Asymmetric Giant Unilamellar Vesicles Research Articles

Using asymmetric giant unilamellar vesicles to investigate the influence of low-melting phospholipid chain length on interleaflet coupling of phase behavior

Using asymmetric giant unilamellar vesicles to investigate the influence of low-melting phospholipid chain length on interleaflet coupling of phase behavior

Experimentally determined leaflet-leaflet phase diagram of an asymmetric lipid bilayer.

We have determined the partial leaflet-leaflet phase diagram of an asymmetric lipid bilayer at ambient temperature using asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs with varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were hemifused to a supported lipid bilayer (SLB) composed of DOPC, resulting in lipid exchange between their outer leaflets. The GUVs and SLB contained a red and green lipid fluorophore, respectively, thus enabling the use of confocal fluorescence imaging to determine both the extent of lipid exchange (quantified for individual vesicles by the loss of red intensity and gain of green intensity) and the presence or absence of phase separation in aGUVs. Consistent with previous reports, we found that hemifusion results in large variation in outer leaflet exchange for individual GUVs, which allowed us to interrogate the phase behavior at multiple points within the asymmetric composition space of the binary mixture. When initially symmetric GUVs showed coexisting gel and fluid domains, aGUVs with less than ~50% outer leaflet exchange were also phase-separated. In contrast, aGUVs with greater than 50% outer leaflet exchange were uniform and fluid. In some cases, we also observed three coexisting bilayer-spanning phases: two registered phases and an anti-registered phase. These results suggest that a relatively large unfavorable midplane interaction between ordered and disordered phases in opposing leaflets (i.e., a midplane surface tension) can overwhelm the driving force for lateral phase separation within one of the leaflets, resulting in an asymmetric bilayer with two uniformly mixed leaflets that is poised to phase-separate upon leaflet scrambling.

Characterization of phosphoinositide domains in asymmetric giant unilamellar vesicles.

Phosphoinositides (PIPs) have been shown to mediate a broad range of cellular events by attracting proteins to specific cellular sites at distinct time points. This spatiotemporal control of protein function is rooted in the rich chemical functionality of the phosphoinositide headgroup, which not only gives rise to selective interactions with proteins but also results in unique physicochemical properties. Several studies have been directed at detailing PIP properties in model symmetric lipid bilayers, however, biological membranes exhibit compositional asymmetry between bilayer leaflets. It is unclear to what extent PIP properties are altered in such an environment. In the last decade, methods have been introduced that enable the fabrication of asymmetric lipid vesicles. We have extended the scope of one of these methods – Ca2+ initiated hemifusion of a symmetric giant unilamellar vesicles (sGUV) with solid supported lipid bilayers (SLB) – to enable the fabrication of asymmetric giant unilamellar vesicles (aGUVs) that exhibit phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the outer leaflet. We found that using Ca2+ as the initiator for sGUV/SLB hemifusion leads to a large disparity of PI(4,5)P2 content in the formed aGUVs. We attribute this disparity to the formation of macroscopic Ca2+/PI(4,5)P2 domains, which leads to sGUVs interacting with SLB regions rich or poor in PI(4,5)P2. In contrast, when Mg2+ is used as initiator, no macroscopic domains form in the SLB and hence, the aGUVs show significantly higher compositional uniformity. We find that Mg2+ concentrations as low as 1 mM are sufficient to initiate the hemifusion process. Using this method to obtain aGUVs, we investigate phosphoinositide domain properties for varying lipid compositions in the leaflet opposite the PIP containing leaflet (raft vs. non-raft lipid mixtures) and we explore the impact of bivalent cations on the lateral morphology of the respective aGUVs.

Phosphatidylinositol-4-phosphate domain properties in asymmetric giant unilammelar vesicles.

