Utilizing adsorbed proteoliposomes trapped in a non-ruptured state on SiO2 for amplified detection of membrane proteins

The quartz crystal microbalance with dissipation (QCM-D) technique was used to monitor the formation of supported phospholipid bilayers (SPBs) on SiO2 using proteoliposomes with reconstituted proton translocating nicotinamide nucleotide transhydrogenase (TH). Exposure of the surface to such proteoliposomes creates a lipid film composed of a mixture of proteolipid bilayers and adsorbed non-ruptured proteoliposomes, where the fraction of the latter is reduced if the TH-liposomes are pretreated with trypsin to remove the water soluble domains of TH [Langmuir 19 (2003) 842]. In the present work, the latter study is complemented by investigating the influence of trypsin treatment of the mixed adlayer (proteolipid bilayer + non-ruptured proteoliposomes) after adsorption on the surface. This demonstrates how trypsin-cleavage induced rupture of adsorbed TH-liposomes can be utilized to detect the presence of less than 0.04 pmol/cm2 of immobilized TH.     

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.     

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.     

Quantitation of cholesterol incorporation into extruded lipid bilayers

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75 mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.     

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.     

A novel strategy for the preparation of liposomes: rapid solvent exchange

During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed.     

Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study.

The helical order parameter of the 26-residue amphiphilic bee venom peptide melittin was measured by polarized attenuated total reflection infrared spectroscopy (ATR-IR) in dry phospholipid multibilayers (MBLs) and when bound to single supported planar bilayers (SPBs) under D2O. Melittin adopted an alpha-helical conformation in MBLs of dipalmitoyl-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), a 4:1 mixture of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and when bound to SPBs of POPC:POPG (4:1). The order parameter of the alpha-helix in the bilayers depended mainly on the type of membrane preparation, and only little on the phospholipid composition of the bilayers. On hydrated SPBs, the helical order parameter was negative, indicating that the alpha-helix long axis of melittin was preferentially oriented parallel to the plane of the supported membrane. However, in dry MBLs, the helical order parameter was positive, indicating that the alpha-helix of melittin was preferentially oriented parallel to the phospholipid fatty acyl chains. It is concluded that the orientation of melittin in membranes depends on the degree of hydration of the model membranes rather than on the technique which is used for its determination. ATR-IR spectroscopy of polypeptides in or associated with supported planar membranes in D2O may become a useful tool for the determination of their orientation in and on membranes.     

Electro-osmosis at the surface of phospholipid bilayer membranes

The electro-osmotic velocity is the velocity of a fluid near an interface produced by an electric field parallel to a surface. The velocity adjacent to fixed phospholipid bilayer membranes was measured by observing the velocity of small vesicles suspended in the fluid. The charge densities of the bilayers ranged from 0 to 1 electronic charge per lipid and experiments were performed at temperatures above and below the transition temperature of the phospholipid bilayer in 1, 10 and 100 mM NaCl solutions. The Helmholtz-Smoluchowski equation correctly predicted the electro-osmotic velocity from the known value of zeta potential of the phospholipid bilayer.     

Partitioning of 1-pyrenesulfonate into zwitterionic and mixed zwitterionic/anionic fluid phospholipid bilayers

Molecular partitioning into biomembranes is of fundamental importance in diverse biochemical processes and reactions. The majority of aqueous/membrane partition data using model membrane systems, is obtained with pure phosphatidylcholine bilayers, being lipid mixtures less used, while studies involving bilayers containing zwitterionic/anionic mixtures of phospholipids are even more scarce. In this study, the solvatochromic effects of 1-pyrenesulfonate observed at 375 nm in aqueous liposome suspensions, and monitored by second derivative absorption spectrophotometry, enabled the determination of its partition constants into defined phospholipid bilayers. We compare, under cautiously settled experimental conditions, the partition of the anionic amphiphile PSA into fluid zwitterionic bilayers of POPC (Kp=6.7 x 10(3), at 25 degrees C), and into fluid mixed zwitterionic/anionic bilayers containing small proportions of anionic phospholipids. At the same temperature, we found increasing K(p) values in parallel with the proportion of POPS mixed with POPC (Kp=3.4 x 10(4) and Kp=7.3 x 10(4), with 5 and 10 mol% of POPS, respectively). Our interpretation is based on the interfacial properties of fluid and flexible mixed zwitterionic/anionic phospholipid bilayers.     

