stalk hypothesis (Markin et al., 1984; Siegel, 1993), one can
question some of the geometric assumptions of this calculation.
However, it is a reasonable result that the bending energy of a
geometrically relaxed stalk can be reduced significantly from the
original estimate that was based on a rigid (circular
torroid) geometry. Even if the bending free energy of the
stalk can be reduced to zero, the interstice or void free energy
attributable to the intrinsic geometric mismatch between lamellar and
nonlamellar lipid structures must persist. This is variously estimated
as several tens of kT, still a formidable energy but much less imposing
than the earlier estimates of the stalk energy.
). The question is whether a better method exists for estimating
this free energy. Kozlovsky and Kozlov (2001) and Kuzmin et al. (2001)
both argue that this energy is better estimated in terms of the energy
needed to bend lamellar structures so sharply that they accommodate the
inherently nonlamellar nature of the proposed stalk structure. This
requires tilt of individual lipid molecules and must occur
at an energy price, as tilting lipid molecules relative to the bilayer
normal expose their hydrophobic regions to water and elongate acyl
chains. Similar to the void energy, the tilt deformation energy can be
estimated from experimental data on phase transitions of lipid liquid
crystals from lamellar-to-nonlamellar states.
use the tilt approach to treat the
deformation of monolayers in terms of a combination of splay (monolayer
bending and/or tilt gradient) and uniform tilt of lipid molecules away
from the local monolayer normal. The "tilt" formalism provides a
means to account for the interstice energy without the assumption of a
void. Figure 8 by Markin and Albanesi illustrates this approach
relative to the traditional void approach. Kozlovsky and Kozlov
describe the stalk in terms of two geometric parameters (width of the
stalk base, interbilayer distance). This approach yields a minimum free
energy for the stalk at an intrinsic curvature of 
0.1
nm
1 of 45-50 kT, which is approximately the
same as one would estimate for the interstice energy based on the void
model, as pointed out by Markin and Albanesi. Curiously, at intrinsic
curvatures of < 0.25 nm1, the stalk of
Kozlovsky and Kozlov becomes thermodynamically stable relative to
planar bilayers.
). The first and
last of these steps involve changes in the topology of membranes and
trapped compartments and thus demand dramatic rearrangements of lipid
and water molecules. These are not likely to find description by the
sort of continuum, macroscopic theories reviewed here, although some
attempts have been made to do so (Kuzmin et al., 2001; Markin and
Albanesi, 2001). The second step, interconversion of fusion
intermediates, probably does not require changes in topology and may be
amenable to such an approach. Nor have we really addressed the problem
of how two membranes become distorted and so closely apposed that the
initial conversion to a fusion intermediate can occur. Most
importantly, experimental tests of these models need to be devised.
Given the highly localized and dynamic nature of membrane fusion
events, this will be very difficult. However, the same rules that
govern the relative energies of different-geometry intermediates must also apply to the relative free energies of lipid molecules in lamellar
and nonlamellar phases. Therefore, it is probably worth some effort to
see which of the new proposals best describes the relative stability
and formation kinetics of complex lipid assemblies such as inverted
cubic phases. Clearly there is much lipid physical chemistry that
remains to be done!
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FOOTNOTES |
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Submitted December 14, 2001, and accepted for publication December 14, 2001.
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REFERENCES |
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Biophys J, February 2002, p. 555-557, Vol. 82, No. 2
© 2002 by the Biophysical Society 0006-3495/02/02/555/03 $2.00
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