Phosphatidylinositol-4-phosphate (PI(4)P) is a precursor for higher phosphorylated phosphoinositide species and mediates directly many cellular processes at the plasma membrane and in the Golgi. Cellular membranes are vertically asymmetric and, with respect to lipid distribution, laterally heterogenous. It has been shown that many physiological functions affected by PI(4)P are associated with clustering of the lipid. Studies using symmetric lipid vesicle model systems have shown that PI(4)P forms domains in the presence of physiological levels of Ca2+ and Mg2+. However, at present it is unclear to what extent PI(4)P domain behavior translates from symmetric to asymmetric lipid bilayer systems. Asymmetric giant unilamellar vesicles (aGUVs) are model systems that allow for the visualization of lipid interactions within a phospholipid bilayer using confocal fluorescence microscopy. Novel techniques have only recently enabled the fabrication of aGUVs. We are using the hemifusion method to obtain aGUVs with PI(4)P containing lipid mixtures in one leaflet and varying lipid mixtures in the opposing leaflet. Our experiments are designed to determine the conditions under which PI(4)P domain formation is favored and we are investigating the registration of PIP domains with raft domains in the opposite leaflet.

Utilizing asymmetric giant unilamellar vesicles to determine how low-melting phospholipids affect interleaflet coupling strength.

Model membranes that mimic cell membrane asymmetry can be used to study coupling between lateral and transverse lipid organization. Theory predicts that, in a leaflet composed of lipids that would otherwise phase separate in a symmetric bilayer, domain formation can be suppressed by strong coupling to a uniformly mixed leaflet. Conversely, when coupled to a leaflet with a tendency to demix, an otherwise miscible leaflet can be induced to demix. Each of these outcomes has been observed in model asymmetric membranes of different composition, suggesting that cells may be able to control domain formation through changes in lipid content and/or asymmetric lipid distribution. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were investigated using confocal fluorescence microscopy; the mixtures containing 14:1-PC, 16:1-PC, and 18:1-PC showed macroscopic phase separation at room temperature, while the mixtures containing 20:1-PC and 22:1-PC did not. Cryogenic electron microscopy, small-angle neutron scattering, and Förster resonance energy transfer confirmed the presence of domains in the first three systems and revealed nanoscopic heterogeneity in the mixture containing 20:1-PC, while the 22:1-PC mixture was uniform. aGUVs were then prepared by calcium-induced hemifusion of the GUVs to a supported lipid bilayer composed of the corresponding low-TM lipid and Chol (80/20 mol%). The aGUVs showed a range of outer leaflet exchange, which revealed a correlation between phase behavior and extent of asymmetry. Domains in the 16:1-PC and 18:1-PC systems were suppressed in aGUVs with more than 50 and 60% asymmetry, respectively, indicating a relatively large free energy cost for antiregistered domains.

Phosphatidylinositol phase behavior in symmetric and asymmetric model membranes.

Phosphatidylinositol (PI) is a precursor to phosphoinositides (PIPs) which are important cellular signaling molecules. While the phase behavior of PI in simple model membranes has been investigated, little is known about its phase behavior in the presence of PIPs. Furthermore, the effect of divalent cations on PI/PIP interaction has not been detailed. Determining PI/PIP phase behavior is of physiological relevance because it potentially affects the lateral distribution of PIPs in cellular membranes and hence the spatiotemporal control of protein function. For diplamitoylphosphatidylinositol (DPPI) we find in pH 7.2 buffer a phase transition temperature of 43.6°C, while in the presence of Ca2+ and Mg2+ the phase transition temperature shifts to 55.8°C and 51.6°C, respectively. In addition, we investigate divalent cation dependent PI/PI(4,5)P2 phase behavior. To study PI phase behavior in asymmetric lipid bilayers, we fabricate asymmetric giant unilamellar vesicles (aGUVs) using the hemifusion method. We describe the conditions to obtain aGUVS with PI in the outer leaflet and characterize their domain morphology.

Investigating the influence of low-melting phospholipids on interleaflet coupling in asymmetric giant unilamellar vesicles

Compositional asymmetry is a characteristic of the plasma membrane whose biological importance is still mostly unknown. Asymmetric model membranes mimicking cell membrane composition offer a useful, highly controlled system for studying the coupling between lateral and transverse lipid organization. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were prepared by electroformation.

