The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

Effects of Spontaneous Bilayer Curvature on Influenza Virus–mediated Fusion Pores

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.     

Membrane mechanics can account for fusion pore dilation in stages.

Once formed, fusion pores rapidly enlarge to semi-stable conductance values. The membranes lining the fusion pore are continuous bilayer structures, so variations of conductance in time reflect bending and stretching of membranes. We therefore modeled the evolution of fusion pores using the theory of the mechanics of deforming homogeneous membranes. We calculated the changes in length and width of theoretical fusion pores according to standard dynamical equations of motion. Theoretical fusion pores quickly achieve semi-stable dimensions, which correspond to energy minima located in a canyon between energy barriers. The height of the barrier preventing pore expansion diminishes along the dimensions of length and width. The bottom of the canyon slopes gently downward along increasing length. As a consequence, theoretical fusion pores slowly lengthen and widen as the dimensions migrate along the bottom of the canyon, until the barrier vanishes and the pore rapidly enlarges. The dynamics of growth is sensitive to tension, spontaneous curvature, bending elasticity, and mobilities. This sensitivity can account for the quantitative differences in pore evolution observed in two experimental systems: HA-expressing cells fusing to planar bilayer membranes and beige mouse mast cell degranulation. We conclude that the mechanics of membranes could cause the phenomenon of stagewise growth of fusion pores.     

Membrane Bending Energy and Fusion Pore Kinetics in Ca-Triggered Exocytosis

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca²⁺-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca²⁺-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca²⁺-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape.     

Stalk mechanism of vesicle fusion

Å mechanism for rupture of a separating bilayer, resulting from vesicle monolayer fusion is investigated theoretically. The stalk mechanism of monolayer fusion, assuming the formation and expansion of a stalk between two interacting membranes is considered. The stalk evolution leads to formation of a separating bilayer and mechanical tension appearance in the system. This tension results in rupture of the separating bilayer and hydrophilic pore formation. Competition between the mechanical tension and hydrophilic pore energy defines the criteria of contacting bilayer rupture. The tension increases with an increase of the absolute value of the negative spontaneous curvature of the outer membrane monolayer, K _s ^o . The pore edge energy decreases with an increase of the positive spontaneous curvature of the inner membrane monolayer, K _s ⁱ . The relations of spontaneous curvatures of outer and inner monolayers, leading to separating bilayer rupture, is calculated. It is demonstrated that his process is possible, provided spontaneous curvatures of membrane monolayers have opposite signs: K _s ^o <0, K _s ⁱ <0. Experimental data concerning the fusion process are analysed.     

Lipids in biological membrane fusion

The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions.Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion.     

The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)- expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.     

The gaussian curvature elastic modulus of N-monomethylated dioleoylphosphatidylethanolamine: relevance to membrane fusion and lipid phase behavior

The energy of intermediates in fusion of phospholipid bilayers is sensitive to kappa(m), the saddle splay (Gaussian curvature) elastic modulus of the lipid monolayers. The value kappa(m) is also important in understanding the stability of inverted cubic (Q(II)) and rhombohedral (R) phases relative to the lamellar (L(alpha)) and inverted hexagonal (H(II)) phases in phospholipids. However, kappa(m) cannot be measured directly. It was previously measured by observing changes in Q(II) phase lattice dimensions as a function of water content. Here we use observations of the phase behavior of N-mono-methylated dioleoylphosphatidylethanolamine (DOPE-Me) to determine kappa(m). At the temperature of the L(alpha)/Q(II) phase transition, T(Q), the partial energies of the two phases are equal, and we can express kappa(m) in terms of known lipid monolayer parameters: the spontaneous curvature of DOPE-Me, the monolayer bending modulus kappa(m), and the distance of the monolayer neutral surface from the bilayer midplane, delta. The calculated ratio kappa(m)/kappa(m) is -0.83 +/- 0.08 at T(Q) approximately 55 degrees C. The uncertainty is due primarily to uncertainty in the value of delta for the L(alpha) phase. This value of kappa(m)/kappa(m) is in accord with theoretical expectations, including recent estimates of the value required to rationalize observations of rhombohedral (R) phase stability in phospholipids. The value kappa(m) substantially affects the free energy of formation of fusion intermediates: more energy (tens of k(B)T) is required to form stalks and fusion pores (ILAs) than estimated solely on the basis of the bending elastic energy. In particular, ILAs are much higher in energy than previously estimated. This rationalizes the action of fusion-catalyzing proteins in stabilizing nascent fusion pores in biomembranes; a function inferred from recent experiments in viral systems. These results change predictions of earlier work on ILA and Q(II) phase stability and L(alpha)/Q(II) phase transition mechanisms. To our knowledge, this is the first determination of the saddle splay (Gaussian) modulus in a lipid system consisting only of phospholipids.     

