pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion.

UNLABELLED: Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. IMPORTANCE: Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs.

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes.

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.

"Entropic traps" in the kinetics of phase separation in multicomponent membranes stabilize nanodomains.

We quantitatively describe the creation and evolution of phase-separated domains in a multicomponent lipid bilayer membrane. The early stages, termed the nucleation stage and the independent growth stage, are extremely rapid (characteristic times are submillisecond and millisecond, respectively) and the system consists of nanodomains of average radius approximately 5-50 nm. Next, mobility of domains becomes consequential; domain merger and fission become the dominant mechanisms of matter exchange, and line tension gamma is the main determinant of the domain size distribution at any point in time. For sufficiently small gamma, the decrease in the entropy term that results from domain merger is larger than the decrease in boundary energy, and only nanodomains are present. For large gamma, the decrease in boundary energy dominates the unfavorable entropy of merger, and merger leads to rapid enlargement of nanodomains to radii of micrometer scale. At intermediate line tensions and within finite times, nanodomains can remain dispersed and coexist with a new global phase. The theoretical critical value of line tension needed to rapidly form large rafts is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles.

A quantitative model for membrane fusion based on low-energy intermediates.

The energetics of a fusion pathway is considered, starting from the contact site where two apposed membranes each locally protrude (as "nipples") toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.

Voltage-induced nonconductive pre-pores and metastable single pores in unmodified planar lipid bilayer.

Electric fields promote pore formation in both biological and model membranes. We clamped unmodified planar bilayers at 150-550 mV to monitor transient single pores for a long period of time. We observed fast transitions between different conductance levels reflecting opening and closing of metastable lipid pores. Although mean lifetime of the pores was 3 +/- 0.8 ms (250 mV), some pores remained open for up to approximately 1 s. The mean amplitude of conductance fluctuations (approximately 500 pS) was independent of voltage and close for bilayers of different area (40,000 and 10 microm(2)), indicating the local nature of the conductive defects. The distribution of pore conductance was rather broad (dispersion of approximately 250 pS). Based on the conductance value and its dependence of the ion size, the radius of the average pore was estimated as approximately 1 nm. Short bursts of conductance spikes (opening and closing of pores) were often separated by periods of background conductance. Within the same burst the conductance between spikes was indistinguishable from the background. The mean time interval between spikes in the burst was much smaller than that between adjacent bursts. These data indicate that opening and closing of lipidic pores proceed through some electrically invisible (silent) pre-pores. Similar pre-pore defects and metastable conductive pores might be involved in remodeling of cell membranes in different biologically relevant processes.

Stalk dynamics and lipid flow upon membrane hemifusion.

A hydrodynamic theory describing the stalk dynamics and lipid flows upon BLM hemifusion was developed. The value of intermonolayer viscosity, etar, for membranes formed from azolectin mixed with lysophosphatidylcholine (7.10(-4) mg/ml) in n-decane, etar approximately 10(-9) g/s, was determined from comparison of the theoretical calculations and literature data. For membranes formed in squalene, the values etar approximately 10(-7) g/s for phosphatidylethanolamine and etar approximately 2-10(-7) g/s for azolectin were obtained. The calculated values are close to the published results of independent experiments which shows that the developed theory describes well the stalk growth and lipid flow.

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.

Lipid flow through fusion pores connecting membranes of different tensions.

When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores.

Theory of electrical creation of aqueous pathways across skin transport barriers.

Experimental studies have shown that application of electrical pulses to human skin that result in U(skin)>30 V for durations of about 1 ms or longer causes a large decrease in electrical resistance within microseconds, followed in seconds by an increase in molecular transport of water-soluble molecules. Local transport regions (LTRs), within which molecular transport is concentrated, mostly form away from the skin's appendages and rete pegs. Theoretical attempts to explain this behavior involve electrically created aqueous pathways ("pores"). For short (about 1 ms) "high voltage" (HV) pulses leading to about U(skin)>50 V, it was hypothesized that such pulses cause electroporation of the multilamellar lipid bilayer membranes of the skin's stratum corneum (SC). Much of the present experimental evidence supports the more specific hypothesis that such pulses create "straight through aqueous pathways", mostly within LTRs, that perforate the SC lipid bilayers and pass through the interiors of hydrated corneocytes. Theoretical estimates of the localized heating within LTRs predict relatively small temperature rises. The theory of LTR formation is incomplete, with both stochastic and deterministic models under consideration. Moderate voltage (MV) pulses leading to about 5

Theory of skin electroporation: implications of straight-through aqueous pathway segments that connect adjacent corneocytes.

Previous in vitro experiments have shown that transdermal high-voltage pulses (Uskin approximately 100 V; duration approximately 1 ms) create local transport regions (LTR) away from appendages in human skin. Quantitative interpretation of the associated ionic and molecular transport led to the view that a large number of aqueous pathways were created, and these connect the corneocytes within an LTR. Here we use the "brick wall" model of the stratum corneum, modified so that morphology important to understanding electrical behavior is emphasized. In this model a minimum-size LTR is regarded as an idealized stack of corneocytes in which the 5-6 multilamellar lipid bilayer membranes between adjacent corneocytes are electroporated. As in artificial planar bilayer and cell membrane electroporation, a distribution of pathway sizes is expected during pulsing, and during recovery after pulsing individual pathway segments are expected to shrink and close randomly, with a time constant tau(seg) that depends on temperature and on lipid composition. Numerical simulations based on stochastic closure of individual segments were used to predict the electrical conductance G(LTR)(t) of a minimum-size LTR after pulsing stops. These theoretical results show that simple exponential decay, G(LTR)(t) = G(LTR)(0)exp(-t/tau(seg)), occurs with minimal fluctuations if the number of pathways is large (np > 10(2)), but for much smaller values the conduction decreases erratically. A "stochastic bottleneck" leading to complete closure is reached only at about np < 3. Thus, for the same number of electrically created pathways, the stratum corneum will remain "open" longer if the pathways are located within an LTR than if the same number of pathways are distributed sparsely over the skin. These predictions are relevant to postpulse transport, including the trapping of linear macromolecules that can hold pathway segments open for prolonged intervals.

Skin appendageal macropores as a possible pathway for electrical current.

The electrical properties of the outermost layer of skin are described by lipid-corneocyte (Zm) and appendageal (Za) impedance, which are connected in parallel. Appendageal macropores are considered as long tubes with distributed electrical parameters. It has been shown that not only Za, but also the macropore resistance Ra and capacitance Ca are frequency dependent. The input of Za in the overall impedance (Z) depends on the space density of active (conductive) macropores n(i), which increase with current density (i) and the duration of iontophoresis. Skin impedance has been demonstrated to decrease under the influence of iontophoretic treatment. Application of the theoretical model to these data provides an estimate of the increase in macropore density during iontophoresis. A comparison of these results with n(i), which was measured directly, shows a strong correlation supporting this unique model.

Electrical properties of skin at moderate voltages: contribution of appendageal macropores.

The electrical properties of human skin in the range of the applied voltages between 0.2 and 60 V are modeled theoretically and measured experimentally. Two parallel electric current pathways are considered: one crossing lipid-corneocyte matrix and the other going through skin appendages. The appendageal ducts are modeled as long tubes with distributed electrical parameters. For both transport systems, equations taking into account the electroporation of lipid lamella in the case the lipid-corneocyte matrix or the walls of the appendageal ducts in the case of the skin appendages are derived. Numerical solutions of these nonlinear equations are compared with published data and the results of our own experiments. The current-time response of the skin during the application of rectangular pulses of different voltage amplitudes show a profound similarity with the same characteristics in model and plasma membrane electroporation. A comparison of the theory and the experiment shows that a significant (up to three orders of magnitude) drop of skin resistance due to electrotreatment can be explained by electroporation of different substructures of stratum corneum. At relatively low voltages (U < 30 V) this drop of skin resistance can be attributed to electroporation of the appendageal ducts. At higher voltages (U > 30 V), electroporation of the lipid-corneocyte matrix leads to an additional drop of skin resistance. These theoretical findings are in a good agreement with the experimental results and literature data.

