Elizabeth K. Mann

Coarsening Dynamics of Domains in Lipid Membranes

We investigate isothermal diffusion and growth of micron-scale liquid domains within membranes of free-floating giant unilamellar vesicles with diameters between 80 and 250 μm. Domains appear after a rapid temperature quench, when the membrane is cooled through a miscibility phase transition such that coexisting liquid phases form. In membranes quenched far from a miscibility critical point, circular domains nucleate and then progress within seconds to late stage coarsening in which domains grow via two mechanisms 1), collision and coalescence of liquid domains, and 2), Ostwald ripening. Both mechanisms are expected to yield the same growth exponent, α = 1/3, where domain radius grows as time(α). We measure α = 0.28 ± 0.05, in excellent agreement. In membranes close to a miscibility critical point, the two liquid phases in the membrane are bicontinuous. A quench near the critical composition results in rapid changes in morphology of elongated domains. In this case, we measure α = 0.50 ± 0.16, consistent with theory and simulation.

Improving Liquid-Crystal-Based Biosensing in Aqueous Phases

Liquid crystal (LC)-based biological sensors permit the study of aqueous biological samples without the need for the labeling of biological species with fluorescent dyes (which can be laborious and change the properties of the biological sample under study). To date, studies of LC-based biosensors have explored only a narrow range of the liquid crystal/alignment layer combinations essential to their operation. Here, we report a study of the role of LC elastic constants and the surface anchoring energy in determining the sensitivity of LC-based biosensors. By investigating a mixture of rod-shape and bent-shape mesogens, and three different alignment layers, we were able to widen the useful detection range of a LC-based sensor by providing an almost-linear mapping of effective birefringence with anionic surfactant concentrations between 0.05 mM and 1 mM (model target analyte). These studies pave the way for optimization of LC-based biosensors and reveal the importance of the choice of both the LC material and the alignment layer in determining sensor properties.

Structure of Ceramide-1-Phosphate at the Air-Water Solution Interface in the Absence and Presence of Ca2+

Ceramide-1-phosphate, the phosphorylated form of ceramide, gained attention recently due to its diverse intracellular roles, in particular in inflammation mediated by cPLA(2)alpha. However, surprisingly little is known about the physical chemical properties of this lipid and its potential impact on physiological function. For example, the presence of Ca(2+) is indispensable for the interaction of Cer-1-P with the C2 domain of cPLA(2)alpha. We report on the structure and morphology of Cer-1-P in monomolecular layers at the air/water solution interface in the absence and presence of Ca(2+) using diverse biophysical techniques, including synchrotron x-ray reflectivity and grazing angle diffraction, to gain insight into the role and function of Cer-1-P in biomembranes. We show that relatively small changes in pH and the presence of monovalent cations dramatically affect the behavior of Cer-1-P. On pure water Cer-1-P forms a solid monolayer despite the negative charge of the phosphomonoester headgroup. In contrast, pH 7.2 buffer yields a considerably less solid-like monolayer, indicating that charge-charge repulsion becomes important at higher pH. Calcium was found to bind strongly to the headgroup of Cer-1-P even in the presence of a 100-fold larger Na(+) concentration. Analysis of the x-ray reflectivity data allowed us to estimate how much Ca(2+) is bound to the headgroup, approximately 0.5 Ca(2+) and approximately 1.0 Ca(2+) ions per Cer-1-P molecule for the water and buffer subphase respectively. These results can be qualitatively understood based on the molecular structure of Cer-1-P and the electrostatic/hydrogen-bond interactions of its phosphomonoester headgroup. Biological implications of our results are also discussed.

Thermotropic liquid crystal films for biosensors and beyond

We briefly review studies of liquid crystal films suspended in submillimeter size grids for biosensing applications and beyond. Due to intense recent research, the sensitivity of liquid crystal films to targeted biologically relevant agents can be increased, and the LC surface can be functionalized to be sensitive only to pre-assigned pathogens. Beyond sensor applications, we show that novel liquid crystal defect structures can be used to manipulate separation and deposition of lipids. Finally, we demonstrate that not only the nematic liquid crystal phase, but also chiral nematic (cholesteric and blue phase) and smectic liquid crystals can be used for sensing and may extend the sensitivity and/or the selection of biomaterials, which can be sensed.