A charge-modified general amber force field for phospholipids: improved structural properties in the tensionless ensemble

Accurately predicting the structural properties of phospholipid with a fully atomistic molecular model is critical for the study of pure phospholipid bilayers, mixed bilayer systems and bilayers containing proteins. The general amber force field (GAFF) has traditionally required the presence of a surface tension parameter to correctly model phospholipid bilayer properties such as area per lipid and order parameters. In this work, the GAFF partial charges for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphochiline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were re-parameterised utilising high-level ab initio calculations and the restrained electrostatic potential method. Simulations of pure POPA, POPC and POPG bilayers using the charge-modified GAFF and no applied surface tension are compared with available experimental data, the original GAFF model and the recent Lipid14 variant. The results indicate a significant improvement in the accuracy of the lipid model for reproducing experimental observables without the need for a surface tension parameter. The successful application of modifying the lipid charge distributions represents an alternative to the use of a surface tension parameter within GAFF, and highlights the importance of the partial charge calculations when modelling lipid bilayers.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip

Painted bilayers containing reconstituted ion channels serve as a well defined model system for electrophysiological investigations of channel structure and function. Horizontally oriented bilayers with easy solution access to both sides were obtained by painting a phospholipid:decane mixture across a cylindrical pore etched into a 200-microm thick silicon wafer. Silanization of the SiO(2) layer produced a hydrophobic surface that promoted the adhesion of the lipid mixture. Standard lithographic techniques and anisotropic deep-reactive ion etching were used to create pores with diameters from 50 to 200 microm. The cylindrical structure of the pore in the partition and the surface treatment resulted in stable bilayers. These were used to reconstitute Maxi K channels in the 100- and 200-microm diameter pores. The electrophysiological characteristics of bilayers suspended in microchips were comparable with that of other bilayer preparations. The horizontal orientation and good voltage clamping properties make the microchip bilayer method an excellent system to study the electrical properties of reconstituted membrane proteins simultaneously with optical probes.     

The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition

The influence of agents, known to affect the membrane dipole potential, phloretin and RH 421, on the multi channel activity of amphotericin B in lipid bilayers of various compositions, was studied. It was shown that the effects were dependent on the membrane’s phospholipid and sphingolipid type. Phloretin enhanced amphotericin B induced steady-state transmembrane current through bilayers made from binary mixtures of POPC (DOPC) and ergosterol and ternary mixture of DPhPC, ergosterol and stearoylphytosphingosine. RH 421 increased steady-state polyene induced transmembrane current through membranes made from binary mixtures of DPhPC (DPhPS) and ergosterol and ternary mixture of DPhPS, ergosterol and stearoylphytosphingosine. It was proposed that the observed effects reflect the fine balance of the interactions between the various components present: amphotericin B, ergosterol, phospholipid, sphingolipid and dipole modifier. The shape of lipid molecules seems to be an important factor impacting the responses of amphotericin B modified bilayers to dipole modifiers. The influence of different phospholipids and sphingolipids on the physical and structural properties of ordered lipid microdomains, enriched in AmB, was also discussed. It was also shown that RH 421 enhanced the antifungal activity of amphotericin B in vitro.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

Lateral diffusion of erythrocyte phospholipids in model membranes comparison between inner and outer leaflet components

