Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.     

Coupling of cholesterol-rich lipid phases in asymmetric bilayers

We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typically found in the outer leaflet of plasma membranes induce liquid-ordered domains in adjacent regions of asymmetric lipid bilayers with apposed leaflets composed of typical inner leaflet lipid mixtures [Kiessling, V., Crane, J. M., and Tamm, L. K. (2006) Biophys. J. 91, 3313-26]. To further examine the nature of transbilayer couplings in asymmetric cholesterol-rich lipid bilayers, the effects on the lipid phase behavior in asymmetric bilayers of different lipid compositions were investigated. We established systems containing several combinations of natural extracted and synthetic lipids that exhibited coexisting liquid-ordered (lo) and liquid-disordered (ld) domains in a supported bilayer format. We find that lo phase domains are induced in all quaternary inner leaflet combinations composed of PCs, PEs, PSs, and cholesterol. Ternary mixtures of PCs/PEs/Chol, PCs/PSs/Chol also exhibit lo phases adjacent to outer leaflet lo phases. However, with the exception of brain PC extracts, binary PC/Chol mixtures are not induced to form lo phases by adjacent outer leaflet lo phases. Higher melting lipid ad-mixtures of PEs and PSs are needed for lo phase induction in the inner leaflet. It appears that the phase behavior of the inner leaflet mixtures is dominated by the intrinsic chain melting temperatures of the lipid components, rather than by their specific headgroup classes. In addition, similar studies with synthetic, completely saturated lipids and cholesterol show that lipid oxidation is not a factor in the observed phase behavior.     

Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.     

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered raft domains

Insulin receptor (IR) is a membrane tyrosine kinase that mediates the response of cells to insulin. IR activity has been shown to be modulated by changes in plasma membrane lipid composition, but the properties and structural determinants of lipids mediating IR activity are poorly understood. Here, using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of IR in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length, which increases both bilayer width and the propensity to form ordered domains. Incorporating purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths also increased IR activity, but more modestly than by increasing lipid acyl chain length in cells. These results suggest that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. These results are consistent with a model in which a combination of bilayer width and ordered domain formation modulates IR activity via IR conformation and accessibility to phosphatases.     

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.     

Differential stiffness and lipid mobility in the leaflets of purple membranes

Purple membranes (PM) are two-dimensional crystals formed by bacteriorhodopsin and a variety of lipids. The lipid composition and density in the cytoplasmic (CP) leaflet differ from those of the extracellular (EC) leaflet. A new way of differentiating the two sides of such asymmetric membranes using the phase signal in alternate contact atomic force microscopy is presented. This method does not require molecular resolution and is applied to study the stiffness and intertrimer lipid mobility in both leaflets of the PM independently over a broad range of pH and salt concentrations. PM stiffens with increasing salt concentration according to two different regimes. At low salt concentration, the membrane Young's normal modulus grows quickly but differentially for the EC and CP leaflets. At higher salt concentration, both leaflets behave similarly and their stiffness converges toward the native environment value. Changes in pH do not affect PM stiffness; however, the crystal assembly is less pronounced at pH > or = 10. Lipid mobility is high in the CP leaflet, especially at low salt concentration, but negligible in the EC leaflet regardless of pH or salt concentration. An independent lipid mobility study by solid-state NMR confirms and quantifies the atomic force microscopy qualitative observations.     

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation

In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ～37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.     

Ordered Raft Domains Induced by Outer Leaflet Sphingomyelin in Cholesterol-Rich Asymmetric Vesicles

Sphingolipid- and cholesterol-rich liquid-ordered (Lo) lipid domains (rafts) are thought to be important organizing elements in eukaryotic plasma membranes. How they form in the sphingolipid-poor cytosolic (inner) membrane leaflet is unclear. Here, we characterize how outer-leaflet Lo domains induce inner-leaflet-ordered domains, i.e., interleaflet coupling. Asymmetric vesicles studied contained physiologically relevant cholesterol levels (∼37 mol %), a mixture of SM (sphingomyelin) and DOPC (dioleoylphosphatidylcholine) in their outer leaflets, and DOPC in their inner leaflets. Lo domains were observed in both leaflets, and were in register, indicative of coupling between SM-rich outer-leaflet-ordered domains and inner-leaflet-ordered domains. For asymmetric vesicles with outer-leaflet egg SM or milk SM, a fluorescent lipid with unsaturated acyl chains (NBD-DOPE) was depleted in both the outer- and inner-leaflet-ordered domains. This suggests the inner-leaflet-ordered domains were depleted in unsaturated lipid (i.e., DOPC) and thus rich in cholesterol. For asymmetric vesicles containing egg SM, outer-leaflet Lo domains were also depleted in a saturated fluorescent lipid (NBD-DPPE), while inner-leaflet Lo domains were not. This indicates that inner- and outer-leaflet Lo domains can have significantly different physical properties. In contrast, in asymmetric vesicles containing outer-leaflet milk SM, which has long acyl chains capable of interdigitating into the inner leaflet, both outer- and inner-leaflet Lo domains were depleted, to a similar extent, in NBD-DPPE. This is indicative of interdigitation-enhanced coupling resulting in inner- and outer-leaflet Lo domains with similar physical properties.     

