The spectral phasor approach to resolving membrane order with environmentally sensitive dyes.

Hyperspectral imaging is a technique that captures a three-dimensional array of spectral information at each spatial location within a sample, enabling precise characterization and discrimination of biological structures, materials, and chemicals, based on their unique spectral features. Nowadays most commercially available confocal microscopes allow hyperspectral imaging measurements, providing a valuable source of spatially resolved spectroscopic data. Spectral phasor analysis quantitatively and graphically transforms the fluorescence spectra at each pixel of a hyperspectral image into points in a polar plot, offering a visual representation of the spectral characteristics of fluorophores within the sample. Combining the use of environmentally sensitive dyes with phasor analysis of hyperspectral images provides a powerful tool for measuring small changes in lateral membrane heterogeneity. Here, we focus on applications of spectral phasor analysis for the probe LAURDAN on model membranes to resolve packing and hydration. The method is broadly applicable to other dyes and to complex systems such as cell membranes.

Operando Scanning Small-/Wide-Angle X-ray Scattering for Polymer Electrolyte Fuel Cells: Investigation of Catalyst Layer Saturation and Membrane Hydration- Capabilities and Challenges.

Polymer electrolyte fuel cells are an essential technology for future local emission-free mobility. One of the critical challenges for thriving commercialization is water management in the cells. We propose small- and wide-angle X-ray scattering as a suitable diagnostic tool to quantify the liquid saturation in the catalyst layer and determine the hydration of the ion-conducting membrane in real operating conditions. The challenges that may occur in operando data collection are described in detail─separation of the anode and cathode, cell alignment to the beam, X-ray radiation damage, and the possibility of membrane swelling. A synergistic development of experimental setup, data acquisition, and data interpretation circumvents the major challenges and leads to practical and reliable insights.

Fluorescent Probe for in Vivo Partitioning into Dynamic Lipid Droplets Enables Monitoring of Water Permeability-Induced Edema.

Lipid droplets (LDs) are intracellular organelles found in most cell types from adipocytes to cancer cells. Although recent investigations have implicated LDs in numerous diseases, the current available methods to monitor them in vertebrate models rely on static imaging using fluorescent dyes, limiting the investigation of their rapid in vivo dynamics. Here, we report a fluorophore chemistry approach to enable in vivo LD dynamic monitoring using a Nernstian partitioning mechanism. Interestingly, the effect of atorvastatin and osmotic treatments toward LDs revealed an unprecedented dynamic enhancement. Then, using a designed molecular probe with an optimized response to hydration and LD dynamics applied to Zebrafish developing pericardial and yolk-sac edema, which represents a tractable model of a human cardiovascular disease, we also provide a unique dual method to detect disease evolution and recovery.

Transportan 10 Induces Perturbation and Pores Formation in Giant Plasma Membrane Vesicles Derived from Cancer Liver Cells.

Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.

Implications of surfactant hydrophobic chain architecture on the Surfactant-Skin lipid model interaction.

Although surfactants have been widely used in skin care and other related applications, our knowledge about how surfactants interact with stratum corneum (SC) lipids remains limited. This work reports how surfactants interact with a lipid SC model by neutron diffraction and molecular dynamics (MD) simulations, focusing on examining the impact of surfactant molecular architecture. The surfactant-SC mixed membrane was constructed by an equimolar mixture of ceramide/cholesterol/fatty acids and surfactant at 1% molar ratio of total lipids. The arrangements of water and surfactant molecules in the membrane were obtained through neutron scattering length density (NSLD) profiles via contrast variation method, meanwhile, MD simulation clearly demonstrated the mechanism of hydration change in the surfactant-model SC mixed membrane. No drastic difference was detected in the repeating distance of the short periodicity phase (SPP) upon adding surfactants, however, it significantly enhanced the membrane hydration and reduced the amount of phase separated crystalline cholesterol, showing a strong dependence on surfactant chain length, branching and double bond. This work clearly demonstrates how surfactant architecture affects its interaction with the SC membrane, providing useful guidance for either choosing an existing surfactant or designing a new one for surfactant-based transdermal application.

Cyclodextrins Exert a Ligand-like Current Inhibitory Effect on the KV1.3 Ion Channel Independent of Membrane Cholesterol Extraction.