Phosphoinositide domain properties in asymmetric lipid vesicles

In cell membranes, the distribution of lipids is laterally heterogeneous and vertically asymmetric. Only within the last decade have model systems been developed which enable the fabrication of asymmetric lipid bilayers. Asymmetric Giant Unilamellar Vesicles (aGUVs) allow for the visualization and quantification of lipid interactions in a phospholipid bilayer using microscopy-based techniques. Many phosphoinositide mediated signaling events have been associated with liquid-ordered (lo) domains of sphingomyelin and cholesterol (rafts).

An integrated microfluidic platform to fabricate single-micrometer asymmetric giant unilamellar vesicles (GUVs) using dielectrophoretic separation of microemulsions.

We present a microfluidic technique that generates asymmetric giant unilamellar vesicles (GUVs) in the size range of 2-14 μm. In our method, we (i) create water-in-oil emulsions as the precursors to build synthetic vesicles, (ii) deflect the emulsions across two oil streams containing different phospholipids at high throughput to establish an asymmetric architecture in the lipid bilayer membranes, and (iii) direct the water-in-oil emulsions across the oil-water interface of an oscillating oil jet in a co-flowing confined geometry to encapsulate the inner aqueous phase inside a lipid bilayer and complete the fabrication of GUVs. In the first step, we utilize a flow-focusing geometry with precisely controlled pneumatic pressures to form monodisperse water-in-oil emulsions. We observed different regimes in forming water-in-oil multiphase flows by changing the applied pressures and discovered a hysteretic behavior in jet breakup and droplet generation. In the second step of GUV fabrication, an oil stream containing phospholipids carries the emulsions into a separation region where we steer the emulsions across two parallel oil streams using active dielectrophoretic and pinched-flow fractionation separations. We explore the effect of applied DC voltage magnitude and carrier oil stream flow rate on the separation efficiency. We develop an image processing code that measures the degree of mixing between the two oil streams as the water-in-oil emulsions travel across them under dielectrophoretic steering to find the ideal operational conditions. Finally, we utilize an oscillating co-flowing jet to complete the formation of asymmetric giant unilamellar vesicles and transfer them to an aqueous phase. We investigate the effect of flow rates on properties of the co-flowing jet oscillating in the whipping mode (i.e., wavelength and amplitude) and define the phase diagram for the oil-in-water jet. Assays used to probe the lipid bilayer membrane of fabricated GUVs showed that membranes were unilamellar, minimal residual oil remained trapped between the two lipid leaflets, and 83% asymmetry was achieved across the lipid bilayers of GUVs.

Investigation of the domain line tension in asymmetric vesicles prepared via hemifusion

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase. To better understand the interleaflet interactions that influence domains, we compared asymmetric model membranes that capture salient properties of the PM with simpler symmetric membranes. Using asymmetric giant unilamellar vesicles (aGUVs) prepared by hemifusion with a supported lipid bilayer, we investigate the domain line tension that characterizes the behavior of coexisting ordered + disordered domains. The line tension can be related to the contact perimeter of the different phases. Compared to macroscopic phase separation, the appearance of modulated phases was found to be a robust indicator of a decrease in domain line tension. Symmetric GUVs of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/cholesterol (chol) were formed into aGUVs by replacing the GUV outer leaflet with DOPC/chol = 0.8/0.2 in order to create a cytoplasmic leaflet model. These aGUVs revealed lower line tension for the ordered + disordered domains of the exoplasmic model leaflet.

Asymmetric Phase Diagram of a Three Component Model Membrane Mixture Containing Sphingomyelin: Coupling of Lateral and Transverse Lipid Organization

Lipid asymmetry is a fundamental aspect of plasma membrane structure. Despite the importance of asymmetry, how the molecular features of individual lipids influence coupling between the leaflets is largely unexplored. We investigated the composition dependent phase behavior of ternary lipid system composed of egg sphingomyelin, DOPC, and cholesterol. Symmetric giant unilamellar vesicles (GUVs) were prepared by electroformation. Asymmetric GUVs were prepared by calcium induced hemifusion of the symmetric GUVs with a supported lipid bilayer composed of DOPC and cholesterol.

