Susan Daniel

Double cushions preserve transmembrane protein mobility in supported bilayer systems.

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.

Influenza Virus-Mediated Membrane Fusion: Determinants of Hemagglutinin Fusogenic Activity and Experimental Approaches for Assessing Virus Fusion

Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.

Effect of roughness geometry on wetting and dewetting of rough PDMS surfaces.

Rough PDMS surfaces comprising 3 μm hemispherical bumps and cavities with pitches ranging from 4.5 to 96 μm have been fabricated by photolithographic and molding techniques. Their wetting and dewetting behavior with water was studied as model for print surfaces used in additive manufacturing and printed electronics. A smooth PDMS surface was studied as control. For a given pitch, both bumpy and cavity surfaces exhibit similar static contact angles, which increase as the roughness ratio increases. Notably, the observed water contact angles are shown to be consistently larger than the calculated Wenzel angles, attributable to the pinning of the water droplets into the metastable wetting states. Optical microscopy reveals that the contact lines on both the bumpy and cavity surfaces are distorted by the microtextures, pinning at the lead edges of the bumps and cavities. Vibration of the sessile droplets on the smooth, bumpy, and cavity PDMS surfaces results in the same contact angle, from 110°-124° to ∼91°. The results suggest that all three surfaces have the same stable wetting states after vibration and that water droplets pin in the smooth area of the rough PDMS surfaces. This conclusion is supported by visual inspection of the contact lines before and after vibration. The importance of pinning location rather than surface energy on the contact angle is discussed. The dewetting of the water droplet was studied by examining the receding motion of the contact line by evaporating the sessile droplets of a very dilute rhodamine dye solution on these surfaces. The results reveal that the contact line is dragged by the bumps as it recedes, whereas dragging is not visible on the smooth and the cavity surfaces. The drag created by the bumps toward the wetting and dewetting process is also visible in the velocity-dependent advancing and receding contact angle experiments.

Condensation on Surface Energy Gradient Shifts Drop Size Distribution toward Small Drops

During dropwise condensation from vapor onto a cooled surface, distributions of drops evolve by nucleation, growth, and coalescence. Drop surface coverage dictates the heat transfer characteristics and depends on both drop size and number of drops present on the surface at any given time. Thus, manipulating drop distributions is crucial to maximizing heat transfer. On earth, manipulation is achieved with gravity. However, in applications with small length scales or in low gravity environments, other methods of removal, such as a surface energy gradient, are required. This study examines how chemical modification of a cooled surface affects drop growth and coalescence, which in turn influences how a population of drops evolves. Steam is condensed onto a horizontally oriented surface that has been treated by silanization to deliver either a spatially uniform contact angle (hydrophilic, hydrophobic) or a continuous radial gradient of contact angles (hydrophobic to hydrophilic). The time evolution of number density and associated drop size distributions are measured. For a uniform surface, the shape of the drop size distribution is unique and can be used to identify the progress of condensation. In contrast, the drop size distribution for a gradient surface, relative to a uniform surface, shifts toward a population of small drops. The frequent sweeping of drops truncates maturation of the first generation of large drops and locks the distribution shape at the initial distribution. The absence of a shape change indicates that dropwise condensation has reached a steady state. Previous reports of heat transfer enhancement on chemical gradient surfaces can be explained by this shift toward smaller drops, from which the high heat transfer coefficients in dropwise condensation are attributed to. Terrestrial applications using gravity as the primary removal mechanism also stand to benefit from inclusion of gradient surfaces because the critical threshold size required for drop movement is reduced.

Tunable physiologic interactions of adhesion molecules for inflamed cell-selective drug delivery

Dysregulated inflammation contributes to the pathogenesis of various diseases. Therapeutic efficacy of anti-inflammatory agents, however, falls short against resilient inflammatory responses, whereas long-term and high-dose systemic administration can cause adverse side effects. Site-directed drug delivery systems would thus render more effective and safer treatments by increasing local dosage and minimizing toxicity. Nonetheless, achieving clinically effective targeted delivery to inflammatory sites has been difficult due to diverse cellular players involved in immunity and endogenous targets being expressed at basal levels. Here we exploit a physiological molecular interaction between intercellular adhesion molecule (ICAM)-1 and lymphocyte function associated antigen (LFA)-1 to deliver a potent anti-inflammatory drug, celastrol, specifically and comprehensively to inflamed cells. We found that affinity and avidity adjusted inserted (I) domain, the major binding site of LFA-1, on liposome surface enhanced the specificity toward lipopolysaccharides (LPS)-treated or inflamed endothelial cells (HMEC-1) and monocytes (THP-1) via ICAM-1 overexpression, reflecting inherent affinity and avidity modulation of these molecules in physiology. Targeted delivery of celastrol protected cells from recurring LPS challenges, suppressing pro-inflammatory responses and inflammation-induced cell proliferation. Targeted delivery also blocked THP-1 adhesion to inflamed HMEC-1, forming barriers to immune cell accumulation and to aggravating inflammatory signals. Our results demonstrate affinity and avidity of targeting moieties on nanoparticles as important design parameters to ensure specificity and avoid toxicities. We anticipate that such tunable physiologic interactions could be used for designing effective drug carriers for in vivo applications and contribute to treating a range of immune and inflammatory diseases.

Generation of Motion of Drops with Interfacial Contact

A liquid drop moves on a solid surface if it is subjected to a gradient of wettability or temperature. However, the pinning defects on the surface manifested in terms of a wetting hysteresis, or first-order nonlinear friction, limit the motion in the sense that a critical size has to be exceeded for a drop to move. The effect of hysteresis can, however, be mitigated by an external vibration that can be either structured or stochastic, thereby creating a directed motion of the drop. Many of the well-known features of rectification, amplification, and switching that are generic to electronics can be engineered with such types of movements. A specific case of interest is the random coalescence of drops on a surface that gives rise to self-generated noise. This noise overcomes the pinning potential, thereby generating a random motion of the coalesced drops. Randomly moving coalesced drops themselves exhibit a directed diffusive flux when a boundary is present to eliminate them by absorption. With the presence of a bias, the coalesced drops execute a diffusive drift motion that can have useful applications in various water and thermal management technologies.

Measuring the partitioning kinetics of membrane biomolecules using patterned two-phase coexistant lipid bilayers.

We report a new method for measuring the partitioning kinetics of membrane biomolecules to different lipid phases using a patterned supported lipid bilayer (SLB) platform composed of liquid-ordered (lipid raft) and liquid-disordered (unsaturated lipid-rich) coexistent phases. This new approach removes the challenges in measuring partitioning kinetics using current in vitro methods due to their lack of spatial and temporal control of where phase separation occurs and when target biomolecules meet those phases. The laminar flow configuration inside a microfluidic channel allows us to pattern SLBs with coexistent phases in predetermined locations and thus eliminates the need for additional components to label the phases. Using a hydrodynamic force provided by the bulk flow in the microchannel, target membrane-bound species to be assayed can be transported in the bilayers. The predefined location of stably coexistent phases, in addition to the controllable movement of the target species, allows us to control and monitor when and where the target molecules approach or leave different lipid phases. Using this approach with appropriate experimental designs, we obtain the association and dissociation kinetic parameters for three membrane-bound species, including the glycolipid G(M1), an important cell signaling molecule. We examine two different versions of G(M1) and conclude that structural differences between them impact the kinetics of association of these molecules to raft-like phases. We also discuss the possibilities and limitations for this method. One possible extension is measuring the partitioning kinetics of other glycolipids or lipid-linked proteins with posttranslational modifications to provide insight into how structural factors, membrane compositions, and environmental factors influence dynamic partitioning.

Membrane Fusion-Competent Virus-Like Proteoliposomes and Proteinaceous Supported Bilayers Made Directly from Cell Plasma Membranes

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Substrate constraint modifies the Rayleigh spectrum of vibrating sessile drops.

In our fluid dynamics video, we demonstrate our method of visualizing and identifying various mode shapes of mechanically oscillated sessile drops. By placing metal mesh under an oscillating drop and projecting light from below, the drops shape is visualized by the visually deformed mesh pattern seen in the top view. The observed modes are subsequently identified by their number of layers and sectors. An alternative identification associates them with spherical harmonics, as demonstrated in the tutorial. Clips of various observed modes are presented, followed by a 10-second quiz of mode identification.

Variations in pH Sensitivity, Acid Stability, and Fusogenicity of Three Influenza Virus H3 Subtypes

ABSTRACT Influenza A virus strains adapt to achieve successful entry into host species. Entry is mediated by the viral membrane protein hemagglutinin (HA), which triggers membrane fusion and genome release under acidic conditions in the endosome. In addition to changes in the receptor binding domain, the acid stability of HA has been linked to the successful transmission of virus between avian and human hosts. However, to fully understand the connection between changes in HA and host tropism, additional factors relevant to HA structure-function and membrane fusion are also likely to be important. Using single-particle-tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which provide detailed information regarding HA pH sensitivity and acid stability and the rate and extent of membrane fusion. This provides a comparative way to characterize and distinguish influenza virus fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn), and A/Brisbane/07/H3N2 (Brisbane). The rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while the fusion of the egg-adapted strains Udorn and X:31 is strongly dependent on pH (and is faster) as the pH decreases. All strains exhibit similar acid stability (the length of time that they remain fusogenic in an acidic environment) at higher pHs, but the egg-adapted strains become less acid stable at lower pHs. Thus, it appears that the laboratory-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH, with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain. IMPORTANCE The ability of influenza virus to release its genome under different acidic conditions has recently been linked to the transmission of influenza virus between different species. However, it is yet to be determined how acid-induced membrane fusion varies with virus strain and influences tropism. The results presented here are the results of an intra-H3-subtype study of acid stability and fusion kinetics. Using a single-particle-tracking (SPT) technique, we show here that the highest pH that initiates fusion is not necessarily the pH at which the kinetics of fusion is fastest and most abundant for a given strain. Strains exhibit different fusion behaviors, as evidenced by their unique kinetic trends; pH sensitivities, as evidenced by the differences when the first fusion events commence; and HA stabilities, as evidenced by the length of time that virions can persist in an acidic environment and still be fusion competent.