Session V45: Soft Matter Physics of Heterogeneous Membranes - Experiments

Structure and mechanics of biological fiber-laden membranes

We present a mechanical model of the plant cell wall viewed as anisotropic 2D soft matter, where a dilute dispersion of cellulose fibrils of variable orientations is on a curved deformable viscoelastic membrane. The model integrates the elastic energy of the curved membrane, the nematic fiber orientation energy, and competing curvophilic and curvophobic interactions mediated by the membrane geometry and the fibrils' orientation. The selected membrane geometry is a straight cylinder, whose cross-sectional shape varies from a circle to a super-ellipse as in many plant species, and its size increases as in plant cell wall during growth. Model predictions indicate that due to curvature--orientation couplings, the fiber orientation displays three modes: axial (at large curvature), helical and transverse (at small curvature). The high curvature also promotes the order of the fibrils. The predicted fiber structure is validated with that in the cell wall of tracheids. To gain insight into the role of fiber structure on the elasticity of fiber-laden membranes, the effect of fiber orientation and order on the effective bending modulus of the fiber-laden membrane cross-section is investigated. The structure--property relations for super-elliptical membranes are established.   

From a single molecule to a membrane of structured ionic polymers: A molecular dynamic simulation study

The association of an A-B-C-B-A co-polymer with an ionizable center and a bulky end block has been investigated using molecular dynamic simulations. The center block consists of a randomly sulfonated polystyrene connected to a flexible poly (ethylene-r-propylene) bridge and end caped with poly (t-butyl styrene). Tailoring the nature of individual segments within a block co-polymer is a potential design tool to form membranes with desired properties. The association mode and the dynamics of the segments control the overall characteristics. The membranes with three sulfonation level for the center block were made by evaporating a common solvent for all blocks. The local structure including size and distribution of the ionic blocks and the continuity of the styrene phase as well as long range correlations were identified at 300 and 500K. The initial membrane structure is affected by the structure in solution. Studies on changes that take place above the glass transition temperature for each of the blocks will also be presented.   

Investigation of the elastic properties of a lipid bilayer by fluorescence interferometry

Freestanding curved lipid bilayers were formed on micron diameter wells fabricated on a silicon chip. The height profile of the lipid bilayers was measured using fluorescence interference contrast microscopy. Dark and bright rings resulted from the interference of emission from the fluorophores in the lipid bilayers with the same light reflected from the bottom surface of the well. By changing the osmotic pressure difference across the bilayers, the relationship between the pressure and the membrane curvature was studied. By using Helfrich theory, the surface tension of the bilayer was extracted. The influence of detergents and antibiotics on the elastic property of lipid bilayers was also investigated.   

Regulating the Size and Stabilization of Lipid Raft-Like Domains and Using Calcium Ions as Their Probe

In this paper, we apply means to probe, stabilize and control the size of lipid raft-like domains in vitro. In biomembranes the size of lipid rafts is ca. 10 - 30 nm. In vitro, mixing saturated and unsaturated lipids results in micro-domains, which are unstable and coalesce. Using solution X-ray scattering, we studied the structure of binary and ternary lipid mixtures in the presence of calcium ions. Three lipids were used: saturated, unsaturated and a hybrid (1-saturated-2-unsaturated) lipid that is predominant in the phospholipids of cellular membranes. Only membranes composed of the saturated lipid can adsorb calcium ions, become charged and therefore considerably swell. The selective calcium affinity was used to show that binary mixtures, containing the saturated lipid, phase separated into large-scale domains. Our data suggests that by introducing the hybrid lipid to a mixture of the saturated and unsaturated lipids, the size of the domains decreased with the concentration of the hybrid lipid, until the three lipids could completely mix. We attribute this behavior to the tendency of the hybrid lipid to act as a line-active co-surfactant that can easily reside at the interface between the saturated and the unsaturated lipids and reduce the line-tension between them.   