Coupling of Leaflet Phase Behavior in Asymmetric Giant Unilamellar Vesicles Prepared by Hemifusion: Investigating the Influence of the Low-Metling Lipid

Coupling of Leaflet Phase Behavior in Asymmetric Giant Unilamellar Vesicles Prepared by Hemifusion: Investigating the Influence of the Low-Metling Lipid

Inter- and Intra-Plane Interactions Control the Existence of Macrodomains in Asymmetric Giant Unilamellar Vesicles

The complexity of the plasma membrane (PM) is in part related to the complexity of its composition. Beyond the hundreds of lipid components in the PM, compositional differences between the exoplasmic and the cytoplasmic leaflets leads to different physical-chemical properties in each leaflet affecting the interaction between them. Model membranes prepared in vitro that reproduce salient properties of the PM can reveal molecular determinants of these interactions. We investigate asymmetric giant unilamellar vesicles (aGUVs) prepared with different lipid compositions by hemifusion. Symmetric GUVs composed of the lipid mixture high-melting lipid (HTm)/DOPC/chol (0.39/0.39/0.22), where HTm saturated lipids ranging from 15-18 carbons, were hemifused with a supported lipid bilayer (SLB) composed of DOPC/chol (0.8/0.2). Fusion of the GUV and SLB outer leaflets promoted a range of lipid exchange, creating asymmetric vesicles. The symmetric GUVs composed of the ternary mixtures exhibited coexistence of liquid-disordered (Ld) and liquid-ordered (Lo) phases. The change in the chain lengths implies different intra-plane interactions in which the domain line tension or the difference in order between the Ld and the Lo phases decreases with the saturated chain length. We found that replacement of the GUV outer leaflet by a composition that forms Ld phase can prevent the formation of macrodomains in a manner that depends on the chain length of the saturated lipid and the percentage of lipid exchange. Interestingly, longer saturated chain length promoted the persistence of macroscopic domains to greater degrees of asymmetry. The domain size transition accompanying changes in the degree of asymmetry can be explained by intra- and inter-leaflet interactions. Our results suggest that cells can control the presence or absence of lipid rafts by controlling the types of lipids present in the two leaflets.

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria.

The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flip-flop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.

Characterization of Asymmetric Phosphoinositide/Lipid Vesicles

Biological membranes are laterally heterogenous and vertically asymmetric. While lateral heterogeneities (domains) have been widely investigated, only recently methodologies have emerged that allow for fabrication of vertically asymmetric lipid vesicles. Several phosphoinositide (PIP) mediated cellular events are sensitive to cholesterol depletion, and it has been speculated that PIPs accumulate in lipid rafts. However, the stearoyl/arachidonoyl acyl chain composition of PIPs is generally unfavorable for accumulation in a liquid-ordered (lo) environment, and in symmetric lipid vesicles, PIPs do not enrich in lo phases. It has been shown, however, that cholesterol promotes phosphoinositide domain formation in ternary PC/PIP/cholesterol lipid mixtures, presumably through an interaction between the PIP headgroup and the cholesterol hydroxyl group. To investigate the potential coupling of outer leaflet raft domains and inner leaflet PIP/cholesterol domains, we have fabricated asymmetric giant unilamellar vesicles and characterized them using fluorescence microscopy. We will present data for different lipid compositions and ionic conditions (e.g., +/- calcium).

A Chlamydia pneumoniae adhesin induces phosphatidylserine exposure on host cells

In mammalian cells, the internal and external leaflets of the plasma membrane (PM) possess different phospholipids. Phosphatidylserine (PS) is normally confined to the inner (cytoplasmic) membrane leaflet. Here we report that the adhesin CPn0473 of the human pathogenic bacterium Chlamydia pneumoniae (Cpn) binds to the PM of human cells and induces PS externalization but unexpectedly not apoptosis. PS externalization is increased in human cells exposed to infectious Cpn cells expressing increased CPn0473 and reduced in exposure to Cpn expressing decreased CPn0473. CPn0473 binds specifically to synthetic membranes carrying PS and stimulates pore formation. Asymmetric giant unilamellar vesicles (GUVs) in which PS is restricted to the inner leaflet reveal that CPn0473 induces PS externalization in the absence of other proteins. Thus our identification of CPn0473 as a bacterial PS translocator capable of specific and apoptosis-independent PS externalization during infection extends the spectrum of mechanisms intracellular pathogens use to enter host cells.