Alignment by Langmuir/Schaefer monolayers of bent-core liquid crystals

Langmuir films of bent-core molecules at the air/water interface are transferred onto a solid surface by the inverse-Langmuir-Schaefer (ILS) technique. Previous work by the authors demonstrated that ILS films of a symmetric bent-core molecule can serve as effective planar alignment layers for a nematic bent-core liquid crystal cell, but the Langmuir films were unstable and formed multilayers at very low pressures. Here, films of bent-core molecules with one hydrophilic end connected to the bent core by a short aliphatic chain are tested as alignment layers. The hydrophilic group led to much more stable Langmuir films, and also to a molecular tilt at the surface which could be controlled by molecular area. This interpretation of the molecular behavior was supported by a combination of atomically accurate molecular dynamics simulations of up to 36 bent core molecules at a water surface and by the continuous variation of tilt induced by ILS films in a rod-like liquid crystal cell. The ILS films were then tested as alignment layers for a bent-core nematic: highly-compressed films induced perpendicular alignment, which provides a significant step towards their practical application.

Inverse Langmuir–Schaefer films of bent-core molecules

Mesostructures in inversed Langmuir–Schaefer films of each of two contrasting bent-core molecules are explored via atomic force microscopy. One of the molecules forms Langmuir films that are optically isotropic and the other those that are optically anisotropic. Both show suboptical mesostructures in the 100 nm range, in spite of appearing optically uniform. The anisotropic films are effective in aligning nematic bent-core samples, but not rod-like liquid crystals such as 5CB.

Gasous Hole Closing in a Polymer Langmuir Monolayer

The hole-closing phenomenon is studied in a polymer Langmuir film with coexisting gaseous and liquid phases both as a test of hydrodynamic theories of a two-dimensional fluid embedded in a three-dimensional one and as a means to accurately determine line tension, an important parameter determining size, shape, and dynamics within these and other membrane model systems. The hole-closing curve consists of both a universal linear regime and a history-dependent nonlinear one. Improved experimental technique allows us to explore the origin of the nonlinear regime. The linear regime confirms previous theoretical work and yields a value lambda = (0.69 +/- 0.02) pN for the line tension of the boundary between the gaseous and liquid phases. The observed hole closing also demonstrates that the two-dimensional polymer gas must be taken as having a small, probably negligible elasticity, so that line-tension measurements assuming that both phases are incompressible should be re-evaluated.

Chiral Nematic Liquid Crystal Microlenses

Nematic liquid crystals (NLCs) of achiral molecules and racemic mixtures of chiral ones form flat films and show uniform textures between circular polarizers when suspended in sub-millimeter size grids and immersed in water. On addition of chiral dopants to the liquid crystal, the films exhibit optical textures with concentric ring patterns and radial variation of the birefringence color. Both are related to a biconvex shape of the chiral liquid crystal film; the rings are due to interference. The curvature radii of the biconvex lens array are in the range of a few millimeters. This curvature leads to a radial variation of the optical axis along the plane of the film. Such a Pancharatnam-type phase lens dominates the imaging and explains the measured focal length of about one millimeter. To our knowledge, these are the first spontaneously formed Pancharatnam devices. The unwinding of the helical structure at the grid walls drives the lens shape. The relation between the lens curvature and material properties such as helical pitch, the twist elastic constant, and the interfacial tensions, is derived. This simple, novel method for spontaneously forming microlens arrays can also be used for various sensors.

Insertion of perilipin 3 into a glycero(phospho)lipid monolayer depends on lipid headgroup and acyl chain species

Lipid droplets (LDs) are organelles that contribute to various cellular functions that are vital for life. Aside from acting as a neutral lipid storage depot, they are also involved in building new membranes, synthesis of steroid hormones, and cell signaling. Many aspects of LD structure and function are not yet well-understood. Here we investigate the interaction of perilipin 3, a member of the perilipin family of LD binding proteins, and three N-terminal truncation mutants with lipid monolayers. The interaction is studied as a function of surface pressure for a series of systematically chosen lipids. We find that the C terminus of perilipin 3 has different insertion behavior from that of the longer truncation mutants and the full-length protein. Inclusion of N-terminal sequences with the C terminus decreases the ability of the protein construct to insert in lipid monolayers. Coupling of anionic lipids to negative spontaneous curvature facilitates protein interaction and insertion. The C terminus shows strong preference for lipids with more saturated fatty acids. This work sheds light on the LD binding properties and function of the different domains of perilipin 3.

Insertion of apoLp-III into a lipid monolayer is more favorable for saturated, more ordered, acyl-chains

Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic α-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic α-helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an α-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic α-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic α-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein-lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.