The physical properties of lipid bilayers with a similar composition to the outer and inner leaflets of the human erythrocyte membrane have been examined in protein-free model systems. The outer leaflet (OL) was represented by a phospholipid mixture containing phosphatidylcholine and sphingomyelin extracted from human erythrocytes, while a mixture of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine represented the inner leaflet (IL). The ratio of cholesterol to phospholipid was varied in both mixtures. The lateral diffusion coefficient of fluorescent phospholipids diluted in such lipid mixtures was determined by the modulated fringe pattern photobleaching technique. Contrast curves with a single exponential decay, indicative of homogeneous samples, were obtained only for temperatures above 15 °C and for a cholesterol to phospholipid molar ratio below 0.8. The rate of lateral diffusion was approximately five times faster in IL than in OL multilayers, in agreement with former results obtained in human erythrocytes (Morrot et al. 1986). Varying the cholesterol to phospholipid ratio from 0 to 0.8 (mol/mol) enabled us to decrease the diffusion constant by only a factor of approximately 2 for both IL and OL mixtures. The order parameter of a spin-labeled phospholipid was determined in the different systems and found to be systematically smaller in IL mixtures than in OL mixtures. The present study indicates that the difference in lipid diffusivity of the two erythrocyte leaflets may be accounted for solely by a difference in phospholipid composition, and may be independent of cholesterol and protein asymmetry.Abbreviations OL outer leaflet - IL inner leaflet - RBC red blood cell - NBD-PC 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] dodecanoyl phosphatidylcholine - NBD-PE 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] dodecanoyl phosphatidylethanolamine - NBD-PS 1-acyl-2-[12-(7-nitrobenz-2-oxy-1,3-diazol-4-yl)amino] dodecanoyl phosphatidylserine - DMPC 1,2 dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2 dimyristoyl-snglycero-3-phosphoserine - PC phosphatidyleholine - C/P cholesterol over phospholipid molar ratio - D lateral diffusion coefficient - S order parameter - ESR electron spin resonance - NMR nuclear magnetic resonance - EDTA ethylene diamine tetraacetic acid - TRIS tris-(hydroxymethyl)amino ethane Offprint requests to: P. F Devaux     

Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers

The miscibility of triolein and cholesteryl oleate with 1-palmitoyl-2-oleoyl phosphatidylcholine was studied at the argon-buffer interface. The surface phase behavior of the system was analogous to that for cholesteryl ester-phospholipid mixtures in that both monolayer and double layer surface phases were formed. By considering the bulk properties of cholesteryl oleatetriolein mixtures and the two-dimensional phase rule, the entire system could be described. Double layer properties suggest that it consists of mostly triolein and phospholipid in the layer adjacent to the aqueous phase. The monolayer phase shows the formation of complexes between the neutral lipids and the phospholipid with stoichiometries nearly identical with those reported for bilayers (Hamilton, J. A., Miller, K. W., and Small, D. M. (1983) J. Biol. Chem. 258, 12821-12826). A second complex with a 3:1 stoichiometry is formed between triolein and cholesteryl oleate independently of interactions with phospholipid. Upon interaction with phospholipid, the triolein-cholesteryl oleate complex loses proportionately more area than either lipid alone. Because the area of complexes with phospholipid is constant, overall neutral lipid miscibility in such complexes is enhanced by the cholesteryl oleate-triolein interaction. Thus, our data explain the apparently nonideal mixing of cholesteryl oleate, triolein, and phospholipid in monolayers and in bilayers.     

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Effect of sterol incorporation on head group separation in liposomes

Electrophoretic mobilities of multilamellar liposomes of varying composition have been measured to determine the effect of incorporated sterols on surface charge density. Liposomes made from mixtures of zwitterionic egg phosphatidylcholine (PC) and anionic egg phosphatidylglycerol (PG) in varying proportions were shown to have electrophoretic mobilities consistent with the anticipated surface charge density. Incorporation of cholesterol up to 50 mole per cent in the bilayer produced no detectable change in surface charge density. Similar results were obtained for lanosterol and epicoprostanol. These results are interpreted to mean that incorporation of the sterols into the bilayers produced no detectable change (less than 3%) in the spacing of charged phospholipids. It is inferred that sterols are incorporated among the fatty acyl chains of these phospholipid bilayers with little or no displacement of the head groups at the surface.     

RNA and DNA interactions with zwitterionic and charged lipid membranes — A DSC and QCM-D study

The aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na⁺) or divalent (Ca²⁺) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC-DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC-DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA-phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.