A mechanism of skin appendage macropores electroactivation during iontophoresis.

A physical mechanism of activation of skin appendage macropores under the influence of an electric field is considered theoretically. The macropore is considered as a long cylinder tube which is closed and flattened out before the electric field is applied. The charging of the capacitance of the macropore walls is the driving force of electroactivation. During this process the free energy of the system decreases, which is energetically favourable and results in water pulled into the tube and to a gradual opening of the tube. It is shown that consideration of the macropore wall conductance leads to a considerable slowdown of electroactivation. The opening time of a separate macropore is estimated. It is equal to 30 min for a macropore 4 mm in length. The dependence of the surface density of activated macropores on time is calculated theoretically. The obtained theoretical results are in a good agreement with the literature data.

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.

Measurement of rapid release kinetics for drug delivery.

A fluorescence measurement system and methods of data analysis were developed to measure rapid kinetics of transdermal transport in vitro. Three variations on the technique were demonstrated, where the receptor compartment concentration was determined by: 1) fluorescence measurements of aliquots removed at discrete time points, 2) continuous fluorescence measurements made directly in the receptor compartment using a custom-made fluorimeter cuvette as a permeation chamber, and 3) continuous fluorescence measurements made in a flow-through cuvette containing receptor solution continuously pumped from a flow-through permeation chamber. In each case, the measured signal was a convolution of the time-dependent molecular flux (the desired information) and the characteristic response of the measurement system. Algorithms for deconvolution of the signal were derived theoretically. For the most complicated case, (3), the experimental confirmation is shown here, proving a time resolution on the order of half a minute.

Mechanism of electroinduced ionic species transport through a multilamellar lipid system.

A theoretical model for electroporation of multilamellar lipid system due to a series of large electrical pulses is presented and then used to predict the functional dependence of the transport of charged molecules. Previously, electroporation has been considered only for single bilayer systems such as artificial planar bilayer membranes and cell membranes. The former have been extensively studied with respect to electrical and mechanical behavior, and the latter with respect to molecular transport. Recent experimental results for both molecular transport and electrical resistance changes in the stratum corneum (SC) suggest that electroporation also occurs in the multilamellar lipid membranes of the SC. In addition, there is the possibility that other skin structures (the "appendages") also experience electroporation. A compartment model is introduced to describe the transport of charged species across the SC, and the predicted dependence is compared with available data. In this model, the SC is assumed to contain many hydrophilic compartments in series separated by boundary bilayers, so that these compartments become connected only upon electroporation. Two limiting cases for the transport of charged molecules are considered: (1) transport along tortuous inter-bilayer pathways in each compartment, followed by transport across individual boundary bilayers due to electroporation, and (2) transport along straight-through pathways in the boundary bilayers with fast mixing in each compartment, which includes the interior space of corneocytes. Both models were fitted to the experimental data. The large electropore radius (rt approximately 200 A) and porated fractional area (ft approximately 10(-3) obtained from the fitting for the tortuous model relative to the more reasonable values obtained for the straight-through model (rs approximately 4 A, fs approximately 10(-6) suggest that the latter is a more realistic description of electroinduced transport of ionized species through the skin.

Electromagnetic fields and cells.

There is strong public interest in the possibility of health effects associated with exposure to extremely low frequency (elf) electromagnetic (EM) fields. Epidemiological studies suggest a probable, but controversial, link between exposure to elf EM fields and increased incidence of some cancers in both children and adults. There are hundreds of scientific studies that have tested the effects of elf EM fields on cells and whole animals. A growing number of reports show that exposure to elf EM fields can produce a large array of effects on cells. Of interest is an increase in specific transcripts in cultured cells exposed to EM fields. The interaction mechanism with cells, however, remains elusive. Evidence is presented for a model based on cell surface interactions with EM fields.

Stalk mechanism of vesicle fusion. Intermixing of aqueous contents.

A 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, Kos. The pore edge energy decreases with an increase of the positive spontaneous curvature of the inner membrane monolayer, Kis. 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: Kos less than O, Kis greater than O. Experimental data concerning the fusion process are analysed.