Molecular dynamics simulations of SOPS and sphingomyelin bilayers containing cholesterol

We performed a molecular dynamics simulation of an asymmetric bilayer that contained different lipid mixtures in its outer and inner leaflets. The outer leaflet contained a mixture of sphingomyelin (SM) with cholesterol and the inner leaflet a mixture of stearoyl-oleoyl-phosphatidylserine (SOPS) with cholesterol. For comparison purposes, we also performed two simulations on symmetric bilayers: the first simulation was performed on a bilayer containing a binary mixture of SOPS with cholesterol; the second contained a mixture of SM with cholesterol. We studied the hydrogen-bonding network of the bilayers in our simulations and the difference in the network properties in the monolayers either with SM or SOPS. We observed that in the asymmetric bilayer the properties of monolayers were the same as in the corresponding monolayers in the symmetric bilayers.     

Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles

Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.     

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.     

Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.     

Molecular studies of the gel to liquid-crystalline phase transition for fully hydrated DPPC and DPPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of saturated lipid bilayers, DPPC and DPPE, near the main phase transition. Though the chemical structure of DPPC and DPPE are largely similar (they only differ in the choline and ethanolamine groups), their transformation process from a gel to a liquid-crystalline state is contrasting. For DPPC, three distinct structures can be identified relative to the melting temperature (Tm): below Tm with "mixed" domains consisting of lipids that are tilted with partial overlap of the lipid tails between leaflet; near Tm with a slight increase in the average area per lipid, resulting in a rearrangement of the lipid tails and an increase in the bilayer thickness; and above Tm with unhindered lipid tails in random motion resulting in an increase in %gauche formed and increase in the level of interdigitation between lipid leaflets. For DPPE, the structures identified were below Tm with "ordered" domains consisting of slightly tilted lipid tails and non-overlapping lipid tails between leaflets, near Tm with minimal rearrangement of the lipids as the bilayer thickness reduces slightly with increasing temperature, and above Tm with unhindered lipid tails as that for DPPC. For DPPE, most of the lipid tails do not overlap as observed to DPPC, which is due to the tight packing of the DPPE molecules. The non-overlapping behavior of DPPE above Tm is confirmed from the density profile of the terminal carbon atoms in each leaflet, which shows a narrow distribution near the center of the bilayer core. This study also demonstrates that atomistic simulations are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the transition state.     

Lipid flip-flop vs. lateral diffusion in the relaxation of hemifusion diaphragms

Molecular dynamics simulations of a solvent-free coarse-grained lipid model are used to characterize the mechanisms by which lipid-bilayer hemifusion diaphragm (HD) intermediates relax, across a range of global compositions of negative intrinsic curvature (NIC) lipids and neutral-curvature lipids. At low concentrations of NIC lipids, rapid fission produces a double bilayer end state through a lateral diffusion-based mechanism enabled by spontaneous rim-pore defects. At moderately higher NIC lipid concentrations, rim pores are absent and stable leaflet three-junctions persist, revealing an HD relaxation mechanism entirely reliant on lipid flip-flop, and end states that are either stable fusion pores or stable HD's. These fusogenic systems exhibit dynamics highly dependent on NIC lipid concentration via an underlying sensitivity of flip-flop rates for neutral lipids on NIC lipid concentration. This work illustrates that HD dynamics may be altered through regulation of lipid composition in the immediate three-junction region. This work further highlights the potential role of flippases in biological fusion and the importance of lipid composition on fusion dynamics.     

Systematic measurements of interleaflet friction in supported bilayers

When lipid membranes curve or are subjected to strong shear forces, the two apposed leaflets of the bilayer slide past each other. The drag that one leaflet creates on the other is quantified by the coefficient of interleaflet friction, b. Existing measurements of this coefficient range over several orders of magnitude, so we used a recently developed microfluidic technique to measure it systematically in supported lipid membranes. Fluid shear stress was used to force the top leaflet of a supported membrane to slide over the stationary lower leaflet. Here, we show that this technique yields a reproducible measurement of the friction coefficient and is sensitive enough to detect differences in friction between membranes made from saturated and unsaturated lipids. Adding cholesterol to saturated and unsaturated membranes increased interleaflet friction significantly. We also discovered that fluid shear stress can reversibly induce gel phase in supported lipid bilayers that are close to the gel-transition temperature.