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition.

The double-membrane cellular envelope of Gram-negative bacteria enables them to endure harsh environments and represents a barrier to many clinically available antibiotics. The outer membrane (OM) is exposed to the environment and is the first point of contact involved in bacterial processes such as signaling, pathogenesis, and motility. As in the cytoplasmic membrane, the OM in Gram-negative bacteria has a phospholipid-rich inner leaflet and an outer leaflet that is predominantly composed of lipopolysaccharide (LPS). We report on a microfluidic technique for fabricating monodisperse asymmetric giant unilamellar vesicles (GUVs) possessing the Gram-negative bacterial OM lipid composition. Our continuous microfluidic fabrication technique generates 50-150 μm diameter water-in-oil-in-water double emulsions at high-throughput. The water-oil and oil-water interfaces facilitate the self-assembly of phospholipid and LPS molecules to create the inner and outer leaflets of the lipid bilayer, respectively. The double emulsions have ultrathin oil shells, which minimizes the amount of residual organic solvent that remains trapped between the leaflets of the GUV membrane. An extraction process by ethanol and micropipette aspiration of the ultrathin oil shells triggers an adhesive interaction between the two lipid monolayers assembled on the water-oil and oil-water interfaces (i.e., dewetting transition), forcing them to contact and form a lipid bilayer membrane. The effect of different inner-leaflet lipid compositions on the emulsion/vesicle stability and the dewetting transition is investigated. We also report on the values for bending and area expansion moduli of synthetic asymmetric model membranes with lipid composition/architecture that is physiologically relevant to the OM in Pseudomonas aeruginosa bacteria.

The Interaction of Proteins with Asymmetric Lipid Bilayers

Cell membranes can be characterized by their thermodynamic, structural and mechanical properties, such as phase behavior, thickness and rigidity. Experimental and computational studies of membrane properties and their effects on the function of proteins embedded in the lipid environment have focused largely on symmetric bilayers, i.e., with the same lipid composition in the two leaflets. However, the mammalian plasma membrane (PM) has an asymmetric lipid distribution, with an outer leaflet having a significant fraction of high-melting lipids, and an inner leaflet containing low-melting and negatively charged lipids. This asymmetry is often ignored in both experimental and modeling studies especially given the difficulty of preparing asymmetric liposomes in vitro. However, the different elastic and phase properties of symmetric bilayer models of the two PM leaflets, as well as the energy that cells spend to maintain their PM transverse structure, make it likely that this compositional asymmetry provides unique bilayer properties of functional significance. Our goal is to evaluate this hypothesis and its consequences both in vitro and in silico, with a systematic study of symmetric and asymmetric bilayers composed of phosphatidylcholine, sphingomyelin, phosphatidylserine and/or phosphatidylethanolamine lipid molecules both in the presence and absence of the transmembrane peptides WALP and Gramicidin. We examine the structural manifestations and mechanisms of interleaflet coupling and their effect on protein-membrane interactions, focusing on changes in bending rigidity, acyl chain order, and surface tension. We then evaluate the implications of these properties for the unique deformation of the asymmetric bilayers around the protein inclusions. Results from computation regarding inter-leaflet coupling are compared to in vitro measurements of the thermodynamics properties of asymmetric giant unilamellar vesicles prepared with a newly developed cyclodextrin-mediated exchange protocol.

Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics.

In this article we detail a robust high-throughput microfluidic platform capable of fabricating either symmetric or asymmetric giant unilamellar vesicles (GUVs) and characterise the mechanical properties of their membranes.