Bilayer Membrane Fusion Research Articles

Liposomal membrane fusion induced by extrusion method

Objective To study the application of extrusion method in inducing liposome membrane fusion or membrane component mixing, and to investigate the effect of different extrusion conditions on liposome membrane fusion rate. Methods N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) labeled 1, 2-dioleoyl lecithin (DOPC) liposomes and non-fluorescently labeled DOPC monolayer liposomes were mixed and extruded. The fluorescence changes before and after the extrusion of the mixed liposomes were observed using laser scanning confocal microscope, and the membrane fusion rate of the mixed liposomes was calculated by fluorescence resonance energy transfer method. Besides, the effects of extrusion times, extrusion pressure and temperature on the fusion rate of liposome membrane were studied. Results The results of laser scanning confocal microscopy showed that the distribution density and intensity of the green fluorescence of N-NBD-PE increased significantly after the extrusion of fluorescently labeled and non-fluorescent labeled DOPC liposomes, which confirmed membrane fusion. After 75 times of extrusion treatments, the liposome membrane fusion rate can reach 26%. The number of extrusions, extrusion pressure and temperature had a significant effect on the fusion rate of the liposome membrane. The higher the number of the extrusions, the smaller the extrusion pressure and the higher the efficiency of the liposome membrane fusion were at physiological temperature. Conclusions Extrusion method can induce liposome membrane fusion and membrane component mixing, and the prepared liposome has a narrower particle size distribution, which is expected to be a new method to induce the bilayer membrane fusion of liposome or lipid vesicle. Key words: Extrusion; Liposome; Lipid bilayer fusion; Fluorescence resonance energy transfer

Rate of hemifusion diaphragm dissipation and ability to form three-junction bound HD determined by lipid composition.

Though the hemifusion diaphragm (HD) is widely accepted as an intermediate in bilayer membrane fusion, lipid contributions toward HD stability and dynamics are still not fully understood. In this paper, we study large, binary, protein-free HD systems at varying compositions of negative intrinsic curvature (NIC) lipids using molecular dynamics (MD) simulations of a solvent-free coarse-grained lipid model. Under MD, initially created HDs are found to relax to three major end states depending on the composition and lipid intrinsic curvature. Low compositions of NIC lipids or weak intrinsic curvature result in double-bilayer end states, and moderate compositions of moderate to strong NIC lipids result in metastable fusion pores. Importantly, high compositions of moderate NIC lipids result in a metastable HD that persists beyond μs time scales. NIC lipids stabilize the HD by filling the junction core around the HD. Sorting of NIC lipids toward the three-junction region occurs in fused-endpoint systems, but no significant sorting was seen in systems that end in a double bilayer indicating that high line tension at the triple junction drives HD dissipation faster than sorting can enrich that junction enough to lower that line tension. The appearance of three end states dependent on the NIC lipid composition highlights the necessity of NIC lipids for non-leaky fusion.

A Molecular Mechanism of Lipid Membrane Fusion

Membrane fusion is an essential molecular event involved in many cellular processes, such as exocytosis, endocytosis, intracellular vesicle trafficking, fertilization, and viral infection to target cells. In spite of extensive studies of membrane fusion, however, the basic molecular mechanisms in biological systems are not well understood. Probably, it is due to the complex nature of biological membranes and the variety of possible molecular pathways for membrane fusion. We have studied the membrane fusion process, particularly ion-induced membrane fusion. Biological membrane fusion seems to occur with either ion-induced or non-ion-induced membrane process, particularly the later case is for virus membrane fusion system. Dr. Chernomordik and his co-workers have studied on non-ion-induced lipid membrane fusion and developed the so-called “Stalk-intermediate model” before total membrane fusion. That fusion model has been well received by many membrane fusion investigators, particularly in the virus fusion field. Stalk formation between two lipid membranes may occur due to undulation of lipid molecules or local binding of the lipid bilayers, which results in the formation of a local region of outer monolayer fusion. The stalk hypothesis can be described by macroscopic models treating bilayers and monolayers as homogeneous elastic surfaces. We have also studied non-ion-induced bilayer membrane fusion. Our membrane fusion theory is based on the interaction energies between the two membranes due to alternation of the membrane surface properties, e.g., hydrophilicity and hydrohobicity of interacting membranes, and then lipid membrane close approach and due to membrane curvature. Although the membrane interaction processes are different between the two models, these membrane fusion properties are the same as those of our ion-induced lipid membrane fusion.