Cyclodextrins (CDs) are cyclic oligosaccharides capable of forming water-soluble complexes with a variety of otherwise poorly soluble molecules including cholesterol and different drugs. Consistently, CDs are widely used in research and clinical practice to deplete cholesterol from cellular membranes or to increase solubility and bioavailability of different pharmaceuticals at local concentrations in the millimolar range. Effects of CDs exerted on cellular functions are generally thought to originate from reductions in cholesterol levels. Potential direct, ligand-like CD effects are largely neglected in spite of several recent studies reporting direct interaction between CDs and proteins including AMP-activated protein kinase, ß-amyloid peptides, and α-synuclein. In this study, by using patch-clamp technique, time-resolved quantitation of cholesterol levels and biophysical parameters and applying cholesterol-extracting and non-cholesterol-extracting CDs at 1 and 5 mM concentrations, we provide evidence for a previously unexplored ligand-like, cholesterol-independent current inhibitory effect of CDs on KV1.3, a prototypical voltage-gated potassium channel with pathophysiological relevance in various autoimmune and neurodegenerative disorders. Our findings propose that potential direct CD effects on KV channels should be taken into consideration when interpreting functional consequences of CD treatments in both research and clinical practice. Furthermore, current-blocking effects of CDs on KV channels at therapeutically relevant concentrations might contribute to additional beneficial or adverse effects during their therapeutic applications.

Impact of macromolecular crowding on the mesomorphic behavior of lipid self-assemblies.

Using LAURDAN fluorescence we observed that water dynamics measured at the interface of DOPC bilayers can be differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG, under conditions where the polymers are not in direct molecular contact with the lipid interface. Specifically, we found that the decrease in water dipolar relaxation at the membrane interface correlates with an increased fraction of randomly oriented (or random coil) configurations in the polymers, as Gelatin > PEG > IgG > HSA. By using the same experimental strategy, we also demonstrated that structural transitions from globular to extended conformations in proteins can induce transitions between lamellar and non-lamellar phases in mixtures of DOPC and monoolein. Independent experiments using Raman spectroscopy showed that aqueous suspensions of polymers exhibiting high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this type of structure for polarizing water and consequently reducing its chemical activity. This effect is in line with one of the tenets of the Association Induction Hypothesis, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins (or other polymers) showing extended conformations. Overall, our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.

Boosting Membrane Hydration for High Current Densities in Membrane Electrode Assembly CO2 Electrolysis.

Despite the advantages of CO2 electrolyzers, efficiency losses due to mass and ionic transport across the membrane electrode assembly (MEA) are critical bottlenecks for commercial-scale implementation. In this study, more efficient electrolysis of CO2 was achieved by increasing cation exchange membrane (CEM) hydration via the humidification of the CO2 reactant inlet stream. A high current density of 755 mA/cm2 was reached by humidifying the reactant CO2 in a MEA electrolyzer cell featuring a CEM. The power density was reduced by up to 30% when the fully humidified reactant CO2 was introduced while operating at a current density of 575 mA/cm2. We reduced the ohmic losses of the electrolyzer by fourfold at 575 mA/cm2 by fully humidifying the reactant CO2. A semiempirical CEM water uptake model was developed and used to attribute the improved performance to 11% increases in membrane water uptake and ionic conductivity. Our CEM water uptake model showed that the increase in ohmic losses and the limitation of ionic transport were the result of significant dehydration at the central region of the CEM and the anode gas diffusion electrode-CEM interface region, which exhibited a 2.5% drop in water uptake.

Spatiotemporal Imaging of Water in Operating Voltage-Gated Ion Channels Reveals the Slow Motion of Interfacial Ions.

Ion channels are responsible for numerous physiological functions ranging from transport to chemical and electrical signaling. Although static ion channel structure has been studied following a structural biology approach, spatiotemporal investigation of the dynamic molecular mechanisms of operational ion channels has not been achieved experimentally. In particular, the role of water remains elusive. Here, we perform label-free spatiotemporal second harmonic (SH) imaging and capacitance measurements of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by aqueous solution on either side. We observe changes in SH intensity upon channel activation that are traced back to changes in the orientational distribution of water molecules that reorient along the field lines of transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. The time scale of these interfacial changes is influenced by the density of ion channels and is subject to a crowding mechanism. Ion transport along cell membranes is often associated with the propagation of electrical signals in neurons. As our study shows that this process is taking place over seconds, a more complex mechanism is likely responsible for the propagation of neuronal electrical signals than just the millisecond movement of ions.

Direct visualization of the lateral structure of giant vesicles composed of pseudo-binary mixtures of sulfatide, asialo-GM1 and GM1 with POPC.

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC18 show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide>asialo-GM1>GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells.