Marjorie L. Longo

Phase Separation of Lipid Membranes Analyzed with High-Resolution Secondary Ion Mass Spectrometry

Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of ∼100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.

AFM for structure and dynamics of biomembranes.

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.

Tailoring the Size Distribution of Ultrasound Contrast Agents: Possible Method for Improving Sensitivity in Molecular Imaging

Encapsulated microbubble contrast agents incorporating an adhesion ligand in the microbubble shell are used for molecular imaging with ultrasound. Currently available microbubble agents are produced with techniques that result in a large size variance. Detection of these contrast agents depends on properties related to the microbubble diameter such as resonant frequency, and current ultrasound imaging systems have bandwidth limits that reduce their sensitivity to a polydisperse contrast agent population. For ultrasonic molecular imaging, in which only a limited number of targeted contrast agents may be retained at the site of pathology, it is important to optimize the sensitivity of the imaging system to the entire population of contrast agent. This article presents contrast agents with a narrow size distribution that are targeted for molecular imaging applications. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters that influence the size distribution and demonstrate initial acoustic testing.

Obstructed Diffusion in Phase-Separated Supported Lipid Bilayers: A Combined Atomic Force Microscopy and Fluorescence Recovery after Photobleaching Approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.

Maintaining monodispersity in a microbubble population formed by flow-focusing.

The dynamic processes impacting the size distributions of lipid-encapsulated microbubbles formed by flow-focusing were observed by video optical microscopy. Parameters studied included the filling gas, gas saturating the surrounding solution, and microbubble size (initial size 2-12 microm) to simulate typical laboratory conditions. Typically, dissolution or growth, followed by Ostwald ripening at a collection cover glass, were observed and quantified. However, in the case of small nitrogen-filled microbubbles surrounded by an air-saturated solution, Ostwald ripening was avoided for at least 9 h. These bubbles had a final size distribution of 1.5 +/- 0.1 microm. This work suggests that lipid-encapsulated microbubbles formed by flow-focusing should be given sufficient time to reach a terminal size before coming into contact with each other. These long-lived mondisperse microbubbles should be of interest in ultrasound contrast agents, microfabrication, food, and research applications.

Trehalose maintains phase separation in an air-dried binary lipid mixture.

Mixing and thermal behavior of hydrated and air-dried mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-d70-sn-glycero-3-phosphocholine (DSPCd-70) in the absence and presence of trehalose were investigated by Fourier transform infrared spectroscopy. Mixtures of DLPC:DSPCd-70 (1:1) that were air-dried at 25 degrees C show multiple phase transitions and mixed phases in the dry state. After annealing at high temperatures, however, only one transition is seen during cooling scans. When dried in the presence of trehalose, the DLPC component shows two phase transitions at -22 degrees C and 75 degrees C and is not fully solidified at -22 degrees C. The DSPCd-70 component, however, shows a single phase transition at 78 degrees C. The temperatures of these transitions are dramatically reduced after annealing at high temperatures with trehalose. The data suggest that the sugar has a fluidizing effect on the DLPC component during drying and that this effect becomes stronger for both components with heating. Examination of infrared bands arising from the lipid phosphate and sugar hydroxyl groups suggests that the strong effect of trehalose results from direct interactions between lipid headgroups and the sugar and that these interactions become stronger after heating. The findings are discussed in terms of the protective effect of trehalose on dry membranes.

Role of Unsaturated Lipid and Ergosterol in Ethanol Tolerance of Model Yeast Biomembranes

We present a combined atomic force microscopy and fluorescence microscopy study of the behavior of a ternary supported lipid bilayer system containing a saturated lipid (DPPC), an unsaturated lipid (DOPC), and ergosterol in the presence of high ethanol (20 vol %). We find that the fluorescent probe Texas Red DHPE preferentially partitions into the ethanol-induced interdigitated phase, which allows the use of fluorescence imaging to investigate the phase behavior of the system. Atomic force microscopy and fluorescence images of samples with the same lipid mixture show good agreement in sample morphology and area fractions of the observed phases. Using area fractions obtained from fluorescence images over a broad range of compositions, we constructed a phase diagram of the DPPC/DOPC/ergosterol system at 20 vol % ethanol. The phase diagram clearly shows that increasing unsaturated lipid and/or ergosterol protects the membrane by preventing the formation of the interdigitated phase. This result supports the hypothesis that yeast cells increase ergosterol and unsaturated lipid content to prevent interdigitation and maintain an optimal membrane thickness as ethanol concentration increases during anaerobic fermentations. Changes in plasma membrane composition provide an important survival factor for yeast cells to deter ethanol toxicity.

Domain Growth, Shapes, and Topology in Cationic Lipid Bilayers on Mica by Fluorescence and Atomic Force Microscopy

Domain formation in mica-supported cationic bilayers of dipalmitoyltrimethylammoniumpropane (DPTAP) and dimyristoyltrimethylammoniumpropane (DMTAP), fluorescently doped with an NBD (((7-nitro-2-1, 3-benzoxadiazol-4-yl)amino)caproyl) phospholipid, was investigated with fluorescence microscopy and atomic force microscopy. Heating above the acyl chain melting temperature and cooling to room temperature resulted in nucleation and growth of domains with distinguishable patterns. Fractal patterns were found for DPTAP, whereas DMTAP domains were elongated and triangular with feathery edges. Reducing the cooling rate or probe concentration for DPTAP bilayers resulted in larger, filled-in domains with more rounded edges. However, for DMTAP, cooling rates mainly affected size and only slightly modified domain morphology. In a saline environment, the domains were dark, and the surrounding continuous region was bright and thus contained the fluorescent probe. However, as the salt concentration was decreased, the dark regions percolated (connected), resulting in bright domains. Atomic force microscopy scans along domain edges revealed that the dark regions in fluorescence images were approximately 1.4 nm thicker than the light regions. Additionally, the dark regions were of bilayer thickness, approximately 4 nm. Comparison of these results in bilayers to well-documented behavior in Langmuir monolayers has revealed many similarities (and some differences) and is therefore useful for understanding our observations and identifying possible growth mechanisms that may occur in domain formation in cell membranes or supported membrane systems.

Modeling Amyloid β-Peptide Insertion into Lipid Bilayers

Inspired by recent suggestions that the Alzheimers amyloid beta peptide (Abeta) can insert into cell membranes and form harmful ion channels, we model insertion of the 40- and 42-residue forms of the peptide into cell membranes using a Monte Carlo code which is specific at the amino acid level. We examine insertion of the regular Abeta peptide as well as mutants causing familial Alzheimers disease, and find that all but one of the mutants change the insertion behavior by causing the peptide to spend more simulation steps in only one leaflet of the bilayer. We also find that Abeta42, because of the extra hydrophobic residues relative to Abeta40, is more likely to adopt this conformation than Abeta40 in both wild-type and mutant forms. We argue qualitatively why these effects happen. Here, we present our results and develop the hypothesis that this partial insertion increases the probability of harmful channel formation. This hypothesis can partly explain why these mutations are neurotoxic simply due to peptide insertion behavior. We further apply this model to various artificial Abeta mutants which have been examined experimentally, and offer testable experimental predictions contrasting the roles of aggregation and insertion with regard to toxicity of Abeta mutants. These can be used through further experiments to test our hypothesis.

Bilayer Structure and Lipid Dynamics in a Model Stratum Corneum with Oleic Acid

The stratum corneum is the uppermost layer of the skin and acts as a barrier to keep out contaminants and retain moisture. Understanding the molecular structure and behavior of this layer will provide guidance for optimizing its biological function. In this study we use a model mixture comprised of equimolar portions of ceramide NS (24:0), lignoceric acid, and cholesterol to model the effect of the addition of small amounts of oleic acid to the bilayer at 300 and 340 K. Five systems at each temperature have been simulated with concentrations between 0 and 0.1 mol % oleic acid. Our major finding is that subdiffusive behavior over the 200 ns time scale is evident in systems at 340 K, with cholesterol diffusion being enhanced with increased oleic acid. Importantly, cholesterol and other species diffuse faster when radial densities indicate nearest neighbors include more cholesterol. We also find that, with the addition of oleic acid, the bilayer midplane and interfacial densities are reduced and there is a 3% decrease in total thickness occurring mostly near the hydrophilic interface at 300 K with reduced overall density at 340 K. Increased interdigitation occurs independent of oleic acid with a temperature increase. Slight ordering of the long non-hydroxy fatty acid of the ceramide occurs near the hydrophilic interface as a function of the oleic acid concentration, but no significant impact on hydrogen bonding is seen in the chosen oleic acid concentrations.