Field Theoretic Study of Bilayer Membrane Fusion III: Membranes with Leaves of Different Composition

We extend previous work on homogeneous bilayers to calculate the barriers to fusion of planar bilayers that contain two different amphiphiles, a lamellar former and a hexagonal former, with different compositions of the two in each leaf. Self-consistent field theory is employed, and both standard and alternative pathways are explored. We first calculate these barriers as the amount of hexagonal former is increased equally in both leaves to levels appropriate to the plasma membrane of human red blood cells. We follow these barriers as the composition of hexagonal formers is then increased in the cis layer and decreased in the trans layer, again to an extent comparable to the biological system. We find that, while the fusion pathway exhibits two barriers in both the standard and alternative pathways, in both cases the magnitudes of these barriers are comparable to one another, and small, on the order of 13 k B T. As a consequence, one expects that once the bilayers are brought sufficiently close to one another to initiate the process, fusion should occur rapidly.

Pathway of Membrane Fusion with Two Tension-Dependent Energy Barriers

Fusion of bilayer membranes is studied via dissipative particle dynamics (DPD) simulations. A new set of DPD parameters is introduced which leads to an energy barrier for flips of lipid molecules between adhering membranes. A large number of fusion events is monitored for a vesicle in contact with a planar membrane. Several time scales of the fusion process are found to depend exponentially on the membrane tension. This implies an energy barrier of about 10k(B)T for intermembrane flips and a second size-dependent barrier for the nucleation of a small hemifused membrane patch.

Field Theoretic Study of Bilayer Membrane Fusion: II. Mechanism of a Stalk-Hole Complex

We use self-consistent field theory to determine structural and energetic properties of intermediates and transition states involved in bilayer membrane fusion. In particular, we extend our original calculations from those of the standard hemifusion mechanism, which was studied in detail in the first article of this series, to consider a possible alternative to it. This mechanism involves non-axial stalk expansion, in contrast to the axially symmetric evolution postulated in the classical mechanism. Elongation of the initial stalk facilitates the nucleation of holes and leads to destabilization of the fusing membranes via the formation of a stalk-hole complex. We study properties of this complex in detail, and show how transient leakage during fusion, previously predicted and recently observed in experiment, should vary with lipid architecture and tension. We also show that the barrier to fusion in the alternative mechanism is lower than that of the standard mechanism by a few kBT over most of the relevant region of system parameters, so that this alternative mechanism is a viable alternative to the standard pathway. We emphasize that any mechanism, such as this alternative one, which affects, even modestly, the line tension of a hole in a membrane, affects greatly the ability of that membrane to undergo fusion.

Tension-induced fusion of bilayer membranes and vesicles

Maintaining the integrity of their protective plasma membrane is a primary requirement of cells. Accordingly, cellular events that breach the membrane are tightly regulated. Artificial vesicles used in drug delivery must also stay intact until they have reached the desired target. In both cases, the intrinsic resistance of the membrane to rupture must be overcome to allow the efflux of the vesicle's contents. Here, we use mesoscopic simulations to study the fusion of 28-nm-diameter vesicles to 50 x 50 nm(2) planar membrane patches over 2 mus. We monitor the time evolution of 93 different fusion attempts. This allows us to construct a global morphology diagram, using the initial tensions of the vesicle and the planar membrane patch as control parameters, and to determine the corresponding fusion statistics. All successful fusion events are observed to occur within 350 ns, which reflects the presence of alternative pathways for the tension relaxation.

Field Theoretic Study of Bilayer Membrane Fusion. I. Hemifusion Mechanism

Self-consistent field theory is used to determine structural and energetic properties of metastable intermediates and unstable transition states involved in the standard stalk mechanism of bilayer membrane fusion. A microscopic model of flexible amphiphilic chains dissolved in hydrophilic solvent is employed to describe these self-assembled structures. We find that the barrier to formation of the initial stalk is much smaller than previously estimated by phenomenological theories. Therefore its creation it is not the rate-limiting process. The relevant barrier is associated with the rather limited radial expansion of the stalk into a hemifusion diaphragm. It is strongly affected by the architecture of the amphiphile, decreasing as the effective spontaneous curvature of the amphiphile is made more negative. It is also reduced when the tension is increased. At high tension the fusion pore, created when a hole forms in the hemifusion diaphragm, expands without bound. At very low membrane tension, small fusion pores can be trapped in a flickering metastable state. Successful fusion is severely limited by the architecture of the lipids. If the effective spontaneous curvature is not sufficiently negative, fusion does not occur because metastable stalks, whose existence is a seemingly necessary prerequisite, do not form at all. However if the spontaneous curvature is too negative, stalks are so stable that fusion does not occur because the system is unstable either to a phase of stable radial stalks, or to an inverted-hexagonal phase induced by stable linear stalks. Our results on the architecture and tension needed for successful fusion are summarized in a phase diagram.

Membrane fusion

Although catalyzed by different proteins, the energy barriers for lipid bilayer fusion in exocytosis, viral fusion, and trafficking seem to be the same as those for the fusion of protein-free phospholipid membranes. To minimize this energy, fusion will proceed through a minimal number of lipid molecules, probably localized in bent non-bilayer intermediates. Experiments on phospholipid bilayer membrane fusion show the pathway of contact, hemifusion, flickering fusion pore formation, and fusion pore enlargement caused by swelling of the vesicle. Lipid curvature determines the barriers to hemifusion and fusion pore formation, while swelling-induced membrane tension drives fusion pore enlargement. Experiments on viral protein-induced cell–cell fusion and exocytosis show the same pathway with the same fundamental effects of lipid curvature and membrane tension. Thus while proteins control these reactions, lipid energetics determine the basic reaction scheme for membrane fusion.

Membrane Fusion in Vesicles of Oligomerizable Lipids

Membrane fusion has been examined in a model system of small unilamellar vesicles of synthetic lipids that can be oligomerized through the lipid headgroups. The oligomerization can be induced either in both bilayer leaflets or in the inner leaflet exclusively. Oligomerization leads to denser lipid headgroup packing, with concomitant reduction of lipid lateral diffusion and membrane permeability. As evidenced by lipid mixing assays, electron microscopy, and light scattering, calcium-induced fusion of the bilayer vesicles is strongly retarded and inhibited by oligomerization. Remarkably, oligomerization of only the inner leaflet of the bilayer is already sufficient to affect fusion. The efficiency of inhibition and retardation of fusion critically depend on the relative amount of oligomeric lipid present, on the concentration of calcium ions, and on temperature. Implications for the mechanism of bilayer membrane fusion are discussed in terms of lipid lateral diffusion and membrane curvature effects.

Fusion in phospholipid spherical membranes. II. Effect of cholesterol, divalent ions and pH.

Effect of cholesterol, divalent ions and pH on spherical bilayer membrane fusion was studied as a function of increasing temperature. Spherical bilayer membranes were composed of natural [phosphatidylcholine (PC) and phosphatidylserine (PS)] as well as synthetic (dipalmitoyl-PC, dimyristoyl-PC and dioleoyl-PC) phospholipids. Incorporation of cholesterol into the membrane (33% by weight) suppressed the fusion temperature and also greatly reduced the percentage of membrane fusion. The presence of 1 mM divalent ions (Ca++, Mg++ or Mn++) on both sides or one side of the PC membrane did not affect appreciably its fusion characteristic with temperature, but the PS membrane fusion with temperature was greatly enhanced by the presence of divalent ions. The variation of pH of the environmental solution in the range of 5.5 approximately 7.0 did not affect the membrane fusion characteristic. However, at pH 8.5, the fusion with respect to temperature was shifted toward the lower temperature by approximately 3degreesC for PC and PS membranes, and at pH 3.0 the opposite situation was observed as the fusion temperature was increased by 6degreesC for PS membranes and by 4degreesC for PC membranes The results seem to indicate that membrane fluidity and structural instability in the bilayer are important for membrane fusion to occur.