Floret-shaped solid domains on giant fluid lipid vesicles induced by pH

Lateral lipid phase separation and domain formation induced by changes in pH is significant in liposome-based drug delivery: environmentally responsive lipid heterogeneities can be tuned to alter collective membrane properties such as drug release and drug carrier reactivity impacting, therefore, the therapeutic outcomes. At the micron-meter scale, fluorescence microscopy on Giant Unilamellar fluid Vesicles (GUVs) shows that lowering pH (from 7.0 to 5.0) promotes the condensation of titratable PS or PA lipids into beautiful floret-shaped solid domains in which lipids are tightly packed via H-bonding and VdWs interactions. Solid domains phenomenologically comprise a circular ``core'' cap beyond which interfacial instabilities emerge resembling leaf-like stripes of almost vanishing Gaussian curvature independent of GUVs' preparation path and in agreement with a general condensation mechanism. Increasing \textit{incompressibility} of domains is strongly correlated with larger \textit{number of thinner stripes }per domain, and increasing \textit{relative} \textit{rigidity} of domains with smaller \textit{core cap areas}. Line tension drives domain ripening, however the final domain shape is a result of enhanced incompressibility and rigidity maximized by domain coupling across the bilayer. Introduction of a transmembrane osmotic gradient (hyperosmotic on the outer lipid leaflet) allows the domain condensation process to reach its maximum extent which, however, is limited by the minimal expansivity of the continuous fluid membrane.   

The influence of membrane stress on phase separation and domain shape in phospholipid vesicles

Phase separation of mixed phospholipid bilayers is of interest due to the potential role of phospholipid rafts in cell adhesion and signaling. Studies of membrane dynamics and the phase diagram itself typically neglect the role of tension, though it is expected that imposition of moderate membrane tensions might mildly shift the phase separation temperature, as anticipated by Clausius Clapeyron. We show here, using a simple binary system (DOPC/DPPC), a more dramatic effect: The tension imposed on giant unilamellar phospholipid vesicles can alter the phase and the domain shape, completely changing the composition of the liquid and solid phases, their proportions, and the transition temperature. The example in this talk demonstrates how striped or patchy hexagonal phases can develop, depending on thermal history and tension. Different incorporation of tracers into the ordered phases suggests fundamental differences in their structure at the molecular level. Rapid quenching and low tensions favor hexagonal patches while increased tension and slower quenching favors a striped phase. For this reason it is believed that the patches contain corrugations such that the structure of the ordered phase is metastable.   

Membrane fluctuations alter the fluidity of clathrin protein lattices

Clathrin is a protein that plays a major role in the creation of membrane-bound transport vesicles in cells. The pinwheel subunits of clathrin assemble into closed, nanoscale assemblies with various shapes and sizes. We develop a model for clathrin, facilitating the study of membrane, surface, and bulk assembly. The clathrin are modeled as pinwheels that form leg-leg associations and resist bending and stretching deformations. Invoking theories of dislocation-mediated melting in two dimensions, we discuss the phase behavior for clathrin. We demonstrate that the generation of defects resembles creation of two dislocations, and we use orientational- and translational-order correlation functions to predict the crystalline-hexatic and hexatic-liquid phase transitions. Accounting for membrane fluctuations, we address the phase behavior of clathrin on a membrane surface. Membrane fluctuations act to soften the elastic coupling between defects in the clathrin lattice, altering the conditions for the crystalline-hexatic phase transition. This effect offers a mechanism for altering the fluidity of protein or polymer films. Furthermore, these results illustrate the pivotal role that molecular elasticity plays in the physical behavior of self-assembling and self-healing materials.   

The structure of unsupported, self-assembled phospholipid bilayers on an artificially nano-patterned surface

We present neutron reflectivity measurements of the in-situ microscopic architecture of phospholipid molecules at the interface between a regularly nano-patterned surface and an aqueous sub-phase using neutron reflectometry. 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) single bilayers were deposited on a patterned silicon substrate. The substrate was patterned with a rectangular array of nano-scaled holes using e-beam nano-lithographic techniques. The goal of these experiments is to produce a set of small freely-suspended bilayers spanning the nanostructured surface. We compare results for films deposited by vesicle adsorption or by the Langmuir--Shafer (L-S) technique. Initial data analysis shows that there are well formed bilayers on the surface. Detailed analysis of the reflectivity curves will be presented to confirm details of the architecture of these bilayer films. Bilayers prepared in this way may serve as model single bilayer systems with freely suspended areas for the study of membrane functionality in biological and biomimetic materials and systems.   