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer.     

Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.     

Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of- phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell- containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time- resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.     

The exocytotic fusion pore interface: a model of the site of neurotransmitter release

infrastructurel techniques have shown that an early event in the exocytotic fusion of a secretory vesicle is the formation of a narrow, water-filled pore spanning both the vesicle and plasma membranes and connecting the lumen of the secretory vesicle to the extracellular environment. Smaller precursors of the exocytotic fusion pore have been detected using electrophysio-logical techniques, which reveal a dynamic fusion pore that quickly expands to the size of the pores seen with electron microscopy. While it is clear that in the latter stages of expansion, when the size of the fusion pore is several orders of magnitude bigger than any known macromolecule, the fusion pore must be mainly made of lipids, the structure of the smaller precursors is unknown. Patch-clamp measurements of the activity of individual fusion pores in mast cells have shown that the fusion pore has some unusual and unexpected properties, namely that there is a large flux of lipid through the pore and the rate of pore closure has a discontinuous temperature dependency, suggesting a purely lipidic fusion pore. Moreover, comparisons of experimental data with theoretical fusion pores and with breakdown pores support the view that the fusion pore is initially a pore through a single bilayer, as would be expected for membrane fusion proceeding through a hemifusion mechanism. Based on these observations we present a model where the fusion pore is initially a pore through a single bilayer. Fusion pore formation is regulated by a macromolecular scaffold of proteins that is responsible for bringing the plasma membrane into a highly curved dimple very close to a tense secretory granule membrane, creating the architecture where the strongly attractive hydrophobic force causes the membranes to form a ‘hemifusion’ intermediate. Membrane fusion is completed by the formation of an aqueous pore after rupture of the shared bilayer. We also propose that the microenvironment of the interface when the pore first opens, dominated by the charged groups on the secretory vesicle matrix and phospholipids, will greatly influence the release of secretory products.     

Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.     

Possible mechanism of membrane fusion

The stalker mechanism of membrane fusion was considered. Initiation and evolution of monolayer and bilayer bridges-stalks between the membranes were studied. From the expression of elastic energy of the stalk the value of spontaneous curvature of its membrane Ks at which the bridge may appear, was found. It was shown that in terms of the stalker mechanism formation of the stalk of the final radius or complete fusion were possible Ks values for realizing this or that variant were found. The energetic barrier of hydrophobic interaction and the barrier of elastic energy which the membranes had to overcome for stalker formation were found. The experimental data on the fusion of small and large liposomes were analysed.     

The exocytotic fusion pore modeled as a lipidic pore.

Freeze-fracture electron micrographs from degranulating cells show that the lumen of the secretory granule is connected to the extracellular compartment via large (20 to 150 nm diameter) aqueous pores. These exocytotic fusion pores appear to be made up of a highly curved bilayer that spans the plasma and granule membranes. Conductance measurements, using the patch-clamp technique, have been used to study the fusion pore from the instant it conducts ions. These measurements reveal the presence of early fusion pores that are much smaller than those observed in electron micrographs. Early fusion pores open abruptly, fluctuate, and then either expand irreversibly or close. The molecular structure of these early fusion pores is unknown. In the simplest extremes, these early fusion pores could be either ion channel like protein pores or lipidic pores. Here, we explored the latter possibility, namely that of the early exocytotic fusion pore modeled as a lipid-lined pore whose free energy was composed of curvature elastic energy and work done by tension. Like early exocytotic fusion pores, we found that these lipidic pores could open abruptly, fluctuate, and expand irreversibly. Closure of these lipidic pores could be caused by slight changes in lipid composition. Conductance distributions for stable lipidic pores matched those of exocytotic fusion pores. These findings demonstrate that lipidic pores can exhibit the properties of exocytotic fusion pores, thus providing an alternate framework with which to understand and interpret exocytotic fusion pore data.     

Dynamics of fusion pores connecting membranes of different tensions

The energetics underlying the expansion of fusion pores connecting biological or lipid bilayer membranes is elucidated. The energetics necessary to deform membranes as the pore enlarges, in some combination with the action of the fusion proteins, must determine pore growth. The dynamics of pore growth is considered for the case of two homogeneous fusing membranes under different tensions. It is rigorously shown that pore growth can be quantitatively described by treating the pore as a quasiparticle that moves in a medium with a viscosity determined by that of the membranes. Motion is subject to tension, bending, and viscous forces. Pore dynamics and lipid flow through the pore were calculated using Lagrange's equations, with dissipation caused by intra- and intermonolayer friction. These calculations show that the energy barrier that restrains pore enlargement depends only on the sum of the tensions; a difference in tension between the fusing membranes is irrelevant. In contrast, lipid flux through the fusion pore depends on the tension difference but is independent of the sum. Thus pore growth is not affected by tension-driven lipid flux from one membrane to the other. The calculations of the present study explain how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. In a similar fashion, swelling of secretory granules after fusion in biological systems could promote pore enlargement during exocytosis. The calculations also show that pore expansion can be caused by pore lengthening; lengthening may be facilitated by fusion proteins.     

Structural intermediates in influenza haemagglutinin-mediated fusion

Fusion pore formation in the haemagglutinin (HA)-mediated fusion is a culmination of a multistep process, which involves low-pH triggered refolding of HA and rearrangement of membrane lipid bilayers. This rearrangement was arrested or slowed down by either altering lipid composition of the membranes, or lowering the density of HA, and/ or temperature. The results suggest that fusion starts with the lateral assembly of activated HA into multimeric complexes surrounding future fusion sites. The next fusion stage involves hemifusion, i.e. merger of only contacting membrane monolayers. Lysophosphatidylcholine reversibly arrests fusion prior to this hemifusion stage. In the normal fusion pathway, hemifusion is transient and is not accompanied by any measurable transfer of lipid probes between the membranes. A temperature of 4degreeC stabilizes this `restricted hemifusion' intermediate. The restriction of lipid flow through the restricted hemifusion site is HA-dependent and can be released by partial cleaving of low pH-forms of HA with mild proteinase K treatment. Lipid effects indicate that fusion proceeds through two different lipid-involving intermediates, which are characterized by two opposite curvatures of the lipid monolayer. Hemifusion involves formation of a stalk, a local bent connection between the outer membrane monolayers. Fusion pore formation apparently involves bending of the inner membrane monolayers, which come together in hemifusion. To couple low pH-induced refolding of HA with lipid rearrangements, it is proposed that the extension of the alpha -helical coiled coil of HA pulls fusion peptides inserted into the HA-expressing membrane and locally bends the membrane into a saddle-like shape. Elastic energy drives self-assembly of these HA-containing membrane elements into a ring-like complex and causes the bulging of the host membrane into a dimple growing towards the target membrane. Bending stresses in the lipidic top of the dimple facilitate membrane fusion.     

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.