Amanda P. Siegel

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

Organotrisulfide: A High Capacity Cathode Material for Rechargeable Lithium Batteries

An organotrisulfide (RSSSR, R is an organic group) has three sulfur atoms which could be involved in multi-electron reduction reactions; therefore it is a promising electrode material for batteries. Herein, we use dimethyl trisulfide (DMTS) as a model compound to study its redox reactions in rechargeable lithium batteries. With the aid of XRD, XPS, and GC-MS analysis, we confirm DMTS could undergo almost a 4 e(-) reduction process in a complete discharge to 1.0 V. The discharge products are primarily LiSCH3 and Li2 S. The lithium cell with DMTS catholyte delivers an initial specific capacity of 720 mAh g(-1) DMTS and retains 82 % of the capacity over 50 cycles at C/10 rate. When the electrolyte/DMTS ratio is 3:1 mL g(-1) , the reversible specific energy for the cell including electrolyte can be 229 Wh kg(-1) . This study shows organotrisulfide is a promising high-capacity cathode material for high-energy rechargeable lithium batteries.

Compartmentalizing a lipid bilayer by tuning lateral stress in a physisorbed polymer-tethered membrane

The current study reports the compartmentalization of a physisorbed polymer-tethered phospholipid bilayer built using subsequent Langmuir–Blodgett (LB) and Langmuir–Schaefer (LS) transfers, where compartmentalization is due to buckling in the bottom (LB) monolayer and polymer-specific stress relaxation processes. Buckling arises from lateral stress within the membrane induced by a high (15–30 mol%) concentration of poly(2-ethyl-2-oxazoline) lipopolymers in the LB monolayer. Epifluorescence microscopy (EPI) and fluorescence recovery after photobleaching (FRAP) experiments confirm the formation of a homogeneous bilayer at low lipopolymer molar concentrations (low lateral stress), but demonstrate the compartmentalization of the bilayer into µm size compartments at elevated lipopolymer concentrations (high lateral stress). Quantitative EPI of the LB monolayer as well as additional atomic force microscopy (AFM) experiments show that bilayer compartmentalization, buckling and partial delamination of the LB monolayer occur without causing notable phospholipid–lipopolymer phase separations, but do preclude bilayer formation above buckled/delaminated regions after LS transfer. As long-time tracking experiments of photostable quantum dot-conjugated lipids in the compartmentalized bilayer system confirm, our membrane system enables the facile adjustment of the permeability of diffusion boundaries between bilayer compartments, thus providing an excellent experimental tool to mimic length-scale dependent diffusion processes observed in cellular membranes. We expect that the fundamental concept of lateral stress regulation and buckling-associated membrane compartmentalization can also be applied to other polymer–lipid composite materials than the one studied in this work.

Polymer Stabilized Lipid Membranes: Langmuir Monolayers

Polymer-tethered membranes combine fascinating structural, dynamic, and viscoelastic properties. Many important insights into these peculiar supramolecular systems can be obtained from studies on polymer-tethered monolayers. This chapter discusses recent experimental findings on polymer-tethered monolayers at the air–water interface. In particular, Langmuir monolayers which are comprised of pure lipopolymers and of binary phospholipid–lipopolymer mixtures are considered. Thermodynamic data as well as structural data based on a host of experimental techniques including X-ray and neutron reflectrometry, infrared reflection absorption spectroscopy, and sum frequency generation spectroscopy provide information on how lipopolymers organize at the air–water interface. This information is followed by a review of the viscoelastic properties of these systems, including the remarkable gelation transition that can be observed in lipopolymers and mixed phospholipid–lipopolymer monolayers. The diffusion properties are also discussed at length, and show that lipid diffusivity is critically dependent on the strength of inter-polymer interactions of lipopolymers.

Pure white-light emitting ultrasmall organic–inorganic hybrid perovskite nanoclusters

Organic-inorganic hybrid perovskites, direct band-gap semiconductors, have shown tremendous promise for optoelectronic device fabrication. We report the first colloidal synthetic approach to prepare ultrasmall (∼1.5 nm diameter), white-light emitting, organic-inorganic hybrid perovskite nanoclusters. The nearly pure white-light emitting ultrasmall nanoclusters were obtained by selectively manipulating the surface chemistry (passivating ligands and surface trap-states) and controlled substitution of halide ions. The nanoclusters displayed a combination of band-edge and broadband photoluminescence properties, covering a major part of the visible region of the solar spectrum with unprecedentedly large quantum yields of ∼12% and photoluminescence lifetime of ∼20 ns. The intrinsic white-light emission of perovskite nanoclusters makes them ideal and low cost hybrid nanomaterials for solid-state lighting applications.

Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy.

Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.

Abstract. The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.

Iterative layer-by-layer assembly of polymer-tethered multi-bilayers using maleimide–thiol coupling chemistry

The current study reports on the layer-by-layer assembly of a polymer-tethered lipid multi-bilayer stack using the iterative addition and roll out of giant unilamellar vesicles (GUVs) containing constituents with thiol and maleimide functional groups, respectively. Confocal microscopy and photobleaching experiments confirm stack integrity and stability over time, as well as the lateral fluidity of individual bilayers within the stacks. Complementary wide-field single molecule fluorescence microscopy and atomic force microscopy experiments show that increasing bilayer-substrate distances are associated with changes in lipid lateral mobility and bilayer morphology. Importantly, the described iterative approach can be employed to assemble multi-bilayer stacks with more than two bilayers, thus further reducing the influence of the underlying solid substrate on membrane behavior. Furthermore, the presence of lipopolymers within the multi-bilayer stacks results in fascinating membrane dynamics and organization properties, with interesting parallels to those found in plasma membranes. In that sense, the described multi-bilayer architecture represents an attractive model membrane platform for a variety of different biophysical studies.

Metric between buckling structures and elastic properties in physisorbed polymer-tethered lipid monolayers

The current study reports the use of buckling structures as a measure of film elasticity on physisorbed, solid-supported polymer-tethered lipid monolayers consisting of phospholipids and poly(ethylene glycol) (PEG) lipopolymers. These monolayer systems, built using the Langmuir–Blodgett (LB) technique, exhibit buckles over a wide range of lipopolymer concentrations. Systematic quantitative analysis of the buckling structures using epifluorescence microscopy and atomic force microscopy reveals that increased lipopolymer concentrations are associated with higher area fractions of buckling regions. Quantitative analyses of buckling profiles show, furthermore, that enhanced lipopolymer concentrations lead to increased buckling widths without notably altering buckling amplitude. On the basis of these experimentally determined buckling parameters, we are able to derive a metric between elastic properties and buckling structures in the polymer-tethered lipid monolayer system. This is achieved by combining mean-field calculations of elastic properties of polymer-tethered lipid membranes with buckling theory of an Euler column. Our findings are significant because they provide new insight into the fascinating materials properties of polymer-lipid supramolecular assemblies and represent a tool for the characterization of elastic properties in complex biomembrane-mimicking film architectures with buckling structures.

Cross-Selectivity Enhancement of Poly(vinylidene fluoride-hexafluoropropylene)-Based Sensor Arrays for Detecting Acetone and Ethanol

Two methods for cross-selectivity enhancement of porous poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP)/carbon black (CB) composite-based resistive sensors are provided. The sensors are tested with acetone and ethanol in the presence of humid air. Cross-selectivity is enhanced using two different methods to modify the basic response of the PVDF-HFP/CB sensing platform. In method I, the adsorption properties of PVDF-HFP/CB are altered by adding a polyethylene oxide (PEO) layer or by treating with infrared (IR). In method II, the effects of the interaction of acetone and ethanol are enhanced by adding diethylene carbonate (DEC) or PEO dispersed in DEC (PEO/DEC) to the film. The results suggest the approaches used in method I alter the composite ability to adsorb acetone and ethanol, while in method II, they alter the transduction characteristics of the composite. Using these approaches, sensor relative response to acetone was increased by 89% compared with the PVDF-HFP/CB untreated film, whereas sensor relative response to ethanol could be decreased by 57% or increased by 197%. Not only do these results demonstrate facile methods for increasing sensitivity of PVDF-HFP/CB film, used in parallel they demonstrate a roadmap for enhancing system cross-selectivity that can be applied to separate units on an array. Fabrication methods, experimental procedures and results are presented and discussed.