Peptide-induced Asymmetric Distribution of Charged Lipids in a Vesicle Bilayer Revealed by Small-Angle Neutron Scattering

Cellular membranes are complex mixtures of lipids, proteins and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoyl phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations, and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state.   

Cooperative assembly in targeted drug delivery

Described as cell analogues, liposomes are self-assembled lipid bilayer spheres that encapsulate aqueous volumes. Liposomes offer several drug delivery advantages due to their structural versatility related to size, composition, bilayer fluidity, and ability to encapsulate a large variety of compounds non-covalently. However, liposomes lack the structural information embedded within cell membranes. Partitioning of unsaturated and saturated lipids into liquid crystalline (L$\alpha )$ and gel phase (L$\beta )$ domains, respectively, affects local molecular diffusion and elasticity. Liposome microdomains may be used to pattern molecules, such as antibodies, on the liposome surface to create concentrated, segregated binding regions. We have synthesized, characterized, and evaluated a series of homogeneous and heterogeneous liposomal vehicles that target inflamed endothelium. These drug delivery vehicles are designed to complement the heterogeneous presentation of lipids and receptors on endothelial cells (ECs). EC surfaces are dynamic; they segregate receptors within saturated lipid microdomains on the cell surface to regulate binding and signaling events. We have demonstrated that cooperative binding of two antibodies enhances targeting by multiple fold. Further, we have shown that organization of these antibodies on the surface can further enhance cell uptake. The data suggest that EC targeting may be enhanced by designing liposomes that mirror the segregated structure of lipid and receptor molecules involved in neutrophil-EC adhesion. This strategy is employed in an atherosclerotic mouse model in vivo.   

Polymer-Protein interaction at air/liquid interfaces: X-ray reflectivity and surface spectroscopy studies

Adsorption of proteins onto a substrate is the first and a critical step that determines the cellular response of substrates. To understand the adsorption and distribution of proteins on surfaces, we employ surface sensitive X-ray scattering and spectroscopic techniques to monitor the adsorption of plasma proteins (fibrinogen) onto surfaces of polymers, poly(DTE carbonate), on aqueous surfaces. Our X-ray measurements provide the density profiles of the polymers-proteins systems on aqueous surfaces, with details on the interactions between the polymers and the protein, and distribution of the protein within and on the polymer surface. The hydrophobic and hydrophilic behaviors of these polymers are modified by incorporating poly(ethylene glycol) (PEG) and by iodinating the tyrosine rings. Our results confirm the inhibition of the adsorption of fibrinogen onto polymer surfaces by PEG, and the counteraction of this influence when the polymers are iodinated.   

Microporous device for local electric recordings on lipid bilayers

Many methods for artificial membrane formation are available. We focus on the reconstitution of lipid bilayers on porous substrates combining the stability of solid supports and the accessibility of both sides of the bilayer of the classical BLM which is necessary for low noise electric experiments. Most commercially available porous substrates however are not suitable for electric experiments or a combination of several measuring techniques. Therefore, we designed a microporous substrate, which meets several demands: We wanted to have the possibility to perform multiple experiments in one, so we chose to divide the device into several individually addressable arrays of pores with separate electrolyte compartments and integrated electronic connections. Also, to perform electrical and fluorescence experiments at the same time, we designed a PDMS sample chamber so that the substrate is accessible to a microscope objective. By having separated electrolyte compartments, we are also able to exchange solutions or introduce chemicals throughout the experiment. Bilayer formation can be probed by impedance spectroscopy and fluorescence microscopy. The function of inserted ion channels can be measured by current recordings.