A stripe-to-droplet transition driven by conformational transitions in a binary lipid-lipopolymer mixture at the air-water interface.

We report the observation of an unusual stripe-droplet transition in precompressed Langmuir monolayers consisting of mixtures of poly(ethylene) glycol (PEG) amphiphiles and phospholipids. This highly reproducible and fully reversible transition occurs at approximately zero surface pressure during expansion (or compression) of the monolayer following initial compression into a two-dimensional solid phase. It is characterized by spontaneous emergence of an extended, disordered stripe-like morphology from an optically homogeneous phase during gradual expansion. These stripe patterns appear as a transient feature and continuously progress, involving gradual coarsening and ultimate transformation into a droplet morphology upon further expansion. Furthermore, varying relative concentrations of the two amphiphiles and utilizing amphiphiles with considerably longer ethylene glycol headgroups reveal that this pattern evolution occurs in narrow concentration regimes, values of which depend on ethylene oxide headgroup size. These morphological transitions are reminiscent of those seen during a passage through a critical point by variations in thermodynamic parameters (e.g., temperature or pressure) as well as those involving spinodal decomposition. While the precise mechanism cannot be ascertained using present experiments alone, our observations can be reconciled in terms of modulations in competing interactions prompted by the pancake-mushroom-brush conformational transitions of the ethylene glycol headgroup. This in turn suggests that the conformational degree of freedom represents an independent order parameter, or a switch, which can induce large-scale structural reorganization in amphiphilic monolayers. Because molecular conformational changes are pervasive in biological membranes, we speculate that such conformational transition-induced pattern evolution might provide a physical mechanism by which membrane processes are amplified.

Thermally induced phase separation in supported bilayers of glycosphingolipid and phospholipid mixtures.

The authors have studied microstructure evolution during thermally induced phase separation in a class of binary supported lipid bilayers using a quantitative application of imaging ellipsometry. The bilayers consist of binary mixtures consisting of a higher melting glycosphingolipid, galactosylceramide (GalCer), which resides primarily in the outer leaflet, and a lower melting, unsaturated phospholipid, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). Three different bilayer compositions of GalCer/DLPC mixtures at 35:65, 20:80, and 10:90 molar ratios were cooled at controlled rates from their high-temperature homogeneous phase to temperatures corresponding to their phase coexistence regime and imaged in real time using imaging ellipsometry. During the thermotropic course of GalCer gelation, we find that two distinct types of morphological features modulate. First, the formation and growth of chain and fractal-like defects ascribed to the net change in molecular areas during the phase transition. The formation of these defects is consistent with the expected contraction in the molecular area during the liquid crystalline to gel-phase transition. Second, the nucleation and growth of irregularly shaped gel-phase domains, which exhibit either line-tension dominated compact shape or dendritic domains with extended interfaces. Quantifying domain morphology within the fractal framework reveals a close correspondence, and the quantization of the transition width confirms previous estimates of reduced phase transition cooperativity in supported bilayers. A comparison of domain properties indicates that thermal history, bilayer composition, and cooling rate all influence microstructure details including shapes, sizes, and distributions of domains and defects: At lower cooling rates and lower GalCer fractions compact domains form and at higher GalCer fractions (or at higher cooling rates) dendritic domains are evident. This transition of domain morphology from compact shapes to dendritic shapes at higher cooling rates and higher relative fractions of GalCer suggests kinetic control of shape equilibration in these phospho- and glycolipid mixtures.

Frustrated phase transformations in supported, interdigitating lipid bilayers.

In free bilayers, the fluid to gel main phase transition of a monofluorinated phospholipid (F-DPPC) transforms a disordered fluid bilayer into a fully interdigitated monolayer consisting of ordered acyl tails. This transformation results in an increase in molecular area and decrease in bilayer thickness. We show that when confined in patches near a solid surface this reorganization proceeds under constraints of planar topography and total surface area. One consequence of these constraints is to limit the complete formation of the energetically favored, interdigitated gel phase. The noninterdigitated lipids experience enhanced lateral tension, due to the expansion of the growing interdigitated phase within the constant area. The corresponding rise in equilibrium transition temperatures produces supercooled lipids that vitrify when cooled further. Ultimately, this frustrated phase change reflects a coupling between dynamics and thermodynamics and gives rise to an unusual phase coexistence characterized by the presence of two qualitatively different gel phases.

Early stages of oxidative stress-induced membrane permeabilization: a neutron reflectometry study.

Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid-liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100-150 A into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data.

Direct visualization of phase transition dynamics in binary supported phospholipid bilayers using imaging ellipsometry.

Via imaging ellipsometry, we study the phase transition dynamics induced by selective gelation of one component in a binary supported phopholipid bilayer. We find the modulation of two attendant morphological features: emergence of extended defect chains due to a net change in the molecular areas and fractal-like domains suggesting weak line tension. A time-lapse analysis of the ellipsometric images reveals the cluster size of 4-20 molecules undergoing gelation indicating weak cooperativity. These results demonstrate the use of ellipsometry for in situ, label-free, non-contact, and large-area imaging of dynamics in interfacial films.

Characterization of physical properties of supported phospholipid membranes using imaging ellipsometry at optical wavelengths.

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.